Preparation and Tumor Cell Uptake of Poly (N-isopropylacrylamide

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Bioconjugate Chem. 2002, 13, 685−692

685

Preparation and Tumor Cell Uptake of Poly(N-isopropylacrylamide) Folate Conjugates Denis Dube´,‡ Mira Francis,† Jean-Christophe Leroux,†,§ and Franc¸ oise M. Winnik*,‡,†

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Department of Chemistry and Faculty of Pharmacy, Universite´ de Montre´al, C. P. 6128, succursale Centre Ville Montre´al QC Canada H3C 3J7. Received September 3, 2001; Revised Manuscript Received January 7, 2002

Folate conjugates (PNIPAM-NH-FA) of a copolymer of N-isopropylacrylamide (NIPAM) and aminoN′-ethylenedioxy-bis(ethylacrylamide) were prepared by an efficient synthesis leading to random grafting, via a short dioxyethylene spacer, of ∼7 folic acid residues per macromolecule. The chemical composition of the copolymer was characterized by 1H NMR and UV/vis spectroscopy. A fluorophorelabeled folate PNIPAM conjugate was tested by in vitro assays performed with cultured KB-31 cells overexpressing the folate receptor. The cellular uptake of the copolymer was found to be temperature dependent and was competitively decreased by free folic acid, indicating that the polymer uptake is mediated specifically by the folate receptor. Hydrophobically modified folate conjugates of NIPAM, amino-N′-ethylenedioxy-bis(ethylacrylamide) copolymers, bearing a small number of n-octadecyl groups were prepared following a modified synthetic procedure for use in future studies of FA-targeted liposomes.

INTRODUCTION

The modification of liposomes with synthetic polymers is a promising approach toward the development of new highly efficient drug carriers. By coating the surface of liposomes with carefully designed water soluble polymers, several major improvements may be achieved, such as increase in liposome circulation time, responsiveness to external stimuli, and specific targeting. For example, it has been recognized several years ago that poly(ethylene glycol) (PEG)-modified liposomes are endowed of exceptional stability in vivo (1). Formulations incorporating small PEG-coated vesicles are used clinically (2). In these systems, the brushlike coat of PEG chains that covers the lipid bilayer is believed to act as a sterically repulsive shield against nonspecific opsonization by plasma proteins. While the PEG chains are particularly effective in this role, other water-soluble polymers have been shown to offer some level of protection, together with additional enabling functions, such as temperatureor pH-responsiveness. Extensively studied systems include complexes between liposomes and the pH-sensitive polyelectrolyte poly(2-ethylacrylic acid) (3) and the temperature-responsive neutral polymer, poly(N-isopropylacrylamide) (PNIPAM) (4). The performance of polymermodified liposomes as drug carriers depends, to a large extent, on the reliable anchoring of the polymer within the lipid bilayer. In the case of PNIPAM, anchoring of the polymer on the bilayer is effected via a small number of long alkyl chains attached to the polymer backbone, an approach pioneered by Ringsdorf and co-workers (5), and employed effectively by several research teams (6, 7, 8). Anchoring PNIPAM chains onto liposome bilayers results in a decrease of the adsorption of serum proteins, at least below the lower critical solution temperature of PNIPAM (9) and increases the liposome circulation times * To whom correspondence should be addressed. Phone (514) 343 6123; fax (514) 343 2362; e-mail francoise.winnik@ umontreal.ca. † Faculty of Pharmacy. ‡ Department of Chemistry. § Canada Research Chair in Drug Delivery.

in vivo (10). The cytotoxicity of PNIPAM and its derivatives still remains to be assessed in depth. Toxicity data on this polymer are scarce. Matsumaru et al. (11) showed that PNIPAM did not present any sign of acute toxicity after intravenous administration to mice. Recently, Fujimoto and co-workers (12) reported that a copolymer of NIPAM and N-methacryloyloxysuccinimide displayed no cytotoxicity toward U-937 cells below 4 mg/mL. We also observed that copolymers of NIPAM, octadecylacrylate, and methacrylic acid generated no acute local and systemic inflammatory response in rat following subcutaneous injection (13). For efficient drug delivery, a carrier system should also promote tumor or lesion targeting and intracellular access. Internalization of the drug carrier may be achieved by coupling to the carrier a normally endocytosed ligand, taking advantage of natural endocytosis pathways. Using this strategy, a variety of ligands, such as antibodies, growth factors, or cytokines, are used to facilitate the uptake of carriers inside target cells (14). In the work reported here, we have sought to take advantage of the mechanism of folic acid (FA) uptake by cells to promote targeting and internalization, a methodology exploited previously to facilitate entry of attached drugs (15, 16), antibodies (17), imaging agents (18), macromolecules (19, 20, 21), or liposomes (22, 23) into cells. Folic acid is transported into cells via potocytosis, a receptor-mediated endocytosis (24, 25, 26). In this process, the ligand-bound receptor is sequestered in caveolae, internalized into postcaveolar plasma vesicles, released from the receptor via an intravesicular reduction in pH, and subsequently transported into the cytoplasm. The ligand-free receptor is then recycled to the cell surface by reopening of the caveolae. The folate receptor is known to be overexpressed on the surface of cancer cells in the case of epithelial malignancies, such as ovarian, colorectal, and breast cancer, whereas in most normal tissue it is expressed in very low levels (27, 28, 29). We are currently assessing applications of folatePNIPAM conjugates as coating of liposomes internalized via receptor-enhanced cellular uptake. We designed a

10.1021/bc010084g CCC: $22.00 © 2002 American Chemical Society Published on Web 03/30/2002

686 Bioconjugate Chem., Vol. 13, No. 3, 2002

Figure 1. Chemical structure of folic acid and of the hydrophobically modified folate polymeric conjugate

family of N-isopropylacrylamide (NIPAM) copolymers carrying a small number of octadecyl groups and folic acid residues grafted along the chain via a short ethylenedioxy chain (Figure 1). The FA conjugates were obtained by amidation, with folic acid, of an aminated precursor copolymer. The folic acid molecule (Figure 1) possesses two carboxyl groups, termed R and γ, which can act as handles for covalent attachment. It has been demonstrated that folate linked via its γ-carboxyl group retains a strong affinity toward its receptor, whereas its R-carboxyl derivatives are not recognized as readily (30, 31). We chose to attach the folate to the distal end of a short ethylenedioxy chain in order to increase the range of accessible receptor sites. We report here the synthesis and characterization of folate-PNIPAM conjugates and a preliminary in vitro evaluation of their targeting potential, using fluorescently labeled folate-PNIPAM conjugates that do not carry hydrophobic chains, which may interfere with the selected assays. Cytotoxicity assays confirmed that the folate-PNIPAM conjugates bind specifically to KB cells without harming the cells. Direct competition experiments with free folate demonstrated that the PNIPAM-folate conjugates effectively target the cells even at folate concentration above normal serum levels. EXPERIMENTAL PROCEDURES

Materials. Water was deionized using a Milli Q water purification system (Millipore). N-Isopropylacrylamide, obtained from Fisher Scientific (Pittsburgh, PA), was recrystallized from acetone/heptane 2/3 v/v. N,N-Azobis(isobutyronitrile), trifluoroacetic acid, 2,2′-(ethylenedioxy)bis(ethylamine), di-tert-butyl dicarbonate, acryloyl chloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), folic acid dihydrate, and 2,4,6trinitrobenzenesulfonic acid (TNBS), were purchased from Aldrich Chemical Co (Milwaukee, WI). N-n-Octadecylacrylamide was prepared as described previously (32). Fluorescein dichlorotriazine (5-DTAF), sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, Triton X-100, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co (St Louis, MO). Bicinchoninic acid (BCA) was obtained from Pierce Co (Rockford,IL). N-BOC-ethylenedioxy-bis(ethylamine) was prepared as described previously (33). Dioxane and tetrahydrofuran (THF) were dried by distillation over sodium. Triethylamine was dried by distillation over potassium hydroxide, Dichloromethane was dried by distillation over calcium hydride. All other solvents were reagent grade and used

Dube´ et al.

as received. Phosphate buffers of pH 4.7 [20 mM NaH2PO4 + HCl (0.1M)], pH 7.4 (50 mM KH2PO4, NaOH 3.91 mmol), and a borate buffer of pH 9.5 (12.5 mM Borax, 0.8 mM NaOH) were prepared from the corresponding salts. Phosphate buffered saline pH 7.2 (PBS) was prepared from 75 mM NaCl, 53 mM Na2HPO4 and 13 mM NaH2PO4. Silica gel (60-200 mesh) was purchased from Baker. The ion-exchange resin AG 501-X8(D) and DOWEX 2 × 8-400 were obtained from BioRad Laboratories and Supelco, respectively. Spectra/Por membranes (3500 MW cutoff) were employed for dialysis. Instrumentation. NMR spectra were recorded on a Bruker ARX-400 400 MHz spectrometer. Infrared spectra were recorded on a Bomem Hartmann & Braun IR spectrometer fitted with a Bomem Grams/32 data analysis software. UV/Visible spectra were measured with a Hewlett-Packard 8452A photodiode array spectrometer. Viscometry measurements were carried out with an Ubbelhode semi-microdilution viscometer at 27 °C. Monomer and Polymer Synthesis. N-BOC-N′-(Ethylenedioxy-bis(ethylacrylamide) (EDBE). A solution of acryloyl chloride (0.7 mL, 8.5 mmol) in dichloromethane (10 mL) was added dropwise into a solution of N-BOCethylenedioxy-bis(ethylamine) (1.94 g, 8.0 mmol) and triethylamine (2.35 mL, 17 mmol) in dichloromethane (40 mL) kept at 0 °C. At the end of the addition, the mixture was kept at 0 °C for 1 h and allowed to warm to room temperature. The mixture was washed with water, 1 N aqueous acetic acid, aqueous saturated sodium bicarbonate, and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The yellow viscous oil which was purified by flash chromatography over silica eluted with hexane/ethyl acetate 1/3 v/v yielding N-BOC-N′-(ethylenedioxy-bis(ethylacrylamide) (1.20 g, 50% yield) as a pale yellow viscous oil. 1H NMR (CDCl3, δ): 1.38 (s, 9H, (CH3)3C), 3.26 (q, 2H, J ) 5 Hz, CH2NHCOO), 3.52 (m, 10 H, CH2 of ethylenedioxy), 4.95 (br s, 1H, NH), 5.58 (dd, 1H, J ) 0.6, 10 Hz, H(Z)HCd), 6.10 (dd, 1H, J ) 10, 17 Hz, H2CdCH), 6,26 (dd, 1H, J ) 0.6, 17 Hz, H(E)HCd) ppm; FTIR (νj, cm-1) 1170 (O-C(CH3)3 stretching), 1527 (CONHR carbonyl stretching), 1711 (urethane carbonyl stretching), 2930 (alkene stretching). Copolymerization of N-Isopropylacrylamide (NIPAM) and N-BOC-N′-Ethylenedioxy-bis(ethylacrylamide). A solution of NIPAM (0.456 g, 4.0 mmol) and N-BOC-N′ethylenedioxy-bis(ethylacrylamide) (0.270 g, 1.0 mmol) in dioxane (10 mL) was degassed for 30 min by vigorous bubbling of nitrogen. The mixture was heated to 60 °C. AIBN (3.4 mg, 0.021 mmol) was added at once. The polymerization mixture was stirred at 60 °C for 18 h. It was cooled to room temperature. The polymer, PNIPAMNHBOC, was isolated by precipitation into diethyl ether (500 mL) and further purified by two precipitations from THF into diethyl ether. It was dried in vacuo (yield: 72 (%); 1H NMR (CDCl3, δ) 1.13 (br s, (CH3)2CHNH), 1.44 (s, (CH3)3C), 3.30-3.62 (m, CH2 of ethylenedioxy group and main chain), 3.99 (br s, (CH3)2CHNH) ppm. Deprotection of PNIPAM-NHBOC. Trifluoroacetic acid (0.18 mL, 2.4 mmol, 10 equiv per BOC equiv) was added to a solution of PNIPAM-NHBOC (150 mg) in dichloromethane (6.0 mL) cooled in an ice/water bath. The reaction mixture was warmed to room temperature and stirred for 18 h. The solvent was evaporated. The polymer, PNIPAM-NH3+CF3COO-, was purified by two precipitations from THF into diethyl ether and dried in vacuo (yield: ∼100%). Dowex 2 × 8-400 (100 mg) was added to a solution of PNIPAM-NH2 (130 mg) in water. The mixture was stirred at room temperature for 2 h.

Notes

The resin was separated by filtration. The polymer was isolated by lyophilization (70 mg, yield: 63%). 1H NMR (D2O, δ) 1.17 (s, (CH3)2CHNH), 3.19-3.74 (m, CH2 of ethylenedioxy group and of main chain), 3.90 (s, (CH3)2CHNH) ppm. Preparation of PNIPAM-NH-FA. A solution of EDC (53 mg, 0.28 mmol) and folic acid (59 mg, 0.12 mmol) in anhydrous DMSO (2 mL) was prepared and stirred at room temperature for 1 h. It was then added to a solution of PNIPAM-NH (100 mg, 0.165 mmol amine groups) in phosphate buffer (25 mL, pH 4.7). The resulting mixture was stirred at room temperature and in the dark for 16 h. It was brought to pH 9.0 by dropwise addition of diluted aqueous NaOH and dialyzed first against pH 7.4 phosphate buffer for 3 days and then against water for 3 days. The polymer was isolated by lyophilization (115 mg, 80%). 1H NMR (D2O, δ) 1.14 (s, (CH3)2CHNH), 3.19-3.76 (m, CH2 of ethylenedioxy and main chain), 3.90 (1H, (CH3)2CHNH), 6.75 (d, J ) 7 Hz, 3′, 5′ H of folate group), 7.63 (d, J ) 7 Hz, 2′, 6′ H of folate group), 8.77 (s, C7-H of folate group) ppm; UV/vis (phosphate buffer pH 7.4) 363 nm; folate content: 0.336 mmol g-1 polymer). PNIPAM-NH-DTAF. A solution of fluorescein dichlorotriazine (3.9 mg, 0.0071 mmol) in aqueous borate buffer (5 mL, pH 9.5) was added to a solution of PNIPAM-NH (85 mg, 0.140 mmol amine) in the same buffer (25 mL). The reaction mixture was stirred at room temperature for 2 h. It was dialyzed against pH 9.5 borate buffer for 16 h and against water for 3 days. The polymer was isolated by lyophilization (70 mg, 71%). 1H NMR (D2O, δ) 1.08 (s, (CH3)2CHNH), 3.19-3.76 (m, CH2 of ethylenedioxy and main chain), 3.90 (1H, (CH3)2CHNH) ppm; UV/ vis (pH 9.5 borate buffer) 490 nm; DTAF content: 0.095 mmol g-1 polymer). PNIPAM-NH-FA-DTAF. The polymer was prepared as described for the preparation of PNIPAM-NH-DTAF, starting from PNIPAM-NH-FA (83 mg); 72% yield; 1.10 (s, (CH3)2CHNH), 3.08-3.64 (m, CH2 of ethylenedioxy), 3.85 (1H, (CH3)2CHNH), 6.74 (d, J ) 7 Hz, folate aromatic protons), 7.62 (d, J ) 7 Hz, folate aromatic protons) ppm; UV/vis (borate buffer pH 9.5) 492 nm; DTAF content: 0.043 mmol g-1 polymer). Preparation of PNIPAM-NH-FA-ODA. The copolymer was prepared following the three-step procedure outlined for the preparation PNIPAM-NH-FA, starting from NIPAM (0.574 g, 4.84 mmol), N-BOC-N′-ethylenedioxy-bis(ethylacrylamide) (0.780 g, 2.5 mmol) and Noctadecylacrylamide (101 mg, 0.308 mmol). PNIPAM-N-t-BOC-ODA: yield: 77%; 1H NMR (CDCl3, δ) 0.87 (t, J ) 17 Hz, H3CC17H34), 1.14 (br s, (CH3)2CHNH), 1.45 (s, (CH3)3C), 2.10 (br, main chain protons and CH2 of octadecyl group), 3.31-3.63 (m, CH2 of ethylenedioxy), 3.99 (br s, (CH3)2CHNH) ppm. PNIPAM-NH-ODA: yield: 59%; 1H NMR (D2O, δ) 0.89 (br t, H3CC17H34), 1.14 (br s, (CH3)2CHNH), 3.193.76 (m, CH2 of ethylenedioxy), 3.90 (br s, (CH3)2CHNH) ppm. PNIPAM-NH-FA-ODA: yield: 50%; 1H NMR (D2O, δ) 0.89 (br t, H3CC17H34), 1.14 (br s, (CH3)2CHNH), 3.193.3.76 (m, CH2 of ethylenedioxy), 3.90 (br s, (CH3)2CHNH) ppm.; UV/vis (phosphate buffer, pH 7.4) 363 nm; folate content 0.196 mmol g-1 polymer. UV/Vis Analysis. Folate Content. The content of folate in the conjugates was determined by quantitative UV spectrophotometry of polymer solutions in aqueous phosphate buffer (pH 7.4) using the molar extinction coefficient value of 6197 mol-1 cm-1 at λ ) 363 nm (34). DTAF Content. The content of DTAF in the conjugates was determined by quantitative UV spectrophotometry

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of polymer solutions in aqueous borate buffer (pH 9.2) using the molar extinction coefficient value of 70000 mol-1 cm-1 at λ ) 490 nm (35). Colorimetric Determination of the Free Amine Groups (36). A known amount of polymer (5 mg) was dissolved in a solution of excess TNBS (0.01 M) in sodium hydrogen carbonate (2.0 mL, 20 g L-1, pH 8.2). The solution was kept for 2 h at 40 °C. It was cooled to room temperature and made up to 10 mL with a water/THF solution (v/v). The amount of TNBS-derivatized amines in a sample was determined from the absorbance at λ 345 nm, using TNBS-derivatized N-BOC-ethylenedioxybis(ethylamine) (345 nm ) 8700 L mol -1 cm -1) as reference compound. A solution of TNBS without added polymer was treated under the same conditions. Its absorbance at 345 nm was subtracted from that of the TNBS-derivatized polymers to correct for its residual absorbance at this wavelength. Cell Culture, Cytotoxicity, and FA Uptake Studies. Cell Lines. KB-31 cells, a human nasopharyngeal epidermal carcinoma cell line overexpressing surface receptors for folic acid (a gift from Dr. Daryl Drummond, Research Institute, California Pacific Medical Center, San Francisco, CA), were maintained in a monolayer culture in folate-free RPMI 1640 medium containing L-glutamine (Gibco laboratories). The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco laboratories) and 1% (v/v) penicillinstreptomycin antibiotics (10000 U mL-1 penicillin G and 10000 µg mL-1 streptomycin, Gibco laboratories) solution (100 U mL-1 penicillin G, 100 µg mL-1 streptomycin). Cultured cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Cytotoxicity Assay. KB-31 cells were seeded in triplicate in 96-well plates at 5 × 104 cells/well. Cells were allowed to adhere for 4 h at 37 °C in a humidified atmosphere of 5% CO2 in air. Solutions of PNIPAM-NH-FA-DTAF or PNIPAM-NH-DTAF in culture medium (0-1 mg mL-1) were added to the cells. After several different incubation times (2-24 h), cell viability was determined by a MTT test, according to the procedure described by Mosmann (37). The test is based on mitochondrial dehydrogenase cell activity as an indicator of cell viability. Ten microliter aliquots of a MTT solution in PBS (5 mg mL-1) were added to each well. After incubation for an additional 4 h at 37 °C, a 2-propanol solution of HCl (100 µL, 0.4 N HCl) was added to each well to ensure solubilization of formazan crystals. The optical density values were determined using a multiwell-scanning spectrophotometer (Bio-Tek Instruments Inc.) at 570 nm. Fluorescence Microscopy. Cells were plated at a concentration of 5 × 105 cells per 35 mm dish in a six-well plastic culture plate 24 h prior to the assay. They were then incubated for 1 h at 37 °C with a solution of PNIPAM-NH-FA-DTAF or PNIPAM-NH-DTAF in culture medium (0.2 mg mL-1). For free folate competition studies, 1 mM folic acid was added to the incubation medium. After incubation, the cells were washed five times with 2 mL of PBS to remove free polymer. Cellassociated fluorescence was viewed with an Axiovert inverted fluorescence microscope (Zeiss) equipped with a filter set that produces excitation in the range 465495 nm and allows observation of fluorescence emission in the range 515-555 nm with a long wave path dichroic mirror and barrier filter. Cells were photographed using a 1310C DVC digital camera (DVC Company Inc.). Quantitation of the Uptake of PNIPAM-NH-FADTAF by KB-31 Cells. Cells were plated at a concentration of 5 × 105 cells per 35 mm dish in a six-well plastic

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Figure 2. Synthetic sequence for the preparation of the copolymer PNIPAM-NH-FA

culture plate 24 h prior to the assay. They were then incubated for 1 h at 37 °C and 2 h at 4 °C with PNIPAMNH-FA-DTAF or PNIPAM-NH-DTAF (0.2 mg mL-1 in culture medium). For free folate competition studies, 1 mM folic acid was added to the incubation medium. After incubation, the cells were washed five times with 2 mL of PBS. The cells were then solubilized in Triton X-100 solution (2 mL, 1% w/v final concentration in PBS). Cell-associated fluorescence was measured using a series 2 Aminco Bowman (Spectronic Instruments Inc.) fluorescence spectrometer. Samples were excited at 492 nm, and fluorescence emission intensity was monitored at 516 nm. The amount of cell-associated polymer was calculated using a predetermined standard curve and calibrated with protein cell content determined by a bicinchoninic acid (BCA) protein assay (Pierce). RESULTS AND DISCUSSION

Preparation and Characterization of Folate Conjugates. Folate-PNIPAM conjugates were prepared by postmodification of a copolymer of N-isopropylacrylamide (NIPAM) and amino-N′-ethylenedioxy-bis(ethylacrylamide) as shown in Figure 2. The copolymer PNIPAMNH was obtained by free radical copolymerization in dioxane of NIPAM and N-BOC-N′-ethylenedioxy-bis(ethylacrylamide) followed by deprotection of the terminal primary amine groups. The composition of the BOCprotected copolymer was determined by 1H NMR spectroscopy, using the singlet at δ 1.14 ppm, assigned to the resonance of the tert-butyl protons of the BOC units, and the broad singlet at δ 3.90 ppm, assigned to the resonance of the methine NIPAM protons, to calculate the molar ratio of EDBE and NIPAM units, respectively (Figure 3). The disappearance of the signal at δ 1.14 ppm in the 1H NMR spectrum of PNIPAM-NH confirmed that the deprotection step went to completion. A primary-aminespecific colorimetric assay was performed with PNIPAMNH to ascertain the composition of the deprotected copolymer. This copolymer is soluble in water and in PBS at all temperatures between 0 and 60 °C, unlike the homopolymer PNIPAM, which in aqueous solution ex-

hibits a cloud point at 31 °C. This enhanced water solubility is attributed to the presence of the hydrophilic ethylenedioxy-containing residues along the polymer chain. Coupling of the folate residue to the polymer was achieved via an EDC-mediated reaction of folic acid and the primary amine functions linked to the polymer. The success of the linkage was confirmed by the presence of signals at δ 6.75-8.77 ppm in the 1H NMR spectrum of PNIPAM-NH-FA and by an absorbance at λ ) 363 nm in the UV spectrum of the copolymer. It is known that carbodiimide-activated folic acid can couple either via the R or the γ carboxyl group of its glutamate residue, although EDC has been shown to favor linkage of the distal γ carboxyl residue. Wang et al. (31) used 1H NMR spectral data to ascertain the regiochemistry of the linkage of folate derivatives and ethylenediamine. Unfortunately, in the 1H NMR spectrum of PNIPAM-NHFA (Figure 3), the relevant signals are masked by broad and strong signals due to the resonances of the protons associated to the polymer residues. Thus, we were unable to determine the regiochemistry of the folate linkage in PNIPAM-NH-FA. It is expected to consist mostly of the γ-linked isomer, due to the reaction conditions selected and also to the steric constraints imposed by the polymer chain. Hydrophobically modified PNIPAM-folate conjugates were obtained via the same synthetic sequence, starting with a copolymer of NIPAM, amino-N′-ethylenedioxy-bis(ethylacrylamide), N-n-octadecylacrylamide (Figure 2). The level of hydrophobic group incorporation in the HMfolate conjugate could not be determined on the basis of the 1H NMR spectrum of its aqueous solution, which exhibited significant line broadening indicative of the formation of micellar structures and concomitant motional restriction of the alkyl chains (38). The octadecyl group content of the precursor, PNIPAM-N-t-BOC-ODA, could be determined accurately from the 1H NMR spectrum of its solution in CDCl3, using the triplet at δ 0.87 ppm, attributed to the resonance of the terminal methyl protons of the octadecyl chain and the broad singlet at δ

Notes

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Figure 3.

Figure 4. CDCl3.

1H

NMR spectrum of PNIPAM-NH-FA in D2O.

1H

NMR spectrum of PNIPAM-NBOC-ODA in

Table 1. Composition and Physical Properties of the Polymers composition (mol %) polymer PNIPAM-NH PNIPAM-NH-FA PNIPAM-NH-ODA PNIPAM-NH-ODA-FA

NIPAMa EDBEa,b ODAa FAc Mvisd 79 79 63 63

21 16 33 30

5 4 4

3

20000 20000 20000 20000

a

From 1H NMR spectra. b From quantitative analysis of primary amine groups (see text). c From UV/vis spectra. d From viscometry in THF, 27 °C, using the relationship [η] ) 10-3.307 Mvis0.79 (Ganachaud, F., et al. (2000) Macromolecules 18, 6738).

4.0 ppm, attributed to the resonance of the NIPAM methine proton (Figure 4). The physical properties and the composition of the PNIPAM-FA-ODA are listed in Table 1. Cellular Uptake of PNIPAM-Folate Conjugates. The cytotoxicity of PNIPAM-NH-FA-DTAF and PNIPAM-NH-DTAF on KB-31 cells was evaluated after

Figure 5. Effect of PNIPAM-NH-FA-DTAF (A) and PNIPAMNH-DTAF (B) concentration on KB-31 cell viability determined by MTT assay after a 2-h (closed columns) or 24-h (open columns) incubation time. Mean ( SD (n ) 3).

incubation at 37 °C for 2 and 24 h (Figure 5). The two polymers exhibited nearly identical toxicity profiles. Cell viability reached a value of approximately 75% and 60% after incubation of 2 and 24 h, respectively, for the highest polymer concentration tested (1 mg/mL). These results corroborate our previous results on the cytotoxicity on macrophage-like J774 cells where a pH-sensitive NIPAM copolymer was evaluated (39) which exhibited an LD50 in excess of 1 mg/mL after a 24-h incubation period. To assess the uptake of PNIPAM-NH-FA by cells overexpressing the folate receptor, the fluorescently

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Figure 7. Uptake of PNIPAM-NH-FA-DTAF or PNIPAMNH-DTAF (0.2 mg/mL) by KB-31cells in the absence or presence of 1 mM folic acid. The cells were incubated at 37 °C for 1 h (closed columns) or 4 °C for 2 h (open columns). Mean ( SD (n ) 3).

Figure 6. Fluorescence micrographs of KB-31 cells incubated at 37 °C for 1 h with PNIPAM-NH-FA-DTAF (A, B) or PNIPAM-NH-DTAF (C) (0.2 mg/mL). Cells were incubated in the absence (A, C) or presence (B) of 1 mM free folic acid.

labeled polymer and its precursor were incubated for 1 h at 37 °C with KB-31 cells. Fluorescence micrographs of cells exposed to the folate-bearing polymer PNIPAMNH-FA-DTAF displayed an intensive and diffuse fluorescence indicating binding of the polymer on the cell membrane (Figure 6A). Some cells displayed a punctuated fluorescence pattern suggesting that the polymer was localized also in endocytic vesicles. In contrast, cells incubated with the fluorescently labeled nontargeted polymer PNIPAM-NH-DTAF displayed only very weak fluorescence attributed to low nonspecific uptake of the polymer (Figure 6B). The role of cell surface folate receptors in polymer binding was assessed further by incubating KB-31 cells with PNIPAM-NH-FA-DTAF in the presence of 1 mM free folic acid used as competitive

inhibitor of cellular uptake. Fluorescence micrographs of the cells incubated for 1 h (Figure 6C) show that the presence of free folic acid effectively inhibited uptake of PNIPAM-NH-FA-DTAF by the cells. The uptake of folate-conjugated and nontargeted polymer by KB-31 cells was quantified by fluorescence spectroscopy. The cells were incubated with each copolymer for 1 h at 37 °C and for 2 h at 4 °C (Figure 7). After incubation at 37 °C, the uptake of PNIPAM-NH-FADTAF was 8.5 times higher than that of PNIPAM-NHDTAF. The level of uptake of the folate-conjugated polymer was reduced by 80% in the presence of 1 mM free folic acid. Incubation at 4 °C resulted in a 2.5-fold decrease in cell association of PNIPAM-NH-FA-DTAF, compared to the uptake upon incubation at 37 °C. The uptake of the nontargeted polymer upon incubation at 4 °C was barely detectable by fluorescence. As the endocytic process is strongly inhibited at 4 °C, fluorescence of cells incubated at this temperature in the presence of labeled polymer can be attributed only to polymer bound on the cell surface. The folate-conjugated polymer still displayed a 19-fold increase in uptake versus its nontargeted counterpart. The addition of 1 mM free folic acid resulted in an 88% decrease of fluorescence, demonstrating the specific receptor-mediated binding of PNIPAM-NH-FADTAF. The fact that 100% inhibition was not achieved upon addition of free folic acid may be an indication of some level of nonspecific uptake of the copolymer. It may also reflect the stronger avidity of the copolymer as a result of multivalent interaction (9, 16), as each polymer chain bears several folic acid residues (40) with an average of seven folic acid molecules. CONCLUSIONS

In this report, we described an efficient and flexible synthesis of noncytotoxic PNIPAM folate conjugates. We have demonstrated that selective recognition of the conjugates by the folate receptors is maintained upon linking of folate to the macromolecule. We recently demonstrated that pH-sensitive amphiphilic NIPAM copolymers could facilitate the transfer of liposomal content from the endosomes/lysosomes to the cytoplasm (39). Therefore, by combining the tumoral targeting ability of folic acid with the unique physicochemical properties of NIPAM copolymers, one could design highly efficient liposomal systems for the cytoplasmic delivery of a variety of active compounds. Indeed, it is now clearly established that folate mediates endocytosis of macromolecules, drugs, and colloidal carriers into vesicles that become rapidly acidified to a pH of about 5 (41). Although

Notes

the folate-targeting approach has been successful in a number of studies (16), endosomally targeted materials, which do not cross easily cell membranes, do not escape from the endosome in the absence of a triggerable membrane destabilization/fusion pathway. Therefore, for the delivery of macromolecules, such as DNA, or of highly hydrophilic drugs, inefficient translocation into the cytosol may compromise the folate targeting approach. To circumvent this problem, folic acid can be conjugated to a copolymer exhibiting endosomolytic properties (e.g., polyethylenimine) (16) or attached to fusiogenic liposomes (42). Future investigations will focus on the design of delivery systems consisting of serum-stable liposomes modified with a pH- and/or temperature-sensitive folateconjugated NIPAM copolymer and their uptake in various cell models overexpressing the folate receptor. ACKNOWLEDGMENT

This work was supported by grants of the Canadian Institutes of Health Research (CIHR) to J.C.L. and of the Natural Sciences and Engineering Research Council of Canada (NSERC) to F.M.W. J.C.L. and M.F. acknowledge scholarships from the Fonds de la Recherche en Sante´ du Que´bec and the Canadian International Development Agency, respectively. LITERATURE CITED (1) Lasic, D., Martin, F., Eds. (1995) Stealth Liposomes, CRC Press, Boca Raton, FL. (2) Drummond, D. C., Meyer, O., Hong, K., Kirpotin, D., and Papahadjopoulos, D. (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol. Rev. 51, 691-743. (3) Thomas, J. L., and Tirrell, D. A. (1992) Polyelectrolytesensitized phospholipid vesicles. Acc. Chem. Res. 25, 336342. (4) Schild, H. G. (1992) Poly (N-Isopropylacrylamide): experiment, theory, and application. Prog. Polym. Sci. 17, 163-249. (5) Ringsdorf, H., Venzmer, J., and Winnik, F. M. (1991) Interaction of hydrophobically modified poly-N-(isopropylacrylamides) with model membranes or playing a molecular accordion. Angew. Chem., Int. Ed. Engl. 30, 315-319. (6) Hayashi, H., Kono, K., and Takagishi, T. (1996) Temperature-controlled release properties of phospholipid vesicles bearing a thermo-sensitive polymer. Biochim. Biophys. Acta 1280, 127-134. (7) Kim. J.-C., Bae, S. K., and Kim, J.-D. (1997) Temperature sensitivity of liposomal lipid bilayers mixed with poly-(Nisopropylacrylamide-co-acrylic acid). J. Biochem. 121, 15-19. (8) Meyer, O., Papahadjopoulos, D., and Leroux, J. C. (1998) Copolymers of N-isopropylacrylamide can trigger pH sensitivity to stable liposomes. FEBS Lett. 42, 61-64. (9) Yamazaki, A., Winnik, F. M., Cornelius, R. M., and Brash, J. L. (1999) Modification of liposomes with N-substituted polyacrylamides: identification of proteins adsorbed from plasma. Biochim. Biophys. Acta 1421, 103-115. (10) Roux, E., Stomp, R., Giasson, S., Pe´zolet, M., Moreau, P., and Leroux, J. C. (2001) Steric stabilization of liposomes by pH-responsive N-isopropylacrylamide copolymer. Pharm. Res. (submitted). (11) Matsumaru, Y., Hyodo, A., Nose, T., Ito, S., Hirano, T., and Ohashi, S. (1996) Applications of thermosensitive polymers as a new embolic material for intravascular neurosurgery. J. Biomater. Sci. Polym. Ed. 7, 795-804. (12) Fujimoto, F., Iwasaki, C., Arai, C., Kuwako, M., and Yasugi, E. (2000) Control of cell death by the smart polymeric vehicle. Biomacromolecules 1, 515-518. (13) Molinaro, G., Adam, A., Zignani, M., Taillefer, J., SchwachAbdellaoui, K., Gurny, R., and Leroux, J. C. (1999) Evaluation of the local and systemic inflammatory responses induced by

Bioconjugate Chem., Vol. 13, No. 3, 2002 691 the subcutaneous injection of different biopolymers. Proceed. Intern. Symp. Control. Relat. Bioact. Mater. 26, 607-608. (14) Duncan, R., and Connors, T., Eds. (1996) Drug targeting in cancer therapy. J. Drug Targeting 3, 317-410. (15) Ladino, C. A., Chari, R. V., Bourret, L. A., Kedersha, N. L., and Goldmacher, V. S. (1997) Folate-maytansinoids: target-selective drugs of low molecular weight. Int. J. Cancer 73, 859-864. (16) Sudimack, J., and Lee, R. J. (2000) Targeted drug delivery via the folate receptor. Adv. Drug Delivery Rev. 41, 147-162. (17) Coney, L. R., Tomasetti, A., Carayannopoulos, L., Frasca, V., Kamen, B. A., Colnaghi, M. L., and Zurawski, V. R. Jr. (1991) Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein. Cancer Res. 51, 6125-6132. (18) Luo, J., Smith, M. D., Lantrip, D. A., Wang, S., and Fuchs, P. L. (1997) Efficient syntheses of pyrofolic acid and pteroyl azide, reagents for the production of carboxyl-differentiated derivatives of folic acid. J. Am. Chem. Soc. 119, 10004-10013. (19) Leamon, C. P., and Low, P. S. (1991) Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis. Proc. Natl. Acad. Sci. U.S.A. 88, 55725576. (20) Mislick, K. A., Baldeschwieler, J. D., Kayyem, J. F., and Meade, T. J. (1995) Transfection of folate-polylysine DNA complexes: evidence for lysosomal delivery. Bioconjugate Chem. 6, 512-515. (21) Kono, K., Liu, M., and Fre´chet, J. M. J. (1999) Design of dendritic macromolecules containing folate or methotrexate residues. Bioconjugate Chem. 10, 1115-1121. (22) Gabizon, A., Horowitz, A. T., Goren D., Tzemach, D., Mandelbaum-Shavit, F., Qazen, M. M. and Zalipsky, S. (1999) Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: in-vitro studies Bioconjugate Chem. 10, 289-298 (and references therein). (23) Anderson, K. E., Stevenson, B. R., and Rogers, J. A. (1999) Folic acid-PEO-labeled liposomes to improve gastrointestinal absorption of encapsulated agents. J. Controlled Release 60, 189-198. (24) Rothberg, K. G., Ying, Y., Kolhouse, J. F., Kamen, B. A., and Anderson, R. G. W.(1992) The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated endocytic pathway. J. Cell. Biol. 110, 637-649. (25) Kamen, B. A., and Capdevilla, A. (1986) Receptor-mediated folate accumulation is regulated by the cellular folate content. Proc. Natl. Acad. Sci. U.S.A. 83, 5983-5987. (26) Anderson, R. G. W., Kamen, B. A., and Rothberg, K. G. (1992) Potocytosis sequestration and transport of small molecules. Science 255, 410-411. (27) Campbell, I. G., Jones, T. A., Foulkes, W. D., and Trowsdale, J. (1991) Folate-binding protein is a marker for ovarian cancer. Cancer Res. 51, 5329-5338. (28) Ross, J. F., Chaudhuri, P. K., and Ratnam, M. (1994) Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo as in established cell linesphysiological and clinical applications. Cancer 73, 2432-2443. (29) Weitman, S. D., Lark, R,. H., Coney, L. R., Fort, D. W., Frasca, V., Zurawski, V. R., Jr., and Kamen, B. A. (1992) Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer Res. 52, 3396-3401. (30) Leamon, C. P., Pastan, I., and Low, P. S. (1993) Cytotoxicity of folate-Pseudomonas exotoxin conjugates towards tumor cells. Contribution of translocation domain. J. Biol. Chem. 269, 24847-24854. (31) Wang, S., Luo, J., Lantrip, D. A., Waters, D. J., Mathias, C. J., Green, M. A., Fuchs, P., and Low, P. S. (1997) Design and synthesis of [111In]DTPA-Folate for use as a tumortargeted radiopharmaceutical. Bioconjugate Chem. 8, 673679. (32) Ringsdorf, H., Venzmer, J., and Winnik, F. M. (1991) Fluorescence studies of hydrophobically modified poly-(Nisopropylacrylamides). Macromolecules 24, 1678-1686. (33) Krapcho, A. P., and Kuell, C. S. (1990) Monoprotected diamines. N-tert-Butoxycarbonyl-R,ω-alkanediamines from R,ω-alkanediamine. Synth. Commun. 20, 2559-2564.

692 Bioconjugate Chem., Vol. 13, No. 3, 2002 (34) Reddy, J. A., and Low, P. S. (1998) Folate-mediated targeting of therapeutic and imaging agents to cancers. Crit. Rev. Ther. Drug Carrier Syst. 15, 587-627. (35) Blakeslee, D. (1977) Immunofluorescence using trichlorotriazine-fluorescein (DTAF). J. Immunol. Methods 17, 361364. (36) Kuijpers, A. J., Engbers, G. H. M., Meyvis, T. K., de Smedt, S. S. C., Demeester, J., Krijgsveld, J., Zaat, S. A. J., Dankert, J., and Feijen, J. (2000) Combined gelatin-chondroitin sulfate hydrogels for controlled release of cationic antibacterial proteins. Macromolecules 33, 3705-3713. (37) Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. (38) Dube´, D. MSc Thesis, Universite´ de Montre´al (2001).

Dube´ et al. (39) Francis, M. F., Dhara, G., Winnik, F. M., and Leroux, J. C. (2001) In vitro evaluation of pH-sensitive polymer/niosome complexes. Biomacromolecules 2, 741-749. (40) The number of folic acid groups per chain, estimated from the viscosity averaged molecular weight of the polymer and the UV-determined folic acid polymer content, is approximately 7. (41) Lee, R. J., Wang, S., and Low, P. S. (1996) Measurement of endosome pH following receptor-mediated endocytosis. Biochim. Biophys. Acta 1312, 237-242. (42) Reddy, J. A., and Low, P. S. (2000) Enhanced folate receptor mediated gene therapy using a novel pH-sensitive lipid formulation. J. Control. Release 64, 27-37.

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