Evaluation of the Potential of Cobalamin Derivatives Bearing Ru(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy

Journal Pre-proof Lipid-core nanoparticles: Classification, preparation methods, routes of administration and recent advances in cancer treatment Pablo Graván, Aixa Aguilera-Garrido, Juan Antonio Marchal, Saúl A. Navarro-Marchal, Francisco Galisteo-González PII: S0001-8686(23)00038-6 DOI: https://doi.org/10.1016/j.cis.2023.102871 Reference: CIS 102871 To appear in: Advances in Colloid and Interface Science Revised date: 3 February 2023 Please cite this article as: P. Graván, A. Aguilera-Garrido, J.A. Marchal, et al., Lipid-core nanoparticles: Classification, preparation methods, routes of administration and recent advances in cancer treatment, Advances in Colloid and Interface Science (2023), https://doi.org/10.1016/j.cis.2023.102871 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2023 Published by Elsevier B.V. Journal Pre-proof Lipid-core nanoparticles: classification, preparation methods, routes of administration and recent advances in cancer treatment. Pablo Graván a,b,c,d,e,f ,†, Aixa Aguilera-Garrido a, †, Juan Antonio Marchal b,c,d,e, f, Saúl A. Navarro-Marchal d,e,g,* and Francisco Galisteo-González a,* a Department of Applied Physics, Faculty of Science, University of Granada, 18071 Granada, Spain. b Department of Human Anatomy and Embryology, Faculty of Medicine, University of of Granada, 18016 Granada, Spain. Instituto de Investigación Biosanitaria de Granada (ibs.GRANADA), 18012 Granada, Spain. d Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical ro c e -p Research (CIBM), University of Granada, 18016 Granada, Spain. Excellence Research Unit Modelling Nature (MNat), University of Granada, 18016 f re Granada, Spain. BioFab i3D - Biofabrication and 3D (bio)printing laboratory, University of Granada, 18100 g lP Granada, Spain. Cancer Research UK Edinburgh Centre, Institute of Genetics and Cancer, University of na Edinburgh, EH4 2XU Edinburgh, U.K. Jo ur † These authors contributed equally to the work. * Co-corresponding authors E-mail: saul.navarro-marchal@ed.ac.uk; galisteo@ugr.es. lipid nanoemulsions, lipid nanocapsules, solid lipid nanoparticles, nanostructured lipid nanocarriers, cancer, preparation methods, routes of administration Nanotechnological drug delivery platforms represent a new paradigm for cancer therapeutics as they improve the pharmacokinetic profile and distribution of chemotherapeutic agents over conventional formulations. Among nanoparticles, lipid-based nanoplatforms possessing a lipid core, that is, lipid-core nanoparticles (LCNPs), have gained increasing interest due to lipid properties such as high solubilizing potential, versatility, biocompatibility, and biodegradability. However, due to the wide spectrum of morphologies and types of LCNPs, there is a lack of consensus regarding their terminology and classification. According to the 1 Journal Pre-proof current state-of-the-art in this critical review, LCNPs are defined and classified based on the state of their lipidic components in liquid lipid nanoparticles (LLNs). These include lipid nanoemulsions (LNEs) and lipid nanocapsules (LNCs), solid lipid nanoparticles (SLNs) and nanostructured lipid nanocarriers (NLCs). In addition, we present a comprehensive and comparative description of the methods employed for their preparation, routes of administration and the fundamental role of physicochemical properties of LCNPs for efficient antitumoral drug-delivery application. Market available LCNPs, clinical trials and preclinical in vivo studies of promising LCNPs as potential treatments for different cancer pathologies are summarized. of 1. Introduction Cancer encompasses a heterogeneous group of disorders defined by an abnormal and ro uncontrolled growth of the cells of a tissue or organ beyond their usual limits.[1] Worldwide, -p an estimated 19.3 million new cancer cases and almost 10.0 million cancer deaths occurred in 2020, being the first or second leading cause of death before the age of 70 years in 112 of 183 re countries.[2] Cancer can develop anywhere in the body, with surgical resection, chemotherapy and radiotherapy being the three main treatment strategies.[3] Chemotherapy consists of the lP administration of an antitumoral drug such as tamoxifen (TMX), paclitaxel (PTX), docetaxel (DTX), doxorubicin (DOX) or methotrexate (MTX), that can kill cancer cells throughout the na body, even those at the edges of tumors, which may not be removed by surgery. However, the poor aqueous solubility and permeability of these drugs have resulted in low bioavailability and Jo ur decreased treatment efficiency.[4] Furthermore, this limitation along with the non-specificity of treatment, results in patients being treated at the maximum tolerated dose of these antitumor compounds, which entails several adverse and off-target effects. These obstacles decrease the therapeutic value of many anticancer drugs and their respective chemotherapy treatment.[5] Nanotechnology stands out as an attractive strategy to avoid the aforementioned problems associated with conventional antitumor drug delivery. Nanosystems can overcome biological barriers and control the release of drugs to target sites, enabling the use of lower doses while decreasing the side effects and increasing the treatment efficiency.[6,7] Anticancer drug-loaded nanoparticles (NPs) possess several advantages compared to conventional chemotherapy due to their reduced toxicity, high-loading capacity, stability, specificity, tolerability and efficacy.[5] Nowadays, nanotechnology plays an important role in the targeted delivery of drugs for cancer treatments. Among NPs, lipid nanoparticles (LNPs) have gained increasing interest due to their potential to load and release drugs from the biopharmaceutical classification system (BCS) of class II 2 Journal Pre-proof (low solubility and high permeability) and class IV (low solubility and low permeability).[8] Due to lipid properties such as high solubilizing potential, flexibility and biodegradability, poorly water-soluble drugs can be loaded into LNPs, increasing their bioavailability. Furthermore, lipids as carriers have the potential to improve oral drug delivery due to their ability to enhance gastrointestinal solubilization and absorption, as well as topical application.[6,9] In the last decade, LNPs have also been intensively studied for enabling gene therapies. LNPs technology is proposed to be a dominant non-viral technology in gene therapy, mainly due to their design flexibility.[10] In August 2018, the first ever approved LNP delivery of nucleic- of acid based drugs was reported by the Food and Drug Administration (FDA) and by the European Commission (EC): Onpattro™ ((Patisiran (ALN-TTR02)), a lipid complex ro encapsulating a siRNA for the treatment of the hereditary transthyretin-mediated -p amyloidosis.[11] In this sense, the COVID-19 vaccines BNT162b2 (Comirnaty®; BioNTech and Pfizer) and Moderna COVID-19 (mRNA-1273) (ModernaTX, Inc; Cambridge, re Massachusetts), which are based on LNPs encapsulating a modified mRNA, are outstanding examples.[12–14] LNPs are also being investigated as the formulation vehicle for mRNA lP vaccines for cancer immunotherapy.[15] Among LNPs for nucleic-acids delivery, liposomes are the most employed systems.[16] na Liposomes represent the first generation of LNPs [17] and, since their discovery in 1965 by Sir Alec Bangham,[18] several studies have been reported. In fact, the FDA has already approved Jo ur liposomal formulations for cancer treatment such as Daunoxome®, Doxil® or Myocet®. Liposomes are spherical lipid vesicles formed mainly by natural or synthetic phospholipids organized in a bilayer structure.[19] In addition, other membrane bilayer constituents, such as cholesterol or hydrophilic polymers conjugated lipids, can be added to their formulation.[20]. Liposomes have an aqueous core in which water-soluble drugs can be dissolved and encapsulated. However, liposomes can also entrap lipophilic drugs into their lipidic bilayer membrane. This structure causes liposomes to have a reduced drug loading capacity for lipophilic drugs compared to LNPs with a lipidic core, i.e., lipid core nanoparticles (LCNPs). Other significant drawbacks related to liposomes are production scalability, the need to use organic solvents during the production and the relatively low stability in biological fluids.[21] LNPs composed of a lipid core i.e., lipid core nanoparticles (LCNPs) (LCNPs) such as microemulsions (MEs) and nanoemulsions (NEs) are also widely employed. MEs were described almost simultaneously to liposomes in 1959 by Schulman.[22] The description and characterization of MEs and, subsequently, NEs, lead to extensive research into this type of 3 Journal Pre-proof drug delivery systems.[23] Furthermore, these emulsion-type LCNPs are considered to be the starting point for the development of solid lipid nanoparticles (SLNs) and nanostructured lipid nanocarriers (NLCs) in the 90s. LCNPs represent an alternative to liposomes for the encapsulation and delivery of hydrophobic drugs. The different systems included in the category of LCNPs have different characteristics in terms of stability and drug release kinetics, which will be discussed in the subsequent sections.[24] Overall, LCNPs offer higher hydrophobic drug loading capacities and physical stability than liposomes.[25] Moreover, production methods for LCNPs include organic solvent-free methods, making easier to scale up their production (Section 3). of In this review, we focus on the description of LCNPs, along with their methods of synthesis, routes of administration and applications as antitumoral drug delivery systems. Since liposomal ro formulations are based on an aqueous core, they will not be included in this review, although -p they are extremely important in LNPs technology development. re 2. Classification LCNPs are differentiated according to the physical state of their lipid core component (Figure lP 1). Accordingly, they are classified in liquid lipid nanoparticles (LLNs), which includes lipid Jo ur 1C) and NLCs (Figure 1D). na nanoemulsions (LNEs) (Figure 1A) and lipid nanocapsules (LNCs) (Figure 1B), SLNs (Figure Figure 1. Schematic representation of LCNPs. They are classified according to the physical state of their lipidic component in LLNs, which include (A) LNEs and (B) LNCs; (C) SLNs and (D) NLCs. Due to the various morphologies and preparation methods existing for LCNPs, the representation presented here could vary. Created with BioRender.com. 4 Journal Pre-proof 2.1. Liquid Lipid Nanoparticles (LLNs) LNPs presenting a liquid lipid core at body temperature are widely described in the literature. LNEs and LNCs are the main types of nanocarriers included in this category. Nanocapsules (NCs) can be defined as vesicular nanosystems with an inner cavity (core) surrounded by a polymeric protective shell, i.e., a core-shell structure.[26,27] The drug or cargo substance can be either confined in the core material or attached to the polymeric shell. Different types of NCs can be formulated according to the nature of their core, which can consist of a liquid phase, either an oily or an aqueous core, a solid phase of a polymeric matrix or a hollow internal structure.[28] In the case of NCs comprising a liquid oily core, generally formed of by vegetable oils or fatty acids, their lipidic component plays an undeniably leading role. The oily core affords a safe and efficient dissolving media for hydrophobic drugs that, in the case ro of oils with therapeutic potentials, can also provide a synergistic and beneficial functionality. -p Furthermore, despite the development of NCs manufactured with an aqueous core, a hollow inner structure or, to a lesser extent, a solid core, oily liquid NCs surrounded by a polymeric re layer are the most widely reported.[29] In fact, some reports directly define NCs as vesicular structures containing an oily core surrounded by a rigid shell, ignoring other feasible lP compositions.[30–33] Therefore, this review includes polymeric oily NCs as part of LCNPs and categorizes this type of nanosystem as LLNs because of their liquid lipidic inner core. na Moreover, there is a need to use a term that includes nanosystems with a core-shell structure, i.e., NCs, based on a liquid lipid core. In this regard, we propose coining a term already LNCs. Jo ur employed in the literature,[34] which accurately describes the structure of these nanocarriers: 2.1.1. Lipid Nanoemulsions (LNEs) NEs are defined as biphasic dispersions of two immiscible liquids, in which one liquid is dispersed within the other in the form of nanodroplets stabilized by an amphiphilic surfactant. While MEs and NEs can appear similar, their stability and structure differ. Contrary to MEs, which are equilibrium systems thermodynamically stable under certain conditions, NEs are non-equilibrium systems that tend to separate into the two phases but possess a high kinetic stability. Interestingly, MEs, as thermodynamically stable systems, are sensitive to conditions of temperature and composition, unlike NEs, which are relatively immune to physical and chemical changes. In the pharmacological and drug delivery field, as some administration routes involve physicochemical changes to the environment, NEs are more suitable for clinical 5 Journal Pre-proof applications. For further insights, readers are referred to reports detailing the differences between NEs and MEs.[35,36] NEs can be either oil in water (O/W) or water in oil (W/O).[37] However, in clinical applications the aqueous dispersion medium is adopted, LNEs, i.e., nanodroplets of oil dispersed in water, are the most investigated and employed. LNEs can be directly employed for active delivery and targeting as drug delivery systems or can act as a template for the preparation of polymeric NPs and LNCs. [38–40] 2.1.2. Lipid Nanocapsules (LNCs) of The term nanocapsule was first employed in 1977 by Couvreur et al.[41,42] Since then, an exhaustive development of this type of nanoparticulated system has been achieved. However, ro the denomination of LNCs was not firmly employed until the 2000s, when it appeared in the -p patent No. WO02688000 by Herault et al.[34] These LNCs were composed of an oily core built up with capric and caprylic acid medium-chain triglycerides (Labrafac®) and a surfactant shell re of a polyethylene glycol(PEG)ylated nonionic surfactant (Solutol®) and lecithin (Lipoid®).[43–46] The patented LNCs are described as nanoemulsion-template NCs lP formulated by a novel phase inversion temperature (PIT) methodology. This technique includes an additional stage of cycling temperatures, which provokes the over-concentration of the na surfactant at the interface of the oil droplets. As a result, a thick surfactant layer is created around the nanodroplets, forming a core-shell structure. These authors state that the major Jo ur difference between LNCs and LNEs comes from the type of energy provided to their formation, thus, LNCs are more rigid than LNEs.[47] Some authors demonstrate the existence of two types of LNCs, based on the structure and composition of their shells: polymer-shelled LNCs and surfactant shelled NCs.[48,49] The former includes a polymer in their shell, whereas the latter are the described and patented LNCs produced by the novel PIT method. Surfactant-shelled LNCs give rise to an interesting debate on whether the use of a polymer is necessary for the preparation of oil-based NCs. The diversity of synthesis methods and the broad range of possible compositions generate a wide number of conformations and formulations of LNCs, which leads to a lack of consensus in the classification of these nanocarriers. From a structural point of view, we consider that any nanocarrier possessing a liquid lipid core and a core-shell structure, regardless of the composition and synthesis method employed, should be considered as LNCs. Furthermore, since a large part of LNCs are prepared from LNEs, it is important to evaluate the transition from LNEs into LNCs, i.e., the characteristics of the nanosystem that will define the 6 Journal Pre-proof subtle line which differentiates them. In a practical sense, the main difference is the inclusion of a polymer during the nanocarrier synthesis. However, given that many surfactants are proper polymers[50,51] and surfactant-shelled LNCs exist, according to some authors, this phenomenon should be accurately evaluated. The most frequent technique employed for studying the structure of NCs and determining their core-shell structure is TEM performed after freeze-fracture. However, because of the nanometer scale of the samples, this method remains very difficult and, often subjective, in determining the wall thickness.[52] Preetz et al. applied atomic force microscopy to study the shell structure of three different preparations: LNCs prepared by the layer-by-layer deposition technique based on a LNE template, the LNE itself of prepared by a high-energy method and PEG-PLA LNCs prepared by interfacial deposition of preformed polymer.[52] The stiffness of the shell of PEG-PLA LNCs and 5-layer LNCs were ro found to be 33.3 and 14.3 %, respectively, higher than the original LNE. This study proved -p atomic force microscopy as a suitable technique to distinguish NCs from NEs. Some reports compare the stability and physicochemical properties of LNEs and LNCs, which essentially re differ in the addition (or not) of polymer during synthesis.[53–55] Clearly, the addition of polymer and the consequent formation of the polymeric shell improve the performance of the lP carrier. These studies indicate that there are significant differences between LNEs and LNCs. However, this is truly evident only when polymers are used. In the absence of polymers, as it na is the case of LNCs prepared by the novel PIT technique, it is necessary to study the rigidity of the shells in order to distinguish between the two types of nanocarriers and, frequently, the limit Jo ur is not well defined. From our point of view, LNCs, defined as a core-shell structure where the core is composed of a liquid lipid phase, can be considered an evolution of LNEs. LNCs are able to maintain the advantages of LNEs, mainly the high loading capacity due to the liquid core, while integrating a stiffer protective barrier, which provides stability and less drug leakage during preparation compared to typically employed surfactants.[56] LNEs can be distinguished from LNCs based on the absence of the core-shell structure and a lower surface thickness and rigidity. However, this difference is not always so clear, as discussed in the previous paragraph. Moreover, it is important to note that LNEs and LNCs can be used for the same applications and some authors employ these terms indistinguishably. In this regard, we consider it appropriate to employ an inclusive designation that consolidates both concepts, as the term LLNs, encompassing both LNEs and LNCs, that accurately and faithfully describes the fundamental characteristics of these nanosystems. 7 Journal Pre-proof 2.2. Solid Lipid Nanoparticles (SLNs) In their attempts to avoid burst release and produce systems for controlled drug release, Speiser and co-workers started to work with small-sized particles. They created micro and nano-sized solid lipid matrixes as drug carriers, which they named as micro and nanopellets.[57] These studies laid the foundation for the later development of SLNs in the same decade. In 1993, Gasco and co-workers patented the production of solid lipid micro-spheres, 50-800 nm in size, through a hot emulsification process.[58] However, Müler and co-workers were the first to name those colloidal systems as SLNs. They described a production method using high pressure homogenization of melted lipid in water at high temperature.[59] of SLNs are made of lipids which are solid at room and body temperature.[60] These form a crystalline and organized structure, which is stabilized by emulsifiers and where the drug is ro entrapped. They were developed to overcome fast degradation and toxicity problems associated -p with LNEs and liposomes, and with polymeric NPs, respectively. The slower diffusion of the drug through the solid matrix allows a sustained drug release from SLNs over a longer period re of time.[61,62] On the other hand, lipids used in SLNs formulations are biocompatible, which reduces their toxicity.[62] lP SLNs present other advantages, such as the possibility of encapsulating lipophilic or hydrophilic drugs with increased solubility, large-scale production and sterilization, and the na existence of production methods where the use of solvents can be avoided.[62] However, the crystalline structure of the lipidic core may represent a disadvantage, since the highly organized Jo ur lipid structure promotes the exclusion of the encapsulated drug. The crystallization of the lipid molecules during the synthesis process leads, in some cases, to low drug encapsulation efficiency and drug loading.[63,64] On the other hand, pure lipids, as found in SLNs, undergo polymorphic changes, i.e., lipids which have recrystallized in a low melting and less stable morphology can change their configurations to a more stable one over time. These modifications involve changes in the crystalline structure which can cause drug expulsion from the lipid core, as well as lead to precipitation of large drug crystals in the aqueous phase.[63] 2.3. Nanostructured lipid carriers (NLCs) SLNs were presented as an avant-garde alternative to LNEs, liposomes, and polymeric NPs. However, problems related to the crystalline lipid core, such as low drug loading, drug exclusion, and slow drug release, lead to the development, in 1999, of a new generation of LCNPs, the so-called NLCs.[64,65] In these particles, the matrix is composed not only of one solid lipid, but of a blend of solid and liquid lipids. The main advantage of NLCs, when 8 Journal Pre-proof compared to SLNs, is an increased loading capacity of actives, which was first shown for retinol.[66] The solid and liquid lipid blend of NLC remains solid at body temperature. The addition of the oil compound distorts the formation of perfect lipid crystals, thus creating imperfections, which increase the uptake capacity for drug or active. This “structure” increases the drug loading capacity. The localization of the drug does not only depend on the structure of the lipid matrix but also on the lipophilicity and structure of the drug itself. An additional advantage of NLCs is a minimized risk of drug expulsion over time. The addition of oil to a solid lipid has recently proven to be able to prevent the re-crystallization of the lipid in a less stable morphology.[67,68] Hence, no changes in morphology occur, and thus, the drug of expulsion from NLCs over time is reduced. Therefore, NLCs generally possess higher physical ro stability than SLNs. -p 3. Preparation methods In this section, synthesis and preparation methods of LNPs are classified. We provide an re overview of the current methodology in this area, highlighting procedural differences between techniques with a shared physical basis but which may need an adaptation depending on the na 3.1. Methods for LNEs preparation lP nature of the lipid component of the carrier. LNEs, being non-equilibrium systems, require an energy input to be formed. This energy can Jo ur proceed from the potential energy stored in the system or from mechanical devices that create powerful disruptive forces. Consequently, two broad categories of techniques for the preparation of LNEs can be distinguished: high-energy methods and low-energy methods. 3.1.1. High-energy methods High-energy methods employ intense disruptive forces which break up the oil and water phases to form nanodroplets. Typically, a coarse emulsion is first produced by mixing both phases. The coarse emulsion is then homogenized, employing mechanical devices such as highpressure homogenizers, high-shear homogenizers, microfluidizers or ultrasonicators (Figure 2). Those techniques use different mechanisms to produce cavitation and shear stress on the sample and break down the particles.[69] The shear stress is the force per area of lateral interaction between the fluid layers.[70] This force causes the deformation of the material by glissade along the plane parallel to the acting stress. Shear stresses are caused by friction between the fluid particles, and they are the consequence of different velocities within the fluid. In turbulent flow, 9 Journal Pre-proof the bigger velocity differences within the particles leads to a greater shear stress in the fluid. Homogenization devices promote shear stress within the fluid to reduce particles size, which ultimately will depend on the type of instrument and their operating conditions, as energy, time, number of cycles and sample formulation and properties. These methods are industrially scalable, but they have some disadvantages such as cost and high temperatures needed for some of the processes, which can inactivate thermolabile drugs and macromolecules, including Jo ur na lP re -p ro of proteins, enzymes and nucleic acids. Figure 2. Representation of typical procedures for LNEs preparation through high-energy methods. First, a coarse emulsion is prepared by mixing both phases. Then, it is subjected to (A) high-pressure homogenization, (B) high-shear homogenization, (C) microfluidization, (D) Ultrasound, or a combination, to obtain a final LNE suspension. Created with BioRender.com. A) High-pressure homogenization This method involves the use of high-pressure homogenizers or piston gap homogenizers. These instruments consist of high-pressure pumps which impel the fluid towards a disruption 10 Journal Pre-proof unit producing several forces, such as hydraulic shear, intense turbulence and cavitation, acting together to generate shear stresses and contributing to particle size reduction (Figure 2A).[71] The droplet size and polydispersity index depend on the pressure, which usually ranges from 50 to 200 MPa, the number of cycles and the temperature of the system, along with the emulsion composition itself. High-pressure homogenization can be processed in high temperature (hot homogenization) or in low temperature (cold homogenization). This method has various advantages such as an easy scale up, short process time and the avoidance of organic solvents. B) High shear homogenization or high-speed stirring of The reduction of particle size in this technique is mainly driven by shear stress. The high shear mixers use a rotor/stator system to produce the shear stresses.[72] This system makes the fluid ro flow between a static platform and an inner-rotary one (Figure 2B). The rotation of the inner -p device acts as an impeller and produces a turbulent flow, which further enhances the shear re stresses.[73] C) Microfluidization lP A microfluidizer is a patented mixing device that uses a high-pressure positive displacement pump (5 to 135 MPa) which repeatedly forces a coarse emulsion through an interaction chamber na consisting of small channels, called microchannels, until the desired particle size is obtained (Figure 2C).[74] Turbulent flow along with cavitation causes droplet disruption and NE Jo ur formation. The bulk emulsion is then filtered to remove large droplets, resulting in a uniform NE. This technique is suitable for its use at industrial scale.[75] D) Ultrasound Ultrasound waves produce changes in the pressure within the fluid over time. At some points in the fluid, pressure reduction is enough to allow fluid evaporation and the formation of bubbles. This phenomenon is known as cavitation. When bubbles collapse, they generate the projection of the liquid and high pressure which can disrupt the fluid droplets or erode the solid surfaces, leading to the formation of smaller particles.[76] The ultrasound waves also produce motion of the fluid and the particles, generating shear stress on the particles surface and contributing to their reduction in size. Ultrasound is a highly effective technique but is not suitable for industrial scale, unlike high-pressure homogenization and microfluidization. Its performance range is limited to laboratory scale and small batches (Figure 2D). 11 Journal Pre-proof 3.1.2. Low-energy methods Nanoemulsification methods involving a low quantity of applied energy rely on the stored internal chemical energy of the system to form nanodroplets. These methods are very attractive because of their low equipment cost.[77] Low-energy methods can be classified according to whether a phase inversion of the surfactant is produced or not. If changes in the surfactant spontaneous curvature happen, they are designated as phase inversion methods. Phase inversion methods employ the chemical energy released by a phase transition produced during the emulsification process. This phase transition can be triggered either by changing the temperature (PIT) or the composition (PIC). If no phase inversion is involved, methods are of termed as spontaneous emulsification. ro A) PIT method -p The PIT method (Figure 3A) was introduced by Shinoda and Saito.[78,79] This method is based on the ability of nonionic surfactants, such as polyethoxylated surfactants, to modify their re affinities for water and oil as a function of the temperature. Polyethoxylated surfactants tend to become lipophilic upon heating as a consequence of the dehydration of polyoxyethylene lP groups.[80] In the PIT method, oil, water and nonionic surfactant are mixed together at its PIT or hydrophobic lipophilic balance (HLB) temperature, where the surfactant exhibits a similar na affinity for the two immiscible phases, forming an unstable emulsion.[81] At a fixed composition, this method consists of a rapid change in temperature from the PIT through rapid Jo ur heating or cooling to generate kinetically stable W/O or O/W NEs, respectively.[82] It is a simple, low-energy consuming and solvent-free method suitable for industrial scale-up. However, a limitation of this procedure is that it can only be applied to surfactants sensitive to changes in temperature. B) PIC method In the PIC method (Figure 3B), also known as emulsion inversion point (EIP) method, the transition in spontaneous curvature is achieved by changes in the composition during emulsification, at constant temperature.[83] The PIC method is a solvent-free technique that yields kinetically stable NEs at room temperatures. PIC is preferred from a scale-up point of view, since it is industrially easier to add one component to a large volume of emulsion rather than to generate a sudden change in temperature. Furthermore, the PIC method is more suitable for thermosensitive components and drugs. Components (water or oil) are added over a mixture of the other components (oil-surfactant or water-surfactant). Typically, water is added 12 Journal Pre-proof progressively and dropwise into the oil phase comprising a W/O emulsion. As the fraction of water increases, surfactant hydrophilic-lipophilic properties begin to balance. When the transition composition is exceeded, phase inversion occurs and a O/W LNE is formed.[84] C) Spontaneous emulsification In this process, two contacting immiscible liquids that are not in equilibrium can form droplets without the need of an external energy input, taking advantage of the chemical potential gradients between both phases. Spontaneous emulsification is driven mainly by the rapid diffusion of a water-miscible solvent present in the organic phase through the aqueous phase, of producing a local supersaturation near the interface that gives rise to the emulsification.[85] Other factors, such as interfacial turbulence and low interfacial tension values, play a secondary ro role in defining colloidal characteristics of the final resulting system.[86] -p Spontaneous emulsification can be produced by the so-called Ouzo effect,[87] also called solvent-displacement method or nanoprecipitation (Figure 3C). In this method, the oily phase re is dissolved in water miscible organic solvents comprising the organic phase. The aqueous phase consists of water and a hydrophilic surfactant. Both phases are mixed under magnetic lP stirring. Oil nanodroplets are instantaneously formed by rapid diffusion of the organic solvent in the aqueous phase and the consequent change in oil solubility.[88] Later, organic solvents na are removed by suitable means, such as vacuum evaporation, and oil droplets remain dispersed in the aqueous phase. Oil viscosity, the HLB of the surfactant and the water solubility of the Jo ur organic solvent are important parameters determining the size and quality of the oil nanodroplets prepared by this process. This methodology has also been reported for freesurfactant systems.[85] However, surfactants stabilize the interface of the formed nuclei and contribute to obtain smaller sizes and/or better polydispersity indexes, as well as provide a higher colloidal stability.[88] With this method, LNEs can be spontaneously fabricated at room temperature with simple equipment. The main drawback is the use of organic solvents, which need to be removed and generates several difficulties during scale-up. Other spontaneous emulsification methods are the dilution of microemulsions technique, also termed the microemulsion method, reported by Taylor and Ottewill,[89] and the dilution of surfactant aggregates.[90] 13 na lP re -p ro of Journal Pre-proof Jo ur Figure 3. Low-energy methods for LNEs preparation. (A) PIT method and (B) PIC methods are based on a phase inversion phenomena, while (C) spontaneous emulsification is driven mainly by the rapid diffusion of a water-miscible organic solvent through the aqueous phase and the subsequent local supersaturation at the interface. Created with BioRender.com. 3.2. Methods for LNCs preparation Techniques describing the manufacturing of LNCs involve the preparation of LNEs.[27] Nanodroplets of emulsions act as bioreactors where a protective layer is formed, generating the core-shell structures of LNCs. As mentioned in section 2.1.2, the core-shell structure of LNCs can be produced with or without the addition of a polymer during their preparation. Surfactantshelled LCNs can be formed through an adapted PIT methodology. When LNCs preparation is based on the formation of a polymeric wall, methods can be classified depending on how the polymeric shell is obtained: by interfacial polymerization of monomers or employing preformed polymers during the preparation. 14 Journal Pre-proof 3.2.1. PIT method for LNCs preparation. The PIT method is normally employed for the preparation of LNEs. However, Herault et al. reported an adapted PIT method for the preparation of LNCs.[91] This novel methodology included an additional stage of cycling temperatures PIT (Figure 4). In this technique, an oily phase (Labafrac) and a water phase in the presence of tensioactives (Lipoïd and Solutol) are first mixed under magnetic stirring. The mixture is then heated from room temperature to 85 ºC and subsequently to 60 ºC at a rate of 4 ºC/min. Three temperature cycles (85-60-85-60-85 ºC) are then applied to achieve phase inversion. The formed emulsion is then rapidly cooled through dilution with cold water to produce LNCs. The temperature cycling produces surfactants to be of trapped and concentrated in the interfacial zone, generating a thick tensioactive shell that acts na lP re -p ro as a barrier to the oil diffusion (i.e., a nanocapsular system).[92] Jo ur Figure 4. Adapted PIT method for the preparation of LNCs. Created with BioRender.com. 3.2.2. Interfacial polymerization This technique is based on the fast polymerization of a monomer at the interface of an emulsion, and can be considered independent of the technique chosen to generate the LNE. The polymerization phenomena can occur either during the emulsion process: A) in situ polymerization (Figure 5A); or once LNEs are prepared: B) polymerization in emulsion (Figure 5B). In situ polymerization was the first reported method of an oil-in-water system where the isobutylcyanoacrylate monomer polymerized at the oil/water interface of the nanodroplets.[93,94] Alkylcyanoacrylates are the most commonly employed monomers for in situ polymerization. In a general procedure, oil, monomer and the active compound to be encapsulated are dissolved in a water-miscible solvent. This organic phase is injected, under magnetic stirring, into an aqueous phase containing a hydrophilic surfactant. Polymerization starts with the addition of the initiator/activator in the continuous phase or induced by UV, ultrasonication or enzymes.[38] This technique presents some major drawbacks: the lack of 15 Journal Pre-proof control of the polydispersity and molecular weight of the polymer obtained, the presence of reactive monomers or oligomers, which can cause unwanted chemical reactions and drug inactivation, as well as the possibility of cross-reactions with the drug.[95,96] To avoid these lP re -p ro of issues during the polymerization process, methods based on preformed polymers are preferred. Figure 5. Methods for the preparation of LNCs through in situ polymerization of monomers. Jo ur with BioRender.com. na (A) interfacial polymerization method and (B) polymerization in emulsion technique. Created 3.2.3. LNCs preparation from preformed polymers For these methods, polymers are dissolved or suspended into the continuous or dispersed phase during LNCs preparation, depending on their nature and solubility. Emulsiondiffusion/evaporation, emulsion-coacervation, solvent-displacement and layer-by-layer are the main methodologies employed to obtain LNCs from preformed polymers. Furthermore, double emulsions, either water in oil in water (W/O/W) or oil in water in oil (O/W/O), can be applied to obtain LNCs. The principle of these double emulsions is associated with emulsiondiffusion/evaporation and coacervation methods.[26] A) Emulsion-diffusion/evaporation The preparation of LNCs using the emulsion-diffusion/evaporation method is based on emulsion of the organic phase into the aqueous one and the subsequent elimination of the organic solvent. This is achieved either by the addition of water and dilution of the system, 16 Journal Pre-proof which provokes the diffusion of the solvent (emulsion-diffusion) (Figure 6A), or through evaporation (emulsion-evaporation) (Figure 6B). LNCs are formed by a combination of polymer precipitation and interfacial deposition phenomena during the diffusion or evaporation event.[28] B) Emulsion-coacervation In the emulsion-coacervation method (Figure 6C), a LNE is employed as a template where the polymeric wall is formed on the surface of the nanodroplets through the formation of a coacervate, which causes polymer precipitation. The physical coacervation process can be of provoked by: electrolytes, the addition of a water miscible non-solvent or a dehydrating agent, ro or temperature modification.[26] -p C) Solvent-displacement method This method, also called interfacial deposition or nanoprecipitation, was first described by Fessi re et al [97] (Figure 6D). It is driven by the Ouzo effect, as in the case of the spontaneous emulsification process presented in the previous section. For instance, for LNCs preparation, lP the preparation procedure only differs in the employment of a polymer to produce the coreshell structure on the surface of LNEs. In this method, a solvent phase consisting of a solution na of polymer, drug, oil and, if needed, a lipophilic tensioactive, is added with moderate stirring to a non-solvent phase, usually water, containing a hydrophilic surfactant.[88] Generally, the Jo ur solvent and non-solvent phases are called organic and aqueous phases, respectively. Polymeric substances and surfactants can be added in the organic or water phase according to their properties. After LNCs formation, the organic solvent is removed. Polymer precipitation and solvent diffusion are the key factors that drive the process of LNCs formation. Polymeric NPs are also prepared with this technique by using an organic phase in the absence of a liquid lipid.[98] D) Layer-by-Layer The Layer-by-Layer method was developed by Sukhorukov et al. (1998).[99] In this method, polymer layers of polycations and polyanions are adsorbed by irreversible electrostatic attraction on the surface of a colloidal template (Figure 6E). The polymer layer can be adsorbed either by incubation in the polymer solution or by decreasing polymer solubility through the addition of a miscible solvent. The procedure is then repeated with multiple polymer layers that are deposited sequentially. Therefore, the main advantage of this technique is that it allows to 17 Journal Pre-proof control the composition and thickness of polymeric shells. When LNEs are employed as template material, LNCs are obtained. Other colloidal templates, such as inorganic particles made of iron oxide, gold, calcium carbonate and silica, are widely employed.[100–102] These materials can be easily removed under mild conditions in order to obtain hollow NCs. The payload entrapment into these hollow NCs is achieved through diffusion (hydrophilic substances) and hydrophobic effect (hydrophobic substances). Polymeric NCs methods that do not lead to the obtention of LNCs, have also been extensively employed and reviewed. Readers are referred to polymeric NCs formulation techniques reported by Mora-Huertas et al.[26] , Jo ur na lP re -p ro of Kothamasu et al.,[103] Vauthier et al.[40] and by Deng et al.[28] 18 Jo ur na lP re -p ro of Journal Pre-proof Figure 6. Methods for the preparation of LNCs by preformed polymers: (A) emulsion-diffusion, (B) emulsion-evaporation, (C) emulsion-coacervation, (D) solvent displacement and (E) layer-by-layer methods. When no oil is employed during the preparation procedure, the layer-by-layer and solvent displacement methods give rise to polymeric NPs and NCs. Created with BioRender.com. 3.3. Methods for SLNs and NLCs preparation Synthesis of SLNs and NLCs usually employs similar methodologies as those described for LNEs. However, the different physical nature of the lipid component precises adaptations in 19 Journal Pre-proof some cases. In this section, we focus on modifications of the previously described methodologies. 3.3.1. High-energy methods The solid nature of the lipid component, where hydrophobic drugs are supposed to be included, usually requires the inclusion of a lipid melting step. The homogenization techniques employ different mechanisms to produce cavitation and shear stress on the sample and break down the particles.[63] of A) High-pressure homogenization There are two different variants of the high-pressure homogenization method: hot ro homogenization and cold homogenization (Figure 7A). In the hot homogenization technique, -p the melted lipids are pre-emulsified with the drug and then introduced into the high-pressure homogenizer, which is kept at a temperature above the lipids melting point. The resulting final re mixture is cooled to let the lipid solidify. During this process, the drug stability can be compromised due to the high temperatures maintained during the process.[63] Schwarz and lP coworkers used this method in 1994 to produce SLNs consisting of the triglyceride trilaurin stabilized with soy lecithin and Poloxamer 188. They compared the particle sizes obtained with na those obtained using ultrasound, stirring, or the combination of both, and found that the most efficient technique, in terms of particle size and size distribution, was the high-pressure hot Jo ur homogenization.[61] The cold homogenization process was later developed to avoid drug instability and degradation due to high temperatures. Sample preparation consists of fast mixing of the drug with melted lipids to achieve drug solubilization. This step is developed at high temperature. Subsequently, the mixture is quickly cooled down to produce a dispersion of the solid drug-lipid mixture. This mixture is then subjected to high-pressure homogenization to produce the small-sized particles.[104] B) High shear homogenization or high-speed stirring In this method, the drug is homogeneously dispersed in the molten lipids. A hot aqueous phase, containing the surfactants, is then added to the molten lipids, and the mixture is homogenized in a high-shear device (Figure 7B).[105] This technique is frequently combined with a final step of ultrasonication to further reduce particle size and narrow size distribution. 20 Journal Pre-proof C) Ultrasound The use of this technique with solid lipids yields large particle sizes and wide size distributions. This is the reason why this technique is usually employed in combination with others, as a further step to reduce the size of preformed NPs (Figure 7C).[105] D) Membrane contractor method In this method, pressure is applied to promote the passing of the melted lipids through a membrane (Figure 7D). The lipids form droplets, whose size depends on the pore size of the membrane. The aqueous phase, which contains the surfactants, flows tangentially to the of membrane and removes the formed lipids droplets. The emulsion is then cooled down to allow ro lipids to solidify and form SLNs or NLCs.[106] -p E) Film ultrasonic method The lipidic component and stabilizers are added to the organic phase. This phase is heated to re dissolve lipids. The organic solvent is then removed under vacuum, usually at a high temperature. The film formed on the evaporating recipient is then mixed with water and lP sonicated, to allow the film to redisperse in the aqueous solution (Figure 7E).[107,108] na F) Solvent emulsification and evaporation In this technique, the hydrophobic ingredients are dissolved in a water-immiscible organic Jo ur solvent, which is added to the aqueous phase containing the surfactant. After emulsification of the organic phase into the water phase, the solvent is removed from the mixture, usually through evaporation, which leads to the precipitation of the hydrophobic component into the water phase, resulting in SLNs/NLCs (Figure 7F). This method was first described by Sjöström and coworkers, and the emulsification of the organic phase was achieved through high shear homogenization followed by high-pressure homogenization.[109] G) Supercritical fluids In this method, a lipid emulsion is first prepared using an organic phase. Supercritical fluids are then employed to extract the organic solvents (Figure 7G). The rapid removal of the solvent leads to lipid precipitation and SLNs or NLCs formation with narrow size distribution.[110] This tecnique achieves higher solvent extraction efficiency than other methods such as evaporation or dilution. CO2 is the most employed supercritical fluid for this method. 21 Jo ur na lP re -p ro of Journal Pre-proof Figure 7. High-energy methods for the preparation of SLNs and NLCs: (A) high-pressure homogenization, (B) high-shear homogenization, (C) ultrasound method, (D) membrane contractor method (E) film ultrasonic method, (F) solvent emulsification and evaporation, and (G) supercritical fluids. Created with BioRender.com. 3.3.3. Low-energy methods SLNs and NLCs can be also obtained using low-energy methods. Those preparation procedures are based on LNEs and LNCs techniques reviewed in the previous sections. The solvent22 Journal Pre-proof displacement method, driven by the Ouzo effect,[111–113] the emulsification-diffusion method,[114,115] the PIT method,[116] and the double emulsion procedure [117,118] have all been employed, without adaptation, to prepare SLNs and NLCs. The coacervation method can be adapted to produce SLNs and NLCs. Furthermore, SLNs and NLCs can be prepared with a ME as the starting point. A) Coacervation method This technique does not require the use of solvents and uses pH to achieve the precipitation of fatty acids from their sodium salt micelles (Figure 8A). The method was first used by Battaglia of et al. to prepare SLNs in 2010.[119] The fatty acids sodium salt solution is briefly dispersed with the polymeric stabilizer solution. The mixture is heated to achieve fatty acid solubilization. ro The pH of the solution is then acidified, leading to the fatty acid precipitation. Ultimately, the -p solution is cooled down to achieve lipid solidification. The starting point of this method is a soap, i.e., a fatty acid sodium salt. In an acidic medium, it exchanges the ionic sodium with a re proton, which makes the molecule more soluble. This process, defined as coacervation, reduces B) Microemulsion method lP the solubility of the molecule and drives precipitation of the fatty acids.[119] . na This method was developed and patented in the nineties by Gasco et al. to produce solid lipid microparticles (Figure 8B). Briefly, the lipids and the cargo are heated above the melting point Jo ur of the lipids. The lipid solution is mixed with an aqueous phase, which is at a temperature equal to or above the lipids melting point and contains the surfactant and co-surfactant. This mixture is kept at a high temperature to obtain the ME. Finally, the hot ME solution is added, under stirring, over a cold-water solution (2 – 10 °C), resulting in lipid solidification and the formation of the microspheres.[120]. Another variant of this technique consists of the cooling of the hot ME without water addition. The cooling process of the solution under stirring drives the solidification of the lipids and the reduction of the particle sizes, yielding SLNs and NLCs with sizes below 300 nm.[121] C) Cold-burst method Recently, Cholakova et al. reported a novel cold-burst process for the preparation of SLNs. In this novel low-energy method, a preformed LNE is cooled so that the dispersed nanodrops freeze into solid lipid particles (Figure 8C). The dispersion is then heated to the lipid melting point provoking lipid particles to spontaneously disintegrates into SLNs. The low surfactant 23 Journal Pre-proof content (<2%) and high drug loading (50%) of this method demonstrates a new strategy for Jo ur na lP re -p ro of scalable emulsification technology.[122] Figure 8. Specific low-energy methods for the preparation of SLNs and NLCs: (A) coacervation method, (B) microemulsion method, and (C) cold-burst method. Created with BioRender.com. 4. Drug administration routes and tumor targeting In a successful treatment, therapeutic agents must pass a series of biological barriers depending on the administration route employed. Nanomaterials used for targeting tumor cells aim to increase the local concentration of drugs in and around tumor cells, thereby reducing potential toxicity toward healthy cells and decreasing the off-target effects of the treatment.[123] Nanocarriers can improve the biodistribution, bioactivity and bioavailability of the encapsulated therapeutic products. The fate of drugs in the organism is no longer determined only by their properties, but also by the type of drug-delivery nanosystem. Table 1 summarizes the main factors and barriers of each route of administration when working with colloidal systems. 24 Journal Pre-proof Table 1. Summary of the main threatening factors and characteristics of each route of administration regarding colloidal drug-delivery systems. Barriers and threats for the colloidal systems Physical Chemical Biological Proteincorona Intravenous Immune system Digestive enzymes Peristalsis pH ProteinFirst pass Mucus layer changes corona metabolism Gastrointestinal Surfactant Enzymatic effect epithelium (bile salts) digestion (intestinal and hepatic) Microbiota activity Distribution in the organism Disadvantages References Systemic circulation Direct route to Qualified person systemic for drug circulation administration [124–127] Systemic circulation Lymphatic drainage Selfadministration Bypass first pass hepatic effect Great absorptive surface High drainage [128–137] Systemic circulation (Hemorrhoidal veins) Lymphatic drainage Proteincorona Tears Immune system Enzymes Proteincorona Intranasal Enzymes Immune system Proteincorona Systemic circulation Direct route to brain Pulmonary Surfactant Proteincorona Surfactant Systemic circulation Lymphatic drainage Immune system Microbiota activity Proteincorona Lymphatic drainage Immune system Proteincorona Systemic circulation Lymphatic drainage Immune system -p Mucus layer Epithelium Vaginal Acidic pH Systemic circulation re Blinking lP Ocular Jo ur Rectal Mucus layer Epithelium Transdermal Epithelium: epidermis, dermis, hypodermis Subcutaneous Extracellular matrix Lymphatic vessels epithelium Enzymes Immune system Proteincorona Lymphatic drainage Intramuscular Extracellular matrix Enzymes Immune system Proteincorona Systemic circulation 25 Advantages Digestive process Low patient Selfcompliance administration Smaller Bypass first absorption [138–140] pass surface owed metabolism to the absence or effect villi and microvilli Continuous and fast tears Selfturnover [139,141,142] administration Ocular structural barriers Bypass bloodbrain barrier (via olfactory [143–146] Control the bulb) aerosol Selfadministration characteristics to control the Great deposition area absorptive of the particles surface Mucus turnover [143,144] High drainage Selfadministration Changes in the SelfpH, administration cervicovaginal Bypass first fluid efflux, pass thickness of the metabolism mucus [141,147,148] effect layer depending Prolonged and on age, continuous hormonal or release physiological state Selfadministration Bypass first pass Interaction with metabolism the extracellular [141,142] effect matrix Prolonged and continuous release SelfSmall volume administration doses Bypass first Interaction with [149,150] pass the extracellular metabolism matrix effect SelfSmall volume [151,152] administration doses ro Enzymes Immune system Microbiota activity na Oral Interfacial changes of Administration route Journal Pre-proof Blood and lymphatic vessels epithelium Lymphatic drainage Bypass first pass metabolism effect 4.1. Intravenous route The intravenous route (IV) is the most common route of administration of nanomaterial-based anticancer drugs, as it is the most direct one to the systemic circulation.[124]. Effectiveness of the treatment is achieved when the administered drug arrives with proper dosage and displays activity in cancer cells. However, this is not easy to achieve. Once NPs enter the bloodstream, they find a complex environment designed to recognize external elements. NPs must overcome different obstacles such as the interaction with plasma proteins and the formation of a protein of corona (PC) or their clearance from the bloodstream by the mononuclear phagocyte system (MPS) and the complement system. In addition, the delivery of NPs to the target tissues can be -p determine their biodistribution and performance. ro classified as passive or active targeting. Furthermore, properties such as size and charge will re 4.1.1. Protein corona formation When nanosystems are in a physiological environment, they quickly adsorb biomolecules, such lP as proteins and lipids, on their surface. This PC can be divided into “hard” and “soft” corona, depending on the strength of the interaction. The PC may change its composition if NPs moves na to another compartment or biological fluid.[153] This corona surrounding the particle changes its original surface charge, size, solubility, aggregation and, therefore, the interaction of NPs Jo ur with cells, thus influencing traffic, biodistribution, and cellular absorption.[125] Furthermore, PC influences macrophage uptake. For instance, opsonins such as IgG, complement factors, and fibrinogen promotes phagocytosis, removal of NPs from the bloodstream and concentration in the liver and spleen, while dyopsonins, such as albumin and apolipoprotein, promote longer circulation times of NPs in the body.[126] While the clearance of many nanocarriers from the bloodstream is a question of minutes, interaction with distant cells may take hours or days. Therefore, the success of the nanocarrier highly depends on its blood circulation lifetime. Furthermore, PC can cover the surface of nanosystems and, therefore, strongly reduce the ability to target and recognize cellular receptors.[154] Hence, understanding the formation of the PC around the NPs is essential in predicting the system performance. 4.1.2. How to Avoid Immune System Clearance NPs can be designed to prevent immune system elimination and increase their circulating halflife in the blood, allowing for a continuous and controlled drug release in the vascular 26 Journal Pre-proof compartment. A relatively successful approach to prolong blood circulation time of NPs is to create a steric/hydrophilic surface barrier of sufficient density. Hydrophilic polymers, such as PEG, or surfactants, such as poloxamers and poloxamines, have been investigated to reduce the adsorption of blood proteins and opsonins and, therefore, increase the half-life of nanosystems. The addition of PEG has been widely used with this purpose and is the most employed method for "masking" NPs. This process is also known as PEGylation.[155]. Several studies have been conducted to determine how a change in the thickness and density of a PEG coating affects opsonization and biodistribution, showing that the degree of protein adsorption depends on the size of the PEG and graft density.[156,157] Poloxamers, also known as Pluronic® and of poloxamines or Tetronic®, are non-ionic block copolymers of hydrophobic propylene oxide (PPO) and hydrophilic ethylene oxide (PEO). Poloxamers consist of a central PPO moiety, ro flanked on both sides by two PEO chains while poloxamines are tetrafunctional block -p copolymers with four coupled PEO-PPO blocks linked by a central bridge of ethylenediamine.[158] The adsorption of these molecules on the surface of NPs through their re hydrophobic PPO fragments provides stability to the suspension by a repulsion effect through a steric stabilization mechanism. NPs designed with poloxamers and poloxamines exhibit lP reduced adsorption of blood proteins and opsonins and, as a result, resist ingestion by phagocytic cells and remain in the systemic circulation for a prolonged period. [159] However, na the foreign nature of synthetic polymers should be considered. For instance, an acquired immune-response to PEG moiety that compromises PEG-NPS performance has been reported. Jo ur [160] To solve these limitations, biomacromolecules such as polysaccharides and proteins have been also employed as coating material for colloids due to their biocompatibility and biodegradability. [161] In addition, an emerging approach in the masking of NPs is cell membrane nanotechnology.[162] This technique, first reported in 2011, [163] consists of the deposition of a bioactive layer of a cell membrane directly onto the surface of NPs. The consequent transference of its lipids, protein and carbohydrates, enables the resultant membrane coated-nanoparticle to take on characteristics of the source cell, such as their biocompatibility and immune-evasion properties, along with tropic and targeting effects.[162,164] 4.1.3. Passive targeting At tumor sites, the vascular barrier is disrupted due to failed rapid growth of blood vessels in angiogenesis, thus enabling nanocarriers to cross and accumulate in the tumor tissue.[165] The gaps between endothelial cells in the tumor vasculature can range up to 2000 nm depending on the tumor type, localization and environment.[165] Moreover, due to poor lymphatic function, 27 Journal Pre-proof NPs are not rapidly cleared and are accumulated in the tumor interstitium.[166] This is known as the enhanced permeability and retention (EPR) effect, which is the basis of passive targeting.[167] This accumulation of the drug at tumor sites is a passive process, and requires prolonged circulation of the drug for appropriate delivery. The accumulation of nanocarriers is essentially dependent on their physicochemical properties such as size, morphology, surface charge and chemistry as will be explained in section 4.1.5 mentioned.[168] Furthermore, biodistribution of the drug is also influenced by blood perfusion, passive interactions with biomolecules along the route and immunological clearance processes such as phagocytosis or of renal clearance.[169,170] 4.1.4. Active targeting ro Active targeting, also known as the ligand-mediated targeted approach, involves affinity-based -p recognition, retention and facilitated uptake by the target cells.[171] Biomolecules such as antibodies, proteins, nucleic acids, peptides, carbohydrates and vitamins are employed as re ligands.[172,173] The target substrates can be: surface molecules expressed in target cells, proteins, sugars, lipids or molecules present in the organs or in the microenvironment of lP cells.[174] Intelligent and targeted systems based on nanomaterials exploit the multivalent nature of ligand interactions with the target antigens. When multiple ligand molecules na accumulate in nanosystems, there is a general increase in the avidity of NPs to their related objective.[175] In addition, the binding of a ligand molecule generally facilitates the binding of Jo ur consequent molecules through cooperative effects, collectively improving binding efficiency and subsequent actions.[176] However, there are other aspect related to the concentration of ligand in the NPs that should be considered. For instance, the concentration and nature of the ligand in the NPs surface will determine the orientation of such ligands at NPs’ surface, and therefore their targeting capabilities.[123] Generally, covalent conjugation methods are employed, but systems with physical adsorption using affinity complexes are also used effectively.[177] The critical aspect of this conjugation is to maintain the stability of the conjugated ligands during the adverse physiological environment.[178] The main angiogenic targets explored by NP systems for therapeutic benefit include vascular endothelial growth factor receptors (VEGFRs), αvβ3 integrins, matrix metalloproteinase receptors (MMPs), and vascular cell adhesion molecule-1 (VCAM-1).[179] Monoclonal antibodies (mAb) were the first and are still the preferred class of targeting molecules since conjugated antibodies enhance uptake and cytotoxic potential of NPs in tumor cells. The first mAb to gain FDA approval for the treatment of cancer was Rituximab in 1997, 28 Journal Pre-proof a chimeric mAb used for the treatment of B-cell non-Hodgkin’s lymphoma. Trastuzumab, in 1998, a humanized mAb used for the treatment of HER2 expressing breast cancer, quickly followed. Cetuximab, which binds to epidermal growth factor receptors (EGFR), was approved for treating colorectal cancer in 2004 and head/neck cancer in 2006. Bevacizumab, a tumor angiogenesis inhibitor that binds to VEGF, was approved for treating colorectal cancer in 2004. Recent studies have tried to encapsulate chemotherapeutic drugs into NPs and then functionalize the particle surface with mAbs to maintain targeting efficacy.[180–182] Some peptide sequences have been also employed due to the high affinity for tumor-associated receptors and, in this context, peptide-based targeting of tumor-associated receptors has of emerged as a potential tumor-specific chemotherapeutic agent. Cell permeating and fusogenic peptides from pathogens or toxins and peptides randomly derived from technologies such as ro phage display, are commonly used for targeting purposes.[183] Among single nuclear -p localization peptides, the trans-activating transcriptional activator peptide has been shown to be an efficient molecule for translocating NPs into cell nuclei via the binding import receptors re importin α and β. In 2012, a peptide was used to conjugate onto mesoporous silica NPs for nuclear-targeted drug delivery of DOX for the first time.[184] lP Ligand conjugation on the NP surface changes the properties of the targeting molecules along with the nanocarrier.[185,186] The ligand-NP conjugation provides a greater targeting capacity na for the resulting nanocarrier, although with a detriment of the rotational and translational freedom of the conjugated ligand.[187] On the other hand, the size, geometry, surface properties Jo ur (charge and hydrophobicity) and the composition of the NPs can also be altered. In some cases, NPs have demonstrated benefits that go beyond simple drug release, such as greater resistance to degradation by nucleases of nucleic acid chains immobilized on the surface of nanomaterials.[188] 4.1.5. Properties affecting NPs performance A) Size and morphology The size and shape of the nanomaterial should be considered when designing NPs, as it affects the way in which the organism “sees” them and, therefore, determines their distribution and pharmacokinetic profile.[189] For spherical particles, smaller sizes represent higher curvatures, which may be problematic for ligand functionalization after synthesis, along with increased toxicity.[190] The kidneys effectively remove, through blood filtration, NPs with diameters smaller than 10-20 nm. Filtration through inter-endothelial slits in the walls of the splenic sinus removes particles of more than 200 nm. These filters suggest that the size of NPs should be 29 Journal Pre-proof greater than 20 nm but not more than 200 nm if prolonged circulation within the body is desired. In addition to the effect on circulation properties and accumulation in tumors,[191] the shape of NPs seems to influence the kinetics of cell internalization by modulating the interactions between the nanomaterial and the cell surface.[192] B) Surface and ligand charge Surface characteristics of NPs, such as charge, chemical moieties, and nature of the materials, can define the NPs toxicity. Most of the currently available studies point out to the surface charge as the main surface-related parameter affecting the toxicity of the system. [193,194] of Positively charged surfaces seem to be more toxic than negatively charged and neutral NPs.[195,196] Moreover, the surface charge can favor or hinder the approach between NPs and ro cells. Thus, surface charge defines the interacting relations of the NPs with the different cells -p in the organism, affecting their uptake and distribution. In addition, surface properties determine the stability of the system in the biological medium. They influence the formation of re the protein-corona and the aggregation or not of the system.[197] The surface is the site of ligand functionalization. From a synthetic perspective, the charge of both the non- lP functionalized NPs and the ligand, can affect conjugation performance and spatial configuration of the ligand on the surface, due to repulsive or attractive forces.[198,199] A chemical spacer na of reasonable length, such as those based on PEG units, can help reduce this effect, but can simultaneously complicate synthesis and increase final particle size.[200] In addition, since Jo ur most ligands are charged molecules, the final surface charge of NPs is determined by the combination of ligand densities, materials, and formulation strategies. C) Hydrophobic/Hydrophilic surface From a colloidal point of view, hydrophilic nanoparticles are more stable than their hydrophobic counterparts. Solvation, i.e. the adhesion of water molecules onto hydrophilic surfaces, makes difficult for hydrophilic particles to touch because of the present “hydration pressure”, whereas “hydrophobic effects” may cause the aggregation of the system.[201] In terms of cellular uptake, hydrophobic NPs are more rapidly internalized. However, they also tend to agglomerate and are earlier removed by the MPS.[201] Hydrophobicity can also affect the presentation of the ligand.[202] This is especially evident for NPs possesing a hydrophobic nucleus since the ligand could be trapped within the core and, thus, not properly exposed on the surface. [203] 30 Journal Pre-proof 4.2. Oral route The oral route is the oldest route used for drug administration due to ease of use, less expensive manufacturing, and high patient compliance. However, in cancer therapies, the oral route is not the most employed due to the physiological conditions and barriers at the gastrointestinal tract (GIT). Poor solubility of some of the chemotherapeutic drugs and their degradation along the GIT lead to low compound bioaccessibility. Moreover, low permeability at the intestinal level further reduces the bioavailability of chemotherapy drugs. On the other hand, this administration route can be used for local drug administration along the GIT or for systemic drug delivery. of With regard to oral administration, the bioavailability of a compound depends on its bioaccessibility and bioactivity. In this case, bioactivity includes absorption at the intestinal ro level and pre-systemic metabolism, which includes intestinal and hepatic first pass metabolism, -p its ability to enter the systemic circulation and maintain its functionality regardless of the interaction with other biological entities, and finally, the ability to reach the target entity.[128] re The first problem that NPs help to solve is the solubilization of hydrophobic drugs. Some of the antitumor drugs currently used in clinics, as well as many bioactive compounds which are under lP investigation as antitumor drugs or as co-adjuvant in cancer therapies, are insoluble in aqueous media. The lipidic core of LCNPs is able to solubilize hydrophobic compounds in a highly Jo ur increasing their solubility. na efficient way. Thus, LCNPs contribute to increased bioaccessibility of those compounds by 4.2.1. GIT absorption The role of the GIT is to break down food into absorbable components and allow the absorption of such nutrients. At the same time, the GIT provides a physical barrier which hinders the entrance of microorganism and toxic compounds into systemic circulation.[204] From the mouth to the anus, the GIT is divided into compartments, each with different functions and physicochemical conditions, posing a threat to orally administered drugs and limiting bioaccessibility and bioavailability. Those adverse conditions comprise ionic strength and physiological medium pH, which are important factors to consider when working with colloidal systems,[129] as well as enzymatic digestion and peristaltic movement, which can further contribute to nanocarrier aggregation. Moreover, bile salts released in the intestine are also concerning when working with NPs due to their ability to displace surfactants from NPs shells.[130–132] Finally, microbiota from the large intestine metabolizes components which cannot be digested by human enzymes, such as complex carbohydrates and proteins.[128] This 31 Journal Pre-proof fact can be used to achieve colon-targeted drug delivery by providing the LCNP with a chitosan, pectin or alginate shell.[205] Shell composition of the nanocarriers can determine the behavior of the system along with the changing conditions on the GIT. For instance, the inclusion of hyaluronic acid (HA) on the shell of albumin-coated LLNs improves the retention of curcumin (CUR) under gastric in vitro simulated digestion conditions.[206] Moreover, we can take advantage of different conditions along the GIT to achieve a controlled release of a loaded compound or to achieve the absorption of NPs in a specified portion of the GIT.[207] If NPs or their loaded compounds achieve to survive the digestion process, then, they have to diffuse through the mucus layer, a viscoelastic gel which acts as a filter and allows some of particles to arrive at the brush border surface, i.e., the absorptive intestinal surface, while hindering pathogens or toxins diffusion.[133] A too weak interaction with the mucus layer will ro lead to the direct transit and excretion of the NPs from the GIT. The inclusion of mucoadhesive -p polymers on the shell is a commonly used strategy to prolong the residence time of the nanocarrier in the GIT and to enhance the absorption of carried drugs at intestinal level. re However, a too strong interaction with the mucus may result in the retention of the nanocarrier and its excretion, together with the mucus, owing to the continuous turnover of this layer.[208] lP On the other hand, mucus penetrating NPs easily diffuse through the mucus layer. These nanocarriers can modify the mucus structure and open ‘gaps’ in the mucus mesh, which allow na them to diffuse. These nanocarriers have coatings which weakly interact with the mucus Jo ur barrier.[209,210] 4.2.2. First pass metabolism Once drugs arrive at the epithelial surface, they must deal with the so-called first pass metabolism, first carried out by the intestinal cells and, later, by the hepatic cells. First pass intestinal metabolism comprises the action of brush border enzymes and intracellular metabolism in the gut cells.[134] Both, enteric and hepatic intracellular metabolisms, follow the same metabolic procedure and are divided into three different phases. Enzymes involved in phases I and II carry out the chemical modification of xenobiotics to make them more chemically-reactive and more soluble.[211] Phase III includes the traffic of molecules into and out of the cells. The main efflux transporter limiting drug absorption both in enterocytes and hepatocytes, is P-glycoprotein (P-gp).[212] During this phase, some of the compounds will be excreted from enteric cells back to the lumen or from hepatocytes to the bile canaliculus. In the case of first pass enteric metabolism, ‘surviving’ compounds will enter portal vein circulation thanks to the mentioned transporter and will be further exposed to suffer first pass hepatic 32 Journal Pre-proof metabolism. The compound which ultimately surpasses hepatic metabolism will enter central vein and systemic circulation.[135] However, for lipids, there is an alternative pathway to enter systemic circulation. This way implies passing across the cells, in the form of chylomicrons, and arriving at the lamina propria. Here, lipids can enter the lymphatic circulation, helping carried-drugs bypass first pass hepatic metabolism and reaching systemic circulation.[134] Once there, the absorbed system will face the same challenges as those of intravenously administered systems (section 4.1). Structural differences between blood vessels and lymphatic vessels are responsible for the differentiated absorption.[136] The more compact structure of blood vessel endothelium leads of to the absorption of molecules mainly through transcellular transport (across the cell), while the less compact structure of lymphatic endothelium makes them more permeable and allows for ro absorption via paracellular transport (through intercellular spaces). Thus, high molecular -p weight molecules and bigger particles, such as colloidal structures (where chylomicrons are included) have preferential access to lymphatic vessels.[137] The lymphatic system plays an re important role in metastasis.[136,213] Hence, NPs and drug circulation through the lymphatic system can promote immune response and become a pathway to reduce metastasis by attacking lP circulating cancer cells and acting on lymphoid metastasis. A schematic and comparative Jo ur presented in Figure 9. na representation of both intravenous and oral administrations and their main challenges are 33 re -p ro of Journal Pre-proof Jo ur na lP Figure 9. Schematic and comparative representation of the oral and intravenous routes of administration for a colloidal drug-delivery systems. When the colloidal nanosystem is administered orally, it is absorbed by the GIT and needs to surpass the (A) digestion process and (B) the enteric epithelial barrier along with the first-pass metabolism. Compounds absorbed into portal vein circulation will suffer (C) hepatic metabolism, and finally, surviving compounds and those absorbed into the lymphatic system will enter (D) systemic circulation. Once in the systemic circulation, the colloidal nanosystem will encompass the (E) protein corona and the (F) macrophage uptake before it can (G) extravasate and arrive at the site of action, where it performs its therapeutic effect. The protein corona and the macrophage uptake are also active biological barriers in the lymphatic system. Created with BioRender.com. 4.3. Mucosal absorption for systemic drug delivery Mucosal administration does not require special training, making patient self-administration possible, which is advantageous when compared to the intravenous route. Mucosal surfaces include all the biological surfaces producing mucus, a viscoelastic gel secretion, to protect a part of the organism in contact with the external medium. Therefore, the digestive system is included in this category. However, considering the relevance and the complexity of the digestive process, we have dedicated a separate section to the oral route of administration (section 4.2). Barriers that NPs encounter during mucosal absorption are similar: mucus protective layer and its clearance rate, epithelial barrier which determines transport (transcellular or paracellular) and, finally, absorption by blood or lymphatic vessels. Nevertheless, the previously described first pass metabolism, a limiting factor in the oral administration route, can be avoided by using other transmucosal administration routes, such 34 Journal Pre-proof as rectal, nasal, pulmonal or vaginal. Each mucosal surface possess different characteristics, such as enzymatic activity, mucus layer thickness, pH and hydration, presence of specific microorganisms or immune system entities (cells, like macrophages, or molecules) and different absorptive surface or different drainage (portal drainage to the liver or systemic vein or lymphatic drainage, which bypasses liver first pass effect).[138,139,214] Drug administration via the skin, vagina, eye and nose are considered topical administration forms when the objective is localized treatment. Topical application of drugs is frequently used to act directly on the deposition area, since it avoids the drawbacks involved in systemic circulation.[141] However, those administration routes also offer a pathway for the systemic of administration of drugs. In this section we will focus on the use of mucosal surfaces for systemic ro drug administration, emphasizing the unique characteristics and challenges of each route. -p 4.3.1. Digestive system-related administration routes: Rectal, sublingual, and buccal administration re Rectal, sublingual, and buccal drug administration are alternative routes involving the digestive system. Rectal administration is a promising route, however not as widely used as the oral route lP due to less patient compliance. Physiological characteristics of the rectum include lower water content and the absence of villi or microvilli, which leads to a smaller absorption surface na compared to the small intestine. The rectum is surrounded by rectal (hemorrhoidal) veins and lymphatic vessels. Moreover, in this part of the large intestine, enzymatic threatening activity Jo ur is very low. It is also interesting to take into account that drainage of the upper part of the rectum occurs in the portal vein, following first pass liver metabolism, while the lower part of the rectum empties into the vena cava, bypassing first pass metabolism, similar to lymphatic drainage.[138,139] 4.3.2. Intranasal administration The respiratory system also provides a window for the entrance of drugs. This administration route involves the inhalation of aerosols. Controlling the characteristics of the aerosolized particles is crucial to ensure drug deposition on the targeted area.[143] Nasal and pulmonary delivery are the most frequently used routes in the respiratory system. In these areas, the mucus layer and its clearance rate are limiting factors for drug absorption, with 20 min being the renewal rate for the nasal mucus layer and 10-20 min for the respiratory tract. [143] On the protective mucus layer, NPs are also exposed to macrophage attack and to surfactants such as phospholipids,[144] which may compromise NPs stability. Pulmonary administration can be 35 Journal Pre-proof driven to local treatment, but since the alveolar region provides a great absorption surface, thanks to ciliated cells, and high drainage to the lymphatic and circulatory systems, drugs may enter systemic circulation quickly, bypassing hepatic metabolism. This provides the possibility of efficient systemic drug administration through the pulmonary system. On the other hand, the nasal route is especially interesting for brain drug administration, since delivery of drugs through the olfactory pathway allows to bypass the blood-brain barrier (BBB).[145] Moreover, the olfactory bulb provides a direct neural route to the brain, avoiding systemic circulation and consequent metabolism.[146] of 4.3.3. Vaginal administration With regard to vaginal administration of drugs, it is necessary to consider the special ro characteristics of this area, such as the acidic pH because of the resident microbiota, the -p thickness of the mucus secretion layer, which can vary depending on age, hormonal and physiological state, and the continuous efflux of cervicovaginal fluid, which can reduce the re retention time of the drug, limiting its absorption.[141] This administration route is mainly used for topical treatment. However, systemic administration of drugs though this route has been lP proved to be effective and, even preferable, in some cases. An example of vaginal administration of compounds at a systemic level is the widely used vaginal ring for hormone na sustained delivery over long periods of time.[147] This route allows prolonged and continuous release, maintaining more constant levels of the drug in the systemic circulation, and requires Jo ur smaller amounts, when compared with the oral route.[148] On the other hand, the extensive vascularization of the vagina, as well as the lymphatic drainage in the area, allow for the absorption of compounds into the systemic circulation, avoiding the first pass hepatic metabolism. [147] 4.3.4. Ocular administration The administration of drugs to the eye is normally used for topical local treatment because the physiological and anatomical barriers to overcome hinder the task. Ocular surface is continuously cleaned by tears, which dilute and reduce the residence time of the administered drugs.[141,142] Besides tear turnover and drainage, ocular administration presents other barriers. The cornea provides a tightly packed cell layer that hinders the passage of hydrophilic and ionic compounds. After crossing the cornea epithelium, lipophilic compounds find the stroma of the cornea, which is a hydrophilic space acting as a barrier and retaining hydrophobic compounds.[139] This is a disadvantage because drug accessibility to the systemic circulation 36 Journal Pre-proof is reduced. However, the retention of hydrophobic compounds provides a drug reservoir and allows sustained drug delivery.[139] 4.4. Other administration routes 4.4.1. Topic application The skin barrier is about 3 mm thick and is divided in three layers: the epidermis, which is the avascularized outer layer, the dermis, widely vascularized and located immediately under the epidermis and, finally, the innermost hypodermis layer. The epidermis is the biological barrier that protects the body from microorganism invasion and preserves body homeostasis. The of dermis acts as thermal protection and the hypodermis provides a protective mechanical barrier. Despite skin drug administration being mostly used for topical application, some drugs are ro permeable through the skin barrier and can achieve systemic circulation. This transdermal route -p bypasses first pass metabolism, allowing the plasma drug level of certain drugs to be re maintained.[141,142] 4.4.2. Intramuscular application lP Intramuscular administration of drugs allows for fast systemic absorption, avoiding first pass hepatic metabolism. However, this absorption will depend on the vascularization and blood na flow of the chosen muscle. Thus, high blood flow will promote faster drug absorption in the systemic circulation.[151] Moreover, muscle choice for drug administration will depend not Jo ur only on previously mentioned blood flow, but also on the volume dose to be administered, which is frequently low (2–5 mL in humans).[152] 4.4.3. Subcutaneous application Subcutaneous administration implies the deposition of the administered formula in the hypodermis.[149] The extracellular matrix of this interstitial area is mainly composed of collagen and hyaluronan. This last molecule forms a gel, which limits the diffusion of the component injected in the area.[150] The subcutaneously administered formula is drained from the extracellular matrix to the circulatory system or the lymphatic system, depending on the size and the physicochemical characteristics of the molecules or the colloidal system. The higher flow rate of the vascular system would provide more efficient drainage from the interstitial area. However, the permeability of vascular endothelium limits absorption, mainly absorbing only small molecules. An alternative drainage route is through the lymphatic system.[150] Lymphatic 37 Journal Pre-proof absorption of the drug, as previously discussed, will allow the drug to enter the systemic circulation and avoid the first pass metabolism effect. However, interactions with the extracellular matrix should be carefully evaluated for the specific system, since high interaction between both would hinder the diffusion and absorption of the NPs. [149] Other factors that must be taken into account are the possible enzymatic degradation or cellular immune system attack. 5. LCNPs in cancer therapy Cancer is considered a heterogeneous disease which includes a variety of subtypes with unique of morphologies and clinical behaviors. The first line chemotherapy treatment for cancer is the use of broad spectrum anticancer drugs such as DOX, DTX, and PTX.[215] These ro antineoplastic compounds present major drawbacks, such as the lack of specificity and their -p rapid clearance from the body. This limitation causes patients to be treated at the maximumtolerated dose of these antitumor compounds, thus suffering several adverse and off-target re effects. Another major cause of treatment failure is multi-drug resistance (MDR). The most studied mechanism of MDR is the overexpression of drug efflux pumps, belonging to the lP adenosine triphosphate binding cassette (ABC) transporters family, which pump drugs from inside the cell to the outside. The main ABC transporters clinically associated with MDR are na P-glycoprotein (Pgp/ABCB1) and MDR related proteins (MRPs/ABCCs).[216] Since anticancer drugs are used at maximum-tolerated doses, a small increase in drug resistance is Jo ur enough to make chemotherapy ineffective and making it impossible to overcome drug resistance by increasing the dose.[217] At present, nanotechnological solutions are employed to surpass those limiting aspects. However, clinical translation of NPs remains a challenge, as it requires a detailed understanding of the physicochemical properties of nanosystems, internal and external structure, chemical reactivity and stability, biodistribution, toxicity and biocompatibility, among other factors, especially for biomedical applications and cancer therapy. In this regard, LCNPs offer significant advantages compared to other nanoparticulate drug-delivery systems. LCNPs can solubilize promising hydrophobic compounds with antitumoral effects with limited application through conventional methodologies, thus allowing their application in cancer nanotherapy. For instance, aromatase inhibitors (AIs), employed in the treatment of estrogenreceptor (ER) positive breast cancers, have poor aqueous solubility. Since about 75% of breast cancers are ER-positive, the development of nanocarriers that can effectively encapsulate and deliver AIs arises as a promising approach to breast cancer treatment. [218] Similarly, 38 Journal Pre-proof LCNPs allow the application of other interesting hydrophobic antitumoral agents such as Camptothecin (CPT), fisetin, melphalan, β-carotene, or Citral.[219–223] Furthermore, lipids can serve as a skeleton for the preparation of interesting lipid-drug conjugates, such as the lipidated C16-DOX prodrug, which can then be included within LCNPs.[224] In addition, lipids which possess bioactive activities can be employed as the core of LCNPs, thus generating LCNPs with interesting inherent properties that can be further loaded with other chemotherapeutics. That is the case of ω-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA). Several reports indicate that DHA is an effective adjuvant of conventional anticancer drugs that improves the antitumoral efficacy while reducing the side of effects of the therapy.[225] Selol, an oily mixture of triglycerides with reported cytostatic effect, ro has also been employed to prepare LNCs co-encapsulating vincristine and DOX.[226] Similarly, we reported the preparation of maslinic acid (MA) SLNs, a plant-derived low water- -p soluble triterpene with antitumor properties, which can be employed as nanocarriers of re hydrophobic compounds.[227] Interestingly, El-Gogary et al. reported the preparation of LNCs and polymeric NPs of ferulic acid, a polyphenolic compound with anticancer properties but lP with low solubility and bioavailability in aqueous media. [228] They reported that LNCs were superior to polymeric NPs both on the physicochemical and cellular level. na LCNPs are widely employed for combinatorial therapy of different chemotherapeutic agents, as they allow the co-encapsulation and combination of different drugs due to their higher drug Jo ur loading capacity compared to other types of nanocarriers. Combination chemotherapy is an attractive strategy in cancer treatment because reduces side effects, since a lower concentration of each drug is needed to get the desired antitumoral effect.[229,230] The combination of chemotherapeutics with agents that can inhibit the MDR effect are very promising. Furthermore, it is reported that LCNPs help in overcoming the MDR phenomenon as they can carry the encapsulated compounds into cells by endocytosis, thus bypassing the Pgp drug efflux mechanism.[231,232] In addition, LCNPs allow for the administration of therapeutic biomacromolecules such as as peptides or RNA/DNA-based agents, which are usually co-administrated with traditional antitumoral drugs. Furthermore, due to their biocompatibility, stability, and versatility, LCNPs can be administered not only through parenteral administration, but also through other not so widely employed routes for NPs administration, such as oral, intranasal, or topical pathways. For instance, they offer significant advantages when using these pathways as they improve oral drug solubility and intestinal permeability, can cross the BBB barrier, and enhance skin 39 Journal Pre-proof penetration. [142,145,227] As mentioned in section 2, they differ in important features such as the release kinetics of their cargo substance or the physicochemical and colloidal properties of the system, mainly due to the different physical state of their lipidic components. In this sense, there are preferred LCNPs for each target tissue and delivery route. From a broader point of view, SLNs and NLCs, more resistant to mechanical forces, are preferred for intranasal and topical administration, whereas LLNs, due to their higher drug-loading capacity, are widely employed for intravenous and oral administration. In this section, we examine and summarize LCNPs, in both the clinical stage or market state of production, as well as those in preclinical state, for the treatment of the most frequent types of of cancer. ro 5.1. Market-available LCNPs and clinical trials -p A literature review was performed on different databases (Medline via PubMed, Cochrane Library, Web of Science, and ClinicalTrials) with the aim of reviewing clinical trials of LCNPs re as cancer treatments. Reports are listed in Table 2. LCNP Superficial basal cell carcinoma Ovarian cancer Jo ur Actinic keratoses Actinic keratoses (face and scalp) Administration Route Cargo substance na Cancer lP Table 2. Clinical trials regarding the use of LCNPs as cancer treatment. Topical 5-ALA Status Identifier Ref Commercial and Phase IV NCT02799069 [233– 241] Phase I NCT05060237 [242] Phase II and Phase NCT02367547 [243– III, active NCT03573401 245] LNE Phase II, terminated NCT02195973 [246,247] PTX Breast cancer Canine lymphoma Carmustine IV 17-AAG Akt-1 Antisense Oligonucleotide Pilot clinical study - [248] Pilot clinical study - [249] Phase I, terminated NCT00319930 [250,251] Phase 1 NCT05267899 [252] Phase 2, terminated NCT03323398 [253] Solid tumors Nonspecified mRNA-2416 mRNA-2752 40 Phase 1 NCT03739931 [254] Journal Pre-proof Oligonucleotide Targeting MYC Phase I, terminated NCT02110563 [255] It should be noted that several clinical trials do not describe the type of lipid nanocarrier employed. These studies are listed as “non-specified” in Table 2. No reports including LNCs, SLNs or NLCs as antitumor drug delivery-systems were found. During this literature search, we only found one market available LCNP formulation: Ameluz® (Biofrontera Pharmaceuticals, Wakefield, MA). Ameluz® was developed in 1998 by Hürlimann et al. as a novel LNE-based gel formulation, containing 10% of 5-aminolaevulinic acid (ALA), for topical treatment of Actinic of keratoses.[233] These disorders are in situ squamous cell carcinomas which need to be treated to prevent their potential progression. Photodynamic therapy with ALA is an accepted treatment ro option for this disease. However, the main problem with ALA formulations is the instability of this active compound in aqueous media. The nanoemulsion formulation, termed as BF-200 and -p patented by Biofrontera Pharmaceuticals (Wakefield, MA),[256] confers improved ALA re stability and skin penetration.[257,258] Several successful clinical studies reported the effectiveness and security of this platform as a treatment for actinic keratoses.[234–241] The lP BF-200 formulation was approved by the FDA in 2016 under the commercial name of Ameluz®. Currently, BF-200 is under clinical trial to evaluate its safety and tolerability as na treatment of actinic keratosis on face and scalp.[242] This LNE-based formulation is also proposed as a photodynamic treatment for nonaggressive basal cell carcinoma (BCC). BCC is Jo ur the most common non-melanoma skin cancer, with superficial BCC (sBCC) being the second most frequent non-aggressive form. Along with excision and surgical procedures, photodynamic therapy has been demonstrated to be an effective therapy alternative for SBCC. Currently, different clinical reports, including a non-controlled single-center clinical study,[243] a Phase II clinical trial [259] and a randomized, intraindividual, non-inferiority, Phase III clinical trial,[245] are assessing the effectiveness and security of this promising formulation. Another interesting LNE platform is a cholesterol-rich nanoemulsion that binds to low-density lipoprotein (LDL) receptors, termed as LDE. LDE is recognized by LDL receptors and can be used to target antineoplastic drugs against cancer cells that overexpress LDL, such as ovarian and breast carcinomas. Dai et al. conducted a pilot study on eight patients with gynecologic carcinoma.[246] These authors studied the pharmacokinetics of LDE associated with PTX oleate, a derivatized form of PTX, and the ability of this nanosystem to concentrate the drug in the tumor sites. Results showed that PTX oleate associated to LDE is stable in the bloodstream, 41 Journal Pre-proof has longer half-life and greater absolute bioavailability, that is, AUC, when compared to the commercial formulation. Furthermore, a Phase II study supports the use of PTX-LDE as thirdline chemotherapy for ovarian cancer. The results also suggest that PTX-LDE can be eligible for clinical trials at first or second line setting in combined chemotherapy.[247] A pilot clinical study of LDE-PTX was also carried out to evaluate the tumoral uptake, pharmacokinetics and toxicity in breast cancer patients.[248] Results showed that LDE-PTX preparation can be advantageous for use in breast cancer treatment as the pharmacokinetic profile is improved, the drug is concentrated in the neoplastic tissue and the toxicity of PTX is reduced. Interestingly, these authors also performed a pilot clinical study of LDE as a carmustine carrier combined of with vincristine (VCR) and prednisone for the treatment of canine lymphoma.[249] LDEcarmustine was shown to be safe and effective in a drug combination protocol, which ro encourages larger studies to investigate the use of this novel formulation. Although not in the -p cancer treatment field, an active Phase III trial is assessing the potential of LDE-PTX as a noninvasive treatment to reduce lesion size and inflammation in patients with aortic and coronary re atherosclerotic disease.[260] Similarly, a prospective, randomized, double-blind, placebocontrolled, Phase III trial is studying LDE associated with MTX as an atherosclerotic disease lP treatment.[261] 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin that na inhibits the Hsp90 family of molecular chaperones, which leads to the proteasomal degradation of client proteins critical in malignant cell proliferation and survival. Therefore, it is a promising Jo ur antitumor compound. Saif et al. undertook an open-label, dose-escalation, safety, pharmacokinetic and pharmacodynamic Phase I trial of CNF1010, a LNE loaded with17AAG,[250] in patients with solid tumors. Unfortunately, the maximum tolerated dose was not formally established and the CNF1010 clinical program is no longer being pursued due to the drug toxicity profile and the development of fully synthetic second and third generation Hsp90 molecules.[251] Several studies under clinical trials are currently evaluating the feasibility of the delivery of genetic materials within LNPs as another interesting approach to treat solid tumors.[252–255] 5.2. Preclinical in vivo LCNPs studies In this section, we present significant reports from 2015 to date regarding the in vivo use of LCNPs in the treatment of the most frequent types of cancer. Medline via PubMed, Scopus and Web of Science were employed as databases. Reports were excluded if they did not include in 42 Journal Pre-proof vivo studies, LCNPs composition was not properly defined or the therapeutic application was not antitumoral treatment. Results are summarized in Table 3. Table 3. Preclinical studies of LCNPs as anticancer treatment. Organ/Cancer LCNP Administration Route Cargo substance Core CSB-INH Triacetin OXA and 5-FU Capryol 90 CS-5-FU Cholesteryloleate, PC, triolein and cholesterol CPT Miglyol 812 PCL, CD and Chitosan [265] Ferulic acid Solutol HS 15 and Epikuron 200 [228] PEGylated PLGA [266] Solutol, Lipoid and Transcutol® HP [267] Poloxamer 188 [268] Oral IV Oral Ferulic acid LNC CUR Colon 5-FU,DOX,OXA, SN38 and IRI ro IV CPT Oral SN38 IV Intraductal C6 ceramide na ClFPh-CHA Prodrug C16-DOX IV Breast Castor oil Poloxamer 407 and [269] Chitosan-TPP Poloxamer 188 and PEG[270] PE Hydrogenated soy PC, [271] Poloxamer 188, and PEG-PE Tween 80 [272] Tween 80 and Eudragit S100 [273] Span 80 and Tween 80 [274] Tween 80, poloxamer 407 and chitosan [275] PEG-35 [224] Ethylis oleas CS-5-FU Tween-80 and Labrasol [264] PTX MCT, LCT and oleic acid Glycerol and PL–100 M [277] DOX and α-linolenic acid α-linolenic acid and cholesterol DAC and PAN Cod liver oil DOX and W198 Oleic acid and soybean oil Lecithin, Tween 80 and FA LPC, PA and carboxylated PEG soy lecithin [278] [279] [280] Chitosan Tween 80 and [281] Captex 300 TPGS soy lecithin, poloxamer [282] MCT, LCT and cholesterol 188 and glycerol Capmul MCM, Captex 300 Simulsol P 23, Poloxamer [219] and Captex 810D 407 and Solutol HS 15 Lipoid-S75 and Zein [218]. Capryol 90 protein CPT IV Tween-80 and Labrasol [264] Phospholipid mixture containing cholesteryloleate, PC, triolein and cholesterol PTX and vitamin E LNC Tween-80 and [262] Transcutol-HP Labrasol, Cremophor EL [263] and Transcutol HP α-TOS CPT Oral PC, cholesterol, DOTAP and PEG-PE conjugate Compritol® ATO 888 and oleic acid Labrafac™ lipophile WL 1349 Monoolein, tributyrin and tricaprylin Ref Cremophor EL and PEG [276] 400 Jo ur IV and IP LNE 5-FU Oral and IP IP Compritol 888 ATO and Precirol 5 ATO lP miRNA Trilaurin and egg yolk PC re CPT IV NLC Glyceryl monooleate -p 5-FU SLN Castor oil, Soybean or Miglyol 812 Labrafac™ lipophile WL 1349 and Labrafil 1944 Cs GMS and CPT-PA conjugate of LNE Shell EXM and RES MTX Maisine 35 –1 PTX and CUR Oleic oil DOX and Selol Selol Caprylic (C8) and Capric (C10) triglycerides Almond oil, Castor oil, and Isopropyl myristate DTX and THQ Honokiol 43 Stearic acid-valine conjugate [283] PEG-PE and Poloxamer [284] 407 PVM/MA-DOX [285] TPGS [286] PEG-PLGA [287] Journal Pre-proof Oral Fisetin PC, cholesteryl oleate and cholesterol CUR GMS Β-carotene GMS, gelucire50/13, and Phospholipid S-100 GMS and Compritol® 888 ATO Raloxifene Intratumoral PTX stearic acid IP DTX Compritol [294] MTX Gelucire and stearyl amine [295] DTX and CUR Compritol and GMS Poloxamer 188 [296] DOX stearic acid Soy lecithin and PEG-PE [297] Melphalan Tristearin Soya lecithin, Polaxamer [221] 188 and PEO-PPO of PEO-PC phospholipid-90 NG, Tween 80 and fucose RGD-HZ-GMS Trilaurin and CholesterolChitosan EXE -p Stearic acid Hydrogenated palm oil and olive oil Precirol® ATO 5 and flaxseed oil re Citral lP Calycosin DOX, DHA and α-TOS na PTX Gambogic acid DOX and CDDP Jo ur DOX and β-lapachone Stearic acid and oleic acid DOX-TS Compritol, DHA and TEA Compritol® 888 ATO, peanut oil and oleic acid Ethyl oleate, cremorphor EL 35and GMS CUR Lycobetaine Oleic acid and soybean oil PTX DL-α-tocopheryl acetate and soybean oil Selol Selol IV LNC IV Lung Erlotinib Lecithin and Transcutol GMS, lyceryl tripalmitate, glyceryl trimyristate, glyceryl tristearate and stearic acid Glyceryl behenate and stearic acid PTX Oral Erlotinib SLN NLC Miglyol and steric acid OmeRx™ DHA 500 TG and Compritol Precirol ATO5 and Maisine 35-1 Compritol 888 ATO and MCT 812 Stearic acid and Precirol® ATO Compritol® 888 ATO, oleic acid and GMS RES DOX and Sclareol LNE [291] 1-tetradecanol Oral Oral [290] Termoporfin Radiolabeled trastuzumab IV [289] DTX CUR NLC [222] GMS and stearic acid DOX IP [288] Trimyristin, Ceramide and TMP-I IV PTX and artemether GMS and stearyl amine IV Transferrin etoposide GMS and stearic acid Intranasal 9-bromo-noscapine Stearic acid 44 PC, DDAB and PE-HA [220] PTX and pEGFP ro SLN HA and chondroitin sulfate Soya lecithin, Poloxamer 188 and Chitosan Tween-80 and Pluronic F68 Phospholipid S-100 and TPGS-1000 Lecithin, Poloxamer 188 and CD Span 80, and Pluronic ® F127 [292] PC, Pluronic ® P85 and [293] Emulsiflex EF-B3 RGD and Myrj52 [298] Chitosan, Epikuron®200, NaTC, Cremophor®RH60 [299] and Pluronic®F68 Lecithin [300] Lipoid S-100, thimerosal, [223] D-Sorbitol and Tween 80 Poloxamer 188, Tween [301] 80, and Tween 20 Tween 80, Span 60, PEG [302] 400 and sucrose stearate Tween 80 Cremophor RH40 and PEG Lecithin, Myrj 52 and RGD Soy PC [303] [304] [305] [306] Soy PC and PEG[307] Succinic Acid Phospholipon® 90 G and [308] Poloxamer 188 Tween 80 and glycerol [309] Tween 80 [310] Lipoid S 75, PEG and Tween 80 PEGylated lecithin and Lipoid E80 [311] [312] Tween 80 and HA [313] PMV/MA [314] DDAB and PEG-Aspartic [315] Acid Soy lecithin, Tween 80, poloxamer 188, and poly(vinyl) alcohol [316] TPGS and soy lecithin [317] Span® 80 and MPEG2000[318] DSPE Soy lecithin and Tween [319] 80 PC and sodium [320] glycocholate Journal Pre-proof ATO-5, MCT, Solutol HS15 and Kolliphor EL Oleic acid and Compritol® 888 ATO Compritol® 888 ATO and Miglyol® 812 PTX and salinomycin PTX and DOX DOX and β-elemene IV pEGFP precirol ATO-5 and olive oil PEG-PE, lecithin, and Tween 80 Lipoid S100, soybean lecithin, tween 80 and Transferrin [323] [324] PTX and DNA GMS and oleic acid Topical Zinc phthalocyanine MCT, Lipoid E80 and DOTAP Oral and IV Piplartine Capmul PG-8 Tween 80 and PE-PEG [329] PTX IV and IP 7-Ketocholesterol IV DTX IP Eugenol and Ace (eugenol acetylated) IV and IP Ferrociphenol and AnsaFerrociphenol and Bcl-2 siRNA Labrafac® Oral DHA-dFdC GMS IV and Intratumoral PTX Stearic acid PTX GMS, Cholesterol and DDAB Subcutaneous Bupivacaine Topical and IV PTX and lidocaine Oral olive oil, cholesterol, α-TOS and oleic acid, Egg lecithin, stearyl amine, albumin and glycerol [332] PCL [333] Kolliphor® HS15 [334] Caprylic (C8) and Capric (C10) triglycerides and sorbitan monoesterate GMS and DDAB Lipid Sefsol R 218 and Geleol Lavender and melaleuca oils Myristyl myristate and Miglyol 812® Tween 20, soy lecithin [335] and TPGS Lecithin, Poloxamer 188, [336] Tyr-3-octreotide-PEG-PE soy PC and HA [337] Octadecylamine, Soy PC, [338] HA and tetraiodothyroacetic acid Pluronic F68 [339] Cremophor R RH40 and [340] bile salt Pluronic F68 [341] Pluronic F68 [342] Kaempferol MCT Egg lecithin and Tween 80 [343] CD73siRNA MCT Lecithin and DOTAP [344] peptide NFL-TBS.40-63 Labrafac® Solutol HS15 and Lipoïd® [345] Anti-Galectin-1 and antiEGFR siRNA Labrafac® Lipoïd® S75-3, Solutol® [346] H15 and Chitosan RES Caprylic (C8) and Capric (C10) triglycerides and sorbitan monoesterate PTX and CpG DNA Captex1 8000 Diphenyl diselenide MCT MTX Caprylic (C8) and Capric (C10) triglycerides and sorbitan monoesterate PTX and CUR Labrafac® Nonpsychotropic cannabinoids Labrafac® CUR Labrafac® Oral and IV IV [331] ro Silymarin Brain Egg PC GMS Jo ur Topical Intracraneal [330] -p DTX PTX and ascorbyl palmitate Intranasal Egg PE andTween 80 re lP na IV Phospholipon 90 G, PEG [325] and FA of Cholesteryl oleate, cholesterol and Miglyol® 812 N, 7-Ketocholesterol, cholesteryl oleate, triolein and cholesterol IP SLN LNC [322] Soylecithin, soy PC and [326] Cetuximab Soy lecithin, Tween-80 [327] and Transferrin Tween 80 and Poloxamer [328] 188 LNC LNE Soybean PE PTX and 5Demethylnobiletin LNE NLC [321] Dynasan 114, Precirol ATO5 and Labrafac lipophileWL 1349, Oleic acid and Compritol® 888 ATO DCT and CUR Melanoma PEO (40) stearate and PEG-PE 45 PCL [347] Lipoid1S75-3, Solutol® [348] H15 and chitosan PCL, Span 80 and Tween [349] 80 PCL [350] Lipoïd® S75-3, Solutol® [351] H15, PEG and chitosan Lipoïd® S75-3 and [352] Solutol® H15 Lipoïd® S75-3 and [353] Solutol® H15 Journal Pre-proof Oral MTX Caprylic (C8) and Capric (C10) triglycerides and sorbitan monoesterate PTX and naringenin Percirol ATO5 and Dynasan 114 DTX GMS and Stearic acid PTX Stearic acid VCR and TMZ 888 ATO SLN IV IR-780 iodide PA Diosgenin CUR Precirol and capmul MCM CUR tripalmitin acid and oleic acid Atorvastatin and CUR Precifac® ATO 5 and Labrasol® VCR and TMZ Stearic acid and COMPRITOL® 888 ATO of Intanasal Stearic acid -p ro IP NLC TMZ COMPRITOL® 888 ATO lP TMZ and VCR DTX IV SLN IV NLC LNE LNC Pancreatic cancer [362] Tween 80, Lipoid S75, Transcutol® HP, HA, FA [363] and cRGDfK and H7K(R2)2 peptides PC, soy lecithin and [364] Lactoferrin and RGD , Cremophor ELP, PC, [365] Soya lecithin and RGD [366] COMPRITOL® 888 ATO PC, soy lecithin and Cremophor ELP [358] DSPE-PEG2000Maleimide and bevacizumab [367] Caprylic/capric triglyceride, Polyoxyethylene stearate and PEG-hydrogenated castor oil Labrafac and Suppocire NC Stearic acid Lipoid s75, Myrj s40 and [368] VEGFR-3 antibody Myrj 52 and lecithin [369] Solutol HS, Myrj 52 and [370] Peptide GX1 Soylecithin, Tween 80 [371] and HA DOTAP, Soya lecithin [372] and Labrafac PG GMS and soybean oil Etoposide GMS and oleic acid Chlorin e6 Oleic acid Folic acid-PEG-PE [373] Dodecafluoropentane Dodecafluoropentane Emulsiflex C-5 [374] GlaB Castor oil PLGA–PEG–DTPA [375] IV PTX Olive oil Aspirine and CUR Stearic acid IV CUR Trilaurin LNC IV Gemcitabine and Baicalein Stearic acid LNE IV DHA-SBT-1214 SLN IV Intragastrical and IP DCA, Epikuron 145, [376] Pluronic F68 and αCD44 poloxamer [377] Epikuron® 200, Tween (20, 40, 80), Cremophor [378] and Pluronic® F68 PC, Tween 80 and HA [379] Lipoid E80®, Tween 80® [380] and PEG-PE Soya lecithin, Tween 80 [381] Stearic acid and GMS and Adenosine TPGs and histamine [382] Trialurin dodecyl carbamate Precirol ATO-5 and Labrafil Soy lecithin and d-α-TOS[383] M 1944CS PEG Fish oil DOX and magnetite NLC Tween 80 5-FU and CDDP DTX and Adenosie Prostate [361] MCT Oral SLN soy lecithin PTX IV IV DSPE-mPEG-2000, Lutrol F188 and RGD [355] peptide Soya lecithin, Tween 80 and Angiopep-2 [356] (conjugated via EDC/NHS) Lecithin, Poloxamer 188 [357] and Tyr-3-octreotide PC, Cremophor ELP, soy [358] lecithin and DDAB PEG-PE, P407-Tween 80 andpeptide (cyclo (Arg- [359] Gly-Asp-d-Tyr-Lys) Lecithin and polysorbate [360] 80 Poloxamer 188 PTX and tanespimycin Jo ur Gastric cancer Ginsenoside Rg3 na LNE [354] GMS and MCT re TMZ and CUR IV PCL Tripterine OXA:Oxaliplatin; 5-Fu:5-fluorouracil; CPT:Camptothecin; PCL:Poly-e-caprolactona; CD:2hydroxypropyl-β-cyclodextrin; CUR:Curcumin; 46 PLGA:poly(lactic-co-glycolic acid); Journal Pre-proof DOX:Doxorubicin; SN38:7-ethyl-10-hydroxycamptothecin; IRI:irinotecan; PA:Palmitic acid; GMS:Glycerin monostearate; PC:Phosphatidylcholine; PE:Phosphatidyl-ethanolamine; PUFA:Polyunsaturated DOTAP:Dioleoyl-3-trimethylammonium fatty acids; trifluorophenyl)carbamoylamino]hexadecenoic PTX:Paclitaxel; MCT:Medium DAC:Decitabine; PEG:Poly(ethylene chain PAN:Panobinostat; glycol); propane; ClFPh-CHA-16:(4-chloro-3- acid; α-TOS;α-tocopherol triglycerides; LCT:Long W198:Bromotetrandrine; chain succinate; triglycerides; TPGS:D-α-tocopheryl polyethylene glycol succinate; EXM:Exemestane; RES:Resveratrol; MTX:Methotrexate; PVM/MA:Poly(methyl vinyl ether-co-maleic anhydride); DTX:Docetaxel; of THQ:Thymoquinone; pEGFP:Plasmid encoding enhanced green fluorescent protein; DDAB:Dimethyldidodecylammonium bromide; HA:Hyaluronic acid; PEO:Poly(ethylene ro oxide); PPO:Poly(propylene oxide); RGD:Arginine-glycine-aspartic tripeptide; HZ:Adipic docosahexaenoyl-2,2-difluorodeoxycytidine; -p acid dihydrazide; DHA:docosahexaenoic acid; CDDP:Cisplatin; DHA-dFdC: 4-(N)VCR:Vincristine; TMZ:Temozolomide; re DTPA:Diethylene triamine pentaacetic acid; CSB-INH-¡:Carvone Schiff base of isoniazid; CS- 5.2.1. Colon cancer lP 5-FU:cholesteryl-succinyl-5-fluorouracil; FA:Folate. na Colorectal cancer is currently the third most common type of cancer and the second most cancer-related cause of death worldwide.[2] In recent years, several LCNPs have been Jo ur described to obtain more effective therapies against colorectal cancer. Among these studies, a frequently studied parameter is drug loading. In this sense, Tsakiris et al. encapsulated six different drugs, three hydrophobic and three hydrophilic, into LCNCs. Apart from using the same synthesis process for the three hydrophobic and the three hydrophilic drugs, drug loading changed depending on the drug. In fact, the formulation with smaller drug loading was chosen for the subsequent in vitro and in vivo assays, since the bioactivity of this encapsulated drug was higher than that of the others.[267] Bhat et al. developed a carvone Schiff base of isoniazid (CSB-INH) loaded LNEs as an orally administered treatment for colorectal cancer. They evaluated the drug release under gastric simulated conditions. Under this acidic condition, they found a higher release of the compound from LNEs than from the insoluble drug suspension. In this case, a higher release means an improvement in terms of bioavailability and, therefore, a higher amount of compound available to be absorbed at the intestinal level. These authors also compared the plasma concentration profile of the compound in rats, after oral administration of drug-loaded LNEs or free drug suspension, and found a higher drug 47 Journal Pre-proof concentration in the plasma of LNEs-fed rats.[262] On the other hand, Yawei et al. evaluated the stability of a camptothecin and palmitic acid conjugate (CPT-PA) in its free form and encapsulated inside SLNs. CPT is very unstable under physiological conditions, especially under reductive ones, which hinder the drug from achieving its target and lead to severe side effects. Moreover, it is a poor water-soluble component and is non-soluble in lipids. The strategy used to protect the compound from premature physiological degradation and increase its solubility was the encapsulation in SLNs by synthetizing a CPT-PA conjugate to improve drug solubility in lipids and drug loading. Unlike the previous study, conjugate release from SLNs was smaller than the release from the suspension under simulated digestion conditions. of In this case, the slower release provided an advantage, since the instability of the free compound would reduce its bioavailability.[268] A fast release of CPT from SLNs under reductive ro conditions was also reported, demonstrating that CPT could be effectively released from the -p conjugate. This fast release under reductive conditions provides a further advantage to the system, since the tumor environment is also reductive, owing to the overexpression of re glutathione.[268] They reported that the encapsulated conjugate could effectively cross an in vitro Caco-2 simulated intestinal epithelium. Bhat et al. and Yawei et al. equate plasma drug lP concentrations and bioavailability.[262,268]. However, according to the bioavailability definition, evaluation of this parameter requires the analysis of the effect on the tumor, because na a higher plasma concentration of the drug without a visible effect on the tumor target would not be relevant in practical terms. In this sense, in vivo assays performed with tumor bearing mice Jo ur to study tumor growth inhibition or tumor drug accumulation provide a better characterization of the nanocarrier.[228,266,267,269–272] For instance, the study of CUR-loaded PEGylated PLGA LNCs in mice showed a prolonged blood circulation time of these LNCs thanks to the PEG coating. Moreover, radio-labelled LNCs were confirmed to accumulate on the tumor site and furthermore, a significant reduction of tumor volumes was observed in CUR-loaded PEGylated PLGA-LNCs treated mice when compared with empty LNCs-treated mice.[266] Similary, the accumulation of LNCs into reticuloendothelial system rich-organs for their subsequent elimination is also common and is one of the factors limiting bioavailability of encapsulated drugs.[266,270] The strategy followed by Jang et al. to reduce LCNPs elimination was to pre-inject tumor bearing mice with empty SLNs before treating animals with CPT-SLNs. This way, they achieved the saturation of the reticuloendothelial system rich-tissues and improved targeting and accumulation of drug-loaded SLNs in tumors.[270] On the other hand, these authors also reported the protective role of SLNs on encapsulated CPT, an in vitro long term sustained release, an improvement in the in vitro cytotoxic effect of CPT-SLNs compared 48 Journal Pre-proof to free CPT, as well as in vivo prolonged blood circulation compared with free CPT and significant tumor growth inhibition.[270] Another interesting strategy to improve drug release at the tumor site is the use of stimuliresponsive NPs. Along these lines, pH-responsive PEG-lipid-derivate SLNs and liposomes are found.[272] These SLNs and liposomes take advantage of the acidic pH of the tumor microenvironment to achieve specific release of the encapsulated microRNA and irinotecan (IRI), respectively, at the tumor site. Moreover, microRNA loaded PEG-coated SLNs and Iriloaded PEG-coated liposomes were further functionalized with tumor targeting peptides, which resulted in improved inhibition of tumor growth and reduction of the side effects and systemic of toxicity on tumor bearing mice. In addition, microRNA-SLNs and IRI-liposomes were administered in a combined treatment, which showed an improvement in in vitro cytotoxicity ro outcomes in HCT116 cells and higher significant reduction of tumor size and side effects in -p mice.[272] Recently, Borderwala et al. prepared NLCs containing 5-fluorouracil (5-FU) as the chemotherapeutic agent and coated with Eudragit S-100, a pH-sensitive polymer found to re provide release in the colonic region.[273] In vitro and in vivo experiments demonstrated the capacity of the prepared NLCs to retain the integrity and to pass through the stomach and dissolved.[273] lP intestine without releasing the drug until reaching the colon, where the coating is na The combination of drugs to enhance anticancer activity is an approach currently applied in clinics. We can find studies where the combined drugs are included in separate nanocarriers, Jo ur which improve drug solubility and/or stability, its cytotoxic effect, drug targeting, or reduce side effects.[267,272] LCPNs offer the possibility to co-encapsulate several drugs in the same nanocarrier, such as the CPT derivative/IRI loaded NLCs coated with HA to target colon adenocarcinoma.[384] However, this study does not include in vivo characterization of the nanocarrier. 49 na lP re -p ro of Journal Pre-proof Jo ur Figure 10. Representative preclinical studies of LCNPs to target colon cancer.[228,262,271,273] 5.2.2. Breast cancer Breast cancer is the most commonly occurring cancer in women.[2] DOX, DTX and PTX are broad spectrum antineoplastic compounds frequently employed in breast cancer chemotherapy.[215] To enhance the chemotherapeutic behavior of these compounds and reduce off-target effects, LCNPs have been applied to encapsulate DOX [224,385], DTX [291], and PTX.[277] Interestingly, Dos Santos Câmara et al. encapsulated the prodrug C16-DOX, a lipidated, inactive and pH-sensitive form of DOX, in castor oil LNEs prepared using a spontaneous emulsification procedure. Once the nanocarrier reached the tumor tissue, its acidic environment cleaved the hydrazone bond of the prodrug, resulting in a localized DOX release. In vivo studies on 4T1 murine cancer model revealed that this nanoformulation allowed for the use of a higher dose of DOX and improved the chemotherapeutic index and tumor control efficacy.[224] Similarly, Burgarelli-Lages et al. entrapped the pH-sensitive doxorubicintocopherol succinate (DOX-TS) prodrug in DHA-based NLCs. The in vitro and in vivo 50 Journal Pre-proof experiments demonstrated better DOX-TS-NLCs pharmacokinetics compared to free DOX and DOX-NLCs, as well as the prevention of short-term cardiotoxic effects of DOX after intravenous injection in 4T1 tumor-bearing mice model.[309] Combination chemotherapy is an attractive strategy for addressing multifaceted challenges associated with cancer.[229] An interesting approach is combinatorial therapy employing established chemotherapeutic agents with compounds that can inhibit the MDR effect. As an example, Cao et al. prepared LNEs of oleic acid containing DOX and bromotetrandrine (W198), a potent P-gp inhibitor that can prevent them from pumping out drugs. In vitro cytotoxicity assays revealed that at the same concentration level, DOX+W198 and DOX/W198- of LNEs exhibited much greater inhibitory effects than DOX solution and DOX-LNEs in MCF7/ADR resistant breast cancer cells. After intravenous injection in MCF-7/ADR-bearing ro xenograft mice, DOX/W198-LNEs demonstrated enhanced tumor uptake and higher plasma -p concentrations along with reduced cardiac toxicity of both drugs.[280] Different LCNPs have also been developed to co-deliver DOX in combination with other drugs to treat breast cancer. re CUR, which possesses P-gp inhibitor properties along with antitumor activity, was included in SLNs;[299] β-lapachone, a novel therapeutic agent that dramatically influences various P-gp- lP related pathways, co-delivered with DOX in NLCs;[307] thymoquinone and Tocopheryl polyethylene glycol 1000 succinate, a soluble natural derivative of Vitamin E, co-delivered with na DTX in LNCs;[286] as well as sello and sclareol.[285,310] Similarly, LCNPs are also employed for the combinatorial therapy of PTX and DTX.[282,284,386] Jo ur Decreasing estrogen levels through the inhibition of aromatase, the enzyme that turns androgens into estrogens, is a selective and effective therapy for hormone-dependent breast cancer patients. Aromatase inhibitors (AIs) are successfully used in the treatment of estrogen receptor (ER) positive breast cancer but have poor aqueous solubility.[387]. Elzoghby et al. prepared protamine-coated LNCs containing the AI Letrozole and the COX-2 inhibitor Celecoxib. COX2 inhibitors can reduce the expression of Prostaglandin E2, which promotes aromatase gene expression, consequently reducing estrogen production in breast cancer cells. The developed LNCs demonstrated antitumor effects in vivo as evidenced by the reduction of tumor volume and aromatase level.[218] This group also reported the development of LNCs coated with a crosslinked shell of zein, a natural hydrophobic protein, for oral codelivery of Exemestane (EXE), a third-generation AI clinically approved, and Resveratrol (RES), a polyphenolic phytoestrogen.[218] Similarly, Singh et al. prepared NLCs of Precirol® ATO 5 and flaxseed oil as the solid and liquid lipid, respectively, encapsulating EXE. An in vivo pharmacokinetic study on female Wistar rats found an increase of 3.9 fold in oral bioavailability of EXE through 51 Journal Pre-proof NLCs compared with EXE suspension.[301] Recently, Jain et al. developed SLNs containing Reloxifene, a second-generation selective estrogen receptor modulator, showing promising results both in vitro and in vivo.[289] LCNPs can be used to solubilize and deliver other promising chemotherapeutic compounds in breast cancer treatment, which have limited clinical application. This is the case of α-tocopherol succinate (α-TOS), a derivative of Vitamin E. Gao et al. designed LNEs mainly composed of ethyl oleate encapsulating α-TOS using an emulsification-evaporation procedure. α-TOS-LNEs showed stronger inhibitory effects on MCF-7 cells compared to free α-TOS solution and, in vivo experiments showed significant improvement on the metabolism time of α-TOS in rats, of both by intravenous and intraperitoneal injection.[276] α-TOS is also applied in combinatorial therapy nanosystems as the NLCs co-encapsulating DOX, DHA and α-TOS proposed by Lages ro et al.[303] In vitro cell studies indicated that DOX, DHA, and α-TOS have synergistic effects -p against 4T1 tumor cells. The in vivo study showed that DHA-DOX-α-TOS-NLCs exhibited the greatest antitumor efficacy by reducing tumor growth in 4T1 tumor-bearing mice and reduced re mice mortality, prevented lung metastasis, and decreased DOX-induced toxicity to the heart and liver. Recently, Arshad et al. designed NLCs containing Calycosin, a novel anti-cancer lP drug under clinical trials, showing significant recovery in mammary glands weight loss, which occurred due to cancer, to their normal level.[302] Similarly, Talaat et al. formulated LNCs na encapsulating fisetin through the layer-by-layer method. Fisetin is a promising flavonol that has proved to inhibit cancer growth without causing toxicity to healthy cells.[220] Compared to the Jo ur free drug, the nanoformulation showed a 4-times decrease of the IC50 in the in vitro cytotoxicity and a superior therapeutic effect in the in vivo model. Another interesting approach is the codelivery of a chemotherapeutic agent and DNA, which can overcome drug resistance, decrease side effects, and achieve enhanced antitumor efficiency.[292] Yu et al. prepared SLNs of glycerol monostearate coated with HA and coencapsulating PTX and pDNA. In vitro experiments on MCF-7 cells and in vivo in breast cancer xenograft BALB/c nude studies, confirmed that the developed SLNs could inhibit the tumor and, at the same time, deliver and transfect genes into cancer cells.[292] Several ligands and targeting moieties can be attached onto the surface of LNPs in order to achieve active targeting in breast cancer treatment. In this sense, Folate (FA) is one of the most employed and studied. FA receptors are upregulated in different types of cancers such as breast, lung and colon. Therefore, FA decorated nanosystems can act as a selective drug delivery system to positive FA receptors cancer cells/tissues.[388] Furthermore, compared to antibody ligands, FA is advantageous due to its smaller size, non-immunogenicity, non-toxicity, ease of 52 Journal Pre-proof handling, stability and low cost.[389] Tripathi et al. prepared FA decorated LNEs of α-linolenic acid encapsulating DOX. In vivo studies in 7,12-dimethylbenz[a] anthracene(DMBA)-induced breast cancer tumor Albino Wister rats revealed that, after tail vein injection, decorated LNEs enhanced antitumor targeting potential and therapeutic safety compared to other non-decorated LNEs and free DOX, thus corroborating the effectiveness of active targeting.[278] Similarly, Poonia et al. reported the synthesis of RES NLCs decorated with FA as the targeting moiety. Cell cytotoxicity experiments revealed high cytotoxic effects of FA-NLCs compared to unmodified NLCs on MCF-7 cells along with enhanced bioavailability and pharmacokinetic behavior in vivo. This study suggested the high potential of targeted NLCs in enhancing the of therapeutic concentration of RES to breast cancer cells.[308] FA is also employed for the active targeting of different LCNPs.[296,304] Bombesin [297], Arginine-glycine-aspartic (RGD) 2-Hydroxypropyl-b-cyclodextrin,[290] ro peptides,[298,305] adenosine,[381] and -p lysophosphatidic acid [279] are also employed as target moieties to decorate and functionalize LCNPs. re In relation to administration routes of LCNPs in breast cancer treatment, most of the developed nanosystems are engineered to reach the tumor tissue by parenteral administration. However, lP LCNPs offer important advantages and can be conveniently used for oral delivery, as, for example, the aforementioned LNCs prepared by Elzoghby et al.[218] or the NLCs developed na by Singh et al.[301] and Nordin et al.[223], encapsulating AIs and Citral, respectively. Baek et al. included CUR in chitosan coated SLNs prepared by hot homogenization.[288] Coated SLNs Jo ur exhibited suppressed burst release in simulated gastric fluid, prevented by the polymer coat, while sustained release was observed in simulated intestinal fluid. Furthermore, the prepared SLNs exhibited increased cytotoxicity and cellular uptake on MCF-7 cells. The lymphatic uptake and oral bioavailability evaluated using male Sprague Dawley rats were found to be 6.3 fold and 9.5 fold higher than that of CUR solution, respectively.[288] Similarly, GarrastazuPereira et al. developed LNEs prepared by the PIT process for the encapsulation of a synthetic derivative of ω-3 polyunsaturated fatty acid, namely CIFPh-CHA.[274] Oral administration in xenografted mice of the drug-loaded LNE was able to significantly reduce tumor mass to ~50% of untreated control at doses of 10 and 40 mg·kg-1.[274] Due to breast cancer usually beginning in the lining of the ducts, intraductal administration arises as a promising administration route to combine efficacy and reduce systemic adverse effects. Migotto et al. developed chitosan-coated positive bioadhesive LNEs encapsulating C6 ceramide as the chemotherapeutic agent. LNEs decreased the IC50 of C6 ceramide in MCF-7 cells by 4.5 fold when compared to the free solution.[275] In vivo experiments of C6-containing 53 Journal Pre-proof LNEs conducted in Female Wistar rats revealed that drug localization, after intraductal Jo ur na lP re -p ro of administration, remained for more than 120 h in the mammary tissue compared to its solution. Figure 11. Representation of interesting preclinical studies of LCNPs for breast cancer treatment.[275,298,301] 5.2.3. Lung cancer Lung cancer is currently the second most common cancer type and the first cancer-related cause of death worldwide.[2] Despite advances in chemotherapeutics to improve survival, median survival remains limited to less than 12 months. Escalation in global lung carcinoma mortality 54 Journal Pre-proof presents a grave concern. Chemotherapy of non-small cell lung cancer, the most common form of lung carcinoma, employs DTX, PTX, DOX and cetuximab, among others, as promising molecules. However, an amalgamation of the issues pertaining to poor safety and toxicity profile, pharmacologic resistance and poor tumor bioavailability of these second generation taxanes calls for strategies that may promote its clinical worth.[390] Combinatorial chemotherapy is also employed in lung cancer treatment. Rawal et al. employed co-delivery of DTX and CUR through the development of FA–appended NLCs (FA-DTX/CUR-NLCs) with promising results, such as significantly better in vivo relative bioavailability of DTX (24.85 fold) with FA-DTX/CUR-NLCs compared with Taxotere®.[325] Immunostaining of the tumor of sections with tumor differentiation biomarkers suggested considerably higher apoptotic, antiproliferative, anti-angiogenic and anti-metastatic potential of FA-DTX/CUR-NLCs compared ro with Taxotere®. In vivo toxicity assessment of the NLCs demonstrated a noteworthy reduction -p in DTX associated side effects.[325] Recently, Khatri et al. prepared FA appended PEGylated SLNs for the encapsulation of PTX and Artemether. In vitro and in vivo experiments concluded re that the anticancer potential of PTX was improved without any renal or hepatic toxicity, which indicated that the developed formulation is able to reduce dose related toxicity of PTX.[318] lP β-elemene (ELE) is an antitumor agent extracted from the chinese medicinal plant Radix Curcumae. Previous studies have shown that ELE exhibited anti-cancer effects in many cancer na cells, especially lung cancer cells, by inducing apoptosis.[323] Cao et al. developed DOX and ELE co-loaded, pH sensitive NLCs (DOX/ELE-NLCs) with greater lung tumor inhibition Jo ur ability.[323] Guo et al. developed cetuximab functionalized, PTX and 5-Demethylnobiletin coloaded NLCs, exhibiting a remarkable in vivo tumor inhibition efficiency, high tumor accumulation amount and low toxicity.[326] Moreover, Zhou et al. demonstrated that PTX and salinomycin active-targeting NLCs killed cancer cells and cancer stem cells (CSCs) at the same time.[321] Additionally, there are other recent studies where LNEs are used as drug nanocarriers against lung cancer. Typically, these LNEs are functionalized with active targeting RGD peptides.[312] LNEs can be used as an imaging agent, targeting different receptors like PARP1,[391] as well as for the treatment of lung cancer, carrying different drugs like CUR or PTX.[311,313] LNCs are also useful in the treatment of lung cancer. Erlotinib (ERL), an EGFR tyrosine kinase inhibitor, is currently available on the market as tablets for oral administration. However, poor oral bioavailability associated with poor solubility and permeability results in limited therapeutic efficacy. In addition, traditional oral delivery of ERL is accompanied by severe side effects including rash, diarrhea, gastrointestinal perforations, ocular lesions, and hematological 55 Journal Pre-proof disorders.[315] Kim et al. developed PEGylated polypeptide LNCs to enhance the anticancer efficacy of ERL in non-small cell lung cancer with high drug entrapment efficiency (∼95%), effective controlled release, efficient internalization via receptor-mediated endocytosis, and improved antitumor efficacy upon intravenous administration.[315] Recently, Rampaka et al. prepared ERL-loaded SLN for oral administration. In vivo pharmacokinetic studies revealed an improvement in bioavailability of ERL around 2.12 fold and a significant reduction in fed to fasted variability.[317] 5.2.4. Melanoma of Melanoma is the most aggressive skin cancer because of its high metastatic rate.[392,393] Current available therapies are ineffective, and dacarbazine, the main chemotherapist used ro against metastatic melanoma, induces a low response rate.[334] Some studies explore the use -p of new compounds such as those reported by Fofaria et al., who developed LNEs to encapsulate piplartine (PL), a hydrophobic anticancer active compound found in black pepper.[329]. The re PL-loaded LNEs achieved solubilization of the compound, provided increased bioavailability compared with the free compound when orally administered and reduced the weight of the lP tumor in tumor bearing mice melanoma model.[329] On the other hand, the encapsulation of diphenyl diselenide [(PhSe)2], a synthetic organoselenium compound that has multiple na pharmacological properties,[394] in LNCs, improved the stability of the compound upon exposure to UV radiation compared to the free form. Moreover, encapsulation significantly Jo ur reduced its cytotoxic effects on healthy cells, proving the safety and highlighting the added value of the nanocarrier.[393] Another approach is the use of LCNPs as the platform for codelivery of synergistic drugs. For instance, Zhou et al. developed palmitate and PTX-loaded SLNs as a dual drug delivery system, which improved the cytotoxic effect in vitro, inducing cell apoptosis, and reduced tumor sizes and density of tumor cells in vivo, compared to the individual drugs.[339] Following this lead, Resnier et al. chose a dual therapy, combining gene therapy with ferrocifen.[334] They managed to encapsulate Bcl-2 siRNA and Ansa-ferrociphenol on the same LNC. The siRNA was able to down-regulate Bcl-2 expression. This protein blocks the oligomerization of Bax and Bak proapoptotic proteins, avoiding the activation of the apoptosis pathway. Therefore, the downregulation of this overexpressed protein in melanoma cells would facilitate apoptosis activation and would reduce chemoresistance in tumor cells. The siRNA-LNCs achieved Bcl2 gene silencing and the Ansa-ferrociphenol-LNCs showed the same cytotoxicity as the free compound. The combination of siRNA-LNCs with the free compound demonstrated the 56 Journal Pre-proof synergistic effect of dual therapy and yielded better results than the combination of siRNALNCs with dacarbazine. In addition, the co-encapsulation of both siRNA and Ansaferrociphenol, improved siRNA encapsulation efficiency. The in vivo outcomes of intravenously administered siRNA/Ansa-ferrociphenol-LNCs also reported a higher tumor size reduction with respect to mice treated with one-drug LNCs.[334] A commonly used strategy to improve chemotherapy effectiveness is the design of targeted nanocarriers to achieve higher drug concentration into the tumor and reduce side effects. In this regard, albumin-decorated DTX-loaded LNEs and HA-coated SLNs have been developed.[107,332] HA is one of the biomolecules frequently used to decorate nanocarriers. of This molecule binds to the CD44 receptor, overexpressed in cancer cells from different cancer types.[337,338] Shi et al. used synergistic dual targeted SLNs based on HA to target CD44 ro receptor and tetraiodothyroacetic acid (tetrac) to target integrin αvβ3, which is overexpressed in -p tumor endothelial cells.[338] The DTX-loaded HA/tetrac SLNs improved the cytotoxic effect in vitro and the cellular uptake of CD44+/ αvβ3+ cells but not of CD44-/ αvβ3- cells. In vivo assays re reported the effective synergistic dual targeting of HA/tetrac SLNs to the tumor environment and cells in xenograft and lung metastasis models. Moreover, HA /tetrac SLNs not only lP provided higher tumor growth inhibitions when compared to the non-targeted system, but also when compared to the HA-coated SLNs and tetrac-coated SLNs.[338] The LDL receptor is also na overexpressed in cancer cells. Kretzer et al. synthesized PTX-loaded LNEs, which specifically bind to the LDL receptor [330] as the 7-ketocholesterol LNEs developed by Favero et al.[331] Jo ur When the skin tumor is superficial, a topical route for drug administration is possible. Drug encapsulation into LCNPs can enhance drug permeability through the skin. An example of this is the one developed by Iqbal et al. using silymarin-loaded SLNs.[331] De Moura et al. prepared DTX and lidocaine co-loaded NLCs embedded into a xanthan-chitosan hydrogel for topical administration. In vivo assays indicated that the hybrid hydrogel was able to inhibit tumor growth in an equivalent manner to the conventional (free DTX) treatment and showed no adverse effects, as revealed by physical, biochemical, and histopathological parameters.[342] Recently, Geronimo et al. prepared NLCs of lavender and melaleuca oils for the encapsulation of Bupivacaine, the most widely used local anesthetic agent in surgery, which presents anticancer properties. In vitro cytotoxicity tests revealed that the optimized NLC increased melanoma cell death and greater in vivo anesthetic activity after subcutaneous application.[341] Furthermore, topical photodynamic therapy is a possible alternative treatment, which requires a photosensitizing agent on the tumor site that will then be excited by a specific wavelength to produce reactive oxygen species in the tumor environment.[328] Dalmolin et al. encapsulated 57 Journal Pre-proof the lipophilic photosensitizing agent zinc phthalocyanine into LNEs and used iontophoresis, i.e., the application of a constant and weak electric field on the skin to generate a voltage gradient, to facilitate the penetration of Zinc Phthalocyanine LNEs through the skin’s inner layers to the tumor site. The iontophoresis process enhanced skin permeability of the emulsions, demonstrating improved permeability when particle size was smaller.[328] A less explored possibility is the use of the intrinsic toxicity of the nanocarriers themselves against tumor cells. Drewes et al. reported the inhibition of melanoma development in mice orally treated with LNCs, both loaded with eugenol or not. This antitumor effect was not associated with systemic toxicity or side effects. In view of these findings, they argue that the of antitumor efficiency of the system is due to the very structure of the carrier and not to its role ro as drug delivery system.[333] -p 5.2.5. Brain cancer Brain cancer is currently the 19th most common cancer type and the 12th cancer-related cause re of death worldwide.[2] Glioblastoma multiforme (GBM) is the most common and malignant type of brain tumor in adults. After initial diagnosis, the median survival of GBM patients is lP about 12–15 months, even with aggressive treatment.[395,396] Unlike other tumors, GBM treatment represents a major challenge, mainly due to its location in the brain, which hinders na complete surgical resection, and the presence of the BBB, which limits drug entry into the central nervous system. Despite all the recent biomedical advances, current treatment is still Jo ur confined to surgical resection, radiotherapy, and chemotherapy epitomized by temozolomide (TMZ). This standard treatment is applied to newly diagnosed GBM patients, but median survival remains unsatisfactory.[397] The development of new drugs has not been sufficient in improving GBM treatment. Most drugs have poor solubility in water, cannot cross the BBB, and require high doses to achieve the effective concentration, leading to toxicity consequences and adverse effects. To overcome these limitations, attempts have been made to develop LCNPs as promising drug-delivery nanocarriers to treat GMB.[398] LCNPs can cross the BBB and are suitable carriers for a wide spectrum of GBM treatments such as large molecules, genes, oligonucleotides, siRNA, and enzymes, with SLNs and NLCs being the most employed.[399] SLNs have proved to cross the BBB carrying DTX and functionalized with peptides such as angiopep-2,[356] or carrying PTX and Tyr-3-octreotide[357] or diosgenin,[360] with promising antitumor effects in orthotopic in vivo models. Interestingly, Wang et al. developed surface-modified with RGD peptides SLNs containing PTX and naringerin peptide for a combinatorial therapy against GMB upon oral administration.[355] The dual drug-loaded SLNs 58 Journal Pre-proof showed significant improvement in drug pharmacokinetics and higher cytotoxicity and chemoprotective effect versus the free drug suspension.[355] Regarding NLCs, different studies have demonstrated their capabilities of crossing the BBB, active targeting, and antitumor in vivo effects with different drugs such as TMZ and CUR, and even using synergic therapy with both drugs.[362,365,366] Di Filippo et al. prepared bevacizumab-coated NLCs encapsulating DTX to target GBM.[367] In vitro anti-tumor assays showed that BVZ-NLC-DTX selectively increased the cytotoxic of DTX in cells overexpressing VEGF (U87MG and A172) though not in healthy cells (PBMCs). An in vivo orthotopic rat model demonstrated that free-DTX was not capable of reducing tumor growth of whereas BVZ-NLC-DTX reduced up to 70% tumor volume after 15-days of treatment. Another example of NLCs as nanocarriers for synergic cancer therapy in GBM is the combination of ro TMZ and VCR-coloaded NLCs functionalized with RGD peptides, which exhibited sustained- -p release behavior, high cellular uptake, high cytotoxicity and synergy effects, increased drug accumulation in the tumor tissue, and notorious tumor inhibition efficiency with low systemic re toxicity.[364] In fact, Wu et al. carried out a study in which they compared SLNs and NLCs for the dual drug delivery of VCR and TMZ. They state that NLCs can deliver VCR and TMZ lP into U87MG cells in an orthotopic brain tumor implant more efficiently, and inhibition efficacy is higher than SLNs. The inhibition efficacies of dual drugs-loaded NLCs in vitro and in vivo na are also higher than single drug-loaded vectors.[358] Finally, Qu et al. conducted a study with the aim of comparing which type of drug delivery nanosystem (SLNs, NLCs, and polymeric Jo ur NPs) was better for GBM chemotherapy using TMZ.[400] They concluded that NLCs exhibited significantly better targeting ability as well as tumor growth inhibition rate.[400] LNCs have been used to treat brain tumors using different strategies. Lollo et al. prepared LNCS loaded with PTX and the immuno-stimulant single-stranded DNA molecule containing methylated cytosine-guanine dinucleotides (CpG), which increased the survival rate of GL261 glioma-bearing mice.[348] Using PTX, Groo et al. developed different LNCs in order to compare their pharmacokinetics and efficacy in a subcutaneous isograft model in rats.[351] MTX is an antifolate drug that has been used for more than a half century in cancer research and clinical treatment. MTX has been studied in a wide range of cancer types, including solid brain tumors.[401] Figueiro et al. observed a decrease in tumor size and an increase in apoptosis in the tumor microenvironment using MTX-LNCs. This treatment decreased the leukocyte number but did not alter toxicological tissue marker expression or metabolic parameters.[354] Furthermore, Pereira et al. employed MTX-LNCs for oral administration.[350] They showed a higher therapeutic efficacy of MTX-LNCs in relation to MTX in GBM treatment, suggesting 59 Journal Pre-proof that low oral doses of MTX-LNCs are a safer and effective alternative to the current expensive and invasive intravenous high-dose MTX regimens. The beneficial effect of MTX-LNCs could be due to the improved ability of LNC-loaded drugs to cross the BBB and the efficient MTXLNCs uptake by cancer and immune cells in the brain.[350] Despite the pharmacological properties of (PhSe)2, some toxic issues limit its therapeutic use, such as the inhibition of enzymes and oxidation of biomolecules.[402,403] In addition, (PhSe)2 is a poorly watersoluble compound,[404] which leads to low oral bioavailability [405] and hinders its administration by other routes, such as the parenteral one. Ferreira et al. demonstrated a decrease in C6 glioma cell viability without causing any adverse effect in astrocyte cells of (healthy control) by employing (PhSe)2-LNCs.[349] Importantly, the (PhSe)2-LNCs had a superior cytotoxic effect compared to its free form, as well as increased nitrite content. ro Intragastric treatment reduced brain tumor size and did not cause alteration in the plasma renal -p and hepatic markers of function or in the parameters of oxidative balance in the brain, liver, or kidneys.[349] re Mucoadhesive-LNEs are also employed intranasally as a brain-tumor drug-delivery system. Colombo et al. demonstrated that LNEs carrying Kaempferol (KPF), an anti-oxidant, anti- lP inflammatory, neuroprotective and anti-tumor agent, showed no toxicity towards nasal mucosa.[343] Ex vivo permeation studies and in vivo biodistribution studies confirmed the na superiority of the developed chitosan-coated LNEs for brain targeting after intranasal administration compared to KPF-LNEs and free KPF. The mucoadhesive LNEs decreased the Jo ur viability of glioma cells by enhancing apoptosis.[343] Similarly, Azambuja et al. prepared cationic LNEs delivering siRNA for CD73, an enzyme responsible for adenosine production involved in a variety of tumor-progression actions, as a gene therapy for GBM treatment.[344] Upon nasal delivery, LNE-siRNA CD73R reduced tumor growth by 60% in glioma-bearing Wistar rats and achieved a 95% decrease in adenosine levels in tumor expression, confirming CD73 silencing.[344] 60 lP re -p ro of Journal Pre-proof 5.2.6. Gastric cancer na Figure 12. Representative studies of LCNPs for brain cancer treatment.[344,346,355] Jo ur Gastric cancer is the fifth most common cancer around the world and the fourth cancer-related cause of death.[2] Among the most recent reviewed literature, we find studies that improve the in vitro and in vivo performance of already used drugs, such as etoposide.[372] This drug inhibits DNA synthesis, but its poor water-solubility limits its bioavailability, and cancer cells show resistance to this compound. The encapsulation in NLCs improved both the solubility and the stability of the drug, contributing to higher in vitro cytotoxicity and better in vivo tumor growth inhibition.[372] A targeting strategy also provides interesting results. Dai et al. functionalized ginsenoside Rg3 LNEs with a vascular endothelial growth factor receptor (VEGFR-3) antibody. The VEGFR-3, together with VEGF-C and VEGF-D, is involved in the metastatic spread of tumor cells throughout lymphatic vessels.[368] VEGFR-3 coated LNEs down-regulated the expression of these three factors and reduced gastric tumor growth in mice when compared with the control group. However, the most remarkable result is that LNEs reduced lymph-node metastasis in mice when compared with the control and 5-FU treated mice. [368] Jian et al. used an 61 Journal Pre-proof oligopeptide, which bound to the blood vessels irrigating the gastric adenocarcinoma, GX1 peptide, to coat PTX-loaded NLCs. The encapsulation of the drug slowed down its release. They also studied the effect of GX1-PTX-NLCs, PTX-NLCs, and PTX on HUVEC cells, as vascular epithelial cells model, and on MKN45 cells, as gastric cancer cells. GX1-PTX-NLCs showed the strongest cytotoxic effect on HUVEC cells, while PTX-NLCs were more cytotoxic for MKN45 cells than the GX1-coated NLCs. GX1-coating also improved the NLCs uptake by HUVEC cells and reported a better inhibition on tumor growth in vivo, compared to PTX-NLCs and free PTX.[370] 5-FU and cisplatin (CDDP) combination is the first line chemotherapy treatment for gastric of cancer. Qu and coworkers developed HA-coated NLCs for the codelivery of these two drugs. They combined both drugs at different proportions and studied the synergistic effects on ro BGC823 human gastric cancer cell line. The in vivo results in mice reported higher tumor -p growth inhibition on 5-FU and CDDP NLCs. Moreover, the HA-coated 5-FU and CDDP LNCs reported the best results regarding tumor growth inhibition, as well as reduced side effects of re treatment.[371] Combinatorial nanotherapy of PTX and tanespimycin co-loaded into SLNs was proposed by Ma et al. The encapsulated SLNs reduced cell viability and colony formation in lP gastric cell lines and could also induce apoptosis in MKN45 cells and inhibit growth of xenografts.[369] na Immunotherapy has become a promising strategy in cancer research. Along these lines, FAcoated chlorin e6-loaded-NLCs is an in-situ vaccine that stimulates dendritic cells and the Jo ur subsequent immunologic response in gastric cancer.[373] Since chlorin e6 is used as a photodynamic therapy agent, this compound is expected to produce cell damage after being excited by the appropriate light wavelength. Tumor cell damage will facilitate the exposure of tumor associated antigens to dendritic cells, which will contribute to immunological activation and cancer vaccination. Their results confirmed the activation of dendritic cells after photodynamic therapy and proved to not only reduce the sizes of the primary tumors, but also of the distant tumor in vivo when mice were treated with FA-coated chlorin e6 -loaded- NLCs. On the other hand, the coating with FA improved the NLCs targeting at the tumor site.[373] 5.2.7. Pancreatic cancer The high mortality rate associated with pancreatic cancer, with a five year survival rate below 7%, makes this cancer the seventh cancer related cause of death.[2] The aggressivity of this illness is linked to the existence of a subpopulation of CSCs within the tumor, which is responsible for metastatic tumor initiation, growth, recurrence and chemotherapy 62 Journal Pre-proof resistance.[406] In light of the relevance of CSCs in pancreatic cancer prognosis, Ingallina and coworkers focused their work on targeting the Hedgehog signaling pathway, which regulates normal cell growth and differentiation.[375] The dysregulation of this pathway leads to tumorigenesis and to more aggressive phenotypes associated with CSCs. Ingallina et al. loaded Glabrescione B (GlaB), a natural compound that binds to the nuclear Hedgehog modulator GLI1, in GlaB-LNCs. [375,407] GlaB-loaded LNCs were more cytotoxic for CSCs compared with the Hedgehog-down regulated-non-stem population. However, the in vivo assays showed accumulation of LNCs on first-pass organs and the concentrations achieved at the tumor site were below the therapeutic threshold. In another study, Navarro-Marchal et al. encapsulated of PTX into olive oil LNCs functionalized with anti-CD44 antibody in an attempt to selectively target pancreatic CSCs.[376] In vitro efficient targeted delivery to PCSCs and the in vivo ro noninvasive imaging cell tracking suggest their promising potential in targeted tumor -p theragnostic.[376] To improve the treatment response of pancreatic tumors, Johnson et al. developed re dodecafluoropentane-loaded LNEs to enhance tumor oxygenation. Their final objective was to raise the effect of tumor radiation therapy, since hypoxia in tumors has proved to reduce lP radiation effectiveness.[374] Dodecafluoropentane binds to O2 and acts as an oxygen carrier. Dodecafluoropentane-LNEs achieved oxygenation of the tumor for a short period of time, na which was enough to attain tumor sensibilization to the radiotherapy. Moreover, the combined treatment of carbogen breathing therapy, radiotherapy, and LNEs provided higher reductions Jo ur of tumor sizes compared with the combined treatment of carbogen breathing therapy and radiotherapy (2 fold reduction) and compared with the non-treated group (25 fold reduction).[374] In terms of chemoprevention, oral administration of aspirin and CUR-loaded SLNs combined with oral administration of free sulforaphane have been proved to suppress the progression of pancreatic intraepithelial neoplasms, the precursor of pancreatic ductal adenocarcinoma.[377] The toxicity of the combined therapy was studied for different time periods, and its lack of toxic effect was proved up to 90 days of administration.[377] In addition to the lack of efficient treatments for pancreatic cancer, little is found in the literature about this subject. This seems to reflect the difficulty involved in the development of effective therapies against this tumor type, partially due to the special characteristics of the cellular population within the tumor. 5.2.8. Prostate cancer 63 Journal Pre-proof Prostate cancer is the fourth most common cancer and the second cancer related cause of death in men.[2] In the most recent literature, the search for effective therapies has led researchers to explore the use of new drugs, such as the new generation toxoid SBT-1214. This drug is active against CSCs, which usually show resistance to other drugs used in clinics.[380] A conjugated omega-3 fatty acid/SBT-1214 prodrug has been effectively encapsulated into LNEs.[380] The LNE showed an efficient uptake by PPT2 cells in both the total population and the enrichedCSCs population. Moreover, the encapsulation of the drug increased its cytotoxic effect. The in vivo assays showed an improvement in tumor growth inhibition in LNEs-treated mice, even at low drug concentrations. In addition, the LNEs treatment inhibited tumor cell proliferation of abilities in in vitro CSCs selective culture conditions.[380] An innovative approach has been recently reported by Liu et al. who developed pH-sensitive ro DOX and superparamagnetic iron oxide nanoparticles (SPIONs)-loaded SLNs.[382] The -p magnetic component provided the possibility of guiding the SLNs toward the desired area due to an external electrostatic field and thermomagnetic therapy. The pH-sensitive coating allowed re for the release at the acidic pH found in the tumor. They confirmed the enhanced cytotoxicity of the pH sensitive DOX/SPIONs-SLNs when the hyperthermia treatment was applied in vitro. lP Moreover, they achieved an in vivo increased accumulation of SLNs in the tumor due to the electrostatic field guidance and the pH-sensitive coating. Tumor growth was effectively na inhibited, and the application of the hyperthermia treatment further improved tumor growth Jo ur inhibition.[382] 6. Conclusions and outlook At present, nanotechnology plays an important role in the targeted delivery of drugs for cancer treatments. LNPs constitute a diverse and extensive group of lipid-based nanotechnological platforms that have been widely studied and employed in the treatment of numerous pathologies due to their versatility and biocompatibility, among other interesting properties. In cancer therapy research, LNPs demonstrate promising potential, as confirmed by the existence of market-available nanopharmaceuticals and numerous clinical trials, with liposomes being the most exploited formulation. In this critical review, LCNPs, defined as LNPs with a lipid core, are classified into LLNs (including LNEs and LNCs), SLNs and NLCs. The great variety of LCNPs types is derived from the large and increasing number of preparation methods. Their production is feasible and effective in traditional laboratories and includes the possibility of large-scale production. Furthermore, due to the fact that their components are usually of GRAS status or accepted by the FDA, they possess a high safety threshold. The selection of ingredients 64 Journal Pre-proof affects the performance of LCNPs, such as their uptake, drug release, or the solubility of drugs as well as their physicochemical properties. Moreover, although LCNPS are well established as safe drug-delivery systems, it is necessary to assess their possible toxicological concerns, especially for new nanoformulations and preparations techniques. As reviewed, LCNPs not only allow for the inclusion of traditional drugs such as hydrophobic chemotherapeutics, but are very promising as carrier systems for larger molecules such as peptides or nucleic acids. At present, the delivery of genetic material in LCNPs is very promising for different biomedical applications. For cancer therapy, biomolecules are mainly included to achieve a combinatorial therapy with chemotherapeutics that develop resistances or of are not effective. Further understanding is required to determine how LCNPs accommodate biomacromolecules, as it remains unclear. Techniques such as freeze-fracture electron ro microscopy (FFEM), NMR, or X-ray scattering are needed to fully understand the structure of -p the formulated complexes. Due to their versatility and adaptability, LCNPs may be introduced into pharmaceutical re formulations administered via different pathways. As for most types of colloidal nanocarriers, intravenous injection is the preferred route of administration for cancer treatment. LCNPs are lP very promising as oral drug delivery systems as they improve oral drug solubility and intestinal permeability. However, the gastrointestinal tract is complex and not easy to replicate in models. na It remains difficult to predict the interactions between NPs and the gut. More sophisticated models are required to study the performance of orally administered nanocarriers. In this regard, Jo ur microfluidics devices could allow the replication of the gastrointestinal environment, including peristaltic movements. Similarly, LCNPs, especially SLNs and NLCs, administered intranasally, establish a promising approach for targeting drugs to the brain. Nevertheless, it is essential to identify factors influencing nasal absorption of the drug such, as mucociliary clearance or enzymatic degradation. LCNPs offer advantages for topical applications such as the use of lipids that enhance skin penetration and modulate drug release. Furthermore, they can be applied directly onto damaged skin due to the safety of their components. In the future, LCNPs development will lead to the adaptation of the formulation methods to encapsulate more complex drugs, especially therapeutic biomacromolecules, and to functionalize particles to achieve target-specific therapy. In this sense, drug-delivery systems to target the brain through intranasal administration is one of the most promising fields in cancer nanotherapy. However, further work is required to study the interactions of LCNPs and biological environments to fully understand and predict their in vivo performance. Despite the presented challenges, LCNPs are a versatile group of nanoparticles that can adapt different 65 Journal Pre-proof formulations to overcome a great number of difficulties related to drug delivery, with increasing interest in the treatment of different types of tumors. Declaration of Competing Interest All authors declare that there is no conflict of interest. Acknowledgements P.G and A.A-G contributed equally to this work. S.A.N-M and F.G-G are co-seniors and cocorresponding authors. P.G acknowledges the Ph.D. student fellowship (FPU18/05336) from of MCIN/AEI/10.13039/501100011033 and FSE and the Ph.D. program of Biomedicine of the University of Granada. The authors thank MCIN / AEI / 10.13039 / 501100011033/ FEDER ro “Una manera de hacer Europa” for funding RTI2018.101309B-C21 and RTI2018.101309B- -p C22 projects, the Chair “Doctors Galera-Requena in cancer stem cell research”. The authors are grateful to Jodi Eckart for the excellent linguistic assistance. The images were created using [3] [4] [5] [6] [7] [8] na [2] Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin 2023;73:17–48. https://doi.org/10.3322/caac.21763. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209–49. https://doi.org/10.3322/caac.21660. Debela DT, Muzazu SG, Heraro KD, Ndalama MT, Mesele BW, Haile DC, et al. New approaches and procedures for cancer treatment: Current perspectives. SAGE Open Med 2021;9:205031212110343. https://doi.org/10.1177/20503121211034366. 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