Antitumor Cream: Transdermal Hydrogel Containing Liposome-Encapsulated Ruthenium Complex for Infrared-Controlled Multimodal Synergistic Therapy.

{"full_text": "RESEARCH ARTICLE\n www.advhealthmat.de\n\n\nAntitumor Cream: Transdermal Hydrogel Containing\nLiposome-Encapsulated Ruthenium Complex for\nInfrared-Controlled Multimodal Synergistic Therapy\nYu-Fei Yan, Xue-Lian Li, Li-Zhen Zeng, Qishuai Liu, Zhongyan Cai, Yanrong Ren,\nXiaoxia Ren,* and Feng Gao*\n\n cancer cases, is the primary cause of\nA transdermal drug delivery cream, which is non-invasive and painless, skin cancer fatalities.[1] In 2024, an esti-\ncontaining a liposome-encapsulated Ru(II) complex (LipoRu) is created for the mated 100640 new melanoma cases and\ntreatment of skin cancer. This formulation capitalizes on the synergistic 8290 deaths are expected.[2] The treat-\nantitumor e\ufb00ects of two-photon excited photodynamic therapy (PDT), ment of melanoma is stage-dependent\n (Figure 1a), taking into account the ge-\nphotothermal therapy (PTT), and chemotherapy. LipoRu exhibits e\ufb00ective\n netic characteristics of the tumor and the\ntumor accumulation, e\ufb03cient cellular uptake, pH-sensitive and overall health of the patient. The treat-\ninfrared-accelerated release, and dual localization to the nucleus and ment for localized melanoma (Stages\nmitochondria. The released Ru(II) complexes within cells exert multiple I and II) primarily consists of surgical\nantitumor mechanisms, such as DNA topoisomerase and RNA polymerase excision with some healthy tissue. Sen-\n tinel lymph node biopsy is conducted on\ninhibition, Type I and II PDT, PTT, DNA photodamage, and apoptosis and\n thicker melanomas to assess for metastasis,\nferroptosis induction. The biodistribution and therapeutic e\ufb03cacy of LipoRu which may necessitate lymphadenectomy\nin vivo are systematically compared via three distinct administration routes: if regional nodes are impacted (Stage\nintratumoral injection, intravenous injection, and transdermal delivery III). Advanced melanoma (Stage IV)\nthrough topical cream application. The positive therapeutic e\ufb00ects of the involves distant metastasis, requiring\nLipoRu cream fabricated here in subcutaneous tumor-bearing mice o\ufb00er targeted therapy (BRAF, MEK, and KIT\n inhibitors), immunotherapy (PD-1/PD-\noptimistic potential for the painless and non-invasive treatment of both\n L1 and CTLA-4 inhibitors, Interleukin-2,\nearly-stage and advanced skin cancers, as well as super\ufb01cially located solid and oncolytic virus), and radiation ther-\ntumors. apy for symptom relief and control.[3]\n Drug injections, whether intra-\n venous (i.v.) or intratumoral (i.t.), are\n inevitable in these treatments and cause\n1. Introduction pain and discomfort. Despite advancements in skin and super\ufb01-\n cial tissue excision surgeries, scarring remains a concern, par-\nSkin cancer is the most frequently identi\ufb01ed malignancy in the ticularly for individuals who are more likely to develop scars,\nUS.[1] Invasive melanoma, though accounting for only 1% of skin which can a\ufb00ect their physical appearance. Non-invasive drug\n delivery technologies are especially noteworthy for treating skin\n cancer in comparison to other types of tumors. Topical creams\nY.-F. Yan, X.-L. Li, L.-Z. Zeng, F. Gao and patches are widely used to treat skin ailments, including in-\nKey Laboratory of Medicinal Chemistry for Natural Resource \ufb02ammation, eczema, warts, and acne. Patches o\ufb00er controlled,\nMinistry of Education sustained release and are suitable for long-term use, while oint-\nSchool of Pharmacy\nYunnan University ments, creams, and gels provide \ufb02exible application, rapid ab-\nEast Outer Ring Road, Kunming 650500, P. R. China sorption, and patient comfort, are bene\ufb01cial for precise and cus-\nE-mail: gaofeng@ynu.edu.cn tomizable dosing, and minimize skin irritation from adhesives.\nQ. Liu, Z. Cai, Y. Ren, X. Ren Hence, we aim to innovate by developing a non-invasive and\nAnimal Research and Resource Center painless transdermal delivery cream for treating super\ufb01cially lo-\nSchool of Life Sciences\n cated or subcutaneous tumors.\nYunnan University\nEast Outer Ring Road, Kunming 650500, P. R. China Coordination complexes exhibit excellent biocompatibility and\nE-mail: renxiaoxia001@ynu.edu.cn biological activity.[4] Phototherapy with metal complexes has\n gained signi\ufb01cant attention, leading to numerous advancements\n The ORCID identi\ufb01cation number(s) for the author(s) of this article in photodynamic therapy (PDT), photothermal therapy (PTT),\n can be found under https://doi.org/10.1002/adhm.202403563 and photoactivated chemotherapy (PACT).[5] These complexes of-\nDOI: 10.1002/adhm.202403563 fer bene\ufb01ts such as enhanced light absorption, prolonged excited\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (1 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 1. a) Single-molecule Ru(II) complexes designed in this study (represented by Ru2), liposome-encapsulated Ru2 (LipoRu), antitumor cream\nfabricated from LipoRu, and di\ufb00erent administration methods targeting the characteristics of melanoma at di\ufb00erent stages. b) Mechanism of action of\nLipoRu for infrared two-photon photodynamic, photothermal, and chemotherapy through light-triggered 1 O2 and O2 \u2022\u2212 generation, apoptosis, ferropto-\nsis, photothermal conversion, and three-pronged cell proliferation inhibition.\n\n\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (2 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\ntriplet state (3 ES) lifetimes, high quantum yields of emission and mal cream demonstrated substantial therapeutic e\ufb03cacy in sub-\nreactive oxygen species (ROS) generation, and structurally tun- cutaneous tumor-bearing mice, highlighting the positive thera-\nable physicochemical properties.[6] Large solid tumors often ex- peutic e\ufb00ects of the LipoRu cream on both early-stage and ad-\nhibit hypoxia, reducing the e\ufb03cacy of oxygen-dependent PDT.[7] vanced skin malignancies, as well as super\ufb01cially situated solid\nOxygen-independent PTT can eradicate tumors via thermal abla- tumors.\ntion and increase intracellular oxygen pressure, thereby enhanc-\ning PDT.[8] Furthermore, ingenious designs have combined pho- 2. Results and Discussion\ntotherapy with other therapies, such as sonodynamic and im-\nmunogenic therapy, yielding synergistic treatments that signif- Three ancillary ligands have been used to construct Ru1\u22123\nicantly improve e\ufb03cacy and reduce the required dosage of thera- (Figure 2a). The hydrophobic ancillary ligands tbubpy and\npeutic agents.[9] dip have been shown to increase the bioavailability of Ru(II)\n Researchers have recently developed metal complexes for the complexes.[8e,f,15] The synthesis and characterization details, in-\nmultimodal synergistic treatment of melanoma.[10] A signi\ufb01cant cluding 1 H and 13 C NMR spectra, HR-MS, IR spectra, and el-\nportion of these molecules is activated by infrared (IR) light, po- emental analysis, are provided in the ESI (Figures S1\u2013S6, Sup-\ntentially overcoming the hinderance of melanin absorption in porting Information).\nvisible light PDT.[11] Intricately designed nanoparticles (NPs) en- The absorption and emission spectra of Ru1\u22123 in aqueous\ncapsulating anti-tumor drugs can e\ufb00ectively accumulate in tu- solution are presented in Figure 2b, with key photophysical\nmors through the enhanced permeability and retention (EPR) data summarized in Table 1. The absorbance followed the Beer-\ne\ufb00ect, exploiting tumor-speci\ufb01c features such as low pH and Lambert law (1\u2212100 \u03bcm), indicating no aggregation. This was\nhigh hydrogen peroxide levels to facilitate the release or ac- further supported by the Tyndall e\ufb00ect experiment (Figure S7,\ntivation of drugs. Lipid nanoparticles (LNPs) represent a sig- Supporting Information). The spectra of Ru1\u22123 remained un-\nni\ufb01cant advancement in therapeutic delivery systems, exempli- changed upon exposure to 450 nm LED or 808 nm laser irra-\n\ufb01ed by recently FDA-approved drugs. LNPs o\ufb00er several advan- diation in both aqueous solution (PBS bu\ufb00er) and cell medium\ntages: enhanced drug stability, improved drug absorption and (Figures S8 and S9, Supporting Information), demonstrating ex-\ndistribution by EPR e\ufb00ect, minimized systemic toxicity through cellent photostability. The absorption bands corresponding to the\ndrug encapsulation, controlled drug release, and reduced dos- metal to ligand charge transfer (MLCT) were observed between\ning frequency.[12] Their components, such as phospholipids and 420 and 480 nm. The excited charge density plots of the pho-\ncholesterol, possess intrinsic high biocompatibility and low toxi- toexcited hole\u2013electron pairs (Figure 2c for Ru2 and Figures S10\ncity. Despite these bene\ufb01ts, current LNP drugs are primarily ad- and S11, Supporting Information, for Ru1 and Ru3) for the most\nministered via injection, and their ability to e\ufb00ectively deliver critical transitions (f > 0.01) and the simpli\ufb01ed Jablonski dia-\ndrugs to the epidermis for skin cancer treatment remains un- graph (Figure 2d) showed that transitions \u2248450 nm were pre-\nknown. dominantly attributed to MLCT from the dmetal to the \ud835\udf0b*main and\n This study integrated above two innovative solutions: a non- intraligand (IL) transitions on the main ligand. Theoretical spec-\ninvasive and painless antitumor cream for transdermal deliv- tra (Figure 2e for Ru2 and Figure S12, Supporting Information,\nery and liposome-encapsulated antitumor therapeutic agents for for Ru1 and Ru3) closely match the experimental data, showing\ntargeted and e\ufb03cient drug uptake by tumor. A series of Ru(II) the reliability of the computational results. Ru1 and Ru2 showed\ncomplexes with multimodal synergistic antitumor e\ufb00ects were similar \u03a6w values to [Ru(bpy)3 ]2+ (Ru0, 4.0%).[16] The primary 1 ES\ndesigned (Figure 1b) to increase the ROS production in hy- transitions (*S1 \u2212*S3 ) for Ru2 involve 1 MLCT (main ligand) and\n 1\npoxic tumor environments and enhance the PCE, hence facil- MLCT+1 IL, whereas for Ru1 and Ru3, they are 3 MLCT (ancil-\nitating the synergistic e\ufb00ects of PDT and PTT by integrating lary ligand) and 1 MLCT+1 LLCT (ligand to ligand charge trans-\n1,1-dioxidothiomorpholine (dotmp), a crucial moiety of Nifur- fer). Moreover, the lowest three 3 ES transitions (T1 \u2212T3 ) involve\ntimox (an antiparasitic drug for Chagas disease), into Ru(II)- 3\n MLCT (ancillary ligand) for Ru1 and Ru3, while for Ru2, the sec-\nbased PSs, as Nifurtimox was recently discovered to gener- ond 3 ES (T2 ) is a combination of 3 MLCT (main ligand) and 3 IL\nate ROS under hypoxic conditions, thereby activating apop- (main ligand). The presence of 3 IL ES is advantageous for PSs.[17]\ntotic pathways.[13] The versatile structure of dotmp also ap- Thus, Ru2 may facilitate more e\ufb03cient energy and electron trans-\npears in the anti-in\ufb02ammatory drug Filgotinib, the anti-HIV fer compared to Ru1 and Ru3.\nmolecule GSK3640254, and the psychiatric disorder modula- The curves and data for the two-photon absorption cross-\ntor Basmisanil.[14] The \ufb02exible six-membered heterocyclic struc- section (TPACS) of Ru1\u22123 were depicted in Figure 2f,g, and\nture of dotmp also has the potential to improve the photother- Table 1. The TPACS maxima vary from 376 to 401 GM (1\nmal conversion e\ufb03ciency (PCE) of metal-based photosensitizers GM = 10\u221250 cm4 s photon\u22121 ), which exceeds those of most re-\n(PSs).[8d] The antitumor activity and mechanisms of the Ru(II) ported metal complexes at their respective TPA wavelengths.[6c,18]\ncomplexes were studied from photophysical, photochemical, and Speci\ufb01cally, at a wavelength of 808 nm, Ru1\u22123 showed remark-\ncell biology perspectives. The selected complex (Ru2) was further ably high TPACS values (286\u2212305 GM), surpassing those of\nencapsulated into liposomes (LipoRu) without any solid particles metal complexes known for their dual PDT/PTT activity.[8d\u2013f]\nand formulated into a topical cream using hydrogel. A system- Consequently, it is anticipated that Ru1\u22123 will exhibit potent PDT\natic comparison of the biodistribution and therapeutic e\ufb03cacy of activity when exposed to 808 nm irradiation.\nLipoRu in vivo was conducted via three distinct administration The quantum yields of 1 O2 and O2 \u2022\u2212 generation in aqueous so-\nroutes: intratumoral injection, intravenous injection, and trans- lution (\u03a6\u0394 ) by Ru1\u22123 were measured using ABDA and DHR123\ndermal delivery through topical cream application. The transder- assays (Figure 2h,i; Figures S13\u2212S18, Supporting Information).\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (3 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 2. Photophysical and photochemical properties of Ru(II) complexes designed in this study (Ru1\u22123) and nanomaterial properties of liposome-\nencapsulated Ru2 (LipoRu). a) Structure of Ru1\u22123. b) Absorption (solid) and emission spectra (dashed, excitation wavelength = 455 nm) of Ru1\u22123\n(10 \u03bcm) in aqueous solution. c) Real space representation of hole (blue) and electron (green) distributions of Ru2 for the main excited state transitions\nresponsible for excitation (Sn ), singlet emission (Fn ) and triplet emission (Tn ). Excitation wavelength (nm), excitation energy (eV), and oscillator strength\n(f) for each hole-to-electron transition are presented. d) Simpli\ufb01ed Jablonski diagram of Ru2 for the energy transfer and main non-radiative decay (heat\nrelease) pathways. e) TDDFT (B3LYP/Lanl2DZ, CPCM for water) calculated absorption spectra and corresponding excited state transitions of Ru2.\nf) TPASCs (\ud835\udf0e) of Ru1\u22123 (50 \u03bcm in anhydrous THF solution) in the range of 760\u2212920 nm. g) Linear \ufb01t of the logarithm plot of two-photon induced\n\ufb02uorescence intensity (F) versus the excitation intensity (I, at 820 nm). h) Singlet oxygen (1 O2 ) generation rates represented by the OD decrease of\nABDA versus irradiation time (min) in the absence and presence of Ru1\u22123 by an 808 nm laser (100 mW cm\u22122 ). [Ru(bpy)3 ]2+ (Ru0), [Ru(bpy)2 (pip)]2+\n(Rupip) and TLD1433 were tested as references. i) Superoxide anion (O2 \u2022\u2212 ) generation represented by the emission intensity increase (I/I0 ) of DHR123\nversus irradiation time (min) in the absence and presence of the tested Ru(II) complex by an 808 nm laser (100 mW cm\u22122 ). j) Transmission electron\nmicroscope (TEM) images of LipoRu. k) Dynamic light scattering (DLS) analysis of particle size (diameter, pH 7.4 and 5.5) and polydispersity index\n(PDI) of LipoRu at pH 7.4. l) Zeta potentials of LipoRu in PBS solution under pH 5.5, 6.4, and 7.4. m) Tyndall e\ufb00ect experiment for 10, 50, and 100 \u03bcm\nof LipoRu in PBS solution. Concentrations represent Ru2 in the PBS solution of the liposome. n) Calibration curve for measuring the concentration and\nencapsulation e\ufb03ciency (EE) of Ru2 in LipoRu. Release kinetics of Ru2 for LipoRu, with or without 808 nm laser (100 mW cm\u22122 ) irradiation, under pH\n5.5, 6.4, and 7.7 at 25 \u00b0C (o) and 37 \u00b0C (p). q) Temperature rising curves of aqueous solutions of Ru1\u22123 (100 \u03bcm in PBS) and LipoRu (50 \u03bcm for Ru2),\nas well as PBS, Ru0 (100 \u03bcm), Rupip (100 \u03bcm), and TLD1433 (100 \u03bcm), irradiated by an 808 nm laser (100 mW cm\u22122 ) for 10 min at 1-min intervals.\n\n\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (4 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\nTable 1. Photophysical and photochemical data of Ru1\u22123.\n\nComplex \ud835\udf06abs /nm (\ud835\udf16 \u00d7104 /M\u22121 cm\u22121 ])a) \ud835\udf06em /nmb) \u03a6w /%c) \ud835\udf0e/GMd) \ud835\udf0e 808 /GMd) \u03a6\u0394TP (1 O2 )/%e) \u03a6\u0394TP (O2 \u2022\u2212 )/%e) \ud835\udf02 808 /%f)\n\nRu1 460(4.78), 427(3.83), 327(10.5), 287(29.5) 603 3.2 376 286 3.14 7.2 4.5\nRu2 469(4.91), 435(4.50), 332(11.0), 288(42.2) 627 3.5 395 305 4.54 15.6 5.2\nRu3 475(5.24), 443(4.93), 319(9.87), 285(16.5) 616 0.66 401 290 4.62 12.3 5.6\na) b)\n Maximum absorption wavelength (\ud835\udf06abs ) and corresponding molar extinction coe\ufb03cient (\ud835\udf16) in aqueous solution; Maximum emission wavelength (\ud835\udf06em ) in aqueous solution;\nc) d)\n Photoluminescence quantum yield in aqueous solution (\u03a6w ); Two-photon absorption cross-section (TPSCS) at maximum MLCT absorption wavelength (\ud835\udf0e) and 808 nm\n e) f)\n(\ud835\udf0e 808 ); Quantum yields of singlet oxygen and superoxide anion under 808 nm excitation (\u03a6\u0394TP ) in aqueous solution; Photothermal conversion e\ufb03ciency (PCE) at 808 nm\n(\ud835\udf02 808 ) in PBS solution.\n\n\n\nRu1\u22123 exhibited exceptional \u03a6\u0394 values under both one-photon The photothermal conversion e\ufb03ciency (PCE, \ud835\udf02 808 in Table 1)\n(OP, 450 nm LED, 50 mW cm\u22122 ) and TP (808 nm laser, 100 mW was measured under 808 nm laser irradiation (Figure 2q; Figures\ncm\u22122 ) irradiation, surpassing the values of Ru0, [Ru(bpy)2 (pip)]2+ S20\u2013S22, Supporting Information). Complexes that contain a dip\n(Rupip), and TLD1433. These \ufb01ndings indicate that Ru1\u22123 have or tbubpy ancillary ligand have a wide range of rotation due to the\nconsiderable promise for a dual PDT mechanism, which involves phenyl and tbu groups, which can dissipate ES energy through\nboth electron transfer (Type I) and energy transfer (Type II). The a non-radiative energy pathway, resulting in a more remarkable\nvalues of \u03a6\u0394TP (1 O2 ) and \u03a6\u0394TP (O2 \u2022\u2212 ) for Ru1\u22123 under 808 nm ir- PCE. As expected, Ru2 and Ru3 exhibited higher PCEs than Ru1\nradiation were summarized in Table 1. Ru2 exhibited the highest without any rotatable group. The superior PCEs of Ru1\u22123 in com-\ncapability for generating ROS, which was attributed to its 3 IL ES parison to Ru0, Rupip, and TLD1433 can be attributed to the fa-\nand high TPACS. Therefore, Ru2 was selected for the preparation vorable vibrational modes of the \ufb02exible six-membered hetero-\nof LNPs and antitumor cream. cyclic structure of dotmp, which enables the more e\ufb03cient dissi-\n The liposome encapsulation of Ru2 was performed by the thin pation of ES energy. The PCE of LipoRu was signi\ufb01cantly higher\n\ufb01lm dispersion method (ESI). The measured apparent diame- than Ru2. Such enhancement in PCE can be attributed to the PS\u2019s\nter of LipoRu was 150 \u00b1 10 nm, as indicated by the transmis- dispersity in the liposomal membrane and its physical interaction\nsion electron microscope (TEM, Figure 2j). The hydrodynamic with phospholipids.[20,21] The PCE improved by liposome encap-\ndiameter of LipoRu was 234 nm as determined by the dynamic sulation and the light-accelerated release of Ru2 will be bene\ufb01cial\nlight scattering (DLS, Figure 2k). The polydispersity index (PDI) to the phototherapeutic e\ufb00ect of LipoRu.\nof LipoRu at pH 7.4 was 0.213, indicating an excellent unifor- The subcellular localization of Ru2 and its liposome LipoRu\nmity in particle size. When the pH value was adjusted to 5.5 in the A375 human melanoma cell line was initially investigated\nand allowed for 24 h, 75% of LipoRu was observed to have a de- by confocal laser scanning microscopy (CLSM). After 15 min in-\ncreased size (d1 = 51.4 nm), while the remaining 25% of LipoRu cubation of Ru2 with the live adherent A375 cells in medium\nretained its previous size (d2 = 234 nm). These \ufb01ndings indicate at 37 \u00b0C, almost all of Ru2 was taken up by the cells. The \ufb02uo-\nthat LipoRu is stable at pH 7.4 but disassembles or releases its rescence images of cells incubated with Ru2 (Figure 3a) showed\ncontents in the acidic environment of the tumor (pH 5.5). Li- apparent colocalization with both the nuclear dye Hoechst33342\npoRu demonstrated a negative and pH-dependent zeta potential and the mitochondrial dye MitoTracker Green (MTG). For Li-\n(Figure 2l). The Tyndall e\ufb00ect assay conducted on LipoRu aque- poRu, in addition to these two locations, Ru2 was also observed\nous solution (Figure 2m) contrasted starkly with those of single near the cell periphery (Figure 3b). Bright-\ufb01eld images depicted\nmolecule Ru1\u22123 solutions (Figure S7, Supporting Information), the presence of a lipid layer enveloping the external cell mem-\nhence verifying the formation of NPs. The absorbance of Ru2 in brane. This phenomenon may be attributed to the adherence or\nthe LipoRu solution adhered to the Beer-Lambert law (Figure 2n), fusion of liposomes with the plasmic membrane. When the in-\nsuggesting a homogenous distribution within the system. The cubation time was increased to 60 min, no Ru2 was observed at\nconcentration of Ru2 in the prepared LipoRu solution was deter- the membrane, indicating that LipoRu may penetrate A375 cells\nmined to be 190 \u00b1 9 \u03bcm, with an encapsulation e\ufb03ciency (EE) of through membrane fusion (Figure 1b)[22] and the uptake of Li-\n92.3% (ESI). poRu via the membrane fusion could be accomplished within\n The release kinetics of LipoRu were determined to de- 60 min.\npend on factors such as pH, temperature, and photoirradiation The cellular localization of Ru1\u22123 and LipoRu in the nucleus\n(Figure 2o,p). The release rate of Ru2 was four times greater and mitochondria was validated by ICP-MS (Figure 3c). The to-\nat 37 \u00b0C than at 25 \u00b0C. LipoRu released Ru2 more e\ufb00ectively tal uptakes of Ru2 and Ru3 were apparently greater than that of\nat the acidity level within tumor cells (pH 5.5) than in the tu- Ru1, due to their lipophilic nature (log P, Table 2), as revealed by\nmor microenvironment (pH 6.4) and blood/normal cells (pH the octanol/water partition assay (Figures S23 and S24, Support-\n7.4),[19] indicating a favorable accumulation and release of Ru2 ing Information).[23] The uptake of LipoRu is 7 times greater than\nwithin tumor cells. Under 808 nm laser irradiation, the re- that of Ru2. This indicates that formulating Ru2 into LNPs signif-\nlease rates of Ru2 under various conditions increased signi\ufb01- icantly enhances its cellular uptake. The data on the subcellular\ncantly. It may be attributed to the photoexcited ROS, which cause localization of the complexes were presented in Table 2. It can\noxidative degradation of the liposome membrane, or the pho- be inferred that Ru1\u22123 target both nucleus and mitochondrion.\ntothermal e\ufb00ects that result in phase transition or membrane Only \u22481% of the complexes were found in the cell membrane,\nrupture.[20] indicating that the membrane was not their target. Although\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (5 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 3. Cellular localization, uptake, endocytic mechanism, intracellular ROS production, and apoptosis- and ferroptosis-relative pathway regulation\nof Ru1\u22123 and LipoRu. Subcellular colocalization of a) Ru2 and b) LipoRu with Hoechst 33 324 and MitoTracker Green (MTG) in live adherent A375\ncells by confocal laser scanning microscopy (CLSM). c) The amount of metal taken up by A375 cells determined by inductively coupled plasma mass\nspectrometry (ICP-MS). Whole: total uptake amounts by whole cell; Mito: mitochondria; Nuc: nuclei; Mem: membrane. The percentages indicate the\nappropriate proportions of each organelle\u2019s intake relative to the overall intake of the whole cell (mean, n = 3). d) Relative amounts of complexes\nRu1\u22123 in the presence of endocytic inhibitor chlorpromazine (CPZ, 10.0 \u03bcg mL\u22121 ), amiloride (100.0 \u03bcg mL\u22121 ), or nystatin (50.0 \u03bcg mL\u22121 ) at 37 \u00b0C or\nwithout inhibitor at 4 \u00b0C taken up by A375 cells (n = 3). e) Normalized absorbance di\ufb00erence between 450 and 620 nm (\u00d7 100) in the presence of\ncomplex and inhibitors of di\ufb00erent cell death mechanisms and irradiated by 808 nm laser determined by the CCK-8 assay (n = 3). Necrostatin-1 (Nec-1),\ntetraethylthiuram disul\ufb01de (TETD), 3-methyladenine (3-MA), benzyloxycarbonyl-Val-Ala-Asp (OMe) \ufb02uoromethylketone (Z-VAD-FMK), and ferrostatin-1\n(Fer-1) were used as the inhibitors of necrosis, pyroptosis, autophagy, apoptosis, and ferroptosis, respectively. (f) Caspase-9 (Cas-9) and -3 (Cas-3)\nactivity in A375 cells after treatment with PBS (control) and Ru1\u22123 and LipoRu in the dark and under 808 nm laser (100 mW cm\u22122 ) irradiation, as well as\ncaspase activity in LipoRu-treated A375 cells under 808 nm (100 mW cm\u22122 ) laser irradiation in the presence of ascorbic acid (Asc, 1.0 mm) and in an ice\nbath during irradiation (n = 3). Relative glutathione (GSH) amounts decreased by Ru1\u22123 in the dark and under 808 nm (100 mW cm\u22122 ) laser irradiation\nin cell-free aqueous solution (g) and A375 (h) cells (n = 3). Lipid peroxidation (LPO) induced by Ru1\u22123 in the dark and under 808 nm (100 mW cm\u22122 )\nlaser irradiation in cell-free aqueous solution (i) and A375 (j) cells (n = 3). H2 O2 and Erastin were tested as the positive controls. bl: below the detection\nlimit. IC50 concentrations under TP excitation conditions were used for the complexes.\n\n\nLipoRu exhibits a distribution ratio of 11.3% in the cell mem- presence of the macropinocytic pathway inhibitor amiloride or\nbrane after 15 min of incubation, the amount of Ru2 remaining the caveolae formation inhibitor nystatin, or when the tempera-\nin the membrane decreased to just 1.6% when the incubation ture was lowered to 4 \u00b0C (Figure 3d), suggesting that their up-\ntime was extended to 60 min. This aligns with the \ufb01nding made take operated by both macropinocytosis and caveolae-mediated\nby CLSM regarding the uptake mechanism of membrane fusion. mechanisms energy-dependently. Tumors frequently encounter\n Endocytosis is also one of the primary uptake mechanisms nutrient-limiting conditions and exhibit robust macropinocyto-\nfor LNPs. Ru1\u22123 and LipoRu exhibited decreased uptake in the sis, a mechanism that aids adaptation to nutrient deprivation by\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (6 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\nTable 2. Biological data regarding DNA, enzyme, and cell of Ru1\u22123.\n\nCompound log Pa) RuMito /%b) RuNuc %b) RuMem %b) Kb /\u00d7105 M\u22121 c) \u0394Tm /\u00b0Cd) IC50-Topo /\u03bcMe) IC50-RNAP /\u03bcMf) IC50-PC /\u03bcMg)\n\nRu1 \u22122.6 46 \u00b1 5 50 \u00b1 5 1.1 \u00b1 0.2 23.2 \u00b1 2.7 5.0 3.6 \u00b1 0.2 0.36 \u00b1 0.06 2.1 \u00b1 0.2\nRu2 2.3 41 \u00b1 5 57 \u00b1 5 1.0 \u00b1 0.2 9.05 \u00b1 1.12 2.7 11.2 \u00b1 0.4 0.42 \u00b1 0.06 1.8 \u00b1 0.2\nRu3 3.0 47 \u00b1 4 53 \u00b1 4 1.2 \u00b1 0.2 18.1 \u00b1 1.5 5.6 12.2 \u00b1 0.4 0.32 \u00b1 0.04 1.0 \u00b1 0.1\na) b)\n Apparent oil-water partition coe\ufb03cients; Distribution of Ru1\u22123 in mitochondria (Mito), nuclei (Nuc), and membrane (Mem) of A375 cells detected by ICP-MS (mean \u00b1 SD,\n c) d) e)\nn = 3); Binding constants of Ru1\u22123 to CT-DNA; Increases in CT-DNA melting points (denaturation temperatures) after complex-binding; IC50 for DNA topoisomerase\n f) g)\ninhibition; IC50 for RNA polymerase inhibition; IC50 for DNA photocleavage. Kb and IC50 were presented as value \u00b1 error.\n\n\n\ninternalizing extracellular proteins and necrotic cell debris. This tity of supercoiled pBR322 DNA (Form I) following incubation\nprocess supports macromolecule synthesis and supplies energy with Ru1\u22123 and exposure to the 808 nm laser. The IC50 values in\nto central carbon metabolic pathways. Ongoing endeavors are be- the micromolar range demonstrated their e\ufb03cient DNA photo-\ning undertaken to discover speci\ufb01c inhibitors of macropinocyto- cleavage activity, which could further boost their ability to directly\nsis for antitumor treatment.[24] The \ufb01ndings that Ru1\u22123 and Li- kill and prevent the proliferation of tumor cells.\npoRu enter tumor cells via the rare macropinocytic pathway may The cytotoxicity of Ru1\u22123 toward A375 cells was tested by the\nprovide signi\ufb01cant support in antitumor activity by leveraging the CCK-8 assay (Table 3), both in the absence and presence of IR\nunique mechanisms of tumor cells to enhance the uptake of an- irradiation (808 nm laser, 100 mW cm\u22122 , light dose = 30.0 J\ntitumor molecules or LNPs. cm\u22122 ). Ru1\u22123 displayed higher phototoxicity indexes (PI) than\n The nuclear targeting of Ru1\u22123 may stem from their DNA the clinically approved PDT drug 5-aminolevulinic acid (5-ALA).\na\ufb03nity, allowing the complexes to participate in DNA-related Cisplatin showed negligible phototoxicity (PI = 1.12). Ru2 and\nchemotherapy and DNA photocleavage within the nucleus. As Ru3 with felicitous lipophilicity and optimal uptake exhibited re-\nrevealed by the spectroscopic titration (Figure S25, Supporting markable PIs. The cytotoxicity decreased signi\ufb01cantly when the\nInformation), binding constant (Kb ) calculation, and DNA ther- irradiation was conducted in the presence of ascorbic acid (Asc,\nmal denaturation experiment (Figure S26, Supporting Informa- 1.0 mm) or in an ice bath, indicating a potent synergy between\ntion), Ru1\u22123 exhibited a moderate to high a\ufb03nity for CT-DNA PDT and PTT with a combination index (CI) below 0.60 (CI < 0.7\n(Table 2), comparable to the reported pip-type Ru(II) complexes. implies a favorable synergistic e\ufb00ect).[27] The photocytotoxicity of\nDNA binders have the ability to impede the activity of RNA tested compounds was marginally impacted when the cell plates\npolymerase (RNAP) or DNA topoisomerase (Topo), enzymes re- were covered with commercial chicken breast skin during irradi-\nsponsible for synthesizing RNA from DNA templates in a pro- ation, assumably due to their outstanding TPACS and the high\ncess known as DNA transcription, or assist in the unwinding penetration of IR light.\nand coiling of DNA during replication and transcription, respec- The intracellular generation of ROS by Ru1\u22123 was ana-\ntively. Topo and RNAP inhibitors can trigger cell death, partic- lyzed by \ufb02ow cytometry. 2\u2032,7\u2032-Dichlorodihydro\ufb02uorescein diac-\nularly in rapidly dividing and proliferation cells, such as cancer etate (DCFH-DA) and Rosup were used as the detector and the\ncells, where fast DNA replication is necessary. Ru1\u22123 exhibited positive control (Figure S30, Supporting Information). ROS were\nremarkably higher inhibitory e\ufb00ects on both Topo and RNAP only detected in the presence of both a Ru(II) complex and light\nin comparison to many other inhibitors (Figures S27 and S28, irradiation (808 nm laser). ROS scavenger Asc completely sup-\nSupporting Information; Table 2).[15,25] Therefore, Ru1\u22123 act as pressed the production of photoinduced ROS, providing evidence\nsubstantial chemotherapeutic agents, in addition to their potent for a PDT mechanism.\nphototherapeutic properties. DNA-binding PSs can induce DNA To explore the possible mechanisms of Ru1\u22123 or LipoRu, the\ncleavage by the produced ROS upon irradiation.[26] Figure S29 photocytotoxicity was further determined in the presence of in-\n(Supporting Information) showed a gradual decrease in the quan- hibitors targeting several antitumor mechanisms. Necrostatin-1\n\n\nTable 3. In vitro (photo)cytotoxicity (IC50 , \u03bcm) and PDT index (PI) of Ru1\u22123, LipoRu, 5-ALA, and cisplatin toward A375 human malignant melanoma\nupon two-photon excitation under various conditions.\n\nComplex IC50,Dark /\u03bcMa) IC50,TP /\u03bcMb) PIc) IC50,TP+Asc /\u03bcMd) IC50,TP+Ice /\u03bcMe) IC50,TP+Ice+Asc /\u03bcMf) CIg) IC50,TP+CBS /\u03bcMh)\n\nRu1 62.6 \u00b1 4.8 11.5 \u00b1 1.0 5.44 18.6 \u00b1 1.7 16.4 \u00b1 1.6 65.7 \u00b1 6.2 0.60 18.4 \u00b1 1.6\nRu2 8.55 \u00b1 0.76 0.143 \u00b1 0.015 59.8 2.65 \u00b1 0.27 1.583 \u00b1 0.16 9.04 \u00b1 0.86 0.52 0.207 \u00b1 0.020\nRu3 2.47 \u00b1 0.23 0.086 \u00b1 0.011 28.7 1.04 \u00b1 0.11 0.652 \u00b1 0.068 4.06 \u00b1 0.38 0.46 0.141 \u00b1 0.012\nLipoRu 6.72 \u00b1 0.63 0.116 \u00b1 0.010 57.9 1.63 \u00b1 0.15 1.62 \u00b1 0.18 6.87 \u00b1 0.65 0.53 0.153 \u00b1 0.016\n5-ALA 154 \u00b1 13 84.5 \u00b1 7.6 1.82 162 \u00b1 15 106 \u00b1 10 176 \u00b1 17 2.1 165 \u00b1 16\nCisplatin 2.14 \u00b1 0.20 2.03 \u00b1 0.19 1.12 2.26 \u00b1 0.21 2.37 \u00b1 0.22 2.19 \u00b1 0.20 3.2 2.21 \u00b1 0.20\na) b) c) d)\n Under dark condition; Upon two-photon excitation by an 808 nm laser (100 mW cm\u22122 , light dose = 30.0 J cm\u22122 ); Phototherapy index (PI) under TP excitation; TP\n e) f)\nexcitation in the presence of ascorbic acid (Asc, 1.0 mm); TP excitation in an ice bath during irradiation; TP excitation under both Asc (1.0 mm) and ice bath treatments;\nh) g)\n Combination index; TP excitation, and the cells were covered with chicken breast skin (1.5 mm in thickness). IC50 was presented in mean \u00b1 SD (n = 3).\n\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (7 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 4. a) The absorption process of the topically applied LipoRu cream. b) Confocal imaging of nude mouse skin applied with 80 \u03bcL of LipoRu cream\nafter 5 min. c) Fluorescence intensity of Ru2 at di\ufb00erent depths of skin (420 \u03bcm in thickness). Inset: Z-stack image of Ru2 distributed within the whole\ndepth of nude mice skin at 10 \u03bcm intervals. d) The amounts of Ru2 that penetrated through the skin (mean \u00b1 SD, n = 3), measured using the Franz\ndi\ufb00usion experiment for LipoRu cream and aqueous solutions of Ru2 and LipoRu.\n\n\n(Nec-1), tetraethylthiuram disul\ufb01de (TETD), 3-methyladenine the amounts of LPO (Figure 3i,j). These \ufb01ndings highlighted the\n(3-MA), benzyloxycarbonyl-Val-Ala-Asp \ufb02uoromethylketone (Z- process of ferroptosis.\nVAD-FMK), and Ferrostatin-1 (Fer-1) were employed to inhibit As presented above, Ru1\u22123 and LipoRu exhibit multimodal an-\nnecrosis, pyroptosis, autophagy, apoptosis, and ferroptosis, titumor mechanisms, including PDT, PTT, chemotherapy, photo-\nrespectively. The antitumor mechanisms of Ru1\u22123 or LipoRu induced apoptosis and ferroptosis, as well as direct DNA photo-\ntoward A375 cells (irradiated by an 808 nm laser) were revealed cleavage. Signi\ufb01cant synergistic e\ufb00ects between PDT and PTT\nto be apoptosis and ferroptosis, rather than other mechanisms have been revealed by the low values of CI. This experimental\n(Figure 3e). \ufb01nding can be rationalized by the enhancement of tumor cell\n Caspase-9 (Cas-9) directly cleaves and activates caspase-3 (Cas- oxygenation through photothermal e\ufb00ect, which facilitates PDT.\n3) and caspase-7 to initiate cell death during apoptosis. The ac- Additionally, the complexes and liposomes can exert chemother-\ntivity of Cas-9 and Cas-3 was quanti\ufb01ed in the Ru1\u22123 or LipoRu- apeutic e\ufb00ects by inhibiting DNA-related enzymes even in the\ntreated A375 cells (Figure 3f). There was no activation of Cas-9 or absence of light. This positively contributes to the drug\u2019s activ-\nCas-3 in dark conditions, except Ru3, which aligns with its higher ity in areas with insu\ufb03cient irradiation, such as deeper-seated\ndark cytotoxicity. Upon irradiation, the caspase activity rose sub- tumor regions.\nstantially while decreasing greatly either in the presence of Asc or To achieve transdermal drug delivery like creams or ointments,\nwhen irradiated in an ice bath. This drop is due to the clearance of a hydrogel formulation containing LipoRu (LipoRu cream) was\nROS or the suppression of heat generated via PTC. Apoptosis in developed. The LipoRu cream exhibited excellent stability, as ev-\ncomplex-treated cells was further detected by \ufb02ow cytometry, with idenced by the absence of any notable alterations in its appear-\npaclitaxel serving as the positive control. Ru1\u22123 clearly induced ance, viscosity (Figure S32, Supporting Information), and pH\napoptosis of A375 cells when exposed to irradiation (Figure S31, (Figure S33, Supporting Information) for a period of 30 days after\nSupporting Information). The ratios of apoptotic cells declined its preparation. In order to verify the transdermal absorption per-\neither in the presence of Asc or when exposed to irradiation in formance of the cream, 80 \u03bcL of LipoRu cream (equivalent to the\nan ice bath, providing support for the photoinduction of apopto- amount used in the subsequent in vivo \ufb02uorescence imaging and\nsis through a dual mechanism involving ROS and heat. phototherapy assay) was applied to the skin at the tumor site of\n Ferroptosis is characterized as the increased oxidative stress in the A375 tumor-bearing mice. The absorption process was con-\ntumor cells caused by the generation of ROS and lowered levels tinually monitored (Figure 4a). Evidently, the LipoRu cream was\nof glutathione (GSH), which leads to the lipid peroxidation (LPO) completely absorbed within a duration of 5 min. To con\ufb01rm the\nof the membrane. The levels of GSH and LPO were investigated transdermal capability of Ru2 liposomes, the distribution of Ru2\nby both chemical and intracellular detections. Ru1\u22123 and LipoRu on the surface of the nude mouse skin and at di\ufb00erent depths\napparently lowered the levels of GSH (Figure 3g,h) and increased after applying the cream was studied using CLSM imaging. Ru2\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (8 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\nwas distributed throughout various depths of the skin, especially plex through urine. This \ufb01nding aligns with the only very early\nin the hair follicles (Figure 4b,c), indicating a possibility of cream report on the in vivo metabolism of Ru complexes.[28]\nabsorption through the hair follicles. Images of each scanned In vivo phototherapy assays were conducted using the same\ndepth were presented in Figure S34 (Supporting Information). three administration methods (Figure 6a) in the BALB/c mouse\n To further investigate the transdermal mechanisms and capac- model with A375 tumor xenografts, based on the successful\nities of the single-molecular complex Ru2, Ru2-encapsulating li- transdermal delivery of LipoRu cream to tumors and the distinct\nposomes LipoRu, and LipoRu cream, in vitro Franz di\ufb00usion ex- tumor accumulation patterns with various administration meth-\nperiments were conducted at 37.0 \u00b1 0.3 \u00b0C (Figure S35, Support- ods. The tumor-bearing mice of drug groups were administrated\ning Information). The thickness of nude mouse skin was mea- LipoRu or LipoRu cream. The irradiation with an 808 nm laser\nsured as 0.42 mm. The amounts of Ru2 that penetrated through (100 mW cm\u22122 , light dose = 30.0 J cm\u22122 ) was performed 1 h after\nthe mouse skin were quanti\ufb01ed using absorption spectroscopy administration to ensure an adequate absorption or accumula-\nof the opposite solution at di\ufb00erent time points (Figure 4d). The tion of LipoRu in the tumors, as con\ufb01rmed by the in vivo imag-\nskin area allowing drug permeation is a circular region with a ing assay. The e\ufb03cacy of PTT was assessed by monitoring the\ndiameter of 10.0 mm (78.5 mm2 ). Ru2 could di\ufb00use into the op- real-time changes in the body temperature of mice using IR ther-\nposite solution (PBS) through the nude mouse skin due to its mal images. In the PBS, LipoRu (i.t.), LipoRu (i.v.), and LipoRu\nhydrophobic character. The di\ufb00usion is signi\ufb01cantly accelerated cream (td) groups, the temperature of the tumor sites increased\nwhen Ru2 is encapsulated in liposomes and further formulated by \u2206T = 4.6, 15.3, 19.5, and 22.4 \u00b0C, respectively, following expo-\ninto a cream (Figure 4d). sure to 808 nm irradiation (Figure 6b). When tumor tissues are\n In vivo and ex vivo \ufb02uorescence imaging studies were further exposed to temperatures exceeding 50 \u00b0C for less than 10 min,\nconducted to test the e\ufb03cacy of di\ufb00erent administration routes of rapid irreversible cell death can result in vessel formation and\nLipoRu on the tumor and distribution to other organs of tumor- protein destruction.[8a] No skin irritation or injury was observed\nbearing nude mice. As shown by \ufb02uorescence imaging for living during the phototherapy, unlike previous studies that employed\nmice (Figure 5a) and collected organs (Figure 5b), intratumoral high-power lasers or xenon lamps.\ninjection achieved an immediate high accumulation of LipoRu in At the end of the experiment (17 d after phototherapy), the sur-\nthe tumor. However, LipoRu concentrated at the injection site and vival rate of mice was 100%. In the PBS and dark groups, the\ndid not spread uniformly throughout the tumor. For more accu- tumor mean volume (V) exceeded 1300 mm3 (Figure 6c; Figure\nrate quanti\ufb01cation, in addition to the \ufb02uorescence intensity data S37, Supporting Information), and no decline in body weight\nprovided by live imaging (Figure 5c), ICPMS was used to analyze was observed in the mice (Figure 6d). This suggests that the in\nthe content of ruthenium in the whole organ (Figure 5d,e). The vivo systemic toxicity of LipoRu is extremely low. The LipoRu+IR\nliver and kidneys received a minor portion of LipoRu in 1 h and groups (Groups IV and VI) exhibited a notable antitumor ef-\nnearly eliminated it in 48 h. The peak accumulation of LipoRu in fect. The results of in vivo phototherapy demonstrate that LipoRu\nthe tumor was observed at 1 h after tail vein injection. A greater cream applied by transdermal delivery achieved similarly e\ufb00ec-\namount of LipoRu accumulated in the liver and kidneys and re- tive therapeutic outcomes in comparison to intravenous injec-\nmained present at 48 h. This indicates that although the EPR ef- tion and slightly better than intratumoral injection. Compared\nfect played a role, there were still challenges in achieving selective to injection methods, the non-invasive administration route of-\naccumulation in the tumor. Remarkably, LipoRu administered via fers advantages in terms of convenience and a milder treatment\ntransdermal cream exhibited similar tumor accumulation as the experience for patients. The variations in the hematoxylin and\nintravenous injection. Nevertheless, the accumulation in the liver eosin (H&E) staining pattern of the tumor sections between the\nand kidneys was signi\ufb01cantly decreased in comparison to intra- LipoRu+IR groups and the other groups suggested that LipoRu-\nvenous injection, and it is comparable to or even lower than intra- mediated therapy could partially eradicate tumor tissues (Figure\ntumoral injection. Applying the cream topically facilitated prefer- S38, Supporting Information). Moreover, the absence of any sub-\nential absorption by the nearby subcutaneous tumors, despite the stantial lesions in the main organs serves as con\ufb01rmation that\nlimited ability of LipoRu to reach the bloodstream through blood the systemic toxicity of LipoRu in vivo is negligible. The superior\nvessel walls. The pathway for the absorbed LipoRu by the skin to therapeutic e\ufb00ects observed in experiments with small tumor-\nenter the bloodstream may involve the subcutaneous tissue and bearing mice still require further validation to determine appli-\nmicrovascular networks. cability across other animals or humans.\n As LipoRu is supposed to enter the bloodstream by transder- The toxic e\ufb00ects of heavy metals on the human body, partic-\nmal delivery, the fate of Ru2 in blood circulation and further di- ularly on the liver and kidneys, pose a serious safety risk. The\ngestion behavior have also been evaluated. The urine and whole amounts of residual metals in the tumors and organs of LipoRu-\nblood of the mice in in vivo \ufb02uorescence imaging studies were treated mice were determined using ICP-MS at the end of the\ncollected, diluted with methanol (1:10 for the urine and 1:500 experiment (Figure 6e). Metal was found in trace amounts only\nfor the whole blood), \ufb01ltered through a 0.22 \u03bcm microporous in livers, kidneys, and tumors. The intravenous injection groups\nmembrane, and analyzed by HRMS-ESI to detect the possible showed a modest increase in liver retention, while the intratu-\nRu species. As the amounts of administrated LipoRu are quite moral injection groups exhibited a higher retention in tumors.\nlow and the dilution of urine and blood samples are unavoidable, The transdermal administration groups demonstrated balanced\nRu species were detected in only one set of samples and identi- and e\ufb00ective clearance. The LipoRu cream groups showed an\n\ufb01ed as Ru2 (Figure S36, Supporting Information). This indicates average residue of 0.3%. The metals were eliminated from the\nthat Ru2 did not undergo chemical reactions during circulation body of A375 mice at higher rates than cisplatin (t1/2 = 58\u221273 h),\nin the body but was excreted intact in the form of the Ru2 com- Photofrin II (t1/2 > 100 h), and Por\ufb01mer Sodium (t1/2 = 250 h)\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (9 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 5. a) In vivo \ufb02uorescence imaging detection of the distribution of LipoRu over time in A375 tumor-bearing mice with three di\ufb00erent administration\nmethods: intratumoral injection (i.t.), tail intravenous injection (i.v.), and transdermal delivery (td) by topical cream application. Mouse without LipoRu\ntreatment was tested as blank. Detection of the time-dependent distribution of LipoRu with di\ufb00erent administration methods based on b) \ufb02uorescence\nimaging and c) numerical radiant \ufb02ux per unit area of dissected tumors and major organs, including the liver (Li), tumor (T), lung (Lu), spleen (S), heart\n(H), and kidneys (K). Ru content in each examined organ (d,e) by ICPMS.\n\n\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (10 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 6. In vivo multimodal therapy of LipoRu toward subcutaneous A375 human melanoma xenograft model BALB/c nude mice. a) Diagram of process\nand summary results of in vivo assays (8 groups). b) Real-time thermal images of mice and tumors during the in vivo phototherapy. c) Histogram of\nmean tumor sizes for A375 tumor-bearing mice in Group I\u2212VIII (mean \u00b1 SD, n = 4). The signi\ufb01cance di\ufb00erence between two groups was assessed by\none-way ANOVA test using OriginPro 2024 software. P<0.05 was the accepted level of signi\ufb01cance (** p < 0.01, *** p < 0.001). d) Histogram of mean\nbody weights for A375 tumor-bearing mice in Group I\u2212VIII (n = 4). e) Ruthenium contents in the collected organs from LipoRu-treated A375 tumor\ngroups (mean \u00b1 SD, n = 8). N.D.: not detected. The amount of ruthenium delivered was calculated by the mean values of total body weight. Normalized\nA450 \u2212A620 (\u00d7 100) in CCK-8 assay of f) human normal kidney cell line HK-2 and g) normal liver cell line HL-7702 incubated with various doses of Ru1\u22123,\nLipoRu, and cisplatin for 24 h ([drug] = 0\u221210 \u03bcm, mean \u00b1 SD, n = 3).\n\n\nbased on the respective half-lives (t1/2 ) of 46 h, 50 h, and 23 h livery through topical cream application. The positive therapeu-\nfor the LipoRu (i.t.), LipoRu (i.v.), and LipoRu cream (td) groups. tic e\ufb00ects of the LipoRu cream fabricated here in subcutaneous\nThe rapid elimination rates of LipoRu can not only reduce the tumor-bearing mice o\ufb00er optimistic potential for the painless\nlevel of metal residue, thereby enhancing safety, but also greatly and non-invasive treatment of both early-stage and advanced skin\ndecrease the occurrence of adverse e\ufb00ects in phototherapy, such cancers, as well as super\ufb01cially located solid tumors.\nas photodermatosis.\n To evaluate the potential hepatotoxic and nephrotoxic e\ufb00ects\nof the residue of the Ru(II) complex, a study was undertaken to 4. Experimental Section\nassess the toxicity of Ru1\u22123 and LipoRu on normal human liver\n(HL-7702) and kidney (HK-2) cells. The in vitro cytotoxicity stud- Animal Ethics Statement: Animal experiments for A375 tumor were\n reviewed and approved by the Institutional Animal Care and Use\nies indicated that Ru1\u22123 and LipoRu had minimal cytotoxic ef-\n Committee (IACUC) at Yunnan University, Kunming, China (Approval\nfects on both cells when not exposed to irradiation (Figure 6f,g). No: YNU20240963). Dr. Xiaoxia Ren (Laboratory Animal Certi\ufb01cate\nRu1\u22123 and LipoRu displayed ignorable toxicity in a range from 1 1118062800091) at Animal Research and Resource Center (accreditation\nto 10 \u03bcm, which is approximately \ufb01ve orders of magnitude higher number SYXK (\u0002) K2021-0002), Yunnan University, Kunming, China, per-\nthan the total residue of LipoRu found in mice. In contrast, cis- formed the experiments and collected the data.\nplatin exhibited a signi\ufb01cant toxicity toward HK-2 and HL-7702 Statistical Analysis: The signi\ufb01cance di\ufb00erence between two groups\ncells. These results allow LipoRu to achieve a minimal risk level was assessed by one-way ANOVA test using OriginPro 2024 software. p\n < 0.05 was the accepted level of signi\ufb01cance (** p <0.01, *** p < 0.001).\nbefore its complete elimination from the organism. Data for cytotoxicity was presented in mean \u00b1 SD (n = 3). Data for in vivo\n therapy was presented in mean \u00b1 SD (n = 4).\n3. Conclusion\nIn summary, we have integrated two innovative solutions: Supporting Information\nliposome-encapsulated antitumor therapeutic agents and a non-\n Supporting Information is available from the Wiley Online Library or from\ninvasive and painless transdermal drug delivery cream. Initially, the author.\nwe developed a series of Ru(II) complexes that exhibited synergis-\ntic antitumor e\ufb00ects through two-photon-excited PDT, PTT, and\nchemotherapy. Subsequently, the selected complex Ru2 was en- Acknowledgements\ncapsulated into liposomes (LipoRu) without solid particles and\n This work was supported by the National Natural Science Foundation\nformulated into a topical cream using hydrogel. The biodistri- of China (22467023 and 22167022), the Yunnan Provincial Science and\nbution and therapeutic e\ufb03cacy of LipoRu in vivo have been sys- Technology Department (202401AS070139 and 202401AT070468), and\ntematically compared via three distinct administration routes: in- the Youth Talents Project of Yunnan Province (YNWR-QNBJ-2018-057).\ntratumoral injection, intravenous injection, and transdermal de- Figure 1 is created with BioRender.com.\n\n\nAdv. Healthcare Mater. 2025, 14, 2403563 2403563 (11 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 21922659, 2025, 3, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202403563 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\nCon\ufb02ict of Interest Chem. Front. 2021, 8, 636; c) C. Xu, K. Y. Pu, Chem. Soc. Rev. 2021,\n 50, 1111; d) S.-J. Tang, Q.-F. Li, M.-F. Wang, R. Yang, L.-Z. Zeng, X.-L.\nThe authors declare no con\ufb02ict of interest. Li, R.-D. Wang, H. Zhang, X. Ren, D. Zhang, F. Gao, Adv. Healthcare\n Mater. 2023, 12, 2301227; e) Y.-A. Deng, S.-J. Tang, M.-F. Wang, X.\n Ren, X.-L. Li, L.-Z. Zeng, D.-N. Ren, M.-R. Wang, W.-L. Xiao, Z.-Y. Cai,\nData Availability Statement D. Zhang, H. Zhang, F. Gao, Inorg. Chem. Front. 2023, 10, 4552; f) M.-\n F. Wang, R. Yang, S.-J. Tang, Y.-A. Deng, G.-K. Li, D. Zhang, D. Chen,\nThe data that support the \ufb01ndings of this study are available from the cor-\nresponding author upon reasonable request. X. Ren, F. 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