Article pubs.acs.org/Biomac

Polymeric Micelles Encapsulating Photosensitizer: Structure/ Photodynamic Therapy Efficiency Relation Laure Gibot,† Arnaud Lemelle,‡ Ugo Till,‡ Béatrice Moukarzel,‡ Anne-Françoise Mingotaud,*,‡ Véronique Pimienta,‡ Pascale Saint-Aguet,§ Marie-Pierre Rols,*,† Mireille Gaucher,∥ Frédéric Violleau,∥ Christophe Chassenieux,⊥ and Patricia Vicendo*,‡ †

Equipe de Biophysique Cellulaire, IPBS-CNRS UMR 5089, 205 route de Narbonne, BP 64182, 31077 Toulouse Cedex, France Université de Toulouse, UPS/CNRS, IMRCP, 118 route de Narbonne, F-31062 Toulouse Cedex 9, France § Technopolym, Institut de Chimie de Toulouse, 118 route de Narbonne, F-31062 Toulouse Cedex 9, France ∥ Université de Toulouse, Institut National Polytechnique de Toulouse − Ecole d’Ingénieurs de Purpan, Département Sciences Agronomiques et Agroalimentaires, UPSP/DGER 115, 75 voie du TOEC, BP 57611, F-31076 Toulouse Cedex 03, France ⊥ LUNAM Université, Université du Maine, IMMM UMR CNRS 6283 Département PCI, Avenue Olivier Messiaen, 72085 Le Mans Cedex 09, France ‡

S Supporting Information *

ABSTRACT: Various polymeric micelles were formed from amphiphilic block copolymers, namely, poly(ethyleneoxide-b-ε-caprolactone), poly(ethyleneoxide-b-D,L-lactide), and poly(ethyleneoxide-b-styrene). The micelles were characterized by static and dynamic light scattering, electron microscopy, and asymmetrical flow field-flow fractionation. They all displayed a similar size close to 20 nm. The influence of the chemical structure of the block copolymers on the stability upon dilution of the polymeric micelles was investigated to assess their relevance as carriers for nanomedicine. In the same manner, the stability upon aging was assessed by FRET experiments under various experimental conditions (alone or in the presence of blood proteins). In all cases, a good stability over 48 h for all systems was encountered, with PDLLA copolymer-based systems being the first to release their load slowly. The cytotoxicity and photocytotoxicity of the carriers were examined with or without their load. Lastly, the photodynamic activity was assessed in the presence of pheophorbide a as photosensitizer on 2D and 3D tumor cell culture models, which revealed activity differences between the 2D and 3D systems.

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INTRODUCTION The recent development of light-irradiation systems in medicine (lasers, optical fibers, endoscopic procedures) has facilitated the emergence of new diagnostic protocols as well as selective therapies based on light-sensitive drugs. Photochemotherapies or photodynamic therapy (PDT) involve the light irradiation of photosensitizers, which generates active molecular species, such as free radicals and singlet oxygen. These short-lived species are highly toxic in biological environments. Moreover, some photosensitizers selectively accumulate in proliferating tissues. This specificity property is used for the treatment of several oncologic1 and ophthalmic diseases.2 At present, the use of PDT has been approved for clinical treatment in the U.S., EU, Canada, Russia, and Japan. The FDA approved the use of PDT in Barrettś esophagitis, obstructive tracheobroncheal carcinoma, using the photosensitizer porfimer sodium (Photofrin) as well as the use of 5-aminolevulinic acid, 5-ALA (Levulan, Kerastick), for actinic keratosis, whereas verteporfrin (Visudyne) may be applied for macular degeneration. In addition, the EU also approved the use of meta-tetrahydroxy-phenyl chlorin (mTHPC, Foscan) for the palliative treatment of advanced cases of head and neck © 2014 American Chemical Society

carcinomas. The difference in the photosensitizer concentration between normal and neoplastic tissues and the ability to focus the light irradiation on the target zone are the key advantages of this technology. Numerous photosensitizers are amphiphilic or hydrophobic compounds, which have a tendency to self-associate in physiologic aqueous environments, leading to a loss of their physical properties. Hence, different delivery systems (liposomes, Cremophor EL, etc.) have been evaluated for the transport of water-insoluble photosensitizers. The use of specific delivery systems can modulate photosensitizers pharmacokinetics and cellular uptake.3 To improve PDT, nanometric formulations of photosensitizers have been assessed because the size of the vector enables its accumulation in solid tumors as a result of the enhanced permeability and retention effect (EPR). One of these formulations uses liposomes, which are multilayered self-assemblies of low-molecular-weight surfactants. Received: January 10, 2014 Revised: February 5, 2014 Published: February 19, 2014 1443

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Early systems used regular liposomes, which tended to be eliminated very quickly from body because of quick lipid exchange and because of their detection by the reticuloendothelial system (RES).3 Second-generation liposomes were modified on their surface with poly(ethyleneglycol) (PEG) chains, which rendered them invisible to RES, thereby enabling them to have long circulating times in blood (t1/2 of tens of hours) and to accumulate efficiently in tumors. However, in some instances, they were observed to be too stable and did not release the drug at all.3 This was, however, not observed in the case of liposomal formulations of mTHPC in conventional and pegylated liposomes developed by Biolitec, the so-called Foslip and Fospeg.4 Another nanometric carrier under consideration is based on silica nanoparticles.5 These have the advantage of being chemically inert and porous, and they do not swell with pH. Various systems have been studied, either by simply entrapping the photosensitizer inside the nanoparticle or grafting it covalently to silica. The PDT results are good and show, in some instances, stronger 1O2 generation in the presence of the nanoparticle. However, a shortcoming of these carriers is their lack of degradability. In the literature, only a few studies have presented the use of polymeric vectors to encapsulate photosensitizers. The very first, to our knowledge, was developed by Leroux and colleagues and used polyion-complex micelles to deliver a phthalocyanine.6,7 A few years later, Kataoka and colleagues also used polyioncomplex micelles to deliver a porphyrin.8,9 Interestingly, these studies revealed that the photosensitivity side effect that led to the photosensitization of the patient over several days disappeared with this new formulation when this was tested in vivo.8,9 Another study published by Allen showed that poly(ethyleneoxide-b-ε-caprolactone)5000−4000 micelles are good candidates for protoporphyrin IX transport.10 On the basis of this analysis, we decided to examine, in a comparative manner, different polymeric micelles as a photosensitizer nanovector and to determine their efficiency in vitro on 2D and 3D tumor models, namely, spheroids.11,12 This model is based on cell-aggregation properties. Three-dimensional cell culture models are more relevant to in vivo cell organization because extracellular matrix as well as homo- or hetero-cell−cell contacts are present. It is indeed known that large tumor spheroids display gradients (e.g., oxygen, nutrients, catabolites, and proliferation) such as those observed in poorly vascularized regions of solid tumors.13,14 Another advantage of spheroids is that they avoid the use of animals, in agreement with the 3R rule on animal experimentation. In the field of nanomedicine, several teams have assessed the use of spheroids to evaluate the penetration of encapsulated drugs in 3D tumor models.15 For instance, Kataoka followed the penetration of encapsulated adriamycin with time in C26 spheroids.16 They showed that the drug remained trapped for 1 h before being released in the spheroid. Very recently, Kwon used both 2D and 3D cell culture models to assess the efficiency of combination of anticancer drugs encapsulated in poly(ethyleneoxide-b-ε-caprolactone) nanovectors.17 In a similar way, Allen compared the action of encapsulated docetaxel and that of taxotere in 2D or 3D cell culture models,18 showing that the cells reacted differently to treatment under 2D or 3D conditions and determined the conditions for growth inhibition of the spheroids. The use of spheroids to evaluate PDT was reviewed in 2006.19 Together, these studies proved to be useful tools to understand the basics of photosensitizer activity on 3D systems. To the best of our knowledge, no study so far has evaluated the therapeutic index of polymeric nanovectors for PDT using spheroids.

Article

MATERIALS AND METHODS

The polymers were purchased from Gearing Scientific, and the molecular weights were characterized by size-exclusion chromatography and 1H NMR. Pheo was obtained from Wako. 1,1′-Dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC) and 3,3′dioctadecyloxacarbocyanine perchlorate (DiOC) fluorophores were obtained from Invitrogen Life Technologies (Saint Aubin, France). PrestoBlue was purchased from Invitrogen. Preparation of Polymer Micelles. Preparation of polymeric micelles was performed by dispersing an acetone polymer solution into ultrapure filtered water as previously described.20,21 Twenty milligrams of the polymer was dissolved in 0.4 mL of acetone. Acetone was preferred over ethanol for solubility reasons. This was added dropwise to 5 mL of ultrapure water (filtered on 0.2 μm RC filters). The solution was left standing for 2 days to remove acetone. Loading of the micelles with photosensitizer or fluorophores was done by adding the latter to the acetone solution. Dynamic Light Scattering (DLS). DLS was carried out at 25 °C on a Malvern Zetasizer NanoZS. Solutions were analyzed without being filtered to characterize the plain samples. Data were analyzed by the general-purpose non-negative least-squares (NNLS) method. The typical accuracy for these measurements was 10−15%. Zeta potential was also measured using Smoluchowski’s model. Simultaneous Static and Dynamic Light Scattering. Static light scattering (SLS) and dynamic light scattering (DLS) measurements were performed on a ALV CGS3 spectrometer operating at λ = 632.8 nm. All LS data were collected at 37 °C. SLS and DLS data were recorded simultaneously for each system. Measurements were made using six different concentrations ranging from 0.04 to 4 mg mL−1 and seven different angles (θ) ranging from 30 to 150°. For these experiments, the solutions were filtered at 0.2 μm. The scattering vector was defined as q = 4πn/λ[sin(θ/2)], in which n is the refractive index of the solvent. The scattered intensity was measured over a period varying from 30 to 80 s to determine both the intensity autocorrelation function, g2(t), for DLS and the mean scattered intensity, I, for SLS. For DLS data, the measured g2(t) was related to the electric-field autocorrelation function, g1(t), using the Siegert relation. g1(t) was analyzed using the REPES routine assuming a continuous distribution of relaxation times, A(log(τ)), according to eq 1. The difference between the measured and calculated baseline for the DLS correlation functions was less than 0.1%.

g1(log(t )) =

∫0

∞

⎛ t⎞ τA(τ ) exp⎜ − ⎟ d[log τ ] ⎝ τ⎠

(1)

For some samples, the resulting distributions exhibited two relaxation processes (Figure S1, Supporting Information). The relaxation times were q2-dependent (Figure S2, Supporting Information), indicating that diffusive motions were probed. The apparent diffusion coefficient (D) was calculated using the relation D = (q2τ)−1. The diffusion coefficient was used to compute the hydrodynamic radii (RH) for the fast relaxation mode according to the Stokes−Einstein equation. For SLS experiments, both the slow and fast modes of relaxation contributed to the total scattered intensity. The Rayleigh ratio for the fast mode (Rθ,fast) of relaxation was calculated according to eq 2

R θ ,fast = A fast (θ)R θ = A fast

Isample(θ) − Isolvent(θ) Ireference(θ)

R reference

(2)

where Afast(θ) is the relative amplitude of the fast mode of relaxation derived from DLS (eq 1), Isample, Isolvent, and Ireference are the scattered intensities at angle θ by the sample, the solvent, and a reference (toluene), respectively, and Rreference is the Rayleigh ratio for toluene. Assuming the concentration of species contributing to the fast mode of relaxation was equal to the overall polymer concentration in solution (i.e., negligible concentration of larger species), the weight-average molecular weight (Mw), second virial coefficient (A2), and radius of gyration (Rg) can be estimated according to

Kc R θ ,fast 1444

=

⎛ q2R g2 ⎞ 1 ⎜ ⎟ + 2A 2 c 1 + M w ⎜⎝ 3 ⎟⎠

(3)

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(no. 2310). HCT-116 and fibroblasts cells were both grown in Dulbecco’s modified Eagles medium (Invitrogen) containing 4.5 g L−1 of glucose as well as L-glutamine and pyruvate and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U mL−1 of penicillin, and 100 μg mL−1 of streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Generation of Healthy and Tumor Spheroids. HCT-116 and fibroblast spheroids were produced by the nonadherent technique as previously described.22,23 Briefly, 5000 cells in suspension were seeded in 150 μL of medium in each well. The plate was centrifuged for 5 min at 4 °C and 300g to accelerate cell sedimentation. Spheroids were cultivated for 5 days at 37 °C in a humidified atmosphere containing 5% CO2 before experiments were performed. Cytotoxicity and Photocytotoxicity of Polymeric Micelles on Adherent Cells. HCT-116 cells were seeded into 96-wells plates. The cytotoxicity of polymers alone (1, 10, and 100 μM) or polymers loaded with pheo (1:30) was assessed after a short (30 min) or long (48 h) incubation time. The phototoxicity of polymers alone or polymers loaded with pheophorbide a (1:30) was assessed after a 30 min incubation at 37 °C followed by photoactivation. Illumination was performed with an overhead projector with a band-pass filter (λ > 400 nm, 8.2 J cm−2).21 Concentrations used for pheophorbide alone were 1:30 of polymers (i.e., 0.033, 0.33, and 3.3 μM). After incubation with nanoparticles or pheo alone, cells were washed, fresh medium was added, and cells were incubated for 24 h. Viability was then assessed with PrestoBlue reagent (Invitrogen), which was read by a spectrophotometer according to the manufacturer’s instructions. For every set of experiments, six biological replicates were produced and analyzed. Statistical differences between values were assessed by Dunnett’s multiple comparison test, which compares each condition with its respective control. All data were expressed as the mean ± SEM, and overall statistical significance was set at p < 0.05. Free or Encapsulated Pheophorbide Penetration in 2D and 3D Cellular Models. For the localization of pheophorbide a in the 2D culture model, cells were grown on coverslips and then incubated at 37 °C for 30 min with 5 μM nanoparticles loaded with Pheo. Once the cells were washed with PBS, the fluorescence was observed using an Olympus FV1000 fluorescence confocal microscope with an excitation wavelength at 578 nm and an emission wavelength at 610 nm. The signal of Pheo was red. Similarly, 5 day old spheroids were incubated with free or encapsulated Pheo and observed by two-photon microscopy on 7MP FLIM microscope (Zeiss, Le Pecq, France). The images were stacked using ImageJ software (NIH, Bethesda, MD, USA). Photodynamic Therapy of 3D Tumor Spheroids. HCT-116 spheroids were incubated with free or encapsulated 1 μM Pheo for 30 min before the first illumination. Spheroids were photoirradiated for 8 min using an overhead projector lamp with a band-pass filter (λ > 400 nm). The total energy received was 8.2 J cm−2 for the duration of 8 min. Then, 8 min illumination took place every 24 h for three illuminations in total. Spheroids treated by photodynamic therapy were observed 24 h after the last illumination (i.e., after 96 h of incubation with polymeric micelles). A blank test showed that the possible residual acetone was not responsible for the observed effect. Cell Viability after Tumor Spheroid PDT. After the PDT procedure, six spheroids and their associated culture media were collected for each condition (control, PEO(2000)-b-PCL(2800)/Pheo, and PEO(2400)-b-PDLLA(2000)/Pheo). After centrifugation to collect both spheroids and detached cell induced by PDT, spheroids were dissociated with trypsin and then incubated with 2 μM calcein AM (Invitrogen, Carlsbad, CA, USA) for 10 min at 37 °C. Calcein AM is known to label viable cells in green. The percentage of viable cells as well as the percentage of cells loaded with Pheo were quantitatively determined by flow cytometry (FACSCalibur, Becton Dickinson, USA).

in which c is the solution concentration and K is a constant, given by eq 4.

K=

2 4π 2nref (dn/dc)2 4 λ NA

(4)

In eq 4, nref is the refractive index of toluene, dn/dc is the refractive index increment of the polymer, and NA is Avogadro’s number. The refractive index increment dn/dc of the copolymer micelles in water was measured with a PSS dndc-2010 instrument at 620 nm and was measured at 37 °C as

PEO(2000)-b-PCL(2800) PEO(5000)-b-PCL(4000) PEO(2400)-b-PDLLA(2000) PEO(3100)-b-PS(2300)

0.167 mL g−1 0.142 mL g−1 0.230 mL g−1 0.383 mL g−1

Kc/Rθ,fast was independent of q2 (Figure S3, Supporting Information); therefore, Kc/Rθ,fast values were averaged for all angles to determine Mw. The reported error for all light-scattering results was 10% of the average value. The size of the objects and the concentrations used did not enable us to characterize the radius of gyration and the second virial coefficient. Transmission Electron Microscopy (TEM). TEM experiments were performed with a Hitachi HT7700 microscope (accelerating voltage of 75 kV). Small amounts of particle suspensions in water were deposited onto a discharged copper grid coated with a carbon membrane and wiped with absorbing paper. A few drops of uranyl acetate solution were deposited onto the grid for 30 s, and the grid was then dried under a lamp for 3 min. AsFlFFF. The asymmetric flow field-flow fractionation instrument was an Eclipse 2 system (Wyatt Technology Europe, Dernbach, Germany). The AsFlFFF channel had a trapezoidal geometry with a length of 17.3 cm, an initial breadth of 1.1 cm, and a final breadth of 0.27 cm. A 250 μm thick Mylar spacer was placed between the ultrafiltration membrane and the upper glass plate. The accumulation wall was an ultrafiltration membrane of regenerated cellulose with a 10 kDa cutoff (Wyatt Technology Europe, Dernbach, Germany). An Agilent 1100 series isocratic pump (Agilent Technologies, Waldbronn, Germany) with an in-line vacuum degasser and an Agilent 1100 autosampler delivered the carrier flow and handled sample injection into the AsFlFFF channel. A 0.1 μm in-line filter (VVLP, Millipore, Germany) was installed between the pump and the AsFlFFF channel. The products were detected with an 18 angles multi-angle light scattering (MALS) DAWN Heleos II (Wyatt Technology, Santa Barbara, CA, USA), an Optilab Rex refractometer (Wyatt Technology, Santa Barbara, CA, USA), and a UV detector (Agilent 1100, λ = 214 or 412 nm). The MALS detectors were normalized with bovine serum albumin (BSA). Calibration of scattering intensity was performed with HPLC-grade filtered toluene. Water, which was filtered with 0.02% sodium azide before use (vacuum filtration system using 0.1 μm Gelman filters), was used as an eluent. For separation, the channel flow rate, Vout, was fixed at 0.3 mL min−1. In focus mode, the flow rate was stabilized 1 min before injection at 1.5 mL min−1. Five microliters of the sample solution was injected into the AsFlFFF channel at a flow rate of 0.2 mL min−1 for 6 min. After injection, 1 min of focus was maintained before the elution started. In elution mode, the cross-flow rate was fixed at 0.4 mL min−1 for 20 min. Cross flow was then stopped to eliminate all particles present in the AsFlFFF system. Dialysis. The solution of Pheo alone or Pheo-loaded polymeric micelles was introduced in a dialysis cell (GE Healthcare Bio-Sciences, MWCO 8 kDa), and the release of Pheo was followed by measuring the optical density of the internal solution at 688 and 669 nm. Stability of Copolymer Micelles in the Presence of Proteins. Micelles were prepared as described above with 2 mol % DiOC and 2 mol % DiIC. To begin the stability follow up, 50 μL of micelle solution was added to 1.95 mL of protein solution (10 mg mL−1 of γ-globulin, 40 mg mL−1 of BSA, and 28 mg mL−1 of α- and β-globulin in 10% FBS), and fluorescence spectra (excitation at 470 nm) were recorded versus time. Cell Culture. The HCT-116 cell line (ATCC no. CCL-247) originated from a human colorectal carcinoma. Human dermal fibroblasts cells were purchased from ScienCell Research Laboratories

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RESULTS AND DISCUSSION

Formation and Characterization of the Polymeric Micelles. Different polymeric self-assemblies were generated by a cosolvent method in water, and the chosen photosensitizer was pheophorbide a (Pheo) (Chart 1). Polymeric micelles were 1445

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Chart 1. Polymers and PS Used

characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Their characteristics are listed in Table 1, and typical DLS and TEM data are shown in Figure 1. Note that during the experimental course PEO(2000)b-PDLLA(2200) had to be replaced with PEO(2400)b-PDLLA(2000) because of availability purposes. Nevertheless, such a modification of the sizes of both blocks had only a slight influence on the size of the polymeric micelles, as evidenced in Table 1. For DLS analysis, two values for the hydrodynamic diameter are given, which correspond to intensity and number averages. Intensity averages are computed without making any calculation hypothesis but are influenced to a larger extent by the presence of big scatterers within the samples that we decided not to filter (see Materials and Methods) in order to investigate their plain behavior. In that sense, intensity averages are more representative of the plain samples. Regardless of the chemical nature of the block copolymers used, the size of the micelles ranged between 20 and 30 nm, and their polydispersity indices were between 0.2 and 0.3 in all cases. For PEO(2000)-b-PDLLA(2200), large aggregates were present with the polymeric micelles, but they were present in a very small amount because their contribution to the signal disappeared when number averages were considered. In the case of Pheo-loaded polymeric micelles, size values obtained from DLS should be taken with caution because part of the incident beam was absorbed by Pheo. In all cases, the loading did not alter the size of the micelles, although, in some instances, it led to a small increase. Zeta potentials were found to be slightly

Figure 1. DLS (A, B) and TEM (C, D) analyses of polymeric micelles: red, PEO(2000)-b-PDLLA(2200); green, PEO(3100)-b-PS(2300); blue, PEO(2000)-b-PCL(2800); black, PEO(5000)-b-PCL(4000); and pink, PEO(2400)-b-PDLLA(2000). (C) PEO(2400)-b-PDLLA(2000) and (D) PEO(3100)-b-PS(2300).

negative in almost all cases and were close to each other, although the values were lower for PEO-PCL systems. They were found to fluctuate depending on the aging of the polymer, probably owing to the presence of carboxylate moieties formed by the slow degradation of polycaprolactone chains. For testing the stability of the micelles, they were also loaded with two fluorophores, namely, DiIO and DiIC. The sizes measured by DLS are also reported in Table 1; they remained on the same order of magnitude and the zeta potential increased only slightly, which was attributed to the positive charge of both fluorophores. TEM measurements (Figure 1) determined the spherical shape of the polymeric micelles. Furthermore, the apparent discrepancy between results from TEM and DLS vanished when averages of the same order are considered (i.e., number averages for both techniques, see Table 1).

Table 1. Characteristics of Polymeric Self-Assemblies polymer

molecular weight

intensity average diameter (DLS) (nm)a

number average diameter (DLS) (nm)

ξ (mV)b

PEO(2000)-b-PCL(2800) PEO(5000)-b-PCL(4000) PEO(2000)-b-PDLLA(2200) PEO(2400)-b-PDLLA(2000) PEO(3100)-b-PS(2300) PEO(2000)-b-PCL(2800)/Pheo 1:30 PEO(5000)-b-PCL(4000)/Pheo 1:30 PEO(2000)-b-PDLLA(2200)/Pheo 1:30 PEO(2400)-b-PDLLA(2000)/Pheo 1:30 PEO(3100)-b-PS(2300)/Pheo 1:30 PEO(2000)-b-PCL(2800) DiIC/DiOC 2 mol % PEO(5000)-b-PCL(4000) DiIC/DiOC 2 mol % PEO(2000)-b-PDLLA(2200) DiIC/DiOC 2 mol % PEO(2400)-b-PDLLA(2000) DiIC/DiOC 2 mol % PEO(3100)-b-PS(2300) DiIC/DiOC 2 mol %

2000−2800 5000−4000 2000−2200 2400−2000 3100−2300 2000−2800 5000−4000 2000−2200 2400−2000 3100−2300 2000−2800 5000−4000 2000−2200 2400−2000 3100−2300

22.0 24.6 27.0/206.0 30.3 19.9 27.2 25.6 24.7/129.0 30.4 21.4 24.5 27.1 20.9/132.0 23.9 19.3

13.7 15.3 17.6 16.8 13.2 13.6 10.6 18.1 10.5 8.5 15.4 16.7 18.1 14.3 10.3

−10 −10 −2.3 −2.4 −1.8 −14.0 −19.5 −6.2 n.d. −14.9 −3.7 −15.0 −2.8 n.d. +0.6

average diameter (TEM) (nm) 13.7 ± 2.7 12.6 ± 2.7 13.8 ± 4.4 n.d. 12.5 ± 2.5 16.8 ± 2.7

11.5 ± 3.5

a

The accuracy of the DLS measurements is estimated to be between 10 and 15%. bThe values of the zeta potential for empty particles was observed to fluctuate depending on the aging of the polymer and ranged between −1.0 and −16.0. 1446

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Figure 2. AsFlFFF characterization of PEO(2400)-b-PDLLA(2000) and PEO(3100)-b-PS(2300) micelles. For all curves, the dotted line is the MALLS signal, the dashed line is the RI signal, and the full-line absorption signal is at 412 nm. (a) Empty PEO(2400)-b-PDLLA(2000), (b) PEO(2400)-bPDLLA(2000)/Pheo, (c) empty PEO(3100)-b-PS(2300), and (d) PEO(3100)-b-PS(2300)/Pheo.

Table 2. Characterization of PEO(3100)-b-PS(2300) and PEO(2400)-b-PDLLA(2000) Polymeric Self-Assemblies by AsFlFFF polymer a

PEO(2000)-b-PCL(2800) PEO(5000)-b-PCL(4000)a PEO(2400)-b-PDLLA(2000) PEO(3100)-b-PS(2300) a

DH (nm)

Mw (g mol−1)

Mn (g mol−1)

Nagg

Đ = Mw/Mn

20.2 26.8 16.2 20.0

960 000 1.72 × 106 3.56 × 105 5.49 × 105

947 000 1.70 × 106 3.47 × 105 5.47 × 105

200 190 80 100

1.01 1.01 1.03 1.00

Data from ref 20; the values of the molecular weights and Nagg have been re-evaluated according to the more precise dn/dc characterization.

corresponding to the polymeric micelles. In some instance, a small peak could also be seen in the light-scattering signal at the end of the elution, demonstrating the presence of a very small amount of larger aggregates (500 nm). However, this was only observed in the intensity average values and not on the number average ones, demonstrating that although it was present, this larger population was minor in terms of concentration. Figure 4 represents the intensity average hydrodynamic size for a Figure 5. Pheo release by dialysis ([Pheo] = 10−6 M).

This result can be related to previous ones showing an instantaneous release of doxorubicin from PEO-PDLLA micelles after injection.26 Because PDT treatments of spheroids were performed over several-day periods, this important fact must be kept in mind. These results allowed computing the affinity constant (K) between Pheo and each type of polymeric micelles by fitting them with a single exponential related to the release of Pheo according to Pheo NP ⇌ Pheofree + NP

K = [Pheofree][Polymer]/[PheoNP] (see Supporting Information for more details) The values found for K are PEO(2400)-b-PDLLA(2000) 2.61 × 10−6 M PEO(2000)-b-PCL(2800) 2.34 × 10−7 M PEO(5000)-b-PCL(4000) 1.57 × 10−7 M PEO(3100)-b-PS(2300) 1.09 × 10−7 M The value obtained for PEO(5000)-b-PCL(4000) is smaller than the one obtained from fluorescence measurements on the same system,27 which was 9.6 × 10−6 M. However, in the fluorescence experiments, the penetration of Pheo was followed over short times, implying that the path followed was not the same. Because the objects are not at thermodynamical equilibrium, this might explain the difference. Lastly, the stability was examined by FRET experiments using two fluorescent probes, namely, 1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DiIC) and 3,3′dioctadecyloxacarbocyanine perchlorate (DiOC). These hydrophobic fluororophores are widely used in biology because of their preferred location in hydrophobic areas.28 Polymeric micelles were prepared in the presence of both probes in the same manner, ensuring their encapsulation and the appearance of a FRET signal (Figure 6). The fluorescence band of DiOC at 503 nm decreased in favor of that of DiIC at 567 nm. Therefore, the ratio between these two bands was a direct indication of the proximity of both probes. The fluorescence spectra of DiIC/ DiOC pairs encapsulated in the different polymeric selfassemblies were followed as a function of time in various media: PBS, 10% fetal bovine serum, cell culture medium, 10 mg mL−1 of γ-globulin, 40 mg mL−1 of BSA, and 28 mg mL−1 of α- and β-globulin. These conditions were used to mimick the

Figure 4. Stability of Pheo-loaded polymeric micelles (1:30 mol/mol) at 37 °C by DLS.

1:50 dilution of the different polymeric micelles. For all of them, a good stability was observed over the experimental period, although the larger population slowly increased in quantity. To assess the quality of the samples, we decided not to consider solutions older than 14 days, although this choice may appear pretty drastic considering the DLS results. The stability was then evaluated by the release of Pheo that was followed by dialysis using solutions of polymeric micelles loaded with 1:30 mol/mol Pheo and a Pheo concentration of 10−6 M. At room temperature, no release was observed over 12 days (data not shown). At 37 °C, a slow release was observed over several days (Figure 5). Whereas unencapsulated Pheo was completely released after 3 days, PEO-PCL micelles still contained half of the starting Pheo at that point. PEO(3100)-b-PS(2300) micelles exhibited the slowest release (30% after 4 days). A strong difference was observed for PEO-PDLLA micelles, which exhibited almost the same dialysis pattern as free Pheo. Because the UV−vis absorption spectra and AsFlFFF characterization showed that Pheo was indeed encapsulated in the PEO(2400)-bPDLLA(2000) micelles (or at least in a hydrophobic environment), this dialysis result proves that Pheo is not strongly retained and is very easily released from the micelles. PEOPDLLA micelles cannot then be considered as efficient vectors. 1449

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b-PS(2300) was the most stable system, remaining almost unchanged after 72 h. The least stable system was PEO-PDLLA, showing only 36% remaining FRET signal after 72 h, in agreement with the results obtained from the dialysis experiments and literature.29 PEO-PCL micelles exhibited a better stability than PEO(2400)-b-PDLLA(2000) for the first 24 h, but they ended at the same remaining FRET ratio after 72 h. These results are consistent with very recent ones on PEO-PCL and PEO(3100)-b-PS(2300) larger nanoparticles, which were shown to be stable for at least 2 h at 37 °C either in cell culture medium or mouse blood plasma.30 In complement, we also carried out FRET experiments where DiOC was encapsulated in the polymeric vectors and DiIC was in the aqueous solution. Indeed, one could argue that in the previous set of experiments DiOC and DiIC could be released as a pair in the aqueous solution, which would still exhibit FRET but outside the vector. Starting from a situation where they were separated from each other, the appearance of FRET with time enabled us to verify the kinetics of penetration of the fluorophores inside the micelles and therefore their stability. Fluorescence of DiIO-loaded polymeric micelles was then measured with time in the presence of a solution of DiIC (PBS or 10% FBS). A typical result is presented in Figure 8. The first spectrum corresponds to that of emitting DiIO; the shoulder at 535 nm is attributed to the serum. With time, a decrease of the DiIO band was observed simultaneously with an increase of a band at 567 nm, demonstrating the appearance of FRET between both probes. For most systems, such a phenomenon started only after 42 h, until which time the polymeric micelles may be considered as keeping their integrity. In the case of PEO(2400)-b-PDLLA(2000) micelles, FRET already appeared after 24 h and grew with time, in agreement with results obtained from the first FRET experiments where both probes were encapsulated within the micelles. Cellular Investigations and Evaluation of the Phototoxic Activity. Before assessing the PDT efficiency of the various systems, the toxicity of the polymeric vectors alone was evaluated using a PrestoBlue test on plated cells. The results are presented in Figure 9. None of the polymeric vectors exhibited

Figure 6. Fluorescence spectra of fluorophores encapsulated in PEO(2000)-b-PCL(2800). λem = 430 nm. Full line, DiIC 2 mol %; dotted line, DiOC 2 mol %; and dashed line, DiIC + DiOC 2 mol %.

Figure 7. Typical fluorescence evolution with time.

different environments encountered by the micelles during medical treatment. Figure 7 shows a typical evolution with time of the FRET signal, and quantitative results are summarized in Table 4, where only the experiments with 10% FBS are presented for clarity reasons. The other experiments are in the Supporting Information. Several experiments were performed at room temperature. The tests performed with PEO(2000)-b-PCL(2800) showed that PBS and α,β-globulins had no influence on the stability of the polymeric micelles. As for other media, γ-globulin led to a partial destabilization over 48 h for PEO(5000)-b-PCL(4000) and PEO(2000)-b-PDLLA(2200). BSA and 10% FBS were observed to be the most disrupting media. In all of these experiments, the PEO(3100)-b-PS(2300) self-assemblies remained stable. Because 10% FBS was found to be the most disrupting medium, further experiments at 37 °C were performed only in this medium. In comparative tests, complete cell culture medium with 10% FBS was shown to exhibit the same behavior as 10% FBS (data not shown), and this was also the case for experiments performed in the presence of cell lysates, thus corroborating this choice. Further experiments assessed the stability of the different micelles at 37 °C in the presence of 10% FBS. An interesting point to note is that experiments performed with stock solutions of polymer micelles (4 mg mL−1) did not show any instability of the system over a 42 h period. This is valuable information regarding solution storage. The subsequent tests were carried out after a 1:100 dilution, corresponding to the concentration used for PDT tests. These tests revealed once again that PEO(3100)-

Figure 8. Appearance of FRET in the case of separated fluorophores using the PEO(2000)-b-PCL(2800) system in 10% FBS at 37 °C. 1450

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Figure 9. Cytotoxicity and phototoxicity of increasing concentrations of polymers alone or loaded with pheophorbide: (A) 30 min incubation, (B) 48 h incubation, and (C) 30 min incubation followed by illumination. White bar, 1 μM polymers or 0.033 μM Pheo alone; gray bar, 10 μM polymers or 0.33 μM Pheo alone; and black bar, 100 μM polymers or 3.33 μM Pheo alone. n = 6. *, p < 0.05; **, p < 0.01.

Table 4. FRET Experiments in 10% FBS [polymer] (mg mL−1)

probe loading (mol %)

[DiIC] (M)

temperature (°C)

kinetics time (h)

I567/I503 (time, h)

PEO(2000)-b-PCL(2800)

0.04

2.0

1.7 × 10−7

37

72

2.2 (0), 1.4 (24)

PEO(5000)-b-PCL(4000)

0.04

2.0

8.6 × 10−8

37

72

PEO(2400)-b-PDLLA(2000)

0.04

2.0

1.8 × 10−7

37

72

PEO(3100)-b-PS(2300)

0.04

2.0

1.5 × 10−7

37

72

PEO(2000)-b-PCL(2800) PEO(5000)-b-PCL(4000) PEO(2000)-b-PCL(2800) PEO(5000)-b-PCL(4000) PEO(2000)-b-PDLLA(2200)

0.04 0.04 0.2 0.2 0.2

2.0 2.0 2.0 3.8 1.8

1.7 × 10−7 8.6 × 10−8 8.5 × 10−7 8.5 × 10−7 8.5 × 10−7

rt rt rt rt rt

24 24 48 48 48

PEO(3100)-b-PS(2300)

0.2

2.3

8.5 × 10−7

rt

48

1.3 (0), 1.04 (24) 1.8 (0), 0.66 (72) 1.06 (0), 0.9 (72) 2.3 (0), 2.0 (24) 1.3(0), 1.2 (24) 3.6 (0), 2.6 (48) 5.1 (0), 2.2 (48) 10.4 (0), 0.9 (48) 2.8 (0), 2.7 (48)

polymer

% remaining FRET (time, h) 63 (24), 44 (48), 31 (72) 80 (24), 62 (48), 34 (72) 36 (24 and 72) 85 (72) 87 (24) 92 (24) 72 (48) 43 (48) 9 (48) 96 (48)

have indeed been described as scaffolds for tissue engineering, 31−35 and they have been shown to facilitate cell adhesion.31−35

toxicity in the range used for PDT. Note that for short incubation times we observed an increase of cellular viability that could be due to an increase of cell number per well. Block copolymers 1451

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Figure 10. Free or encapsulated pheophorbide localization within tumor cells cultivated in 2D or 3D. (A) Plated cells incubated for 30 min at 37 °C with pheophorbide 5 μM were observed by confocal microscopy. Confocal images are the maximum-intensity projections of the complete z section. (B) Red fluorescence and phase contrast pictures are merged to localize pheophorbide. (C) Living spheroids incubated for 30 min at 37 °C with pheophorbide 5 μM were observed by two-photon microscopy. Pictures represented a stack of approximately the first 150 μm of the spheroid. Pheophorbide fluorescence is red.

penetrate both adherent healthy and tumor cells (Figure 10). In HCT-116 cells and fibroblasts (data not shown), the fluorescence was concentrated in the cytosol regardless of the polymeric micelle used. This is consistent with a study by Tang et al. in which pheophorbide fluorescence overlapped with the pattern of the mitochondria stained by MitoTracker Green.36

To compare the drug delivery efficiency of the different nanosystems, cellular penetration of free and loaded Pheo was investigated by fluorescence microscopy. As shown in Figure 10, control pictures showed no autofluorescence of adherent cells and spheroids, indicating that the red fluorescence signal came from Pheo. Free Pheo as well as encapsulated Pheo was shown to 1452

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After 30 min of incubation at 37 °C, the micelles ensured an increase of Pheo cell penetration compare to that of free Pheo. As shown in Figure 10 and after integration of the cellular fluorescence (Table 5), PEO-PDLLA copolymer micelles

our protocol, HCT-116 spheroids were incubated with the photosensitizer either in the presence or absence of polymeric micelles, which were then submitted to three cycles of PDT, with each cycle lasting 24 h. At the end of the PDT treatment, macroscopic observations of the spheroids morphology by transmission widefield microscopy showed a ring of cells detaching from the spheroid surface (Figure 11). Detaching

Table 5. Fluorescence Integration of 2D Images polymer

total intensity

PEO(2400)-b-PDLLA(2000) PEO(3100)-b-PS(2300) PEO(2000)-b-PCL(2800) PEO(5000)-b-PCL(4000) Pheo alone

1.9 × 106 1.3 × 106 1.3 × 106 0.9 × 106 0.7 × 106

appeared to be the most efficient delivery system under these conditions compared to PEO-PCL nanoparticles. Cells treated with Pheo/PEO(2400)-b-PDLLA(2000) micelles have a strong heterogeneous fluorescence that seems to be concentrated in large clusters in the cytoplasm. Dialysis experiments have shown that pheophorbide may escape more easily from PEO-PDLLA than from the other nanosystems. Moreover, Chen et al.28 showed that this type of nanovector may disrupt in the vicinity of cell membrane and release the encapsulated drug in the cell culture medium. All of these results suggest that when Pheo is encapsulated in PEO-PDLLA it may be released in contact with the cell membrane and enter the cells possibly through the association of some monomers of PEOPDLLA. In this form, Pheo a may not be aggregated, and its fluorescence would not be quenched. By contrast, with PEOPCL, the fluorescence of the internalized Pheo in cells is less important than that of with PEO-PDLLA micelles. Large clusters were not observed, but a diffuse fluorescence was seen in the cytoplasm. This cellular repartition may be explained by the fact that Pheo may diffuse through the micelle and the cell membrane without being in contact with the cell culture medium, as we have recently shown in a related work.27 Moreover, dialysis experiments showed a better release of Pheo a from PEO-PDLLA nanoparticles than from PEO-PCL micelles. With PEO(3100)-bPS(2300) micelles, the fluorescence of Pheo was similar to that observed with PEO(2000)-b-PCL(2800) micelles, but the repartition into large clusters was similar to that observed with PEO(2400)-b-PDLLA(2000). Moreover, PEO(3100)-b-PS(2300) micelles had the lowest yield of Pheo release in the dialysis experiments. These results suggest that Pheo, encapsulated in PEO(3100)-b-PS(2300) micelles, may enter cells by different processes, such as diffusion (for released Pheo) through the cell membrane because of the hydrophobicity of Pheo or by endocytosis that is either associated or not with the micelle. If we compare the phototoxic efficiency of the different nanosystems, then Pheo/PEO(2000)-b-PCL(2800) is the most efficient. Pheo/PEO-PCL is 3.3 and 1.3 times more phototoxic than free Pheo and Pheo-loaded PEO-PDLLA, respectively. Moreover, there is no correlation between the intracellular fluorescence of Pheo and its phototoxic activity. It could be expected that the Pheo/PEO-PDLLA system would be the most efficient. This discrepancy between the cellular penetration of Pheo and its phototoxic activity may be explained by the different subcellular localization of Pheo depending of the nanovector. The PEO-PCL drug delivery mechanism does not seem to modify the cellular localization of Pheo. At a high concentration of Pheo (100 μM), all of the systems are similar. Photodynamic Therapy on 3D Tumor Spheroids. Lastly, PDT experiments were performed using HCT-116 spheroids. In

Figure 11. Macroscopic observation of tumor spheroids incubated with different polymeric micelles loaded with pheophorbide a after PDT. HCT-116 spheroids were observed by transmission widefield microscopy after three illuminations to photoactivate the sensitizer.

cells were more abundant when tumor spheroids were treated with Pheo-loaded PEO(2400)-b-PDLLA(2000) and PEO(3100)-b-PS(2300) micelles. We took advantage of Pheo’s fluorescent property to quantify simultaneously its loading in cells and their viability by flow cytometry (Figure 12). Whereas about 100% of the cells within the control spheroid were viable, only 45% remained viable after PDT treatment with Pheo/PEO(2400)-b-PDLLA(2000) micelles. Approximately 96% of the spheroid cells were loaded with Pheo under these conditions, indicating that PDLLA was the best nanocarrier for enhancing Pheo cell penetration, even in a 3D 1453

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nanomedicine. In the presence of various blood components, this stability remained very good and is compatible with their use by injection followed by a passive concentration increase in tumors owing to their enhanced permeability and retention effect, which is known to occur over a few days. Lastly, PDT of spheroids enabled the corroboration of results obtained on 2D cell culture and showed that encapsulation of Pheo in the suggested polymers yielded a strong increase in photocytotoxicity. However, small differences for the nanovectors were highlighted, with PEO(2000)-b-PCL(2800) being the most efficient in 2D cell culture, whereas PEO(2400)-b-PDLLA(2000) was the best for 3D tests. Part of the discrepancy might be explained by the different durations for each type of experiment. As explained in the text, in spite of the good results for PEO-PDLLA, it cannot be considered as a good nanovector because of its early release of the cargo. PEO-PCL has been shown to be more efficient in 2D cell culture compared to PEO-PS, but here again, 3D experiments proved that PEO-PS led to the highest spheroid dissociation. Therefore, after this proof of concept, in vivo experiments will now be performed to assess the efficacy of such vectors for PDT under true physiological conditions, which will provide feedback for the in vitro tests.

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Figure 12. Quantification of viable and Pheo-loaded tumor cells after PDT. Percentage of viable cells (white bars) and Pheo-loaded cells (black bars) were simultaneously determined by flow cytometry after PDT of the tumor spheroids. Scale bar = 100 μm. The graph represents the results from six different spheroids.

ASSOCIATED CONTENT

S Supporting Information *

Characterization data for the polymers, example of a bimodal DLS analysis (PEO(5000)-b-PCL(4000)); typical evolution of 1/τ with the scattering vector (PEO(5000)-b-PCL(4000)); example of evolution of Kc/Rθ with the scattering vector for the fast mode (PEO(5000)-b-PCL(4000)); calculation of affinity constants from dialyses; and FRET ratio evolution. This material is available free of charge via the Internet at http://pubs.acs.org.

cellular structure. This result is likely related to the finding of Pheo release from its PEO-PDLLA carrier in the dialyses experiments. Considering the results presented in this article, it is reasonable to assume that during the PDT tests Pheo is slowly but fully released from the PEO-PDLLA vector. The release of Pheo thus appears to be an essential step in the treatment. This can be linked to other results obtained by our team that showed that penetration of Pheo from PEO(5000)-b-PCL(4000) systems in HCT-116 cells occurs by a direct transfer of Pheo from its carrier to the membrane cell, thus leading to a penetration without the vector.27 PDT was observed to be more efficient with PEO(2400)-bPDLLA(2000) than Pheo alone, which might appear to be surprising based on the behavior in the dialysis experiments. This result might indicate that the release of Pheo from PEO-PDLLA vectors leads to unaggregated Pheo, possibly because of an autoorganization of Pheo with some PEO-PDLLA monomers. In this form, Pheo is therefore more efficient for PDT, whereas Pheo alone is expected to yield aggregated species in water. The difference between the phototoxic activity of the PEOPCL and PEO PDLLA systems observed on cell monolayers and spheroids may be easily explained by the different drug delivery mechanism that is involved for both types of micelles. It is interesting that when Pheo is delivered by PEO-PCL micelles it seems to be more active. Indeed, as shown by Figure 12, around 30% of the cells were labeled with Pheo, leading to 25% cell death against 100% labeled and 60% cell death for spheroids treated with Pheo-loaded PEO(2400)-b-PDLLA(2000) micelles, respectively.

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AUTHOR INFORMATION

Corresponding Authors

*(A.-F.M.) E-mail: [email protected]; Fax: (33) (0) 5 61 55 81 55; Tel: (33) (0) 5 61 55 62 72. *(M.-P.R.) E-mail: [email protected]; Fax: (33) (0) 5 61 17 59 94; Tel: (33) (0) 5 61 17 58 11. *(P.V.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the French ANR P2N Copopdt. The EU (FEDER-35477, Nano-objets pour la biotechnologie) is greatly acknowledged for financial support. Christophe Mingotaud is acknowledged for helpful discussions. We thank Bruno Payré and Dominique Goudounèche from CMEAB for help with TEM.

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CONCLUSIONS This study has shown that all four systems evaluated were stable from a physical chemistry standpoint either upon aging or dilution. This fact is essential for their possible use as vectors in 1454

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NOTE ADDED AFTER ASAP PUBLICATION This article posted ASAP on March 3, 2014. The caption for Figure 10 has been revised. The correct version posted on March 21, 2014.

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