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Dihydroperfamine, an alkaloid from haplophyllum glabrinum
Biocompatible, stimuli-responsive hydrogel of chemically crosslinked b-cyclodextrin as amoxicillin carrier Arpita Roy,1 Priti Prasanna Maity,2 Santanu Dhara,2 Sagar Pal
1
1
Polymer Chemistry Laboratory, Department of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad 826004, India Biomaterials and Tissue Engineering Laboratory, School of Medical Science and Technology, Indian Institute of Technology,
2
Kharagpur, Kharagpur 721302, India Correspondence to: S. Pal (E - mail: [email protected]); S. Dhara (E - mail: [email protected])
ABSTRACT: A novel pH-responsive, chemically crosslinked hydrogelator (cl-b-CD/pVP) has been fabricated using b-cyclodextrin (bCD) and N-vinyl pyrollidone (N-VP) in presence of diurethane dimethacrylate (DUDMA) crosslinker/azobisisobutyronitrile initiator through free radical polymerization. Various grades of cl-b-CD/pVP have been synthesized and the best grade has been considered with higher crosslinking density, higher gel strength, and lower % swelling ratio. The hydrogelator has been characterized by FTIR, 1 H and 13C NMR spectroscopy, TGA, and FESEM analyses. The hydrogelator demonstrates pH-responsive behavior, which has been confirmed by swelling behavior and gel characteristics at various pH (at 37 8C). Using hen egg lysozyme, degradation experiment has been performed, which confirms the biodegradable nature of the hydrogel. The in vitro cytotoxicity study and live–dead assay suggest that the hydrogelator is cytocompatible toward MG-63 cells. Finally, the hydrogelator shows excellent efficacy as an antibiotic (amoxiC 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 135, 45939. cillin) carrier. V
KEYWORDS: biomaterials; crosslinking; oligosaccharides
Received 28 August 2017; accepted 16 October 2017 DOI: 10.1002/app.45939
INTRODUCTION
In recent years, the development of controlled release drug delivery systems (CRDDS) has become prominent in biomedical applications. Drug release at an anticipated rate has great benefits over the conventional drug delivery and is more effective to maintain the drug concentration level in blood, to minimize the harmful side effects, enhance the efficiency time, improve patient compliance, as well as to increase the bioavailability.1 Several drug delivery systems have been developed in this purpose over last few years, among them the simplest one is the hydrogel based matrix device, where the drug is incorporated in polymeric network.2 The composition and structure of the polymeric system has an effective role to control the drug release. Porous polymeric hydrogels are rapidly employed for delayed and sustained drug release over the years mainly to control the rate of drug delivery by diffusion mechanism or simultaneously by diffusion mechanism with the erosion of matrix.3,4 Now a days, “stimuli-responsive gels” have become one of the most important device for drug delivery applications.5–7 The phase transition of these gels are highly influenced by some external stimulating factors like pH,8,9 temperature,10,11 ionic strength,12,13 and magnetic/electric filed.14,15 These types of gels
possess some unique striking properties like desired morphology, adjustable dimension, high porosity, and hence forth able to absorb high amount of water in its three-dimensional network structure. All these favorable properties make hydrogels extremely useful for biomedical applications.16–18 Hydrogels can be defined as physically or chemically crosslinked 3D polymeric networks that gets swelled by absorbing large amount of water in presence of water or other biological fluids, while it does not dissolve in the media.8,19 Polymeric hydrogels have versatile uses in biomedical field and drug delivery.20,21 Recently, biopolymer or modified biopolymerbased hydrogels are broadly used as drugs carrier to control the rate of drug release.17 Polysaccharides, oligosaccharides, or biopolymers-based hydrogels are more favorable for the use of drug delivery system over synthetic polymers owing to their biodegradability, easy to functionalization, and nontoxicity.22–24 However, natural biopolymers have some shortcomings such as uncontrolled hydration rate, microbial contamination, and decrease of viscosity on storage.25 These drawbacks can be evaded by functionalization through crosslinking on the backbone of polysaccharides/oligosaccharides that provides several enhanced characteristics including the development of controlled drug delivery device.26 Besides, the crosslinking density of a chemically
Additional Supporting Information may be found in the online version of this article. C 2017 Wiley Periodicals, Inc. V
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Table I. Synthesis Details, Crosslinking Density, Gel Strength, Yield Strength, and % ESR of Various cl-b-CD/pVP Hydrogelators Amount of b-cyclodextrin 5 8.8107 3 1024 mol Cross linker concentration 5 1.06 3 1023 mol
Hydrogelators
Initiator concentration (mol 3 1024)
Monomer concentration (mol 3 1022)
Crosslinking density (CD) (quantitative)
Gel strength
Yield stress
% ESR (pH 7.4)
cl-b-CD/pVP1
0.60
4.72
0.37
1.92
1799
231.6 6 11
cl-b-CD/pVP2
0.91
4.72
0.47
2.01
1959
226.2 6 11
cl-b-CD/pVP3
1.21
4.72
0.84
2.48
2754
174.0 6 8
cl-b-CD/pVP4
1.53
4.72
0.21
1.46
1585
291.6 6 14
cl-b-CD/pVP5
1.21
5.66
0.67
2.36
2323
192.4 6 9
cl-b-CD/pVP6
1.21
6.61
0.58
2.20
2133
221.4 6 11
crosslinked hydrogel has a crucial role on release rate—higher is the crosslinking density, higher would be the gel strength that would enhance the sustained release characteristics.27 Hydrogels based on polysaccharides/oligosaccharides have been developed and reported.19–22 One of the most widely used oligosaccharide is b-cyclodextrin, which received great attention in the field of drug delivery.28 Chemically modified bcyclodextrin-based drugs carrier have already been developed and reported.29,30 However, it is still imperative to develop new and novel b-cyclodextrin-based hydrogel with improved stimuli-responsive behavior, nontoxic, and biocompatible device that can deliver the drugs at more desired rate. In this concern, herein, for the first time we have explored the synthesis of a pH-sensitive novel hydrogel through crosslinking of poly(N-vinyl pyrollidone) on b-cyclodextrin using diurethane dimethacrylate (DUDMA) crosslinker via free radical polymerization. The hydrogel demonstrates excellent gel characteristics with superior controlled release behavior toward the release of antibiotic (amoxicillin) that opens up a new pathway for its use in real time applications EXPERIMENTAL
Materials b-Cyclodextrin (b-CD) was purchased from Alfa Aesar (Heysham, UK). N-Vinyl pyrollidone (N-VP), amoxicillin trihydrate, and lysozyme hydrochloride were obtained from TCI Chemie (Tokyo, Japan). Diurethane dimethacrylate was procured from Sigma-Aldrich (Schnelldorf, Germany). Azobisisobutyronitrile (AIBN) was supplied by Loba Chemie Pvt. Ltd. (Mumbai, India). Acetone was acquired from E-Merck (I) Pvt. Ltd. (Mumbai, India). Guar gum was a gift sample from Hindustan Gum & Chemicals Ltd. (Haryana, India). For all experimental works, Milli Q water was used. Synthesis of Crosslinked Cl-b-CD/pVP Hydrogelator Free radical polymerization was employed for the synthesis of clb-CD/pVP hydrogelators in presence of AIBN initiator. In a three-necked round bottom (RB) flask, 8.8107 3 1024 mol of bCD was dissolved in 25 mL DMSO at room temperature. Then, the RB was placed in a silicon oil bath maintained at 60–70 8C. The reaction mixture within the RB was under continuous
stirring at a speed of 350 rpm. To create inert atmosphere, Ar gas was purged into RB. After 20 min, required amount of AIBN (Table I) dissolved in 2 mL DMSO was poured into it. Then (after 10 min), requisite amount of monomer (N-VP; Table I) was mixed with the prior solution in RB flask. After 30 min of monomer addition, 1.06 3 1023 mol of DUDMA was added to the reaction mixture and stirring was continued at the same temperature and speed (60–70 8C and 350 rpm) for 4 h. The termination of polymerization reaction was done with addition of 5 mL (0.1%, v/w) saturated hydroquinol solution. The resultant product of the reaction was cooled down to normal temperature, precipitated in acetone and kept overnight for complete precipitation. Finally, the product was centrifuged and the obtained gel (i.e., cl-b-CD/pVP hydrogel) was dried in a vacuum oven at 40 8C till constant weight. The probable synthesis scheme for the formation of cl-b-CD/pVP hydrogelator is depicted in Supporting Information Scheme S1. Characterization cl-b-CD/pVP hydrogelator was characterized by CHN analysis, FTIR, 1H NMR and 13C NMR spectroscopy, TGA, FESEM, and EDX analyses. Detailed procedure has been given in Supporting Information. Determination of Swelling/Deswelling/Reswelling Ratio and Swelling Kinetics of b-Cyclodextrin and Crosslinked b-CD/pVP Hydrogels Swelling characteristics of b-CD was explored at pH 1.2 and 7.4 media at 37 8C. The pH sensitivity of different cl-b-CD/pVP hydrogels were determined by calculating % equilibrium swelling ratio (% ESR) at 37 8C. Deswelling and reswelling characteristics were also studied to ensure the reversible nature of the crosslinked hydrogel. The detailed descriptions of swelling, deswelling, and reswelling studies are described in Supporting Information. The Voigt model [eq. (1)] was used to assess the rate of swelling, deswelling, and reswelling.31 Swelling, deswelling, and reswelling equilibrium ratio and corresponding s values have been reported in Supporting Information Table S2. 2t (1) St 5Se 12e =s where St and Se signify swelling of the sample at time t and equilibrium, respectively. “s” implies rate parameter of swelling.
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The more is the “s” value, the less would be the rate of swelling/deswelling.11,22,24 Rheological Characteristics The gel properties of synthesized cl-b-CD/pVP hydrogels were evaluated in swollen state at pH 7.4 buffer after attaining equilibrium swelling (7 h) at 37 8C. The rheological parameters were measured using an Advanced Rheometer (Bohlin Gemini2, Malvern, UK). To determine the yield stress32 and gel strength,32 amplitude sweep measurements were performed using continuous variation of shear stress from 1 to 5000 Pa. The frequency sweep experiment of hydrogel was accomplished in the range of 1–20 Hz with a constant stress of 1 Pa at 37 8C. The shear viscosity of hydrogels was assessed at different shear rates within 0.1–1000 s21 range at 37 8C. Further to investigate the pH-responsive behavior, the gel strength of the best hydrogel was determined at different buffer media (pH 1.2 and 7.4). Cell Viability Study and Morphological Assessments The pellet of hydrogelator powder with equal weight (pellets used for 1, 3, and 5 days analyses) was prepared using hydraulic press and detailed cell seeding procedure has been explained in Supporting Information. Cytotoxicity and cell proliferation assay were evaluated by ever known MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay method. The experimental procedure is given in Supporting Information. Live–Dead Assay Cell viability of the swollen cl-b-CD/pVP hydrogel sample and amoxicillin loaded cl-b-CD/pVP hydrogel based tablet were assessed using Live–Dead staining kit (Invitrogen, Pleasanton, CA 94566 USA) performed after 1, 3, and 5 days post culture. The cell–hydrogel construct was initially washed with PBS and then incubated for 30 min at normal temperature in a solution containing 2 mM calcein acetomethoxy (AM) and 4 mM ethidium homodimer. After 30 min of incubation at room temperature, the cell–hydrogel assembly was observed using a fluorescent microscope (Oberkochen, Carl Zeiss, Germany) with excitation filters of 450–490 nm (green, Calcein AM) and 510– 560 nm (red, ETD-1) using ZEN software. Biodegradation Test An enzymatic biodegradation of cl-b-CD/pVP3 hydrogelator film was performed using lysozyme from hen egg white.11,12,24,33–35 The detailed procedure has been elucidated in Supporting Information. Preparation of Tablets and In Vitro Amoxicillin Release Study Preparation of Tablets. b-Cyclodextrin and various cl-b-CD/ pVP hydrogelators (450 mg) were used to prepare amoxicillin (500 mg) tablets. The procedure for the fabrication of tablet is described in Supporting Information. In Vitro Amoxicillin Release Study. During amoxicillin release, partial disintegration of some tablets occurred. By using eq. (2),17 the amount of erosion (D) was obtained: D ðt Þð%Þ5
Wi 2 Wd ðt Þ 2Wd ð12Mt =M1 Þ 3100 Wi
(2)
where initial dry weight of the tablet is Wi and the dry weight at time t is denoted by Wd(t), Wd is the amount of amoxicillin
initially present in tablet, and Mt/M1 be the fraction of amoxicillin released at time t. The in vitro release of amoxicillin was observed for first two hours at pH 1.2 and afterward at pH 7.4 up to 24 h. The detailed experimental procedure has been given in Supporting Information. To determine the release mechanism, Korsemeyer–Peppas model27 was employed. RESULTS AND DISCUSSION
Synthesis of Cl-b-CD/pVP Hydrogelator Synthesis of chemically crosslinked b-cyclodextrin-based biocompatible hydrogel was employed with the help of free radical polymerization. A probable enlightenment toward the development of the hydrogel is centered on the assumption that in presence of inert (argon) atmosphere, AIBN generates 2-cyano prop-2-yl radical, which creates free radical active sites on the cyclic oligosaccharide b-cyclodextrin (R1, Supporting Information Scheme S1). These radicals react with N-VP, leading to the formation of embedded network (R2, Supporting Information Scheme S1). Owing to the poly-functionality of the crosslinker DUDMA, a macroradical was generated with four reactive sites and these sites were then connected with the R2 (Supporting Information Scheme S1) to develop a 3D crosslinked hydrogelator network. Various grades of cl-b-CD/pVP were synthesized to explore the consequences of reaction parameters. The best grade (i.e., cl-b-CD/pVP3) was optimized on the basis of higher crosslinking density, higher gel strength, and lower % ESR (Table I). Moreover for comparison, only poly(N-vinyl pyrollidone) was synthesized via free radical polymerization in presence of AIBN. Consequences of Reaction Parameters. For the optimization of hydrogelators, the initiator concentration was altered from 0.60 3 1024 to 1.53 3 1024 mol. It was found that cl-b-CD/pVP3 exhibits lower % ESR and higher crosslinking density (Table I). This may be explained as at relatively higher initiator concentration, free radical reactive sites would be generated on the b-CD backbone along with on the grafted network. This in turn would be crosslinked to produce hydrogelator with the structure having fewer but longer grafted/crosslinked chains, resulting higher crosslinking density and lower % ESR. However, with further increase in initiator concentration, more free radicals would be generated on oligosaccharide moiety. This will make the hydrogel structure with the presence of more but shorter grafted/crosslinked chains that will make the gel structure less compact, ensuing lower crosslinking density. On the contrary, the monomer concentration (N-VP) was changed from 4.72 3 1022 to 6.61 3 1022 mol. It was perceived that with increase in monomer concentration, crosslinking density was decreased, i.e., % ESR was increased. This is possibly because of the formation of homopolymers at higher monomer concentration.9 Characterization CHN Analysis. The CHN analysis of b-cyclodextrin shows that it contains 47.3% C and 10% H and does not contain any nitrogen. While, for cl-b-CD/pVP3 hydrogelator, % C, % H, and % N were found to be 52.8, 9.2, and 6.0, respectively. The presence of N in the hydrogelator suggests the existence of N containing monomer and crosslinker in the 3D polymeric network.
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FTIR Spectroscopy. FTIR spectrum of b-CD is shown in Supporting Information Figure S1a. It is obvious from Supporting Information Figure S1a that stretching vibrations of OAH and CAH bonds were found at 3304 and 2919 cm21, respectively. The bands at 1152 and 1016 cm21 were responsible for CAOAC stretching vibration. In case of N-VP (Supporting Information Fig. S1b), the band at 2892 cm21 was due to CAH stretching of ACH3, ACH2A groups. Besides, the bands at 1692, 1614, and 1261 cm21 were also observed in Supporting Information Figure S1b, mainly due to C@O stretching, C@C stretching, and CAN stretching, correspondingly. From the FTIR spectrum of DUDMA (Supporting Information Fig. S1c), the bands were found at 3358, 2955, 1706, 1522, and 1240 cm–1, which are corresponding to NAH stretching, CAH stretching vibrations of ACH3, ACH2A groups, C@O stretching, NAH bending, and CAN stretching vibrations, respectively. The FTIR spectrum of cl-b-CD/pVP3 hydrogelator has been represented in Supporting Information Figure S1d. The bands at 3478 and 1657 cm21 are responsible for NAH stretching and merged carbonyl C@O vibrations of ester and ketone groups, respectively. This suggests the successful incorporation of crosslinker and monomer units onto oligosaccharide backbone. Supporting Information Figure S1e demonstrates the FTIR spectrum of amoxicillin. In this spectrum, the bands at 3443, 3160, 2949, 1762, 1685, 1572, 1466, and 1247 cm21 are owing to NAH stretching, OAH stretching, CAH stretching, C@O stretching of ACOOH group, C@O stretching of amide, NAH bending, ACH2 scissoring, and CAN stretching vibrations, respectively. Finally, the FTIR spectrum of amoxicillin containing dried tablet is shown in Supporting Information Figure S1f. It is obvious that all the characteristics bands of amoxicillin (Supporting Information Fig. S1e) were also found in Supporting Information Figure S1f with slight shifting to lower wavenumber. This is probably due to the presence of H-bonding as well as physical interactions between hydrogelator and the drug (as proposed in Supporting Information Fig. S2), suggesting the proper fabrication of drug with the hydrogel in tablet formulation. 1
H Nuclear Magnetic Resonance Spectroscopy. In the 1H NMR spectrum of b-CD (Supporting Information Fig. S3), the peaks at d 5 5.7 ppm are due to anomeric protons (H1). Peaks between d 5 3.3 and 3.6 ppm are assigned to H2–H6 protons of b-cyclodextrin. The peaks at d 5 4.4, 4.8 ppm are responsible for AOH (2) and AOH (3, 6) protons, respectively. Supporting Information Figure S4 represents the 1H NMR spectrum of NVP. Here, the peaks between d 5 6.9 and 7.0 ppm are attributed to H3 of monomer, whereas the peaks at d 5 4.4 ppm are due to vinylic protons (H1 and H2). d 5 3.4, 2.4, 2.0 ppm are responsible for ring protons H4, H6, and H5, respectively. From the 1H NMR spectrum of DUDMA (Supporting Information Fig. S5), the peaks at d 5 5.7 and 6.0 ppm are due to H2 and H3 vinylic protons, respectively. Peaks between d 5 7.1 and 7.2 ppm are due to H6 proton, i.e., ANH proton of DUDMA. Peaks at d 5 4.2 and 3.4 ppm are due to methylene protons (H4 and H5) close to electronegative O atoms and H7, H12 methylene protons that are adjacent to electronegative N atoms, respectively. Peaks between d 5 2.8 and 3.0 ppm are due to H10 (as it is 1:1 mixture of H and ACH3). H1 methyl protons
demonstrate the peak at d 5 0.8 ppm. Whereas, H9 methyl protons provide the peak at d 5 1.9 ppm. Peaks between d 5 1.2 and 1.3 ppm are attributed to H8, H11 methyl protons. Figure 1(a) represents the 1H NMR spectrum of cl-b-CD/pVP3 hydrogelator. Peaks between d 5 3.3–3.6 and 5.7 ppm are because of the H1, oligosaccharides ring protons (H2–H5) and anomeric proton (H6), respectively. The peaks between d 5 3.2–3.3, 3.1, 2.5, and 2.0–2.1 ppm are owing to H8, H11, H7, H9, and H10 protons, respectively. The decrease of the peak intensity in the range of d 5 4.4–4.8 ppm indicates the reactivity of the hydroxyl protons of b-cyclodextrin moiety. The peaks at d 5 7.5, 2.2, 1.6, 1.3, and 0.9 ppm are due to H16 (ANH protons), H13, H18, H20 methylene protons, H19, and H12 protons. d 5 4.1 and 3.8 ppm are due to the H14, H15 methylene protons adjacent to O atoms and H17, H22 methylene protons nearby N atom, respectively. While, the presence of peaks of H8, H9, H10, and H11 protons indicate the existence of pVP in the crosslinked hydrogel. Besides, the peaks for H12, H14, H15, H16, H17, H18, H19, and H20 protons imply the presence of crosslinker (DUDMA) in the hydrogel network. The crosslinking density (CD) has been calculated using the intensities of characteristic peaks of b-cyclodextrin (IH6) and DUDMA crosslinker (IH12).36 It is supposed that one third of intensity (integration value) of three protons (H12) of DUDMA would be comparable to the intensity (integration value) of H6 proton. The crosslinking density was calculated using eq. (3): 0:33 3IH12 crosslinking density ðCDÞ5 (3) IH6 It has been observed that IH6 5 1.00 (assuming as base value for one proton of b-cyclodextrin) and IH12 5 2.53 (for three proton of DUDMA) [Fig. 1(a)]. Hence, the crosslinking density of clb-CD/pVP3 hydrogelator was found to be 0.84. The 1H NMR spectra of other hydrogels (i.e., cl-b-CD/pVP1, cl-b-CD/pVP2, cl-b-CD/pVP4, cl-b-CD/pVP5, and cl-b-CD/pVP6) developed in this study are shown in Supporting Information Figures S6– S10. Also the IH6 and IH12 values of all these hydrogelators are reported in Supporting Information Table S1. Crosslinking densities of various synthesized hydrogels have been determined using eq. (3) and the results are reported in Table I. It is apparent that cl-b-CD/pVP3 hydrogelator is characterized with highest crosslinking density and therefore is considered as optimized grade of hydrogelator of the series. 13
C Nuclear Magnetic Resonance Spectroscopy. Solid state 13C NMR spectrum of b-CD (Supporting Information Fig. S11) discloses four characteristic peaks. Peak at d 5 104.2 ppm is due to the presence of anomeric C atom (C-1), 82.4 ppm is assigned to C-5 atom, 74.6 ppm is for ring carbon atoms, i.e., for C-2 to C-4 and 61.4 ppm can be assigned to carbon atom of ACH2OH (C-6). Supporting Information Figure S12 represents the solution state 13C NMR spectrum (DMSO-d6) of N-VP. The peaks at d (ppm) 5 172.8, 128.9, 93.8, 43.7, 30.9, and 16.9 can be attributed to lactum AC@O group (C-6), vinylic sp2 carbon (i.e., C-2 and C-1 atoms), and ring C atoms C-3, C-5, and C-4, respectively. Crosslinker DUDMA has many characteristic peaks in solution state 13C NMR spectrum (DMSO-d6) (Supporting Information Fig. S13). Peaks at d (ppm) 5 166.8 and 156.5 are
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Figure 1. (a) 1H NMR spectrum of cl-b-CD/pVP3 hydrogelator in DMSO-d6 and (b) solid state 13C NMR spectrum of cl-b-CD/pVP3 hydrogelator. [Color figure can be viewed at wileyonlinelibrary.com]
responsible for AC@O (C-4 atom) of ester group and C-7 (AC@O group), respectively. Peaks at 136.2 and 126.3 ppm are attributed to the sp2 carbon atoms, i.e., C-2 and C-3. Peaks at 63.6 and 62.1 ppm are due to the methylene carbon (C-5 and C-6) attached to O atom. Other methylene groups like C-8, C9, C-12, and C-13 are characterized with the peaks at d 5 48.4, 46.2, 42.4, 39.9, and 37.4 ppm. Another peak at 27.5 ppm can be assigned to C-11. Peaks due to sp3 methyl (C-1 and C-10)
groups were found at 18.6 ppm. In case of cl-b-CD/pVP3 [Fig. 1(b), solid state NMR spectrum], there are some additional peaks along with the peaks (d 5 104.2, 83.3, 74.5, and 62.0 ppm) of b-cyclodextrin. Peak at 176.2 ppm is the characteristics peak of amide carbon (C-9) of pVP and ester carbon (C-13) of DUDMA. Peak at d 5 157.3 ppm is responsible for the AC@O group of DUDMA, where the AC@O group attached to O atom at one end and N atom at the other end, i.e., C-16. This
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Figure 2. FESEM images of (a) b-cyclodextrin and (b) cl-b-CD/pVP 3 hydrogelator.
indicates the presence of crosslinker in the hydrogelator moiety. Peaks at 43.3 and 42.0 ppm are assigned to the methylene carbon (i.e., C-12, C-14, and C-15) and peaks at 37.8 and 37.3 ppm are due to the methylene carbons C-17 and C-18 of crosslinker. Since the methylene carbons are attached to electronegative O and N atoms, therefore the characteristics peaks were observed at a higher d value than normal methylene carbons. The C-7, C-8, C-10, and C-11 peaks were found at d 5 32.7 ppm. A sharp peak was observed at 19.6 ppm, which is due to the methyl carbon (C-19 and C-20). Besides, due to the polymerization, the sp2 carbon atoms (C-1 and C-2) of N-VP and DUDMA were converted to sp3 carbon in the hydrogelator and hence the peaks responsible for vinylic sp2 carbon (d 5 128.9, 93.8 ppm for N-VP and 136.3, 126.3 ppm for DUDMA) were absent with the presence of additional peaks at 43.3 and 42.0 ppm (C-12, C-14, and C-15). This observation clearly authenticates the grafting and crosslinking of poly(vinyl pyrollidone) and poly (DUDMA) on b-CD moiety. TGA. Thermogravimetric analysis of b-CD displays two distinct zones of weight loss (Supporting Information Fig. S14a). The first decomposition zone is owing to the minute amount of moisture present in cyclic oligosaccharide. The second region of weight loss (280–370 8C) is attributed to the degradation of oligosaccharide backbone. Supporting Information Figure S14b shows the thermogram of cl-b-CD/pVP3 hydrogelator containing two new additional zones of weight loss at 220–270 8C and 390–500 8C range. These two zones are responsible for the decomposition of DUDMA and pVP fragment from the 3D hydrogelator network, respectively. FESEM Analysis. Figure 2 displays the FESEM images of b-CD and cl-b-CD/pVP3 hydrogelator. From Figure 2(a), it is apparent that b-CD has stone like smooth surface morphology, while on crosslinking the surface morphology became porous [Fig. 2(b)] with network like structure for cl-b-CD/pVP3 hydrogelator. Determination of Swelling, Deswelling, and Reswelling Ratio and Swelling Kinetics of b-Cyclodextrin and Crosslinked b-CD/pVP Hydrogels It was found that b-CD is soluble in aqueous media. While in case of cl-b-CD/pVP hydrogels, a steady increment in % swelling was observed (Supporting Information Figs. S15a and S15b) and after 7 h, the hydrogels reached to its equilibrium swelling. The synthesized hydrogels also showed a higher % equilibrium swelling than b-cyclodextrin. This perhaps due to
the increase in hydrophilicity upon modification of bcyclodextrin by means of grafting of pVP moiety onto the bcyclodextrin backbone and afterward chemically crosslinking by DUDMA. cl-b-CD/pVP3 experienced with lowest % ESR that may be due to the denser hydrogel network owing to its highest crosslinking density (Table I). The pH-responsive behavior of the synthesized hydrogels were studied at pH 1.2 and pH 7.4 (Supporting Information Fig. S15a,b). It was found that % ESR for all the hydrogels in acidic medium (pH 1.2) is lower than that of the alkaline medium, i.e., pH 7.4. This difference may be explained as in the acidic medium (pH 1.2), hydrophilic moieties of the synthesized hydrogel network were protonated, which hampered the H-bond formation with H2O molecules, causing lower % ESR. While in basic media (pH 7.4), the hydrophilic groups were exist in unprotonated state, which helps the formation of more H-bonds with H2O molecules, resulting higher % ESR.22 Deswelling and reswelling experiments of cl-b-CD/pVP3 were performed (at 37 8C) to explore the reversible nature of the hydrogel. The results (Figure 3) clearly support the reversible nature of the cl-b-CD/ pVP3 hydrogel. The water absorption rate of cl-b-CD/pVP3 hydrogel in different buffer solutions was analyzed using Voigt model.22,37 It has been observed that the rate parameter value (s) of cl-b-CD/pVP3 for swelling at pH 7.4 and 37 8C is slightly lower than that of reswelling at the same physiological condition (Supporting Information Table S2). The close value of s for reswelling with swelling further confirmed the reversible nature of the hydrogel. Rheological Characteristics Rheological characteristics of various cl-b-CD/pVP hydrogels were studied. In the viscomertic mode (Supporting Information Fig. S16), it is obvious that with increase in shear rate, the shear viscosity gradually decreases. This implies the non-Newtonian shear thinning behavior of various hydrogels.11 It has also been found that cl-b-CD/pVP3 has maximum shear viscosity, which can be attributed to its maximum crosslinking density. Supporting Information Figure S17 represents the result of dynamic frequency sweep experiment at 1–20 Hz. From the result, it is apparent that all the hydrogels have higher elastic modulus (G0 ) than viscous modulus (G00 ) and both the moduli were increased with increase in frequency, representing the elastic nature and gel state of the synthesized cl-b-CD/pVP hydrogels.11,21,31 From Supporting Information Figure S18 (i.e., amplitude sweep
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of this cell was assessed by evaluating the metabolic activity of cells grown in TCP and cl-b-CD/pVP3. The calculated absorbance values from MTT assay were converted to cell population rate using standard curve. It is obvious from Figure 4(i)a that cells grown on cl-b-CD/pVP3 hydrogel pellets were greater than TCP. After duration of 5 days, the no. of cells grown on cl-bCD/pVP3 hydrogel and TCP were 6.75 3 105 and 3.31 3 105, respectively. This may be due to the three-dimensional porous morphology of the hydrogel pellet that acts as a solid surface for cell growth. Therefore, the result ascertained that the prepared hydrogel is capable of cell adhesion and cytocompatible in nature and this soft material can be used as a biomedical drug carrier.
Figure 3. Swelling, deswelling, and reswelling plots of cl-b-CD/pVP3 hydrogel at pH 7.4 and 37 8C. [Color figure can be viewed at wileyonlinelibrary.com]
experiment at frequency 1 Hz), it has been found that here also G0 is greater than G00 . This further supports the gel nature of the cl-b-CD/pVP hydrogels. It has also been observed that with increase in shear stress, both G0 and G00 were decreased steadily and after a certain stress value (yield stress) both elastic modulus (G0 ) and loss modulus (G00 ) declined rapidly. The yield stress (r) and gel strength of various cl-b-CD/pVP hydrogels are given in Table I and it is apparent that the hydrogel with highest crosslinking density (i.e., cl-b-CD/pVP3) was characterized with maximum gel strength and yield stress. Again from Supporting Information Figure S19, it has been observed that both yield stress as well as gel strength is higher in case of cl-b-CD/ pVP3 swollen hydrogel at pH 1.2 than pH 7.4. This may be explained as hydrogel gets more swelled in alkaline buffer media than acidic buffer media, thus at pH 7.4 the gel network acquired relaxed structure causing a lower gel strength (2.48) and yield stress (2754 Pa). While at pH 1.2, cl-b-CD/pVP3 hydrogel swells less, which offers more compact structure causing a higher value of gel strength (2.66) and yield stress (4017 Pa). This endorsed the pH-responsive characteristics of the fabricated hydrogel. Cell Viability Study and Morphological Assessment Cytotoxicity and cell proliferation results anticipate the cellular compatibility of the prepared hydrogel for controlled drug delivery applications. The MG 63 cells were grown on TCP and cl-b-CD/pVP3 hydrogel pellet for 1, 3, and 5 days. The viability
The cellular attachment on hydrogel pellet and TCP as control were evaluated by rhodamine–phalloidin and DAPI staining at specific time intervals [Fig. 4(i)b]. On day 1, the cells with low population show no well-extended cytoplasm skeletons. But on third and fifth day, the cellular population were more in cl-b-CD/pVP3 hydrogel pellet in comparison to TCP and cells with well-structured cytoskeleton is obvious on both the cases. cl-b-CD/pVP3 hydrogel pellet has higher no. of cell population may be due to boosted metabolic rate as well as owing to the three-dimensional network structure of the hydrogel, which offers more superficial area for cell adhesion and cell proliferation.38 These images corroborates that the hydrogel cl-b-CD/pVP3 specimen was noncytotoxic to cells and can be implemented as biomedical controlled release vector. Live–Dead Assay Adhesion and viability of MG 63 cells on cl-b-CD/pVP3 hydrogel discs and amoxicillin loaded cl-b-CD/pVP3 hydrogel based tablet were evaluated by live–dead cell staining [Fig. 4(ii)]. After first day, third day, and fifth day cultured, it was observed that both cl-b-CD/pVP3 hydrogel and cl-b-CD/pVP3 hydrogel based tablet have well cell adhesion and proliferation. From Figure 4(ii), it has also been observed that there were very minute number of dead cells (Red)39 and dense live-cell sheet (green)40 on cl-b-CD/pVP3 hydrogel. However, in amoxicillin-loaded hydrogel, there are almost no dead cells present. Therefore, a large number of viable cells were evident and hence this hydrogel is a promising biocompatible material for drug delivery, and after drug loading the drug and the drug loaded hydrogel also has no adverse effect on cell. Biodegradation Test Supporting Information Figure S20 designates the biodegradation results of cl-b-CD/pVP hydrogelators after 3, 7, 14, 21, and 28 days. Lysozyme degrades the cyclic oligosaccharides backbone by means of the enzymatic hydrolysis of the glycosidic bonds of the hexameric sugar ring.34,35 The successive weight loss (Supporting Information Fig. S20) indicates the biodegradable nature of the cl-b-CD/pVP hydrogelator. Besides, from the FTIR spectrum (Supporting Information Fig. S21) of the degraded product (after 14 and 28 days), it has been found that the peak for CAOAC stretching is less intense for the degraded product after 14 days and is absent for the product after 28 days of degradation. This observation further established the
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Figure 4. (i) (a) Cell viability outcomes of TCP and cl-b-CD/pVP3 hydrogel obtained from MTT assay at 1, 3, and 5 days and (b) morphology of MG 63 cells determined by rhodamine–phalloidin and DAPI staining after 1, 3, and 5 days. (ii) Live–dead assay results of cl-b-CD/pVP 3 hydrogel and drug loaded cl-b-CD/pVP3 hydrogel based tablet at 1, 3, and 5 days. [Color figure can be viewed at wileyonlinelibrary.com]
biodegradability of the cl-b-CD/pVP hydrogel in presence of lysozyme chloride. Also, from the FESEM images of 14 and 28 days of degraded cl-b-CD/pVP hydrogelator, it is found that the porous network like morphology of the hydrogelator was deformed and degraded (Supporting Information Fig. S22), which further supports the degradable nature of the hydrogelator. In Vitro Amoxicillin Release Study In Vitro Amoxicillin Release Study. In vitro amoxicillin release rate is typically dependent on the swelling property of the hydrogel and on its chemical structure.41 There are three main steps of drug release, first step follows the dissolution of drug molecules present on the surface of the tablet to the releasing media. Second step involves the diffusion of water in the tablet matrices and release of drug molecule by diffusion and the final step is the release of drug by means of erosion of the hydrogel matrix.22 Hydrogel matrix with higher % ESR would have more surface area. Hence, the diffusion rate or the release rate of drug from the inside of that matrix would be faster.9 As the hydrogel has a higher swelling rate at pH 7.4 than in pH 1.2, so the hydrogels exhibit higher rate of amoxicillin release at pH 7.4 and in acidic medium (i.e., for first 2 h) the amount of amoxicillin release from the hydrogel matrices are insignificance (Fig. 5). From the in vitro drug release study of b-cyclodextrin and pVP copolymer (without crosslinker; Fig. 5), it is apparent that
pVP copolymer (without crosslinker) released the whole drug in 11 h and 100% drug was released from b-cyclodextrin in 10 h. While, the drug release from the different grades of the developed hydrogel matrices were more controlled and cl-b-CD/ pVP3 demonstrates most sustained drug release (Fig. 5) behavior. It released 56% amoxicillin in 24 h. This is owing to its higher crosslinking density, lower % ESR as well as lesser rate of erosion (Supporting Information Table S3). This represents a good compatibility between amoxicillin drug molecules and clb-CD/pVP hydrogelator network, as the less is the % erosion the more is the interaction between the hydrogel matrix and drug molecule. Mechanism of Drug Release. In case of tablet formulation with hydrogel matrix system, the drug is released in a controlled way following three key steps. First the hydrogel matrix gets hydrated, then relaxation of the polymer occurs and finally the transportation of dissolved drug takes place by diffusion or erosion of the matrix.22,42 Three types of diffusion mechanisms have been suggested for the release of drug from the polymeric material: Fickian diffusion, non-Fickian diffusion, and Super Case II diffusion.43,44 Korsemeyer–Peppas model27 has been used for the evaluation of the mechanism of amoxicillin release from cl-b-CD/pVP hydrogel based matrix. For this purpose amoxicillin release data were fitted in eq. (4).
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indicate the gelling nature of the hydrogel. The swelling, deswelling, and reswelling studies of the synthesized hydrogel convey the reversible nature of cl-b-CD/pVP. Cell study implies that the hydrogel is nontoxic toward MG-63 cells. Biodegradation test via hen egg lysozyme authenticates that cl-b-CD/pVP hydrogel is biodegradable in nature. Finally, the in vitro drug release study suggests that cl-b-CD/pVP released amoxicillin in a controlled way and may be a suitable alternative as amoxicillin/other antibiotics carrier in real clinical application. ACKNOWLEDGMENTS
Authors earnestly acknowledge CRF, IIT(ISM), Dhanbad, for various instrumental facilities.
REFERENCES
1. Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. Rev. 1999, 99, 3181. 2. Osuna, B.; Ferrero, C.; Jimenez-Castellanos, M. R. Eur. J. Pharm. Biopharm. 2008, 69, 285. 3. Saltzman, W. M.; Radomsky, M. L. Chem. Eng. Sci. 1991, 46, 2429. 4. Sylman, J. L.; Neeves, K. B. J. Chem. Educ. 2013, 90, 918. 5. Chen, L.; Tian, Z.; Du, Y. Biomaterials 2004, 25, 3725. Figure 5. Amoxicillin release profile from cl-b-CD/pVP hydrogels at 37 8C. [Color figure can be viewed at wileyonlinelibrary.com]
Mt 5Kt n M1
(4)
where Mt/M1 is the part of amoxicillin release in t time, “K” is a constant that designates characteristic of the drug–hydrogel matrix system, and “n” is diffusion exponent. This n value stands for different modes of release mechanisms. When n 0.45, it suggests Fickian diffusion. This is the case where the diffusion rate is lower than the relaxation rate and in this case diffusion process controls release rate. If the value of n is in between 0.45 and 0.89, then it suggests nonFickian diffusion mechanism, where the drug release rate depends on diffusion and erosion of the hydrogel matrix simultaneously. When value of n > 0.89, the main mode of drug release is Super Case II mechanism, where erosion of matrix is the major factor for the drug release.17 The “n” values for different hydrogel matrices, pVP polymer (without crosslinker), and b-CD are given in Supporting Information Table S3. The obtained n value was in between 0.45 and 0.89. Therefore, release of amoxicillin from the formulated tablets is based on non-Fickian diffusion mechanism. CONCLUSIONS
From above findings and discussions, it is evident that novel stimuli-responsive, biocompatible chemically crosslinked, and porous hydrogels were synthesized using b-cyclodextrin and poly(N-vinyl pyrollidone), where DUDMA was utilized as crosslinker. The formation of crosslinked network was confirmed by various characterization techniques. Rheological characteristics
6. Ninawe, P. R.; Parulekar, S. J. Ind. Eng. Chem. Res. 2012, 51, 1741. 7. Nita, L. E.; Nistor, M. T.; Chiriac, A. P.; Neamtu, I. Ind. Eng. Chem. Res. 2012, 51, 7769. 8. Jeong, J. H.; Schmidt, J. J.; Cha, C.; Kong, H. Soft Matter 2010, 6, 3930. 9. Das, D.; Das, R.; Ghosh, P.; Dhara, S.; Panda, A. B.; Pal, S. RSC Adv. 2013, 3, 25340. 10. Huynh, D. P.; Im, G. J.; Chae, S. Y.; Lee, K. C.; Lee, D. S. J. Control. Release 2009, 137, 20. 11. Patra, P.; Rameshbabu, A. P.; Das, D.; Dhara, S.; Panda, A. B.; Pal, S. Polym. Chem. 2016, 7, 5426. 12. Das, R.; Das, D.; Ghosh, P.; Dhara, S.; Panda, A. B.; Pal, S. RSC Adv. 2015, 5, 27481. 13. Azzam, F.; Moreau, C.; Cousin, F.; Menelle, A.; Bizot, H.; Cathala, B. Langmuir 2014, 30, 8091. 14. Kim, S. J.; Kim, H.; Park, S. J.; Kim, S. Sens. Actuators A: Phys. 2004, 115, 146. 15. Meenach, S. A.; Hilt, J. Z.; Anderson, K. W. Acta Biomater. 2010, 6, 1039. 16. Patil, N.; Roy, S. G.; Haldar, U.; De, P. J. Phys. Chem. B 2013, 117, 16292. 17. Wang, J.; Loh, K. P.; Wang, Z.; Yan, Y.; Zhong, Y.; Xu, Q. H.; Ho, P. C. Angew. Chem., Int. Ed. 2009, 48, 6282. 18. Taylor-Pashow, T. M. L.; Rocca, J. D.; Huxford, R. C.; Lin, W. Chem. Commun. 2010, 46, 5832. 19. Das, D.; Das, R.; Mandal, J.; Ghosh, A.; Pal, S. J. Appl. Polym. Sci. 2014, 131, 40039. 20. Das, D.; Pal, S. RSC Adv. 2015, 5, 25014.
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21. Lai, W. F.; Shum, H. C. ACS Appl. Mater. Interfaces 2015, 7, 10501.
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22. Das, D.; Ghosh, P.; Dhara, S.; Panda, A. B.; Pal, S. ACS Appl. Mater. Interfaces 2015, 7, 4791.
34. Das, D.; Patra, P.; Ghosh, P.; Rameshbabu, A. P.; Dhara, S.; Pal, S. Polym. Chem. 2016, 7, 2965.
23. Huang, X.; Nayak, B. R.; Lowe, T. L. J. Polym. Sci. Part A: Polym. Chem. 2004, 42, 5054.
35. Freier, T.; Koh, H. S.; Kazazian, K.; Shoichet, M. S. Biomaterials 2005, 26, 5872.
24. Das, D.; Ghosh, P.; Ghosh, A.; Haldar, C.; Dhara, S.; Panda, A. B.; Pal, S. ACS Appl. Mater. Interfaces 2015, 7, 14338.
36. Das, D.; Rameshbabu, A. P.; Ghosh, P.; Patra, P.; Dhara, S.; Pal, S. Carbohydr. Polym. 2017, 171, 27.
25. Vijan, V.; Kaity, S.; Biswas, S.; Isaac, J.; Ghosh, A. Carbohydr. Polym. 2012, 90, 496.
37. Rana, V.; Rai, P.; Tiwary, A. K.; Singh, R. S.; Kennedy, J. F.; Knill, C. J. Carbohydr. Polym. 2011, 83, 1031.
26. Singh, B.; Sharma, N. Carbohydr. Polym. 2008, 74, 489. 27. Korsmeyer, R. W.; Peppas, N. A. J. Memb. Sci. 1981, 9, 211.
38. Panja, S.; Maji, S.; Maiti, T. K.; Chattopadhyay, S. A. ACS Appl. Mater. Interfaces 2015, 7, 24229.
28. Zhang, M.; Xiong, Q.; Chen, J.; Wang, Y.; Zhang, Q. Polym. Chem. 2013, 4, 5086.
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29. Vishwakarma, N. K.; Patel, V. K.; Hira, S. K.; Ramesh, K.; Srivastava, P.; Mitra, K.; Singh, S.; Chattopadhyay, D.; Maiti, P.; Misra, N.; Manna, P. P.; Ray, B. RSC Adv. 2015, 5, 15547.
40. Lutzke, A.; Pegalajar-Jurado, A.; Neufeld, B. H.; Reynolds, M. M. J. Mater. Chem. B 2014, 2, 7449.
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41. Bardajee, G. R.; Pourjavadi, A.; Soleyman, R. Colloids Surf. A 2011, 392, 16. 42. Kiortsis, S.; Kachrimanis, K.; Broussali, T.; Malamataris, S. Eur. J. Pharm. Biopharm. 2005, 59, 73.
31. Omidian, H.; Hashemi, S. A.; Sammes, P. G.; Meldrum, I. Polymer 1998, 39, 6697.
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1
1
Polymer Chemistry Laboratory, Department of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad 826004, India Biomaterials and Tissue Engineering Laboratory, School of Medical Science and Technology, Indian Institute of Technology,
2
Kharagpur, Kharagpur 721302, India Correspondence to: S. Pal (E - mail: [email protected]); S. Dhara (E - mail: [email protected])
ABSTRACT: A novel pH-responsive, chemically crosslinked hydrogelator (cl-b-CD/pVP) has been fabricated using b-cyclodextrin (bCD) and N-vinyl pyrollidone (N-VP) in presence of diurethane dimethacrylate (DUDMA) crosslinker/azobisisobutyronitrile initiator through free radical polymerization. Various grades of cl-b-CD/pVP have been synthesized and the best grade has been considered with higher crosslinking density, higher gel strength, and lower % swelling ratio. The hydrogelator has been characterized by FTIR, 1 H and 13C NMR spectroscopy, TGA, and FESEM analyses. The hydrogelator demonstrates pH-responsive behavior, which has been confirmed by swelling behavior and gel characteristics at various pH (at 37 8C). Using hen egg lysozyme, degradation experiment has been performed, which confirms the biodegradable nature of the hydrogel. The in vitro cytotoxicity study and live–dead assay suggest that the hydrogelator is cytocompatible toward MG-63 cells. Finally, the hydrogelator shows excellent efficacy as an antibiotic (amoxiC 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 135, 45939. cillin) carrier. V
KEYWORDS: biomaterials; crosslinking; oligosaccharides
Received 28 August 2017; accepted 16 October 2017 DOI: 10.1002/app.45939
INTRODUCTION
In recent years, the development of controlled release drug delivery systems (CRDDS) has become prominent in biomedical applications. Drug release at an anticipated rate has great benefits over the conventional drug delivery and is more effective to maintain the drug concentration level in blood, to minimize the harmful side effects, enhance the efficiency time, improve patient compliance, as well as to increase the bioavailability.1 Several drug delivery systems have been developed in this purpose over last few years, among them the simplest one is the hydrogel based matrix device, where the drug is incorporated in polymeric network.2 The composition and structure of the polymeric system has an effective role to control the drug release. Porous polymeric hydrogels are rapidly employed for delayed and sustained drug release over the years mainly to control the rate of drug delivery by diffusion mechanism or simultaneously by diffusion mechanism with the erosion of matrix.3,4 Now a days, “stimuli-responsive gels” have become one of the most important device for drug delivery applications.5–7 The phase transition of these gels are highly influenced by some external stimulating factors like pH,8,9 temperature,10,11 ionic strength,12,13 and magnetic/electric filed.14,15 These types of gels
possess some unique striking properties like desired morphology, adjustable dimension, high porosity, and hence forth able to absorb high amount of water in its three-dimensional network structure. All these favorable properties make hydrogels extremely useful for biomedical applications.16–18 Hydrogels can be defined as physically or chemically crosslinked 3D polymeric networks that gets swelled by absorbing large amount of water in presence of water or other biological fluids, while it does not dissolve in the media.8,19 Polymeric hydrogels have versatile uses in biomedical field and drug delivery.20,21 Recently, biopolymer or modified biopolymerbased hydrogels are broadly used as drugs carrier to control the rate of drug release.17 Polysaccharides, oligosaccharides, or biopolymers-based hydrogels are more favorable for the use of drug delivery system over synthetic polymers owing to their biodegradability, easy to functionalization, and nontoxicity.22–24 However, natural biopolymers have some shortcomings such as uncontrolled hydration rate, microbial contamination, and decrease of viscosity on storage.25 These drawbacks can be evaded by functionalization through crosslinking on the backbone of polysaccharides/oligosaccharides that provides several enhanced characteristics including the development of controlled drug delivery device.26 Besides, the crosslinking density of a chemically
Additional Supporting Information may be found in the online version of this article. C 2017 Wiley Periodicals, Inc. V
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Table I. Synthesis Details, Crosslinking Density, Gel Strength, Yield Strength, and % ESR of Various cl-b-CD/pVP Hydrogelators Amount of b-cyclodextrin 5 8.8107 3 1024 mol Cross linker concentration 5 1.06 3 1023 mol
Hydrogelators
Initiator concentration (mol 3 1024)
Monomer concentration (mol 3 1022)
Crosslinking density (CD) (quantitative)
Gel strength
Yield stress
% ESR (pH 7.4)
cl-b-CD/pVP1
0.60
4.72
0.37
1.92
1799
231.6 6 11
cl-b-CD/pVP2
0.91
4.72
0.47
2.01
1959
226.2 6 11
cl-b-CD/pVP3
1.21
4.72
0.84
2.48
2754
174.0 6 8
cl-b-CD/pVP4
1.53
4.72
0.21
1.46
1585
291.6 6 14
cl-b-CD/pVP5
1.21
5.66
0.67
2.36
2323
192.4 6 9
cl-b-CD/pVP6
1.21
6.61
0.58
2.20
2133
221.4 6 11
crosslinked hydrogel has a crucial role on release rate—higher is the crosslinking density, higher would be the gel strength that would enhance the sustained release characteristics.27 Hydrogels based on polysaccharides/oligosaccharides have been developed and reported.19–22 One of the most widely used oligosaccharide is b-cyclodextrin, which received great attention in the field of drug delivery.28 Chemically modified bcyclodextrin-based drugs carrier have already been developed and reported.29,30 However, it is still imperative to develop new and novel b-cyclodextrin-based hydrogel with improved stimuli-responsive behavior, nontoxic, and biocompatible device that can deliver the drugs at more desired rate. In this concern, herein, for the first time we have explored the synthesis of a pH-sensitive novel hydrogel through crosslinking of poly(N-vinyl pyrollidone) on b-cyclodextrin using diurethane dimethacrylate (DUDMA) crosslinker via free radical polymerization. The hydrogel demonstrates excellent gel characteristics with superior controlled release behavior toward the release of antibiotic (amoxicillin) that opens up a new pathway for its use in real time applications EXPERIMENTAL
Materials b-Cyclodextrin (b-CD) was purchased from Alfa Aesar (Heysham, UK). N-Vinyl pyrollidone (N-VP), amoxicillin trihydrate, and lysozyme hydrochloride were obtained from TCI Chemie (Tokyo, Japan). Diurethane dimethacrylate was procured from Sigma-Aldrich (Schnelldorf, Germany). Azobisisobutyronitrile (AIBN) was supplied by Loba Chemie Pvt. Ltd. (Mumbai, India). Acetone was acquired from E-Merck (I) Pvt. Ltd. (Mumbai, India). Guar gum was a gift sample from Hindustan Gum & Chemicals Ltd. (Haryana, India). For all experimental works, Milli Q water was used. Synthesis of Crosslinked Cl-b-CD/pVP Hydrogelator Free radical polymerization was employed for the synthesis of clb-CD/pVP hydrogelators in presence of AIBN initiator. In a three-necked round bottom (RB) flask, 8.8107 3 1024 mol of bCD was dissolved in 25 mL DMSO at room temperature. Then, the RB was placed in a silicon oil bath maintained at 60–70 8C. The reaction mixture within the RB was under continuous
stirring at a speed of 350 rpm. To create inert atmosphere, Ar gas was purged into RB. After 20 min, required amount of AIBN (Table I) dissolved in 2 mL DMSO was poured into it. Then (after 10 min), requisite amount of monomer (N-VP; Table I) was mixed with the prior solution in RB flask. After 30 min of monomer addition, 1.06 3 1023 mol of DUDMA was added to the reaction mixture and stirring was continued at the same temperature and speed (60–70 8C and 350 rpm) for 4 h. The termination of polymerization reaction was done with addition of 5 mL (0.1%, v/w) saturated hydroquinol solution. The resultant product of the reaction was cooled down to normal temperature, precipitated in acetone and kept overnight for complete precipitation. Finally, the product was centrifuged and the obtained gel (i.e., cl-b-CD/pVP hydrogel) was dried in a vacuum oven at 40 8C till constant weight. The probable synthesis scheme for the formation of cl-b-CD/pVP hydrogelator is depicted in Supporting Information Scheme S1. Characterization cl-b-CD/pVP hydrogelator was characterized by CHN analysis, FTIR, 1H NMR and 13C NMR spectroscopy, TGA, FESEM, and EDX analyses. Detailed procedure has been given in Supporting Information. Determination of Swelling/Deswelling/Reswelling Ratio and Swelling Kinetics of b-Cyclodextrin and Crosslinked b-CD/pVP Hydrogels Swelling characteristics of b-CD was explored at pH 1.2 and 7.4 media at 37 8C. The pH sensitivity of different cl-b-CD/pVP hydrogels were determined by calculating % equilibrium swelling ratio (% ESR) at 37 8C. Deswelling and reswelling characteristics were also studied to ensure the reversible nature of the crosslinked hydrogel. The detailed descriptions of swelling, deswelling, and reswelling studies are described in Supporting Information. The Voigt model [eq. (1)] was used to assess the rate of swelling, deswelling, and reswelling.31 Swelling, deswelling, and reswelling equilibrium ratio and corresponding s values have been reported in Supporting Information Table S2. 2t (1) St 5Se 12e =s where St and Se signify swelling of the sample at time t and equilibrium, respectively. “s” implies rate parameter of swelling.
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The more is the “s” value, the less would be the rate of swelling/deswelling.11,22,24 Rheological Characteristics The gel properties of synthesized cl-b-CD/pVP hydrogels were evaluated in swollen state at pH 7.4 buffer after attaining equilibrium swelling (7 h) at 37 8C. The rheological parameters were measured using an Advanced Rheometer (Bohlin Gemini2, Malvern, UK). To determine the yield stress32 and gel strength,32 amplitude sweep measurements were performed using continuous variation of shear stress from 1 to 5000 Pa. The frequency sweep experiment of hydrogel was accomplished in the range of 1–20 Hz with a constant stress of 1 Pa at 37 8C. The shear viscosity of hydrogels was assessed at different shear rates within 0.1–1000 s21 range at 37 8C. Further to investigate the pH-responsive behavior, the gel strength of the best hydrogel was determined at different buffer media (pH 1.2 and 7.4). Cell Viability Study and Morphological Assessments The pellet of hydrogelator powder with equal weight (pellets used for 1, 3, and 5 days analyses) was prepared using hydraulic press and detailed cell seeding procedure has been explained in Supporting Information. Cytotoxicity and cell proliferation assay were evaluated by ever known MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay method. The experimental procedure is given in Supporting Information. Live–Dead Assay Cell viability of the swollen cl-b-CD/pVP hydrogel sample and amoxicillin loaded cl-b-CD/pVP hydrogel based tablet were assessed using Live–Dead staining kit (Invitrogen, Pleasanton, CA 94566 USA) performed after 1, 3, and 5 days post culture. The cell–hydrogel construct was initially washed with PBS and then incubated for 30 min at normal temperature in a solution containing 2 mM calcein acetomethoxy (AM) and 4 mM ethidium homodimer. After 30 min of incubation at room temperature, the cell–hydrogel assembly was observed using a fluorescent microscope (Oberkochen, Carl Zeiss, Germany) with excitation filters of 450–490 nm (green, Calcein AM) and 510– 560 nm (red, ETD-1) using ZEN software. Biodegradation Test An enzymatic biodegradation of cl-b-CD/pVP3 hydrogelator film was performed using lysozyme from hen egg white.11,12,24,33–35 The detailed procedure has been elucidated in Supporting Information. Preparation of Tablets and In Vitro Amoxicillin Release Study Preparation of Tablets. b-Cyclodextrin and various cl-b-CD/ pVP hydrogelators (450 mg) were used to prepare amoxicillin (500 mg) tablets. The procedure for the fabrication of tablet is described in Supporting Information. In Vitro Amoxicillin Release Study. During amoxicillin release, partial disintegration of some tablets occurred. By using eq. (2),17 the amount of erosion (D) was obtained: D ðt Þð%Þ5
Wi 2 Wd ðt Þ 2Wd ð12Mt =M1 Þ 3100 Wi
(2)
where initial dry weight of the tablet is Wi and the dry weight at time t is denoted by Wd(t), Wd is the amount of amoxicillin
initially present in tablet, and Mt/M1 be the fraction of amoxicillin released at time t. The in vitro release of amoxicillin was observed for first two hours at pH 1.2 and afterward at pH 7.4 up to 24 h. The detailed experimental procedure has been given in Supporting Information. To determine the release mechanism, Korsemeyer–Peppas model27 was employed. RESULTS AND DISCUSSION
Synthesis of Cl-b-CD/pVP Hydrogelator Synthesis of chemically crosslinked b-cyclodextrin-based biocompatible hydrogel was employed with the help of free radical polymerization. A probable enlightenment toward the development of the hydrogel is centered on the assumption that in presence of inert (argon) atmosphere, AIBN generates 2-cyano prop-2-yl radical, which creates free radical active sites on the cyclic oligosaccharide b-cyclodextrin (R1, Supporting Information Scheme S1). These radicals react with N-VP, leading to the formation of embedded network (R2, Supporting Information Scheme S1). Owing to the poly-functionality of the crosslinker DUDMA, a macroradical was generated with four reactive sites and these sites were then connected with the R2 (Supporting Information Scheme S1) to develop a 3D crosslinked hydrogelator network. Various grades of cl-b-CD/pVP were synthesized to explore the consequences of reaction parameters. The best grade (i.e., cl-b-CD/pVP3) was optimized on the basis of higher crosslinking density, higher gel strength, and lower % ESR (Table I). Moreover for comparison, only poly(N-vinyl pyrollidone) was synthesized via free radical polymerization in presence of AIBN. Consequences of Reaction Parameters. For the optimization of hydrogelators, the initiator concentration was altered from 0.60 3 1024 to 1.53 3 1024 mol. It was found that cl-b-CD/pVP3 exhibits lower % ESR and higher crosslinking density (Table I). This may be explained as at relatively higher initiator concentration, free radical reactive sites would be generated on the b-CD backbone along with on the grafted network. This in turn would be crosslinked to produce hydrogelator with the structure having fewer but longer grafted/crosslinked chains, resulting higher crosslinking density and lower % ESR. However, with further increase in initiator concentration, more free radicals would be generated on oligosaccharide moiety. This will make the hydrogel structure with the presence of more but shorter grafted/crosslinked chains that will make the gel structure less compact, ensuing lower crosslinking density. On the contrary, the monomer concentration (N-VP) was changed from 4.72 3 1022 to 6.61 3 1022 mol. It was perceived that with increase in monomer concentration, crosslinking density was decreased, i.e., % ESR was increased. This is possibly because of the formation of homopolymers at higher monomer concentration.9 Characterization CHN Analysis. The CHN analysis of b-cyclodextrin shows that it contains 47.3% C and 10% H and does not contain any nitrogen. While, for cl-b-CD/pVP3 hydrogelator, % C, % H, and % N were found to be 52.8, 9.2, and 6.0, respectively. The presence of N in the hydrogelator suggests the existence of N containing monomer and crosslinker in the 3D polymeric network.
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FTIR Spectroscopy. FTIR spectrum of b-CD is shown in Supporting Information Figure S1a. It is obvious from Supporting Information Figure S1a that stretching vibrations of OAH and CAH bonds were found at 3304 and 2919 cm21, respectively. The bands at 1152 and 1016 cm21 were responsible for CAOAC stretching vibration. In case of N-VP (Supporting Information Fig. S1b), the band at 2892 cm21 was due to CAH stretching of ACH3, ACH2A groups. Besides, the bands at 1692, 1614, and 1261 cm21 were also observed in Supporting Information Figure S1b, mainly due to C@O stretching, C@C stretching, and CAN stretching, correspondingly. From the FTIR spectrum of DUDMA (Supporting Information Fig. S1c), the bands were found at 3358, 2955, 1706, 1522, and 1240 cm–1, which are corresponding to NAH stretching, CAH stretching vibrations of ACH3, ACH2A groups, C@O stretching, NAH bending, and CAN stretching vibrations, respectively. The FTIR spectrum of cl-b-CD/pVP3 hydrogelator has been represented in Supporting Information Figure S1d. The bands at 3478 and 1657 cm21 are responsible for NAH stretching and merged carbonyl C@O vibrations of ester and ketone groups, respectively. This suggests the successful incorporation of crosslinker and monomer units onto oligosaccharide backbone. Supporting Information Figure S1e demonstrates the FTIR spectrum of amoxicillin. In this spectrum, the bands at 3443, 3160, 2949, 1762, 1685, 1572, 1466, and 1247 cm21 are owing to NAH stretching, OAH stretching, CAH stretching, C@O stretching of ACOOH group, C@O stretching of amide, NAH bending, ACH2 scissoring, and CAN stretching vibrations, respectively. Finally, the FTIR spectrum of amoxicillin containing dried tablet is shown in Supporting Information Figure S1f. It is obvious that all the characteristics bands of amoxicillin (Supporting Information Fig. S1e) were also found in Supporting Information Figure S1f with slight shifting to lower wavenumber. This is probably due to the presence of H-bonding as well as physical interactions between hydrogelator and the drug (as proposed in Supporting Information Fig. S2), suggesting the proper fabrication of drug with the hydrogel in tablet formulation. 1
H Nuclear Magnetic Resonance Spectroscopy. In the 1H NMR spectrum of b-CD (Supporting Information Fig. S3), the peaks at d 5 5.7 ppm are due to anomeric protons (H1). Peaks between d 5 3.3 and 3.6 ppm are assigned to H2–H6 protons of b-cyclodextrin. The peaks at d 5 4.4, 4.8 ppm are responsible for AOH (2) and AOH (3, 6) protons, respectively. Supporting Information Figure S4 represents the 1H NMR spectrum of NVP. Here, the peaks between d 5 6.9 and 7.0 ppm are attributed to H3 of monomer, whereas the peaks at d 5 4.4 ppm are due to vinylic protons (H1 and H2). d 5 3.4, 2.4, 2.0 ppm are responsible for ring protons H4, H6, and H5, respectively. From the 1H NMR spectrum of DUDMA (Supporting Information Fig. S5), the peaks at d 5 5.7 and 6.0 ppm are due to H2 and H3 vinylic protons, respectively. Peaks between d 5 7.1 and 7.2 ppm are due to H6 proton, i.e., ANH proton of DUDMA. Peaks at d 5 4.2 and 3.4 ppm are due to methylene protons (H4 and H5) close to electronegative O atoms and H7, H12 methylene protons that are adjacent to electronegative N atoms, respectively. Peaks between d 5 2.8 and 3.0 ppm are due to H10 (as it is 1:1 mixture of H and ACH3). H1 methyl protons
demonstrate the peak at d 5 0.8 ppm. Whereas, H9 methyl protons provide the peak at d 5 1.9 ppm. Peaks between d 5 1.2 and 1.3 ppm are attributed to H8, H11 methyl protons. Figure 1(a) represents the 1H NMR spectrum of cl-b-CD/pVP3 hydrogelator. Peaks between d 5 3.3–3.6 and 5.7 ppm are because of the H1, oligosaccharides ring protons (H2–H5) and anomeric proton (H6), respectively. The peaks between d 5 3.2–3.3, 3.1, 2.5, and 2.0–2.1 ppm are owing to H8, H11, H7, H9, and H10 protons, respectively. The decrease of the peak intensity in the range of d 5 4.4–4.8 ppm indicates the reactivity of the hydroxyl protons of b-cyclodextrin moiety. The peaks at d 5 7.5, 2.2, 1.6, 1.3, and 0.9 ppm are due to H16 (ANH protons), H13, H18, H20 methylene protons, H19, and H12 protons. d 5 4.1 and 3.8 ppm are due to the H14, H15 methylene protons adjacent to O atoms and H17, H22 methylene protons nearby N atom, respectively. While, the presence of peaks of H8, H9, H10, and H11 protons indicate the existence of pVP in the crosslinked hydrogel. Besides, the peaks for H12, H14, H15, H16, H17, H18, H19, and H20 protons imply the presence of crosslinker (DUDMA) in the hydrogel network. The crosslinking density (CD) has been calculated using the intensities of characteristic peaks of b-cyclodextrin (IH6) and DUDMA crosslinker (IH12).36 It is supposed that one third of intensity (integration value) of three protons (H12) of DUDMA would be comparable to the intensity (integration value) of H6 proton. The crosslinking density was calculated using eq. (3): 0:33 3IH12 crosslinking density ðCDÞ5 (3) IH6 It has been observed that IH6 5 1.00 (assuming as base value for one proton of b-cyclodextrin) and IH12 5 2.53 (for three proton of DUDMA) [Fig. 1(a)]. Hence, the crosslinking density of clb-CD/pVP3 hydrogelator was found to be 0.84. The 1H NMR spectra of other hydrogels (i.e., cl-b-CD/pVP1, cl-b-CD/pVP2, cl-b-CD/pVP4, cl-b-CD/pVP5, and cl-b-CD/pVP6) developed in this study are shown in Supporting Information Figures S6– S10. Also the IH6 and IH12 values of all these hydrogelators are reported in Supporting Information Table S1. Crosslinking densities of various synthesized hydrogels have been determined using eq. (3) and the results are reported in Table I. It is apparent that cl-b-CD/pVP3 hydrogelator is characterized with highest crosslinking density and therefore is considered as optimized grade of hydrogelator of the series. 13
C Nuclear Magnetic Resonance Spectroscopy. Solid state 13C NMR spectrum of b-CD (Supporting Information Fig. S11) discloses four characteristic peaks. Peak at d 5 104.2 ppm is due to the presence of anomeric C atom (C-1), 82.4 ppm is assigned to C-5 atom, 74.6 ppm is for ring carbon atoms, i.e., for C-2 to C-4 and 61.4 ppm can be assigned to carbon atom of ACH2OH (C-6). Supporting Information Figure S12 represents the solution state 13C NMR spectrum (DMSO-d6) of N-VP. The peaks at d (ppm) 5 172.8, 128.9, 93.8, 43.7, 30.9, and 16.9 can be attributed to lactum AC@O group (C-6), vinylic sp2 carbon (i.e., C-2 and C-1 atoms), and ring C atoms C-3, C-5, and C-4, respectively. Crosslinker DUDMA has many characteristic peaks in solution state 13C NMR spectrum (DMSO-d6) (Supporting Information Fig. S13). Peaks at d (ppm) 5 166.8 and 156.5 are
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Figure 1. (a) 1H NMR spectrum of cl-b-CD/pVP3 hydrogelator in DMSO-d6 and (b) solid state 13C NMR spectrum of cl-b-CD/pVP3 hydrogelator. [Color figure can be viewed at wileyonlinelibrary.com]
responsible for AC@O (C-4 atom) of ester group and C-7 (AC@O group), respectively. Peaks at 136.2 and 126.3 ppm are attributed to the sp2 carbon atoms, i.e., C-2 and C-3. Peaks at 63.6 and 62.1 ppm are due to the methylene carbon (C-5 and C-6) attached to O atom. Other methylene groups like C-8, C9, C-12, and C-13 are characterized with the peaks at d 5 48.4, 46.2, 42.4, 39.9, and 37.4 ppm. Another peak at 27.5 ppm can be assigned to C-11. Peaks due to sp3 methyl (C-1 and C-10)
groups were found at 18.6 ppm. In case of cl-b-CD/pVP3 [Fig. 1(b), solid state NMR spectrum], there are some additional peaks along with the peaks (d 5 104.2, 83.3, 74.5, and 62.0 ppm) of b-cyclodextrin. Peak at 176.2 ppm is the characteristics peak of amide carbon (C-9) of pVP and ester carbon (C-13) of DUDMA. Peak at d 5 157.3 ppm is responsible for the AC@O group of DUDMA, where the AC@O group attached to O atom at one end and N atom at the other end, i.e., C-16. This
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Figure 2. FESEM images of (a) b-cyclodextrin and (b) cl-b-CD/pVP 3 hydrogelator.
indicates the presence of crosslinker in the hydrogelator moiety. Peaks at 43.3 and 42.0 ppm are assigned to the methylene carbon (i.e., C-12, C-14, and C-15) and peaks at 37.8 and 37.3 ppm are due to the methylene carbons C-17 and C-18 of crosslinker. Since the methylene carbons are attached to electronegative O and N atoms, therefore the characteristics peaks were observed at a higher d value than normal methylene carbons. The C-7, C-8, C-10, and C-11 peaks were found at d 5 32.7 ppm. A sharp peak was observed at 19.6 ppm, which is due to the methyl carbon (C-19 and C-20). Besides, due to the polymerization, the sp2 carbon atoms (C-1 and C-2) of N-VP and DUDMA were converted to sp3 carbon in the hydrogelator and hence the peaks responsible for vinylic sp2 carbon (d 5 128.9, 93.8 ppm for N-VP and 136.3, 126.3 ppm for DUDMA) were absent with the presence of additional peaks at 43.3 and 42.0 ppm (C-12, C-14, and C-15). This observation clearly authenticates the grafting and crosslinking of poly(vinyl pyrollidone) and poly (DUDMA) on b-CD moiety. TGA. Thermogravimetric analysis of b-CD displays two distinct zones of weight loss (Supporting Information Fig. S14a). The first decomposition zone is owing to the minute amount of moisture present in cyclic oligosaccharide. The second region of weight loss (280–370 8C) is attributed to the degradation of oligosaccharide backbone. Supporting Information Figure S14b shows the thermogram of cl-b-CD/pVP3 hydrogelator containing two new additional zones of weight loss at 220–270 8C and 390–500 8C range. These two zones are responsible for the decomposition of DUDMA and pVP fragment from the 3D hydrogelator network, respectively. FESEM Analysis. Figure 2 displays the FESEM images of b-CD and cl-b-CD/pVP3 hydrogelator. From Figure 2(a), it is apparent that b-CD has stone like smooth surface morphology, while on crosslinking the surface morphology became porous [Fig. 2(b)] with network like structure for cl-b-CD/pVP3 hydrogelator. Determination of Swelling, Deswelling, and Reswelling Ratio and Swelling Kinetics of b-Cyclodextrin and Crosslinked b-CD/pVP Hydrogels It was found that b-CD is soluble in aqueous media. While in case of cl-b-CD/pVP hydrogels, a steady increment in % swelling was observed (Supporting Information Figs. S15a and S15b) and after 7 h, the hydrogels reached to its equilibrium swelling. The synthesized hydrogels also showed a higher % equilibrium swelling than b-cyclodextrin. This perhaps due to
the increase in hydrophilicity upon modification of bcyclodextrin by means of grafting of pVP moiety onto the bcyclodextrin backbone and afterward chemically crosslinking by DUDMA. cl-b-CD/pVP3 experienced with lowest % ESR that may be due to the denser hydrogel network owing to its highest crosslinking density (Table I). The pH-responsive behavior of the synthesized hydrogels were studied at pH 1.2 and pH 7.4 (Supporting Information Fig. S15a,b). It was found that % ESR for all the hydrogels in acidic medium (pH 1.2) is lower than that of the alkaline medium, i.e., pH 7.4. This difference may be explained as in the acidic medium (pH 1.2), hydrophilic moieties of the synthesized hydrogel network were protonated, which hampered the H-bond formation with H2O molecules, causing lower % ESR. While in basic media (pH 7.4), the hydrophilic groups were exist in unprotonated state, which helps the formation of more H-bonds with H2O molecules, resulting higher % ESR.22 Deswelling and reswelling experiments of cl-b-CD/pVP3 were performed (at 37 8C) to explore the reversible nature of the hydrogel. The results (Figure 3) clearly support the reversible nature of the cl-b-CD/ pVP3 hydrogel. The water absorption rate of cl-b-CD/pVP3 hydrogel in different buffer solutions was analyzed using Voigt model.22,37 It has been observed that the rate parameter value (s) of cl-b-CD/pVP3 for swelling at pH 7.4 and 37 8C is slightly lower than that of reswelling at the same physiological condition (Supporting Information Table S2). The close value of s for reswelling with swelling further confirmed the reversible nature of the hydrogel. Rheological Characteristics Rheological characteristics of various cl-b-CD/pVP hydrogels were studied. In the viscomertic mode (Supporting Information Fig. S16), it is obvious that with increase in shear rate, the shear viscosity gradually decreases. This implies the non-Newtonian shear thinning behavior of various hydrogels.11 It has also been found that cl-b-CD/pVP3 has maximum shear viscosity, which can be attributed to its maximum crosslinking density. Supporting Information Figure S17 represents the result of dynamic frequency sweep experiment at 1–20 Hz. From the result, it is apparent that all the hydrogels have higher elastic modulus (G0 ) than viscous modulus (G00 ) and both the moduli were increased with increase in frequency, representing the elastic nature and gel state of the synthesized cl-b-CD/pVP hydrogels.11,21,31 From Supporting Information Figure S18 (i.e., amplitude sweep
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of this cell was assessed by evaluating the metabolic activity of cells grown in TCP and cl-b-CD/pVP3. The calculated absorbance values from MTT assay were converted to cell population rate using standard curve. It is obvious from Figure 4(i)a that cells grown on cl-b-CD/pVP3 hydrogel pellets were greater than TCP. After duration of 5 days, the no. of cells grown on cl-bCD/pVP3 hydrogel and TCP were 6.75 3 105 and 3.31 3 105, respectively. This may be due to the three-dimensional porous morphology of the hydrogel pellet that acts as a solid surface for cell growth. Therefore, the result ascertained that the prepared hydrogel is capable of cell adhesion and cytocompatible in nature and this soft material can be used as a biomedical drug carrier.
Figure 3. Swelling, deswelling, and reswelling plots of cl-b-CD/pVP3 hydrogel at pH 7.4 and 37 8C. [Color figure can be viewed at wileyonlinelibrary.com]
experiment at frequency 1 Hz), it has been found that here also G0 is greater than G00 . This further supports the gel nature of the cl-b-CD/pVP hydrogels. It has also been observed that with increase in shear stress, both G0 and G00 were decreased steadily and after a certain stress value (yield stress) both elastic modulus (G0 ) and loss modulus (G00 ) declined rapidly. The yield stress (r) and gel strength of various cl-b-CD/pVP hydrogels are given in Table I and it is apparent that the hydrogel with highest crosslinking density (i.e., cl-b-CD/pVP3) was characterized with maximum gel strength and yield stress. Again from Supporting Information Figure S19, it has been observed that both yield stress as well as gel strength is higher in case of cl-b-CD/ pVP3 swollen hydrogel at pH 1.2 than pH 7.4. This may be explained as hydrogel gets more swelled in alkaline buffer media than acidic buffer media, thus at pH 7.4 the gel network acquired relaxed structure causing a lower gel strength (2.48) and yield stress (2754 Pa). While at pH 1.2, cl-b-CD/pVP3 hydrogel swells less, which offers more compact structure causing a higher value of gel strength (2.66) and yield stress (4017 Pa). This endorsed the pH-responsive characteristics of the fabricated hydrogel. Cell Viability Study and Morphological Assessment Cytotoxicity and cell proliferation results anticipate the cellular compatibility of the prepared hydrogel for controlled drug delivery applications. The MG 63 cells were grown on TCP and cl-b-CD/pVP3 hydrogel pellet for 1, 3, and 5 days. The viability
The cellular attachment on hydrogel pellet and TCP as control were evaluated by rhodamine–phalloidin and DAPI staining at specific time intervals [Fig. 4(i)b]. On day 1, the cells with low population show no well-extended cytoplasm skeletons. But on third and fifth day, the cellular population were more in cl-b-CD/pVP3 hydrogel pellet in comparison to TCP and cells with well-structured cytoskeleton is obvious on both the cases. cl-b-CD/pVP3 hydrogel pellet has higher no. of cell population may be due to boosted metabolic rate as well as owing to the three-dimensional network structure of the hydrogel, which offers more superficial area for cell adhesion and cell proliferation.38 These images corroborates that the hydrogel cl-b-CD/pVP3 specimen was noncytotoxic to cells and can be implemented as biomedical controlled release vector. Live–Dead Assay Adhesion and viability of MG 63 cells on cl-b-CD/pVP3 hydrogel discs and amoxicillin loaded cl-b-CD/pVP3 hydrogel based tablet were evaluated by live–dead cell staining [Fig. 4(ii)]. After first day, third day, and fifth day cultured, it was observed that both cl-b-CD/pVP3 hydrogel and cl-b-CD/pVP3 hydrogel based tablet have well cell adhesion and proliferation. From Figure 4(ii), it has also been observed that there were very minute number of dead cells (Red)39 and dense live-cell sheet (green)40 on cl-b-CD/pVP3 hydrogel. However, in amoxicillin-loaded hydrogel, there are almost no dead cells present. Therefore, a large number of viable cells were evident and hence this hydrogel is a promising biocompatible material for drug delivery, and after drug loading the drug and the drug loaded hydrogel also has no adverse effect on cell. Biodegradation Test Supporting Information Figure S20 designates the biodegradation results of cl-b-CD/pVP hydrogelators after 3, 7, 14, 21, and 28 days. Lysozyme degrades the cyclic oligosaccharides backbone by means of the enzymatic hydrolysis of the glycosidic bonds of the hexameric sugar ring.34,35 The successive weight loss (Supporting Information Fig. S20) indicates the biodegradable nature of the cl-b-CD/pVP hydrogelator. Besides, from the FTIR spectrum (Supporting Information Fig. S21) of the degraded product (after 14 and 28 days), it has been found that the peak for CAOAC stretching is less intense for the degraded product after 14 days and is absent for the product after 28 days of degradation. This observation further established the
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Figure 4. (i) (a) Cell viability outcomes of TCP and cl-b-CD/pVP3 hydrogel obtained from MTT assay at 1, 3, and 5 days and (b) morphology of MG 63 cells determined by rhodamine–phalloidin and DAPI staining after 1, 3, and 5 days. (ii) Live–dead assay results of cl-b-CD/pVP 3 hydrogel and drug loaded cl-b-CD/pVP3 hydrogel based tablet at 1, 3, and 5 days. [Color figure can be viewed at wileyonlinelibrary.com]
biodegradability of the cl-b-CD/pVP hydrogel in presence of lysozyme chloride. Also, from the FESEM images of 14 and 28 days of degraded cl-b-CD/pVP hydrogelator, it is found that the porous network like morphology of the hydrogelator was deformed and degraded (Supporting Information Fig. S22), which further supports the degradable nature of the hydrogelator. In Vitro Amoxicillin Release Study In Vitro Amoxicillin Release Study. In vitro amoxicillin release rate is typically dependent on the swelling property of the hydrogel and on its chemical structure.41 There are three main steps of drug release, first step follows the dissolution of drug molecules present on the surface of the tablet to the releasing media. Second step involves the diffusion of water in the tablet matrices and release of drug molecule by diffusion and the final step is the release of drug by means of erosion of the hydrogel matrix.22 Hydrogel matrix with higher % ESR would have more surface area. Hence, the diffusion rate or the release rate of drug from the inside of that matrix would be faster.9 As the hydrogel has a higher swelling rate at pH 7.4 than in pH 1.2, so the hydrogels exhibit higher rate of amoxicillin release at pH 7.4 and in acidic medium (i.e., for first 2 h) the amount of amoxicillin release from the hydrogel matrices are insignificance (Fig. 5). From the in vitro drug release study of b-cyclodextrin and pVP copolymer (without crosslinker; Fig. 5), it is apparent that
pVP copolymer (without crosslinker) released the whole drug in 11 h and 100% drug was released from b-cyclodextrin in 10 h. While, the drug release from the different grades of the developed hydrogel matrices were more controlled and cl-b-CD/ pVP3 demonstrates most sustained drug release (Fig. 5) behavior. It released 56% amoxicillin in 24 h. This is owing to its higher crosslinking density, lower % ESR as well as lesser rate of erosion (Supporting Information Table S3). This represents a good compatibility between amoxicillin drug molecules and clb-CD/pVP hydrogelator network, as the less is the % erosion the more is the interaction between the hydrogel matrix and drug molecule. Mechanism of Drug Release. In case of tablet formulation with hydrogel matrix system, the drug is released in a controlled way following three key steps. First the hydrogel matrix gets hydrated, then relaxation of the polymer occurs and finally the transportation of dissolved drug takes place by diffusion or erosion of the matrix.22,42 Three types of diffusion mechanisms have been suggested for the release of drug from the polymeric material: Fickian diffusion, non-Fickian diffusion, and Super Case II diffusion.43,44 Korsemeyer–Peppas model27 has been used for the evaluation of the mechanism of amoxicillin release from cl-b-CD/pVP hydrogel based matrix. For this purpose amoxicillin release data were fitted in eq. (4).
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indicate the gelling nature of the hydrogel. The swelling, deswelling, and reswelling studies of the synthesized hydrogel convey the reversible nature of cl-b-CD/pVP. Cell study implies that the hydrogel is nontoxic toward MG-63 cells. Biodegradation test via hen egg lysozyme authenticates that cl-b-CD/pVP hydrogel is biodegradable in nature. Finally, the in vitro drug release study suggests that cl-b-CD/pVP released amoxicillin in a controlled way and may be a suitable alternative as amoxicillin/other antibiotics carrier in real clinical application. ACKNOWLEDGMENTS
Authors earnestly acknowledge CRF, IIT(ISM), Dhanbad, for various instrumental facilities.
REFERENCES
1. Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. Rev. 1999, 99, 3181. 2. Osuna, B.; Ferrero, C.; Jimenez-Castellanos, M. R. Eur. J. Pharm. Biopharm. 2008, 69, 285. 3. Saltzman, W. M.; Radomsky, M. L. Chem. Eng. Sci. 1991, 46, 2429. 4. Sylman, J. L.; Neeves, K. B. J. Chem. Educ. 2013, 90, 918. 5. Chen, L.; Tian, Z.; Du, Y. Biomaterials 2004, 25, 3725. Figure 5. Amoxicillin release profile from cl-b-CD/pVP hydrogels at 37 8C. [Color figure can be viewed at wileyonlinelibrary.com]
Mt 5Kt n M1
(4)
where Mt/M1 is the part of amoxicillin release in t time, “K” is a constant that designates characteristic of the drug–hydrogel matrix system, and “n” is diffusion exponent. This n value stands for different modes of release mechanisms. When n 0.45, it suggests Fickian diffusion. This is the case where the diffusion rate is lower than the relaxation rate and in this case diffusion process controls release rate. If the value of n is in between 0.45 and 0.89, then it suggests nonFickian diffusion mechanism, where the drug release rate depends on diffusion and erosion of the hydrogel matrix simultaneously. When value of n > 0.89, the main mode of drug release is Super Case II mechanism, where erosion of matrix is the major factor for the drug release.17 The “n” values for different hydrogel matrices, pVP polymer (without crosslinker), and b-CD are given in Supporting Information Table S3. The obtained n value was in between 0.45 and 0.89. Therefore, release of amoxicillin from the formulated tablets is based on non-Fickian diffusion mechanism. CONCLUSIONS
From above findings and discussions, it is evident that novel stimuli-responsive, biocompatible chemically crosslinked, and porous hydrogels were synthesized using b-cyclodextrin and poly(N-vinyl pyrollidone), where DUDMA was utilized as crosslinker. The formation of crosslinked network was confirmed by various characterization techniques. Rheological characteristics
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J. APPL. POLYM. SCI. 2017, DOI: 10.1002/APP.45939