Effect of Polyethylene Glycol on Properties and Drug Encapsulation

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Effect of Polyethylene Glycol on Properties and Drug Encapsulation− Release Performance of Biodegradable/Cytocompatible Agarose− Polyethylene Glycol−Polycaprolactone Amphiphilic Co-Network Gels Arvind K. Singh Chandel,†,‡ Chinta Uday Kumar,† and Suresh K. Jewrajka*,†,‡ †

Reverse Osmosis Membrane Division, CSIR and ‡Academy of Scientific and Innovative Research-AcSIR, Central Salt and Marine Chemicals Research Institute, Gijubhai Badheka Marg, Bhavnagar, Gujarat 364002, India S Supporting Information *

ABSTRACT: We synthesized agarose−polycaprolactone (Agr-PCL) bicomponent and Agr−polyethylene glycol−PCL (Agr-PEG-PCL) tricomponent amphiphilic co-network (APCN) gels by the sequential nucleophilic substitution reaction between amine-functionalized Agr and activated halide terminated PCL or PCL-b-PEG-b-PCL copolymer for the sustained and localized delivery of hydrophilic and hydrophobic drugs. The biodegradability of the APCNs was confirmed using lipase and by hydrolytic degradation. These APCN gels displayed good cytocompatibility and blood compatibility. Importantly, these APCN gels exhibited remarkably high drug loading capacity coupled with sustained and triggered release of both hydrophilic and hydrophobic drugs. PEG in the APCNs lowered the degree of phase separation and enhanced the mechanical property of the APCN gels. The drug loading capacity and the release kinetics were also strongly influenced by the presence of PEG, the nature of release medium, and the nature of the drug. Particularly, PEG in the APCN gels significantly enhanced the 5-fluorouracil loading capacity and lowered its release rate and burst release. Release kinetics of highly water-soluble gemcitabine hydrochloride and hydrophobic prednisolone acetate depended on the extent of water swelling of the APCN gels. Cytocompatibility/blood compatibility and pH and enzyme-triggered degradation together with sustained release of drugs show great promise for the use of these APCN gels in localized drug delivery and tissue engineering applications. KEYWORDS: agarose−polycaprolactone-PEG APCN gel, effect of PEG, cytocompatibility and blood compatibility, biodegradability, high loading capacity, triggered and sustained release

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INTRODUCTION Amphiphilic co-networks (APCNs) are a rapidly emerging class of nanophasic soft materials.1−10 APCNs are composed of chemically cross-linked hydrophilic and hydrophobic macromolecules. These materials swell and interact with both hydrophilic and hydrophobic solvents and materials. Hence, APCN behaves as both hydrophilic and hydrophobic gels and may be termed a APCN gel. APCN gels are mostly prepared by macromonomer method via polymerization of a telechelic macromonomer containing at least two polymerizable groups with a selected low-molecular-weight monomer.1,2,4,5 Chemical cross-linking (covalent bonds) of hydrophilic and hydrophobic polymer chains provides unique nanophase morphology that was earlier investigated in detail.6,11−14 Physically cross-linked APCN gel was also formed by stereocomplexation of polylactide.15 Preparation of APCN gel via the strong hydrogen bonding between functional three-armed star polyisobutylene (PIB) and telechelic polyethylene glycol (PEG) was also reported earlier.16 The PIB-PEG gel behavior depended on the molecular weight of PEG. In contrast, hydrogels consist of © XXXX American Chemical Society

hydrophilic segments that are useful for controlled release of hydrophilic drugs,17,18 tissue engineering,19,20 and regenerative medicine20 applications. There are some concerns with hydrogel systems for drug delivery and tissue engineering applications. The loading capacity and homogeneous distribution of a hydrophobic drug into hydrogels may be limited owing to a lack of solubilization of the hydrophobic drug into the hydrophilic phase. The high water content of most hydrogels often results in the rapid release of drugs. The low mechanical strength of water-swollen hydrogels also limits their use in tissue engineering of relatively high mechanical strength tissues. APCN gels are a suitable class of material for delivery of both hydrophilic and hydrophobic drugs owing to the interaction of APCNs with the drugs. The APCNs are also superior materials compared to hydrogels for specific tissue engineering Received: November 5, 2015 Accepted: January 13, 2016

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DOI: 10.1021/acsami.5b10675 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

2(1H)-pyrimidinone (DMPU, 98%, Aldrich), 4-(chloromethyl) benzoyl chloride (Cl-Bz-Cl, 98%, TCI), polycaprolactone diol (HOPCL-OH, Mn = 2000 and 530 g/mol, Aldrich) and polyethylene glycols (Mn = 4000 and 1500 g/mol, Aldrich) were used as received. Rose Bengal dye (RB, Spectro Chem, India), prednisolone acetate (TCI), 5-fluorouracil (5-Flu, TCI), and gemcitabine hydrochloride (TCI) were used as received. All solvents were from Spectro Chem, India, and were distilled before use. Caprolactone (CL, TCI) was dried with calcium hydride and distilled under reduced pressure before use. The catalyst 2-ethylhexanoate [Sn(Oct)2] was from Aldrich and was used as received. Synthesis of Multifunctional Agr-Amine (Agr-NMe2), Benzyl Methyl Chloride Terminated PCL (ClCH2Ph-PCL-PhCH2Cl), and Benzyl Methyl Chloride Terminated Triblock Copolymer of PEG and PCL (ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl). Agr-NMe2 was prepared by the reaction of hydroxyl groups of Agr with tertiaryamine carbonylimidazole derivative. A typical example is as follows. CDI (11.5 g, 0.070 mol) was dissolved in dry THF (40 mL) which was added to a 100 mL round-bottomed flask equipped with a gas inlet, a septum cap, and a magnetic stir bar. 3-(Dimethyl amino)1propylamine (7 g, 0.07 mol) was injected into the flask by syringe while the temperature in the flask was maintained below 50 °C. The reaction mixture was then stirred for 16 h at room temperature. The resulting tertiaryamine carbonylimidazole solution was isolated by evaporation of THF. The resulting yellow oily mixture was used without further purification. 1H NMR confirms the formation of amine carbonylimidazole with yield as high as 91%. Agr (10 g) was added to a 500 mL round-bottomed flask with gas inlet and magnetic stirring bar. Dry NMP (300 mL) was then added to the flask under nitrogen and heated for 24 h at 70 °C. After complete dissolution of Agr, amine carbonylimidazole (6 g, 0.030 mol) was added, and finally DMPU (0.4 mL) was injected. The mixture was stirred for 72 h at 80 °C. Next, the reaction mixture was precipitated in methanol (200 mL) under vigorous stirring. The obtained mass was again dissolved and reprecipitated in methanol to remove any unreacted reactants. The solid mass was dried under vacuum for 48 h at 70 °C and characterized by 1H NMR (Figure S1). The degree of tertiary amine substitution was calculated to be 0.40 (Supporting Information). Appearance of an extra band at 1717 cm−1 for carbonyl stretching vibration of − O−CO−NH− in the IR spectrum of AgrNMe2 compared to IR spectrum of neat Agr also confirmed formation of Agr-NMe2 (Figure S2). Two OH-PCL-OH (molecular weights 2000 and 530 g/mol) were converted to corresponding ClCH2Ph-PCL-PhCH2Cl by following our earlier procedure.44 On the other hand, ClCH2Ph-PCL-b-PEG-b-PCLPhCH2Cl was synthesized by a two-step process. First was the synthesis of OH-PCL-b-PEG-b-PCL-OH (Supporting Information). The gel permeation chromatography (GPC)-derived molecular weight (Mn,GPC) and polydispersity index (PDI) of this triblock copolymer were 8100 g/mol and 1.28, respectively (Figure S3). The second step is the esterification of OH-PCL-b-PEG-b-PCL-OH by Cl-Bz-Cl. The 1H NMR derived molecular weight (Mn,NMR) of ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl was calculated to be ca. 11 900 g/mol (Figure S4). This copolymer thus contains 34% (w/ w) PEG and 68% (w/w) PCL as obtained by 1H NMR analysis (Figure S4). 1H NMR (CDCl3, δ/ppm) 1.42 (−CH2− of PCL), 2.34 (−CH2−CO− of PCL), 3.5 (CH2−O of PEG), 4.09 (−CH2OOC of PCL), 4.25 (−CH2OOC−Ph), 4.6 (−Ph−CH2−Cl), and 7−8 (aromatic proton). Synthesis of APCN Gels by Sequential Nucleophilic Substitution Reaction. APCN gels were synthesized by reacting Agr-NMe2 with ClCH2Ph-PCL-PhCH2Cl or ClCH2Ph-PCL-b-PEG-bPCL-PhCH2Cl in DMF followed by slow evaporation of solvent. A typical example of preparation of APCN gel by precursors amount 1:1 (w/w) is as follows. Purified Agr-NMe2 (1 g, 0.0013 mol tertiary amine) was dissloved in DMF (15 mL) and strirred for 8 h at 60 °C. ClCH2Ph-PCL-PhCH2Cl (1 g, 0.001 mol activated halide) or ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl (1 g, 1.7 × 10−4 mol activated halide) was also separatley dissolved in DMF (2 mL). The ClCH2PhPCL-PhCH2Cl or ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl solution

applications because of the better mechanical property of the water-swollen APCN. Importantly, the mechanical property and extent of water swelling of APCNs can be controlled by adjusting the hydrophilic to hydrophobic ratio. Thus, APCNs have wider applications such as in nanotemplates for organic/inorganic nanohybrids,21 organic solvent superabsorbant,22 membranes for water desalination via electrodyalysis,23 supports for high-efficiency enzyme catalysis,24 antifouling and antimicrobial coatings,25,26 stimuliresponsive biomaterials such as controlled drug release matrices,27−30 and scaffolds for tissue engineering.31,32 Biostable and biocompatible APCNs showed promising results for application in an insulin delivery system.33,34 Biodegradable and biocompatible APCNs have attracted great attention because of their biomedical applications. Poly(methyl methacrylate) (PMMA) and poly(N,N′-dimethyl aminoethyl)methacrylate (PDMA) triblock and statistical copolymers-based degradable APCNs were synthesized by the use of degradable cross-linker.35 It is highly desirable to combine biocompatible/biodegradable hydrophilic and hydrophobic polymers for the synthesis of APCN gels for safe biomedical applications. Agarose (Agr) is hydrophilic biocompatible/biodegradable polymer obtained from seaweed. Hydrophobic polycaprolactone (PCL) or amphiphilic copolymer of PCL and polyethylene glycol (PEG) is also biocompatible/ biodegradable. PCL, PEG, and Agr are currently used in FDAapproved devices. Hence, APCN gels of Agr and PCL or PCLb-PEG-b-PCL copolymer will be interesting biomaterials for biomedical applications. Earlier, polysaccharide-based hydrogels were mostly reported for biomedical applications.36−39 As far as the use of PCL is considered, the film surface of PCL was grafted by zwitterionic poly(3-dimethyl(methacryloyloxyethyl) ammoniumpropanesulfonate) for the enhancement of surface hemocompatibility.40 Enzyme-degradable PEG- and PCL-based APCN gels were synthesized by photoinitiated copolymerization of mixture of methacrylate terminated PCL-b-PEG-b-PCL and PEG.41 Degradable poly(2-hydroxyethyl methacrylate)-coPCL APCN gel was synthesized by ATRP of HEMA in the presence of PCL-based ATRP initiator and methacrylateterminated PCL cross-linker for a tissue engineering scaffold.42 PEG-PIB based APCN gel was synthesized by coupling hydroxyl functional three-armed PIB and PEG by the help of diisocyanate.43 Earlier, we reported the synthesis of APCN gels by combining Agr-based graft copolymer (containing PMMA and PDMA side chains) and PCL.44 To the best of our knowledge, APCN gels composed of Agr and PCL or Agr, PCL, and PEG for biomedical applications have not yet been reported. Herein, we demonstrate the synthesis of APCN gels by combining a new type of tertiary amine containing Agr (AgrNMe2) and activated-chloride-terminated PCL or PCL-b-PEGb-PCL for localized drug delivery. The effect of PEG on phase separation behavior, mechanical property, swelling, loading/ release behavior, and cytotoxicity of the APCN gels has been systematically studied. All the APCN gels exhibited acid-, base-, and lipase-catalyzed degradation, along with sustained release of drugs. We have also demonstrated the injectability of the drugloaded powdery APCN gels through a hypodermic syringe.

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EXPERIMENTAL SECTION

Materials. Agarose (Agr, Type I-A low EEO, Aldrich), 3(dimethylamino)-1-propylamine (DMPA, 99%, Aldrich), 1,1′-carbonyldiimidazole (CDI, 95%, TCI), 1,3-dimethyl-3,4,5,6-tetrahydroB

DOI: 10.1021/acsami.5b10675 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Synthesis of Precursors and Representative APCN Gela

a Synthesis pathways for (A) Agr-NMe2, (B) ClCH2Ph-PEG-b-PCL-b-PEG-PhCH2Cl, and (C) APCN. (D) Strucure and injection of milled waterswollen soft APCN gel particles through hypodermic syringe of needle size 20-G.

and were milled by mechanical motor to obtain a fine powder. These paricles were then subjected to seiving with 160 μm mesh filter. The size of the particles were analyzed by microscope. These particles (0.2 g) were dispersed in water (1 mL) and vigorously hand-shaked to obtain dispersion. The solution was injectable through hypodermic syringe of needle size 20-G. Characterizations, Degradation, Drug Loading, Drug Release Experiments, Cytocompatibility, and Blood Compatibility. Swelling of APCN gels was determined as reported earlier.1,34,44 Briefly, DMF extracted gel films (5 cm × 5 cm) were accurately weighed and transferred to milli-Q water. The equilibrium water swelling was determined periodically (30 min) by removing the water adsorbed to the film surfaces by blotting with tissue paper and weighing. Equilibrium water swelling was recorded when the weight of the swollen films became unchanged at 30 °C. The percentage of water swelling is expressed by the following equation:

was then added into the solution of Agr-NMe2 slowly under stirring at room temperature. The complete transfer of the solution was ensured by transferring the left over solution by additional DMF (1 mL). The admixture was stirred for 10 min at 60 °C and then poured into flat Petri dish. The DMF was then evaporated at 70 °C for 12 h. After evaporation of DMF, the entrapped DMF was again removed in vacuum oven at 60 °C for 24 h to obtain dried APCN film. The thickness of the film was 0.22−0.3 mm. Similarly, all the APCN films were prepared as described above. Determination of Actual Composition of APCN Gels by TwoStep Extraction. The actual amount of Agr and PCL in the APCN gels was determined by a sequential solvent extraction process.28,44 The first extraction was performed with acetone to remove unreacted PCL, whereas the second extraction was performed with DMF to remove any unreacted Agr. Typical solvent extraction process is as follows. APCN films (5 cm × 5 cm) in triplicate were submerged in acetone (30 mL) and were gently stirred for 24 h at 30 °C. Then, acetone was collected in a pre-weighed, round-bottomed flask. Next, fresh acetone was poured in the beakers, and the APCNs were extracted again. This procedure was repeated thrice. The collected acetone was evaporated by rotary evaporator, and the weight of the mass was recorded. The collected mass was PCL as confirmed by 1H NMR. The acetone-extracted APCN films were similarly subjected to DMF extraction. The weight of the collected mass from DMF solution was recorded. This mass was due to unreacted Agr as confirmed by 1H NMR. Preparation of Injectable Water-Dispersed APCN Gel Particles. The extracted APCN films were frozen by liquid nitrogen

water swelling (%) =

m ws − mdry mdry

× 100

where mws and mdry are the masses of the water-swollen and the dry films, respectively. Toluene swelling was also measured by the above procedure. Extractable, differential scanning calorimetry (DSC), stress−strain property, IR, solid-state 13C NMR, and atomic force microscopy (AFM) were performed using standard procedures as described previously.44 Degradation of APCN gels,35,44 loading of RB, and loading and release profiles of hydrophilic (5-Flu and gemcitabine hydrochloride) and hydrophobic (prednisolone acetate) drugs were performed using standard protocol (Supporting Information).44 C

DOI: 10.1021/acsami.5b10675 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Extracted APCN gels were used in all the experiments. Cytocompatibility of APCN gels and degraded species were determined by standard MTT assay using HeLa cell line for 24 h. Polystyrene tissue culture (96 well-plates) was used as standard for cell viability calculations (Supporting Information).44 Blood compatibility experiments were performed using phosphate buffer solution (PBS) and Triton-X as negative and positive controls, respectively (Supporting Information).

Agr and PCL. This facilitated the reaction between Agr-NMe2 and ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl. The DMF-extracted and purified Agr-PCL(5:2), AgrPCL(1:1), Agr-PEG-PCL(5:2), and Agr-PEG-PCL(1:1) were subjected to IR (Figure 1A) and solid-state 13C NMR (Figure 1B) analyses to confirm the presence of Agr, PCL, and PEG in the respective APCN gels. The IR band at 1730 cm−1 appeared for all the APCN gels because of carbonyl stretching (−CO) vibration of backbone ester linkage of PCL chains. The intensity ratio of 1730 cm−1 band to 1637 cm−1 band was enhanced for Agr-PEG-PCL(1:1) and Agr-PCL(1:1) (Figure 1A, spectra a and c) compared to that of Agr-PEG-PCL(5:2) and Agr-PCL(5:2) (Figure 1A, spectra b and d) because of an enhanced amount of PCL in the former two APCNs. The appearance of an IR band at 931 cm−1 for all the APCNs is due to C−O−C bridge of 3,6-anhydrogalactose of Agr. The AgrPEG-PCL(1:1) and Agr-PEG-PCL(5:2) show an enhanced intensity band at 1177 cm−1, which is ascribed to the C−O−C starching vibration of − O−CH2−CH2 units of PEG. The band at 1550 cm−1 is ascribed to the stretching vibration of + N(CH3)2 group generated because of nucleophilic substitution reaction between −N(CH3)2 of Agr and phenyl methyl chloride end groups of PCL or PCL-b-PEG-b-PCL. This band is absent in the IR spectra of Agr and Agr-NMe2 (Figure S2). Figure 1B shows the solid-state 13C NMR spectra of the APCN gels. The spectra show signals (a−c) at δ values of 25, 32, and 37 ppm because of backbone carbon of PCL chain. The strong signals (d and f) at δ values of 70−80 ppm and signal g near 100 ppm are due to various carbons of Agr backbone. The intensity of the signal near 70 ppm (g) was enhanced for AgrPEG-PCL(5:2) and Agr-PEG-PCL(1:1) compared to that for Agr-PCL(5:2) and Agr-PCL(1:1), respectively, because of the presence of PEG backbone carbon (−O*CH2−) in the former two APCNs. Low-intensity signals at 125−140 ppm (h) may be ascribed to the aromatic carbon (from halide cross-linkers). Signal k, near 170 ppm, is due to carbonyl carbon (−*CO) of ester group of PCL backbone. Hence, IR and 13C NMR spectra clearly indicate formation of bi- (Agr-PCL) and tricomponent (Agr-PEG-PCL) APCNs. The IR spectra of the APCN gels did not show C−Cl stretching vibration, which indicated near complete substitution reaction through nucleophilic attack at −CH2−Cl by tertiary amine of Agr groups. Because the reaction between tertiary amine and activated halide is fast and facile in DMF, an number −NMe2 moieties of Agr-NMe2 equivalent to that of activated chloride units of PCL or PCL-PEG-PCL became quaternized (Scheme 1). Effect of PEG on Sol Fraction, Swelling, and Phase Morphology of the APCN Gels. 1H NMR and IR spectra of soluble masses obtained by DMF extraction of APCNs confirmed the presence of both PCL and Agr (Figure S5). The sol fraction values (22−43%, Table 2) of these APCNs were higher than those (5−10%) obtained with the APCNs synthesized by Agr-g-poly(methyl methacrylate)-b-poly(dimethyl amino ethyl)methacrylate (Agr-b-PMMA-b-PDMA) and ClCH2-PhPCL-Ph-CH2Cl.44 This is attributed to the comparatively lower miscibility between Agr and PCL or PCLb-PEG-b-PCL compared to that of Agr-b-PMMA-b-PDMA and PCL. This is because the presence of side chain ester groups (PMMA and PDMA) in the Agr-g-PMMA-b-PDMA enhanced the miscibility between Agr-g-PMMA-b-PDMA and PCL and facilitated the cross-linking reaction. Notably, sol fraction values

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RESULTS AND DISCUSSION Synthesis and Composition of APCN Gels. We synthesized and selected Agr-NMe2, ClCH2Ph-PCL-PhCH2Cl, and ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl precursors with well-defined molecular weight and functionality for further synthesis of APCN gels owing to their biocompatibility and biodegradability. ClCH2Ph-PCL-PhCH2Cl (Mn of PCL = 2000 g/mol) was selected for the synthesis of APCNs. This is because there is no effect of relatively low molecular weight ClCH2Ph-PCL-PhCH2Cl (Mn of PCL = 530 g/mol) on the sol fraction of the resulted APCN gels. In contrast, the relatively high molecular weight ClCH2Ph-PCL-PhCH2Cl (Mn,GPC of PCL = 4700 g/mol) enhanced the sol fraction of the resulted gels because of the enhanced phase separation between relatively high molecular PCL and Agr during synthesis of the gels. Furthermore, when the Mn of PEG middle block of ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl was 1500 g/mol, the sol fraction of the resulted gels was increased compared to that of ClCH2Ph-PCL-b-PEG-b-PCL-PhCH2Cl with Mn of PEG middle block = 4000 g/mol. Scheme 1 illustrates the synthesis of precursors and representative APCN gel. Hence, Agr-PCL or Agr-PEG-PCL APCN gels of different compositions were synthesized by sequential nucleophilic substitution reaction between Agr-NMe2 (degree of amine substitution ca. 0.40) and ClCH2Ph-PCL-PhCH2Cl (Mn of PCL = 2000 g/mol, PDI = 1.20) or ClCH2Ph-PCL-b-PEG-bPCL-PhCH2Cl (Mn of middle PEG block =4000 g/mol, Mn,GPC of copolymer = 8100 g/mol, and PDI = 1.28; Table 1). The Table 1. Abbreviations, Synthesis Conditions, and Actual Compositions of the APCN Gels Agr/PCL or Agr/PCL-PEG-PCL (%, w/w)a

actual amount in APCN gel (%, w/w)c

APCN gel

reaction mixture

actual compositionb

Agr

PCL

PEG

Agr-PEG-PCL(1:1) Agr-PEG-PCL(5:2) Agr-PCL(1:1) Agr-PCL(5:2)

1:1 1:0.4 1:1 1:0.4

1:0.55 1:0.21 1:0.35 1:0.15

65 83 74 86

23 11 26 14

12 6 0 0

a

Functional polymers and copolymers. bCalculated from two-step (acetone and DMF) extraction process and. cFrom two-step extraction and composition of PCL-b-PEG-b-PCL copolymer.

APCN films were milled to obtain fine particles. Soft waterswollen APCN particles remained dispersed after hand shaking in water for about 5 min and were injectable through hypodermic syringe of needle size 20-G (Scheme 1).44 On the basis of individual component solubility, the actual composition of the APCN gels was determined by sequential extraction with acetone and DMF (Table 1). The degree of PCL incorporation in the APCN gels was lower than that of incorporation of PCL-b-PEG-b-PCL because of enhanced miscibility between Agr and PCL-b-PEG-b-PCL over that of D

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Figure 1. (A) IR spectra and (B) solid-state 13C NMR spectra of APCN gels.

Table 2. DMF Sol Fraction and Equilibrium Swelling of Different APCN Gels swelling (%) co-network Agr-PEG-PCL(1:1) Agr-PEG-PCL(5:2) Agr-PCL(1:1) Agr-PCL(5:2) Agr-PEG(1:1)

sol fraction (%) 28 18 40 23 10

(±2) (±2) (±3) (±3) (±2)

water 178 192 154 163 240

(±6) (±7) (±5) (±4) (±4)

toluene 22 15 36 28

(±1) (±2) (±1) (±2)

obtained with Agr-PEG-PCL(1:1) and Agr-PEG-PCL(5:2) were lower than that of Agr-PCL(1:1) and Agr-PCL(5:2) because of comparatively easier encounter between Agr and PCL-b-PEG-b-PCL (Table 2). Thus, PEG lowers the phase separation between Agr and PCL during the reaction by virtue of providing amphiphilicity and H-bonding with Agr (vide infra). This was further confirmed by reaction of ClCH2PhPEG-PhCH2Cl and AgrNMe2 which gave fully transparent chemically cross-linked model hydrogel [Agr-PEG (1:1)] with extractability as low as 10% and equilibrium water swelling ca. 240% (Table 2, entry 5). All the APCN gels attained equilibrium water swelling within ca. 6 h (Figure S6). The equilibrium water swelling of PCL-bPEG-b-PCL-containing APCN gels (entries 1 and 2, Table 2) were higher than those of PCL-containing APCN gels (entries 3 and 4, Table 2) usually because of a greater amount of hydrophilic Agr and PEG in the former APCN gels (Table 2). The water swelling of these APCNs remained unaltered with the change of water pH. The toluene swelling was obviously higher in the PCL-containing APCNs than in corresponding PCL-b-PEG-b-PCL-containing APCNs because of the presence of a higher amount of PCL in the former APCNs. The hydrophobic (PCL) and hydrophilic (Agr and PEG) phases of representative Agr-PEG-PCL(1:1) undergo swelling in toluene and in water without provoking macroscopic phase separation. This was evident by the transparency of DMF extracted Agr-PEG-PCL(1:1) films in both dry (Figure 2A) and swollen (Figure 2B,C) states, whereas DMF-extracted AgrPCL(1:1) films showed relatively less transparency in dry (Figure 2D) and in swollen (Figure 2E,F) states. This indicated

Figure 2. Digital pictures of APCN films showing comparative transparency. (A) Dry, (B) water-swollen, and (C) toluene-swollen Agr-PEG-PCL(1:1) films. (D) Dry, (E) water-swollen, and (F) toluene-swollen Agr-PCL(1:1) films. (G and H) RB-absorbed AgrPEG-PCL(1:1) and Agr-PCL(1:1) films, respectively. (I and J) Phasemode AFM images (5 × 5 μ) of Agr-PEG-PCL(1:1) and AgrPCL(1:1), respectively. The thin film was deposited on mica surface for AFM analysis. The roughness values of corresponding AFM height images were 10−12 nm.

co-continuous nanophase separated morphology with greater degree of phase mixing in Agr-PEG-PCL(1:1) than in AgrPCL(1:1). Furthermore, the RB-absorbed Agr-PEG-PCL(1:1) film (Figure 2G) showed better transparency than that of AgrPCL(1:1) (Figure 2H). The strong absorption of RB also indicated presence of quaternized nitrogen in the APCNs. Phase-mode AFM images of representative APCNs showed phase separation with appearance of brighter and darker phases. The phase image (Figure 2I) of Agr-PEG-PCL(1:1) showed relatively low degree of phase separation of brighter domains compared to that of (Figure 2J) Agr-PCL(1:1) in which the size of brighter domains was relatively larger. DSC (Figure S7) and DMA (tan δ vs temperature plots, Figure S8) analyses also indicated enhanced phase mixing in the PEG containing APCNs. In the DSC thermograms, there is a difference in the region ca. 50−85 °C. The glass transition temperature (Tg) of Agr phase of the APCN gels appears at this E

DOI: 10.1021/acsami.5b10675 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces broad region. The effect was prominent at ca. 55−82 °C for neat Agr, Agr-PCL(1:1), and mechanical blend of Agr+PCL (1:1, w/w), whereas the change was prominent at 50−74 °C for Agr-PEG-PCL(1:1). This is attributed to the effect of PEG that enhanced the phase miscibility in the APCN gels (containing PEG). The Tg of neat Agr appeared at broad temperature region that can be identified by the change of DSC thermograms in the region as reported earlier.45 Melting endotherms of semicrystalline neat PCL (Mn = 2000 g/mol) appeared at ca. 30−45 °C. These endotherms decreased and broadened for Agr-PCL(1:1) and almost vanished in Agr-PEG-PCL(1:1). This also indicated better phase miscibility in the Agr-PEG-PCL(1:1). The decrease of crystallization in Agr-PCL(1:1) may be attributed to the effect of glassy Agr phase on PCL in the APCN gel. Thus, the effect of Agr on PCL was more pronounced in Agr-PEG-PCL(1:1) because of enhanced miscibility between PCL and Agr in the presence of PEG. The lower amount of PCL in the Agr-PEGPCL(1:1) compared to that of Agr-PCL(1:1) also might have affected the crystallization in the APCN gels.9 Furthermore, in the tan δ plots, the Tg (for hard matrix) of Agr-PEG-PCL(1:1) appeared at ca. 43 °C, whereas the Tg of Agr-PCL(1:1) appeared at 55 °C. The Tg of amorphous PCL matrix appeared at −62 and −70 °C for Agr-PEG-PCL(1:1) and Agr-PCL(1:1), respectively, as seen from storage modulus−temperature plots. This result also supports the DSC observations. Effect of PEG on Tensile Stress−Strain Property of the APCNs. The ultimate tensile properties of water-swollen PEGPCL-containing APCNs also showed considerable improvements. The tensile stress and strain at break (Figure 3)

APCN gels. The reason for enhanced mechanical property of mixed polydimethylsiloxane (PDMS)/hexamethylene oxide based polyurethane compared to that of PDMS-based polyurethane was discussed earlier on the basis of enhanced miscibility between PDMS and hard segment in the presence of hexamethylene oxide, which facilitated stress transfer from PDMS to hard segments.46 Higher amounts of PCL in the APCN gels may also contribute to enhance stress−strain property of Agr-PEG-PCL(1:1) also showed 1.5−1.9 times higher storage modulus than that of Agr-PCL(1:1) at wide range of temperature as confirmed by DMA thermograms (Figure S8) due to the same reason as described above.46 Effect of PEG on Degradation of APCN Gels. Figure 4 shows the degradation profiles of representative Agr-PEG-

Figure 4. Degradation of Agr-PCL(1:1) and Agr-PEG-PCL(1:1) in PBS (pH 7.4), in PBS (pH 7.4, containing lipase 0.5 mg/mL), and in PBS (pH 5) for up to 60 days at 37 °C. Each experiment was conducted in triplicate and average data are plotted.

PCL(1:1) and Agr-PCL-(1:1) at pH 7.4 and 5 and in the presence of lipase (pH 7.4). The rate of degradation was enhanced in PEG-PCL-containing APCNs in the respective catalyzed processes. The enhanced water swelling of PEG-PCLcontaining APCNs facilitates the diffusion of H+ or lipase in these co-network matrices and thereby enhanced the hydrolytic/enzymatic degradation compared to that of PCLcontaining APCNs. The rate of degradation was also higher in the presence of lipase than at pH 5. This is because lipase catalyzed the degradation of PCL by hydrolysis of ester bonds,41,42 and the Agr backbone also undergoes normal hydrolytic degradation.44 In contrast, in acidic medium, the degradation of Agr backbone was catalyzed compared to degradation in medium of pH 7.4.44 At pH 9, the degradation rate slightly increased compared to that at pH 7.4 because of comparatively higher degree of hydrolytic degradation of PCL under basic condition (data not shown).42 Such type of enhanced degradation in acidic and enzymatic media might influence the release of entrapped drugs from the APCNs matrices. The species formed by degradation were lowmolecular-weight PCL and Agr as confirmed by 1H NMR of the obtained lyophilized mass (spectrum not shown). Effect of PEG on Loading and Release of Hydrophilic 5-Fluorouracil and Gemcitabine Hydrochloride. There is a dramatic effect of PEG on loading and release of 5-Flu. PEGPCL-containing APCNs exhibited 1.6−1.8 times higher 5-Flu loading capacity than did PCL-containing APCNs. The 5-Flu loading capacity of APCNs increased with time, and equilibrium loading was attained after ca. 35 h of incubation at 30 °C with initial solution drug concentration of 8 mg/mL (Figure 5A). The loading capacity of APCNs attained equilibrium within 100 and 20 h when the concentrations of

Figure 3. Tensile stress−strain properties of APCN gels in their equilibrium water-swollen state.

decreased in the following order for water-swollen APCNs: Agr-PEG-PCL(1:1) > Agr-PEG-PCL(5:2) > Agr-PCL(1:1) > Agr-PCL(5:2). The higher tensile stress (ca. 0.8−1 MPa) and strain (ca. 30−95%) at break of PEG-PCL-containing APCNs (curves c and d) compared to that of corresponding PCLcontaining APCNs (0.3−0.6 MPa stress and 20−30% strain; curves a and b) may be due to the relatively low degree of phase separation between Agr and PCL in the former APCNs than that of later APCNs. The low degree of phase separation probably helps to transfer the applied stress from water-swollen Agr and PEG phases to semicrystalline PCL phase in the AgrPEG-PCL(1:1) and Agr-PEG-PCL(5:2). In contrast, the transfer of stress from swollen Agr to PCL is relatively difficult in Agr-PCL(1:1) and Agr-PCL (5:2) because of relatively enhanced phase separation between PCL and Agr in these F

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Figure 5. (A) 5-Flu loading capacity of different APCN gels with time at 30 °C. (B and C) Cumulative release of 5-Flu with time from different APCN gels in PBS of pH 7.4 (plots in B) and (C) in PBS of pH 7.4, 5, and with lipase (pH 7.4). Drug release experiments were conducted in triplicate at temperature ca. 37 °C and averages are plotted. Inset: digital pictures of (a−d) unloaded and (a′−d′) 5-Flu-loaded Agr-PCL(5:2), AgrPEG-PCL(5:2), Agr-PCL(1:1), and Agr-PEG-PCL(1:1) films, respectively, showing relative transparency.

indicates good correlation between degradation profiles and drug release profiles of the APCNs. (Compare Figure 4 and Figure 5C.) We next focused our efforts on investigating the underlying reasons for the observed effect of PEG on 5-Flu loading capacity and release kinetics. Higher 5-Flu loading capacity and its sustained and slow release from PEG-PCL-containing APCNs compared to that from PCL-containing APCN gels may be usually attributed to the high degree of 5-Flu solubilization in the former APCNs. PEG enhances the miscibility between Agr and PCL in the APCNs and provides better spreading of hydrophilic channels, which in turn solubilize 5-Flu to a greater extent. This is more likely because possible interaction between PEG (in water) and 5-Flu is ruled out as evident by its similar release kinetics from water solution and PEG solution (Figure S9). Thus, the barrier provided by the dialysis membrane and neat soluble PEG was negligibly small. The absorbance maxima of UV−visible spectra of 5-Flu in presence and absence of PEG appeared at same wavelength, which also indicated no probable interaction with PEG (Figure S10). Furthermore, the PEG-PCL-containing APCNs before and after loading with 5-Flu showed similar relative transparency (digital images b, b′, d, and d′, Figure 5), whereas the relative transparency decreased significantly in 5-Flu-loaded PCL-containing APCNs compared to that of corresponding unloaded gels (digital images a, a′, c, and c′, Figure 5). This fact once again indicated a high degree of solubilization of 5-Flu by the PEG-PCL-containing APCNs matrices. The DSC thermo-

this drug were 4 and 10 mg/mL, respectively (data not shown). The cumulative release of 5-Flu was also almost 1.6- to 2-fold slower from PEG-PCL-containing APCNs than that from PCLcontaining APCNs in the respective catalyzed processes (Figure 5B,C). The drug release profiles showed initial burst release from all the Agr-based APCN gels. The extent of burst release of 5-Flu from our gels was much higher than the release of theophylline from earlier reported PIB-based APCNs.29,30 This may be due to release of loosely bound drug from the Agrbased APCN gels.44 The comparatively higher loading of 5-Flu in our APN gels compared to theophylline loading in the PIBbased APCNs also contributed to the burst release from Agrbased APCN gels. Our 5-Flu-loaded films and dispersed particles of Agr-PCL (1:1) and Agr-PEG-PCL(1:1) each exhibited similar release profiles (at pH 7.4), which indicated a high degree of reproducibility of the release kinetics that was not influenced by the shape of the APCNs (Figure 5C). The release kinetics of 5-Flu was accelerated in the presence of lipase or at pH 5. For example, the cumulative release of 5-Flu from representative Agr-PEG-PCL(1:1) was 38, 27, and 18% after 300 h of release in the presence of lipase (pH 7.4), at pH 5, and at pH 7.4, respectively (Figure 5C). A similar effect was observed in the case of Agr-PCL(1:1), and the cumulative release of 5-Flu was 48, 44, and 38% in the presence of lipase, at pH 5, and at pH 7.4, respectively. This is due to acceleration of degradation of the APCNs in the presence of lipase or at pH 5, which caused triggered release of drug. The higher rate of drug release in the presence of lipase compared to that at pH 5 and 7 G

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ACS Applied Materials & Interfaces grams of 5-Flu-loaded Agr-PEG-PCL(1:1) showed melting peak maxima of 5-Flu at ca. 225 °C, whereas this peak maxima appeared at ca. 240 °C for 5-Flu-loaded Agr-PCL(1:1) (Figure S11). The heat capacity (Cp) values were 3.2 and 4.24 J/(g K) for 5-Flu-loaded Agr-PEG-PCL(1:1) and Agr-PCL(1:1), respectively. These data confirmed better solubilization of 5Flu in the PEG-PCL-containing APCNs. However, there is almost no effect of PEG on equilibrium gemcitabine hydrochloride loading capacity of the APCNs (Figure S12A). The release of gemcitabine hydrochloride was exactly opposite that of 5-Flu (Figure S12B). Higher release rate of gemcitabine hydrochloride from Agr-PEG-PCL(1:1) compared to that from Agr-PCL(1:1) is due to higher water swelling of the former APCN compared to that of latter. Hence, loading content and release of hydrophilic drug depend on nature of drugs as well as gel matrix. For example, water solubility of 5-Flu and gemcitabine hydrochloride are ca. 10 and 25 mg/mL at 25 °C. No influence of PEG on gemcitabine loading is attributed to the much higher solubility of gemcitabine hydrochloride within both the APCNs by the hydrophilic Agr phase as evidence from similar transparency of gemcitabine-loaded APCs compared to that of unloaded APCNs (digital pictures a and b, Figure S12). Hence, when loading is similar because of a similar degree of solubilization of drug by the APCNs, the release is mainly influenced by the extent of water swelling, which is the case for gemcitabine hydrochloride. Effect of PEG on Loading and Release of Hydrophobic Prednisolone Acetate. The loading and release of hydrophobic prednisolone acetate were governed by the amount of hydrophobic component and extent of water swelling of the APCN gels. The equilibrium prednisolone acetate loading capacity of Agr-PCL(5:2) and Agr-PCL(1:1) was higher than that of corresponding PEG-PCL-containing APCNs because of the high degree of solubilization of the drug by PCL-rich phase (Figure 6A). This is because the amount of PCL (14 and 26%, respectively) in Agr-PCL(5:2) and Agr-PCL(1:1) is higher than that of Agr-PEG-PCL(5:2) and Agr−PEG-PCL(1:1) (11 and 23%, respectively). This was confirmed by the relatively greater degree of phase separation of prednisolone acetate in the AgrPEG-PCL(1:1) film (Figure 6, picture b) compared to that of Agr-PCL(1:1) film (Figure 6, picture a). The high degree of solubilization of prednisolone acetate by Agr-PCL(5:2) and Agr-PCL(1:1) matrices and their comparatively lower water swelling slowed down the release of this drug from these APCNs (Figure 6B,C). Sustained release of prednisolone acetate occurred from all the APCNs. Triggered release of prednisolone acetate also occurred in the presence of lipase and at pH 5 compared to that at pH 7.4, as was observed for 5-Flu release. Stability of Drug in the APCNs and Drug Encapsulation−Release Mechanism. The drug molecules remained stable inside the APCN gels as confirmed by UV−visible and IR analyses. The drug-loaded films were stored in air at room temperature for six months; then, the release experiments were conducted for UV−visible analysis. The absorbance maximum of released 5-Flu (λmax = 266 nm) and prednisolone acetate (λmax= 247 nm) remained same as that of pure drugs. The IR spectra of loaded drugs taken after six months of loading also exhibited spectra similar to that of free drugs (Figure S13). For example, the −CO stretching (1639 cm−1) band of 5-Flu remained intact in the APCNs. The broadening of this band in APCN is due to merging of this band with the −CO

Figure 6. (A) Prednisolone acetate loading capacity of different APCN gels with time at 30 °C. (B and C) Cumulative release of prednisolone acetate with time from different gels (B) in PBS of pH 7.4 and (C) in PBS of pH 7.4 and 5 and with lipase (pH 7.4). Drug release experiments were conducted in triplicate at ca. 37 °C, and averages are plotted. Inset: digital pictures of prednisolone acetate loaded waterswollen (a) Agr-PCL(1:1) and (b) Agr-PEG-PCL(1:1).

stretching vibration band of ester group of PCL presence in the APCN (Figure S13A). The C−F band also appeared at same positions (ca. 1245 cm−1). The IR spectra of loaded prednisolone acetate also showed all characteristic bands (−CO stretching band of ester and −CO of carbonyl carbon) of neat prednisolone acetate (Figure S13B). The facile loading of hydrophilic and hydrophobic drugs in the APCN gels is attributed to the solubilization of the drug in the gel matrices by physical absorption.29,30 The hydrophobic phase provides a medium for solubilization of a hydrophobic drug, whereas the hydrophilic phases solubilizes the hydrophilic drugs. The chemical interaction of drugs and APCN gel matrices was ruled out, as evident from the IR analyses as discussed above. After the initial burst release from all three types of drugs, the regression coefficient values (R2) obtained from the zero-order kinetic model were greater than those from the first-order kinetic model (Tables S1 and Table S2). This indicates zeroorder release kinetics of 5-Flu (Table S1) after the initial burst. The diffusion exponent (n) values obtained from the Korsmeyer−Peppas model47 are between 0.63 and 0.64, which indicates that non-Fickian diffusion mechanism predominates, i.e., combination of diffusion and erosion of the matrix. The R2 values obtained with the Hixson−Crowell model48 are greater than that of the Higuchi model.49 Cell Viability and Hemocompatibility. Initial MTT assay indicated a high degree of HeLa cell viability by these APCNs. The high degree of cell viability and no loss of cell viability compared to biocompatible polystyrene tissue culture plate (control) indicate cytocompatibility of our APCNs (Figure 7A). There is not much difference in cell viability among the APCNs, which indicates no notable effect of PEG on cell viability. This is because the precursors, e.g., Agr and PCL, are well-known biocompatible polymers. The species formed by degradation of representative Agr-PCL(1:1) and Agr-PEGPCL(1:1) after 30 days of degradation also exhibited no loss in H

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Figure 7. (A) HeLa cell viability and (B) hemocompatibility in terms of hemolysis of different APCN gels. The cell viability was measured by the standard MTT assay and was expressed as a relative to that of cells grown in the presence of polystyrene tissue culture plate as control for 24 h of incubation. In all experiments, n = 3, and SD is shown as bar.

cell viability (not included in Figure 7). The excellent HeLa cell compatibility of the APCN gels and its degraded species is attributed to the cytocompatible nature of the APCNs. Hemocompatibility of biomaterials is also an essential criterion for in vivo applications. Hemolysis value in the range of 10−25% is acceptable for blood-contacting applications.44 Generally, hemolysis less than 20% is considered to be hemocompatible. Hemocompatibility of our APCNs was also determined by treating the samples with RBC (5 mg samples with 100 μL RBC stock solution; Figure 7 B). A low degree of hemolysis (4−6%) once again indicates the possible blood compatibility of our APCN gels. The species formed by degradation of APCN gels also showed 5−8% hemolysis, which indicates the possible applicability of these materials in directblood-contacting applications.

select proper APCN for localized delivery applications on the basis of the required release amount and nature of drug.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b10675. Experimental details of degradation, drug loading and release experiments, loading of RB, 1NMR spectra, IR spectra, GPC traces, equilibrium water swelling of APCN gels, DSC and DMA thermograms, loading capacity with time, cumulative release of gemcitabine hydrochloride, drug release kinetics data, UV−visible spectra of 5fluorouracil in water and in PEG solution, and MTT assay and blood compatibility experiments. (PDF)

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CONCLUSIONS We have demonstrated the synthesis of agarose−polycaprolactone (Agr-PCL) bicomponent and agarose−polyethylene glycol−polycaprolactone (Agr-PEG-PCL) tricomponent amphiphilic co-network gels utilizing sequential nucleophilic substitution reaction between multiamine functional agarose as nucleophile and activated-chloride-terminated PCL or PCLb-PEG-b-PCL as electrophile. The sol fraction decreased when chloride-terminated PCL-b-PEG-b-PCL was used as electrophile instead of chloride-terminated PCL because of enhanced miscibility between triblock copolymer and Agr compared to that of Agr and PCL. PEG reduced the degree of phase separation between Agr and PCL in the Agr-PCL-PEG gels, which in turn enhanced mechanical stress−strain property and 5-fluorouracil loading content compared to those of Agr-PCL gels. The 5-fluorouracil release rate and burst release from AgrPCL-PEG gels was slower compared to those from Agr-PCL gels, owing to a high degree of solubilization of 5-fluorouracil in the former gels. The diffusion-mediated release of other hydrophilic drugs such as gemcitabine hydrochloride and representative hydrophobic drug prednisolone acetate was governed by swelling of the APCN gels. The release of all three types of drugs followed almost zero-order kinetics after initial burst. The degradation of APCNs and release of drugs were triggered in the presence of lipase or at acidic pH. In addition to sustained release behavior, all types of APCN gels exhibited good cytocompatibility and hemocompatibility. Thus, one can

AUTHOR INFORMATION

Corresponding Author

*Fax: +912782566511. Tel.: +912782566511. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS CSIR-CSMCRI Registration No. 108/2015. We thank the Department of Science and Technology (project grant number EMR/2015/000843), Government of India for financial support. AKSC thanks CSIR, India for providing research fellowship. We thank Centralized Analytical Facility-CSMCRI for all round analytical support. We thank reviewers for their constructive comments and suggestions which have been incorporated in the manuscript.

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REFERENCES

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DOI: 10.1021/acsami.5b10675 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.5b10675 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX