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Luminescent Iridium-Peptide Nucleic Acid Bioconjugate as Photosensitizer for Singlet Oxygen Production toward a Potential Dual Therapeutic Agent.
A novel bioorganometallic PNA conjugate ( Ir-PNA ) was
synthesized by covalently bonding a model PNA tetramer to a luminescent
bis-cyclometalated Ir(III) complex that acted as a photosensitizer
under light irradiation to generate singlet oxygen ( 1 O 2 ). The conjugate was prepared using an Ir complex bearing
the 1,10-phenanthroline ligand functionalized with either a free primary
amine ( Ir-NH 2 ) or a carboxyl
group ( Ir-COOH ) for the conjugation to PNA. The photophysical
studies on the Ir-COOH and the Ir-PNA demonstrated
that the luminescent properties were maintained after the conjugation
of the Ir fragment to PNA. Furthermore, the abilities to produce 1 O 2 of Ir-COOH and Ir-PNA were confirmed in a cuvette under visible light irradiation employing
1,5-dihydroxynaphthalene as a reporter, and the measured singlet oxygen
quantum yield (Φ Δ ) supported the Ir-PNA conjugate efficacy as a photosensitizer (Φ Δ = 0.54). Two-photon absorption microscopy on HeLa cells revealed
that Ir-PNA localized in both the cytosol and nucleus,
suggesting its potential as an intracellular carrier for PNA. Cytotoxicity
assays by MTT tests showed that Ir-PNA was nontoxic in
the absence of light, but induced cell death (EC 50 = 18
μM) after UV irradiation. Overall, the Ir-PNA conjugate
represents a promising system for the intracellular delivery of the
PNA and its application in PDT.
## Introduction
1 Introduction Photodynamic therapy (PDT)
is a noninvasive medical treatment that
is able to produce reactive oxygen species (ROS) exploiting light
and is categorized into Type I and Type II PDT. 1 − 3 The latter consists
of exciting molecular oxygen from its triplet ground state ( 3 O 2 ) to the cytotoxic singlet excited state ( 1 O 2 ) by using a photosensitizer (PS). First, the PS is
excited to the singlet state (S 1 ) by suitable light irradiation,
followed by intersystem crossing (ISC) to the triplet excited state
(T 1 ), where its energy is transferred to 3 O 2 . Conversely, Type I PDT occurs through electron or proton
transfer between the PS and the O 2 , leading to the formation
of hydroxyl or superoxide radicals. Among many different PSs, 4 luminescent
organometallic complexes provide several advantages compared to organic
fluorophores primarily due to their accessible, low-energy excited
triplet states. This feature enables several desirable photophysical
properties, including long lifetimes, high photostability, and large
Stock shifts, making them highly suitable for biological applications.
Additionally, they exhibit minimal background interference, especially
if the organometallic compounds can be excited via two-photon absorption
(TPA). 5 − 7 In this context, different transition metal complexes,
especially those containing Ru, Ir, Re, and Pt, are gaining attention
as PSs due to their exceptional photophysical, photochemical, and
photobiological properties. 8 In particular,
cyclometalated Ir(III) complexes are suitable as PS for 1 O 2 production in PDT due to the accessibility of triplet
excited states, usually 3 MLCT. 9 Many of these compounds possess a positive charge, which promotes
their solubility in water, allows interaction with the plasma membrane,
and enhances their cellular uptake. Although Ir(III) compounds often
exhibit shorter absorption wavelengths compared to their Ru(II) counterparts,
they typically show very large Stokes shifts and are much more effectively
quenched in the presence of oxygen. This is strictly related to an
excellent energy transfer process in Type II PDT, which allows high
singlet oxygen quantum yields. In addition, most Ir(III) complexes
are characterized by a high two-photon absorption cross section, 7 making them useful as two-photon PDT agents that
can obtain deeper tissue penetration with lower cellular damage. Overall,
these unique photophysical features along with their good cell permeability
make cyclometalated Ir(III) complexes valuable systems as imaging
and sensing probes. Moreover, the conjugation of these complexes to
targeting vehicles (i.e., small regulatory peptides) 10 or to oligonucleotides 11 − 14 leads to drug conjugates with
enhanced pharmacological properties that find applications in targeted
theranostic and antisense technology. In particular, the covalent
linkage of Ir(III) polypyridyl complexes to DNA oligonucleotides represents
an emerging area for innovative applications of nucleic acids as nanobiosensors
for target DNA detection, 12 and photoredox
systems for studying DNA-mediated hole and electron transport. 13 , 14 To the best of our knowledge, no examples of cyclometalated
Ir(III)
complexes conjugated with synthetic mimics of natural nucleic acids
have been reported so far, taking into account that chemically modified
nucleic acids display improved biological properties compared with
natural DNA/RNA (e.g., enzymatic stability, binding affinity) and
are valuable tools in therapeutics, clinical diagnostics and nanotechnology. 15 Among them, peptide nucleic acids (PNAs) belong
to the family of xeno-nucleic acids, in which the ribose-phosphate
backbone is replaced by the synthetic polyamide-based aminoethylglycine
( aeg ) unit. 16 This modification
allows for a significant increase in the enzymatic stability of PNAs
toward nucleases and proteases, 17 and the
neutral aeg backbone also ensures strong hybridization
properties toward complementary natural nucleic acids, due to the
lack of electrostatic repulsion between PNAs and DNA or RNA strands. 18 However, the great potential of PNAs especially
as therapeutic agents is still limited due to their poor cell- or
tissue-specific delivery. Diverse approaches have been reported to
address the problem of inefficient PNA delivery, including the covalent
conjugation of PNA to luminescent organometallic complexes. This represents
a useful strategy because it not only enhances the intrinsic PNA properties
but also endows PNAs with new additional features depending on the
nature of the metal complex. 19 Examples
of PNA oligomers covalently linked to d 6 or d 8 transition-metal complexes have been reported (e.g., mono 20 and dinuclear 21 rhenium(I)
complexes, tris(bipyridine)ruthenium(II) 22 and diamminedichloroplatinum(II) 23 complexes),
and for some of these conjugates the metal complex has proven to be
a good carrier for PNA to eukaryotic cells and a nontoxic luminescent
tag for PNA labeling. Nevertheless, none of these metal-PNA conjugates
have been explored as photosensitizers for PDT, despite some of them
having the potential to exhibit all the necessary characteristics
for ROS production under appropriate light irradiation. Indeed, very
few studies on PNA–PS conjugates have been reported so far,
exclusively utilizing organic photosensitizers. 24 To bridge this gap, this work aims to synthesize and characterize
a novel bio-organometallic PNA conjugate, consisting of a model PNA
tetramer covalently tethered to a luminescent bis-cyclometalated Ir(III)
complex, which can generate singlet oxygen upon light irradiation. Covalently integrating the emissive Ir(III) complex into the PNA
structure could offer several advantages: (i) it enables the tracking
of PNA fate in cells otherwise invisible through fluorescence microscopy,
as well as through other analytical investigation techniques; (ii)
the Ir complex can positively influence the low ability of PNA to
cross cell membrane; (iii) the conjugation can create a synergistic
effect, combining the therapeutic action of singlet oxygen generated
by the Ir complex with the precise genetic targeting capability of
the PNA. Herein, we report the synthesis of the Ir-PNA conjugate
( Figure 1 ), which has
been obtained using two different bis-cyclometalated Ir(III) complexes Ir-NH 2 and Ir-COOH ( Figure 1 ). Both complexes
contain, as a third ligand, a 1,10-phenantroline endowed with a proper
functional group (i.e., a free primary amine or a carboxylic group)
for the covalent conjugation to the model PNA tetramer. The photophysical
properties of the novel Ir-COOH complex and the Ir-PNA conjugate have been fully investigated and exploited
to study the uptake and intracellular localization in HeLa cells by
confocal microscopy. The capabilities of the Ir-COOH and
the Ir-PNA to act as PSs producing 1 O 2 were measured as well, and in vitro tests
were performed to assess the cytotoxicity in the dark (no irradiation)
and after irradiation with UV light. Figure 1 Structure of the Ir-PNA conjugate,
the Ir-NH 2 , and the Ir-COOH complexes.
## Results and Discussion
2 Results and Discussion 2.1 Design and Synthesis of the Ir-PNA Conjugate Considering the advantages of solid-phase synthesis, the preparation
of the Ir-PNA conjugate was envisaged through a stepwise
solid-phase approach. The Ir complex was conjugated at the N -terminal end of the resin-supported PNA tetramer via amide
bond formation, followed by the release of the conjugate from the
solid support ( Scheme 1 ). Scheme 1 General Strategy for the Synthesis of the Ir-PNA Conjugate To verify the outcome of the amidation step,
two complementary
synthetic routes were tested: (a) the amide bond formation between
the N -terminal end of the PNA1 with
the carboxylic group on the Ir-COOH complex ( Scheme 1 a); (b) the amide
bond formation between the carboxylic function in the properly modified PNA2 and the primary amine group of the Ir-NH 2 complex ( Scheme 1 b). The Ir-NH 2 complex was prepared
according to the literature, 25 while the Ir-COOH complex was synthesized through the reaction between
the new ligand Phen-COOH with the Ir dimer precursor [Ir(ppy) 2 Cl] 2 , following a procedure akin to that used for the synthesis
of the Ir-NH 2 ( Scheme 2 ). Scheme 2 Schematic Procedure
for: (a) the Synthesis of the Phen-COOH Ligand; (b) the
Synthesis of the Ir-COOH Complex First of all, the ligand 4-((4-(1,10-phenanthrolin-4-yl)butyl)amino)-4-oxobutanoic
acid ( Phen-COOH ) was prepared starting from 4-(1,10-phenanthrolin-4-yl)butan-1-amine
( Phen-NH 2 ). 26 A literature-based procedure was partially followed, 27 employing triethylamine (TEA) to achieve complete
deprotonation of the amine, and utilizing an equimolar amount of succinic
anhydride in dry dichloromethane (DCM), resulting in the formation
of a terminal carboxylic group ( Scheme 2 a). The 1 H NMR spectrum showed a signal
pattern consistent with the formation of the desired product ( Figure S1 ). Notably, the amide NH(5′)
signal at 7.45 ppm showed a scalar correlation with the methylene
(4′) in the α position as observed in a 2D 1 H COSY experiment ( Figure S2 ). To complete
the NMR characterization, both direct and long-range 1 H– 13 C scalar correlation 2D experiments were acquired and reported
in the Supporting Information ( Figures S3 and S4 ). Next, the Phen-COOH ligand underwent a reaction with
the Ir dimer precursor [Ir(ppy) 2 Cl] 2 in a mixture of THF and
H 2 O, using a method similar to that employed for the synthesis
of the Ir-NH 2 , 25 but with an increased amount of THF ( Scheme 2 b), which resulted in a significantly higher
yield of 96%. The resulting Ir-COOH complex was fully
characterized by mono and bidimensional 1 H and 13 C NMR experiments (see Figures S5–S10 ) and electrospray ionization mass spectrometry in positive mode
(ESI + MS, Figure S11 ). NMR spectroscopy
allowed ascertaining that the Ir-COOH complex was produced
as a zwitterion, where the remaining positive charge of Ir(III) is
neutralized by the deprotonated carboxylate terminal moiety. According to Scheme 1 , before proceeding with the conjugation of the Ir complex to the
PNA tetramer in the solid phase, the stability of the Ir-NH 2 was tested under the acidic conditions
normally used for the cleavage of PNA from the resin. Hence, the behavior
of a solution of Ir-NH 2 dissolved
in a mixture of trifluoroacetic acid (TFA)/ m -cresol
9:1 at room temperature was followed over time, monitoring the presence
of the MLCT bands, which remained unvaried over 1.5 h ( Figure S12 ). Thus, this test confirmed the stability
of this family of Ir complexes, presumably due to the tight chelation
of the three ligands, in agreement with the results previously obtained
for similar cyclometalated Ir(III) complexes. 10 Then, the Ir-COOH and the Ir-NH 2 were conjugated to the PNA starting from the
resin-supported,
fully protected PNA1-Fmoc and PNA2 , which
were prepared according to Fmoc/Bhoc (fluorenylmethyloxycarbonyl/benzhydryloxycarbonyl)
manual solid phase synthesis as previously reported. 28 The first route to synthesize the Ir-PNA conjugate
involved the removal of the Fmoc group with a solution of piperidine,
followed by the coupling reaction between the free primary amine group
of PNA1 and the carboxylic group of the Ir-COOH complex in the presence of O -(7-aza-1 H -benzotriazole-1-yl)- N,N,N,N -tetramethyluronium
hexafluorophosphate (HATU) as a condensing agent and N -ethyldiisopropylamine (DIPEA) as base in N -methylpyrrolidone
(NMP) for 4 h at room temperature ( Scheme 3 a). Scheme 3 Synthesis of the Ir-PNA Conjugate through Two Complementary
Routes Next, the treatment of the resin with a solution
of TFA/ m -cresol provided the removal of the N -Bhoc
protecting groups from the nucleobases and the concomitant cleavage
from the resin of Ir-PNA , which was purified by reverse-phase
high-performance liquid chromatography (RP-HPLC). The Ir-PNA identity and purity were confirmed by high-resolution electrospray
ionization mass spectrometry (HR-ESI + MS) and RP-HPLC analysis,
respectively ( Figures S13 and S14 ). Alternatively, the resin-supported, fully protected PNA2 was treated with a solution of HATU and DIPEA in NMP for 8 min and
subsequently reacted with a solution of Ir-NH 2 in NMP for 4 h at room temperature ( Scheme 3 b). After the TFA-mediated
deprotection of N -Bhoc groups and release of the
conjugate from the resin, the RP-HPLC trace of the crude Ir-PNA was found to be very similar to that obtained following pathway
(a), demonstrating the same outcome for both complementary procedures
that can be used for the PNA conjugation of this class of bis-cyclometalated
Ir(III) complexes. 2.2 Photophysical Characterization of Ir-COOH
and Ir-PNA The photophysical data for the Ir-COOH complex and the corresponding Ir-PNA conjugate are
summarized in Table 1 and depicted in Figure 2 and Figures S15 and S16. Figure 2 UV–vis
absorption (solid lines) and photoluminescence spectra
(dashed lines) of the PNA (blue trace), the Ir-COOH (red
traces) and the Ir-PNA (black trace) in methanol at room
temperature in aerated conditions. The inset shows a magnification
of the MLCT bands centered at 377 and 415 nm. Table 1 UV-vis and Photoluminescence Data
at Room Temperature for the Compounds Ir-PNA (in Aerated
Methanol; λ ex = 405 nm), Ir-COOH (in
Aerated DCM, Acetonitrile, Methanol, and Water; λ ex = 420 nm) and Ir-NH 2 (in Aerated
DCM, Acetonitrile, and Water; λ ex = 400 nm) species solvent λ abs (nm) λ em (nm) Φ τ (ns) k r (s –1 ) b k nr (s –1 ) b Ir-PNA MeOH 377 582 0.039 13.5 (6%) 4.8 × 10 5 12.0 × 10 6 415 80.3
(94%) 470 Ir-COOH CH 2 Cl 2 381 571 0.084 128.3 6.5
× 10 5 7.1 × 10 6 415 472 CH 3 CN 374 585 0.027 56.9 4.7
× 10 5 1.7 × 10 7 415 470 MeOH 377 590 0.034 33.0 (10%) 4.9
× 10 5 1.4 × 10 7 415 69.2 (90%) 470 H 2 O 374 600 0.051 31.8 (3%) 4.5 × 10 5 8.4 × 10 6 412 113.4 (97%) 469 Ir- NH 2 a CH 2 Cl 2 383 574 0.052 153 3.4 × 10 5 6.2 ×
10 6 417 CH 3 CN 376 591 0.017 56 3.1 × 10 5 1.8 × 10 7 414 H 2 O 378 604 0.033 2 (2%) 2.8 × 10 5 8.3 × 10 6 417 116 (98%) a Literature data from ref ( 25 ). b k r and k nr indicate the radiative and nonradiative decay
constants of the excited states, respectively, and are computed on
the most significant lifetime component. UV–vis absorption spectra of the Ir-COOH , acquired
in different solvents, display intense absorption bands in the ultraviolet
region at about 220–300 nm, whose position is independent of
the solvent polarity, and attributable to spin-allowed π-π*
ligand-centered 1 LC transitions. According to the literature
data, 29 − 35 the bands lying in the range 220–250 nm receive larger contributions
from ppy-centered transitions, while those between 250 and 290 nm
are mainly centered on the phenanthroline ligand. In the visible region,
the Ir-COOH complex displays less intense absorption
bands in the range 350–420 nm which are mainly attributed to
spin-allowed 1 MLCT [dπ(Ir)-π*(phen)] transition,
due to the excitation from filled t 2g orbitals of the Ir(III)
center to a vacant π* orbital of the phenanthroline ligand,
acting as acceptor. However, it should be considered that in Ir(III)
cyclometalated compounds, as a consequence of the large covalency
of the Ir–C- bond, the HOMO is not completely localized on
the d orbitals of the metal center but can be significantly delocalized
over the cyclometalating ligands, with a large participation also
of the Ir–C- σ bond orbital. 30 , 31 , 36 Therefore, the absorption bands observed
in the visible region could be assigned to the σ bond-to-ligand
charge transfer ( 1 SBLCT) transitions, i.e. the ppy-to-phen
charge transfer ( 1 LLCT) transitions (see Figure S15 ). Finally, the absorption bands at the lowest energy
(at about 470 nm) are assigned to spin-forbidden CT transitions, which
steal the intensity from the spin-allowed transitions thanks to the
large spin–orbit coupling induced by the heavy iridium center.
The CT character of these absorption bands is confirmed by the observed
solvatochromic effect, affording a progressive blue shift on going
from the least polar (DCM) to the most polar (water) solvent used
in these measurements (see Table 1 and Figure S15 ). All of
these features were expected and showed to be in line with the many
orthometalated mononuclear Ir(III) complexes of the [Ir(N^C) 2 (N^N)] + family described in the literature. The
conjugation of the Ir complex to the PNA generates only small
modifications of the absorption spectrum in the ultraviolet range,
affording a new band at 210 nm due to the PNA backbone transitions
and an increase of the absorption at 260 nm due to the 1 LC transitions of the purine bases ( Figure 2 ). Moreover, as expected, the visible part
of these absorption spectra is not modified either by the presence
of the PNA oligomer in the Ir-PNA or by the free primary
amine instead of the carboxylic group in Ir-NH 2 (see Figure 2 and Table 1 ). 25 Indeed, the presence of the aliphatic
chain hampers any electronic conjugation between the PNA, or the functional
groups (COOH and NH 2 ), and the 1,10-phenanthroline ligand
involved in the electronic transitions. Upon excitation at 405
nm, the Ir-COOH and Ir-PNA display photoluminescence
emission bands in the yellow-orange range
of the visible spectrum, attributable to a metal-to-ligand charge
transfer transition having triplet character ( 3 MLCT) (see Figure S16 ). The broad and structureless shape
of these emission bands suggests the absence of the role of the π-π*
LC transitions in the emission process, further confirmed by the excitation
spectra ( Figure S17 ) and the monoexponential
decay profile of the lifetimes observed in DCM and acetonitrile (ACN)
solutions. The slight blue shift of the emission band observed in
the DCM solution for Ir-COOH and Ir-NH 2 , with respect to the analogous Ir complex containing
the unsubstituted 1,10-phenanthroline ligand (Ir-Phen, λ em = 582 nm in DCM), 33 reflects
the weak electron donor ability of the aliphatic substituent on the
phenanthroline ligand, thus confirming the charge transfer character
of the emission. 30 , 33 , 35 Moreover, both Ir-COOH and Ir-NH 2 display a red-shift of the emission maximum passing
from the least polar (DCM) to the most polar (water) solvent, 37 in agreement with the commonly observed trend
of excited states having charge transfer character (see Table 1 ). The red shift of the emission
maximum is usually associated with a progressive decrease of the photoluminescent
quantum yield (PLQY) and a shortening of lifetimes, in agreement with
the energy gap law. 38 Nevertheless, it
is interesting to note that in the case of Ir-COOH and Ir-NH 2 , the lifetimes and PLQYs do
not follow this trend. As for the Ir-NH 2 complex, 25 also Ir-COOH displays higher PLQY in polar solvents such as water or methanol
than in the less polar solvent acetonitrile, together with longer
and biexponential lifetimes (see Table 1 ). This behavior suggests that, in these polar solvents,
the Ir-COOH is also present as a nanosized aggregate,
as already observed for the previously reported Ir-NH 2 complex, where the complex may be affected
by a more rigid and less polar surrounding. To confirm this hypothesis,
we measured the photophysical properties of Ir-COOH dissolved
in ACN mixed with increasing quantities of H 2 O (see Figure S18 and Table S1 ). As the water content
increases, a progressive red-shift of the emission maximum is observed,
followed by an increase in fluorescence quantum yield and a corresponding
lengthening of lifetimes, which become progressively biexponential
( Figure S19 ). This behavior supports the
hypothesis that, in highly polar solvents, the emission is mainly
governed by nanoaggregates. The quantity of these nanoaggregates increases
with higher water content, although they do not account for the entire
amount of the compound present in the solution. It is to be noted
that the slight shortening of the main lifetime component observed
in neat water—corresponding to a 2-fold increase in the k nr constant (see Table S1 )—strongly suggests that water
acts as a classical quencher, capable of competing with nonradiative
deactivation processes, as previously reported for other dyes. 39 The progressive formation of nanoaggregates
was confirmed by DLS
measurements ( Figure S20 ). In neat acetonitrile
and H 2 O/ACN mixtures with ratios of 1:9, scattering signals
were either absent or insufficient to be detected. However, on increasing
the water content, an increase in scattered photons was observed and,
parallelly, the fitting of the correlation functions returned dimensional
distributions with larger populations. Also for the MeOH solution
of Ir-COOH , DLS measurements confirmed the presence of
a population centered at 300 nm, alongside a numerically prevailing
population attributable to the single molecules in solution ( Figure S21 ). It is important to note that
nanoaggregates larger than 50 nm are
undetectable by 1 H NMR spectroscopy. Accordingly, the 1 H NMR spectrum of the Ir-COOH complex in methanol- d 4 ( Figure S5 ) displayed
only sharp signals, which are characteristic of the nonaggregated
molecular fraction, providing only a partial view of the overall system. The formation of these nanoaggregates seems to be further favored
by the presence of the hydrophobic PNA chain in the Ir-PNA conjugate, thus accounting for an 8 nm blue shift of the emission
maximum in water and the further increase in PLQY together with the
main component of the lifetime. Indeed, the short component (τ1
= 33.0 and 13.5 ns for the Ir-COOH and the Ir-PNA , respectively), observed in the biexponential emission decay kinetics,
can be attributed to the free molecules (which are more affected by
the presence of oxygen and more exposed to the polar solvent), while
the longer component (τ2 = 69.2 and 80.3 ns for the Ir-COOH and the Ir-PNA , respectively), is attributed to the
nanoaggregates. 2.3 Photochemical Stability of the Ir-COOH and
the Ir-PNA To evaluate the durability of the Ir-PNA conjugate under extended exposure to light in the presence of oxygen,
we conducted a photochemical stability test. This assessment was needed
as a critical requirement for the potential use of Ir-PNA as a photosensitizer. Hence, the compound was dissolved in methanol,
and the solution was saturated with O 2 , bubbling the gas
through the solution for 5 min. Subsequently, the solution was subjected
to visible light exposure, using a monochromatic LED light (λ
= 400 nm, 15.2 mW/cm 2 ) and UV–vis absorption spectra
were recorded every few minutes for a total period of irradiation
of 20 min ( Figure S22 ). Analysis of the
superimposed absorption spectra obtained at various time points demonstrated
the high photostability of the compound. The same stability test was
replicated for the Ir-COOH complex, yielding similar
outcomes ( Figure S23 ). 2.4 Photoreaction of the Ir-COOH and the Ir-PNA
with 1,5-Dihydroxynaphtalene as an Indirect Reporter of Singlet Oxygen
Production Previously, the ability of the Ir-NH 2 complex to serve as a PS inducing the generation
of singlet oxygen was evaluated. 25 This
assessment was based on the use of the singlet oxygen indirect reporter
1,5-dihydroxynaphthalene (DHN). 34 The same
photoreaction assay was conducted in this work, under irradiation
with monochromatic LED light (λ = 400 nm, 15.2 mW/cm 2 ) within a cuvette for both Ir-COOH and Ir-PNA complexes, to compare their performances as PS using the estimation
of their singlet oxygen quantum yields Φ Δ ,
which are reported in Table 2 . Table 2 Estimated Quantum Yields of Singlet
Oxygen Production (φ Δ ) and Kinetic Constant
( k obs ) of the Studied Compounds in Methanol species k obs (min –1 ) ν (10 –6 M min –1 ) I λ=400 (10 –7 mol min –1 ) Φ Δ Ir(ppy) 3 0.0401 6.82 7.00 0.50 a Ir-PNA 0.0158 2.60 2.48 0.54 Ir-COOH 0.0146 2.44 2.83 0.44 a From ref ( 34 ). A schematization of the selective and quantitative
photoreaction
of DHN with 1 O 2 is reported in Scheme 4 . Scheme 4 Depiction of the
Photochemical Conversion of DHN in Juglone The PS is excited to a singlet excited state
upon irradiation with
sufficiently energetic photons; then, it undergoes intersystem crossing
leading to the population of a triplet excited state, and finally,
through energy transfer, the PS promotes the excitation of molecular
oxygen from its triplet ground state to the singlet cytotoxic excited
state. The resulting photoproduced 1 O 2 , in the
presence of an excess of DHN, undergoes a quantitative reaction as
illustrated in Scheme 4 , ultimately forming the oxidized species Juglone. Figure 3 Evolution of UV–vis absorption spectra of DMC/methanol 9:1
solutions containing DHN (a) Ir-COOH (1.27 × 10 –5 M), (b) Ir-PNA (1.12 × 10 –5 M), (c) Ir(ppy) 3 (1.18 × 10 –5 M) irradiated for several minutes (λ = 400 nm). (d) Comparison
of the semilogarithmic plots of DHN consumption as a function of the
irradiation time registered for the investigated sensitizers, with A t and A 0 referring to the typical DHN absorbance value (at 316 nm),
registered at irradiation times t and 0, respectively. The comparison of Φ Δ determined
for the Ir-COOH complex (0.44) and the Ir-PNA conjugate
(0.54) shows that, in these conditions, 1 O 2 is
generated even more efficiently after the conjugation of the Ir complex
to the PNA chain. This suggests that the PS properties of the Ir complex
are neither compromised nor affected by its conjugation with the PNA
tetramer. 2.5 Fluorescence Microscopy Localization Study
by Two-Photon Absorption Cellular uptake and intracellular
localization in HeLa cells of both Ir-COOH and Ir-PNA compounds were monitored by two-photon-excitation (TPE)
microscopy. HeLa cells were treated with 25 μM of each compound
and incubated for 21 h. After this incubation period, the cells were
observed under the microscope. Notably, TPE at 720 nm was utilized
to excite the iridium compounds and acquire microscopy images ( Figure 4 ) as described in
the Experimental Part. The control exhibited a slight autofluorescence
( Figure 4 a,b) detectable
in the blue channel (480 ± 30 nm), predominantly attributed to
NADH (λ em = 460 nm), as documented in previous literature
for TPE excitation. 40 Both the Ir-PNA and the Ir-COOH compounds ( Figure 4 c–f) displayed luminescence in the
green and red channels (535 ± 50 and 600 ± 40 nm, respectively),
consistent with their broad emission spectra ( Figure 2 ). Images captured at the highest magnification
( Figure 4 d and 4 f) were acquired through z-scan techniques to confirm
that the signal originated from within the cell rather than the outer
membrane. Clearly, the emission resulting from the treatment with
the Ir-PNA conjugate originated from all visible cells
within the observed frame. In contrast, with the Ir-COOH complex, emission predominantly arose from a pool of treated HeLa
cells. This discrepancy implies that the internalization process may
be more facilitated for the PNA adduct compared to the neutral form
of the Ir-COOH complex, which is primarily present at
physiological pH (being deprotonated, the terminal COOH group balances
the residual positive charge of the Ir(III) metal center). Moreover,
apart from the positive charge retained by the Ir-PNA conjugate, this has a higher lipophilic character, which could also
enhance its internalization. Nevertheless, the Ir-PNA conjugate appeared to form aggregate-like spots, probably due to
its limited solubility in water, mainly accumulating in the cytosol
as well as in the nuclei, where a green/red emission is observable.
On the contrary, the Ir-COOH complex showed, where present,
a more homogeneous distribution at the cytoplasm level without any
emission coming from the nuclei. Further studies will be dedicated
in the near future to better characterize the molecular mechanisms
through which the Ir-PNA conjugate is internalized in
cells, and colocalization studies will also be carried out. Figure 4 TPE microscopy
images at two distinct magnifications (a, c, and
e = 100×, 300 × 300 μm 2 field of view;
b, d, and f = 50×, 150 × 150 μm 2 field
of view) of vehicle-treated HeLa cells (control, panels a and b),
HeLa cells treated with the Ir-PNA (panels c and d) and
HeLa cells treated with the Ir-COOH (panels e and f)
for 21 h. The z-scan images (panels d and f) are the superposition
of several images acquired along the optical axis at 1 μm intervals. 2.6 PDT Treatment on HeLa Cells The effectiveness
of the Ir-COOH complex alone and its conjugate with PNA
in inducing photocytotoxicity and cytotoxicity in the dark were assessed
and compared employing HeLa cells. The cells were treated separately
with increasing doses of both compounds either in the dark or under
light exposure. HeLa cells were incubated for 24 h to promote internalization,
as suggested by the previous TPE microscopy observations (see paragraph
2.5). Potential dark cytotoxicity was assessed by using an MTT assay.
The results, presented in Figure 5 , indicate that the Ir-COOH complex induced
significant dark cytotoxicity, in a dose-dependent manner. On the
other hand, cells treated with the Ir-PNA conjugate maintained
high cell viability (>90%) in the dark across all the tested concentrations.
This characteristic is crucial for a photosensitizer to be considered
for further in vivo applications. Figure 5 Dose–response
plots for the Ir-PNA conjugate
(panel a) and the Ir-COOH complex (panel b) in the dark
(black curves) and under UV irradiation. One-way ANOVA corrected for
multiple comparison by Sidak’s post hoc analysis was performed,
*** p < 0.001; n = 4. This level of toxicity for the iridium complexes
was already observed
in previous studies conducted on HeLa cells, where the similar complex Ir-NH 2 displayed higher cytotoxicity
in the dark compared to the corresponding conjugate with an amide-based
polymer. 25 Concurrently, the same experiment was
performed by irradiating
the samples for 30 min, using a low-intense UV- lamp emitted by a
polychromatic irradiation source (5.2 mW/cm 2 in the 200–800
nm range, see emission profile in Figure S24 ). To avoid any overheating due to the lamp exposure, the temperature
during the irradiation time was constantly monitored and maintained
at 37 °C. The dose–response curves for the Ir-COOH and the Ir-PNA compounds (shown in Figure 5 , panels (a) and (b), respectively)
were fitted nonlinearly using a sigmoidal function to determine dark
and light EC 50 values. These values represent the effective
concentration required to reduce cell viability to 50% in the dark
and after irradiation ( Table 3 ). Both compounds showed higher cytotoxicity upon UV light
exposure. However, the light EC 50 was 1 order of magnitude
higher for the Ir-PNA conjugate compared to the Ir-COOH complex, indicating greater phototoxicity when the
Ir complex was not bound to the PNA. Considering the insights gained
from TPE microscopy images, the lower photocytotoxicity of the Ir-PNA conjugate could be attributed to its tendency to form
aggregates, confined in vesicle-like structures (see Figure 4 ). These aggregates might potentially
trap singlet oxygen produced during irradiation and hinder it from
reaching target biomolecules effectively. On the other hand, the amplified
cytotoxicity showed by Ir-COOH could be possibly due
to the additional contributions of the intrinsic toxicity in the dark. Table 3 Cytotoxicity of the Ir-PNA and Ir-COOH Compounds on Hela Cells in the Dark and
upon Irradiation, Expressed in Terms of Dark EC 50 and Light
EC 50 species dark EC 50 light
EC 50 PI a Ir-PNA >50 μM 18 μM >2.8 Ir-COOH >50 μM 3.5 μM >14.3 a PI: phototherapeutic index = dark-EC 50 /light-EC 50 . Additionally, the lower limit phototherapeutic
indexes (PI) were
calculated from the dark to light EC 50 values (see Table 3 ), reflecting the
amplification of cytotoxic activity by light treatment. Our complexes
exhibit low phototherapeutic indexes if compared to other orthometalated
Ir complexes of the same [Ir(N^C) 2 (N^N)] + family, 41 , 7 which show PI even 1 order of magnitude higher than ours. This could
be due to many different factors, such as cellular uptake as well
as subcellular localization within a particularly sensitive subcellular
organelle. Nevertheless, making a fully reliable comparison is challenging,
as certain variables should be standardized, such as the administered
light dose, the incubation time both before and after irradiation,
and, of course, provided that the comparison of different molecular
effects is conducted within the same cell line. Our data are preliminary,
and a complete biological study has not been carried out yet.
## Design and Synthesis of the Ir-PNA Conjugate
2.1 Design and Synthesis of the Ir-PNA Conjugate Considering the advantages of solid-phase synthesis, the preparation
of the Ir-PNA conjugate was envisaged through a stepwise
solid-phase approach. The Ir complex was conjugated at the N -terminal end of the resin-supported PNA tetramer via amide
bond formation, followed by the release of the conjugate from the
solid support ( Scheme 1 ). Scheme 1 General Strategy for the Synthesis of the Ir-PNA Conjugate To verify the outcome of the amidation step,
two complementary
synthetic routes were tested: (a) the amide bond formation between
the N -terminal end of the PNA1 with
the carboxylic group on the Ir-COOH complex ( Scheme 1 a); (b) the amide
bond formation between the carboxylic function in the properly modified PNA2 and the primary amine group of the Ir-NH 2 complex ( Scheme 1 b). The Ir-NH 2 complex was prepared
according to the literature, 25 while the Ir-COOH complex was synthesized through the reaction between
the new ligand Phen-COOH with the Ir dimer precursor [Ir(ppy) 2 Cl] 2 , following a procedure akin to that used for the synthesis
of the Ir-NH 2 ( Scheme 2 ). Scheme 2 Schematic Procedure
for: (a) the Synthesis of the Phen-COOH Ligand; (b) the
Synthesis of the Ir-COOH Complex First of all, the ligand 4-((4-(1,10-phenanthrolin-4-yl)butyl)amino)-4-oxobutanoic
acid ( Phen-COOH ) was prepared starting from 4-(1,10-phenanthrolin-4-yl)butan-1-amine
( Phen-NH 2 ). 26 A literature-based procedure was partially followed, 27 employing triethylamine (TEA) to achieve complete
deprotonation of the amine, and utilizing an equimolar amount of succinic
anhydride in dry dichloromethane (DCM), resulting in the formation
of a terminal carboxylic group ( Scheme 2 a). The 1 H NMR spectrum showed a signal
pattern consistent with the formation of the desired product ( Figure S1 ). Notably, the amide NH(5′)
signal at 7.45 ppm showed a scalar correlation with the methylene
(4′) in the α position as observed in a 2D 1 H COSY experiment ( Figure S2 ). To complete
the NMR characterization, both direct and long-range 1 H– 13 C scalar correlation 2D experiments were acquired and reported
in the Supporting Information ( Figures S3 and S4 ). Next, the Phen-COOH ligand underwent a reaction with
the Ir dimer precursor [Ir(ppy) 2 Cl] 2 in a mixture of THF and
H 2 O, using a method similar to that employed for the synthesis
of the Ir-NH 2 , 25 but with an increased amount of THF ( Scheme 2 b), which resulted in a significantly higher
yield of 96%. The resulting Ir-COOH complex was fully
characterized by mono and bidimensional 1 H and 13 C NMR experiments (see Figures S5–S10 ) and electrospray ionization mass spectrometry in positive mode
(ESI + MS, Figure S11 ). NMR spectroscopy
allowed ascertaining that the Ir-COOH complex was produced
as a zwitterion, where the remaining positive charge of Ir(III) is
neutralized by the deprotonated carboxylate terminal moiety. According to Scheme 1 , before proceeding with the conjugation of the Ir complex to the
PNA tetramer in the solid phase, the stability of the Ir-NH 2 was tested under the acidic conditions
normally used for the cleavage of PNA from the resin. Hence, the behavior
of a solution of Ir-NH 2 dissolved
in a mixture of trifluoroacetic acid (TFA)/ m -cresol
9:1 at room temperature was followed over time, monitoring the presence
of the MLCT bands, which remained unvaried over 1.5 h ( Figure S12 ). Thus, this test confirmed the stability
of this family of Ir complexes, presumably due to the tight chelation
of the three ligands, in agreement with the results previously obtained
for similar cyclometalated Ir(III) complexes. 10 Then, the Ir-COOH and the Ir-NH 2 were conjugated to the PNA starting from the
resin-supported,
fully protected PNA1-Fmoc and PNA2 , which
were prepared according to Fmoc/Bhoc (fluorenylmethyloxycarbonyl/benzhydryloxycarbonyl)
manual solid phase synthesis as previously reported. 28 The first route to synthesize the Ir-PNA conjugate
involved the removal of the Fmoc group with a solution of piperidine,
followed by the coupling reaction between the free primary amine group
of PNA1 and the carboxylic group of the Ir-COOH complex in the presence of O -(7-aza-1 H -benzotriazole-1-yl)- N,N,N,N -tetramethyluronium
hexafluorophosphate (HATU) as a condensing agent and N -ethyldiisopropylamine (DIPEA) as base in N -methylpyrrolidone
(NMP) for 4 h at room temperature ( Scheme 3 a). Scheme 3 Synthesis of the Ir-PNA Conjugate through Two Complementary
Routes Next, the treatment of the resin with a solution
of TFA/ m -cresol provided the removal of the N -Bhoc
protecting groups from the nucleobases and the concomitant cleavage
from the resin of Ir-PNA , which was purified by reverse-phase
high-performance liquid chromatography (RP-HPLC). The Ir-PNA identity and purity were confirmed by high-resolution electrospray
ionization mass spectrometry (HR-ESI + MS) and RP-HPLC analysis,
respectively ( Figures S13 and S14 ). Alternatively, the resin-supported, fully protected PNA2 was treated with a solution of HATU and DIPEA in NMP for 8 min and
subsequently reacted with a solution of Ir-NH 2 in NMP for 4 h at room temperature ( Scheme 3 b). After the TFA-mediated
deprotection of N -Bhoc groups and release of the
conjugate from the resin, the RP-HPLC trace of the crude Ir-PNA was found to be very similar to that obtained following pathway
(a), demonstrating the same outcome for both complementary procedures
that can be used for the PNA conjugation of this class of bis-cyclometalated
Ir(III) complexes.
## Photophysical Characterization of Ir-COOH
and Ir-PNA
2.2 Photophysical Characterization of Ir-COOH
and Ir-PNA The photophysical data for the Ir-COOH complex and the corresponding Ir-PNA conjugate are
summarized in Table 1 and depicted in Figure 2 and Figures S15 and S16. Figure 2 UV–vis
absorption (solid lines) and photoluminescence spectra
(dashed lines) of the PNA (blue trace), the Ir-COOH (red
traces) and the Ir-PNA (black trace) in methanol at room
temperature in aerated conditions. The inset shows a magnification
of the MLCT bands centered at 377 and 415 nm. Table 1 UV-vis and Photoluminescence Data
at Room Temperature for the Compounds Ir-PNA (in Aerated
Methanol; λ ex = 405 nm), Ir-COOH (in
Aerated DCM, Acetonitrile, Methanol, and Water; λ ex = 420 nm) and Ir-NH 2 (in Aerated
DCM, Acetonitrile, and Water; λ ex = 400 nm) species solvent λ abs (nm) λ em (nm) Φ τ (ns) k r (s –1 ) b k nr (s –1 ) b Ir-PNA MeOH 377 582 0.039 13.5 (6%) 4.8 × 10 5 12.0 × 10 6 415 80.3
(94%) 470 Ir-COOH CH 2 Cl 2 381 571 0.084 128.3 6.5
× 10 5 7.1 × 10 6 415 472 CH 3 CN 374 585 0.027 56.9 4.7
× 10 5 1.7 × 10 7 415 470 MeOH 377 590 0.034 33.0 (10%) 4.9
× 10 5 1.4 × 10 7 415 69.2 (90%) 470 H 2 O 374 600 0.051 31.8 (3%) 4.5 × 10 5 8.4 × 10 6 412 113.4 (97%) 469 Ir- NH 2 a CH 2 Cl 2 383 574 0.052 153 3.4 × 10 5 6.2 ×
10 6 417 CH 3 CN 376 591 0.017 56 3.1 × 10 5 1.8 × 10 7 414 H 2 O 378 604 0.033 2 (2%) 2.8 × 10 5 8.3 × 10 6 417 116 (98%) a Literature data from ref ( 25 ). b k r and k nr indicate the radiative and nonradiative decay
constants of the excited states, respectively, and are computed on
the most significant lifetime component. UV–vis absorption spectra of the Ir-COOH , acquired
in different solvents, display intense absorption bands in the ultraviolet
region at about 220–300 nm, whose position is independent of
the solvent polarity, and attributable to spin-allowed π-π*
ligand-centered 1 LC transitions. According to the literature
data, 29 − 35 the bands lying in the range 220–250 nm receive larger contributions
from ppy-centered transitions, while those between 250 and 290 nm
are mainly centered on the phenanthroline ligand. In the visible region,
the Ir-COOH complex displays less intense absorption
bands in the range 350–420 nm which are mainly attributed to
spin-allowed 1 MLCT [dπ(Ir)-π*(phen)] transition,
due to the excitation from filled t 2g orbitals of the Ir(III)
center to a vacant π* orbital of the phenanthroline ligand,
acting as acceptor. However, it should be considered that in Ir(III)
cyclometalated compounds, as a consequence of the large covalency
of the Ir–C- bond, the HOMO is not completely localized on
the d orbitals of the metal center but can be significantly delocalized
over the cyclometalating ligands, with a large participation also
of the Ir–C- σ bond orbital. 30 , 31 , 36 Therefore, the absorption bands observed
in the visible region could be assigned to the σ bond-to-ligand
charge transfer ( 1 SBLCT) transitions, i.e. the ppy-to-phen
charge transfer ( 1 LLCT) transitions (see Figure S15 ). Finally, the absorption bands at the lowest energy
(at about 470 nm) are assigned to spin-forbidden CT transitions, which
steal the intensity from the spin-allowed transitions thanks to the
large spin–orbit coupling induced by the heavy iridium center.
The CT character of these absorption bands is confirmed by the observed
solvatochromic effect, affording a progressive blue shift on going
from the least polar (DCM) to the most polar (water) solvent used
in these measurements (see Table 1 and Figure S15 ). All of
these features were expected and showed to be in line with the many
orthometalated mononuclear Ir(III) complexes of the [Ir(N^C) 2 (N^N)] + family described in the literature. The
conjugation of the Ir complex to the PNA generates only small
modifications of the absorption spectrum in the ultraviolet range,
affording a new band at 210 nm due to the PNA backbone transitions
and an increase of the absorption at 260 nm due to the 1 LC transitions of the purine bases ( Figure 2 ). Moreover, as expected, the visible part
of these absorption spectra is not modified either by the presence
of the PNA oligomer in the Ir-PNA or by the free primary
amine instead of the carboxylic group in Ir-NH 2 (see Figure 2 and Table 1 ). 25 Indeed, the presence of the aliphatic
chain hampers any electronic conjugation between the PNA, or the functional
groups (COOH and NH 2 ), and the 1,10-phenanthroline ligand
involved in the electronic transitions. Upon excitation at 405
nm, the Ir-COOH and Ir-PNA display photoluminescence
emission bands in the yellow-orange range
of the visible spectrum, attributable to a metal-to-ligand charge
transfer transition having triplet character ( 3 MLCT) (see Figure S16 ). The broad and structureless shape
of these emission bands suggests the absence of the role of the π-π*
LC transitions in the emission process, further confirmed by the excitation
spectra ( Figure S17 ) and the monoexponential
decay profile of the lifetimes observed in DCM and acetonitrile (ACN)
solutions. The slight blue shift of the emission band observed in
the DCM solution for Ir-COOH and Ir-NH 2 , with respect to the analogous Ir complex containing
the unsubstituted 1,10-phenanthroline ligand (Ir-Phen, λ em = 582 nm in DCM), 33 reflects
the weak electron donor ability of the aliphatic substituent on the
phenanthroline ligand, thus confirming the charge transfer character
of the emission. 30 , 33 , 35 Moreover, both Ir-COOH and Ir-NH 2 display a red-shift of the emission maximum passing
from the least polar (DCM) to the most polar (water) solvent, 37 in agreement with the commonly observed trend
of excited states having charge transfer character (see Table 1 ). The red shift of the emission
maximum is usually associated with a progressive decrease of the photoluminescent
quantum yield (PLQY) and a shortening of lifetimes, in agreement with
the energy gap law. 38 Nevertheless, it
is interesting to note that in the case of Ir-COOH and Ir-NH 2 , the lifetimes and PLQYs do
not follow this trend. As for the Ir-NH 2 complex, 25 also Ir-COOH displays higher PLQY in polar solvents such as water or methanol
than in the less polar solvent acetonitrile, together with longer
and biexponential lifetimes (see Table 1 ). This behavior suggests that, in these polar solvents,
the Ir-COOH is also present as a nanosized aggregate,
as already observed for the previously reported Ir-NH 2 complex, where the complex may be affected
by a more rigid and less polar surrounding. To confirm this hypothesis,
we measured the photophysical properties of Ir-COOH dissolved
in ACN mixed with increasing quantities of H 2 O (see Figure S18 and Table S1 ). As the water content
increases, a progressive red-shift of the emission maximum is observed,
followed by an increase in fluorescence quantum yield and a corresponding
lengthening of lifetimes, which become progressively biexponential
( Figure S19 ). This behavior supports the
hypothesis that, in highly polar solvents, the emission is mainly
governed by nanoaggregates. The quantity of these nanoaggregates increases
with higher water content, although they do not account for the entire
amount of the compound present in the solution. It is to be noted
that the slight shortening of the main lifetime component observed
in neat water—corresponding to a 2-fold increase in the k nr constant (see Table S1 )—strongly suggests that water
acts as a classical quencher, capable of competing with nonradiative
deactivation processes, as previously reported for other dyes. 39 The progressive formation of nanoaggregates
was confirmed by DLS
measurements ( Figure S20 ). In neat acetonitrile
and H 2 O/ACN mixtures with ratios of 1:9, scattering signals
were either absent or insufficient to be detected. However, on increasing
the water content, an increase in scattered photons was observed and,
parallelly, the fitting of the correlation functions returned dimensional
distributions with larger populations. Also for the MeOH solution
of Ir-COOH , DLS measurements confirmed the presence of
a population centered at 300 nm, alongside a numerically prevailing
population attributable to the single molecules in solution ( Figure S21 ). It is important to note that
nanoaggregates larger than 50 nm are
undetectable by 1 H NMR spectroscopy. Accordingly, the 1 H NMR spectrum of the Ir-COOH complex in methanol- d 4 ( Figure S5 ) displayed
only sharp signals, which are characteristic of the nonaggregated
molecular fraction, providing only a partial view of the overall system. The formation of these nanoaggregates seems to be further favored
by the presence of the hydrophobic PNA chain in the Ir-PNA conjugate, thus accounting for an 8 nm blue shift of the emission
maximum in water and the further increase in PLQY together with the
main component of the lifetime. Indeed, the short component (τ1
= 33.0 and 13.5 ns for the Ir-COOH and the Ir-PNA , respectively), observed in the biexponential emission decay kinetics,
can be attributed to the free molecules (which are more affected by
the presence of oxygen and more exposed to the polar solvent), while
the longer component (τ2 = 69.2 and 80.3 ns for the Ir-COOH and the Ir-PNA , respectively), is attributed to the
nanoaggregates.
## Photochemical Stability of the Ir-COOH and
the Ir-PNA
2.3 Photochemical Stability of the Ir-COOH and
the Ir-PNA To evaluate the durability of the Ir-PNA conjugate under extended exposure to light in the presence of oxygen,
we conducted a photochemical stability test. This assessment was needed
as a critical requirement for the potential use of Ir-PNA as a photosensitizer. Hence, the compound was dissolved in methanol,
and the solution was saturated with O 2 , bubbling the gas
through the solution for 5 min. Subsequently, the solution was subjected
to visible light exposure, using a monochromatic LED light (λ
= 400 nm, 15.2 mW/cm 2 ) and UV–vis absorption spectra
were recorded every few minutes for a total period of irradiation
of 20 min ( Figure S22 ). Analysis of the
superimposed absorption spectra obtained at various time points demonstrated
the high photostability of the compound. The same stability test was
replicated for the Ir-COOH complex, yielding similar
outcomes ( Figure S23 ).
## Photoreaction of the Ir-COOH and the Ir-PNA
with 1,5-Dihydroxynaphtalene as an Indirect Reporter of Singlet Oxygen
Production
2.4 Photoreaction of the Ir-COOH and the Ir-PNA
with 1,5-Dihydroxynaphtalene as an Indirect Reporter of Singlet Oxygen
Production Previously, the ability of the Ir-NH 2 complex to serve as a PS inducing the generation
of singlet oxygen was evaluated. 25 This
assessment was based on the use of the singlet oxygen indirect reporter
1,5-dihydroxynaphthalene (DHN). 34 The same
photoreaction assay was conducted in this work, under irradiation
with monochromatic LED light (λ = 400 nm, 15.2 mW/cm 2 ) within a cuvette for both Ir-COOH and Ir-PNA complexes, to compare their performances as PS using the estimation
of their singlet oxygen quantum yields Φ Δ ,
which are reported in Table 2 . Table 2 Estimated Quantum Yields of Singlet
Oxygen Production (φ Δ ) and Kinetic Constant
( k obs ) of the Studied Compounds in Methanol species k obs (min –1 ) ν (10 –6 M min –1 ) I λ=400 (10 –7 mol min –1 ) Φ Δ Ir(ppy) 3 0.0401 6.82 7.00 0.50 a Ir-PNA 0.0158 2.60 2.48 0.54 Ir-COOH 0.0146 2.44 2.83 0.44 a From ref ( 34 ). A schematization of the selective and quantitative
photoreaction
of DHN with 1 O 2 is reported in Scheme 4 . Scheme 4 Depiction of the
Photochemical Conversion of DHN in Juglone The PS is excited to a singlet excited state
upon irradiation with
sufficiently energetic photons; then, it undergoes intersystem crossing
leading to the population of a triplet excited state, and finally,
through energy transfer, the PS promotes the excitation of molecular
oxygen from its triplet ground state to the singlet cytotoxic excited
state. The resulting photoproduced 1 O 2 , in the
presence of an excess of DHN, undergoes a quantitative reaction as
illustrated in Scheme 4 , ultimately forming the oxidized species Juglone. Figure 3 Evolution of UV–vis absorption spectra of DMC/methanol 9:1
solutions containing DHN (a) Ir-COOH (1.27 × 10 –5 M), (b) Ir-PNA (1.12 × 10 –5 M), (c) Ir(ppy) 3 (1.18 × 10 –5 M) irradiated for several minutes (λ = 400 nm). (d) Comparison
of the semilogarithmic plots of DHN consumption as a function of the
irradiation time registered for the investigated sensitizers, with A t and A 0 referring to the typical DHN absorbance value (at 316 nm),
registered at irradiation times t and 0, respectively. The comparison of Φ Δ determined
for the Ir-COOH complex (0.44) and the Ir-PNA conjugate
(0.54) shows that, in these conditions, 1 O 2 is
generated even more efficiently after the conjugation of the Ir complex
to the PNA chain. This suggests that the PS properties of the Ir complex
are neither compromised nor affected by its conjugation with the PNA
tetramer.
## Fluorescence Microscopy Localization Study
by Two-Photon Absorption
2.5 Fluorescence Microscopy Localization Study
by Two-Photon Absorption Cellular uptake and intracellular
localization in HeLa cells of both Ir-COOH and Ir-PNA compounds were monitored by two-photon-excitation (TPE)
microscopy. HeLa cells were treated with 25 μM of each compound
and incubated for 21 h. After this incubation period, the cells were
observed under the microscope. Notably, TPE at 720 nm was utilized
to excite the iridium compounds and acquire microscopy images ( Figure 4 ) as described in
the Experimental Part. The control exhibited a slight autofluorescence
( Figure 4 a,b) detectable
in the blue channel (480 ± 30 nm), predominantly attributed to
NADH (λ em = 460 nm), as documented in previous literature
for TPE excitation. 40 Both the Ir-PNA and the Ir-COOH compounds ( Figure 4 c–f) displayed luminescence in the
green and red channels (535 ± 50 and 600 ± 40 nm, respectively),
consistent with their broad emission spectra ( Figure 2 ). Images captured at the highest magnification
( Figure 4 d and 4 f) were acquired through z-scan techniques to confirm
that the signal originated from within the cell rather than the outer
membrane. Clearly, the emission resulting from the treatment with
the Ir-PNA conjugate originated from all visible cells
within the observed frame. In contrast, with the Ir-COOH complex, emission predominantly arose from a pool of treated HeLa
cells. This discrepancy implies that the internalization process may
be more facilitated for the PNA adduct compared to the neutral form
of the Ir-COOH complex, which is primarily present at
physiological pH (being deprotonated, the terminal COOH group balances
the residual positive charge of the Ir(III) metal center). Moreover,
apart from the positive charge retained by the Ir-PNA conjugate, this has a higher lipophilic character, which could also
enhance its internalization. Nevertheless, the Ir-PNA conjugate appeared to form aggregate-like spots, probably due to
its limited solubility in water, mainly accumulating in the cytosol
as well as in the nuclei, where a green/red emission is observable.
On the contrary, the Ir-COOH complex showed, where present,
a more homogeneous distribution at the cytoplasm level without any
emission coming from the nuclei. Further studies will be dedicated
in the near future to better characterize the molecular mechanisms
through which the Ir-PNA conjugate is internalized in
cells, and colocalization studies will also be carried out. Figure 4 TPE microscopy
images at two distinct magnifications (a, c, and
e = 100×, 300 × 300 μm 2 field of view;
b, d, and f = 50×, 150 × 150 μm 2 field
of view) of vehicle-treated HeLa cells (control, panels a and b),
HeLa cells treated with the Ir-PNA (panels c and d) and
HeLa cells treated with the Ir-COOH (panels e and f)
for 21 h. The z-scan images (panels d and f) are the superposition
of several images acquired along the optical axis at 1 μm intervals.
## PDT Treatment on HeLa Cells
2.6 PDT Treatment on HeLa Cells The effectiveness
of the Ir-COOH complex alone and its conjugate with PNA
in inducing photocytotoxicity and cytotoxicity in the dark were assessed
and compared employing HeLa cells. The cells were treated separately
with increasing doses of both compounds either in the dark or under
light exposure. HeLa cells were incubated for 24 h to promote internalization,
as suggested by the previous TPE microscopy observations (see paragraph
2.5). Potential dark cytotoxicity was assessed by using an MTT assay.
The results, presented in Figure 5 , indicate that the Ir-COOH complex induced
significant dark cytotoxicity, in a dose-dependent manner. On the
other hand, cells treated with the Ir-PNA conjugate maintained
high cell viability (>90%) in the dark across all the tested concentrations.
This characteristic is crucial for a photosensitizer to be considered
for further in vivo applications. Figure 5 Dose–response
plots for the Ir-PNA conjugate
(panel a) and the Ir-COOH complex (panel b) in the dark
(black curves) and under UV irradiation. One-way ANOVA corrected for
multiple comparison by Sidak’s post hoc analysis was performed,
*** p < 0.001; n = 4. This level of toxicity for the iridium complexes
was already observed
in previous studies conducted on HeLa cells, where the similar complex Ir-NH 2 displayed higher cytotoxicity
in the dark compared to the corresponding conjugate with an amide-based
polymer. 25 Concurrently, the same experiment was
performed by irradiating
the samples for 30 min, using a low-intense UV- lamp emitted by a
polychromatic irradiation source (5.2 mW/cm 2 in the 200–800
nm range, see emission profile in Figure S24 ). To avoid any overheating due to the lamp exposure, the temperature
during the irradiation time was constantly monitored and maintained
at 37 °C. The dose–response curves for the Ir-COOH and the Ir-PNA compounds (shown in Figure 5 , panels (a) and (b), respectively)
were fitted nonlinearly using a sigmoidal function to determine dark
and light EC 50 values. These values represent the effective
concentration required to reduce cell viability to 50% in the dark
and after irradiation ( Table 3 ). Both compounds showed higher cytotoxicity upon UV light
exposure. However, the light EC 50 was 1 order of magnitude
higher for the Ir-PNA conjugate compared to the Ir-COOH complex, indicating greater phototoxicity when the
Ir complex was not bound to the PNA. Considering the insights gained
from TPE microscopy images, the lower photocytotoxicity of the Ir-PNA conjugate could be attributed to its tendency to form
aggregates, confined in vesicle-like structures (see Figure 4 ). These aggregates might potentially
trap singlet oxygen produced during irradiation and hinder it from
reaching target biomolecules effectively. On the other hand, the amplified
cytotoxicity showed by Ir-COOH could be possibly due
to the additional contributions of the intrinsic toxicity in the dark. Table 3 Cytotoxicity of the Ir-PNA and Ir-COOH Compounds on Hela Cells in the Dark and
upon Irradiation, Expressed in Terms of Dark EC 50 and Light
EC 50 species dark EC 50 light
EC 50 PI a Ir-PNA >50 μM 18 μM >2.8 Ir-COOH >50 μM 3.5 μM >14.3 a PI: phototherapeutic index = dark-EC 50 /light-EC 50 . Additionally, the lower limit phototherapeutic
indexes (PI) were
calculated from the dark to light EC 50 values (see Table 3 ), reflecting the
amplification of cytotoxic activity by light treatment. Our complexes
exhibit low phototherapeutic indexes if compared to other orthometalated
Ir complexes of the same [Ir(N^C) 2 (N^N)] + family, 41 , 7 which show PI even 1 order of magnitude higher than ours. This could
be due to many different factors, such as cellular uptake as well
as subcellular localization within a particularly sensitive subcellular
organelle. Nevertheless, making a fully reliable comparison is challenging,
as certain variables should be standardized, such as the administered
light dose, the incubation time both before and after irradiation,
and, of course, provided that the comparison of different molecular
effects is conducted within the same cell line. Our data are preliminary,
and a complete biological study has not been carried out yet.
## Conclusions
3 Conclusions To the best of our knowledge,
this study marks the first instance
of a conjugate between an iridium complex and a PNA tetramer, and
although the literature contains notable examples of studies on PNA-metal
complex conjugates, none have exploited these conjugates for PDT applications.
The conjugation did not compromise the emissive properties of the
iridium complex or its ability to produce singlet oxygen. On the contrary,
the iridium complex bound to the PNA, in a cuvette under a nonpolar
environment (DCM/MeOH 9:1), showed a higher singlet oxygen yield Φ Δ (0.54) compared to the one of Ir-COOH (0.44).
Furthermore, the singlet oxygen generated under irradiation did not
appear to damage the PNA tetramer, as confirmed by the UV–vis
spectra of irradiated Ir-PNA , which remained unchanged. More importantly, biological tests on HeLa cells demonstrated that
the Ir-COOH complex enters cells more efficiently when
conjugated to PNA, as all of the cells in the TPE microscope images
exhibited emission in the green/red channels. In contrast, the free Ir-COOH complex was not internalized in a significant portion
of the cells, which displayed only autofluorescence in the blue channel.
This reduced cellular uptake may be due to a combination of lower
lipophilicity and a neutral net charge of the Ir-COOH complex, which at pH > 2–3 exists as a zwitterion (with
the
deprotonated carboxyl group balancing the residual +1 net charge of
the Ir(III) bis-orthometalated ion). In comparison, the Ir-PNA conjugate shows enhanced lipophilicity due to the presence of the
tetramer and retains the residual positive net charge of the metal
ion, which is not counterbalanced in the adduct. Nonetheless, starting
from 25 μM dose on, the Ir-COOH complex exhibited
increased cytotoxicity in the dark compared to the Ir-PNA , which, on the contrary, did not cause cellular death under the
same conditions. This level of toxicity for the iridium complexes
was already observed in previous studies conducted on HeLa cells.
When irradiated, both Ir-based compounds showed an increase in cytotoxicity,
more pronounced for Ir-COOH, possibly due to the additional
contributions of the intrinsic toxicity in the dark and its light-triggered
cytotoxicity. Conversely, the Ir-PNA remained nontoxic
in the dark and exhibited cytotoxicity only upon irradiation. Overall, the Ir complex appears to transfer its beneficial properties
to the conjugate by helping PNA to enter cells, while the PNA seems
to mitigate the negative characteristics of the complex in terms of
dark cytotoxicity. Based on these results, our next step will
be to select an appropriate
PNA sequence capable of acting as an antisense agent, and after conjugating
it with the Ir-COOH complex, we will test the corresponding
Ir-PNA conjugate on different cell lines to study the potential synergistic
effect in inducing the death of diseased cells of the conjugate with
respect to the two separate components.
## Experimental Section
4 Experimental Section 4.1 Synthesis of the Phen-COOH Ligand and the
Ir(III) Complexes Ir-NH 2 and Ir-COOH Commercial
reagents were used without further purification. 4-(4′-Aminobutyl)-1,10-Phenanthroline
( Phen-NH 2 ) was synthesized as
previously described. 26 [Ir(ppy) 2 Cl] 2 was prepared using a published method with IrCl 3 ·3H 2 O as a precursor (BASF). 42 All
the manipulations for the syntheses of both Phen-NH 2 , [Ir(ppy) 2 Cl] 2 and Ir-NH 2 were performed under nitrogen using oven-dried
Schlenk-type glassware. THF was distilled from sodium/benzophenone
just before its use. NMR experiments were acquired on a Bruker DRX400
spectrometer equipped with a Bruker 5 mm BBI Z-gradient probe head
with a maximum gradient strength of 53.5 G/cm (π/2 pulse: 1 H 8.5 μs, 13 C 13 μs) operating at 400.13
and 100.62 MHz for 1 H and 13 C NMR, respectively.
The ESI + MS analyses were recorded with a Thermo Fisher
LCQ Fleet ion trap mass spectrometer. Elemental C, H, and N analyses
were performed on a PerkinElmer CHN 2400 instrument. 4.2 Solid Phase Synthesis of the Ir-PNA Conjugate The resin-supported PNA1-Fmoc and PNA2 were prepared by solid phase Fmoc/Bhoc synthesis, as previously
described. 28 The protected PNA monomers
(Fmoc-PNA-T–OH; Fmoc-PNA-C(Bhoc)–OH; Fmoc-PNA-G(Bhoc)–OH;
and Fmoc-PNA-A(Bhoc)–OH) were purchased from ASM Research Chemicals
GmbH (Hannover, Germany). The H-rink amide ChemMatrix was purchased from Merck. Polypropylene one-way syringes (1.5 or
4 mL) and corresponding PTFE frits, used as reaction vessels, were
purchased from SepaChrom (Milan, Italy). Solid phase syntheses were
performed by using an orbital rotator shaker at 500 rpm. Centrifugation
steps were performed using a 0.6 LISA centrifuge. The RP-HPLC purification
of the Ir-PNA conjugate was performed on an Agilent 1200
Series system, using the semipreparative column Luna C18 (250 ×
10 mm, 5 μm) at a flow rate of 3 mL/min. Solvent A (0.1% TFA
in water) and solvent B (0.1% TFA in acetonitrile) were used in the
following gradient: 0% B (5 min), B 0–50% (30 min), B 50–100%
(1 min), B 100% (5 min), B 100–0% (1 min), and B 0% (3 min).
This method was used with detection by UV (220, 260, 280, 360, and
366 nm). The RP-HPLC analyses of the purified Ir-PNA were
performed using the analytical column Luna C18 (150 × 4.6 mm,
5 μm) at a flow rate of 1 mL/min, using the same gradient reported
for the semipreparative column. High-resolution electrospray ionization
mass spectrometry (HR-ESI + MS) analyses were acquired in
a positive polarity with a Synapt G2-Si QToF instrument (Waters) interfaced
through a ZsprayTM ESI-probe for electrospray ionization (Waters).
Data were processed with a MassLynxTM v4.2 software (Waters). The
concentrations of Ir-PNA solutions were determined by
measuring the absorbance at 260 nm with an Agilent 8453 UV/vis spectrophotometer.
The molar extinction coefficient of the PNA tetramer was calculated
according to standard protocol. 43 4.3 Photophysical Characterization UV–vis
absorption spectra were acquired on a single beam Agilent model 8543
spectrophotometer equipped with a diode array detector using a 1 cm
path length quartz cuvette at room temperature. Molar absorption coefficients
of MLCT transitions were measured by weighting a known amount of Ir
complex and quantitatively dissolving it in a known MeOH volume. The
stock solution was then used to prepare diluted solutions at different
molar concentrations (in the range 6 × 10 –5 –1.7 × 10 –4 M), which were employed
to acquire UV–vis absorption spectra, from which the absorbance
values at 377 and 415 nm were collected. The slopes of the straight
lines obtained by plotting the absorbances vs molarity returned the
ε (M –1 cm –1 ). The estimated
experimental errors are 2 nm on the absorption maxima and 5% on the
molar absorption coefficients. Emission spectra were acquired by using
an Edinburgh FLS980 spectrofluorimeter equipped with a 450 W xenon
arc lamp. Emission spectra were corrected for source intensity (lamp
and grating) and emission spectral response (detector and grating)
by standard correction curves. The estimated experimental errors are
2 nm on the PL bands maxima. Time-resolved measurements were performed
using the time-correlated single-photon counting (TCSPC) option on
the FLS980. The pulsed excitation source, an LED at 404 nm, was mounted
directly on the sample chamber, and the emission was collected by
a multichannel plate MCP-PMT Hamamatsu H10720 –01 single photon-counting
detector. The photons collected at the detector were correlated by
a time-to-amplitude converter (TAC) to the excitation pulse. The data
analysis was performed using the commercially available F980 software
(Edinburgh Instruments). The goodness of the data fitting was assessed
by minimizing the reduced chi-squared function (χ 2 ). The uncertainty on fluorescence lifetimes is estimated to be ±0.05
ns. Photoluminescence quantum yields (Φ) were collected
for an optically diluted solution (<10 –5 M) using
wavelength scanning with a Hamamatsu C11347 –11 Quantaurus-QY
absolute PL quantum yield spectrometer, equipped with a xenon light
source (150 W), a monochromator, and a Spectralon integrating sphere,
and employing the commercially available U6039–05 PLQY measurement
software (Hamamatsu Photonics Ltd., Shizuoka, Japan). The photoluminescence
quantum yields were measured by exciting the samples between 350 and
430 nm. 4.4 Two-Photon Excitation Microscopy Localization
Study Two-photon excitation (TPE) fluorescence microscopy
experiments were conducted using a 720 nm excitation beam generated
by a mode-locked Ti:sapphire laser (Mai Tai HP, Spectra Physics).
The laser emits pulses with a duration of 120 fs full width at half-maximum
and a repetition frequency of 80 MHz, delivering 20 mW of power at
the sample plane. The optical setup comprises a confocal scanning
head (M610 scanning mirrors module, ISS) integrated onto an upright
optical microscope (BX51, Olympus), equipped with a high working distance
objective (XLPlan, NA = 1.05, wd = 2 mm, 25x water immersion, Olympus).
Fluorescence signals, collected in epifluorescence geometry by the
same objective, were directed to a nondescanned detection unit and
then transmitted to three Hamamatsu analog output photomultipliers
(HC125–02, Hamamatsu). Sample emissions were filtered using
480/30, 535/50, and 600/40 band-pass filters to eliminate scattering
contributions. Fluorescence data were processed by using the ISS Vista
Vision Suite software (ISS). The microscopy images presented in this
paper are the result of 4 averaged scans, with a residence time of
10 μs per pixel. The field of view for the 512 × 512 pixel
images ranged from 300 to 50 μm 2 , depending on the
zoom factor employed. Z-scan images were acquired at 1 μm intervals. 4.5 Photochemical Stability Study The
photochemical stability test and photoreaction with DHN in the presence
of investigated systems were monitored by employing a Jasco V-650
spectrophotometer. The experiments were performed by directly irradiating
a 3 mL quartz cuvette with a monochromatic LED light with a maximum
emission centered at 400 nm (BRIDGELUX, USA). The average full emission
intensity reaching the cuvette corresponded to ca. 15.2 mW/cm 2 , as regularly checked with a Thorlabs PM200 optical power
meter equipped with a thermal Thorlabs S302C power sensor. The normalized
emission spectrum of the LED is reported in Figure S25 . 4.6 Biological Study For the MTT tests,
formazan formation was measured spectrophotometrically at 570 nm using
the microplate reader (PerkinElmer, EnSpire, Waltham, MA, USA). The
phototoxicity tests on HeLa cells were carried out by using a low-intensity
and mainly UV-light-emitting polychromatic irradiation source, with
an average intensity emission of ca. 5.2 mW/cm 2 in the
200–800 nm range. The normalized emission spectrum of the employed
irradiation source is reported in Figure S24 . 4.7 Synthesis of the [Ir(ppy) 2 (Phen-NH 2 )]Cl Complex (Ir-NH 2 ) [Ir(ppy) 2 (Phen-NH 2 )]Cl was prepared by slightly varying a literature
procedure. 25 [Ir(ppy) 2 Cl] 2 (88.6 mg, 0.0827 mmol) was suspended in 20 mL of freshly
distilled THF, obtaining a yellow suspension in which 40.8 mg of Phen-NH 2 (0.163 mmol) was added. After
the addition of 10 mL of H 2 O Milli-Q, the brown solid aggregates
of Phen-NH 2 were completely dissolved.
The mixture was then heated at 70 °C under an inert atmosphere,
and after a few minutes, the cloudy yellow suspension turned completely
clear. Once the set temperature was reached, it turned emerald-green,
denoting a slight defect of ligand. After heating the mixture for
5 h, a nominal slight excess of Phen-NH 2 (3.2 mg, 0.0127 mmol) was added, turning the color from emerald
green to yellow-greenish. The reaction mixture was left under stirring
at 70 °C for another 2 h and then at room temperature overnight.
The clear yellow-greenish solution, which under UV lamp irradiation
showed a yellow-orange emission, was evaporated by vacuum to dryness.
The obtained fine solid was dissolved in a few mL of dry THF and treated
with an excess of diethyl ether (Et 2 O, 2×), obtaining
a fine yellow precipitate. Yield 94% (124 mg). 1 H NMR (D 2 O, 300 K, 9.4 T): δ phen-NH 2 ligand 8.62
(1H, CH(9)), 8.35 (1H, CH(5)), 8.32 (1H, CH(7)), 8.20 (1H, CH(2)),
8.18 (1H, CH(6)), 7.72 (1H, CH(8)), 7.60 (1H, CH(3)), 3.30 (2H, CH(δ)),
2.93 (2H, CH 2 (α)), 1.83 (2H, CH 2 (γ)),
1.73 (2H, CH 2 (β)); δ phenylpyridine ligands
8.1 (2H, CH(6′)), 7.86 (2H, CH(3″)), 7.74 (2H, CH(5′)),
7.43 (2H, CH(3′)), 7.1 (2H, CH(4″)), 6.96 (2H, CH(5″)),
6.81 (2H, CH(4′)), 6.47 (2H, CH(6″)). The complete NMR
characterization of this complex can be found in ref [ 25 ]. UV–vis (MeOH)
MLCT absorptions: λ abs = 377 nm (ε = 5102 M –1 cm –1 ) and 415 nm (ε = 2517
M –1 cm –1 ). 4.8 Synthesis of the Phen-COOH Ligand Succinic anhydride (19.9 mg, 0.199 mmol) was dissolved in freshly
distilled CH 2 Cl 2 (1 mL) and treated with TEA
(56 μL, 2.1 equiv). Phen-NH 2 (50.0 mg, 0.199 mmol) was added to the solution. The mixture was
refluxed under stirring for 6 h and then at room temperature overnight.
The next day, the mixture presented a small amount of a light brown
sticky solid at the bottom of the flask and a light yellow clear supernatant
solution. Then, the supernatant was isolated and the sticky solid
was washed with a few mL of CH 2 Cl 2 (2×)
warming at 40 °C. The crude product, present in the combined
supernatants, was characterized by NMR in CH 2 Cl 2 /CDCl 3 . 1 H NMR (CH 2 Cl 2 /CDCl 3 , 300 K, 9.4 T): δ 8.88
(1H, C H (9) dd, J = 4.5 Hz, 1.9 Hz), 8.7 (1H, C H (2) d, J = 4.7 Hz), 8.0 (1H, C H (7) dd,
J = 8.2 Hz, 1.9 Hz), 7.8 (1H, C H (5) d J = 9.2 Hz),
7.6 (1H, C H (6)d J = 9.2 Hz), 7.3 (1H, C H (8) dd J = 8.2 Hz, 4.5 Hz), 7.2 (1H, C H (3) d J =
6.6 Hz), 7.45 (1H, CON H (5′)), 3.0 (2H, C H 2 (4′) m), 2.9 (2H, C H 2 (1′) t J = 19.2 Hz), 2.2 (4H, C H 2 (7′ and 8′) m), 1.5 (2H, C H 2 (2′) m), 1.4 (2H, C H 2 (3′) m). 13 C NMR (CH 2 Cl 2 /CDCl 3 , 300 K, 9.4 T): δ 178.4
( C OO(9′)), 173.5 ( C ONH(6′)),
149.8 ( C H(9)), 149.5 ( C H(2)), 148.4
( C q (1a)), 146.4 ( C q (10a)), 145.9 ( C q (4a)), 135.6
( C H(7)), 128.2 ( C q (6a)),
127.5 ( C q (4)), 125.9 ( C H(6)), 122.8 ( C H(3)), 122.6 ( C H(8)),
122.2 ( C H(5)), 38.4 ( C H 2 (4′)), 32.9 ( C H 2 (7′ and
8′)), 31.8 ( C H 2 (1′)), 29.4
( C H 2 (3′)), 27.5 ( C H 2 (2′)). Et 2 O was added to the combined
supernatants, which caused the precipitation of a whitish solid. The
mixture was evaporated to dryness, the residue was washed with CH 2 Cl 2 /Et 2 O, and the resulting whitish
crude precipitate was isolated and vacuum-dried. Yield 49.6%. Elemental
Analysis: Found = C, 62.52%; H, 6.62%; N, 10.74% (calcd for (C 20 H 21 N 3 O 3 )(C 4 O 4 H 6 ) 0.45 (C 6 H 15 NCl) 0.6 : C, 62.70%; H, 6.77%; N, 10.36%). 4.9 Synthesis of the Ir-COOH Complex To a suspension of Phen-COOH (37 mg, 0.105 mmol) in
freshly distilled THF (20 mL) in a Schlenk tube, the precursor [Ir(ppy) 2 Cl] 2 was added (48.1 mg, 0.045 mmol, 0.43 equiv)
under stirring at room temperature. The mixture was heated at 70 °C
under magnetic stirring and an inert atmosphere. Neither the Ir precursor
nor the functionalized phenanthroline was soluble, even when the mixture
was heated at reflux. Hence, after 30 min at 70 °C, the mixture
was cooled down at room temperature and added with 6 mL of H 2 O Milli-Q. Immediately the heterogeneous mixture became more homogeneous,
and after a few minutes at 70 °C the mixture turned clear and
yellow. The mixture was left under stirring at 70 °C for ca.
5 h and then was left overnight at room temperature. The obtained
clear yellow mixture, which under UV lamp irradiation showed a yellow-orange
emission, was evaporated by vacuum to dryness, and the residue was
redissolved in a few mL of dry THF and treated with an excess of Et 2 O (2x) to give a fine yellow precipitate. Yield 96.3% (80.2
mg, of which 3.1 mg are triethylammonium succinate). 1 H NMR (MeOD, 300 K, 9.4 T): δ phen-COOH
ligand 8.76 (1H, C H (9) dd, J = 8.2 Hz, 1.9 Hz), 8.52
(1H, C H (5) d, J = 9.2 Hz), 8.37 (1H, C H (7) dd, J = 5.3 Hz, 1.9 Hz), 8.32 (1H, C H (6) d,
J = 9.2 Hz), 8.24 (1H, C H (2) d, J = 5.6 Hz), 7.90
(1H, C H (8) dd J = 8.2 Hz, 5.3 Hz), 7.79 (1H, C H (3) d, J = 5.6 Hz), 3.36 (2H, C H 2 (1″) t, J = 8.9 Hz), 3.29 (2H, C H 2 (4″) t, J = 6.8 Hz), 2.54 (2H,
C H 2 (8″) t, J =
7.0 Hz), 2.43 (2H, C H 2 (7″) t, J = 7.0 Hz), 1.86 (2H, C H 2 (3″) m), 1.70 (2H, C H 2 (2″) m); δ phenylpyridine ligands
8.13 (1H, C H (2′) ps d, J =
8.2 Hz), 7.87 (1H, C H (9′) ps d J = 7.9 Hz), 7.81 (1H, C H (3′) ps t, J = 7.7 Hz), 7.45 (1H, C H (5′) ps d, J = 5.9 Hz), 7.10 (1H, C H (8′) ps t, J = 7.5 Hz), 6.95 (1H, C H (7′) ps t, J = 7.7 Hz), 6.91 (1H, C H (4′) ps t, J = 6.4 Hz), 6.41 (1H, C H (6′)
dd, J = 7.4, 2.8 Hz). 13 C NMR (MeOD, 300 K, 9.4 T): δ phen-COOH ligand 176.2 ( C OO(9″)), 173.8 ( C ONH(6″)), 153.1 (C q (4)), 150.9 (C q (6a)), 150.6 (CH(7)), 149.9 ( C H(2)), 147.2 ( C q (10a)), 138.2
( C H(9)), 131.7 (( C q (1a)),
130.6 ( C q (4a)), 127.9 ( C H(6)), 126.4 ( C H(3)), 124.7 ( C H(8)),
124.6 ( C H(5)), 38.3 ( C H 2 (4″)), 31.4 ( C H 2 (1″)),
31.3 ( C H 2 (7″)), 30.5 ( C H 2 (8″)), 29.0 ( C H 2 (2″)),
27.2 ( C H 2 (3″)). δ phenylpyridine
ligands 168.1 (C q (12′)), 150.2 (C q (11′)),
148.6 ( C H(5′)), 144.3 ( C q (10′)), 138.3 ( C H(3′)), 131.7
( C H(6′)), 130.2 ( C H(7′)),
124.6 ( C H(9′)), 123.0 ( C H(4′)),
122.4 ( C H(8′)), 119.6 ( C H(2′)).
ESI + MS, m / z : 890.3 [M+K–H] + , 874.5 [M+Na–H] + , 852.7 [M] + , 752.6 [M–C(O)CH 2 CH 2 COOH] + . UV–vis spectroscopy of Ir-COOH in MeOH: ε
(λ abs = 377 nm) = 5.1 × 10 3 M –1 cm –1 . ε (λ abs = 415 nm) = 2.5 × 10 3 M –1 cm –1 . 4.10 Synthesis of the Ir-PNA Conjugate through
Coupling Reaction between PNA1-Fmoc and Ir-COOH The PNA1-Fmoc resin (40 mg, 0.3 mmol/g, 12 μmol) was swollen
in CH 2 Cl 2 for 45 min, then was treated with
a 20% v/v solution of piperidine in NMP (1 mL, twice
for 8 min). A solution of HATU (14 mg, 36 μmol, 3 equiv) in
NMP was added to a solution of the Ir-COOH (36 mg, 36
μmol, 3 equiv) and DIPEA (21 μL, 120 μmol, 10 equiv)
in NMP, and the resulting solution was shaken for 2 min. Then, this
brown solution was transferred to the resin that was shaken for 4
h at room temperature. The resin was washed with NMP, and the simultaneous
deprotection of nucleobases and the cleavage were performed by treating
the resin with a mixture of TFA/ m -cresol (9/1, v/v , 1 mL) for 1.5 h. The collected filtrate was concentrated
by bubbling N 2 onto the solution, and the residue was poured
onto cold Et 2 O to precipitate the crude Ir-PNA as a yellow solid. The solid was centrifuged, washed with Et 2 O, dried under a vacuum, and purified by RP-HPLC. HR-ESI + MS, m / z : found (calculated)
for C 85 H 91 N 29 O 14 Ir: 1932.6938
(1932.6906) [M] + , 1000.8215 (1000.8231) [M+3Na-2H] 2+ , 997.8154 (997.8191) [M+Na+K–H] 2+ , 989.8307
(989.8322) [M+2Na–H] 2+ , 978.8405 (978.8412) [M +
Na] 2+ , 966.8480 (966.8487) [M + H] 2+ , 665.5449
(665.5485) [M+Na+K] 3+ , 660.2238 (660.2239) [M+2Na] 3+ , 658.2186 (658.2212) [M+K+H] 3+ , 652.8962 (652.8966)
[M+Na+H] 3+ , 644.9016 (644.9015) [M+2H] 3+ . Analytical
RP-HPLC: t R = 29.6 min. 4.11 Synthesis of the Ir-PNA Conjugate through
Coupling Reaction between PNA2 and Ir-NH 2 After
the swelling in CH 2 Cl 2 for 45 min, PNA2 resin (40 mg, 0.3 mmol/g, 12 μmol) was shaken with a solution
of HATU (14 mg, 36 μmol, 3 equiv) and DIPEA (21 μL, 120
μmol, 10 equiv) in NMP for 8 min. Then, the brown solution of
the Ir-NH 2 (28 mg, 36 μmol,
3 equiv) was transferred to the resin containing the solution of HATU
and DIPEA and shaken for 4 h at room temperature. The resin was washed
with NMP, and after the cleavage, the crude Ir-PNA was
obtained as a yellow solid, and its RP-HPLC analysis showed a very
similar trend observed for the conjugation with the Ir-COOH . Analytical RP-HPLC: t R = 29.6 min. 4.12 Stability Test on Ir-NH 2 under
Cleavage Conditions A tiny amount of the Ir-NH 2 complex was dissolved in 3 mL of a TFA/ m -cresol 9:1 mixture, and the solution was monitored by
UV–vis spectroscopy, recording subsequent spectra over a period
of 1.5 h, mimicking the contact time and the conditions used during
the cleavage of the PNA from the resin. UV–vis spectra are
reported in Figure S12 . 4.13 Photochemical Stability of Ir-COOH and Ir-PNA The Ir-COOH complex and the Ir-PNA conjugate,
both dissolved in methanol, were diluted to 2.5 mL by using a CH 2 Cl 2 –methanol mixture (9:1) in a quartz cuvette.
In this way, the same concentration used during the photoreaction
of the two photosensitizers with DHN was achieved. The solutions were
irradiated with a monochromatic LED light (400 nm, 15.2 mW/cm 2 ) for increasing times ranging from 1 to 25 min. Potential
variations were monitored by UV–vis spectroscopy ( Figures S22 and S23 ). 4.14 Photoreaction of Ir-COOH and Ir-PNA with
DHN In a quartz cuvette, 15 μL of a 1.68 × 10 –3 M methanol solution of the Ir-PNA adduct
was diluted to 2.25 mL with a CH 2 Cl 2 -methanol
9:1 mixture. 250 μL of a 1.44 × 10 –3 M
solution of DHN was added in the same solvent mixture, to obtain a
1:10 PS-DHN molar ratio. Subsequently, the solution was saturated
with O 2 by bubbling it for 5 min and exposed to monochromatic
LED light (400 nm, 15.2 mW/cm 2 ) for durations ranging from
30 s to 25 min. Thus, the variations in DHN and Juglone absorption
bands were monitored using UV–vis spectroscopy ( Figure 3 of the main text); in particular,
the decreasing absorbance of the DHN band at 316 nm was used for the
determination of the initial rate constant ( k obs ) of the oxidation of DHN to Juglone. The same procedure
was carried out for the Ir-COOH complex and for the Ir(ppy) 3 , whose 1 O 2 quantum yield (Φ Δ ) in CH 2 Cl 2 -methanol 9:1, obtained from the literature, 34 was used as a standard to calculate the Φ Δ values for the Ir-PNA and the Ir-COOH . While the equation rate of the overall reaction of juglone production
can be described by eq 1 , by applying the steady-state approximation to the reactive 1 O 2 species, the DHN consumption reaction in the
initial stages ( v ) follows pseudo-first-order kinetics,
as described by eq 2 . 1 2 The 1 O 2 production ability of the investigated species can thus be
indirectly monitored by evaluating the corresponding k obs rate constant, which can be estimated by plotting,
as a function of the irradiation time, the characteristic DHN absorbance
value ( A ), registered at 316 nm, according to the
following integrated eq ( eq 3 ): 3 The fitted absorbance
values (with A t and A 0 referring to the A values measured
at times t and 0, respectively) were thus obtained
from regularly acquired UV–vis spectra of the reaction mixture
over a fixed irradiation time, as depicted in Figure 3 . Panel (d) of Figure 3 shows a comparison of the linear decrease
of ln(A t /A 0 ) vs time acquired for the investigated
systems. The quantum yield for singlet oxygen 1 O 2 formation (Φ Δ ) can then be determined by
using eq 4 : 4 where v i is the initial rate, I indicates
the photons absorbed by the sensitizer, and the apex std labels the
values for the adopted standard, which in our case corresponds to
the Ir(ppy) 3 complex, whose generation of 1 O 2 in DCM/methanol 9:1 is known and fixed to Φ Δ = 0.50. 34 The values of ν i were obtained as the product of k obs and [DHN] 0 (with [DHN] 0 in the 1.65–1.70
× 10 –4 M range), while the value of I was calculated as I = I source (400 nm) × (1–10 A (400 nm) ) where I source (400
nm) is the intensity of the 400 nm LED emitted light incident on the
sample, while A (400 nm) is the corresponding absorbance
of the sensitizer at such wavelength. 4.15 Cell Culture and Treatment HeLa
cells were cultured in DMEM culture medium (EuroClone, ECB7501) supplemented
with 1 mM l -glutamine (EuroClone, ECB3004D), penicillin-streptomycin
(SERVA 31749.04, 35500.01), and 10% supplemented with 10% fetal bovine
serum (GIBCO, A4766801) at 37 °C in 5% CO 2. For fluorescence
microscopy analysis, HeLa cells were seeded at 200,000 cells/well
in 6-well plates (Euroclone, ET3006) and treated with 25 μM Ir-COOH or Ir-PNA , and methanol as vehicle control
for 24 h. For cell viability assay, HeLa cells were seeded at 25,000
cells/well in 48-well plates (Euroclone, ET3048) and treated for 24
h with different doses of the Ir-COOH and the Ir-PNA (1–5–10–25–50 μM). Untreated cells
and cells treated with methanol (vehicle) were used as negative controls. 4.16 Viability Tests in the Dark and in Light MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)
(M5655, Sigma-Aldrich) assay was performed to determine the cell viability
and the potential cytotoxicity of the Ir-COOH and the Ir-PNA both in the dark and under irradiation using a nail
UV lamp. After 24 h of treatment, cells were exposed to light for
30 min, and after 6 h of incubation at 37 °C, the medium was
replaced with 300 μL of MTT solution. Following incubation for
30 min at 37 °C, MTT was removed and 500 μL of 2-propanol
was added to each well to dissolve formazan crystals. The same experiments
were performed in the dark as the control. Formazan formation was
measured spectrophotometrically at 570 nm using the microplate reader
(PerkinElmer, EnSpire, Waltham, MA, USA). Light EC 50 values
were obtained by analyzing the fitting curves by OriginPro2021 (Copyright
© 1991–2021 OriginLab Corporation) software using the
vertical cursor instrument for reading the data at 50% of cell viability. Statistical analysis: Curve-fitting was performed with OriginPro2021
software, and statistical data analysis was performed using GraphPad
Prism 9 (GraphPad Software Inc., San Diego, CA). Data were normalized
to untreated control. Results were plotted using nonlinear regression
with three parameters. One-way ANOVA corrected for multiple comparisons
by Sidak′s post hoc analysis was performed, *** p < 0.001; n = 4.
## Synthesis of the Phen-COOH Ligand and the
Ir(III) Complexes Ir-NH
4.1 Synthesis of the Phen-COOH Ligand and the
Ir(III) Complexes Ir-NH 2 and Ir-COOH Commercial
reagents were used without further purification. 4-(4′-Aminobutyl)-1,10-Phenanthroline
( Phen-NH 2 ) was synthesized as
previously described. 26 [Ir(ppy) 2 Cl] 2 was prepared using a published method with IrCl 3 ·3H 2 O as a precursor (BASF). 42 All
the manipulations for the syntheses of both Phen-NH 2 , [Ir(ppy) 2 Cl] 2 and Ir-NH 2 were performed under nitrogen using oven-dried
Schlenk-type glassware. THF was distilled from sodium/benzophenone
just before its use. NMR experiments were acquired on a Bruker DRX400
spectrometer equipped with a Bruker 5 mm BBI Z-gradient probe head
with a maximum gradient strength of 53.5 G/cm (π/2 pulse: 1 H 8.5 μs, 13 C 13 μs) operating at 400.13
and 100.62 MHz for 1 H and 13 C NMR, respectively.
The ESI + MS analyses were recorded with a Thermo Fisher
LCQ Fleet ion trap mass spectrometer. Elemental C, H, and N analyses
were performed on a PerkinElmer CHN 2400 instrument.
## Solid Phase Synthesis of the Ir-PNA Conjugate
4.2 Solid Phase Synthesis of the Ir-PNA Conjugate The resin-supported PNA1-Fmoc and PNA2 were prepared by solid phase Fmoc/Bhoc synthesis, as previously
described. 28 The protected PNA monomers
(Fmoc-PNA-T–OH; Fmoc-PNA-C(Bhoc)–OH; Fmoc-PNA-G(Bhoc)–OH;
and Fmoc-PNA-A(Bhoc)–OH) were purchased from ASM Research Chemicals
GmbH (Hannover, Germany). The H-rink amide ChemMatrix was purchased from Merck. Polypropylene one-way syringes (1.5 or
4 mL) and corresponding PTFE frits, used as reaction vessels, were
purchased from SepaChrom (Milan, Italy). Solid phase syntheses were
performed by using an orbital rotator shaker at 500 rpm. Centrifugation
steps were performed using a 0.6 LISA centrifuge. The RP-HPLC purification
of the Ir-PNA conjugate was performed on an Agilent 1200
Series system, using the semipreparative column Luna C18 (250 ×
10 mm, 5 μm) at a flow rate of 3 mL/min. Solvent A (0.1% TFA
in water) and solvent B (0.1% TFA in acetonitrile) were used in the
following gradient: 0% B (5 min), B 0–50% (30 min), B 50–100%
(1 min), B 100% (5 min), B 100–0% (1 min), and B 0% (3 min).
This method was used with detection by UV (220, 260, 280, 360, and
366 nm). The RP-HPLC analyses of the purified Ir-PNA were
performed using the analytical column Luna C18 (150 × 4.6 mm,
5 μm) at a flow rate of 1 mL/min, using the same gradient reported
for the semipreparative column. High-resolution electrospray ionization
mass spectrometry (HR-ESI + MS) analyses were acquired in
a positive polarity with a Synapt G2-Si QToF instrument (Waters) interfaced
through a ZsprayTM ESI-probe for electrospray ionization (Waters).
Data were processed with a MassLynxTM v4.2 software (Waters). The
concentrations of Ir-PNA solutions were determined by
measuring the absorbance at 260 nm with an Agilent 8453 UV/vis spectrophotometer.
The molar extinction coefficient of the PNA tetramer was calculated
according to standard protocol. 43
## Photophysical Characterization
4.3 Photophysical Characterization UV–vis
absorption spectra were acquired on a single beam Agilent model 8543
spectrophotometer equipped with a diode array detector using a 1 cm
path length quartz cuvette at room temperature. Molar absorption coefficients
of MLCT transitions were measured by weighting a known amount of Ir
complex and quantitatively dissolving it in a known MeOH volume. The
stock solution was then used to prepare diluted solutions at different
molar concentrations (in the range 6 × 10 –5 –1.7 × 10 –4 M), which were employed
to acquire UV–vis absorption spectra, from which the absorbance
values at 377 and 415 nm were collected. The slopes of the straight
lines obtained by plotting the absorbances vs molarity returned the
ε (M –1 cm –1 ). The estimated
experimental errors are 2 nm on the absorption maxima and 5% on the
molar absorption coefficients. Emission spectra were acquired by using
an Edinburgh FLS980 spectrofluorimeter equipped with a 450 W xenon
arc lamp. Emission spectra were corrected for source intensity (lamp
and grating) and emission spectral response (detector and grating)
by standard correction curves. The estimated experimental errors are
2 nm on the PL bands maxima. Time-resolved measurements were performed
using the time-correlated single-photon counting (TCSPC) option on
the FLS980. The pulsed excitation source, an LED at 404 nm, was mounted
directly on the sample chamber, and the emission was collected by
a multichannel plate MCP-PMT Hamamatsu H10720 –01 single photon-counting
detector. The photons collected at the detector were correlated by
a time-to-amplitude converter (TAC) to the excitation pulse. The data
analysis was performed using the commercially available F980 software
(Edinburgh Instruments). The goodness of the data fitting was assessed
by minimizing the reduced chi-squared function (χ 2 ). The uncertainty on fluorescence lifetimes is estimated to be ±0.05
ns. Photoluminescence quantum yields (Φ) were collected
for an optically diluted solution (<10 –5 M) using
wavelength scanning with a Hamamatsu C11347 –11 Quantaurus-QY
absolute PL quantum yield spectrometer, equipped with a xenon light
source (150 W), a monochromator, and a Spectralon integrating sphere,
and employing the commercially available U6039–05 PLQY measurement
software (Hamamatsu Photonics Ltd., Shizuoka, Japan). The photoluminescence
quantum yields were measured by exciting the samples between 350 and
430 nm.
## Two-Photon Excitation Microscopy Localization
Study
4.4 Two-Photon Excitation Microscopy Localization
Study Two-photon excitation (TPE) fluorescence microscopy
experiments were conducted using a 720 nm excitation beam generated
by a mode-locked Ti:sapphire laser (Mai Tai HP, Spectra Physics).
The laser emits pulses with a duration of 120 fs full width at half-maximum
and a repetition frequency of 80 MHz, delivering 20 mW of power at
the sample plane. The optical setup comprises a confocal scanning
head (M610 scanning mirrors module, ISS) integrated onto an upright
optical microscope (BX51, Olympus), equipped with a high working distance
objective (XLPlan, NA = 1.05, wd = 2 mm, 25x water immersion, Olympus).
Fluorescence signals, collected in epifluorescence geometry by the
same objective, were directed to a nondescanned detection unit and
then transmitted to three Hamamatsu analog output photomultipliers
(HC125–02, Hamamatsu). Sample emissions were filtered using
480/30, 535/50, and 600/40 band-pass filters to eliminate scattering
contributions. Fluorescence data were processed by using the ISS Vista
Vision Suite software (ISS). The microscopy images presented in this
paper are the result of 4 averaged scans, with a residence time of
10 μs per pixel. The field of view for the 512 × 512 pixel
images ranged from 300 to 50 μm 2 , depending on the
zoom factor employed. Z-scan images were acquired at 1 μm intervals.
## Photochemical Stability Study
4.5 Photochemical Stability Study The
photochemical stability test and photoreaction with DHN in the presence
of investigated systems were monitored by employing a Jasco V-650
spectrophotometer. The experiments were performed by directly irradiating
a 3 mL quartz cuvette with a monochromatic LED light with a maximum
emission centered at 400 nm (BRIDGELUX, USA). The average full emission
intensity reaching the cuvette corresponded to ca. 15.2 mW/cm 2 , as regularly checked with a Thorlabs PM200 optical power
meter equipped with a thermal Thorlabs S302C power sensor. The normalized
emission spectrum of the LED is reported in Figure S25 .
## Biological Study
4.6 Biological Study For the MTT tests,
formazan formation was measured spectrophotometrically at 570 nm using
the microplate reader (PerkinElmer, EnSpire, Waltham, MA, USA). The
phototoxicity tests on HeLa cells were carried out by using a low-intensity
and mainly UV-light-emitting polychromatic irradiation source, with
an average intensity emission of ca. 5.2 mW/cm 2 in the
200–800 nm range. The normalized emission spectrum of the employed
irradiation source is reported in Figure S24 .
## Synthesis of the [Ir(ppy)
4.7 Synthesis of the [Ir(ppy) 2 (Phen-NH 2 )]Cl Complex (Ir-NH 2 ) [Ir(ppy) 2 (Phen-NH 2 )]Cl was prepared by slightly varying a literature
procedure. 25 [Ir(ppy) 2 Cl] 2 (88.6 mg, 0.0827 mmol) was suspended in 20 mL of freshly
distilled THF, obtaining a yellow suspension in which 40.8 mg of Phen-NH 2 (0.163 mmol) was added. After
the addition of 10 mL of H 2 O Milli-Q, the brown solid aggregates
of Phen-NH 2 were completely dissolved.
The mixture was then heated at 70 °C under an inert atmosphere,
and after a few minutes, the cloudy yellow suspension turned completely
clear. Once the set temperature was reached, it turned emerald-green,
denoting a slight defect of ligand. After heating the mixture for
5 h, a nominal slight excess of Phen-NH 2 (3.2 mg, 0.0127 mmol) was added, turning the color from emerald
green to yellow-greenish. The reaction mixture was left under stirring
at 70 °C for another 2 h and then at room temperature overnight.
The clear yellow-greenish solution, which under UV lamp irradiation
showed a yellow-orange emission, was evaporated by vacuum to dryness.
The obtained fine solid was dissolved in a few mL of dry THF and treated
with an excess of diethyl ether (Et 2 O, 2×), obtaining
a fine yellow precipitate. Yield 94% (124 mg). 1 H NMR (D 2 O, 300 K, 9.4 T): δ phen-NH 2 ligand 8.62
(1H, CH(9)), 8.35 (1H, CH(5)), 8.32 (1H, CH(7)), 8.20 (1H, CH(2)),
8.18 (1H, CH(6)), 7.72 (1H, CH(8)), 7.60 (1H, CH(3)), 3.30 (2H, CH(δ)),
2.93 (2H, CH 2 (α)), 1.83 (2H, CH 2 (γ)),
1.73 (2H, CH 2 (β)); δ phenylpyridine ligands
8.1 (2H, CH(6′)), 7.86 (2H, CH(3″)), 7.74 (2H, CH(5′)),
7.43 (2H, CH(3′)), 7.1 (2H, CH(4″)), 6.96 (2H, CH(5″)),
6.81 (2H, CH(4′)), 6.47 (2H, CH(6″)). The complete NMR
characterization of this complex can be found in ref [ 25 ]. UV–vis (MeOH)
MLCT absorptions: λ abs = 377 nm (ε = 5102 M –1 cm –1 ) and 415 nm (ε = 2517
M –1 cm –1 ).
## Synthesis of the Phen-COOH Ligand
4.8 Synthesis of the Phen-COOH Ligand Succinic anhydride (19.9 mg, 0.199 mmol) was dissolved in freshly
distilled CH 2 Cl 2 (1 mL) and treated with TEA
(56 μL, 2.1 equiv). Phen-NH 2 (50.0 mg, 0.199 mmol) was added to the solution. The mixture was
refluxed under stirring for 6 h and then at room temperature overnight.
The next day, the mixture presented a small amount of a light brown
sticky solid at the bottom of the flask and a light yellow clear supernatant
solution. Then, the supernatant was isolated and the sticky solid
was washed with a few mL of CH 2 Cl 2 (2×)
warming at 40 °C. The crude product, present in the combined
supernatants, was characterized by NMR in CH 2 Cl 2 /CDCl 3 . 1 H NMR (CH 2 Cl 2 /CDCl 3 , 300 K, 9.4 T): δ 8.88
(1H, C H (9) dd, J = 4.5 Hz, 1.9 Hz), 8.7 (1H, C H (2) d, J = 4.7 Hz), 8.0 (1H, C H (7) dd,
J = 8.2 Hz, 1.9 Hz), 7.8 (1H, C H (5) d J = 9.2 Hz),
7.6 (1H, C H (6)d J = 9.2 Hz), 7.3 (1H, C H (8) dd J = 8.2 Hz, 4.5 Hz), 7.2 (1H, C H (3) d J =
6.6 Hz), 7.45 (1H, CON H (5′)), 3.0 (2H, C H 2 (4′) m), 2.9 (2H, C H 2 (1′) t J = 19.2 Hz), 2.2 (4H, C H 2 (7′ and 8′) m), 1.5 (2H, C H 2 (2′) m), 1.4 (2H, C H 2 (3′) m). 13 C NMR (CH 2 Cl 2 /CDCl 3 , 300 K, 9.4 T): δ 178.4
( C OO(9′)), 173.5 ( C ONH(6′)),
149.8 ( C H(9)), 149.5 ( C H(2)), 148.4
( C q (1a)), 146.4 ( C q (10a)), 145.9 ( C q (4a)), 135.6
( C H(7)), 128.2 ( C q (6a)),
127.5 ( C q (4)), 125.9 ( C H(6)), 122.8 ( C H(3)), 122.6 ( C H(8)),
122.2 ( C H(5)), 38.4 ( C H 2 (4′)), 32.9 ( C H 2 (7′ and
8′)), 31.8 ( C H 2 (1′)), 29.4
( C H 2 (3′)), 27.5 ( C H 2 (2′)). Et 2 O was added to the combined
supernatants, which caused the precipitation of a whitish solid. The
mixture was evaporated to dryness, the residue was washed with CH 2 Cl 2 /Et 2 O, and the resulting whitish
crude precipitate was isolated and vacuum-dried. Yield 49.6%. Elemental
Analysis: Found = C, 62.52%; H, 6.62%; N, 10.74% (calcd for (C 20 H 21 N 3 O 3 )(C 4 O 4 H 6 ) 0.45 (C 6 H 15 NCl) 0.6 : C, 62.70%; H, 6.77%; N, 10.36%).
## Synthesis of the Ir-COOH Complex
4.9 Synthesis of the Ir-COOH Complex To a suspension of Phen-COOH (37 mg, 0.105 mmol) in
freshly distilled THF (20 mL) in a Schlenk tube, the precursor [Ir(ppy) 2 Cl] 2 was added (48.1 mg, 0.045 mmol, 0.43 equiv)
under stirring at room temperature. The mixture was heated at 70 °C
under magnetic stirring and an inert atmosphere. Neither the Ir precursor
nor the functionalized phenanthroline was soluble, even when the mixture
was heated at reflux. Hence, after 30 min at 70 °C, the mixture
was cooled down at room temperature and added with 6 mL of H 2 O Milli-Q. Immediately the heterogeneous mixture became more homogeneous,
and after a few minutes at 70 °C the mixture turned clear and
yellow. The mixture was left under stirring at 70 °C for ca.
5 h and then was left overnight at room temperature. The obtained
clear yellow mixture, which under UV lamp irradiation showed a yellow-orange
emission, was evaporated by vacuum to dryness, and the residue was
redissolved in a few mL of dry THF and treated with an excess of Et 2 O (2x) to give a fine yellow precipitate. Yield 96.3% (80.2
mg, of which 3.1 mg are triethylammonium succinate). 1 H NMR (MeOD, 300 K, 9.4 T): δ phen-COOH
ligand 8.76 (1H, C H (9) dd, J = 8.2 Hz, 1.9 Hz), 8.52
(1H, C H (5) d, J = 9.2 Hz), 8.37 (1H, C H (7) dd, J = 5.3 Hz, 1.9 Hz), 8.32 (1H, C H (6) d,
J = 9.2 Hz), 8.24 (1H, C H (2) d, J = 5.6 Hz), 7.90
(1H, C H (8) dd J = 8.2 Hz, 5.3 Hz), 7.79 (1H, C H (3) d, J = 5.6 Hz), 3.36 (2H, C H 2 (1″) t, J = 8.9 Hz), 3.29 (2H, C H 2 (4″) t, J = 6.8 Hz), 2.54 (2H,
C H 2 (8″) t, J =
7.0 Hz), 2.43 (2H, C H 2 (7″) t, J = 7.0 Hz), 1.86 (2H, C H 2 (3″) m), 1.70 (2H, C H 2 (2″) m); δ phenylpyridine ligands
8.13 (1H, C H (2′) ps d, J =
8.2 Hz), 7.87 (1H, C H (9′) ps d J = 7.9 Hz), 7.81 (1H, C H (3′) ps t, J = 7.7 Hz), 7.45 (1H, C H (5′) ps d, J = 5.9 Hz), 7.10 (1H, C H (8′) ps t, J = 7.5 Hz), 6.95 (1H, C H (7′) ps t, J = 7.7 Hz), 6.91 (1H, C H (4′) ps t, J = 6.4 Hz), 6.41 (1H, C H (6′)
dd, J = 7.4, 2.8 Hz). 13 C NMR (MeOD, 300 K, 9.4 T): δ phen-COOH ligand 176.2 ( C OO(9″)), 173.8 ( C ONH(6″)), 153.1 (C q (4)), 150.9 (C q (6a)), 150.6 (CH(7)), 149.9 ( C H(2)), 147.2 ( C q (10a)), 138.2
( C H(9)), 131.7 (( C q (1a)),
130.6 ( C q (4a)), 127.9 ( C H(6)), 126.4 ( C H(3)), 124.7 ( C H(8)),
124.6 ( C H(5)), 38.3 ( C H 2 (4″)), 31.4 ( C H 2 (1″)),
31.3 ( C H 2 (7″)), 30.5 ( C H 2 (8″)), 29.0 ( C H 2 (2″)),
27.2 ( C H 2 (3″)). δ phenylpyridine
ligands 168.1 (C q (12′)), 150.2 (C q (11′)),
148.6 ( C H(5′)), 144.3 ( C q (10′)), 138.3 ( C H(3′)), 131.7
( C H(6′)), 130.2 ( C H(7′)),
124.6 ( C H(9′)), 123.0 ( C H(4′)),
122.4 ( C H(8′)), 119.6 ( C H(2′)).
ESI + MS, m / z : 890.3 [M+K–H] + , 874.5 [M+Na–H] + , 852.7 [M] + , 752.6 [M–C(O)CH 2 CH 2 COOH] + . UV–vis spectroscopy of Ir-COOH in MeOH: ε
(λ abs = 377 nm) = 5.1 × 10 3 M –1 cm –1 . ε (λ abs = 415 nm) = 2.5 × 10 3 M –1 cm –1 .
## Synthesis of the Ir-PNA Conjugate through
Coupling Reaction between PNA1-Fmoc and Ir-COOH
4.10 Synthesis of the Ir-PNA Conjugate through
Coupling Reaction between PNA1-Fmoc and Ir-COOH The PNA1-Fmoc resin (40 mg, 0.3 mmol/g, 12 μmol) was swollen
in CH 2 Cl 2 for 45 min, then was treated with
a 20% v/v solution of piperidine in NMP (1 mL, twice
for 8 min). A solution of HATU (14 mg, 36 μmol, 3 equiv) in
NMP was added to a solution of the Ir-COOH (36 mg, 36
μmol, 3 equiv) and DIPEA (21 μL, 120 μmol, 10 equiv)
in NMP, and the resulting solution was shaken for 2 min. Then, this
brown solution was transferred to the resin that was shaken for 4
h at room temperature. The resin was washed with NMP, and the simultaneous
deprotection of nucleobases and the cleavage were performed by treating
the resin with a mixture of TFA/ m -cresol (9/1, v/v , 1 mL) for 1.5 h. The collected filtrate was concentrated
by bubbling N 2 onto the solution, and the residue was poured
onto cold Et 2 O to precipitate the crude Ir-PNA as a yellow solid. The solid was centrifuged, washed with Et 2 O, dried under a vacuum, and purified by RP-HPLC. HR-ESI + MS, m / z : found (calculated)
for C 85 H 91 N 29 O 14 Ir: 1932.6938
(1932.6906) [M] + , 1000.8215 (1000.8231) [M+3Na-2H] 2+ , 997.8154 (997.8191) [M+Na+K–H] 2+ , 989.8307
(989.8322) [M+2Na–H] 2+ , 978.8405 (978.8412) [M +
Na] 2+ , 966.8480 (966.8487) [M + H] 2+ , 665.5449
(665.5485) [M+Na+K] 3+ , 660.2238 (660.2239) [M+2Na] 3+ , 658.2186 (658.2212) [M+K+H] 3+ , 652.8962 (652.8966)
[M+Na+H] 3+ , 644.9016 (644.9015) [M+2H] 3+ . Analytical
RP-HPLC: t R = 29.6 min.
## Synthesis of the Ir-PNA Conjugate through
Coupling Reaction between PNA2 and Ir-NH
4.11 Synthesis of the Ir-PNA Conjugate through
Coupling Reaction between PNA2 and Ir-NH 2 After
the swelling in CH 2 Cl 2 for 45 min, PNA2 resin (40 mg, 0.3 mmol/g, 12 μmol) was shaken with a solution
of HATU (14 mg, 36 μmol, 3 equiv) and DIPEA (21 μL, 120
μmol, 10 equiv) in NMP for 8 min. Then, the brown solution of
the Ir-NH 2 (28 mg, 36 μmol,
3 equiv) was transferred to the resin containing the solution of HATU
and DIPEA and shaken for 4 h at room temperature. The resin was washed
with NMP, and after the cleavage, the crude Ir-PNA was
obtained as a yellow solid, and its RP-HPLC analysis showed a very
similar trend observed for the conjugation with the Ir-COOH . Analytical RP-HPLC: t R = 29.6 min.
## Stability Test on Ir-NH
4.12 Stability Test on Ir-NH 2 under
Cleavage Conditions A tiny amount of the Ir-NH 2 complex was dissolved in 3 mL of a TFA/ m -cresol 9:1 mixture, and the solution was monitored by
UV–vis spectroscopy, recording subsequent spectra over a period
of 1.5 h, mimicking the contact time and the conditions used during
the cleavage of the PNA from the resin. UV–vis spectra are
reported in Figure S12 .
## Photochemical Stability of Ir-COOH and Ir-PNA
4.13 Photochemical Stability of Ir-COOH and Ir-PNA The Ir-COOH complex and the Ir-PNA conjugate,
both dissolved in methanol, were diluted to 2.5 mL by using a CH 2 Cl 2 –methanol mixture (9:1) in a quartz cuvette.
In this way, the same concentration used during the photoreaction
of the two photosensitizers with DHN was achieved. The solutions were
irradiated with a monochromatic LED light (400 nm, 15.2 mW/cm 2 ) for increasing times ranging from 1 to 25 min. Potential
variations were monitored by UV–vis spectroscopy ( Figures S22 and S23 ).
## Photoreaction of Ir-COOH and Ir-PNA with
DHN
4.14 Photoreaction of Ir-COOH and Ir-PNA with
DHN In a quartz cuvette, 15 μL of a 1.68 × 10 –3 M methanol solution of the Ir-PNA adduct
was diluted to 2.25 mL with a CH 2 Cl 2 -methanol
9:1 mixture. 250 μL of a 1.44 × 10 –3 M
solution of DHN was added in the same solvent mixture, to obtain a
1:10 PS-DHN molar ratio. Subsequently, the solution was saturated
with O 2 by bubbling it for 5 min and exposed to monochromatic
LED light (400 nm, 15.2 mW/cm 2 ) for durations ranging from
30 s to 25 min. Thus, the variations in DHN and Juglone absorption
bands were monitored using UV–vis spectroscopy ( Figure 3 of the main text); in particular,
the decreasing absorbance of the DHN band at 316 nm was used for the
determination of the initial rate constant ( k obs ) of the oxidation of DHN to Juglone. The same procedure
was carried out for the Ir-COOH complex and for the Ir(ppy) 3 , whose 1 O 2 quantum yield (Φ Δ ) in CH 2 Cl 2 -methanol 9:1, obtained from the literature, 34 was used as a standard to calculate the Φ Δ values for the Ir-PNA and the Ir-COOH . While the equation rate of the overall reaction of juglone production
can be described by eq 1 , by applying the steady-state approximation to the reactive 1 O 2 species, the DHN consumption reaction in the
initial stages ( v ) follows pseudo-first-order kinetics,
as described by eq 2 . 1 2 The 1 O 2 production ability of the investigated species can thus be
indirectly monitored by evaluating the corresponding k obs rate constant, which can be estimated by plotting,
as a function of the irradiation time, the characteristic DHN absorbance
value ( A ), registered at 316 nm, according to the
following integrated eq ( eq 3 ): 3 The fitted absorbance
values (with A t and A 0 referring to the A values measured
at times t and 0, respectively) were thus obtained
from regularly acquired UV–vis spectra of the reaction mixture
over a fixed irradiation time, as depicted in Figure 3 . Panel (d) of Figure 3 shows a comparison of the linear decrease
of ln(A t /A 0 ) vs time acquired for the investigated
systems. The quantum yield for singlet oxygen 1 O 2 formation (Φ Δ ) can then be determined by
using eq 4 : 4 where v i is the initial rate, I indicates
the photons absorbed by the sensitizer, and the apex std labels the
values for the adopted standard, which in our case corresponds to
the Ir(ppy) 3 complex, whose generation of 1 O 2 in DCM/methanol 9:1 is known and fixed to Φ Δ = 0.50. 34 The values of ν i were obtained as the product of k obs and [DHN] 0 (with [DHN] 0 in the 1.65–1.70
× 10 –4 M range), while the value of I was calculated as I = I source (400 nm) × (1–10 A (400 nm) ) where I source (400
nm) is the intensity of the 400 nm LED emitted light incident on the
sample, while A (400 nm) is the corresponding absorbance
of the sensitizer at such wavelength.
## Cell Culture and Treatment
4.15 Cell Culture and Treatment HeLa
cells were cultured in DMEM culture medium (EuroClone, ECB7501) supplemented
with 1 mM l -glutamine (EuroClone, ECB3004D), penicillin-streptomycin
(SERVA 31749.04, 35500.01), and 10% supplemented with 10% fetal bovine
serum (GIBCO, A4766801) at 37 °C in 5% CO 2. For fluorescence
microscopy analysis, HeLa cells were seeded at 200,000 cells/well
in 6-well plates (Euroclone, ET3006) and treated with 25 μM Ir-COOH or Ir-PNA , and methanol as vehicle control
for 24 h. For cell viability assay, HeLa cells were seeded at 25,000
cells/well in 48-well plates (Euroclone, ET3048) and treated for 24
h with different doses of the Ir-COOH and the Ir-PNA (1–5–10–25–50 μM). Untreated cells
and cells treated with methanol (vehicle) were used as negative controls.
## Viability Tests in the Dark and in Light
4.16 Viability Tests in the Dark and in Light MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)
(M5655, Sigma-Aldrich) assay was performed to determine the cell viability
and the potential cytotoxicity of the Ir-COOH and the Ir-PNA both in the dark and under irradiation using a nail
UV lamp. After 24 h of treatment, cells were exposed to light for
30 min, and after 6 h of incubation at 37 °C, the medium was
replaced with 300 μL of MTT solution. Following incubation for
30 min at 37 °C, MTT was removed and 500 μL of 2-propanol
was added to each well to dissolve formazan crystals. The same experiments
were performed in the dark as the control. Formazan formation was
measured spectrophotometrically at 570 nm using the microplate reader
(PerkinElmer, EnSpire, Waltham, MA, USA). Light EC 50 values
were obtained by analyzing the fitting curves by OriginPro2021 (Copyright
© 1991–2021 OriginLab Corporation) software using the
vertical cursor instrument for reading the data at 50% of cell viability. Statistical analysis: Curve-fitting was performed with OriginPro2021
software, and statistical data analysis was performed using GraphPad
Prism 9 (GraphPad Software Inc., San Diego, CA). Data were normalized
to untreated control. Results were plotted using nonlinear regression
with three parameters. One-way ANOVA corrected for multiple comparisons
by Sidak′s post hoc analysis was performed, *** p < 0.001; n = 4.