Folate-Conjugated Thermoresponsive Block Copolymers - American

Feb 21, 2008 - Priyadarsi De, Sudershan R. Gondi, and Brent S. Sumerlin*. Department of Chemistry, Southern Methodist University, 3215 Daniel Avenue, ...
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Biomacromolecules 2008, 9, 1064–1070

Folate-Conjugated Thermoresponsive Block Copolymers: Highly Efficient Conjugation and Solution Self-Assembly Priyadarsi De, Sudershan R. Gondi, and Brent S. Sumerlin* Department of Chemistry, Southern Methodist University, 3215 Daniel Avenue, Dallas, Texas 75275-0314 Received November 14, 2007; Revised Manuscript Received January 17, 2008

A combination of controlled radical polymerization and azide-alkyne click chemistry was employed to prepare temperature-responsive block copolymer micelles conjugated with biological ligands with potential for active targeting of cancer tissues. Block copolymers of N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with an azido chain transfer agent (CTA). Pseudo-first-order kinetics and linear molecular weight dependence on conversion were observed for the RAFT polymerizations. Cu(I)-catalyzed coupling with propargyl folate resulted in folic acid residues being efficiently conjugated to the R-azido chain ends of the homo and block copolymers. Temperatureinduced self-assembly resulted in aggregates capable of controlled release of a model hydrophobic drug. Cu(I)catalyzed azide-alkyne cycloaddition has proven superior to conventional methods for conjugation of biological ligands to macromolecules, and the general strategy presented herein can potentially be extended to the preparation of folate-functionalized assemblies with other stimuli susceptibility (e.g., pH) for therapeutic and imaging applications.

Introduction Stimuli-responsive polymers in aqueous media typically undergo a change in character of functional groups, e.g. from hydrophilic to hydrophobic. In the unique case of a “smart” block copolymer in which one block is hydrophilic and the other stimuli-responsive, the copolymer character can be tuned to be either double-hydrophilic or amphiphilic, depending on the presence or absence of the stimulus.1 Selective desolvation of the responsive block leads to reversible self-assembly into aggregates such as polymeric micelles, vesicles, or higher-order morphologies. While a variety of stimuli have been employed to induce solution self-assembly,2 temperature-responsive polymers are among the most widely studied, as sensitivity is relatively easy to introduce and potential in vitro and in vivo applications can be envisioned.3 Most thermoresponsive polymers demonstrate a critical solution temperature, at which the phases of polymer and solution are discontinuously changed according to concentration. For example, aqueous solutions of poly(N-isopropylacrylamide) (PNIPAM) exhibit sharp phase transitions around 32 °C.4 Above this lower critical solution temperature (LCST), the polymer exists as molecularly dissolved unimers. Upon heating, chain dehydration and intermolecular aggregation generally leads to insolubility and eventual precipitation. If the responsive polymer is part of a block copolymer with a hydrophilic component, micellization occurs such that intermolecular aggregates of insoluble blocks are stabilized by a corona composed of the hydrophilic polymer. Block copolymers of N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA) are example systems in which the poly(N,Ndimethylacrylamide) (PDMA) block remains hydrophilic above the LCST of the responsive PNIPAM block.5 In these cases, heating results in polymeric micelles with hydrophobic PNIPAM cores and hydrophilic PDMA coronas. * Corresponding author. E-mail: [email protected]. Telephone: +1 (214) 768-8802. Fax: +1 (214) 768-4089.

A variety of drugs, genes, and proteins6 can be integrated into hydrophobic micelle cores and released upon micelle disassembly triggered by an appropriate stimulus. Many areas of drug delivery, most notably those related to cancer treatment, benefit from targeted delivery to specific tissues. The delivery of therapeutics to tumors via polymeric systems has traditionally relied on passive targeting mechanisms, with the most common example arising from the enhanced permeability and retention effect.7 Often it is advantageous to enhance specificity through an active mechanism by which a biological signal on the delivery vehicle periphery is recognized by cell surface ligands such that preferential absorption and/or endocytosis can occur.8 Herein, we report a strategy to prepare materials potentially capable of active targeting by decorating supramolecular assemblies with moieties known to interact with receptors overexpressed on many cancer cell membranes. Folic acid-based ligands are widely employed for targeted delivery of therapeutics and imaging agents to inflammation sites or cancer tissues.9 Folate binding protein, a membrane-associated receptor with high affinity (Kd ≈ 10-10 M) for folate and its conjugates, is generally absent or minimally accessible in normal tissue but selectively upregulated on the surface of many types of human cancer cells, including those of the ovary, kidney, lung, mammary gland, brain, and other organs.10 Conjugation of biological signals to the outer surface of preformed polymeric micelles is of great value when designing carrier systems with the potential for receptor-mediated drug delivery.11 An alternative method of micelle surface functionalization is folate conjugation via the end groups of telechelic micelle-forming block copolymers. Kataoka et al. demonstrated that in the case of folate-receptor-mediated endocytosis, the number and distribution of micelle surface ligands can have a significant affect on cytotoxicity, biodistribution, and anticancer activity.12 Therefore, precise control during synthesis over the degree of conjugation is a substantial benefit when designing systems for potential therapeutic applications.13 When the targeting ligand is conjugated to end groups of polydisperse

10.1021/bm701255v CCC: $40.75  2008 American Chemical Society Published on Web 02/21/2008

Folate-Conjugated Thermoresponsive Block Copolymers

block copolymers prior to micellization, many conventional conjugation strategies are sterically biased toward coupling with shorter chains.14 Therefore, upon self-assembly to micelles, the targeting ligands become buried within the corona and not easily accessible to cell surface receptors. Ligand conjugation to the end group of the corona-forming block must be highly efficient to maximize its chemical availability. In fact, inefficient folate conjugation can lead to unreacted ligand molecules that competitively hamper binding between folate-bound micelles and folate-binding proteins.15 Additionally, it is advantageous for ligand conjugation methodologies to be applicable to a wide variety of block copolymer systems with minimal chance for side reactions with other functional groups contained in the polymer structure. Therefore, an efficient and orthogonal functionalization strategy is highly beneficial during postpolymerization modification with folate. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) results in highly specific and efficient preparation of 1,4disubstituted 1,2,3-triazole products under moderate reaction conditions.16,17 The reaction is tolerant to aqueous or organic media, and little or no side reactions are observed. The practicality and versatility of CuAAC led to its inclusion in the class of efficient and specific organic reactions, commonly termed “click chemistry,” as coined by Sharpless et al.18 CuAAC19 and other click reactions are efficient and versatile tools for preparing or modifying tailor-made polymers20 and an ideal approach for preparing telechelic macromolecules.21 The particular combination of reversible addition-fragmentation chain transfer (RAFT) polymerization22 and click chemistry efficiently leads to functional telechelics, as demonstrated by our group21b,23 and others.24 RAFT polymerization is an excellent method for the preparation of well-defined acrylamido polymers,25,26 and in this study, we employed RAFT to prepare homopolymers and block copolymers of NIPAM and DMA. Polymerization with an azido-functionalized RAFT agent, followed by postpolymerization modification with alkynefunctionalized folic acid, resulted in telechelic polymers with terminal biological ligands. The thermoresponsive nature of PNIPAM allowed block copolymer self-assembly into folateconjugated nanoaggregates at elevated temperatures. Because CuAAC has previously proven preferable to conventional methods for conjugation of biological ligands to macromolecules,27 the general strategy presented herein can potentially be extended to the preparation of folate-functionalized micelles with other stimuli susceptibility (e.g., pH) for therapeutic and imaging applications.

Experimental Section Materials. 1,4-Dioxane (Alfa Aesar, 99+%) and N,N-dimethylacrylamide (DMA, Fluka, 98%) were passed through a column of basic alumina prior to polymerization. 2,2′-Azobisisobutyronitrile (AIBN, Sigma, 98%) was recrystallized from ethanol, and N-isopropylacrylamide (NIPAM, TCI America) was recrystallized twice using hexanes. The RAFT chain transfer agent (CTA) 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl-propionic acid 3-azidopropyl ester (1) (C12-CTAN3) was prepared as previously reported from our group.21b Folic acid (FA, Fisher Bioreagents), N-hydroxysuccinimide (Alfa Aesar, 98%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, Acros, 98%), propargylamine (TCI America, 95%), N,N-dimethylformamide (DMF, Aldrich 99.9%), N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, Aldrich, 99%), dipyridamole (DIP, TCI America), CDCl3 (Cambridge Isotope, 99% D), and dimethylsulfoxide-d6 (DMSOd6, Cambridge Isotope, 99.9% D) were used as received. Copper bromide (CuBr, Aldrich, 98%) was stirred in glacial acetic acid for

Biomacromolecules, Vol. 9, No. 3, 2008 1065 15 h at 24 °C, filtered, washed with glacial acetic acid, and then dried under vacuum. All other chemicals were purchased from VWR and used without further purification. Synthesis of Alkyne-Functionalized Folic Acid (Propargyl Folate) (2). The synthesis of propargyl folate was accomplished by a method derived from literature reports of folate conjugation.28 Folic acid (1.0 g, 0.0022 mol) was dissolved in DMF (10 mL) and cooled in a water/ ice bath. N-Hydroxysuccinimide (260 mg, 0.0025 mol) and EDC (440 mg, 0.0025 mol) were added, and the resulting mixture was stirred in the ice bath for 30 min to give a white precipitate. A solution of propargylamine (124 mg, 2.25 mmol) in DMF (5.0 mL) was added, and the resulting mixture was allowed to warm to room temperature and stirred for 24 h. The reaction mixture was poured into water (100 mL) and stirred for 30 min to form a precipitate. The orange-yellow precipitate was filtered, washed with acetone, and dried under vacuum for 6 h to yield 1.01 g of product (93% yield). 1H NMR (DMSO-d6, ppm): 8.64 (s, PtC7 H, 1H), 8.29–8.24 (d, PtC6-CH2NH-Ph, 1H, J ) 5.2 Hz), 8.04–8.02 (d, -CONHCHCO2H, 1H, J ) 7.8 Hz), 7.67–7.65 (d, Ph-C2H and Ph-C6H, 2H, J ) 8.3 Hz) 6.93 (br s, NH2, 2H), 6.65–6.63 (d, Ph-C3H and Ph-C5H, 2H, J ) 8.4 Hz), 4.49–4.48 (d, PtC6-CH2NH-Ph, 2H, J ) 5.2 Hz), 4.32–4.30 (m, -CONHCHCO2H, 1H), 3.84–3.81 (m, -CONH-CH2CtCH, 2H), 3.07–3.05 (t, -CONHCH2CtCH, 1H, J ) 2.6 Hz), 2.88 (s, -CONH-CH2CtCH, 1H), 2.72 (br s, OH, 1H), 2.31–2.20 (m, -CH2CO2H, 2H), 1.98–1.96 (m, -CHCH2CH2, 1H), 1.87–1.85 (m, -CHCH2CH2, 1H). IR (KBr, cm-1): 3284 (CONH), 2366 (CtC), 1701 (CdO), 1608 (CONH), 1518 (CdCs), 1297, 1189 (C-O-C), 1127, 839, 617 (C-Cb). Synthesis of Azido-Terminated Poly(N-isopropylacrylamide) (N3PNIPAM) (3). Polymerization of NIPAM was conducted at 70 °C under a nitrogen atmosphere, employing C12-CTA-N3 (1) as the RAFT CTA and AIBN as the primary radical source. A typical RAFT polymerization procedure was as follows. NIPAM (1.27 g, 11.2 mmol), C12-CTA-N3 (101 mg, 0.224 mmol), 1,3,5-trioxane (50.4 mg, 0.559 mmol, internal standard), AIBN (1.84 mg, 0.0112 mmol; 1 mL solution of 9.2 mg AIBN in 5 mL 1,4-dioxane), and 1,4-dioxane (3.9 mL) were sealed in a 20 mL vial equipped with a magnetic stir bar. The solution was purged with nitrogen for 40 min, and the reaction vial was placed in a preheated reaction block at 70 °C. Samples were removed periodically by syringe to determine molecular weight and polydispersity index (PDI) by gel permeation chromatography (GPC) and monomer conversion by 1H NMR spectroscopy. The polymerization was quenched by cooling in liquid nitrogen and exposing the solution to air. The solution was concentrated under vacuum, and the polymer was precipitated into cold ether. The polymer was reprecipitated four times from THF/ether and dried under vacuum at room temperature for 12 h. For the CuAAC reaction, R-azido-terminated PNIPAM was synthesized with [NIPAM]:[C12-CTA-N3]:[AIBN] ) 30:1:0.05 (reaction volume ) 10 mL, reaction time ) 2 h). Yield: 62%; Mn ) 2700 g/mol; PDI ) 1.15. Synthesis of Azido-Terminated Poly(N,N-dimethylacrylamide) (N3-PDMA) (4). Polymerization of DMA was conducted as previously reported.21b Synthesis of Azido-Terminated PDMA-b-PNIPAM (N3-PDMAb-PNIPAM) (5). The R-azido-terminated PDMA-b-PNIPAM block copolymer was synthesized by RAFT polymerization in 1,4-dioxane at 70 °C under a nitrogen atmosphere using N3-PDMA (4) as a macroCTA in a 20 mL vial equipped with a magnetic stir bar; [NIPAM] (285 mg, 2.52 mmol):[N3-PDMA] (530 mg, 0.126 mmol):[AIBN] (1.0 mg, 0.0061 mmol) ) 20:1:0.05 (reaction volume ) 5 mL, reaction time ) 2 h). The polymerization was quenched by cooling in liquid nitrogen and exposing the solution to air. The solution was concentrated under vacuum, and N3-PDMA-b-PNIPAM was precipitated into cold ether. Finally, the polymer was reprecipitated (×4) from THF/ether and dried under vacuum at room temperature for 12 h (conversion ) 58%; Mn ) 6050 g/mol; PDI ) 1.15). Click Reaction of Azido-Terminated Polymers with Propargyl Folate (2). The azido-terminated polymers were reacted with propargyl folate (2) in a manner analogous to the following example procedure.

1066 Biomacromolecules, Vol. 9, No. 3, 2008 A solution of N3-PDMA-b-PNIPAM (5) (Mn ) 6050 g/mol, 484 mg, 0.0800 mmol) in DMF (7 mL), and PMDETA (8.7 mg, 0.050 mmol) was purged with nitrogen for 60 min and transferred via syringe to a vial equipped with a magnetic stir bar containing CuBr (7.2 mg, 0.050 mmol) and propargyl folate (2) (42 mg, 0.088 mmol) under a nitrogen atmosphere. The reaction mixture was stirred at 26 °C for 22 h in the absence of oxygen. The reaction mixture was exposed to air, and the solution was passed through a column of neutral alumina. DMF was removed under vacuum, and the product was precipitated into hexanes. The resulting folate-terminated block copolymer (FA-PDMA-bPNIPAM) (6) was dissolved in THF and filtered to remove excess propargyl folate. THF was removed, and then the polymer was dissolved in deionized (DI) water and dialyzed for 6 h using a membrane with a molecular weight cutoff of 1000 Da. The polymers were isolated by lyophilization. Model Drug Release Study. FA-PDMA-b-PNIPAM (6) (1.0 mg) and dipyridamole (DIP) (0.2 mg) were dissolved in THF (0.5 mL). DI water (10 mL) was added dropwise, and the solution was stirred at 50 °C for 6 h to incorporate the drug into the hydrophobic core of the nanoaggregates. The solution (2.5 mL) was divided, and the absorbance of DIP was measured at λ ) 415 nm via UV–vis spectroscopy at 25 and 37 °C. Control measurements were also conducted by observing the time-dependent reduction in DIP absorbance in deionized water in the absence of block copolymer. The absorbance at both 25 and 37 °C was measured for each time point, and the value was subtracted from that observed in the block copolymer solution. Instrumentation. Gel permeation chromatography (GPC) was conducted in DMF with 50 mM LiBr at 55 °C with a flow rate of 1.0 mL/min (Viscotek GPC pump, refractive index detector (λ ) 660 nm) and UV–vis detector (λ ) 254 nm); ViscoGel I-series G3000 and G4000 mixed-bed columns (0–60 × 103 and 0–400 × 103 g/mol, respectively); calibration based on polystyrene standards. 1H and 13C NMR spectroscopy was conducted with a Bruker Avance 400 spectrometer operating at 400 and 100 MHz, respectively. FTIR spectra were obtained with a Nicolet Magna-IR 560 ESP spectrometer. Dynamic light scattering (DLS) was conducted with a Malvern Zetasizer Nano-S equipped with a 4 mW, 633 nm He-Ne laser and an Avalanche photodiode detector at an angle of 173°. The temperature of the polymer solutions with concentration of 0.1 wt % was regulated within an error of (0.1 °C. UV–vis spectroscopy was conducted with an Ocean Optics UV–vis spectrometer (USB2000, Ocean Optics Inc.).

De et al. Scheme 1. Reversible Addition-Fragmentation Chain Transfer Homopolymerization and Block Copolymerization of N-Isopropylacrylamide (NIPAM) and N,N-Dimethylacrylamide (DMA) with Azido-Functionalized Chain Transfer Agents

equilibrium was established.29 The correlation between theoretical and experimental Mn values was excellent throughout the conversion ranges, demonstrating the controlled nature of the polymerizations. Combination of these data indicates a constant concentration of propagating radicals, efficient initiation, and a lack of degradative chain transfer. Narrow and unimodal molecular weight distributions (Mw/Mn < 1.2) were observed by GPC, with the refractive index traces shifting smoothly toward lower elution volume with increasing conversion (Figure

Results and Discussion RAFT Polymerization of NIPAM and DMA. A significant benefit of RAFT is its ability to control polymerizations of most vinyl monomers. Recently, our group reported the synthesis of C12-CTA-N3 (1), which proved efficient in the preparation of R-azido-terminated polymers that were subsequently reacted via CuAAC to yield well-defined functional telechelics21b or hyperbranched polymers.23 The highly efficient and specific functionalization demonstrated by these methods led us to employ CuAAC to conjugate folate residues to azido-terminated double-hydrophilic block copolymers. RAFT polymerizations of NIPAM and DMA with C12-CTAN3 (1) as the chain transfer agent resulted in R-azido-functionalized polyacrylamides (Scheme 1). Both homopolymerizations were conducted with [monomer]:[C12-CTA-N3]:[AIBN] ) 50:1:0.05 at 70 °C. Conversions were determined by 1H NMR spectroscopy in CDCl3 by comparing the integration of the vinyl protons of unreacted monomer and OCH2 protons of the 1,3,5trioxane internal standard. In both cases, pseudo-first-order kinetic plots (Figure 1A) were linear, and as seen in Figure 1B, Mn increased linearly with conversion. As is common for many RAFT polymerizations, a small induction period of approximately 30 min was observed after which the main RAFT

Figure 1. (A) Pseudo-first-order kinetic plot and (B) number-average molecular weight (Mn) vs monomer conversion for the reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA) with azido-functionalized chain transfer agents (C12-CTA-N3 (1)). (C) Gel permeation chromatography (GPC) traces as a function of time for the RAFT polymerization of NIPAM in 1,4-dioxane at 70 °C with [NIPAM]:[C12-CTA-N3 (1)]:[AIBN] ) 50:1:0.05; [NIPAM] ) 2.24 M. (D) GPC traces as a function of time for the RAFT polymerization of DMA in DMF at 70 °C with [DMA]:[C12-CTA-N3 (1)]:[AIBN] ) 50:1:0.05; [DMA] ) 2.23 M.

Folate-Conjugated Thermoresponsive Block Copolymers Table 1. Data from the Reversible Addition-Fragmentation Chain Transfer Homopolymerization and Block Copolymerization of N-Isopropylacrylamide (NIPAM) and N,N-Dimethylacrylamide (DMA) with Azido-Functionalized Chain Transfer Agents polymer N3-PNIPAM (3) N3-PDMA (4) N3-PDMA-bPNIPAM (5) FA-PNIPAM FA-PDMA FA-PDMA-bPNIPAM (6)

conva Mn,GPCb Mn,NMRc Mn,theo LCSTf (%) (g/mol) Mw/Mnb (g/mol) (g/mol) (°C) 62 63 58

2700 4200 6050 3540 4730 6350

1.15 1.11 1.15 1.21 1.22 1.18

2500 4100

2550d 3570d 5510d

26

3500 5160

e

22

3180 4680e 6530e

36

34

a Monomer conversion as determined by 1H NMR spectroscopy. Determined by gel permeation chromatography in N,N-dimethylformamide with conventional calibration based on polystyrene standards. c Determined by 1H NMR spectroscopy. d Mn,theo ) ([monomer]0/[chain transfer agent (CTA)]0) × monomer conversion + CTA molecular weight). e Mn,theo ) Mn,GPC of N3-polymer + 478.4 g/mol (for propargyl folate (FA) (2)). f Lower critical solution temperature (LCST) as determined by dynamic light scattering. (Mn: number-average molecular weight; Mn,theo: theoretical Mn; Mw: weight-average molecular weight; N3-PNIPAM: R-azido-functionalized poly(N-isopropylacrylamide) (3); N3-PDMA: R-azido-functionalized poly(N,N-dimethylacrylamide) (4); N3-PDMA-b-PNIPAM: R-azido-functionalized PDMA-b-PNIPAM block copolymer (5). b

1C,D). Experimental molecular weights were determined by GPC and NMR spectroscopy. The latter (Mn,NMR) was determined from integration of the NIPAM repeat unit signals at 3.82 ppm (-CH(CH3)2) and the signals at 4.03 ppm of the CTA end groups (N3-CH2-CH2-CH2-O-). Similarly for N3-PDMA (4), Mn,NMR was determined by comparing the area of the backbone repeat unit signals (CH) at 2.2–2.6 ppm with those of the CTA end groups (N3-CH2-CH2-CH2-O-) at 4.04 ppm. The theoretical, NMR, and GPC molecular weights were in good agreement (Table 1). Azido-terminated block copolymers of PDMA and PNIPAM were prepared by employing N3-PDMA (4) as a macroCTA for polymerization of NIPAM with [NIPAM]:[N3-PDMA]: [AIBN] ) 20:1:0.05 in 1,4-dioxane at 70 °C (Scheme 1). A conversion of 58% was obtained in 2 h, resulting in N3-PDMAb-PNIPAM (5) block copolymer with Mn ) 6050 g/mol and Mw/Mn ) 1.15 (Figure 2). The azido-terminated homopolymers and block copolymer were analyzed by FTIR spectroscopy, and retention of the terminal azide groups was confirmed by the presence of signals at 2106 cm-1 (Figure 3). End Group Functionalization via CuAAC. End group modification with folate was accomplished by CuAAC. The azido-functionalized PNIPAM (3), PDMA (4), and PDMA-bPNIPAM (5) were reacted at 26 °C in DMF with propargyl folate according to Scheme 2. Although a ligand is not necessary for sufficient solubility of CuBr in DMF,30 PMDETA was employed to ensure enhanced reaction rates and increased end group functionalization. After catalyst removal, the end-functionalized polymers were purified by reprecipitation and lyophilization to remove unconjugated propargyl folate starting material. Figure 2 shows the GPC curves of PNIPAM before (N3PNIPAM (3)) and after (FA-PNIPAM) the conjugation reaction. Peak symmetry was conserved, while a slight shift to higher molecular weight was observed after the addition of propargyl folate (2) (FW ) 478.4 g/mol) to the N3-PNIPAM (3) (Mn ) 2700 g/mol). The molecular weight shift was not as prominent after the click reaction with N3-PDMA (4) and N3-PDMA-bPNIPAM (5), most likely a result of the higher molecular weights of the starting materials (Mn ) 4200 and 6050 g/mol, respectively) leading to decreased chromatographic resolution (Figure 2).

Biomacromolecules, Vol. 9, No. 3, 2008 1067 1

H NMR spectroscopy was used to confirm end group functionalization of N3-PNIPAM (3) after reaction with propargyl folate by comparing the peak of the proton on the pteridine ring at 8.64 ppm with the signal of the PNIPAM CH(CH3)2 protons at 3.82 ppm (Figure 4). From the ratio of these two peak areas, an Mn,NMR ) 3500 g/mol was obtained, which is in excellent agreement with the Mn,GPC ) 3540 g/mol, indicating near-quantitative functionalization of N3-PNIPAM (3) with folate. Also, 1H NMR spectroscopy demonstrated the disappearance of the peak at 4.03 ppm related to N3-CH2-CH2-CH2O-, indicating quantitative conversion of the terminal azido moiety. Similarly, for the functionalization of N3-PDMA-bPNIPAM (5) with folate, disappearance of the peak at 4.04 ppm related to N3-CH2-CH2-CH2-O- indicated quantitative conversion of the terminal azido moiety. The signal from the triazole ring proton could not accurately be integrated for the click reactions of N3-PNIPAM (3) and N3-PDMA-b-PNIPAM (5) because of overlap with the -NH signal from PNIPAM (6.9–7.8 ppm). However, the triazole ring proton in the 1H NMR spectrum for the click reaction of N3-PDMA (4) with propargyl folate was observed at 7.65 ppm (Supporting Information). The integration ratio of this peak (1H) to the peak at 8.62 ppm (from the folic acid PtC7 H, 1H) was 0.93, indicating approximately one triazole per folate residue. The Mn,NMR ) 5160 g/mol was obtained by comparing the area of the backbone -CH peak at 2.2–2.6 ppm with those of the triazole ring protons at 7.65 ppm. By comparing this value with Mn,GPC ) 4730 g/mol, conjugation efficiency of N3-PDMA (4) with propargyl folate (2) was estimated to be 92%. Successful conjugation was further evidenced by absence of the azide absorbance peaks at 2106 cm-1 in the IR spectrum (Figure 3). Two control experiments were carried out to confirm the mechanism of conjugation. In the first experiment, N3-PNIPAM (3), CuBr, and PMDETA were stirred in DMF at 26 °C. In the second experiment, N3-PNIPAM (3) and propargyl folate (2) were stirred in DMF at 26 °C for 22 h. In both cases, no change in molecular weight or molecular weight distribution was observed by GPC. The terminal azido groups remained unchanged, as evidenced by persistence of the characteristic FTIR absorption at 2106 cm-1. 1H NMR analysis indicated no change in the spectra before and after treatment. These experiments indicate that with the conjugation conditions employed, consumption of the azido end groups only occurs via CuAAC with the propargyl folate (2). Solution Behavior. The temperature-responsive aqueous solubility of PNIPAM is well-known.4 Above the LCST of approximately 32 °C, disruption of the delicate hydrophilic/ hydrophobic balance causes collapse and aggregation of the dehydrated macromolecules.31 DLS was used to detect the LCST of the PNIPAM (co)polymers by observing the temperatureinduced increase in hydrodynamic size resulting from intermolecular aggregation (Table 1). N3-PNIPAM (3) demonstrated a transition at approximately 26 °C, much less than the typical LCST of PNIPAM. LCST transitions can be affected by several variables including electrolyte concentration, incorporation of comonomers, and molecular weight. The latter is especially important for polymers of relatively short chain length because of the increased contribution of end groups. In the case of N3PNIPAM (3) (Mn ) 2700 g/mol; PDI ) 1.15), the ω-chain terminus contains a dodecyl trithiocarbonate moiety by virtue of the RAFT mechanism. We attribute the reduced LCST of this polymer to the presence of these hydrophobic end groups.23 Indeed, aminolysis of the thiocarbonylthio end groups,32 result-

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De et al.

Figure 2. Gel permeation chromatography traces of (A) R-azido-functionalized poly(N-isopropylacrylamide) (N3-PNIPAM) (3) and the product obtained after coupling with propargyl folate (FA-PNIPAM); (B) R-azido-functionalized poly(N,N-dimethylacrylamide) (N3-PDMA) (4) and the product obtained after coupling with propargyl folate (FA-PDMA); (C) N3-PDMA macroCTA, N3-PDMA-b-PNIPAM (5), and the product obtained after coupling with propargyl folate (FA-PDMA-b-PNIPAM) (6).

Figure 3. FTIR spectra of (A) R-azido-functionalized poly(N-isopropylacrylamide) (N3-PNIPAM) (3); (B) R-azido-functionalized poly(N,Ndimethylacrylamide) (N3-PDMA) (4); (C) N3-PDMA-b-PNIPAM (5), and (D) folic acid-PDMA-b-PNIPAM (6).

Figure 4. 1H NMR spectra of (A) propargyl folate (FA, 2), (B) N3PNIPAM (3), and (C) FA-PNIPAM.

Scheme 2. CuI-Catalyzed Cycloaddition of Azido-Terminated Polymers and Propargyl Folate (2)

Figure 5. Hydrodynamic size distributions of FA-PDMA-b-PNIPAM (6) (Mn ) 6350 g/mol; Mw/Mn ) 1.18) at (A) 26 °C (9) and (B) 46 °C (b) in 0.1% aqueous solution.

ing in reduction of the dodecyl trithiocarbonate end groups to thiols, resulted in polymers with an LCST of 31 °C. The N3-PDMA-b-PNIPAM (5) block copolymer demonstrated an LCST of 36 °C. In this case, the slight increase in LCST was most likely a result of the adjacent hydrophilic PDMA block. After conjugation with folic acid, the block copolymer underwent self-assembly at 34 °C to yield aggregates of approximately 46 nm. Size distributions obtained from DLS measurements above and below the LCST for FA-PDMA-bPNIPAM (6) are shown in Figure 5. These experiments suggest that FA-PDMA-b-PNIPAM (6) self-assembles to form multimolecular aggregates. Variable temperature NMR spectroscopy confirmed the hydrophilic to hydrophobic transition of the PNIPAM block upon heating above the LCST, while the PDMA-FA portion of the block copolymer remained hydrophilic (Supporting Information). While we cannot be fully certain of the aggregate morphology, it is reasonable to assume the block copolymers form micelles in aqueous solution with hydrophobic

PNIPAM cores and hydrophilic PDMA-FA exterior shells. Selfassembly in the presence of unconjugated PDMA-b-PNIPAM free of folate residues allows facile tuning of the degree of surface functionalization, an important consideration for potential applications in controlled/targeted delivery of therapeutics and imaging agents. Controlled Release. Dipyridamole (DIP), a model hydrophobic drug with release easily monitored by UV–vis spectroscopy, was loaded into N3-PDMA-b-PNIPAM (Supporting Information) and FA-PDMA-b-PNIPAM (6) aggregates.26,33 After correcting for the slight solubility of DIP in pure water, the loading capacity of the aggregates was estimated to be approximately 5.5 w/w%, corresponding to a loading efficiency of 27%. Release kinetics were determined at 25 and 37 °C by monitoring the reduction in absorbance of the solubilized drug upon release and subsequent precipitation.26 DIP loading was accomplished by stirring the drug in an aqueous solution of the block copolymer at 50 °C. Upon lowering the solution temper-

Folate-Conjugated Thermoresponsive Block Copolymers

Biomacromolecules, Vol. 9, No. 3, 2008 1069

Universities (Ralph E. Powe Junior Faculty Enhancement Award) for partial support of this research. Supporting Information Available. FTIR and NMR spectra, LCST data, and additional drug release kinetics. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes

Figure 6. Dipyridamole release from FA-PDMA-b-PNIPAM (6) (Mn ) 6350 g/mol; Mw/Mn ) 1.18) at 25 °C (9), 37 °C (b), and FA-PDMAb-PNIPAM (6) upon cooling to 25 °C after 12 days at 37 °C (O) in 0.01% aqueous solution. Inset: Release kinetics at 25 °C (9), 37 °C (b) up to 16 h. Release was normalized for the slight temperaturedependent water solubility of DIP.

ature to 25 °C, the block copolymer aggregates dissociated to unimers and burst release led to DIP precipitating from solution. On the other hand, release of DIP from a solution containing self-assembled aggregates at 37 °C was considerably slower (Figure 6). After normalizing for the slight temperaturedependent water solubility of DIP, the release profiles above and below the critical aggregation temperature indicated release of the drug was temperature responsive and that self-assemblies at 37 °C were capable of controlled release over a significantly extended period. After gradual release of DIP over the course of approximately 12 days, a reduction in temperature to 25 °C resulted in rapid, near-quantitative release of the remaining encapsulated drug (Figure 6, open circle). Thus, in addition to the gradual controlled release of DIP at 37 °C, responsive dissociation of the block copolymers led to temperature-induced release.

Conclusion An azido-functionalized CTA successfully mediated the RAFT polymerizations of NIPAM and DMA, resulting in R-azido terminal homopolymers and block copolymers. The azido chain ends were coupled with propargyl folate via CuAAC, demonstrating a feasible route to efficiently conjugate folic acid residues to telechelic homo and block copolymers. The orthogonal nature of click chemistry techniques facilitates this functionalization potentially being extended to a variety of responsive copolymers. These temperature-responsive bioconjugates may potentially be useful as drug delivery vehicles that contain ligands to facilitate cancer tissue-specific targeting and subsequent internalization by endocytosis. Self-assembly in the presence of nonconjugated block copolymer allows tuning of the degree of functionalization of the aggregate surface, a factor that can significantly affect in vivo efficacy.12 This general process of preparing folate-decorated nanoaggregates is being extended to block copolymers covalently tethered to anticancer drugs and to block copolymers with pH sensitivity, both of which facilitate extension to potential therapeutic applications. Acknowledgment. Acknowledgment is made to SMU, the Donors of the American Chemical Society Petroleum Research Fund (45286-G7), the Defense Advanced Research Projects Agency (HR0011-06-1-0032), and Oak Ridge Associated

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