Light Induced Functionalization of PCL-PEG Block Copolymers for the

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Biomacromolecules 2009, 10, 966–974

Light Induced Functionalization of PCL-PEG Block Copolymers for the Covalent Immobilization of Biomolecules Vincent Pourcelle,† He´le`ne Freichels,‡ François Stoffelbach,‡ Rachel Auze´ly-Velty,§ Christine Je´roˆme,‡ and Jacqueline Marchand-Brynaert*,† Universite´ catholique de Louvain, Unite´ de Chimie Organique et Me´dicinale (CHOM), Baˆtiment Lavoisier, place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium, Center for Education and Research on Macromolecules (CERM), Universite´ de Lie`ge, Sart-Tilman B6, B-4000 Lie`ge, Belgium, and Centre de Recherches sur les Macromolećules Ve´ge´tales (CERMAV-CNRS), BP53, 38041 Grenoble cedex 9, France Received January 7, 2009; Revised Manuscript Received January 22, 2009

Functionalized poly-ε-caprolactone-block-polyethyleneglycol (PCL-PEG) amphiphilic copolymers were prepared to be constituents of nanocarriers used for the targeting of specific cells. Hence, we conceived a smooth and simple photografting methodology on these copolymers using a bifunctional molecular clip (O-succinimidyl-4(p-azido-phenyl)butanoate). We prepared PCL-PEGs with pendent N-hydroxysuccinimide esters and studied the grafting with 3H-lysine, which radioactivity was counted by LSC. Several parameters were investigated, such as behavior of homopolymers, initial concentrations, irradiation, and incubation durations. Evidences of a “PEG directed photografting” are discussed and this selectivity could be improved by a selective solvent technique. The photografting on different PCL-PEGs revealed a dependency of the rates to the crystallinity of the copolymers. Several controls by SEC, DLS, and TEM of the treated copolymers were realized. Lastly, the coupling of R-Dmannopyranoside ligand was performed, reaching amounts of 5400 nmol/g of PCL-PEG. This derivatized PCLPEG enters in the preparation of nanocarriers used for the targeting of antigen presenting cells.

Introduction At the beginning of the 20th century, the medicine Nobel laureate, Paul Ehrlich, theorized the concept of the “magic bullets”, that is, agents that would “be able to exert their full action exclusively on the parasite harbored within the organism”.1,2 Since then the addressing of drugs directly to their site of action has remained a tremendous challenge. One of the most highlighted strategies that appeared in the last decades was the useofsmartnanocarriersmadeofamphiphilicblockcopolymers.3-5 In aqueous media they form core/shell micelles trapping hydrophobic or low soluble compounds in a lipophilic core stabilized by a surrounding hydrophilic shell, which can be used for the coupling of targeting units.6-10 In term of drug delivery, those systems offer several advantages such as prolonged circulation half-life, ability to bypass natural barriers, “passive targeting”, protection of the drug until it has attained its target and sustained release of their contents.11 We were interested in the conception of tailor-made degradable targeted nanovectors made of PLGA, PLGA-PEG, and PCL-PEG block copolymers in which the targeting units will be borne by one block.12 PCLPEG seemed to be the candidate of choice: first, because it is the most resistant to aqueous treatments; second, because PCL itself offers a good biocompatibility combined with good permeability to many therapeutic drugs and good compatibility with a large range of other polymers.13 In spite of all these advantages, PCL-PEG suffers from a lack of functional group that limits its applications as fine-tunable * To whom correspondence should be addressed. Tel.: +32-10-472740. Fax:+32-10-474168. E-mail: [email protected]. † Universite´ catholique de Louvain. ‡ Universite´ de Lie`ge. § Centre de Recherches sur les Macromolećules Ve´ge´tales (affiliated with Universite´ Joseph Fourier and member of the Institut de Chimie Molećulaire de Grenoble).

biomaterial.14 To overcome this drawback, several strategies have been investigated. They mainly consist in the development of original ring opening polymerization (ROP) with either chainend functionalized PEG7-10 or modified ε-CL monomers,15-17 but also in postpolymerization modifications.18-21 Conceptions of new polymerizable functional lactones, or of end-activatedPEG, are long and tricky problems to solve.22,23 Moreover, the copolymers obtained have pendent groups or chain-end functions that could react with a very limited amount of chemicals. Basically it means that a new ROP strategy should be conceived for each application. In comparison, methods occurring downstream of the ROP are seen as the most straightforward approaches. But as far as aliphatic polyester block copolymers are concerned, soft methodologies need to be employed to avoid cleavage and separation of the two blocks. Among the most employed techniques, such as aminolysis,18,19 electron beam graft polymerization,20 or anionic derivatization,21 none is adaptable to copolymers because they induce reaction of the polymer backbone. Thus, there is a need to develop general derivatization methods on PCL-PEG copolymers that avoid lyses and harsh conditions but could offer a control of derivatization rates. In the last years, some of us have developed a simple and smooth grafting methodology based on the introduction of activated ester functions on polymer devices by irradiation of a bifunctional photolinker (O-succinimidyl-4-(p-azido-phenyl)butanoate), coated or intimately mixed within the material.24,25 The irradiated aryl-azide liberates a highly reactive nitrene that inserts randomly in the polymer backbone. Further treatment with NH2-terminated compounds allows the covalent coupling of biomolecules or molecular probes via an amide linkage resulting from N-hydroxysuccinimide (NHS) displacement in very soft conditions. In the literature, aryl-nitrene based photoimmobilization has been exemplified for surface grafting of

10.1021/bm900027r CCC: $40.75  2009 American Chemical Society Published on Web 02/18/2009

Light Induced Functionalization of PCL-PEG

several polymeric devices,26-29 but to the best of our knowledge, it was used once on an aliphatic polyester (polylactide acid)30 and never on amphiphilic block copolymers. This approach might offer several benefits. It is a simple and direct postpolymerization chemical treatment employing smooth conditions that should preserve the main properties of the copolymers (molar mass, HLB). Furthermore, the introduction of NHSactivated esters opens the door to a large bunch of possible derivatizations. Lastly, depending on the quantities of aryl-azide introduced, we might be able to control the rates of grafted reactive functions. We had already succeeded in the grafting of GRGDS (GlyArg-Gly-Asp-Ser) pentapeptide on the surface of nanocarriers by the same methodology.12,31 Here we present a deeper investigation of the grafting conditions and extend it to other ligands (such as sugar derivative). First we describe the synthesis and characterization of several PCL-PEG block copolymer with long to moderate chain lengths. The grafting of the photolinker is then studied with a tritiated probe (L-[4,5-3H] lysine) that mimics the coupling of amine-terminated compounds and can easily be quantified with a scintillation counter (LSC analysis). Several parameters have been studied and optimized such as irradiation time, washings, incubation condition with the probe and selectivity toward both blocks. The factors that may link the grafting rates to the main properties of copolymers are also discussed. The treated PCL-PEGs have been characterized to control the iniquity of the method toward their main material characteristics (micellization ability, chain cleavage). At last, we illustrate the potential of this versatile way of derivatization with the grafting of a mannose derivative valuable for dendritic cells targeting.

Experimental Section Materials. Reagents and solvents were of analytical grade and purchased from Acros (Beerse, Belgium) and Sigma-Aldrich-Fluka (Bornem, Belgium). O-Succinimidyl-4-(p-azidophenyl)-N-butanoate (molecular clip) was prepared according to a procedure already described.24 Water (HPLC grade) was obtained with a Milli-GQ system (Millipore, Bedford U.S.A.). The radiolabeled L-[4,5-3H] lysine monohydrochloride in aqueous solution was purchased form Amersham Biosciences (Little Chalf-ont, U.K.) with a specific activity of 89.0 Ci/mmol. Monomethoxypoly(ethylene glycol) (PEG) of different molecular weights (5000, 6000, and 9500 g/mol) were purchased from Aldrich. ε-Caprolactone (Aldrich, 99%) was dried over CaH2 and distilled before use. Phosphate buffer (PB; 0.1 M, pH 8) was prepared from Na2HPO4 (16.86 g, 94.72 mmol) and NaH2PO4 (0.826 g, 5.98 mmol) in Milli-GQ water (1 L). The lysine solution (10-3 M) for coupling and LSC analysis was prepared as follows: 187.5 µL of radioactive lysine and 250 µL of 0.1 M lysine solution [lysine monohydrochloride (0.1826 g) in water (10 mL)] were dissolved in 25 mL of PB solution. Before each experiment glass plates were cleaned by immersion in a piranha solution (H2SO4/H2O2 (3:1), 15 min) rinsed with water, acetone, and dried in oven. The reactive and rinsing solutions containing the polymer samples were shaken (200 rpm) with an Edmund Bu¨hler stirrer (model KL-2). The IR spectra were taken on a Perkin-Elmer 600 instrument; thin films were obtained by dissolving copolymers in CH2Cl2 and casting on KBr plates; frequency absorption are given in cm-1. The 1H and 13C NMR spectra were taken on a Bruker 300 spectrometer operating at 300 and 75 MHz, respectively, and Bruker DRX400 operating at 400 and 100 MHz, respectively. Chemical shift δ are given in part per million (ppm) in reference to the solvent: 7.26 ppm for CDCl3, 1.93 ppm for CD3CN, 4.79 ppm for D2O. The PCL/ PEG ratios were calculated from the integration of the 1H NMR peaks at 3.65 ppm for PEG and at 4.02 ppm for PCL in CDCl3 and peaks at 3.55 ppm for PEG and at 4.01 ppm for PCL in CD3CN.

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Table 1. Composition, Molecular Weight Distribution, and Micellization of PCL-PEG Block Copolymers DLSc a

b

copolymer

Mn PEG

Mn PCL

total Mn

PDI (Mw/Mn) (SEC)

RH (nm)

PDI

PCL-PEG1 PCL-PEG2 PCL-PEG3

9500 6000 5000

22800 21200 13200

32300 27200 18200

1.30 1.30 1.40

35 35 29

0.08 0.12 0.08

a Calculated from 1H NMR spectrum in CDCl3 at 25 °C by comparing the intensity of the terminal methyl group (CH2OCH3, 3.2 ppm) with the methylene protons (OCH2CH2O, 3.6 ppm). b Calculated from 1H NMR spectrum in CDCl3 at 25 °C by comparing the intensity of the methylene protons of PEG peak at 3.6 ppm, with the peak of the R-methylene protons of PCL at 4.05 ppm. c Average hydrodynamic radius and size distribution as measured by DLS.

Copolymers Synthesis and Characterization. Preparation of PCL-PEGs.32 Typical procedure for the synthesis of poly-ε-caprolactone-block-polyethyleneglycol(PCL-PEG)copolymer:5gofmonomethoxypoly (ethylene glycol) (Me-PEG-OH, Mw ) 9500 g/mol, 0.53 mmol) was dried by three azeotropic distillations from toluene before use. The monomer, ε-caprolactone (12 mL, 0.12 mol), was then added, and the Schlenk ampule was put in an oil bath at 130 °C until all the components were melted. Then, 0.1 mL of a solution of stannous octanoate (0.06 M in toluene) was added, and the ampule was left in the bath for several days. The advancement of reaction was checked by 1H NMR. At the end of the polymerization process, the polymer formed (PCL-PEG1) was dissolved in THF, precipitated in diethyl ether, filtered, dried under vacuum at room temperature for 24 h, and characterized by 1H NMR and SEC. Following the same protocol, various PEG macroinitiators have been used to initiate the copolymerization, the block length of PCL being adapted by simply playing on the ε-CL/PEG macroinitiator ratio. Accordingly, PCL-PEG2 and PCL-PEG3 were obtained from MePEG-OH, with Mw ) 6000 g/mol (5 g) and 5000 g/mol (5 g), and ε-CL amounts of 19 and 15.4 mL, respectively. 1 H NMR (CDCl3): δ (ppm) 1.38 (m, 2H, O-(CH2)2-CH2-(CH2)2CdO), 1.61-1.65 (m, 4H, O-CH2-CH2-CH2-CH2-CH2-CdO), 2.29 (t, 2H, O-(CH2)4-CH2-CdO), 3.62 (s, 4H, O-CH2-CH2-O), 4.05 (t, 2H, CH2-CH2-C(O)-O-CH2); 9500(PEG)-22800(PCL); SEC: Mn ) 32300, PDI ) 1.30 (Data of PCL-PEG1, see Table 1). Size Exclusion Chromatography (SEC). SEC was carried out in THF at 45 °C at a flow rate of 1 mL/min using a SFD S5200 autosampler liquid chromatograph equipped with a SFD refractometer index detector 2000. Columns (HP PL gel 51 m 105 Å, 104 Å, 103 Å, 100 Å) were calibrated with polystyrene standards. Differential Scanning Calorimetry (DSC). DSC was carried out with a TA DSC Q 100 thermal analyzer calibrated with indium. Glass transition and melting temperatures were measured, after a first cooling (-80 °C) and heating (100 °C) cycle. Thermograms were recorded during the second heating cycle at 10 °C/min. Percentages of crystallinity were obtained by integration of the DSC curve with references of 134.9 J/g and 189.8 J/g for a 100% of crystallinity for PCL and PEG, respectively. Preparation of Micellar Solutions. The following typical procedure was applied for the preparation of micellar solutions: 5 mg of amphiphilic copolymer was dissolved in 1 mL of acetone. After complete dissolution, 10 mL of water was added dropwise over a period of 1 h. The mixture was shaken for 2 h before adding rapidly 10 mL of water. After another hour of shaking, acetone was eliminated by leaving the reactor open for one night under the hood. DLS Measurements. Dynamic light scattering (DLS) measurements were performed on a Malvern CGS-3 equipped with a He-Ne laser (633 nm). A bath of filtered toluene surrounded the scattering cell and measurements were performed at an angle of 90°, at a controlled temperature of 25 °C. The polydispersity index (PDI) corresponds to the µ2/Γ12 ratio, where µ2 is the second cumulant and Γ1 is the first cumulant. The DLS data were also analyzed by the CONTIN routine, a method which is based on a constraint inverse Laplace transformation

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Figure 1. SEC chromatograms of PCL-PEG3 derivatized with lysine. Plain lines are for native copolymer and dashed lines are for grafted copolymer. SEC chromatograms of PCL-PEG1 and 2 are provided as Supporting Information.

of the data and which gives access to a size distribution histogram for the aggregates. All the micellar solutions were filtered over a 0.45 µm filter before DLS measurements. TEM Imaging. Samples for transmission electron microscopy (TEM) were prepared by slow evaporation of a drop of the DLS solution deposited on a TEM grid coated by a thin Formvar film. Finally, the samples were stained by an aqueous solution of phosphotungstic acid (0.1 w/w %). They were analyzed by a Philips CM100 apparatus equipped with a CCD Gatan 673 camera connected to a computer (Krontron KS100 software). Activation of Polymers by Photografting of the Molecular Clip. Standard Protocol for the ActiVation of PCL-PEGs or PCL Homopolymer. Polymer powders were solubilized in CH2Cl2 or in CH3CN (40 mL/g) with the desired amount of molecular clip (0.1-1.2 mmol/g, see text) and the solutions were cast on clean glass plates (1 mL per plate). After solvent evaporation, the samples were dried under vacuum to constant weight. Polymer samples were removed from the plates as shavings. These were irradiated at 254 nm, under an argon atmosphere, for the required time, in a homemade reactor (rotating quartz flask of 15 mL; 3 UV lamps of 8 W placed at a distance of 4.5 cm). The samples were washed by shaking in isopropanol/ethyl acetate (19:1 (v:v), 80 mL/g; three times) at room temperature. Standard Protocol for the ActiVation of PEG Homopolymer. The above protocol was applied, except for the washings that were done in acetone at 0 °C (80 mL/g; three times). Preparation of Control Samples. Several samples were prepared to control the nonspecific adsorption versus covalent grafting at each step of the derivatization protocol. Nonirradiated. Standard protocol was followed, omitting the UV irradiation. Blank. Standard protocol was followed, omitting the UV irradiation and the molecular clip. Irradiated Blank. Standard protocol was followed, omitting the molecular clip. Procedure for the Grafting Rate Measurement by LSC. Coupling of [3,4-3H] Lysine on PCL-PEG and PCL. For any single experiment, three polymer samples (activated, nonactivated, blank) were treated in parallel. They were immersed into 2 mL of a 1 mM labeled lysine solution and shaken for the required time at room temperature. The lysine solution was removed by suction and the samples were washed with slightly acidic (pH 5-6) deionized water (six times for 10 min and one time overnight) and rinsed with MeOH on a filter. They are placed in individual PE vials, vaccuum-dried at 40 °C to constant weight, dissolved in AQUALUMA (5 mL), and counted by LSC. Results are the means of at least three independent experiments. Samples recovery after treatments was superior to 90% for PCL-PEG1 and PCL homopolymer and superior to 80% for PCL-PEG2-4 (as compared to initial mass). Coupling of [3,4-3H] Lysine on PEG. Three PEG samples (activated, nonactivated, blank) were individually treated by immersion in 2 mL

Figure 2. TEM image of micelles (0.025 wt %) in aqueous solution for PCL-PEG2 before (A) and after (B) photografting.

of the 1 mM labeled lysine solution for 1 h at room temperature under shaking. The mixture was acidified with 1 mL of 5 mM HCl and extracted with CH2Cl2 (3 × 10 mL). The organic phase was concentrated under vacuum to precipitate the PEG. Then samples were washed at 0 °C with acetone (10 times, 5 mL), vacuum-dried at 40 °C to constant weight, introduced into individual PE vials, dissolved in AQUALUMA (5 mL), and counted by LSC. Results are the mean of at least three independent experiments. Samples recovery after treatments was superior to 80% (as compared to initial mass). RadioactiVity Measurement. We used 20 mL polyethylene (PE) vials (Milli-Q20, Packard-Bioscience, San Diego, CA, U.S.A.), AQUALUMA cocktail (Lumac, Basel), and a TriCarb 1600 TR liquid scintillation analyzer (Perkin-Elmer instrument, San Diego, CA) according to the standard protocol25 (details given as Supporting Information).

Results and Discussion Preparation and Characterization of PCL-PEG Block Copolymers. Three PCL-PEG diblock copolymers (Table 1) were prepared following a conventional method, that is, starting with R-methoxy, ω-hydroxy polyethyleneglycol as macroinitiator for the ring-opening bulk polymerization of ε-caprolactone at 130 °C, and with stannous octanoate as catalyst.32 These native copolymers have been characterized by SEC (Figure 1) and NMR and their micelles size in water by DLS (Table 1) and TEM (Figure 2A). These characteristics will be compared later on with the ones measured after photochemical posttreatment to confirm the copolymers integrity. Conception of a Photografting Protocol on Amphiphilic Copolymers. To obtain N-hydroxysuccinimide (NHS)-activated copolymers, we used a bifunctional photolinker that has been already exemplified on several surfaces, inorganic24 as well as organic,25 namely, O-succinimidyl-4-(p-azido-phenyl)butanoate, designed as the “molecular clip” or ArN3 throughout the paper. According to the literature, photolysis at 254 nm of aryl azide implies a cascade of reactions depicted in (Scheme 1).33,34



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Scheme 1. Photochemistry of Phenyl Azide

Insertion into X-H bonds (X ) C, N, O) of a substrate (i.e., copolymers) results from reactions of singlet (path a, minor process) or triplet nitrene intermediates (path b, major process), while several side reactions lead to soluble small molecules (azepine, aniline, azo-dimer). On the other part of the molecular clip, the NHS ester remains unchanged and can further be derivatized by amine bearing compounds. In the last several years some of us have developed a surface reactivity assay based on the coupling of L-[4,5-3H] lysine as a probe mimicking the coupling of small biological compounds such as peptides or peptidomimetics (Scheme 2). After incubation in the probe solution followed by washings, the remaining radioactivity associated to the sample is counted in a liquid of scintillation (LSC). This measure gives an estimation of the reactivity of the solvent-accessible surface. Whereas we had validated this methodology on several surfaces of rigid materials (PEEK, PET, PBT, PVDF, Si, Ge),24,25,35-37 our aim was to adapt it to soft materials such as biodegradable amphiphilic copolymers. A new procedure convenient for block copolymers was conceived. The molecular clip was intimately mixed within the polymer matrix by dissolution in a common solvent of both blocks (dichloromethane for instance) followed by casting on clean glass plates. Solvent traces were removed under vacuum and the solid mixture was exposed to UV irradiation (254 nm) in a homemade quartz reactor under inert atmosphere to avoid oxidation of triplet nitrene or photooxidation/degradation of the polymer. To get rid of the side products, samples were dipped in a good solvent of the clip

and nonsolvent of the copolymer (isopropanol for instance). The NHS-activated polymer was then immersed in a 1 mM solution of 3H-lysine in phosphate buffer (PB), washed to remove ungrafted materials, dried, and analyzed by LSC. For each experiment control samples were treated in parallel to estimate the amounts of 3H-lysine that are trapped or adsorbed on the polymer but not covalently bound. Those free molecules could have diverse origins: adsorbed lysine, unreacted azide, or photoderivative residues that have reacted with lysine. The first control samples suffered all the protocol except irradiation: they give us an estimation of the amount of free molecules trapped and are identified as “nonirradiated samples”. The second ones of native copolymers incubated in 3H-lysine: they assess the rates of adsorbed lysine and are noted as “blank samples”. Both samples lead to an overall assessment of the unspecific adsorption. Aliphatic polyesters are sensitive to both hydrolysis and photodegradation leading to the formation of polar groups (i.e., carboxylate) and, as a consequence, in our radiolabeling methodology, to the rise of unspecific adsorption.25 This could be assayed by a third series of control samples having suffered all the protocol with omitting the molecular clip. The so-called “irradiated blanks” allowed us to determine initial experimental conditions so as to minimize the modifications of copolymer backbone. Accordingly, the parameters “at minima” were chosen as follows: (i) irradiation time of 10 min;28,30 (ii) molecular clip concentration of 0.1 mmol/g of polymer to avoid a photosensitizer effect38 or a blooming effect of the additive at

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Scheme 2. Photografting Assessment with Radiolabeled Lysine

Pourcelle et al. Table 2. LSC Results with Parameters “at Minima” on Homopolymers and PCL-PEG1 LSC method Aa samples

nmol/gb

grafting yieldc

PCL homopolymer irradiated nonirradiated blank correctedd PEG homopolymer irradiated nonirradiated blank correctedd PCL-PEG1 irradiated nonirradiated blank correctedd

216.91 ( 21.01 45.95 ( 8.27 40.38 ( 12.59 170.97 ( 22.58 1274.40 ( 95.18 160.80 ( 88.48 71.17 ( 63.19 1113.60 ( 129,95 378.16 ( 64.94 31.55 ( 7.94 14.11 ( 6.50 309.93 ( 68.81

0.16%

a

0.13% 0.98% 0.85% 0.29% 0.24%

Cast from dichloromethane. Mean of three analyses. c Chemical grafting yield ) (grafting amount (LSC))/(initial amount of molecular clip). d (LSC amount on irradiated sample) - (LSC amount on nonirradiated sample).

the sample surface;39 (iii) incubation time of 1 h in PB with 3 H-lysine; and (iv) limited number of washings (control of the remaining radioactivity in the sewage). Grafting on Homopolymers. We considered the grafting of homopolymers separately on both poly-ε-caprolactone (PCL) and polyethyleneglycol (PEG). Submitted to the protocol described above, PCL was relatively easy to manipulate and presented no practical difficulty. From the comparison of the irradiated samples and the nonirradiated ones (Table 2) it appears that there is much more grafting than unspecific adsorption; moreover the standard deviation is reasonable acknowledging for a reproducible grafting on this polymer. Because all the samples suffered numerous washings, a relevant gap between the fully treated ones and the controls applies for covalently bound probes. Hence, the “real” amount of tritiated tag could be assessed by subtracting the value of the nonirradiated samples to the fully treated ones giving a “corrected” amount of 171.0 ( 22.6 nmol · g-1. The chemical grafting yield could then be defined by the ratio of corrected lysine rates on the initial amount of clip introduced. As far as the corrected amount represents the functionalization of the accessible surface, it does not represent an absolute grafting yield but only a relative quantification of the derivatization that can be reached in the conditions of the experiment. For PCL the grafting yield is quite low (0.13%), which is mostly due to its poor solubility in the aqueous media we used for the radiolabeling. Thus, this result gives us an estimation of the number of NHS functions available at the interface between the grafted PCL chains and water.

b

A modified protocol was applied for the grafting of polyethyleneglycol (PEG) homopolymer. Because it is soluble in water, we had to extract it with dichloromethane and to wash the excess of lysine with acetone. The results collected are presented in Table 2. Here again we obtained a clear difference between irradiated samples and controls applying for a positive effect of the clip insertion with a value of 1113.6 ( 130.0 nmol · g-1 of covalently bound lysine and a grafting yield of 0.85%. The standard deviation is higher than for PCL but still is significantly small in regards of the rates obtained demonstrating a good reproducibility of the method. The significant difference of grafting rates in favor of PEG might be due to the better solubility of PEG in the radiolabeling solution but other factors should be considered. Most notably, we have to take in consideration the wellknown better reactivity of nitrene toward C-H bond next to ether functions than C-H bond of hydrocarbon chain. This chemoselectivity was explained by the stabilization of the radical formed on carbons adjacent to the oxygen allowing further recombination with the amino radical.40,41 The behavior of native homopolymers under UV is also of crucial importance. In the absence of a photosensitizer PEG is known to be blind toward UV,38 so it was not surprising to find that for irradiation time up to 40 min no change occurred in the rates of adsorbed tritiated tags. Whereas for PCL an increase of lysine rates by 1.3- and 7.2-fold factors was observed for irradiation times of 10 and 20 min, respectively. This result is coherent with the fact that polyesters undergo photodegradations leading to an increase of unspecific adsorption.25 Nevertheless, for irradiation times of 10 min, it is still negligible enough to confirm that observed differences between irradiated and nonirradiated samples are unambiguously due to clip insertion rather than formation of polar groups. These first results acknowledged the fact that PEG is an interesting candidate for the photografting of aryl-azide on copolymers and because of its good solubility in aqueous media is more easily functionalized than PCL. Grafting on Block Copolymers. We investigated the grafting on PCL-PEG1 (Table 1) in the same initial conditions; the results obtained are presented in Table 2 and Figure 3. An amount of covalently grafted lysine of 309.9 ( 68.8 nmol · g-1 was reached with an acceptable standard deviation, corresponding to 0.24% of grafting yield. Hence, this assay, with parameters “at minima”, seems suitable to functionalize PCL-PEG1 block copolymer. Nevertheless, the robustness of the method and the


Figure 3. LSC results on PEG, PCL, and PCL-PEG1 (ArN3 concentration of 0.1 mmol/g, 10 min irradiation, incubation 1 h in 1 mM lys, optimized washings).

Figure 4. Corrected LSC amounts and grafting yields on PCL-PEG1 for increasing molecular clip (ArN3) content (10 min irradiation, incubation 1 h in 1 mM lys, optimized washings).

factors that are influent on the grafting rates were studied to optimize the procedure for the coupling of bioactive molecules. The choice of the possible limiting factors was based on our own knowledge of aryl azide chemistry24,25 and on reported studies.27 Initial Molecular Clip Concentration. Limitations might rise in front of high contents of molecular clip due to the formation of side products (see Scheme 1), photosensitizing,38 and saturation effects.39 It also may induce damage to the copolymer itself due to an increase of cleavable hemiaminal bonds on PEG backbone enhancing the sensitivity to hydrolysis. We assayed the photografting at increasing concentrations of ArN3. As shown by Figure 4, a quasi proportional relationship appeared between the initial amount of molecular clip and the resulting value of lysine covalently bound; moreover, there is a good reproducibility as proven by the grafting yields obtained that are almost constant at any of the tested concentrations (Figure 4). It seems obvious that on this copolymer (PCL-PEG1) and for concentrations of ArN3 less than 1 mmol/g, the grafting is still efficient with no visible disturbing effect of high concentrations. Indeed, this factor was critical on other block copolymers we attempted to graft with ArN3, namely, PLA-PEG, PLGAPEG, and smaller PCL-PEGs, as discussed later in this paper. Incubation Time in Labeled Lysine. The incubation time in labeled lysine was varied from 1 to 24 h. It seems that an incubation of 1 day is necessary to have a full functionalization of the copolymer. After 24 h, the amount of lysine on control samples (nonirradiated and blank) is not significantly different from the amount of lysine recorded after one hour of incubation, applying for a true positive effect of the molecular clip grafting


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Figure 5. LSC results for different incubation times in radiolabeled 1 mM lysine solution (ArN3 concentration of 0.1 mmol/g, 10 min irradiation, optimized washings).

even after 24 h and not only on an increase of unspecific adsorption (Figure 5). Irradiation Time. An UV exposure of 10 min should not be long enough for a complete conversion of all azide molecules into nitrenes.25,27,28,30 The irradiation time also depends on the absorption properties of the material and should be adjusted to limit degradation. For that purpose, we followed, by FT-IR, the disappearance of the azide stretching absorption band at 2110 cm-1, inside the copolymer matrix, for increasing irradiation times. A sample with a concentration of 0.1 mmol of ArN3 per gram of copolymer (PCL-PEG1) was prepared, submitted to UV exposure for successive periods of 2 min, and controlled by FT-IR. The total disappearance of the azide band was observed between 18 and 20 min of irradiation, indicating the photolysis completion. Finally, the grafting rate of lysine was of 1548.1 nmol/g with a grafting yield of 1.3%, a value 5 times higher than for 10 min of irradiation. To control the effect of a longer UV exposition time, we irradiated a native sample of PCLPEG1 for 20 and 40 min. A rise of the unspecific adsorption rates of lysine was observed heightened by a factor of 2 after 20 min and by a factor of 7 after 40 min. Despite this increase, it is still negligible in regards of the grafting rates obtained for the fully treated samples. Hence, 20 min of irradiation at 254 nm seemed to be a good compromise between a total conversion of azide and polymer preservation. PEG Selectivity. As suggested by literature and results obtained on PEG homopolymers, we expected that the insertion of the molecular clip should more or less be “PEG-directed”. Moreover, as evidenced by Lazare et al. in semicrytalline PCLPEGs, additives are concentrated in the softer parts.39 This is explained by the different crystallization behavior of the two blocks. PCL crystallizes first followed by PEG leading to an imperfect crystallization of this latter.42 Consequently, during crystallization of PCL block, the impurities (such as ArN3) are expulsed from the spherulites toward the softer PEG domains. As long as we are below the saturation solubility these expulsed entities stay trapped in PEG. Considering the applications of our functionalized copolymers (e.g., drug delivery), it is of great interest to graft mostly on these hydrophilic domains. So we tried to take advantage of what happens during evaporation and recrystallization by the choice of a solvent that would favor the presence of the clip in PEG, but volatile enough to be easily removed. A study by 1H NMR evidenced a clear selectivity of acetonitrile for PEG. For instance, we measured that for CH3CN solutions of PCL-PEG1 only 46% of PCL segments are solvated (see Supporting Information). Consequently, we modified the protocol by

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Table 3. Functionalization of the PCL-PEG1 by Photografting of O-Succinimidyl 4-(p-azido-phenyl)butanoate. Comparison of methods B and C. LSC method Ba c

e

samples

nmol/g

grafting yield

PCL-PEG1 irradiated nonirradiated blank correctedg

1016.37 ( 53.43 76.36 ( 48.66 4.61 ( 2.75 940.00 ( 21.80

0.83%

LSC method Cb

grafting improvement

nmol/g

grafting yielde 0.09%

×3.1

107.82 ( 60.0 25.00 ( 12.09 20.67 ( 0.17 82.82 ( 48.11

0.76%

f

d

grafting improvementf

×0.27

0.07%

Cast from acetonitrile. Grafting on NPs + CH3CN introduction of ArN3. Mean of three analyses. Mean of two analyses. Chemical grafting yield ) (grafting amount (LSC))/(initial amount of molecular clip). f Comparison with method A. g (LSC amount on irradiated sample) - (LSC amount on nonirradiated sample). a

b

c

d

e

Figure 6. LSC results on PCL-PEG1with different methods to introduce ArN3. Method A, cast from dichloromethane; method B, cast from acetonitrile; method C, grafting on NPs + CH3CN introduction (ArN3 concentration of 0.1 mmol/g, 10 min irradiation, incubation 1 h in 1 mM lys, optimized washings).

Figure 7. Corrected LSC results for various PCL-PEG block copolymers (10 min irradiation, incubation 1 h in 1 mM lys, optimized washings); plain lines and full marks are for lysine rates. Dashed lines and empty marks are for grafting yields.

replacing dichloromethane (method A), used for the introduction of the molecular clip with acetonitrile. The results (method B) obtained are presented in Table 3 and compared to method A in Figure 6. An improvement was observed, and the amount of lysine covalently bound on PCL-PEG1 was multiplied by a factor of 3. With those results in hand, it could be concluded that we should have enhanced the selectivity toward PEG blocks. We also tried to graft lysine on micelles formed from pure PCL-PEG1, via an acetonitrile impregnation of the molecular clip (details in Supporting Information). Unfortunately, this method C failed (Figure 6) to give acceptable yields, certainly because of a poor solubility of the micelles aggregate in CH3CN. Grafting on Different PCL-PEGs. We extended our methodology to several PCL-PEGs varying in their blocks length. In comparison with PCL-PEG1, PCL-PEG2 has shorter PEG chain but equivalent PCL chain length and PCL-PEG3 has both parts shorter than the two other copolymers (Table 1). Keeping the same initial parameters of the protocol we tested different concentrations of molecular clip varying between 0.2 and 1.2 mmol per gram of copolymer. Results of Figure 7 show clearly that for all copolymers at low ArN3 concentration (0.2 mmol/g) the rates obtained are equivalent. With increasing ArN3 content, shorter PCL-PEGs lead to lower rates of grafting and at high concentration there is a clear fall of the amount of covalently bound lysine. The reproducibility of this behavior has been controlled with PCL-PEG4, a copolymer batch (Mn 15200-4600, PDI 1.3) very similar to PCL-PEG3. As expected, high concentrations of azide lead to several limiting problems explaining the fall recorded for PCL-PEG2-4, which was not observed for PCL-PEG1. To understand this difference of reactivity we have to take into consideration that the process occurs in a rigid bulk where diffusion is limited. This particular case of photoimmobilisation was overlooked in the literature where it is mostly practiced on

surfaces. Only one publication refers to the reactivity of nitrene in a solid matrix.43 In this study, Reiser et al. had irradiated under inert atmosphere polystyrene samples containing a low concentration of 1-azidonaphtalene. A careful analysis of the photoreaction products highlighted that a decrease of the matrix’ hardness leads to a drastic increase of side-products (such as primary amine). A noteworthy explanation lies in the fact that triplet nitrene leads to insertion reactions via a two step process (cf. Scheme 1). During the first step the nitrene is converted into aminyl-radical by H-abstraction forming a macroradical on the polymer chain. Then the probability of the two species to couple is proportional to the time they will be kept in contact. It means that in a rigid matrix, with a frozen diffusion, the two radicals would be able to couple, whereas in a flabby environment, they will likely move out from each other, leading to side products such as primary amine after another H-abstraction or diazo compounds by reactions with amino-radical or nitrene (cf. Scheme 1). Thus, in solid matrix the nitrene reactivity might be controlled mainly by diffusion. In our case, the relationship between grafting yields and materials rigidity could be easily assumed since we handle semicrystalline copolymers. It is known that for PCL-PEGs the longer are the chains the higher is the crystallinity.42 Hence, it seems logical that longer copolymers lead to higher grafting rates, which is actually the order of grafting we observed: PCLPEG1 > PCL-PEG2 > PCL-PEG3-4 (for crystallinity measurements of fully treated polymers see Table 4). Interestingly, it is to be noticed that attempts to graft, by the same methodology, amorphous polyester block-copolymers such as PLA-PEGs and PLGA-PEGs failed (results not shown). Optimized Protocol for the Coupling of Biomolecules. Finally at this stage we were able to propose an optimized protocol for the coupling of molecules of interest (biomolecules) on PCL-PEG2 and PCL-PEG3: clip amount around 0.6 mmol/g



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Scheme 3. Synthesis the Mannolysated PCL-PEG

Table 4. Crystallinity and Micellization of PCL-PEG Block Copolymers Derivatized with Lysine d

crystallinity a

PCL-PEG1 PCL-PEG2 PCL-PEG3

b

DLS c

% PEG

% PCL

% total

RH (nm)

PDI

55.16 49.21 38.52

56.78 57.58 50.71

56.31 55.73 50.71

34 35 36

0.06 0.1 0.15

a Degree of crystallinity of PEG block calculated, based on the heat of melting of a 100% crystalline PEG (189.8 J/g) by integration of the DSC curve. b Degree of crystallinity of PCL block calculated, based on the heat of melting of a 100% crystalline PCL (134.9 J/g) by integration of the DSC curve. c Total degree of crystallinity of the copolymer, calculated by (% crystallinity (PEG) × wt % PEG in the copolymer) + (% crystallinity (PCL) × wt % PCL in the copolymer). d Average hydrodynamic radius and size distribution as measured by DLS.

(Figure 7), acetonitrile as casting solvent to enhance PEG selectivity, irradiation at 254 nm for 20 min, and incubation for 24 h in a 1 mM PB solution of the compound of interest, followed by washings. Under these conditions, PCL-PEG3 led to an amount of covalently bound lysine of 2945.8 nmol/g with a grafting yield of 0.5%. Comparatively to the initial conditions “at minima”, it represents an improvement of 6.4 times. Final Control of Copolymers Properties. Because we manipulate copolymers that are sensitive to both hydrolysis and photodegradation we have to control their main characteristics after full treatment. For that purpose we grafted nonlabeled lysine using the optimized conditions and prepared micellar solutions. The results obtained confirmed that our methodology is safe toward the copolymers (Table 4). Polydispersities and molar masses before and after treatment measured by size exclusion chromatography are unchanged proving that there is no major chain cleavage and/or photocross-linking reactions (Figure 1). DLS measurements of micelles solutions of lysinefunctionalized PCL-PEGs return almost the same results as for native PCL-PEGs (Table 4). Moreover, TEM pictures of those solutions (Figure 2B, e.g PCL-PEG2) confirm the formation of spherical objects of around 70 nm diameter that are truly monodisperse with no secondary aggregates. The TEM calculations are coherent with DLS and literature results for the same kind of systems.44 Reported studies on stability of PCL-PEG block copolymers claimed that chain cleavages begin after several hours of irradiation under powerful UV-lamps.38 Concerning the hydrolysis, changes in term of mass decrease were significant only after several weeks of immersion in aqueous solvent at pH 7-8 and relatively long copolymers were reported to be more resistant toward water penetration.45 Accordingly, our smooth protocol is well adequate to achieve functionalization of PCLPEGs ready to be employed for biomedical applications or targeted drug delivery systems. Application to the Coupling of a Targeting Ligand. This functionalization process was finally exemplified for the grafting of mannose on a pre-existing PCL-PEG block copolymer. Mannose is a ligand of interest in the formulation of nanocarriers for oral delivery for instance in oral vaccination. Indeed,

dendritic cells that capture and process antigens before presenting them to T-cells have a crucial role in the early immune answer. They are known to have mannose receptors and could be targeted by mannosylated nanocarriers.46,47 With the developed strategy it was possible to conceive tailor-made targetable systems in which the formulation could be adjusted independently of the ligands introduction. The formulation that fit best the requirement of oral vaccination was constituted of PLGAPEG, PLGA, and PCL-PEG in the proportions 70/15/15.12,31 As aforementioned, the photografting on amorphous copolymers failed and PCL-PEG was the best candidate to be functionalized for the introduction of targeting units. In this aim, coupling of a mannose derivative, namely, 2-aminoethyl-R-D-mannopyroside (MannOH-NH2, synthesis provided as Supporting Information), was attempted on PCL-PEG3 (Scheme 3). MannOH-NH2 was coupled using the optimized procedure for photoactivation (insertion of NHS activated ester) and coupling (formation of amide bond). The efficiency of the process was controlled with a spectrophotometric assay.48 We found a corrected amount of 5424.5 ( 1.6 nmole of MannOH-NH2 covalently grafted per gram of copolymer (colorimetric assay provided as Supporting Information). This result is 1.8 times higher than the one obtained for this copolymer, with the radiolabeled lysine coupled in the same conditions. Nevertheless, it is coherent with the fact that LSC results are underestimated due to chemical and surface quenching effects of the polymer chains in the liquid of scintillation (see Supporting Information). The mannosylated PCL-PEG was thereafter used for the preparation of targeted nanocarriers for the oral delivery of vaccine materials, which will be the purpose of a dedicated paper.49

Conclusion We were interested in the derivatization of PCL-PEGs via a direct, smooth, and practical methodology that avoids degradation of these sensible copolymers. We developed a photoimmobilization-based strategy using an aryl azide molecular clip and monitoring the grafting with 3H-radiolabeled lysine. We validated the grafting on PCL-PEGs with rather long chains and optimized the main parameters. Several convergent effects led to a preferential grafting on PEG segments, which was acknowledged by playing on solvent selectivity. The extension of the method to shorter PCL-PEGs exemplified a dependence of the grafting yields to the copolymers crystallinity, certainly due to the diffusion dependent two-step insertion mechanism of triplet nitrene. We found out that the smaller the PCL-PEGs the lower are the grafting yields. Nevertheless, we validated the whole process on small PCL-PEG, that is, PCL-PEG3, with coupling of 2900 nmol of lysine/g and of 5400 nmol/g of a mannose derivative. Those rates were attested to be enough for using the derivatized PCL-PEGs in the composition of nanoparticles displaying ligands on their external shell.12,31 Thus, our mannosylated PCL-PEGs were used for the formulation of targeted nanocarriers of vaccine materials.49 So we have in hand a postpolymerization derivatization methodology convenient for sensible copolymers as far as they are partially crystalline. It

974


fulfills the main prerequisites for such kinds of functionalizations, such as inducing no degradation of the copolymer backbone and displaying ligands on the hydrophilic segments. Acknowledgment. This work has been supported by the “Region Wallonne” (Belgium), for the project VACCINOR (WINNOMAT Contract No. 415661). Jacqueline MarchandBryanert is senior research associate of F.R.S-FNRS (Belgium). CERM is indebted to the FNRS and IAP (VI-27) “Functional Supramolecular Systems” for financial support. The international collaboration has been supported by the CGRI-FNRS-CNRS cooperation program. Supporting Information Available. Figures: (i) SEC chromatograms of PCL-PEG1 and PCL-PEG2 derivatized with lysine; (ii) Synthesis of 2-aminoethyl-R-D-mannopyroside (MannOH-NH2). Methodological precisions about (i) the general procedure for the photografting; (ii) radioactivity measurement; (iii) irradiation time determination; (iv) protocol for the coupling of biological compounds; (v) NMR study of PCL-PEG1 in CD3Cl; (vi) micelle grafting strategy; (vii) synthesis of 2-aminoethyl-R-D-mannopyroside (MannOH-NH2); and (viii) the measure of grafted MannOH-NH2 amount by colorimetric assay. This material is available free of charge via the Internet at http:// pubs.acs.org.

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BM900027R

Light Induced Functionalization of PCL-PEG Block Copolymers for the

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