β-Cyclodextrin based pH and thermo-responsive biopolymeric hydrogel as a dual drug carrier

Arpita Roya, Priti Prasanna Maityb, Anirbandeep Bosec, Santanu Dhara*b and Sagar Pal*a
aPolymer Chemistry Laboratory, Department of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad-826004, India. E-mail: sagarpal@iitism.ac.in
bBiomaterials and Tissue Engineering Laboratory, School of Medical Science & Technology, Indian Institute of Technology, Kharagpur-721302, India
cTAAB Biostudy Services, Jadavpur, Kolkata-700032, India

Received 5th September 2018 , Accepted 4th December 2018

First published on 5th December 2018


Herein, a novel biocompatible and stimuli-responsive network gel has been developed by grafting and crosslinking poly(N-isopropyl acrylamide) and poly(methacrylic acid) on cyclic oligosaccharide β-cyclodextrin [β-CD-cl-(PNIPAm-co-PMAc)]. Various characterizations (such as NMR spectroscopy, CHN analysis, LCST measurement, and FESEM analysis) have been performed to confirm the formation of copolymer. Swelling characteristics reveal that the developed hydrogel demonstrates dual responsive behaviour (pH and thermo-responsive). The copolymer shows viscoelastic properties and is characterized with excellent yield stress as well as compressive stress. The network hydrogel displays a non-cytotoxic nature towards MG-63 cell lines. The simultaneous in vitro release of metronidazole and ofloxacin from the gel endorses that it released both the colonic drugs simultaneously at the preferred pH (colonic pH 7.4) and body temperature (37 °C) at a desired rate. Finally, the β-CD-cl-(PNIPAm-co-PMAc) gel is probable to be promising for targeted and sustained release of metronidazole and ofloxacin as is obvious from in vivo analysis.


1. Introduction

Stimuli responsive hydrogels have received noteworthy attention as therapeutics, such as in drug delivery applications, skin care, bone regeneration and so on.1–3 Sustained release of antibiotics is essential, and may be achieved using stimuli responsive polymeric gels.4 Sustained release of entrapped drugs is highly influenced by the structure and composition of the network polymer, which is mainly due to the attachment of drugs with gel networks by means of physical interactions.5,6 Hydrogels, chemically or physically crosslinked 3D polymeric networks, are swelled by absorbing large amounts of water or biological fluid,7–9 while they do not dissolve in the media. Smart composition of hydrogels may be stimulated through phase transition by external environmental stimuli like pH,10–14 temperature,15,16 light,17 ionic strength,18,19 and magnetic20 or electric field.21 Temperature responsive polymers like poly(N-isopropylacrylamide) (PNIPAm) based hydrogels are capable of swelling more below LCST and shrinking above LCST. PNIPAm has been studied widely as a temperature sensitive hydrogel for biomedical applications.22 In the present context, dual-responsive hydrogels are more useful in biomedical and pharmaceutical applications as it is believed that temperature and pH are the most affected environmental stimuli for typical biological and physiological systems.23 Hence for real applications, hydrogels that are able to respond to more than one stimulus simultaneously are more desirable.

Polymeric hydrogels containing carboxyl or amino groups are pH sensitive, since they respond to pH changes in biological systems.23 In principle, pH-responsive hydrogels control the rate of drug release depending on the swelling rate at different pH values.24 For example, poly(acrylic acid),25 poly(methacrylic acid)26 and poly(itaconic acid)27 based hydrogels have been introduced as pH-responsive gels to carry drugs and deliver them at a preferred rate. For practical application, combinations of temperature and pH sensitive hydrogel systems have been studied widely, in which the gel properties can be tuned by varying the external stimuli. Poly(N-isopropyl acrylamide-co-poly methacrylic acid) hydrogel demonstrated different swelling behaviour with respect to temperature and pH.28 The dual stimuli responsive hydrogels are capable of exhibiting different swelling behaviours depending on the variation of pH and temperature. This is mainly because of the presence of two different types of moiety in the three dimensional hydrogel network, which respond to the external stimuli. By virtue of the dual responsive nature, it is possible to release the loaded drug at a predetermined rate at a targeted area.

Network hydrogels derived from biopolymers and modified biopolymers have been investigated widely,4,29,30 owing to their biodegradability, biocompatibility and economical viability. Crosslinked biopolymeric gels are usually fabricated to overcome certain limitations of biopolymers and modified biopolymers like unrestrained hydration rates, bacterial contamination, reduction of viscosity on storage and so on. Chemical crosslinking helps to enhance the gel strength and thereby release the enclosed drugs at an anticipated rate.31

β-Cyclodextrin (β-CD) is one of the most common oligosaccharides which is used for pharmaceutical applications.32,33 β-CD based pH sensitive hydrogels have already been developed and reported.34 Still there is ample scope to improve the functional characteristics of β-CD, by preparing a stimuli-responsive crosslinked gel as a device for sustained and targeted delivery of model drugs. Henceforth, here our main attention is on developing a novel chemically crosslinked, dual stimuli responsive biocompatible hydrogel using β-CD for its potential application in the targeted delivery of colonic drugs (metronidazole and ofloxacin). The synthesized hydrogel, which contains PNIPAm and PMAc units in its crosslinked 3D network, demonstrates excellent stimuli responsive behaviour in terms of both pH and temperature. The crosslinked gel is found to be significant for targeted and sustained release of dual colonic drugs, owing to its excellent gel strength, which arises due to the covalent attachment of ethylene glycol diacrylate (EGDA, crosslinker) with the modified β-CD. Because of the formation of a stronger gel as well as its better compressive stress, it can avoid the dissolution problem during drug delivery. Further, because of its biocompatible nature, β-CD-cl-(PNIPAm-co-PMAc) hydrogel may be useful for real life applications. The crosslinked β-CD-cl-(PNIPAm-co-PMAc) is adequately stable, stimuli responsive and has a porous network, which enables its efficacy to transport two colonic drugs (metronidazole and ofloxacin) simultaneously with a controlled rate. Metronidazole is an antibiotic, generally used for the treatment of intestinal amoebiasis.35 Ofloxacin is also an antibiotic, and is active for both Gram positive and Gram negative bacteria.36 The mode of action of ofloxacin occurs mainly by blocking the bacterial cells from dividing or repairing. Hence, it kills the bacteria. Metronidazole destroys the bacteria by destroying its DNA. So, a combination of both of the drugs can be useful for the treatment of infections like diarrhoea or dysentery more effectively.

2. Experimental section

2.1. Chemicals

β-CD was procured from Alfa Aesar, England. NIPAm, MAc and ofloxacin were purchased from TCI Chemie, Japan. EGDA and metronidazole were supplied by Sigma-Aldrich, USA. Azobisisobutyronitrile (AIBN) was obtained from Loba Chemie Pvt. Ltd, Mumbai, India. Methanol and acetone were supplied by E-Merck (I) Pvt. Ltd, Mumbai, India. All experimental work was carried out using double distilled water.

2.2. Synthesis of crosslinked hydrogels

2.2.1. Synthesis of β-CD-cl-PNIPAm. NIPAm was crosslinked on a β-CD backbone by a free radical pathway consuming AIBN as initiator. The experimental procedure has been described in the ESI.
2.2.2. Synthesis of β-CD-cl-PMAc. β-CD-cl-PMAc xerogel was synthesised using free radical polymerization in the presence of AIBN initiator and EGDA crosslinker. The detailed methodology has been given in the ESI.
2.2.3. Synthesis of β-CD-cl-(PNIPAm-co-PMAc) hydrogel. β-CD-cl-(PNIPAm-co-PMAc) hydrogel was prepared via radical polymerization through grafting and crosslinking NIPAm and MAc simultaneously on the oligosaccharide. At first, 8.8107 × 10−4 moles of β-CD was dissolved in 30 mL DMSO at ambient temperature. After that, the temperature was increased and maintained at 60–65 °C using a silicone oil bath. The reaction was continued under an inert atmosphere (N2) with constant stirring of 400 rpm. After 10 min, 0.91 × 10−4 moles of AIBN solution (dissolved in 5 mL DMSO) was added to the prior solution. After 10 min, 3.53 × 10−2 moles of NIPAm was mixed with the reaction mixture. The reaction was allowed to proceed for 1 h. After 1 h, 4.73 × 10−2 moles of MAc monomer was added and the reaction was continued for half an hour, after which 6.42 × 10−4 moles of crosslinker (EGDA) was added and the polymerization as well as crosslinking reaction was continued for another 4 h. Finally, the reaction was terminated using hydroquinone solution. Then the product obtained was allowed to cool down at room temperature, and was immersed in a methanol–water mixture (70[thin space (1/6-em)]:[thin space (1/6-em)]30, v/v) for 3 h. Then it was left in acetone for one night for the complete removal of homopolymers, unreacted monomers and other impurities. The gel was separated from acetone and was dried under vacuum (60 °C).

To evaluate the effect of β-CD towards a cell cytotoxicity study, a pure copolymer hydrogel composed of PNIPAm and PMAc [i.e. cl-(PNIPAm-co-PMAc)] has also been synthesised using the same composition.

2.3. Characterization

The developed materials were characterized by FTIR, 1H NMR and 13C NMR spectral analyses, CHN analysis, FESEM analysis and DLS studies. Details of the instruments used and techniques are described in the ESI.

2.4. Evaluation of lower critical solution temperature (LCST) of β-CD-cl-(PNIPAm-co-PMAc) hydrogel

LCST is known as the temperature under which all the compositions of a mixture are miscible. Above LCST, phase transition occurs and the transmittance of that solution became 50% of the initial transmittance.16,37 The visual transmittance of the solution of β-CD-cl-(PNIPAm-co-PMAc) hydrogel in double distilled water was spectrophotometrically studied by a UV-vis-NIR spectrophotometer (model: Agilent Carry 5000 UV-VIS) at 563 nm.38 The heating rate was adjusted at 1 °C min−1.

2.5. Swelling experiment

The swelling characteristics of different grades of β-CD-cl-PNIPAm and β-CD-cl-PMAc were studied at pH 7.4 and physiological temperature 37 °C. On the other hand, the swelling properties of β-CD-cl-(PNIPAm-co-PMAc) hydrogel were evaluated in acidic (pH: 1.2) and mild alkaline (pH: 7.4) media at 37 °C. The pH and thermo responsive nature of β-CD-cl-(PNIPAm-co-PMAc) hydrogel was explored by determining % equilibrium swelling ratio (% ESR) at different pH and temperatures (25 °C and 37 °C). The detailed procedure is given in the ESI. To measure the degree of swelling, the Voigt model39 (eqn (1)) was employed.
 
St = Se(1 − et/τ) (1)
where Se and St designate swelling of the hydrogel at equilibrium and time t, respectively. ‘τ’ stands for the rate parameter of swelling. The ‘τ’ value is inversely proportional to the rate of swelling/deswelling.16,40,41

To examine the reversible features of the synthesised β-CD-cl-(PNIPAm-co-PMAc) copolymer, deswelling and reswelling experiments have also been performed. % ESR, % DSR, % ERSR and corresponding ‘τ’ values are given in Table S4, ESI.

2.6. Rheological studies

The gel properties of synthesised β-CD-cl-PNIPAm, β-CD-cl-PMAc and β-CD-cl-(PNIPAm-co-PMAc) hydrogel were studied in a fully swollen state at 37 °C [after attaining equilibrium swelling (∼14 h) at pH 7.4]. The rheological measurements were performed using an advanced rheometer (model: Bohlin Gemini-2, Malvern, UK). Yield stress12 was calculated from dynamic amplitude sweep measurements using a nonstop deviation of shear stress from 150 to 10[thin space (1/6-em)]000 Pa at a constant frequency of 1 Hz. The frequency sweep experiment of β-CD-cl-(PNIPAm-co-PMAc) hydrogel was studied with a frequency variation of 1–20 Hz at a constant stress (1 Pa) at 37 °C. Again to explore the thermo and pH responsive behaviour, the yield stress of β-CD-cl-(PNIPAm-co-PMAc) hydrogel was calculated in acidic (pH 1.2) and mild alkaline (pH 7.4) media and also at temperatures above and below the LCST i.e. 37 °C and 25 °C, respectively.

2.7. Compressive test

The compressive stress of three hydrogel samples [i.e. β-CD-cl-PNIPAm, β-CD-cl-PMAc and β-CD-cl-(PNIPAm-co-PMAc)] was measured using a universal testing machine (UTM) Hounsfield – H25KS at ambient temperature with a crosshead speed of 2 mm min−1. The methodology has been discussed in the ESI.

2.8. Cell viability experiment and morphological evaluations

Solution of β-CD in double distilled water, equal quantities of fully swollen β-CD-cl-(PNIPAm-co-PMAc) hydrogel, cl-(PNIPAm-co-PMAc) hydrogel and TCP were taken for MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and rhodamine–phalloidin and DAPI staining (used for 1, 3 and 5 day studies). The methodology has been illustrated in the ESI.

2.9. Live dead assay testing

The in vitro cytocompatibility of β-CD, cl-(PNIPAm-co-PMAc) hydrogel, β-CD-cl-(PNIPAm-co-PMAc) hydrogel and metronidazole and ofloxacin loaded β-CD-cl-(PNIPAm-co-PMAc) gel based tablets was studied using a Live–Dead staining kit (Invitrogen, Pleasanton, CA 94566, USA). The detailed procedure has been illustrated in the ESI.

2.10. In vitro metronidazole and ofloxacin (in combination) release study

The simultaneous in vitro release of both metronidazole and ofloxacin was performed in tablet formation. The tablet preparation technique has been described in the ESI. The pH responsive release of colon targeted dual drugs metronidazole and ofloxacin (in combination) from β-CD and β-CD-cl-(PNIPAm-co-PMAc) based tablets was spectrophotometrically determined using a UV-vis spectrophotometer (model: Shimadzu, Japan; UV 1800). The release study was carried out with the help of drug dissolution apparatus (Lab India, Model DS 8000) at 37 °C and 25 °C, with a constant rotation of 60 rpm. The release behaviour was investigated for 24 h, in which the first 2 h was studied in stomach pH (i.e. pH 1.2), and the other 22 h in colonic pH (i.e. pH 7.4). To determine the % cumulative release of the drugs, 5 mL buffer was withdrawn after a requisite interval and absorbance was measured. To study the kinetics and release mechanism, the data acquired from the drug delivery study were fitted to zero order42 and first order43 kinetic models and the Korsemeyer–Peppas model.44 Details of the models are illustrated in the ESI.

2.11. In vivo release study

A single dose bioavailability study was carried out for both metronidazole and ofloxacin drugs from β-CD-cl-(PNIPAm-co-PMAc) and β-CD based tablets. The study was carried out in 6 healthy albino rabbits weighing 1.5 kg to 2.5 kg. The protocol of the study was approved by the Institutional Animal Ethics Committee (IAEC of TAAB Biostudy Services, Registration No. 1938/PO/Rc/S/17/CPCSEA). The detailed methodology has been discussed in the ESI.

3. Results and discussion

3.1. Synthesis of β-CD-cl-(PNIPAm-co-PMAc) xerogel

Initially β-CD-cl-PNIPAm (Scheme S1, ESI) and β-CD-cl-PMAc (Scheme S2, ESI) xerogel were synthesized, the details of which have been discussed in the ESI. The optimized grades were selected with higher % crosslinking and lower % ESR (Tables S1 and S2, ESI). Considering the optimized concentration of reactants as well as the reaction conditions, the β-CD-cl-(PNIPAm-co-PMAc) xerogel has been prepared. β-CD-cl-(PNIPAm-co-PMAc) xerogel was synthesised via simultaneous polymerization and grafting of NIPAm and MAc moieties on pristine β-CD. The reaction possibly proceeds through the following steps: AIBN was decomposed to form a free radical (2-cyano prop-2-yl radical) in an inert atmosphere. Then the 2-cyano prop-2-yl radical in turn generates free radical reactive sites on the β-CD backbone (R1, Scheme 1), which was thereafter reacted with NIPAm and developed a macro-radical i.e. β-CD-PNIPAm (R2, Scheme 1). R2 was then copolymerized with MAc to produce another macro-radical β-CD-(PNIPAm-co-PMAc) (R3, Scheme 1). Afterward the β-CD-(PNIPAm-co-PMAc) macro-radical (i.e. R3) was crosslinked with EGDA as proposed in Scheme 1. By virtue of its (i.e. EGDA) multiple functionality, macro-radicals containing four active sites were generated, which were then attached to R3, leading to the formation of a three dimensional polymeric network β-CD-cl-(PNIPAm-co-PMAc) (P, Scheme 1). It is essential to mention that the reaction was terminated using saturated hydroquinone solution.
image file: c8qm00452h-s1.tif
Scheme 1 Probable synthetic route for the formation of β-CD-cl-(PNIPAm-co-PMAc) hydrogel.

3.2. Characterization

3.2.1. 1H NMR spectroscopy. The 1H NMR spectrum of β-CD-cl-PNIPAm 4 xerogel (Fig. S1, ESI) shows the peak at δ = 5.7 ppm, which is due to anomeric protons (H1)45 of β-CD. The peak at δ = 3.3 ppm is for the ring protons (H2–H6) and peaks at 4.4 and 4.8 ppm are attributed to the protons of alcohol groups present in β-CD37 i.e. OH 2, OH 3, and OH 6, respectively. The N–H proton (H11) of the PNIPAm moiety shows chemical shifts at 7.3 ppm and other protons of PNIPAm chains are present in β-CD-cl-PNIPAm xerogel (i.e. H7, H8 and H10, H9, H12 and H13 demonstrate chemicals shifts at 3.5, 2.3, 1.9, 3.6 and 1.0 ppm, respectively). Signals at 1.9 and 3.8 ppm are assigned to H14 and H15 (protons from crosslinker). The absence of shifts at 5.4 and 6.1 ppm (vinylic protons of NIPAm monomer)16 and generation of new signals at 1.9 ppm due to methylene protons, which are generated in the polymerization reaction, confirms the successful polymerization of NIPAm and crosslinker.

All the characteristic peaks of β-CD45 are present in the 1H spectrum of β-CD-cl-PMAc xerogel (Fig. S2, ESI) along with some new peaks. In this spectrum, signals at δ = 2.3 and 3.6 ppm are for H10 and H11 (protons from crosslinker), respectively. Shifts at δ = 2.6, 2.0 and 12.3 ppm are as a result of H7, H8 and an acid proton (H9) of the PMAc component. Here also the shifts due to vinylic protons of MAc are absent and a new peak appeared at 2.6 ppm, which is due to newly formed methylene protons. This suggests the successful polymerization of MAc on the β-CD backbone.

The 1H NMR spectrum of β-CD-cl-(PNIPAm-co-PMAc) xerogel (Fig. 1) demonstrates all the peaks of β-CD, β-CD-cl-PNIPAm and β-CD-cl-PMAc parts. The less intense signals in the range of 4.4–4.8 ppm imply that the hydroxyl protons of β-CD have taken part in the polymerization reaction. The presence of peaks at 3.5, 2.3, 7.1, 3.6, and 1.0 ppm are attributed to H7, H8, H9, H10, and H11 of the PNIPAm part and signals at 2.6, 1.9, and 12.3 are for H12, H13 and H14 of the PMAc part, which manifests the successful polymerization of both NIPAm and PMAc chains on β-CD. Additionally, the presence of chemical shifts due to the H15 and H16 protons of EGDA at 1.8 and 3.8 further confirm the successful addition of crosslinker units in the 3D xerogel network.


image file: c8qm00452h-f1.tif
Fig. 1 1H NMR spectrum of β-CD-cl-(PNIPAm-co-PMAc) xerogel in DMSO-d6.

The crosslinking density of β-CD-cl-(PNIPAm-co-PMAc) xerogel has been evaluated by measuring the integration of representative peaks of both β-CD (IH1) and EGDA (IH16).46 It has been assumed that one fourth of the integration value of four equivalent protons (H16) of EGDA would correspond to the integration value of one H1 proton of β-CD.45 The crosslinking density was determined using eqn (2):

 
image file: c8qm00452h-t1.tif(2)
It has been found that IH1 = 1.00 (presuming base value for one proton of β-CD) and IH16 = 2.9 (for four protons of EGDA) (Fig. 1). Hence the calculated value of crosslinking density of the developed dual responsive gel was found to be 0.72.

3.2.2. Solid state 13C-NMR spectral analysis. Fig. S3, ESI shows the 13C NMR spectrum of β-CD-cl-PNIPAm 4 xerogel, in which all of the characteristic peaks of β-CD are present45 (i.e. C1 at 103.9 ppm, C2–C4 at 73.8 ppm, C5 at 81.0 ppm and C6 at 62.4 ppm). The carbonyl carbons of ester and amide groups are characterised with a chemical shift at 174.9 ppm, while C15 gives a signal at 22.7 ppm. Besides, C7–C12 and C14 carbons altogether demonstrate a signal at 41.9 ppm. From the 13C NMR spectrum of β-CD-cl-PMAc 5 xerogel (Fig. S4, ESI), it has been observed that C1, C2–C4, C5 and C6 shows peaks at δ = 103.3, 73.1, 84.0 and 62.9 ppm, respectively. The presence of these signals suggests the existence of the β-CD unit in cl-β-CD/PMAc 5 xerogel. The acid carbonyl carbon of the PMAc unit demonstrates a chemical shift at 181.2 ppm. Other carbon atoms of the PMAc unit i.e. C7, C8 and C9 provide characteristic peaks at 45.4, 55.0 and 17.5 ppm, respectively, which confirm the presence of the PMAc moiety in the crosslinked gel. The presence of EGDA in β-CD-cl-PMAc xerogel is characterised with signals at 45.5, 31.0 and 62.2 ppm, which are corresponding to C11, C12 and C13, respectively.

The 13C NMR spectrum of β-CD-cl-(PNIPAm-co-PMAc) xerogel (Fig. 2) demonstrates all of the characteristics carbon peaks of β-CD (C1 at 103.6 ppm, C2–C4 at 73.7 ppm, C5 at 81.5 ppm and C6 at 63.4 ppm), PNIPAm (C7 at 45.3 ppm, C8 and C10 at 40.4 ppm, C9 at 176.6 ppm and C11 at 23.0 ppm), PMAc (C12 at 40.4 ppm, C13 at 55.0 ppm, C14 at 17.9 ppm and C15 at 180.4 ppm) and EGDA (C16, C17, C18 and C19 at 45.3, 40.4, 176.6, and 60.0 ppm respectively) fragments. The presence of all the components in the polymeric xerogel β-CD-cl-(PNIPAm-co-PMAc) suggests the successful development of the crosslinked polymer. Moreover during the course of polymerization, the vinylic sp2 carbon atoms of NIPAm, MAc and EGDA were converted to methylenic sp3 carbon atoms. The peaks due to the vinylic carbon atoms of monomers and crosslinker disappeared in the newly formed xerogel, while new peaks at 45.5 and 40.4 ppm (C-7, 16, 12) appeared due to newly generated methylenic sp3 carbon atoms. This result undoubtedly authenticates the fruitful polymerization and crosslinking of both the monomers and crosslinker on the β-CD backbone.


image file: c8qm00452h-f2.tif
Fig. 2 Solid state 13C NMR spectrum of β-CD-cl-(PNIPAm-co-PMAc) hydrogel.
3.2.3. CHN analysis. CHN analysis results reveal that β-CD does not contain any N.45 The β-CD-cl-PMAc 5 xerogel also contain no N, since neither MAc nor EGDA contain any nitrogen atoms (Table S3, ESI). However, for β-CD-cl-PNIPAm 4 xerogel and β-CD-cl-(PNIPAm-co-PMAc) xerogel, it has been found that both the compounds contain N, which may be due to the presence of the nitrogen containing PNIPAm moiety in both of the gels.
3.2.4. FESEM analysis. It was found that β-CD possesses a smooth surface morphology.45 FESEM images of β-CD-cl-PNIPAm 4 xerogel (Fig. S5a, ESI) show that the surface has a fish scale like structure, while β-CD-cl-PMAc 5 xerogel (Fig. S5b, ESI) demonstrates a smooth and slightly porous morphology. On the other hand, β-CD-cl-(PNIPAm-co-PMAc) xerogel (Fig. 3a) exhibits a 3D network-like highly porous surface morphology.
image file: c8qm00452h-f3.tif
Fig. 3 (a) FESEM morphology of freeze-dried gel, (b) transmittance (%) at different temperatures (°C), (c) nature of swelling of the crosslinked hydrogel at different pH and temperatures, and (d) DLS results at different conditions for β-CD-cl-(PNIPAm-co-PMAc) hydrogel.
3.2.5. Determination of LCST of β-CD-cl-(PNIPAm-co-PMAc) hydrogel. Fig. 3b illustrates the % transmittance curve plotted against temperature for the β-CD-cl-(PNIPAm-co-PMAc) hydrogel dispersed solution. The transmittance became 50% of its initial transmittance at 33 °C. At this temperature phase separation has taken place, and it is considered as the LCST of β-CD-cl-(PNIPAm-co-PMAc) hydrogel.37

This behaviour of the synthesised gel has been observed due to the presence of the PNIPAm moiety in the crosslinked network. PNIPAm has hydrophilic (amide carbonyl part) as well as hydrophobic (isopropyl part) units. Below LCST, hydrophilic force predominates over hydrophobic force. Moreover, the hydrophilic groups of β-CD-cl-(PNIPAm-co-PMAc) hydrogel took part in intermolecular H-bonding with water molecules. This results in higher swelling of the hydrogel.37,41 However, above LCST, the hydrophilic groups of β-CD-cl-(PNIPAm-co-PMAc) hydrogel take part in intramolecular H-bonding. Therefore intermolecular hydrogen bonding with water is hindered, which results in lower swelling.37

3.2.6. Swelling study. The swelling characteristics of different grades of β-CD-cl-PNIPAm and β-CD-cl-PMAc hydrogels were investigated at pH 7.4 at 37 °C and it has been observed that both of the hydrogels exhibit a continuous increase in % swelling with an increase in time. β-CD-cl-PNIPAm hydrogels reached equilibrium at ∼10 h (Fig. S6a, ESI), while β-CD-cl-PMAc hydrogels attained equilibrium at ∼16 h (Fig. S6b, ESI). Amongst various grades, β-CD-cl-PNIPAm 4 and β-CD-cl-PMAc 5 exhibit the lowest % ESR, which may be due to the more rigid network structure of the corresponding hydrogels (due to the highest % crosslinking, Tables S1 and S2, ESI).
pH responsive swelling behaviour. The effect of pH on the swelling patterns of β-CD-cl-(PNIPAm-co-PMAc) hydrogel is represented by Fig. 3c. It was observed that the synthesized dual responsive gel demonstrates a higher swelling rate at pH 7.4 than pH 1.2 at 37 °C. This may be because of the fact that in acidic media, the hydrophilic parts of the gel (i.e. –COOH, –NHCO–) became protonated and hence cannot form H bonds with the media. This leads to a lower % ESR. However, when the gel was dipped in the alkaline media, these groups remain in an unprotonated state, which favours intermolecular H bonding with water molecules, leading to a higher % ESR.
Thermoresponsive swelling. The temperature dependent swelling characteristics of the hydrogel are presented in Fig. 3c. Here, it is obvious that the hydrogel showed a higher degree of swelling at 25 °C than at 37 °C (at pH 7.4). This is likely due to the presence of both hydrophilic and hydrophobic moieties in the 3D network structure. Below LCST (25 °C), the hydrophilic force governs, which facilitates intermolecular hydrogen bonding of hydrophilic groups of the hydrogel with the water molecules, resulting in higher % ESR. On the other hand, above LCST (37 °C), the hydrophobic force dominates and the developed gel produced intramolecular H bonding, which hampers intermolecular H bond formation with water molecules, leading to a lower % ESR.

In order to study the reversible properties of the synthesized hydrogel, deswelling and reswelling experiments were carried out (Fig. S7, ESI) and the calculated values of τ (Table S4, ESI) for swelling and reswelling manifest the reversible nature of the synthesized hydrogel.

3.2.7. DLS analysis. DLS analysis was performed (Fig. 3d) to further support the temperature and pH responsive properties of the fabricated β-CD-cl-(PNIPAm-co-PMAc) hydrogel. It has been observed that due to lower swelling of the gel at 37 °C as compared to 25 °C, the hydrodynamic diameter is smaller (580 nm) at 37 °C than at 25 °C (837 nm). This supports the thermo-responsive behaviour of the hydrogel. Moreover, the pH responsive swelling nature is supported by the measurement of hydrodynamic diameter at pH 1.2 and pH 7.4 at 37 °C. It has been observed that the hydrodynamic diameter at pH 7.4 is relatively larger (580 nm) than that at pH 1.2 (356 nm), since the crosslinked gel swells more at pH 7.4 than at pH 1.2.
3.2.8. Rheological studies. Rheological plots of dynamic frequency sweep measurement from 1–20 Hz at 1 Pa shear stress (Fig. S8a, ESI) reveal that with an increase in frequency, there are slight increments for both G′ and G′′. Moreover, the storage modulus (G′) is greater than the loss modulus (G′′), which supports the viscoelastic nature of the hydrogel. In the case of amplitude sweep experiments for the swollen hydrogels at pH 7.4 and 37 °C (Fig. S8b, ESI), it is apparent that initially G′ is higher than G′′ and after application of a certain stress, there is a sharp decrease in both G′ and G′′. This may be as a consequence of the breakdown of the 3D gel network. This point is recognised as the yield stress (σ) of the hydrogel. The yield stress has been calculated for β-CD-cl-PNIPAm 4, β-CD-cl-PMAc 5 and β-CD-cl-(PNIPAm-co-PMAc) hydrogel and it has been observed that the yield stress of β-CD-cl-(PNIPAm-co-PMAc) hydrogel is higher than that of β-CD-cl-PNIPAm 4 (Fig. S9b, ESI) and β-CD-cl-PMAc 5 hydrogels (Fig. S9a, ESI). This may be due to an increase in the chain length as well as branching nature of β-CD-cl-(PNIPAm-co-PMAc) that exhibits higher yield stress (Table S5, ESI). From Fig. S8c, ESI, it has been found that as the shear rate was increased, shear viscosity decreased progressively. This supports the non-Newtonian shear thinning nature of the synthesised β-CD-cl-(PNIPAm-co-PMAc) hydrogel,16 which is beneficial for use as an injectable gel under high shear rate.

Since the synthesised gel is pH and temperature responsive, it is thus expected to exhibit a different yield stress corresponding to its swelling behaviour. The less swelling, the more rigid the network, which has been characterized with higher yield stress (Fig. 4). The values of yield stress of β-CD-cl-(PNIPAm-co-PMAc) hydrogel with the variation of temperature and pH have been reported in Table S5, ESI.


image file: c8qm00452h-f4.tif
Fig. 4 Plots of G′, G′′ at different shear stresses for β-CD-cl-(PNIPAm-co-PMAc) hydrogel swollen at different pH and temperatures.
3.2.9. Compressive test. The stress vs. strain curves, presented in Fig. S10, ESI, exhibit a nonlinear and exponential nature for the synthesised hydrogels. All of the three hydrogels showed excellent resistance to deformation under uniaxial compressive strain applied in the z direction. The compressive stresses at ∼80% strain for β-CD-cl-PMAc 5 and β-CD-cl-PNIPAm 4 were 1.59 kPa and 2.11 kPa, respectively. However, the β-CD-cl-(PNIPAm-co-PMAc) hydrogel is characterized with higher compressive stress (3.0 kPa). The compressive stress of β-CD-cl-(PNIPAm-co-PMAc) gel was improved nearly 50% as compared to β-CD-cl-PMAc 5 and 33% compared to β-CD-cl-PNIPAm 4 alone (Fig. S10, ESI).
3.2.10. Cell viability experiment and morphological evaluations. An in vitro cell viability experiment was carried out using an MG63 cell line to find out the level of toxicity of the synthesized hydrogel. Tissue culture plate (TCP), β-CD, cl-(PNIPAm-co-PMAc) hydrogel and β-CD-cl-(PNIPAm-co-PMAc) hydrogel with the same amount of MG63 cells were allowed to grow for 1, 3 and 5 days. The cellular viability was calculated by measuring the metabolic activity of the cultivated cells on TCP and β-CD as well as on the hydrogels. With the help of a standard curve, the OD values that were found from MTT assay were changed to cell population. In Fig. 5a, it is shown that the number of cells present on β-CD-cl-(PNIPAm-co-PMAc) hydrogel (10 × 105, 12.9 × 105, and 18 × 105 respectively), β-CD (9.89 × 105, 12.7 × 105, and 17.8 × 105 respectively), and cl-(PNIPAm-co-PMAc) hydrogel (5.2 × 105, 8.0 × 105, and 9.3 × 105 respectively) after 1, 3 and 5 days are higher than the number of cells grown on TCP after 1, 3 and 5 days (4.8 × 105, 7.4 × 105, and 8.8 × 105 respectively). This may be explained as follow: by virtue of its porous and floppy network-like structure, the hydrogel offers more surface area for cell growth as compared to TCP. These results establish the nontoxic character of the synthesized β-CD-cl-(PNIPAm-co-PMAc) hydrogel and hence it can be used as a colon targeted drug carrier.
image file: c8qm00452h-f5.tif
Fig. 5 (a) MTT assay results of TCP, β-CD, cl-(PNIPAm-co-PMAc) hydrogel and β-CD-cl-(PNIPAm-co-PMAc) hydrogel obtained after 1, 3, and 5 days and (b) structure of MG 63 cells on TCP, TCP in the presence of β-CD solution, cl-(PNIPAm-co-PMAc) hydrogel and β-CD-cl-(PNIPAm-co-PMAc) hydrogel achieved from rhodamine–phalloidin and DAPI staining after 1, 3, and 5 days.

Rhodamine–phalloidin and DAPI staining on TCP, TCP in the presence of β-CD, cl-(PNIPAm-co-PMAc) and the synthesized β-CD-cl-(PNIPAm-co-PMAc) hydrogel after 1, 3 and 5 days cell seeding (Fig. 5b) demonstrates that on day 1, there are lower numbers of cells present. However on day 3 and day 5, cells have good cytoplasm morphology and as compared to TCP, a large number of cells were present in the developed hydrogel. This may be due to the fact that swollen β-CD-cl-(PNIPAm-co-PMAc) hydrogel has a larger space for cell growth and adhesion as compared to TCP, which enhanced the metabolic rate, leading to a high population of cells.20 From rhodamine–phalloidin and DAPI staining analysis after 1, 3 and 5 days, it is also obvious that in the presence of β-CD, cl-(PNIPAm-co-PMAc) seeded cells also have good cytoplasm morphology, demonstrating the biocompatible nature of the materials.

3.2.11. Live dead assay. Live dead assays of β-CD, cl-(PNIPAm-co-PMAc) hydrogel, β-CD-cl-(PNIPAm-co-PMAc) hydrogel and dual drug (metronidazole and ofloxacin) containing hydrogel based tablet show (Fig. S11, ESI) that MG 63 cells have nice adhesion and viability on all of the samples. It is also apparent that the samples after 1, 3 and 5 days of experiment exhibit enormous numbers of live cells (green colour).47 There are no red dead cells48 present for the samples for day 1 and day 3. However, a negligible amount of red dead cells were observed at day 5 for β-CD-cl-(PNIPAm-co-PMAc) hydrogel. Therefore, the presence of huge numbers of viable cells on the hydrogel and hydrogel based tablet surface clearly indicates that the synthesized hydrogel may be used as a matrix for drug delivery and even after the incorporation of drugs, the material has no toxic effect on cells.
3.2.12. In vitro metronidazole and ofloxacin (in combination) release study. The probable interactions between the hydrogel and the drugs have been explained with the help of FTIR spectral analysis (explanation has been given in the ESI, Fig. S12 and S13, ESI).

In vitro release of both metronidazole and ofloxacin from the developed hydrogel matrix depends on the arrangement of different groups of the hydrogel network and the extent of swelling.49 There are three crucial steps for the release of drugs from hydrogel based drug delivery systems. The first step is dissolution, where the drugs present in the superficial areas of the tablet are released to the media. In the next stage, water is diffused inside the tablet and drug molecules present inside the tablet are diffused out to the releasing media. At the last stage, the drug is released through erosion of the matrix.16 The more swelling there is of the hydrogel matrix, the more effective surface area there will be and hence the release rate of the drug from the matrix to the media would be higher.40


pH responsive drug release study. The developed β-CD-cl-(PNIPAm-co-PMAc) hydrogel has a higher swelling rate at colonic pH (7.4) as compared to stomach pH (1.2) and thus released an insignificant amount of both colonic drugs (i.e. metronidazole and ofloxacin) at stomach pH for the first 2 h (Fig. 6). However, the release rate was significantly higher at colonic pH. In the case of the β-CD based tablet, it released the whole amount of drugs within 11h, while the β-CD-cl-(PNIPAm-co-PMAc) hydrogel based tablet released only ∼65% metronidazole and ∼53% ofloxacin in 24 h at colonic pH (Fig. 6).
image file: c8qm00452h-f6.tif
Fig. 6 Metronidazole and ofloxacin release profile from β-CD-cl-(PNIPAm-co-PMAc) hydrogel and β-CD based tablets. Results represented are mean ± S D, n =3.

Temperature responsive drug release study. The developed β-CD-cl-(PNIPAm-co-PMAc) hydrogel exhibits LCST at 33 °C. So, below LCST (i.e. at 25 °C) the hydrogel exhibits higher swelling as compared to above LCST (i.e. at 37 °C). As a result, the fabricated β-CD-cl-(PNIPAm-co-PMAc) hydrogel exhibits a higher rate of drug release at 25 °C as compared to 37 °C. At 25 °C, the β-CD-cl-(PNIPAm-co-PMAc) hydrogel matrix releases ∼82% metronidazole and ∼76% ofloxacin in 24 h (Fig. 6), which is higher than at 37 °C. Therefore, it is apparent that the synthesised hydrogelator released both the drugs simultaneously in a sustained manner at body temperature.

Thus, in vitro drug delivery study suggests that the developed hydrogels act as worthy carrier of colonic drugs metronidazole/ofloxacin and delivered at an anticipated rate.


3.2.12.1. Kinetics and mechanism of drug release. The data obtained from the release of both metronidazole and ofloxacin drugs from β-CD-cl-(PNIPAm-co-PMAc) and β-CD based tablets were fitted to zero order,42 1st order43 and Korsemeyer–Peppas models.44 The R2 (correlation coefficient) value for the zero order kinetic model is higher than that for the first order kinetic model (Table S6, ESI). From the Korsemeyer–Peppas model, it has been found that for the release of both of the drugs, the ‘n’ (diffusion coefficient) value lies between 0.45 and 0.89 (Table S6, ESI). Thus, the release of both of the drugs from tablet formulations follows a zero order kinetic model and non-Fickian diffusion mechanism.
3.2.12.2. In vivo release study. The pharmacokinetic study was conducted on rabbits for both metronidazole and ofloxacin drugs using β-CD-cl-(PNIPAm-co-PMAc) and β-CD based tablets. The plasma concentrations of both the drugs in different dosage forms are represented in Fig. 7. The results demonstrate that β-CD-cl-(PNIPAm-co-PMAc) based formulations have excellent sustained release properties as compared to β-CD based tablets, which was further supported with in vivo pharmacokinetic data (Fig. S7, ESI). The results thus obtained from the bioavailability study surely indicate the sustained release of dual drugs from β-CD-cl-(PNIPAm-co-PMAc) tablets, which is probably due to the formation of crosslinked networks.
image file: c8qm00452h-f7.tif
Fig. 7 In vivo release of (a) metronidazole and (b) ofloxacin from β-CD and β-CD-cl-(PNIPAm-co-PMAc) based tablets. Results represented are mean ± S D, n =3.

4. Conclusion

From the above observations and discussions, it is evident that a dual-responsive (pH and temperature) biocompatible hydrogelator was successfully synthesised from β-CD. 1H NMR and 13C NMR spectral analyses confirmed the development of the copolymeric gel, while FESEM analysis suggests the formation of a gel with an excellent porous network. The rheological characteristics revealed the viscoelastic nature of the hydrogel. Swelling characteristics manifest the formation of a reversible gel that demonstrates dual-responsive behaviour, which was further supported by DLS data. The synthesised gelators are non-cytotoxic, as evidenced from MTT, rhodamine-DAPI and Live–Dead assay analyses. Finally, both in vitro and in vivo studies revealed that β-CD-cl-(PNIPAm-co-PMAc) is a probable worthy carrier towards controlled release of metronidazole and ofloxacin, simultaneously.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The first author (A. R.) sincerely acknowledges the financial support of DST INSPIRE Fellowship (from Department of Science & Technology, New Delhi, India for the research grant, Award letter No. – IF170018). The authors also acknowledge SAIF, IISc Bangalore for providing solid state 13C NMR spectroscopy results. The authors also thankfully acknowledge the kind help of TAAB Biostudy Services, Jadavpur, Kolkata – 700032, India for providing in vivo analysis results.

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Footnote

Electronic supplementary information (ESI) available: Table S1. Synthesis details, % crosslinking and % ESR of various β-CD-cl-PNIPAm hydrogelators. Scheme S1. Probable polymerization reaction scheme for the synthesis of β-CD-cl-PNIPAm hydrogelator. Table S2. Synthesis details, % crosslinking and % ESR of various β-CD-cl-PMAc hydrogelators. Scheme S2. Probable polymerization reaction scheme for the synthesis of β-CD-cl-PMAc hydrogelator. Fig. S1. 1H NMR spectrum of β CD-cl-PNIPAm 4 hydrogelator in DMSO-d6. Fig. S2. 1H NMR spectrum of β-CD-cl-PMAc 5 hydrogelator in DMSO-d6. Fig. S3. Solid state 13C NMR spectrum of β-CD-cl-PNIPAm 4 hydrogelator. Fig. S4. Solid state 13C NMR spectrum of β-CD-cl-PMAc 5 hydrogelator. Table S3. CHN analysis result. Fig. S5. Surface morphology obtained from FESEM analyses of (a) β-CD-cl-PNIPAm 4 xerogel and (b) β-CD-cl-PMAc 5 xerogel. Fig. S6. Swelling characteristics of various grades of (a) β-CD-cl-PNIPAm hydrogel and (b) β-CD-cl-PMAc hydrogel at pH 7.4 and 37 °C (results represented are mean ± SD, n = 3). Fig. S7. Swelling, deswelling, and reswelling plots of β-CD-cl-(PNIPAm-co-PMAc) hydrogel at pH 7.4 and 37 °C. Table S4. % ESR for swelling and reswelling, %DSR and τ values for swelling, deswelling and reswelling of β-CD-cl-(PNIPAm-co-PMAc) hydrogel at different pH and temperatures. Fig. S8. Rheological plots of swollen of β-CD-cl-(PNIPAm-co-PMAc) hydrogel (a) G′, G′′ vs. frequency at 1 Pa shear stress (b) G′, G′′ vs. shear stress at 1 Hz frequency and (c) viscometric mode at pH 7.4 and 37 °C. Table S5. Yield stress and gel strength of β-CD-cl-PMAc 5, β-CD-cl-PNIPAm 4 and β-CD-cl-(PNIPAm-co-PMAc) hydrogel at different pH and temperatures. Fig. S9. Rheological G′, G′′ vs. shear stress at 1 Hz frequency plots of swollen (a) β-CD-cl-PMAc 5 hydrogel and (b) β-CD-cl-PNIPAm 4 hydrogel. Fig. S10. Compressive stress analysis of different hydrogels. Fig. S11. Calcein Et–Br staining results of β-CD-cl-(PNIPAm-co-PMAc) hydrogel and drug loaded β-CD-cl-(PNIPAm-co-PMAc) hydrogel based tablet after 1, 3, and 5 days. Fig. S12. FTIR spectra of (a) β-CD-cl-(PNIPAm-co-PMAc) hydrogel, (b) metronidazole, (c) ofloxacin, and (c) tablet. Fig. S13. Feasible interactions between β-CD-cl-(PNIPAm-co-PMAc) hydrogel and metronidazole and ofloxacin drug. Table S6. Metronidazole and ofloxacin release kinetics and mechanism data. Table S7. In vivo comparative pharmacokinetics parameters of metronidazole and ofloxacin based tablets in rabbit plasma. See DOI: 10.1039/c8qm00452h

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