Multifunctional Poly[N-(2-hydroxypropyl)methacrylamide] Copolymers

Article pubs.acs.org/Macromolecules

Multifunctional Poly[N‑(2-hydroxypropyl)methacrylamide] Copolymers via Postpolymerization Modification and Sequential Thiol−Ene Chemistry Nora Francini, Laura Purdie, Cameron Alexander,* Giuseppe Mantovani,* and Sebastian G. Spain*,† School of Pharmacy, University of Nottingham, University Park, Nottingham. NG7 2RD. U.K. S Supporting Information *

ABSTRACT: Poly[N-(2-hydroxypropyl)methacrylamide] is a promising candidate material for biomedical applications. However, synthesis of functional pHPMA via compolymerization results can lead to variations in monomer composition, molar mass, and dispersity making comparison difficult. Postpolymerization modification routes, most commonly aminolysis of poly[active ester methacrylates], have alleviated some of these problems, but ester hydrolysis can lead to other problems. Here we report the synthesis of multifunctional pHPMA via a simple two-step derivatization of pHPMA homopolymer using readily available standard reagents and atom-efficient procedures. First, treatment with allyl isocyanate yields the corresponding carbamate with predictable incorporation of side-chain functionality. Allyl-pHPMA can then be derivatized further via radical thiol−ene reactions to generate pHPMA with multiple diverse functionalities but without adverse effects on the molecular weight and dispersity of the polymer. The applicability of the method to production of biologically relevant materials is demonstrated by cytocompatibility and cell labeling experiments with easily prepared ligand-functionalized pHPMA in the HCT 116 model cell line.

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INTRODUCTION Poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA) is a hydrophilic polymer that has found many applications in nanomedicine and remains one of the few synthetic polymers to have reached clinical trial.1,2 Like its more common counterpart poly[ethylene glycol] (PEG), its applicability lies in its ability to act as a carrier and/or shield for therapeutics, thus providing protection from unfavorable biological interactions. pHPMA also has advantages over PEG including ease of synthesis via radical polymerization and the potential to add multiple functionalities as pendent groups, the latter allowing combinations of drugs, targeting moieties and other labels to be combined on a single polymeric backbone akin to the idealized prototypical drug delivery system proposed by Ringsdorf.3 Recent advances in controlled radical polymerization have been applied to pHPMA synthesis resulting in greater control over molecular weight, dispersity and architecture, and thus allowing access to more complex materials.2 However, until recently, the introduction of functional pendent groups was limited to copolymerization with functional or reactive monomers. Although successful in part, this route presents difficulties in comparing different functionalities as new polymers must be synthesized with associated variations in degree of polymerization, etc., with the additional complication of differing reactivity ratios affecting composition and monomer sequence.4−6 More recently, postpolymerization derivatization of reactive polymer precursors, has been investigated for the synthesis of copolymers including pHPMA.7,8 The most widely © XXXX American Chemical Society

used is poly[pentafluorophenyl methacrylate] (pPFPMA) as its greater solubility in organic solvents allows for ease of synthesis via controlled polymerization techniques such as RAFT.9 Treatment of pPFPMA with an excess of primary amine(s) results in a high degree of conversion to the respective poly[methacrylamide] allowing access to a variety of previously unavailable materials.10,11 However, complete conversion of pPFPMA to pHPMA can prove to be challenging,12 with competitive hydrolysis resulting in the formation of unreactive methacrylic acid moieties. This side reaction that has recently been thoroughly investigated by Zentel et al., who found the presence of 3 vol % water caused no detectable structural changes as measured by inverse gated 13C NMR spectroscopy. However, pHPMA synthesized from pPFPMA in the presence of 3 vol % water was found to have a ζ-potential of −15.8 ± 2.0 mV whereas polymer synthesized under anhydrous conditions, or directly from HPMA mononmer, had near neutral values.13 For certain biomedical applications, for example those involving delivery of drugs to target sites at or in cell membranes, small variations in charge can result in large differences in uptake,14−17 making such changes in charge unacceptable. Similarly, where a material with a net positive charge is desired greater functionality must be introduced,18 and intra- and intermolecular electrostatic interactions can lead to instability. Received: March 3, 2015 Revised: April 7, 2015

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DOI: 10.1021/acs.macromol.5b00447 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 1. Synthesis of Functional pHPMA via Post-Polymerization and Thiol−Ene Chemistrya

a

Reagents: (i) allyl isocyanate, triethylamine, anhydrous DMAc; (ii) R-SH, 2,2-dimethoxy-2-phenylacetophenone (DMPA), hν (365 nm), DMSO.

form the corresponding N-allyl carbamate. To determine optimal conditions a series of reactions was performed with varying solvent, base, temperature and reaction time (Table S1). Modification of up to 70% of hydroxyl moieties could be achieved when an excess of isocyanate (2 equiv) and elevated temperatures (60 °C) were used. However, the high conversion was accompanied by the development of a large high molecular weight shoulder evident in the SEC chromatogram (Figure S2B) indicating some degree of cross-linking occurring between chains. Optimal reaction conditions were found to be 20 °C in DMAc as the solvent, using 5 equiv of triethylamine. As allyl isocyanate is prone to hydrolysis, polymer solutions were dried in vacuo directly before addition of the isocyanate and base. Successful modification was confirmed by 1H NMR spectroscopy with the appearance of resonances at 5.82 and 5.08 ppm corresponding to the vinyl protons of the allyl moieties (Figure 1A). SEC chromatograms (Figure 1B) after introduction of the allyl functionality showed only a small increase in molecular weight and dispersity, suggesting that no significant cross-linking or branching occurred under the reaction conditions employed (see Tables 1 and 2). Different levels of allyl functionality were introduced, by reacting P1−P3 with different (allyl isocyanate):(hydroxyl) molar ratios: 0.5, 0.75, 1, and 2. Comparison of the vinyl peaks integrals to those of the CHOH and CHOCO signal at 4.88 ppm was used to determine conversion of pHPMA hydroxyl groups into the desired carbamate moieties. An efficiency of reaction of ca. 50% was observed for modification reactions (Figure 1C) except for the lowest stoichiometry (0.5 equiv) where efficiency was ∼30%. The latter could be attributable to a proportionally greater extent of hydrolysis of allyl isocyanate by residual water at lower levels of functionalization: i.e., at low (allyl isocyanate):pHPMA ratios, water:isocyanate ratios were higher leading to increased incidence of hydrolysis side-reactions. However, unlike for poly[active ester] functionalizable polymers, the hydrolysis of allyl isocyanate does not result in unwanted functionalities in the final polymer product, instead yielding small molecule byproducts (amines and ureas) which can be easily removed during the subsequent purification steps. The efficiency of postpolymerization modification was found to be independent of the polymer chain length, in accord to what has been observed with pPFPMA-based materials12 under the range of polymer molecular weights investigated. No significant

Here we describe a new and versatile methodology for the synthesis of multifunctional pHPMA by postpolymerization modification of a pHPMA homopolymer. The 2° hydroxyl group of pHPMA is reacted with commercially available allyl isocyanate to yield allyl-pHPMA. The allyl moieties may then be derivatized further with a wide range of functionalities via radical thiol−ene chemistry, generating new families of polymers quickly and easily (Scheme 1).

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RESULTS AND DISCUSSION Synthesis of pHPMA-Based Polymers. A series of pHPMA homopolymers was synthesized by aqueous RAFT polymerization as described by McCormick et al.19 In all cases high monomer conversion (>80%) and low dispersity (Đ ≲ 1.14) were achieved, as determined by 1H NMR spectroscopy and size exclusion chromatography (SEC) respectively (P1− P3, Table 1). To minimize potential side reactions in later Table 1. Characterization of poly[N-(2hydroxypropyl)methacrylamide precursors Mn (kDa) sample P1 P2 P3 P4

DPn

theora

SECb

Đb

% thiolc

85 165 240

12.2 23.7 34.3





17.0 30.3 45.9 20.6

1.11 1.14 1.11 1.60

d d 0.32 ND

Calculated considering the initial [HPMA]/[CTA] ratio and final conversion. bDetermined by SEC using DMF + 0.1% LiBr as the mobile phase calibrated with PMMA standards. cResidual thiol after CTA residue removal using AIBN, as determined by Ellman’s assay. ND=not determined. dBelow the detection limit. a

derivatization steps the dithioester end groups were removed using the procedure of Perrier et al.20 Removal was confirmed by UV−vis spectroscopy and Ellman’s assay with 99%), methacryloyl chloride (97%), (4-cyanopentanoic acid)-4-dithiobenzoate (CPADB,), 4,4′azobis(4-cyanovaleric acid) (V-501, >97%), azobis(isobutyronitrile) (AIBN, 98%), allyl isocyanate (98%), 1-thio-β-D-glucose sodium salt, benzyl mercaptan (99%), cysteamine (>98%), 6-bromohexanoyl chloride (97%), 2,4-dimethylpyrrole (97%), boron trifluoride diethyl etherate (redist.), and triethylamine (TEA, >98%) were purchased from Sigma-Aldrich (Poole, U.K.). Piperidine (>99%), anhydrous (99%) were purchased from Acros Organics (Geel, Belguim). 2-(7-Aza-1Hbenzotriazole-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate (HATU, 98%) was purchased from Fluorochem Ltd. (Hadfield, UK). H-Cys(Trt)-2-ClTrt resin was purchased from Novabiochem. Fmoc amino acids were purchased from Novabiochem, Sigma-Aldrich and Acros Organics. All other chemicals and solvents were analytical or HPLC grade and purchased from Fisher Scientific. AIBN was recrystallized from MeOH, and methacryloyl chloride was distilled under Ar flow, before use. All other chemicals were used as received unless otherwise stated. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 400 MHz spectrometer. All chemical shifts are reported in ppm (δ) relative to tetramethylsilane or referenced to the chemical shifts of residual solvent resonances. Multiplicities are described with the following abbreviations: s = singlet, br = broad, d = doublet, t = triplet, m = multiplet. Infrared spectroscopy was performed using Thermo Scientific Nicolet IR 200 FT-IR. Analysis of spectra was performed with Omnic 8.0 (Thermo Fisher Scientific Inc.) Liquid and oily samples were analyzed as thin films between NaCl disks. Solid samples were prepared by grinding the 0.5−1 mg of analyte with potassium bromide and pressed to form a thin disk. Mass spectrometry was carried out using a Micromass LCT ToF with electrospray ionization and OpenLynx software. Samples were prepared in 1:1 MeCN/water containing 0.1% v/v formic acid. Size exclusion chromatography (SEC) was performed on a Polymer Laboratories GPC 50 system equipped with a refractive index detector. Separations were achieved with a pair of PLgel Mixed-D (5 μm bead, 7.8 × 300 mm) columns with a matching guard (7.8 × 50 mm) and DMF containing either 0.1% LiBr or 0.5% NH4BF4 as eluent at a flow rate of 1 mL min−1. Calibration was performed using narrow PMMA standards (Polymer Laboratories) in the molecular weight range 1− 1200 kDa. Molecular weights and dispersity values were calculated using Cirrus GPC 3.0. Aqueous SEC was performed on Shimadzu Prominence UPLC system fitted with a DGU-20A5 degasser, LC20AD low pressure gradient pump, CBM-20A LITE system controller, SIL-20A autosampler and an SPD-M20A diode array detector. Separations were performed on a PL Aquagel−OH 30 column (7.5 × 300 mm) with a mixture of 3:7 MeOH:PBS at a flow rate of 1.0 mL min−1 as the mobile phase. For thiol−ene reactions UV irradiation was performed using either a UVP XX-15 lamp fitted with 2 × 15W 365 nm tubes or a BEIYI Beauty nail gel curing lamp equipped with 4 × 9W tubes as described by Haddleton et al.25 Synthesis of N-(2-hydroxypropyl)methacrylamide (HPMA). HPMA was prepared as previously described.26,27 Briefly, 1-amino-2propanol (30 g, 0.40 mol) was dissolved in CH2Cl2 (800 mL), followed by the addition of solidNaHCO3 (36 g, 0.44 mol). Freshly distilled methacryloyl chloride (40 g, 0.38 mol) was dissolved in CH2Cl2 (60 mL) and added to the suspension dropwise over the course of 1 h, at 0 °C. The reaction was allowed to proceed for 3 h at room temperature. The suspension was filtered and the solvent removed under reduced pressure. The crude residue was recrystallized from acetone twice and the crystals dried under vacuum (42 g, 0.29 mol, 73%). 1 H NMR (400 MHz, CDCl3): δ = 6.48 (br, 1H, NH), 5.73−5.69 (m, 1H, vinyl Z to Me−CC), 5.37−5.30 (m, 1H, vinyl E to Me− E

DOI: 10.1021/acs.macromol.5b00447 Macromolecules XXXX, XXX, XXX−XXX

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2: m/z = 449.1747, 897.3378; [M]+, theor, 449.1693; [(RGDC−)2 + H]+, theor, 897.3302. General Procedure for the Derivatization of P1b via Thiol− Ene Chemistry. Typically, P1b (15 mg, 36 μmol of alkene) was dissolved in ∼25 μL of DMSO-d6. DMPA (25 μL aliquot of 40 mg mL−1 solution in DMSO-d6), thiol and TCEP (1:1 ratio to thiol) were added as detailed in Table 3. The solution was purged with nitrogen for 5 min before UV irradiation at 365 nm. When sequential modifications were performed, DMPA was added with each thiol. Products were purified by precipitation in diethyl ether 3 times and traces of solvents were removed under reduced pressure. Reaction involving hydrophilic thiols were further dialyzed in water (membrane MWCO 3.5 kDa) for 24 h and the products subsequently lyophilized. NMR analysis of all functionalized polymers is reported in the Supporting Information (Figures S6−S15). Stability of BODIPY-SH under radical thiol−ene conditions. BODIPY-SH (1.2 mg, 3.49 μmol) and P1a (10 mg, 11.2 μmol of alkene) were dissolved in MeOH (100 μL) together with DMPA (0.3 mg, 11.7 μmol), before addition of water (100 μL). The solution was then transferred in 25 μL aliquots in 1.5 mL glass vials and flushed with nitrogen for 5 min. Samples were irradiated at 365 nm and reactions stopped by removing the vial from UV irradiation at various time points. PBS (1 mL) was added to each sample before analysis using SEC chromatography equipped with fluorescence detector (λex 485 nm, λem 506 nm). At 0 min, BODIPY-SH was not completely soluble and therefore the sample was centrifuged prior to analysis. Cell Culture. HCT 116 cells were obtained from ATCC. Cells were maintained in RPMI-1640 media supplemented with 10% heat inactivated fetal calf serum and 2 mM L-glutamine in a humidified incubator with 5% CO2. Viability Assay. Cells were seeded on 96-well plates at 104 cells/ well 24 h prior to use. Cells were incubated with P14 diluted in OptiMEM media (Life Technologies) at the stated concentrations for 24 h. The media was aspirated and the cells washed once with DPBS. The cells were then incubated with Alamar Blue reagent for 1 h and fluorescence was measured using a Molecular Devices Flexstation 3 plate reader (λex = 585 nm, λem = 610 nm). Viability values are reported relative to untreated cells (100% viability). Confocal Microscopy. HCT-116 cells were seeded on coverglass slides. After 24 h they were incubated with P14 at 100 μg/mL in OptiMEM for 2 h. Cells were counterstained with CellMask Deep Red Plasma membrane stain, (Molecular Probes, 1 in 1000 dilution) and Hoechst 33342, (Sigma-Aldrich, 2 μM) for 5 min at 37 °C. Cells were visualized using a Leica LSM710 confocal microscope.

the course of 5 min. The mixture was heated under reflux for 3 h and then volatiles were removed under reduced pressure. The residue was redissolved in CH2Cl2 (10 mL) before addition of hexane (100 mL), and allowed to precipitate overnight at −18 °C. The supernatant was decanted off and the residue washed with cold hexane (2 × 20 mL). The residue was dissolved in toluene (30 mL) and allowed to stir for 10 min after addition of dry triethylamine (729 mg, 7.2 mmol). After this time, boron trifluoride diethyl etherate (1.02 g, 7.2 mmol) was added and mixture heated to 70 °C for 3 h. The reaction mixture was cooled at room temperature, and washed with water (40 mL) and brine (3 × 40 mL). The organic phase was dried over MgSO4 and the solvent removed under reduced pressure. The oily crude mixture was finally purified by flash column chromatography on silica (60 Å, 35−70 μm) using CH2Cl2:hexane 1:1 mixture as the mobile phase. 1 H NMR (400 MHz, CDCl3): δ = 6.08 (s, 2H, CHarom), 3.45 (t, J = 6.6 Hz, 2H, CH2Br), 2.98 (m, 2H, CCH2), 2.54 (s, 6H, 2CH3), 2.43 (s, 6H, 2CH3), 1.95 (broad, 2H, CH2), 1.95−1.26 (broad, 6H, CH2). 13 C NMR (400 MHz, CDCl3): δ = 153.59 (2C, CH3CN), 146.61 (1C, C(CH2)5), 140.21 (2C, CCH), 131.37 (2C, NCC(CH2)5), 121.47 (2C, CHCN), 33.90 (1C, CCCH2), 32.71 (1C, CH2Br), 29.28 (1c, CH3), 28.65 (1c, CH3), 28.39 (1c, CH3), 28.06 (1c, CH3), 16.26 (2C, CH2alk), 14.36 (1C, CH2alk). FT-IR: ν = 3581, 2253, 1550, 1614, 1201, 907, 732 cm−1. ESI−TOF−MS: m/z = 397.1312, [M + H]+, theor, 397.1257. Synthesis of BODIPY-SH. BODIPY-Br (100 mg, 0.25 mmol) and potassium thioacetate (35 mg, 0.30 mmol) were dissolved in acetone (20 mL) and stirred under reflux for 2 h. Volatiles were removed under reduced pressure and the residue was dissolved in CH2Cl2 (20 mL) and washed with water (3 × 40 mL). The organic layer was dried over MgSO4 and solvent removed under reduced pressure to obtain the thioacetate derivate, which was used for the following step without further purification. BODIPY thioacetate was dissolved in absolute ethanol (5 mL) and the solution bubbled with nitrogen for 30 min. Potassium carbonate (70 mg, 0.51 mmol) was then added and the solution heated to 30 °C for 4 h under nitrogen atmosphere. The reaction was monitored by 1H NMR spectroscopy until complete disappearance of the signal corresponding to the methyl protons (COCH3) of the thioacetate at 2.35 ppm. The solution was poured into saturated ammonium chloride solution (10 mL) and extracted with dichloromethane (3 × 10 mL). The organic layer was dried over MgSO4 and the solvent evaporated under reduced pressure. The crude mixture was purified by flash column chromatography using a CH2Cl2:hexane 1:1 mixture as the mobile phase. 1 H NMR (400 MHz, CDCl3): δ = 6.07 (s, 2H, CHarom), 2.98 (m, 2H, CH2SH), 2.72 (m, 2H, CCH2), 2.54 (s, 6H, 2 × CH3), 2.43 (s, 6H, 2 × CH3), 1.78 (broad, 2H, CH2), 1.78−0.95 (broad, 6H, 3 × CH2). 13C NMR (400 MHz, CDCl3): δ = 153.92 (2C, CH3CN), 146.05 (1C, C(CH2)5), 140.17 (2C, CCH), 131.39 (2C, NCC(CH2)5), 121.64 (2C, CHCN), 53.42 (1C, CH2SH), 38.53 (1C, C CCH2), 31.50 (1C, CH3), 28.90 (1C, CH3), 28.78 (1C, CH3), 28.29 (1C, CH3), 16.44 (2C, CH2alk), 14.45 (1C, CH2alk). ESI−TOF−MS: m/z = 390.3474, [M + K]+; theor, 389.1431. Synthesis of RGDC Peptide (7). Standard solid phase peptide synthesis procedure was used to synthesize RGDC peptide.29 N-Fmoc amino acids: Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH and Fmoc-Arg(Pbf)-OH were coupled in sequence to H-Cys(Trt)-2-ClTrt resin (500 mg, 0.32 mmol) using HATU as the coupling agent. After cleavage and deprotection the crude peptide was precipitated into cold ether. Purification was performed on an Agilent 971-FP automated flash purification system using a BIOTAGE KP-C18-HS 12 g column. Elution was performed using a linear gradient from 2−40% MeCN in water containing 0.1% TFA over 40 min and further increasing from 40 to 90% MeCN in water containing 0.1% TFA over 20 min at a flow rate of 5 mL min−1. After purification, the peptide was subjected to analytical reverse phase HPLC with a linear gradient from 5−90% MeCN in water containing 0.1% TFA over 40 min at a flow rate of 1 mL min−1. Two major peaks were identified in the chromatogram (Figure S5) which were collected and analyzed by mass spectrometry. ESI−TOF−MS (positive). Peak 1: m/z = 450.1681; [M + H]+ requires 450.1765. Peak

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ASSOCIATED CONTENT

S Supporting Information *

Removal of dithioester end group from RAFT polymers, synthesis of allyl-pHPMA, stability of BODIPY-SH, HPLC analysis of RGDC peptide, thiol−ene chemistry on allylpHPMA, and further characterization including SEC chromatograms and NMR spectra and other supporting figures. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (C.A.). *E-mail: [email protected] (G.M.). *E-mail: s.g.spain@sheffield.ac.uk (S.G.S.). Present Address †

Department of Chemistry, University of Sheffield, Dainton Building, Sheffield S3 7HF, U.K.

Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acs.macromol.5b00447 Macromolecules XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS We wish to thank the Engineering and Physical Sciences Research Council (EPSRC Leadership Fellowship and Grant EP/H005625/1 to C.A.), PsiOxus Therapeutics, and the University of Nottingham for funding.

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REFERENCES

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DOI: 10.1021/acs.macromol.5b00447 Macromolecules XXXX, XXX, XXX−XXX