Liquid Prepolymer-Based In Situ Formation of Degradable Poly

Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Liquid Prepolymer-Based in Situ Formation of Degradable Poly(ethylene glycol)-Linked-Poly(caprolactone)-Linked-Poly(2dimethylaminoethyl)methacrylate Amphiphilic Conetwork Gels Showing Polarity Driven Gelation and Bioadhesion Bhingaradiya Nutan, Arvind K. Singh Chandel, and Suresh K. Jewrajka*

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Membrane Science and Separation Technology Division, Academy of Scientific and Innovative Research, CSIR-Central Salt and Marine Chemicals Research Institute G. B. Marg, Bhavnagar, Gujarat 364002, India S Supporting Information *

ABSTRACT: Amphiphilic conetwork (APCN) gels suffer from lack of direct injectability due to use of organic solvent, prolonged crosslinking/polymerization process and immiscibility between hydrophilic and hydrophobic prepolymers. On the basis of prepolymers compatibility and polarity, we report the use of an advanced prepolymer liquid system for in situ construction of APCN gels. Solid elastic poly(ethylene glycol)linked-poly(ε-caprolactone)-linked-poly(2-dimethylaminoethyl)methacrylate (PEG-l-PCL-l-PDMA) APCN gels were formed upon addition of an appropriate amount of PDMA diluted in nonreactive sacrificial liquid PEG into a compatible blend of activated halide terminated PEG and PCL liquids. Compatibility among the prepolymers allowed favorable gelation. The polarity of the prepolymer liquid greatly influenced the gelation time. PEG-l-PCL-l-PDMA APCN gels were cytocompatible/ biodegradable and showed storage modulus in the range of 50−200 kPa and bioadhesive strength of 40−90 kPa. The fluorescence experiments showed that the hydrophobic probe, pyrene was distributed in both hydrophilic and hydrophobic phases of the APCN gels. These APCNs exhibited sustained release of hydrophobic and hydrophilic drugs. Effects of polarity, composition, and molecular weight of the liquid prepolymers on the gelation time, rheological property, and swelling behavior of the APCN gels have been investigated in details. KEYWORDS: solvent free liquid prepolymers, injectable conetwork gels, polarity-driven gelation, mechanical property, bioadhesive property, encapsulation and release property

1. INTRODUCTION

Several methodologies for the synthesis of APCN gels are reported in the literature. APCN gels are synthesized by the copolymerization of hydrophobic and hydrophilic monomers in the presence of bifunctional monomers, crosslinking of amphiphilic copolymers,40 reacting multifunctional hydrophobic and hydrophilic polymers with small molecular weight crosslinkers,41 copolymerizations of telechelic macromonomers with small molecular weight monomers,42 and crosslinking of amphiphilic copolymers by functional hydrophilic or hydrophobic polymer.12−15 Unlike hydrogels, APCN gels show better mechanical property, controlled water swelling, and encapsulation of both hydrophilic and hydrophobic therapeutics. This is due to the presence of both hydrophobic and hydrophilic phases in the APCN matrices.12−19,43,44 On the other hand, an important advantage of hydrogels over APCN gels is that the prepolymers of the former class of macromolecular entity are injectable under physiological conditions owing to their water solubility.43−51 Although the mechanical

Amphiphilic conetwork (APCN) gels are the relatively new class of polymer network composed of hydrophilic and hydrophobic polymer chains. The hydrophobic units are arranged in a segment but not randomly distributed in hydrophilic units. Such an arrangement of hydrophilic and hydrophobic polymer chains creates a driving force for nanophase separation.1−8 Nanophase-separated co-continuous morphology of hydrophilic and hydrophobic phases makes the APCNs suitable for encapsulation and release of both hydrophobic and hydrophilic therapeutics.9−19 For example, Papaphilippou et al. reported the synthesis of pH-, temperature-, and magnetic field-responsive APCN for controlled release of therapeutics.19 APCNs are also used as materials for soft contact lenses,20−22 delivery of therapeutics,23 tissue engineering scaffolds,24−26 antifouling coatings,27−29 temperature-responsive,30,31 pH-responsive,32 and chiral separation membranes,33 membrane for pervaporation,34 electrodialysis membranes,35,36 and phase transfer reactions.37 Biostable and biocompatible APCNs showed promises for use as controlled delivery of insulin.38,39 © XXXX American Chemical Society

Received: August 24, 2018 Accepted: October 15, 2018 Published: October 15, 2018 A

DOI: 10.1021/acsabm.8b00461 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials

Table 1. Compositions, Mn, and Gelation Time of Prepolymers, and Gel Fraction and pK Values of Resultant APCN Gels APCN

PEG/PCL (Mn, g/mol)

DMA/active halidea (mol/mol)

PEG/PCL (w/w)

PCL-l-PDMA-1 PEG-l-PCL-l-PDMA-2 PEG-l-PCL-l-PDMA-3 PEG-l-PCL-l-PDMA-4 PEG-l-PDMA-5 PEG(2k)-l-PCL-l-PDMA-6 PEG(4k)-l-PCL-l-PDMA-7 PEG-l-PCL(2k)-l-PDMA-8 PEG(4k)-l-PCL(2k)-PDMA-9d PEG(2k)-l-PCL-l-PDMA-10 PEG(4k)-l−PCL-PDMA-11 PCL−PEG-PCL-l-PMDAf

−/870 850/870 850/870 850/870 850/− 2500/870 4200/870 850/2500 4200/2500 2500/870 4200/870 550/700

1.05 1.07 1.07 1.05 1.05 1.59 1.78 1.57 4.0 1.07e 1.07e 1.33

0:100 25:75 50:50 75:25 100:0 50:50 50:50 50:50 50:50 50:50 50:50 50:50

pK of APCN

2.32 2.61 2.34 2.35

gelation timeb (min)

gel fractionc (w/w)

13 13 11 12 7 12 13 12 18 12 12 24

97 97 97 96 96 97 97 96 97 96 97 97

a

14% w/w PDMA crosslinker and 14% w/w liquid PEG (600 g/mol) diluent (with respect to total amount of liquid prepolymer components). bBy rheological time sweep experiment. cDMF extraction. d50% w/w diluent. e7% w/w PDMA crosslinker to achieve DMA to active halide ratio (mol/ mol) close to 1. fCl-PCL-b-PEG-b-PCL-Cl copolymer instead of Cl-PEG-Cl and Cl-PCL-Cl with total 50% PEG diluent.

and good bulk compatibility (miscibility) to avoid macro-phase separation during gelation reaction with tertiary amine functional macromolecule (PDMA). Instead of the use of a mixture of reactive PEG and PCL, a reactive triblock copolymer (PCL-b-PEG-b-PCL) could be employed for the in situ construction of APCN gel. According to the best of our knowledge, organic solvent-free/water-free, injectable prepolymers for the in situ construction of biodegradable APCNs have not been reported yet. Controlled in situ gelations of prepolymer liquids, and good bioadhesive strength, mechanical properties, and sustained release property of our APCN gels showed great promise for use in biomedical applications.

and water-swelling properties of APCNs could be controlled by adjusting the ratio (w/w) of hydrophilic to hydrophobic polymer chains, prepolymers of APCNs are not injectable at physiological conditions. This is due to the use of organic solvent, prolonged crosslinking/polymerization process, the requirement of temperature for crosslinking reaction, and immiscibility between hydrophilic and hydrophobic prepolymers. Amphiphilic copolymers containing predominant weight fraction of hydrophilic polymer are dispersible (micelle formation) in water. However, copolymers containing a high fraction of hydrophobic polymer are not dispersible in water. Low water dispersibility restricts the injectability of amphiphilic prepolymers for in situ preparation of APCN gels. An injectable prepolymer system is required for in situ construction and to extend the use of APCN gels in biomedical applications such as injectable cell and drug delivery system. Injectable gel system is beneficial for biomedical application due to avoid of the invasive surgical procedure. Thus, the main objective of this present work is the development of an injectable liquid prepolymer system without the use of organic solvents or water for in situ construction of cytocompatible/biodegradable APCN gels. The other objective of this work is to tune the gelation time, mechanical property, and water swelling behaviors of the APCN gels. Herein, we postulated that reactive liquid hydrophilic and hydrophobic prepolymers could be useful for in situ construction of APCN if these prepolymers are compatible (miscible) in the absence of a solvent. Low molecular weight (Mn) poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) are liquid as well as miscible in bulk.52 With the abovementioned initial lead, we designed a waterless/organic solvent-less injectable system to in situ construct APCN gels. Herein, we have demonstrated that the addition of poly(2dimethylamino)ethyl methacrylate (diluted in liquid PEG) into the liquid bulk blend of activated chloride terminated (low molecular weights) PEG and PCL formed biodegradable/ cytocompatible APCN gels. The design of the gelation system was based on the tunable gelation time of prepolymers depending on their polarity. In this regard, nucleophilic substitution reaction for the gelation of prepolymers was introduced to create charge species in the transition state and the product side. The end-functional reactive PEG and PCL (prepolymers) were selected based on their cytocompatibility

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(ethylene glycol) samples (PEG, 4000 g/mol, 2000 g/mol, and 600 g/mol, Sigma-Aldrich), poly(ε-caprolactone)diols (HO-PCL-OH, 550 g/mol, and 2000 g/mol, Sigma-Aldrich), 4(cholromethyl)benzoyl chloride (Cl-Bz-Cl, > 98%, TCI), triethylamine (>99%, TCI), pyrene (TCI), phosphate buffer solution (PBS, 98%, Sigma-Aldrich), and dialysis membrane (D0530, MWCO 2000 g/mol, Sigma-Aldrich) were used as received. All the solvents were from Spectro Chem, India, and were distilled before use. The monomers, ε-caprolactone (ε-CL, Aldrich), and 2-(dimethylamino)ethyl methacrylate (DMA, 98%, Aldrich) were distilled under reduced pressure before use. 2.2. Synthesis of Halide Terminated PEG (Cl-PEG-Cl), PCL (Cl-PCL-Cl), and Triblock Copolymer (Cl-PCL-b-PEG-b-PCL-Cl). Cl-PEG-Cl and Cl-PCL-Cl of different molecular weight (Mn) were synthesized by the esterification reaction of PEG and PCL with Cl-BzCl (Supporting Information).12,17 Well-defined structures of the polymers were confirmed by the 1H NMR spectroscopy. The Mn of the prepolymers was determined by 1H NMR spectroscopy (Figures S1 and S2, Supporting Information). The Mn values of these polymers were the sum of Mn of backbone and end groups. The calculated Mns of Cl-PEG-Cl polymers were 850 g/mol, 2500 g/mol, and 4200 g/ mol, respectively. The 1H NMR spectroscopy determined Mns of ClPCL-Cl polymers were 870 g/mol and 2500 g/mol, respectively. The calculated Mns of the backbone of the polymers were close as mentioned in the commercial samples. First, OH-PCL-b-PEG-b-PCL-OH was synthesized by ring opening polymerization of ε-CL using PEG as an initiator in the presence of Sn(Oct)2 as a catalyst. Esterification of hydroxyl end of the copolymer with Cl-Bz-Cl gave Cl-PCL-b-PEG-b-PCL-Cl (Supporting Information). The Mn of the copolymer was calculated to be 1500 g/mol. The Mns of PEG and PCL backbone in the copolymer were 550 g/mol and 700 g/mol, respectively, as determined by 1H NMR (Figure S3, Supporting Information). The PDI value of the copolymer was 1.3 as B


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Figure 1. (A) Liquid prepolymer injectable APCN gel showing the transparency of prepolymer and resultant gel. Cross-sectional SEM images of (B) PEG-l-PCL-l-PDMA-3, (C) PEG-l-PCL-l-PDMA-4, (D) PEG(2k)-l-PCL-l-PDMA-6, (E) PEG(4k)-l-PCL-l-PDMA-7, and (F) PEG(4k)-lPCL(2k)-PDMA-9 APCN gels. determined by gel permeation chromatography (GPC). A multifunctional crosslinker, PDMA (Mn = 12000 g/mol, and PDI = 1.31) was synthesized by the reversible addition−fragmentation chain transfer (RAFT) polymerization.17 2.3. Liquid Prepolymer Based in Situ Formation of APCN Gels. First, a stock solution of PDMA was prepared by dissolving PDMA (12 000 g/mol, 0.12 g) in 0.6 mL of acetone. To this solution, a nonreactive liquid diluent PEG (600 g/mol, 0.12 g) was added. The admixture was then evaporated in a rotary evaporator to remove acetone completely. The removal of acetone was confirmed by 1H NMR spectroscopy. This liquid is termed as A. Next, liquids Cl-PEGCl (850 g/mol, 0.2 g) and Cl-PCL-Cl (870 g/mol, 0.2 g) were mixed together in a vial. This liquid mixture is termed as B. Both the liquids were transparent. The liquid A (0.16 g) was then added to liquid B (0.4 g). The liquid prepolymer was mixed by hand shaking for 20 s to obtain transparent injectable liquid (C), which underwent gelation within 11 min at 37 °C. The liquid prepolymer system containing Cl-PEG-Cl or Cl-PCL-Cl of relatively high Mn was prepared as follows. Semisolid Cl-PEG-Cl (2500 g/mol or 4200 g/mol, 0.2 g) was dissolved in liquid Cl-PCL-Cl (850 g/mol, 0.2 g) to obtain viscous liquid B. Addition of the required amount of A into B gave crosslinkable liquid prepolymer. The amounts of PDMA crosslinker and PEG diluent were each 14% w/w in the liquid prepolymer (Cl-PEG-Cl, Cl-PCL-Cl, PDMA, and PEG

diluent). The compositions of liquid prepolymers are depicted in Table 1 (vide infra). Injectable APCN gel from Cl-PCL-b-PEGb-PCL-Cl and PDMA was prepared using PEG diluent to lower the initial viscosity of the prepolymer (50% of total prepolymer). The ClPEG-Cl to Cl-PCL-Cl ratio (w/w) was varied, while the amount of PDMA and total diluent (PEG) was kept constant to obtain APCNs with varying compositions. 2.4. Rheological Properties and Evaluation of Gelation Time. The gelation time of prepolymers and rheological property of directly injected and fully water swollen premade APCNs were evaluated using Physica MCR 301(Anton Paar) rheometer using a probe with 49.973 mm diameter.12,17 For determination of gelation time, each prepolymer liquid as described above was injected in the parallel plat of rheometer. The time lag between mixing the prepolymers and injection was about 20 s. First, time sweep experiments were performed at a constant frequency of 1 Hz at 37 °C. The measurements were performed at the viscoelastic region. The time at which storage modulus (G′) was equal to the loss modulus (G″) was noted (gelation time). Frequency sweep experiments were performed after time sweep experiments for about 40 min when the G′ value was almost constant. Frequency sweep experiments were performed by scanning in the range of 0.1 to 100 Hz at 1% strain and at temperature 37 °C. C


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Figure 2. Equilibrium water and toluene swelling of APCN gels as a function of (A) composition (Mns of reactive PEG and PCL were 850 g/mol and 870 g/mol), (B) Mn of PEG (Mn of PCL = 870 g/mol), and (C) Mn of PCL (Mn of PEG = 850). (Panel D) Digital photographs of in situ formed and fully water swollen APCN gels showing relative volume change. (E) DSC thermograms of different APCN gels and (F) AFM phase image of a representative PEG-l-PCL-l-PDMA-3. Similarly, the frequency sweep experiment of fully water swollen premade APCNs was performed. The premade samples were submerged in water for 24 h to remove the diluent. After removal of PEG diluent, the APCNs were allowed to attain equilibrium swelling for further use in frequency sweep experiments. Averages of 3−4 samples were taken. Strain response experiments were performed at a frequency of 1 Hz. The variation of G′ and G″ with the variation of applied strain was determined with premade fully water-swollen APCNs. The adhesive strength of the in situ APCN gels was determined by applying the liquid prepolymer in between mucus of goat skin as described earlier.17 2.5. Evaluation of Effect of Nonreactive Sacrificial Diluents (PEG, HO-PCL-OH, PEG+HO-PCL-OH, and Glycerol) on Gelation Time and Modulus. Effect of dielectric constant or polarity of the diluents on the gelation time of prepolymer was evaluated. The above-mentioned liquid prepolymers contained 14% w/w nonreactive

PEG diluent with respect to total prepolymer components. The PEG diluent was replaced by equivalent weight of glycerol, liquid OH-PCLOH (530 g/mol), and liquid PEG+HO-PCL-OH separately, and the gelation kinetics and modulus of the APCNs were evaluated by injecting the liquids in a parallel plate of the rheometer as described earlier (Section 2.3). The values of dielectric constant of water, glycerol, PEG (Mn= 600 g/mol), and PCL (Mn = 530 g/mol) were about 80, 58, 42, and 11.6 at ∼30 °C. The polarity or dielectric constant of the diluent followed the order water > glycerol > PEG > PCL. Water was used as diluent only when reactive PEG was used alone. The extent of the polarity of different prepolymer liquids was qualitatively obtained by the pyrene encapsulation study (Supporting Information). 2.6. Sol Fraction, Swelling, and Accelerated Degradation Study. Sol fraction and swelling of the APCN gels were determined using standard protocol (Supporting Information). The extent of D


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ACS Applied Bio Materials degradation of APCNs was evaluated at accelerated degradation conditions. In this study, each day may be equal to approximately 1 week in real-time degradation (pH 7.4 at physiological conditions).53 First, the APCNs were extracted with acetone to remove PEG diluent and other unreacted prepolymers if any. The extracted and dried mass (0.4 g each) of APCNs was taken in individual glass vials and filled with PBS of pH 12 (100 mL). The vials were incubated in an incubator shaker (INFORS AGCH-4103) at 100 rpm and at temperature 37 °C. At a specific time interval, the gels were removed from the media and poured in deionized water to remove buffer salts. The thoroughly water rinsed gels were placed in acetone for 12 h to extract water and then dried under vacuum oven at temperature 37 °C to obtain a constant weight. The mass loss was calculated by the following equation:

Degradation (%) =

mb − ma × 100 mb

PCL(2k)-PDMA-9. The above order of porosity and pore size of the APCN gels may be attributed to the increase of Mn of reactive PEG or PCL. Relatively high Mn PCL and PEG create more free volume (low crosslinking density) within the networks of PEG(4k)-l-PCL-l-PDMA-7 and PEG(4k)-l-PCL(2k)-PDMA-9 than that of other APCN gels. Presumably, once the network was formed by crosslinking reaction, it remained in frozen state and retained its porosity. 3.2. Swelling Behavior and Phase Morphology of APCN Gels. In situ formed APCN gels swell in water and in organic solvents. Water swelling decreased and toluene swelling increased with increasing amount of PCL fraction in the APCNs (Figure 2A). Variation of swelling was not exactly linear with the composition of the gels. This is due to the compositional and structural variation among the APCNs.55 Water swelling of the APCNs significantly increased when the Mn of reactive PEG increased from 850 g/mol to 4200 g/mol (Figure 2B). This is due to increase of chain mobility and decrease of crosslinking of the conetwork with increasing Mn of reactive PEG. Furthermore, PEG solvation in toluene decreases with increasing its M n. This indicates low contribution of PEG chains (Mn = 2500−4200 g/mol) toward swelling of APCN gels in toluene. Reverse situation occurred when the Mn of reactive PCL increased from 870 g/mol to 2500 g/mol (Figure 2C). This provides an opportunity to tune the swelling behavior of the APCNs by both changing the composition and Mn of polymers without changing the amount of multifunctional crosslinker (PDMA). The PEG(2k)-l-PCLl-PDMA-6, PEG(4k)-l-PCL-l-PDMA-7, and PEG-l-PCL(2k)-lPDMA-8 (Table 1, entries 6−8) should exhibit pH-responsive water-swelling since these conetworks contain unreacted amine moieties.56−58 However, pH-responsive water-swelling was observed only in PEG(4k)-l-PCL-l-PDMA-7 (Figure 2B). This is attributed to the relatively high pK value of this APCN compared to the others (Table 1). Presence of relatively high Mn PEG chains enhances water-swelling and porosity of PEG(4k)-l-PCL-l-PDMA-7. This leads to relatively easy diffusion of H+ ions toward the unreacted DMA moieties of the APCN. Thus, the pK value of unreacted DMA moieties depends on the composition of prepolymer liquid, Mn of reactive PEG, and porosity of the formed APCN gels. Change of pK of DMA moieties with the composition of our APCNs is consistent with the previous reports.13,58 The conetworks maintained transparency in as injected as well as fully water-swollen states (Figure 2D). Extent of volume change (14−35%) between the two states was low in PCL-l-PDMA-1, PEG-l-PCL(2k)-l-PDMA-8, and PEG-l-PCLl-PDMA-3. However, this change was relatively high (95− 200%) in PEG(2k)-l-PCL-l-PDMA-6 and PEG(4k)-l-PCL-lPDMA-7 APCNs and PEG-l-PDMA-5 hydrogel. The DSC thermograms of the APCNs showed the appearance of Tg at −18 °C (Figure 2E). The Tg value of both PEG and PCL is about −65 °C, while it is about 19 °C for PDMA (Figure S5A, Supporting Information). Good compatibility among the three components leads to the appearance of single Tg in the APCN gels.52,57 Earlier Tg of miscible blend of PEG and PCL was also found to be about −24 °C.52 Melting peak of PCL was observed at 20 °C in DSC thermograms of HO-PCL-OH (Mn = 530 g/mol) homopolymer, blend of nonreactive PEG (Mn = 750 g/mol) and HOPCL-OH (Mn = 850 g/mol), and blend of PEG (Mn = 750), HO-PCL-OH (Mn = 850 g/mol), and PDMA. HO-PCL-OH with relatively high Mn (Mn= 2000 g/mol) exhibited two

(1)

where mb is the dry mass and ma is the residual dry mass of the sample after a specified time of degradation. Averages of three independent measurements were recorded for each type of samples. Determination of glass transition temperature and in situ encapsulation of pyrene in the APCNs are included in the Supporting Information. Hemocompatibility and cell viability experiments were conducted with these APCNs using standard protocols (Supporting Information).12,13,16

3. RESULTS AND DISCUSSION 3.1. In Situ Formation of APCN Gels by Liquid Prepolymers. Halide end-functional PEG and PCL are compatible with each other in their low Mn range and form a transparent injectable liquid at 37 °C. Addition of PDMA (diluted with 14% w/w of nonreactive PEG) into a mechanical blend of halide end-functional reactive PEG (Mn = 850 g/mol) and PCL (870 g/mol) formed a homogeneous liquid, which turned into a transparent APCN gel within about 11 min (Figure 1A). Differential scanning calorimetric study (DSC) and transparency of the prepolymer liquids indicated good compatibility among the reactive PEG, PCL, and PDMA (vide infra).52 Compositions and Mns of the prepolymer liquids were varied to obtain a series of APCN gels. The Mn of either PCL or PEG was varied wherein the polymer of relatively high Mn was solubilized in low Mn polymer. This is an organic solventfree/water-free system, except the use of nonreactive sacrificial diluent (PEG, Mn = 600 g/mol) to lower the viscosity of the prepolymer system. Typical nucleophilic substitution reaction takes place among the DMA moieties of PDMA and halide moieties of PEG and PCL.17,54 Instead of reactive PEG and PCL, a halide end-functional triblock copolymer, namely PCLb-PEG-b-PCL, was also used (Figure 1A). Table 1 shows the composition, Mn, and gelation time of different prepolymer liquids. Table 1 also shows gel fraction, pK values of DMA moieties, and abbreviations of the APCNs. High gel fraction (96−97%) of these APCNs in DMF indicates an efficient crosslinking reaction. The DMF soluble fraction was found to be PEG and PCL. FT-IR spectroscopic analysis of the DMF extracted samples showed that the actual composition of the APCNs was close to the composition of the prepolymers (Figure S4 and Table S1, Supporting Information). PEG-lPCL-l-PDMA-3 and PEG-l-PCL-l-PDMA-4 (SEM images B and C, Figure 1) showed dense morphology, while PEG(2k)-lPCL-l-PDMA-6, PEG(4k)-l-PCL-l-PDMA-7, and PEG(4k)-lPCL(2K)-l-PDMA-9 (SEM images D−F, Figure 1) showed relatively porous morphology. Specifically, the relative porosity and average pore size followed the order for the APCN gels, PEG-l-PCL-l-PDMA-3 ≈ PEG-l-PCL-l-PDMA-4 < PEG(2k)-lPCL-l-PDMA-6 < PEG(4k)-l-PCL-l-PDMA-7 < PEG(4k)-lE


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Figure 3. Variation of gelation time as a function of (A) composition of the prepolymer liquids, and Mn of reactive (B) PEG, and (C) PCL. Prepolymer liquids contain reactive PEG, PCL, PDMA (15% w/w), and a nonreactive PEG diluent (14% w/w). (D) Schematic illustration showing stabilization of transition state of gelation reaction in polar medium. Variation of gelation time of prepolymer liquids containing reactive (E) PEG (Mn = 850 g/mol) and PDMA, and (G) PEG, PCL, and PDMA with the addition of different nonreactive diluents (14% w/w) separately. (F, H) Real-time monitoring of gelation process (E and G) by the rheometer.

melting peaks maxima at 38 and 47 °C, respectively, due to its semicrystalline character. The PEG of Mn 4000 g/mol showed melting peak at around 62 °C in the blend (Figure S5B, Supporting Information). In contrast to above results, melting peaks of PEG and PCL were not visible in the DSC thermograms of the APCN gels (Figure 2E). Co-continuous morphology of the conetworks greatly suppressed the crystalline fraction of PCL and PEG chains in the conetworks. Significant suppression of crystalline fraction of poly(tetrahydrofuran) phase in the poly(tetrahydrofuran)−l-poly(vinyl imidazole) APCN was observed earlier.42,59 Phase mode AFM image (Figure 2F) of a representative PEG-l-PCL-l-

PDMA-3 shows no microphase separation, which confirms the observed single Tg from DSC measurement (Figure 2E). 3.3. Effect of Polarity of Prepolymers on Gelation Time. Variation of gelation time as a function of composition, Mn, and polarity of the prepolymers was evaluated. The crossover point between G′ and G″ during APCN formation was monitored by injecting the prepolymer liquids in the rheometer. Rheometer time profiles monitoring the progress of gelation have been included in the Supporting Information (Figure S6). Gelation time increased with increasing reactive PCL fraction in the prepolymers (Figure 3A). In this prepolymer set, Mns of reactive PEG and PCL were 850 g/ F


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Figure 4. Maximal G′ values of in situ formed APCNs as a function of (A) composition, and Mn of reactive (B) PEG and (C) PCL, respectively. The frequency sweep (under 1% strain) experiments were performed after time sweep experiments when the G′ values became constant. (D) Illustration showing change of relative volume with increasing Mn of prepolymers. (E−G) Maximal G′ values of fully water swollen APCNs as determined by frequency sweep experiment (Figure S9, Supporting Information). Total nonreactive PEG (14% w/w) diluent and amount of PDMA (14%, w/w) were kept constant in each experiment (Table 1, entries 1−5 for bar diagrams A and E, and entries 6−8 for bar diagrams B, C, F, and G).

transient species.60 The above mechanism and the effect of polarity of prepolymer liquids on gelation time were corroborated by preparing a model hydrogel with reactive PEG (850 g/mol) and PDMA under similar experimental conditions as mentioned in Figure 2A for APCN gels. Aqueous PBS or glycerol was used as a nonreactive diluent for the preparation of hydrogel (Figure 3E,F). The aqueous- and glycerol-based prepolymer liquids undergo gelation within about 2 and 2.4 min, respectively. Gelation time increased to about 7 min for prepolymer liquid containing PEG as nonreactive diluent. The gelation time increased significantly with increasing amount of either nonreactive PEG or aqueous PBS (Figure S7, Supporting Information). Akin to the above experiments, the gelation time of prepolymer liquids of representative PEG-l-PCL-l-PDMA-3 APCN gel containing nonreactive diluents, namely HO-PCL-OH, HO-PCL-OH +PEG mixture, PEG, and glycerol, respectively, was determined (Figures 3G,H).61 The gelation time decreased with increasing polarity of the diluents. The above experiments provide strong leads regarding controlling of gelation time. Polarity of prepolymer liquids follows the order for the

mol and 870 g/mol, respectively, and a nonreactive PEG diluent (Mn = 600 g/mol) was used. Our gelation reaction follows second-order kinetic in respect to the concentration of tertiary amine and halide moieties. The rate of gelation depends on the concentration of reactive constituents, the polarity of prepolymer liquid, and nucleophilicity of DMA moieties. The DMA to active halide ratio (mol/mol) in this set of prepolymer liquids is similar (Table 1, entries 1−5). Hence, lowering of gelation time with increasing PEG fraction in the prepolymer liquids is attributed to the increase of polarity of the prepolymer liquids. Gelation time marginally increased with increasing Mn of reactive PEG or PCL (Figure 3B,C). Polarity of the prepolymer liquids remained unchanged when these liquids contained 1:1 (w/w) reactive prepolymers. The polarity of prepolymer liquid also remained unchanged when the Mn of reactive PCL was in the range 870−2500 g/mol. On the basis of polarity of prepolymer liquids, we propose a reaction mechanism of our gelation system (Figure 3D). It is seen that the charge of transient species increased relative to the starting compounds. This leads to the significant increase in reaction rate in polar medium through stabilization of G


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Figure 5. (A) Maximal bioadhesive strength of different APCN gels (varying composition), a representative hydrogel, a commercial fibrin glue, and a reported dopamine-based glue. Variation of bioadhesive strength of APCN gels with the variation of Mn of reactive (B) PEG and (C) PCL. The bioadhesive strength was measured by applying solutions in between two goat mucous skins in the present study.

in water for 48 h to reach equilibrium swelling. This situation will attain during the application of APCN gels in a biological system. The fully water-swollen APCN gels showed relatively low value of G′ (Figures 4E−G) to that of in situ formed APCNs, except PCL-l-PDMA-1 and PEG-l-PCL(2k)-l-PDMA8 (Figures 4G). This is due to the increasing extent of swelling of the other APCNs in water compared to that of in situ formed condition. The increased water swelling causes plasticizing effect in the matrices. On the other hand, water swelling of PCL-l-PDMA-1 and PEG-l-PCL(2k)-l-PDMA-8 was much lower than that of other APCNs. The G′ value of fully water-swollen PEG-l-PCL-l-PDMA-3 was about 44 kPa. Earlier, an injectable hydrogel of dopamine-terminated PEG prepolymer showed G′ of 60 kPa but not in fully hydrated state.62 The polarity of the diluents did not affect the G′ values of the APCN gels (Figure 3F and H). The PEG-l-PCL-lPDMA-3 showed the highest modulus presumably due to the formation of homogeneous phase morphology in this APCN as discussed earlier. Crosslinked hydrophobic PCL domains segregated from the hydrophilic domains when APCN remained in water swollen state. This reinforced the mechanical property of the PEG-l-PCL-l-PDMA-3. Representative PCL−PEG-PCL-l-PMDA APCN (Table 1, entry 12) formed by reacting Cl-PCL-b-PEG-b-PCL-Cl and PDMA (in nonreactive diluent PEG, 50% w/w of total prepolymer) showed G′ value of 120 kPa, while its G′ value decreased to 21 kPa in its fully water-swollen state. The gelation time of the prepolymer liquid of this APCN was relatively high (24 min) due to use of a higher amount of diluent (Figure S11, Supporting Information). It may be noted that the G′ values of different prepolymer systems at the beginning of gelation reaction were different due to different viscosity of the starting prepolymer liquids and the rate of crosslinking reaction (Figure 3F,H and Figure S6, Supporting Information, vide supra). The time lag between prepolymers mixing and injection into the rheometer was about 20 s for all experiments. Hence, a prepolymer with a greater degree of polarity showed the faster progress of reaction at this time lag, which resulted in formation of relatively viscous prepolymer liquid with a greater value of G′ at the start of rheological measurements. Bioadhesive strength of a gel material depends on the functionality of prepolymer constituents, charge of prepolymers, and presence of dangling polymer chain.63−65 The mechanical property and water swelling behavior of the formed APCN gels also affect the bioadhesive strength.63−65 The PEGl-PCL-l-PDMA-3 APCN showed maximal bioadhesive strength

diluents, water > glycerol > PEG > HO-PCL-OH. The change of polarity of prepolymer liquids containing different diluents was qualitatively measured by comparing the environment of pyrene probe in the prepolymer liquids (vide infra).61 3.4. Rheological Properties and Bioadhesive Strength of APCN Gels. Strain sweep profiles of the fully water-swollen APCNs showed nearly constant values of G′ and G″ under a low applied strain (linear viscoelastic region). The decrease of G′ and increase of G′′ was observed at high strain (critical strain). The critical strain of APCNs increases with increasing Mn of reactive PEG or PCL. This is usually due to the increase of relaxation time of polymer chains upon increasing applied strain. The extent of water-swelling also influenced the critical strain of the APCN (Figure S8, Supporting Information). After time sweep experiment for 40 min, frequency sweep experiments were conducted to evaluate the G′ values of in situ formed APCNs (Figure S9, Supporting Information). G′≫ G′′ in the frequency range of 0.1−100 Hz indicated the formation of stable APCN gels. The G′ values of the APCN gels depend on composition as well as Mn of reactive PEG and PCL (Figure 4A−C). A 50:50 (w/w) of reactive PEG (Mn = 850 g/mol) and PCL (Mn = 870 g/mol) produced PEG-l-PCL-l-PDMA-3 with G′ value as high as >200 kPa (Figure 4A). The G′ values of APCNs (Table 1, entries 6−11) decreased as Mn of either PEG or PCL increased (Figure 4B,C). This is attributed to the decrease of crosslinking density in the APCN gels with increasing Mn of the prepolymer. This was further corroborated by the fact that PEG(2k)-l-PCL-l-PDMA-10 and PEG(4k)-l-PCL-l-PDMA-11 APCNs (Table 1, entries 10 and 11) formed by reacting equivalent amount of PDMA (DMA to halide ratio, mol/mol) with mixture of reactive PEG (Mn = 2500 g/mol or 4200 g/mol), and PCL (Mn = 870 g/mol) showed similar values of G′ (∼100 and 48 kPa) to that of PEG(2k)-l-PCL-l-PDMA-6 and PEG(4k)-l-PCL-l-PDMA-7 (Figure S10, Supporting Information). Illustration of Figure 4D helps to visualize the proposed structure of the formed APCN gels from reactive prepolymers of different Mn. The free volume in the conetworks increases with increasing Mn of reactive PEG or PCL. Thus, crosslinking density of APCN gel decreased with increasing Mn of reactive PEG or PCL. The proposed illustration is albeit in an ideal condition, considering a complete consumption of halide ends of either PEG or PCL through the sequential nucleophilic substitution reaction with DMA moieties. Next, the in situ formed conetworks were extracted with water to remove PEG diluent. The gels were allowed to swell H


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Figure 6. (A) Fluorescence spectra of pyrene (λex = 346 nm) encapsulated in prepolymers of PEG-l-PCL-l-PDMA-3 APCN containing HO-PCLOH, PEG, and glycerol diluents separately (spectra a−c) and prepolymer of a PEG-l-PDMA-5 hydrogel containing water diluent (spectrum d). (B) Fluorescence spectra of pyrene encapsulated in as obtained APCNs prepared by the addition of respective diluents (spectra a−c) and hydrogel containing water as diluent (spectrum d). (C) Fluorescence spectra of pyrene entrapped in diluent free (dried) APCN and hydrogel. UV−visible spectra of (D) pyrene and (E) rhodamine-6G entrapped in APCN. Time-dependent release of pyrene from PEG-l-PCL-l-PDMA-3 at pH (F) 7.4 and (G) 5, respectively. (H) Digital images of surface, and cross-sectional parts of the APCN, and printable prepolymer.

than the later APCN (35 kPa) in fully water swollen state. Presence of PEG chains of low Mn between the cross-linking points helped to enhance the bioadhesive strength of the hydrogel. Good adhesive property of our APCN gels on goat mucus is attributed to the primary adhesion of prepolymer liquids due to both electrostatic interactions as well as the interaction of functional groups of the prepolymers with the mucus. The prepolymer liquids contain amine groups, which provide electrostatic interaction with the mucin fiber,63 while the esters and ethers groups of prepolymers helped to form a hydrogen bond with the mucus. The formed APCN gels were strongly adhered on the mucus layer due to low water swelling,

of about 90 kPa when applied on goat mucous (Figure 5A). Bioadhesive strength of APCN gels decreased with increasing Mn of reactive PEG and PCL (Figure 5B,C). This is due to the lowering of modulus of the formed APCN gels. Bioadhesive strength (70−90 kPa) of our APCNs and PEG-l-PDMA-5 hydrogel was much higher than that of polydextran aldehyde glue (∼4 kPa), dopamine-based glue (58.2 kPa), and a commercially available fibrin glue (5.6 kPa).64,65 Presence of hydrophobic PCL has apparently feeble effect on the bioadhesive strength of the conetworks as confirmed by the close adhesive strength of PEG-l-PDMA-5 hydrogel (80 kPa) and PCL-l-PDMA-1 APCN (77 kPa) (Figure 5A), although the G′ value (10 kPa) of the former hydrogel was much lower I


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Figure 7. (A) Accelerated degradation of APCNs at pH 12 and at temperature 37 °C. Hemolysis of red blood cells with the (B) prepolymer liquids (directly injected) and (C) different premade APCN gels. (D) MTT assay showing HeLA cell viability on the APCNs. The fluorescence image showing cell morphology on different gels: (E) PCL-l-PDMA-1, (F) PEG-l-PCL-l-PDMA-3, (G) PEG-l-PDMA-5, and (H) control (polystyrene tissue culture plate).

APCN gels. Pyrene was also solubilized in different diluents to evaluate its environment. The I1/I3 values of pyrene in neat nonreactive HO-PCL-OH, PEG, and glycerol were measured to be 1.40, 1.48, and 1.5, respectively (Figure S12A, Supporting Information). This value was 1.7 in neat water. The I1/I3 values of pyrene were 1.43, 1.45, and 1.48, respectively, in the prepolymer liquids of PEG-l-PCL-lPDMA-3 APCN containing HO-PCL-OH, PEG, and glycerol diluents separately (Figure 6A, spectra a−c). The I1/I3 value of pyrene increased in the prepolymer of APCN containing HOPCL-OH as diluent compared to that of neat HO-PCL-OH liquid due to additional presence of more polar components (PEG and PDMA) in the former system. In contrast, the I1/I3 value of pyrene decreased in the prepolymers containing PEG, and glycerol as diluents compared to that of individual neat diluents. This indicates that neat PEG, and glycerol are more polar with higher dielectric constant than that of the prepolymer liquids. The I1/I3 value of pyrene in the prepolymer of PEG-l-PDMA-5 hydrogel containing water as

good mechanical properties, and presence of cationic charge centers. The cationic charge centers were created in the APCN gels via quaternization reaction of tertiary amine with halide ends of PEG and PCL. The increase of adhesion strength of cationic polymer than that of nonionic polymer was reported in the literature.63 Low water swelling of these APCN lowers the slippage of the mucilage, which provides mechanical support for the adhesion. 3.5. Environment of Hydrophobic Probe and Encapsulation/Release Property of APCN Gels. The intensity ratio of first (I1) to third (I3) emission bands of a hydrophobic fluorescence probe, pyrene, provides reliable information about the local dielectric property and hydrophobic drug encapsulation efficiency of micelles54 and polymer matrices.17,61,66 Hence, pyrene was used as probe molecule to evaluate the dielectric and encapsulation properties of the APCN gels. Pyrene was first solubilized in a mixture of reactive PEG and PCL. Addition of PDMA (diluted with nonreactive PEG) into the reactive polymer yielded pyrene loaded homogeneous J


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high efficiency for hydrophobic drug encapsulation and controlled release behavior. Photographs of panel H show homogeneous distribution of pyrene on the surface and inner section of the formed PEG-l-PCL-l-PDMA-3 APCN. This model experiment indicates that the in situ formed APCN is able to homogeneously incorporate hydrophobic molecules for controlled release application. The pyrene encapsulation experiments further support our claim regarding variation of gelation time of the prepolymer with the extraneous addition of various nonreactive diluents as discussed earlier. Furthermore, in situ formed PEG-l-PCL-l-PDMA-3 exhibited controlled release of hydrophobic (griseofulvin and pyrene) and hydrophilic (doxorubicin) drugs with nearly zero-order kinetics (R2 = 0.991 to 0.995) (Table S2, Supporting Information) due to compact structure (Figure 1B) and lower extent of swelling (Figure 2A). The low degree of swelling and compact nature of this APCN prevent the burst release of drugs. The drug molecules were encapsulated into prepolymer liquids for the experiments (Figure S13, Supporting Information).17 3.6. Degradation Behavior, Hemocompatibility, and Cell Viability. Accelerated degradation behavior of the APCN gels and a representative hydrogel was determined at pH 12 and at temperature 37 °C (Figure 7A). Each day of time frame is considered to be equal to about 7 days in physiological condition.53 The PEG-l-PDMA-5 hydrogel degraded in a faster rate. On the other hand, PCL-l-PDMA-1 APCN showed the slowest rate of degradation. The degradation is mainly caused by the hydrolysis of ester groups of DMA units involved in the cross-linking reaction (Figure 1A, marked by a circle). The positively charged nitrogen atoms lower the electron density of ester carbonyl groups of quatermized DMA moieties. This facilitates hydrolytic cleavage under the above-mentioned condition.17,53 The degradation of ester linkages of PCL backbone may also be possible.17,54 Except charge in carbonyl carbon, hydrolytic cleavage of ester linkages also depends on the water swelling and porosity of the gels. Relatively high water swelling of hydrogel compared to that of APCN gels causes faster degradation of the former gel. APCN gels of greater degree of both water swelling and porosity undergo faster degradation except PEG-lPCL(2k)-l-PDMA-8 (Figure 7A). The PEG-l-PCL(2k)-lPDMA-8 contains PCL chains of relatively high Mn, which undergo relatively fast degradation (backbone cleavage) in the test conditions, in addition to cleavage of ester linkages of quaternized DMA groups (close to the quaternized nitrogen atoms). The prepolymer liquids and formed APCN gels exhibited hemocompatibility with hemolysis

Liquid Prepolymer-Based In Situ Formation of Degradable Poly

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