Photochemical Properties and Activity of Water-Soluble Polymer/C 60 Nanohybrids for Photodynamic Therapy
Marie Hurtgen, Antoine Debuigne, Maryse Hoebeke,* Catherine Passirani,*
Nolwenn Lautram, Ange Mouithys-Mickalad, Pierre-Henri Guelluy, Christine Je´roˆme, Christophe Detrembleur*
1. Introduction
Photodynamic therapy (PDT) has developed as a non- invasive clinical treatment of various dermatological, ophthalmic, and cardiovascular diseases ranging from minor affections to solid tumors.[1] PDT destroys target cells when a photosensitizer (PS) is irradiated in the presence of oxygen, which generates highly reactive singlet oxygen (1O2). Singlet oxygen then attacks cellular compo- nents, which activates an immune response against targeted cells. Although the most widely used PS are cyclic tetrapyrroles such as porphyrins and phthalocyanines,[2]
[60] fullerene or C60has received much attention as PS[3,4]
due to its long-lived triplet state and high quantum yield of singlet oxygen production [F(1O2)],[5]which are important Dr. M. Hurtgen, Dr. A. Debuigne, Prof. C. Je´roˆme,
Prof. C. Detrembleur
Department of Chemistry, Center for Education and Research on Macromolecules (CERM), B6A, University of Lie`ge, 4000 Lie`ge, Belgium
E-mail: christophe.detrembleur@ulg.ac.be Prof. M. Hoebeke, Dr. P.-H. Guelluy
Laboratory of Biomedical Spectroscopy, B5A, University of Lie`ge, 4000 Lie`ge, Belgium
E-mail: m.hoebeke@ulg.ac.be Prof. C. Passirani, N. Lautram
INSERM U 646, University of Angers, Rue A. Boquel 10, 49100 Angers, France
E-mail: catherine.passirani@univ-angers.fr Dr. A. Mouithys-Mickalad
Center for Oxygen Research and Development, B6A, University of Lie`ge, 4000 Lie`ge, Belgium
Water-soluble star-like poly(vinyl alcohol)/C
60and poly{[poly(ethylene glycol) acrylate]-co- (vinyl acetate)}/C
60nanohybrids are prepared by grafting macroradicals onto C
60and are assessed as photosensitizers for photodynamic therapy. The photophysical and biological properties of both nanohybrids highlight key
characteristics influencing their overall efficiency.
The macromolecular structure (linear/graft) and nature (presence/absence of hydroxyl groups) of the polymeric arms respectively impact the photodynamic activity and the stealthiness of the nanohybrids. The advantages of both nano- hybrids are encountered in a third one, poly[(N- vinylpyrrolidone)-co-(vinyl acetate)]/C
60, which has linear grafts without hydroxyl groups, and shows a better photodynamic activity.
PVOH/C
60P(PEGA- co -VAc)/C
60light
1
O
2*
1
O
2*
3
O
23
O
2Cell death
requirements for PDT. However, the poor solubility of C60in water is an obstacle to its use in PDT. To solve this problem, a simple approach consists in grafting water-soluble and biocompatible polymers onto C60. This strategy offers the opportunity to selectively target diseased cells through the enhanced permeation and retention (EPR) effect, which allows a preferential accumulation of macromolecules in tumor tissues compared to healthy ones.[6,7] Various reactions were employed to functionalize C60with water- soluble polymers, such as the addition of amino-terminated poly(ethylene glycol) (PEG) onto C60,[8–11]the cycloaddition of a PEG-modified diazo compound onto C60[12]or the radical copolymerization of N-vinylpyrrolidone (NVP) with a polymerizable derivative of C60.[13] The photodynamic activities of the resulting polymer/C60nanohybrids were assessed using different methods but fundamental photo- physical properties like triplet state life-time (tT) andF(1O2) were not reported, making comparisons impossible.
Recently, cobalt-mediated radical polymerization (CMRP)[14,15] has developed as an efficient tool for the synthesis of (co)polymers based on hydrophilic poly(vinyl alcohol) (PVOH),[16] poly(N-vinylpyrrolidone) (PNVP),[17]
poly(N-vinylcaprolactam)[18] and poly(acrylic acid).[19]
Among these, PVOH[20] and PNVP-based copolymers[21]
were grafted onto C60to yield potential PSs. Also, a quasi- diblock poly[(PEG acrylate)-co-(vinyl acetate)] [P(PEGA-co- VAc)] copolymer was recently made available[22]and could be dissolved in aqueous medium due to the presence of PEG grafts. CMRP therefore appears as a useful technique for the preparation of water-soluble polymers and their easy grafting onto C60, which points at a potential use in PDT.
Motivated by the hydrosolubility and biocompatibility of PVOH and PEG, we investigate here the photophysical properties and the photodynamic activity of PVOH/C60and of a new P(PEGA-co-VAc)/C60nanohybrid. Both materials are assessed as PSs through photophysical measurement of their triplet state properties and singlet oxygen quantum yields. Also, their photo induced cell toxicity and complement system activation are quantified by in vitro assays. The comparison of both nanohybrids results in the identification of structural parameters positively impact- ing the PDT activity, e.g., the presence of linear rather than graft polymer arms on the C60 core and the absence of hydroxyl groups. These characteristics are encountered in a previously synthesized nanohybrid, poly[(N-vinylpyrroli- done)-co-(vinyl acetate)]/C60,[21] which therefore shows the best activity among all three nanohybrids.
2. Experimental Section
2.1. Materials
Vinyl acetate (Vac,>99%, Acros) was dried over calcium hydride, degassed by several freeze-thawing cycles before being distilled
under reduced pressure and stored under argon. Poly(ethylene glycol) methyl ether acrylate (PEGA,Mn454 gmol1, DPPEG¼8–
9, Aldrich) was dissolved in dry toluene, dried by vacuum distillation of the water/toluene azeotrope (3 times) and stored at208C under argon. 1,2,4-trichlorobenzene (TCB) was dried over molecular sieves. All the solvents were degassed by argon bubbling for 30 min. Buckminsterfullerene (C60) (99.5%, Aldrich), 2,20- azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70,>96%, Wako), bis(acetylacetonato)cobalt(II) [Co(acac)2] (>98%, Acros), potassium hydroxide (KOH,>90%, Aldrich), 2,2,6,6-tetramethylpiperidine-1- oxy (TEMPO) (98%, Aldrich) and rose Bengal (RB) (95%, Aldrich) were used as received.
2.2. Characterizations
Size exclusion chromatography (SEC) of polymer samples was carried out in THF (flow rate: 1 mLmin1) at 408C with a Waters 600 liquid chromatograph equipped with a 410 refractive index detector and Styragel HR columns (four columns HP PL gel 5mm, 105A˚ , 104A˚ , 103A˚ , 102A˚ ). Polystyrene standards were used for calibration. Dual detection was used for samples containing fullerenes with a 410 refractive index and a SFD S3240 UV-vis detector (l¼290 nm).
1H NMR spectra were recorded at 298 K with a Bruker Spectro- meter (250 MHz) in CDCl3.
UV-vis absorption spectra were recorded with a Hewlett Packard 8453 spectrometer.
Inductively coupled plasma mass spectrometry (ICP-MS) was carried out with a Spectrometer Elan DRC-e Perkin-Elmer SCIEX.
Samples were prepared by dissolving a small amount (10 mg) of polymer in 5mL of HNO3(65%). This solution was heated until complete dissolution and then diluted with 50 mL of bidistilled water at room temperature before analysis.
Dynamic light scattering (DLS) experiments were carried out at 258C on a Malvern Zetasizer Nano Series DTS 1060 (Malvern Instruments S.A., Worcestershire, UK) with a polymer concentra- tion of 2 mgmL1in milliQ water. Data were processed by the Dispersion Technology Software 5.10, performing the cumulant method to extract thez-average diameter of the particles.
2.3. Synthesis of Poly(vinyl alcohol)/C60Nanohybrids Polymerizations and grafting experiments were performed by classical Schlenk techniques under argon. Liquids were transferred with syringes and stainless steel capillaries. The synthesis of poly(vinyl acetate) was carried out as follows. An alkylcobalt(III) adduct in CH2Cl2 solution (11.4 mL, 1 mmol) was prepared as described previously,[23]added into a round bottom flask capped by a three-way stopcock and evaporated to dryness under reduced pressure. After addition of degassed vinyl acetate (41.5 mL, 38.8 g, 0.45 mol), the reaction mixture was heated at 408C under stirring for 4h (monomer conversion estimated gravimetrically¼25%).
Part of the pink PVAc-Co(acac)2was deactivated by reaction with TEMPO prior to precipitation in heptane and characterization (Mn;SEC¼10 900 g/mol;Mw=Mn¼1.06). The rest of the polymer- ization medium was dried under reduced pressure and PVAc- Co(acac)2(7.5 g; 0.94 mmol) was dissolved in dry and degassed TCB
(65 mL) and added to a solution of C60(85 mg, 0.114 mmol) in dry and degassed TCB (30 mL). The reaction mixture was stirred at 308C for 20 h. The resulting mixture was then precipitated in cold heptane, dissolved in THF and filtered onto silica and 0.45mm teflon filters to remove the cobalt complex. In order to remove the ungrafted PVAc chains from the PVAc/C60nanohybrid, the dried reaction product was dissolved in ethanol/water 70/30 (v/v) and separated by centrifugal filtration using Amicon Ultra-15 centri- fugal filters (MWCO¼30 000 Da, Millipore). The centrifugation/
addition of solvent cycle was repeated until no free PVAc was detected by SEC in the filtered PVAc/C60nanohybrids. The final PVAc/C60, recovered by solvent evaporation, was then analyzed by SEC with RI and UV detections (Mn;SEC¼54 500 g/mol;
Mw=Mn¼1.10). The PVOH/C60 nanohybrid was prepared by hydrolysis of PVAc/C60 by KOH in methanol, as described else- where.[20,21]The C60weight percentage in the PVOH/C60nanohybrid was estimated as 2.6 wt%, assuming that C60is functionalized by an average of five polymer chains. The residual cobalt content in PVOH/C60was determined by ICP-MS as 40 ppm.
2.4. Synthesis of Poly(PEGA-co-VAc)/C60Nanohybrids The P(PEGA-co-VAc)-Co(acac)2 precursor was synthesized as described previously.[22] Briefly, Co(acac)2 (0.074 g, 0.288 mmol) and the alkylcobalt(III) adduct in CH2Cl2(7 mL, 0.961 mmol) were added into a round bottom flask capped by a three-way stopcock.
CH2Cl2was evaporated to dryness under reduced pressure. Dry and degassed VAc (16.2 mL, 15.1 g, 0.175 mol) and PEGAe (3.84 mL, 4.18 g, 9.22 mmol) were added and the copolymerization was carried out at 308C for 20 h (PEGA conv.¼100%, VAc conv.¼20%
determined by 1H NMR). Part of the P(PEGA-co-VAc)-Co(acac)2
was deactivated by reaction with TEMPO prior to precipitation in heptane and characterization by SEC (Mn;SEC¼8 700 g/mol;
Mw=Mn¼1.12). The rest of the polymerization medium (17g, 0.817 mmol of polymer chains) was added with dry and degassed TCB (20 mL) and the residual VAc monomer was evaporated under reduced pressure. The resulting P(PEGA-co-VAc)-Co(acac)2polymer in TCB was then added with a solution of C60(73.5 mg, 0.102 mmol) in dry and degassed TCB (30 mL). The reaction mixture was stirred at 308C for 20h. All purifications are identical to those performed on PVAc/C60, except that the purified P(PEGA-co-VAc)/C60nanohybrid was also filtered onto a Chelex 100 column in methanol to remove cobalt traces. The final P(PEGA-co-VAc)/C60was dialyzed against water, recovered by freeze-drying, and then analyzed by SEC with RI and UV detections (Mn;SEC¼40 100 g/mol;Mw=Mn¼1.25).
The residual cobalt content in P(PEGA-co-VAc)/C60was evaluated by ICP-MS to 47 ppm.
2.5. Flash Photolysis
Nanosecond laser flash photolysis measurements were carried out using a laser photolysis equipment described previously.[24]Briefly, the excitation source was a Q-switched Nd/YAG laser (Quantel YG 441) of 2 ns full width at half maximum with third harmonic (355 nm) generation. The 355 nm beam was directed onto one side of a square silica cell containing the sample. The variations were monitored at right angles to the excitation in a crossbeam
arrangement using a xenon flash lamp, a monochromator, a photomultiplier and a digitized oscilloscope interfaced with a microcomputer. The time and the spectral resolutions of this set-up were 2 ns and 1 nm, respectively. The differential absorption spectra of PVOH/C60in DMSO and P(PEGA-co-VAc)/C60in water ([C60]¼105M) were recorded at the end of a laser pulse either in argon-flushed or air-equilibrated solutions. The triplet state absorption spectra were determined according to a previously reported procedure[25,26] using AT¼DAþaA0 where AT is the triplet state absorbance,A0the ground state absorbance andathe fraction of ground state molecules converted into triplet at timet.
For different values ofatried, the resulting absolute absorption spectrum of the triplet was compared with that of the ground state.
An acceptable value ofawas selected under the assumption that the triplet absorption spectrum must be positive and different from that of the ground state.
2.6. Singlet Oxygen Quantum Yields
The ability of the PVOH/C60and P(PEGA-co-VAc)/C60nanohybrids to produce singlet oxygen upon irradiation was tested following the 9,10-anthracenedipropionic acid (ADPA) bleaching method.[27]
ADPA readily reacts with the singlet oxygen produced by the nanohybrids and this photo-oxidation can be quantified by monitoring the decrease of ADPA’s 400 nm absorption band by UV-vis spectroscopy. The absolute value of the singlet oxygen quantum yield of both nanohybrids [Fnanohybrid(1O2)] was calcu- lated using Equation 1[28]with RB as a standard:
Fnanohybridð1O2Þ ¼FRBð1O2Þ IRB
Inanohybrid
knanohybrid
kRB
(1)
whereFRB(1O2) is the singlet oxygen quantum yield of RB (¼0.76 in water at pH¼7).[29]kRBorknanohybridare the rate constants of ADPA photo-oxidation in the presence of RB or the nanohybrid and are given by ln([ADPA]/[ADPA]0)¼ kt. The concentration of ADPA was monitored by UV-vis spectroscopy at 400 nm.Inanohybrid
and IRBare the total light intensities absorbed by the nanohybrid or RB, and are determined according to[30]
I¼ Z
IlampðlÞ ð110AðlÞÞdl (2)
whereIlamp(l) is the emission spectrum of the lamp andA(l) the absorbance of the nanohybrid or RB. Blank UV-vis spectra were recorded from a phosphate buffer solution (0.050M, pH¼7) containing the nanohybrid or RB. ADPA in phosphate buffer was then added (final [ADPA]¼0.122103M) and the mix- ture was irradiated with a white lamp (150 W tungsten filament lamp, 350–800 nm). Both PVOH/C60 and P(PEGA-co-VAc)/C60
([C60]¼20106M) were tested as described above. RB (2106M) was also tested for comparison.
2.7. Cell Viability Assays
Human promyelocytic leukemia cells (HL-60, American Type Culture Collection) were cultured in Iscove’s modified Dulbecco’s medium
(IMDM) supplemented with 20% fetal bovine serum (FBS), penicillin- streptomycin and Fungizone (Invitrogen) and maintained at 378C with a 5% CO2incubator balanced with air. The cellular toxicity of PVOH/C60and P(PEGA-co-VAc)/C60was evaluated in the dark and upon irradiation by means of cell viability assays using Trypan blue as a staining agent.[31]The stock solution of Trypan blue (Sigma, Germany) was 1 mol% in phosphate-buffered saline (PBS) at pH¼7.4. HL-60 cells were seeded at a density of 5105cellsmL1 in IMDM. After a 24 h incubation step with the nanohybrids ([C60]¼5, 10 or 20106M), the cells were centrifuged at 250gfor 10 min at 378C and the supernatant was discarded. The cells were then suspended in HBSS buffer (Invitrogen) and either kept in the dark or irradiated at room temperature with a white light.
The luminous flux received by the cells was measured as 78 000 lx with a luxmeter. The cells were then centrifuged, re-suspended in complete IMDM and incubated for another 24 h. Thereafter, 400mL of the cell suspension was added with Trypan blue (100mL) and 10mL of the resulting mixture was analyzed by light microscopy for cell counting. Results are expressed as the average of three measure- ments from different cell batches.
2.8. Complement Activation Test (CH50)
The complement activation was measured as the lytic capacity of a normal human serum (NHS) towards antibody-sensitized sheep erythrocytes after exposure to the nanohybrids.[32]Aliquots of NHS were incubated with increasing concentrations of nanohybrids.
The amount of serum causing 50% hemolysis after exposure to the nanohybrids was determined (‘‘CH50 units’’) for each nanohybrid.
NHS was provided by the ‘‘Etablissement Franc¸ais du Sang’’
(Angers, France) and stored as aliquots at 808C until use.
Veronal-buffered saline (VBS) containing 0.15103MCa2þand 0.5103MMg2þ(VBSþþ) was prepared as reported elsewhere.[33]
Firstly, sheep erythrocytes were sensitized by rabbit anti-sheep erythrocytes antibodies (Se´rum he´molytique, Biome´rieux, Marcy- l’Etoile, France) and diluted by the VBS at a final concentration of 2109cellsmL1. Increasing amounts of nanohybrids (highest concentration: 1.25 mgmL1) were added to NHS diluted in VBSþþ such that the final dilution of NHS in the mixture was 1/4 (v/v) in a final volume of 1 mL. After 1 h of incubation at 378C under gentle agitation, the suspension was diluted 1/25 (v/v) in VBSþþ, and aliquots of 8 different dilutions were added to a given volume of sensitized sheep erythrocytes. After 45 min of incubation at 378C, the reaction mixture was slightly centrifuged at 2000 rpm for 10 min. The absorption of the supernatant was determined at 414 nm with a microplate reader and compared to the results obtained with control serum in order to evaluate the amount of hemolyzed erythrocytes. Positive and negative controls were made in each series of experiments in order to account for any difference in the hemoglobin response from a given erythrocyte preparation.
Furthermore, corrections for particle light-scattering and sponta- neous erythrocyte hemolysis were estimated by UV-vis measure- ments using blanks containing only particles and only erythro- cytes, respectively. In order to compare nanohybrids of different average diameters, results were expressed in terms of surface area (i.e., surface of the nanohybrids exposed to the human serum), rather than concentration. Indeed, the adsorption of complement proteins on a particle depends on both the concentration and size of
the latter. These two parameters are gathered under a single one, the surface area, which is therefore used to normalize the results with respect to concentration and size. The surface area of the nanohybrids is calculated according to the formula:S¼3mr1r1, whereSis the surface area (cm2),mthe weight of nanohybrid (mg) in 1 mL,rthe average radius (cm) determined by DLS, andrthe volumetric mass (mgcm3) of the nanohybrids estimated at 106mgcm3.[34]The experimental data are the average of three independent experiments with a 10% standard deviation.
3. Results and Discussion
3.1. Synthesis of PVOH/C60and P(PEGA-co-VAc)/C60 Nanohybrids
With the aim of developing new polymer/C60nanohybrids for PDT, CMRP was selected as a tool to prepare well-defined water-soluble polymer (or precursors) that are prone to radical addition onto C60. Among polymers available by CMRP, poly(vinyl acetate) was chosen as a precursor of poly(vinyl alcohol), widely used in the biomedical field.[35]
In addition, P(PEGA-co-VAc) was selected because of its potential stealthy behavior that should confer long blood circulation to the nanohybrid.
The synthesis of PVOH/C60involved the polymerization of vinyl acetate by CMRP followed by radical addition onto C60 and hydrolysis of PVAc/C60 into PVOH/C60, as described elsewhere.[20,21] Noticeably, the purification procedure was significantly improved here since special care was taken to remove the ungrafted polymer chains from the polymer/C60nanohybrid by centrifugal filtration on appropriate membranes. The P(PEGA-co-VAc)/C60nano- hybrids were prepared by polymerizing a PEGA/VAc mixture by CMRP[22]and grafting the resulting P(PEGA- co-VAc) macroradicals onto C60. The same purification steps were carried out to remove ungrafted chains. Also, cobalt traces were removed by filtration on a chelating ion exchange resin. Since PEGA and VAc have extremely different reactivities, their copolymerization results in P(PEGA-co-VAc) copolymers having a strong gradient composition and a quasi-diblock structure.[22]Therefore, the corresponding P(PEGA-co-VAc)/C60nanohybrid can be seen as C60core surrounded by an outer shell of PEG grafts and an inner shell of PVAc (Figure 1).
The macromolecular characteristics of PVOH/C60 and P(PEGA-co-VAc)/C60 nanohybrids used in this study are summarized in Table 1. The numbers of polymer arms attached to C60were determined by comparing the peak molecular weight of the nanohybrids (Mp, determined by SEC relative to standards) to that of the polymer precursor before grafting onto C60. Since SEC underestimates the molecular weight of star macromolecules, Mp, nanohybrid/ Mp, precursoris therefore somewhat smaller than the actual number of grafts.[36]In a parallel study,[37]it was shown
thatMp, nanohybrid/Mp, precursor¼4.7 and 6.3 corresponds to 6 and 8 polymer chains attached, respectively. This was determined based on the ratios of reagents involved and on the well-documented observation[38,39]that only even numbers of polymer chains are grafted onto C60 via a radical mechanism. In the present case, Mp, nanohybrid/ Mp, precursor¼5 for PVOH/C60and 4.6 for P(PEGA-co-VAc)/
C60. By comparison of Mp, nanohybrid/Mp, precursorwith the actual number of grafts,[37]the structures were assigned as [PVOH]6-8/C60(a mixture of 6 and 8 arms) and [P(PEGA-co- VAc)]6/C60. Therefore, 3 to 4 double bonds of C60 were opened during the grafting reaction to accommodate those 6-8 polymer chains.
Both nanohybrids could be dissolved in water and their size was determined by DLS (Table 1). The z-average hydrodynamic diameter (Dh) of PVOH/C60(11040 nm) is bigger than that of P(PEGA-co-VAc)/C60(216 nm) because PVOH is known to aggregate in water through inter- molecular hydrogen bonds.[41]
Since both nanohybrids are water-dispersible, they could be subjected to a thorough evaluation of their photo- physical properties and photoinduced cytotoxicity, as an assessment for use in PDT.
3.2. Triplet State
The photodynamic activity of a PS is closely related to its ability to produce reactive oxygen species like singlet oxygen (1O2) upon irradiation. Since1O2is initially formed by energy transfer from the excited triplet state of the PS to ground-state oxygen, the demand of high1O2 quantum yield [F(1O2)] includes the prerequisite of adequately high triplet state quantum yield (FT) and a relatively long
R
OH 6-8 n
OAc 6 R p
O
MeO 8 O m
Figure 1.Schematic structures of PVOH/C60and P(PEGA-co-VAc)/
C60nanohybrids used in this study.
Table1.Characteristicsofpolymer/C60nanohybrids(n.d.:notdetermined). SamplePolymerprecursorPolymer/C60nanohybrid Mna) [gmol1 ]Mw=Mna)CompositionMna) [gmol1 ]Mw=Mna)Mp,nanohybrid/ Mp,precursor
xb) C60c) [wt%]Dhd) [nm] [(VAc)n]x/C60109001.06n¼127545001.1056–81.3n.d. [(VOH)n]x/C60n.d.n.d.n¼12727900e) n.d.n.d.6–82.6e) 11040 [(PEGA)n-co-(VAc)p]x/C6080001.13n¼8;p¼59401001.254.661.8216 [(NVP)n-co-(VAc)p]x/C60f) 93001.33n¼78;p¼8183001.77341.5206 a)DeterminedbySECinTHFwithaPScalibration.Accordingtoapreviousreport,[40]Mn(PVAc)determinedbySECinTHFwithapolystyrenecalibrationunderelutionconditions describedintheExperimentalSectionisveryclosetotheabsolutemolecularweight;b)x¼numberofpolymergraftsonC60;c)C60¼720/Mn100%;d)z-averagediameterdetermined byDLS;e)DeterminedbycalculatingthemasslossuponquantitativehydrolysisofPVAcintoPVOH;f)Datafromref.[21]
lifetime of the triplet states (tT).[29]The ground state and triplet properties of polymer/C60nanohybrids were inves- tigated by UV-vis spectroscopy and nanosecond flash photolysis, respectively. The spectra for PVOH/C60 were recorded in DMSO in order to avoid aggregation occurring for PVOH in water and subsequent scattering phenomena.
On the other hand, P(PEGA-co-VAc)/C60 was properly dissolved in water. The ground state absorption spectra of both nanohybrids (Figure 2) are characterized by monotonously decreasing absorption coefficients between 300 and 700 nm and are typical of functionalized full- erenes.[42,43]
Triplet state absorption spectra recorded under anaero- bic conditions (squares in Figure 2) show one broad absorption peaking at 640–660 nm. This band corresponds toT1!Tntransitions usually observed in C60adducts.[43–46]
Further confirmation that the transient state observed in Figure 2 corresponds to the triplet state is given by its quenching in the presence of oxygen. Indeed, at any wavelength, the decay of the signal was monoexponential with time, indicating a single deactivation pathway for the
triplet state, i.e., deactivation to the singlet ground state.
Under anaerobic conditions, the lifetime of the triplet state (tT,Ar) was determined as 10ms for both nanohybrids, which is similar to tTfor some small derivatives of C60[44]and tetrapyrrole-based PSs encapsulated in liposomes.[25,26]tT
can also probe possible self-quenching phenomena since the triplet lifetime is generally inferior to 0.1ms in aggregates of C60.[47] As tTof both nanohybrids is two orders of magnitude greater, it seems unlikely that they aggregate through their C60moieties, thanks to the steric stabilization of the polymeric grafts.
The presence of oxygen led to a decrease of the triplet lifetime down to 2ms, indicating that the triplet state can be deactivated by another pathway corresponding to the quenching by ground state O2 to generate singlet O2 according to: 3C60þ3O2!1C60þ1O2. The triplet state characteristics of both nanohybrids are summarized in Table 2.
3.3. Singlet Oxygen Quantum Yield
While a long-lived triplet state is a prerequisite for a good photodynamic activity, the singlet oxygen quantum yield, F(1O2), better reflects the ability of the PS to produce reactive oxygen species. The production of singlet oxygen was determined by monitoring the concentration decrease of a substrate (anthracene-9,10-dipropionic acid, ADPA) that is converted into an endoperoxide by reaction with photogenerated 1O2. F(1O2) for the nanohybrids was determined by comparing the decay slopes of ADPA in the presence of the nanohybrids to that in the presence of a standard, RB, according to Equation 1 in the Experimental Section.
The nanohybrids did not induce any oxidation of ADPA in the dark, whereas they generated1O2under illumination, as evidenced by the decrease of [ADPA] (Figure 3).F(1O2) was determined as 0.12 for PVOH/C60and 0.13 for P(PEGA- co-VAc)/C60. This value is similar for both nanohybrids, which indicates that their C60cores are substituted in a 0
300 400 500 600 700 800 900 0.000 0.005 0.010 0.015
λ (nm) ε.10-3(L.mol-1.cm-1)
10 20 30 40
Absorbance
0
300 400 500 600 700 800 900 0.000 0.005 0.010 0.015
λ (nm) ε.10-3(L.mol-1.cm-1)
10 20 30 40
Absorbance
0 10 20 30 40 50
300 400 500 600 700 800 900 0.00 0.01 0.02 0.03 0.04
λ (nm)
ε.10-3(L.mol-1.cm-1) Absorbance
0 10 20 30 40 50
300 400 500 600 700 800 900 0.00 0.01 0.02 0.03 0.04
λ (nm)
ε.10-3(L.mol-1.cm-1) Absorbance
a)
b)
Figure 2.Ground state () and triplet state (&) absorption spec- tra of (a) PVOH/C60in DMSO and (b) P(PEGA-co-VAc)/C60in water at the end of the laser pulse ([C60]¼105M).
Table 2.Triplet state properties of polymer/C60nanohybrids (n.d.:
not determined).
Sample lmax
T1!Tn
[nm]
tT,
Ara)
[ms]
tT,
airb)
[ms]
F (1O2)
PVOH/C60 640 10 2 0.12
P(PEGA-co-VAc)/C60 660 10 2 0.13
P(NVP-co-VAc)/C60 n.d. n.d. n.d. 0.50c)
a)Solution deoxygenized by argon bubbling; b)Air-equilibrated solution;c)Data calculated by comparison with PVOH/C60in ref.[21]
similar way. Indeed, the substitution pattern of C60 is known to influence photophysical properties such as F(1O2).[48]Also, theseF(1O2) are similar to those measured for small derivatives of C60, e.g., C60[C(COOH)2]5[44]and are therefore somewhat smaller than expected for a C60having 3–4 double bonds affected. This relatively lowF(1O2) may be ascribed to quenching of1O2by the PVOH or PEG polymer arms.[49]RegardingF(1O2), the most promising nanohybrid prepared by the grafting of Co(acac)2end-capped polymers onto C60 is the P(NVP-co-VAc)/C60 nanohybrid prepared previously,[21]whoseF(1O2) is now estimated as 0.50 by comparison with the ADPA decay slope for PVOH/C60
(Table 2). This can be related to the electronic structure of C60
where only 2 double bonds were disturbed to accommodate the 4 P(NVP-co-VAc) grafts.
3.4. Photoinduced Cytotoxicity
The photodynamic activity of PVOH/C60 and P(PEGA-co- VAc)/C60 nanohybrids was evaluated by means of cell viability assays under irradiation. HL-60 cells were selected as a human cancer cell line and were incubated overnight with different concentrations of nanohybrids and subse- quently exposed to increasing light doses. The percentage of living cells was then determined by the Trypan blue staining technique (Figure 4).
Control experiments without nanohybrids revealed that no significant decrease of cell viability could be ascribed to the irradiation procedure itself. Both nanohybrids were deprived of intrinsic cytotoxicity in the dark. The photo- cytotoxicity induced by PVOH/C60 is both concentration and light dose dependent (Figure 4a), which is the expected behavior for a candidate PS. In our experimental conditions,
the irradiation of 20mM PVOH/C60
induced a 55% cell mortality.
Although direct comparison with other polymer/C60nanohybrids can- not be made due to different photo- dynamic activity measurement pro- tocols, this concentration lies in an acceptable range for use in PDT.[11,13]
Surprisingly, P(PEGA-co-VAc)/C60 did not induce any significant cyto- toxicity, whatever the concentration or the irradiation time (Figure 4b).
Since both nanohybrids have almost identical F(1O2), the lack of photo- dynamic activity of P(PEGA-co-VAc)/
C60 might result from a different interaction with the cell components and/or from a shielding effect of the pendant PEG grafts. Indeed, PEG is known to limit cellular internaliza- tion,[32] which would hamper the photodynamic effect.
Also, the graft structure around C60might prevent1O2from diffusing to the solution and oxidizing cellular components.
Finally, the collapse of hydrophobic PVAc segments at the inner-shell of the nanohybrid might also somehow prevent
1O2to reach its targets. This effect was not evidenced in theF(1O2) measurement probably because the substrate -2
-1.5 -1.0 -0.5 0.0
0 5 10 15 20 25
time (min) ln(A/A0)
PVOH/C60, dark
PVOH/C60, irradiated P(PEGA-co-VAc)/C60, dark
P(PEGA-co-VAc)/C60, irradiated
Rose Bengal, irradiated -2
-1.5 -1.0 -0.5 0.0
ln(A/A0)
PVOH/C60, dark
60
P(PEGA-co-VAc)/C60, dark
P(PEGA-co-VAc)/C60
Figure 3.Evolution of ADPA absorbance in the presence of PVOH/C60(2105M), P(PEGA-co- VAc)/C60(2105M) and rose Bengal (2106M) in the dark or under irradiation.
20 40 60 80 100
Control 5 μM
Cell viability (%)
dark 15' irrad 30' irrad 60' irrad
20 40 60 80 100
Cell viability (%)
dark 15' irrad 30' irrad 60' irrad a)
b)
10 μM 20 μM
Control 5 μM 10 μM 20 μM 20
40 60 80 100
Control 5 μM
dark 15' irrad 30' irrad 60' irrad
20 40 60 80 100
10 μM 20 μM
Control 5 μM 10 μM 20 μM
Figure 4.Cytotoxicity of (a) PVOH/C60and (b) P(PEGA-co-VAc)/C60
nanohybrids on HL-60 cells in the dark or upon irradiation.
(ADPA) could diffuse near the C60core to react with1O2, which might not be the case for cellular targets. Such trapping effects, induced either by the collapsed/solvated state of the polymer or by its structure, have already been observed in dendrimer/C60nanohybrids.[50]
For the sake of comparison, the previously studied P(NVP- co-VAc)/C60nanohybrid induced a 55% higher cell mortality than PVOH/C60,[21]which makes it the most photo-cytotoxic candidate among the three water-soluble nanohybrids.
3.5. Stealth Properties
When injected intravenously, foreign bodies such as synthetic nanoparticles are subject to the immune system activation, in particular to the complement system, a set of plasma proteins that will induce their opsonization and removal from the bloodstream.[32]The stealthiness, i.e., the ability of the nanohybrids to resist protein adsorption and opsonization is therefore a key point to assess when long- circulating PSs are targeted.[51]Indeed, the PS shall not be removed from the bloodstream until it has accumulated in the tumor. However, this issue is hardly addressed in the evaluation of existing or new PSs. The ability of PVOH/C60 and P(PEGA-co-VAc)/C60 nanohybrids to resist protein adsorption was studied by a quantitative measurement of the adsorption of human serum proteins on the nanohybrids according to the so-called CH50 test.[32]After incubation of the nanohybrids with human serum, proteins that did not adsorb onto the nanohybrids can be quantified since they induce the lysis of sensitized sheep erythrocytes added secondly to the medium. The so-released hemoglobin can therefore be used as a dye in a colorimetric titration.
The CH50 unit is the concentration of complement units in the serum able to cause 50% hemolysis of a fixed volume of sheep erythrocytes after exposure to the nanohybrids.
The experimental results are expressed as the percentage of the CH50 unit consumed as a function of the nanohybrid surface (Figure 5).
Although PVOH/C60and P(PEGA-co-VAc)/C60were tested within the same concentration range, the surface area of PVOH/C60exposed to the solution was smaller [0–700 vs.
0–3600 cm2mL1 for P(PEGA-co-VAc/C60)] because PVOH/C60aggregates in water and forms bigger particles [110 nm vs. 21 nm for P(PEGA-co-VAc)/C60], as determined by DLS). Therefore, the surface area of PVOH/C60exposed to the solution was much lower than for P(PEGA-co-VAc)/C60 at the same concentration, which explains the gap between both curves along the x-axis. Also, further increasing the amount of PVOH/C60 to reach higher surface areas was pointless since the complement activation was already 100%. For both molecules, the CH50 consumption increases with increasing nanohybrid concentration in the serum, which is an expected behavior since the surface area in contact with proteins is higher. Protein adsorption is much lower for P(PEGA-co-VAc)/C60than for PVOH/C60that already adsorbs proteins at low exposed surface areas. This discrepancy might be ascribed to the higher size of PVOH/
C60aggregates since bigger particles are less stealthy than small ones.[52]In addition, the complement system might be further activated by hydroxyl functions in PVOH/C60.[32]
Although PVOH is a complement activator, it is non toxic and possesses many reactive groups allowing chemical coupling with ligands for tumor active targeting and is therefore a valuable polymer for drug delivery purposes.
Also, the complement activation does not always reflect the plasma clearance measured in vivo,[32] and PVOH/
phthalocyanine conjugates for PDT were shown to have a long plasma half-life (6.8 h).[53]
The comparatively lower complement activation induced by P(PEGA-co-VAc)/C60is ascribed to the PEG chains at the outer-shell that are known to be protein-repellent due to their hydrophilicity and high chain mobility.[32]
Since it turned out to be the most efficient PS of all three, P(NVP-co-VAc)/C60 prepared previously[21] was therefore tested for complement activation. Its activation profile is similar to that of P(PEGA-co-VAc)/C60 and therefore indicates a low to moderate complement activa- tion. This behavior is in agreement with the observed long blood circulation time of PNVP.[54]P(NVP-co-VAc)/C60 is therefore the most efficient PS of the three, since it has a four times higher singlet oxygen quantum yield and is a moderate complement system activator. Comparisons with other polymer/C60nanohybrids designed for PDT[8–13]
will only be possible when their fundamental photo- physical properties, i.e., singlet oxygen quantum yield, are determined.
4. Conclusion
Water-soluble PVOH/C60 and P(PEGA-co-VAc)/C60 nano- hybrids prepared by CMRP were assessed as PSs for PDT.
0 20 40 60 80 100
0 500 1000 1500 2000 2500 3000 3500 4000 Nanohybrids surface area (cm2/mL)
CH50 units consumpsion(%)
PVOH/C60
P(PEGA-co-VAc)/C60 P(NVP-co-VAc)/C60
0 20 40 60 80 100
0 500 1000 1500 2000 2500 3000 3500 4000
2/mL)
CH50 units consumpsion(%)
PVOH/C60
P(PEGA-co-VAc)/C60 P(NVP-co-VAc)/C60
60
-co-VAc)/C60 P(NVP-co-VAc)/C60
Figure 5. Consumption of CH50 units induced by the water- soluble polymer/C60nanohybrids.
Their photophysical properties and photodynamic activities were determined by flash photolysis and in vitro assays, respectively. The triplet states of both nanohybrids were shown to be quenched by ground state oxygen, thereby generating singlet oxygen, a reactive oxygen species that is toxic towards target cells. The singlet oxygen production upon irradiation was similar in both materials, indicating a similar functionalization of the C60cores. Importantly, in vitro toxicity tests performed on cancer cells revealed that the two nanohybrids are not cytotoxic in the absence of irradiation. A concentration and light dose dependent photocytotoxicity was observed for PVOH/C60upon irra- diation, but PVOH is a complement system activator. On the other hand, no photoinduced cytotoxicity was detected for P(PEGA-co-VAc)/C60, which could be ascribed to the trapping effect of the PEG grafts. Therefore, the molecular structure (linear/graft) and the nature (presence/absence of hydroxyl groups) of the polymeric arms impact the photodynamic activity and the stealth properties of the nanohybrid. The identification of such key characteristics may help to direct further efforts in the field. To date, the most efficient nanohybrid prepared by CMRP remains P(NVP-co-VAc)/C60 that has linear hydrosoluble arms without hydroxyl groups.[21]Regardless of the nature of the polymers grafted on C60, the performances of the nanohybrids prepared by CMRP would undoubtedly be improved by decreasing the number of grafts, which would induce a lesser perturbation of the electronic system of C60 and decrease the possible steric crowding around C60.
Acknowledgements: The authors are grateful to the ‘‘Belgian Science Policy’’ for the financial support in the frame of the Interuniversity Attraction Pole Programme (PAI VI/27) and to the National Fund for Scientific Research (F.R.S.-FNRS). A.D. and C.D.
are research associate and senior research associate by the F.R.S.-FNRS, respectively.
Received: July 18, 2012; Revised: August 30, 2012; Published online: November 29, 2012; DOI: 10.1002/mabi.201200251 Keywords: biological applications of polymers; cobalt-mediated radical polymerizations; fullerenes; graft copolymers; photo- dynamic therapy
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