Strychnos Potatorum L. Seed Polysaccharide-Based Stimuli-Responsive Hydrogels and Their Silver Nanocomposites for the Controlled Release of Chemotherapeutics and Antimicrobial Applications.

Natural Strychnos potatorum L. (SPL) polysaccharide-based dual-responsive semi-IPN-type (SPL-DMA) hydrogels have been fabricated using dimethylaminoethyl methacrylate by simple free radical polymerization. Furthermore, a facial and eco-friendly method has been developed for the green synthesis of silver nanoparticles on SPL-DMA hydrogel templates (SPL-DMA-Ag) using an aqueous leaf extract of Carissa spinarum (as a bioreducing agent). SPL-DMA and SPL-DMA-Ag were characterized using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetry analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and evaluated network parameters. 5-Fluorouracil and doxorubicin were successfully encapsulated, and in vitro drug release studies were performed at pH values of 1.2 and 7.4 and at 25 and 37 °C. To understand the drug release mechanism of SPL-DMA hydrogels, various kinetic parameters were calculated. Biocompatibility and anticancer activities of SPL-DMA hydrogels were proved by an antioxidant activity study and in vitro cell viability studies against HeLa and 3T3-L1 cell lines. SPL-DMA-Ag hydrogels were used for antibacterial application.

Introduction

In recent years, polysaccharide-based biomaterials have been extensively used in the fields of pharmaceuticals,[1−3] food,[4,5] and environment[6] due to their biocompatibility, biodegradability, easy availability, and cost-effectiveness. The best source of polysaccharides is plant materials/living organisms, which contain a structurally diverse class of biomacromolecules; these are obtained by either whole biosynthesis or chemical synthesis.[7] Biomacromolecules possess a broad range of physiochemical properties, which are highly useful for food, medical, and biological applications.[1−6] Hence, the use of polysaccharide-based biomaterials is gaining huge importance over fully synthetic materials.

To improve the bioavailability of chemotherapeutic agents, various types of carriers such as micelles, liposomes, micro/nanoparticles, beads, films, and hydrogels have been developed to overcome their inherent drawbacks.[8] Compared to other types of carriers, hydrogels have been widely investigated owing to their easy fabrication, high stability, and significant encapsulation efficiency.[9] Hydrogels are water-swellable 3D polymeric networks that exhibit a significant stimuli-responsive behavior in an external environment, i.e., pH, temperature, ionic environment, light, and electric and magnetic fields. In addition, hydrogel properties such as functionality, swelling, porosity, mechanical strength, opacity, shape, and flexibility can be altered based on the requirement.[9] These versatile properties of hydrogels enable them to be used extensively for biomedical applications, especially in the development of artificial organs and in wound healing and controlled drug/gene delivery. Nowadays, synthetic polymer hydrogels are fully replacing polysaccharide-based hydrogels because they are biocompatible and biodegradable, which are the prime criteria to choose hydrogels as anticancer drug delivery devices, which in turn eliminate surgical operations after the loaded drug is exhausted in the implanted material.[10−14]

In the present work, we hypothesize the use of Strychnos potatorumL. (SPL) seed polysaccharide for fabrication of a stimuli-responsive semi-IPN-type hydrogel, which is responsive to tumor microenvironment for chemotherapeutic drug delivery and templates for green synthesis of silver nanoparticles for antimicrobial (wound healing) applications. SPL is a moderate tree that belongs to the family of Loganiaceae and is mostly found in Sri Lanka, Burma, and the southern and central parts of India.[15−18] All parts of this tree have a significant medical value; hence, it is widely used as a medicine in the Indian traditional system (Ayurveda) for the treatment of diseases such as eye and urinary tract infections.[19−21] However, the seeds of SPL polysaccharide consist of a 1:1.7 ratio of a mixture of galactomannan and galactan moieties; the chemical structure of this polysaccharide is β-(1 → 4)-linked D-galctopyranose bearing side chains with mannopyranosyl.[15−18]

The prime aim of this study is to fabricate new SPL polysaccharide-based stimuli-responsive hydrogels and their silver nanocomposites. So far, various types of hydrogel-based drug delivery devices using commercially available natural polymers such as guar gum, gum ghatti, gum karaya, xanthan gum, alginate, carboxymethyl cellulose, pectin, and chitosan have been reported.[9] There have been no reports on SPL polysaccharide-co-dimethylaminoethyl methacrylate (SPL-DMA)-based semi-IPN hydrogels so far; in addition, Carissa spinarum aqueous leaf extract is used as a green reducing agent for the synthesis of silver nanoparticles in the SPL-DMA semi-IPN hydrogel networks. But to the best of our knowledge, this is the first report on fabrication of hydrogels from SPL polysaccharide for in vitro drug release of chemotherapeutics, cancer cell viability studies, antioxidant activity, and antimicrobial applications.

Experimental Section

Materials

S. potatorumL. seeds were purchased from Sirigiri Venkappa Ayurveda store, Nandyala, Andhra Pradesh, India. Dimethylaminoethyl methacrylate (DMAEMA), acrylamide, N,N-methylene bisacrylamide (MBA), and ammonium persulphate (APS) were purchased from Sigma-Aldrich Chemical Co. 5-Fluorouracil, silver nitrate, sodium hydroxide, and hydrochloric acid were purchased from SD Fine Chemicals, Mumbai, India. Doxorubicin hydrochloride (DOX) was received from LC laboratories. All of the chemicals were used as received, and double-distilled water (DDW) was used for all experiments.

Isolation of S. potatorumL. (SPL) Polysaccharide

The polysaccharide was isolated from SPL seeds as per the procedure adopted by Matteo Adinolfi et al.[15] Briefly, SPL seeds were washed twice with double-distilled water, then crushed into small pieces, and ground well into a fine powder with a mixer grinder. Next, 5 gm of the seed powder was taken in a 500 mL beaker, 250 mL of distilled water was added, and the mixture was stirred overnight, followed by centrifugation at 8000 rpm for 10 min. The supernatant solution was filtered with Whatman cellulose acetate filters. The filtrate was added to a 1:1 tris buffer saturated with phenol and chloroform. The resulting emulsion was stirred for 1 h, followed by centrifugation at 8000 rpm for 10 min. The aqueous layer was separated, then mixed with equal volumes of chloroform, followed by centrifugation as per the above conditions, and finally added to three-fold of ethanol to obtain a white precipitate of polysaccharide. The precipitate was stored in a refrigerator at 0 °C for further use. The polysaccharide is characterized using Fourier transform infrared (FTIR), 1H NMR, and 13C NMR spectroscopies (Figure S1).

Fabrication of SPL-DMA Semi-IPN Hydrogels

To fabricate SPL-DMA semi-IPN hydrogels, a simple redox polymerization technique was employed using ammonium persulphate (APS) as an initiator and N,N-methylene bisacrylamide (MBA) as a cross-linker. The polymerization reaction was carried out in a 50 mL beaker, which consisted of 5 mL of DDW; to this beaker, specific amounts of 2 wt % SPL, 1 wt % APS, and 2 wt % MBA aqueous solutions were added and stirred to obtain a homogeneous solution, which was then purged with N2 gas for 10 min. Finally, DMAEMA was added and stirred thoroughly for 10 min and then for 180 min at room temperature (30 °C) to obtain a semitransparent hydrogel. The formed SPL-DMA semi-IPN hydrogels were immersed in DDW water for 48 h, and the water was changed every 6 h to remove any unreacted species. Finally, hydrogel discs were dried at 45 °C and stored at room temperature for further use.

Green Synthesis of Silver Nanocomposite Hydrogels

Preparation of C. spinarum Aqueous Leaf Extract (CSLE)

Fresh leaves of C. spinarum plant were collected from the premises of Yogi Vemana University, Vemana Puram, Kadapa, Andhra Pradesh, India (14.468552, 78.714183), in May 2019. The collected leaves were washed thoroughly with running tap water and then DDW to remove any adhered dust particles. Further, these leaves were air-dried for 1 week and kept in a hot air oven for 48 h at 40 °C. The dried leaves were ground into a fine powder. Then, 1 g of leaf powder was transferred into a 250 mL beaker containing 100 mL of DDW, and the system was continuously stirred at 100 °C. The aqueous leaf extract was filtered using Whatman filter paper (No. 4), and a clear solution obtained was collected and stored at 4 °C for further experiments. The digital photographs of the SPL tree, SPL seeds, SPL seed powder, and SPL-DMA hydrogels are shown in Figure S1.

Green Synthesis of SPL-DMA-Ag Nanocomposite Hydrogels

In the first step, SPL-DMA semi-IPN hydrogel samples were equilibrated with DDW by the transfer of accurately weighed 1 mg of the dried sample into a 50 mL beaker containing 25 mL of DDW. The second step involves silver ionization of the SPL-DMA hydrogel by transfer of the DDW equilibrated hydrogel into the 50 mL beaker containing 25 mL of 5 mM AgNO3 aqueous solution. Finally, the silver-ionized hydrogel is transferred into the 50 mL beaker containing 20 mL of C. spinarum aqueous CSLE, and reduction of silver ions into silver nanoparticles begins to occur, indicated by the change of the hydrogel color to dark brown. The reduction process is allowed for 4 h to complete the reaction. The digital photographs of C. spinarum leaves and SPL-DMA-Ag hydrogel nanocomposite are shown in Figure S1.

Swelling Studies

Swelling profiles of fabricated SPL-DMA semi-IPN hydrogels were determined by immersing a preweighed hydrogel in DDW at room temperature (30 °C).[2] The swollen hydrogels were weighed at regular intervals after blotting the surface-adhered liquid with tissue paper. The swelling ratio of hydrogels was calculated using the following relationshipwhere W0 is the dried/initial weight of the hydrogel and W is the weight of the swollen hydrogel at time t.

Equilibrium swelling at various pH values (1, 3, 5, 7, 9, and 11) and temperatures (25, 30, 35, and 40 °C) was also determined to prove the dual-responsive behavior of the hydrogel. The equilibrium swelling ratio was then calculated by the following relationshipwhere We is the weight of the swollen hydrogel at equilibrium time. All of the swelling experiments were performed in triplicate, and the data points represent the mean ± stranded deviation.

Encapsulation Studies of 5-Fluorouracil

5-Fluorouracil (5-FU), an anticancer drug, was loaded into SPL-DMA semi-IPN hydrogels by a simple physical absorption method. 5-FU solubility of 13 mg/mL deionized water, which increases with addition of sodium salt (NaOH).[2] Dried hydrogels were transferred into a known concentration of an aqueous sodium salt of 5-FU solution and allowed to swell for 36 h at room temperature. Finally, incubated gels were removed from the solution and dried at room temperature for 24 h and then for 48 h at 45 °C. To evaluate the encapsulation efficiency of 5-FU, 10 mg of drug-loaded SPL-DMA semi-IPN hydrogels was placed in a phosphate buffer solution, pH 7.4, for about 48 h and crushed well with an agate mortar. The solution was then filtered to separate the polymeric gel and rinsed with buffer. In the case of DOX encapsulation, the dried SPL-DMA hydrogel (about 200 mg) was transferred to 25 mL (1 mg/mL) of DOX solution equilibrated for 24 h at room temperature to load the drug into the hydrogel. The drug-loaded swollen hydrogel was washed rapidly with double-distilled water to remove excess drug molecules on the surface. The 10 mg of dried drug-loaded gel was placed in phosphate buffer solution (PBS), and complete DOX entrapment was allowed for 24 h. The solution was filtered, and the removed polymeric gel was washed twice with buffer solution.

The filtrate was analyzed using a UV–visible spectrophotometer (LAB INDIA, UV-3092, Mumbai, India) at a wavelength λmax of 269 nm for 5-FU and λmax of 490 nm for DOX. The % 5-FU/DOX loading and % encapsulation efficiency of 5-FU/DOX were calculated using the following relationship.

Drug Release Studies

5-FU and DOX release studies were performed with a tablet dissolution tester (LAB INDIA, DS-8000, India) at 37 °C with a speed of 100 rpm in pH 1.2 and 7.4 buffer solutions (GIT conditions). For all formulations, 100 mg of the drug-encapsulated SPL-DMA semi-IPN hydrogel was taken in each basket, which was already filled with 500 mL of buffer. At predetermined intervals, 2 mL of the sample was taken out and the release of 5-FU was analyzed by adsorption at a wavelength λmax of 269 nm for 5-FU and λmax of 490 nm for DOX using the UV–visible spectrophotometer (LAB INDIA, UV-3092, Mumbai, India). The amounts of 5-FU and DOX were calculated using the drug standard calibration curve. After each sampling, 2 mL of fresh buffer was added to maintain a constant volume of the solution. Each experiment was carried out in triplicate, and the final results were calculated as an average with mean ± standard deviation.

Antioxidant Assay

SPL-DMA hydrogel antioxidant activity tests were carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. In the DPPH scavenging assay, a SPL-DMA hydrogel sample of 10 mg was placed in a test tube with a methanolic solution of DPPH radical, and a DPPH solution without the hydrogel sample and with ascorbic acid was taken as a reference in another test tube. The reaction solution was kept in the dark, and the absorbance was measured at λmax 517 nm using ultraviolet–visible spectroscopy. The experiments were carried out in triplicate.

Cell Viability Studies

Human cervix adenocarcinoma cell line (HeLa) and mouse embryo fibroblast (3T3-L1) cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose supplemented with 10% fetal bovine serum (FBS) along with 1% 100 U/mL penicillin/streptomycin (Gibco, Invitrogen Ltd.). Cells were maintained in a humidified incubator at 37 °C under a 5% CO2 atmosphere.The cell viability of samples was analyzed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and HeLa and 3T3-L1. The cells (HeLa and 3T3-L1) were seeded at a density of 10 000 cells/cm2 and were allowed to attach in a culture medium (100 μL) for 24 h. The culture medium was discarded from the well plates and incubated with different concentrations of pure SPL-DMA hydrogel, 5-FU-loaded hydrogel, DOX-loaded hydrogel, pure 5-FU, and pure DOX. Then, 100 μL of MTT solution (0.5 mg/mL in DDW) was added and incubated for 1 h at 37 °C. Finally, the MTT reagent was removed, and then 100 μL of DMSO was added to solubilize the formed formazan crystals. The solution was swirled homogeneously for about 20 min with a shaker. The optical density was measured using a microplate reader at 570 nm (Bio-T Instruments, Inc.). All experiments conducted were analyzed in triplicate, and data were presented as near-standard deviation.

Antimicrobial Studies

The antimicrobial activity of SPL-DMA semi-IPN hydrogel silver nanocomposites (SPL-DMA-Ag) was investigated using the disc diffusion method. Antimicrobial studies of SPL-DMA-Ag samples were performed on bacteria, i.e., Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Klebsiella pneumonia (ATCC 700603). All of the required reagents and agar media were sterilized at 121 °C and 6.8 kg pressure for 30 min in an autoclave. Bacterial strains were procured from the Department of Microbiology, Yogi Vemana University, India, and used in the present study. Mueller Hinton agar (M173) plates were inoculated with Gram-positive and Gram-negative bacterial suspensions. After solidification of the media solution in Petri dishes, 0.1 mL of an 18 h old bacterial suspension culture was spread using a sterile spreader on the solid surface. The 5 mg/5 mL sample aqueous solutions were prepared and 30 μL was added to each plate; then, the plates were inoculated for 20 h at 37 °C. The zone formed around the well is measured in millimeters and compared with a standard (tetracycline).

Characterization

To identify the chemical structure and functional groups of isolated SPL polysaccharide, SPL-DMA semi-IPN hydrogel, 5-FU/DOX-loaded SPL-DMA semi-IPN hydrogel, 5-FU, DOX, CSLE, and CSLE-reduced SPL-DMA-Ag semi-IPN hydrogel, they were analyzed using Fourier transform infrared (FTIR, Perkin Elmer Spectrum 2 model) spectroscopy. In addition, SPL polysaccharide was analyzed using a Bruker NMR spectrometer at a probe temperature of 298 K and operated using a 400 MHz instrument. The green synthesis of silver from silver ions was monitored using the UV–visible spectrometer (LAB INDIA, UV-3092, Mumbai, India) at about λmax of 425. To understand the uniform distribution and presence of 5-FU/DOX in the SPL-DMA semi-IPN hydrogel network and the structure of the green synthesized silver nanoparticles from CSLE, samples are analyzed using an X-ray diffractometer (Rigaku, Minflex 600, Japan). X-ray diffractograms were recorded at 2θ values from 10 to 80° under Cu Kα radiation (λ = 1.5406). Also, differential scanning calorimetry (DSC) (TA Instruments, SDT Q600, U.K.) was used to better understand the uniform distribution and presence of 5-FU/DOX throughout the hydrogel network. The DSC and TGA thermograms were recorded under a N2 atmosphere in the range of 0–600 °C with a heating rate of 10 °C/min. Scanning electron microscopy (SEM) images of SPL-DMA, drug-loaded SPL-DMA, and SPL-DMA-Ag semi-IPN hydrogels were taken using a JEOL, JSM-IT500 InTouchScope. Samples were sputtered with gold (thickness of ∼10 nm) and placed on a copper stub, and SEM images were scanned with an accelerating voltage of 15 kV. Also, energy-dispersive X-ray (EDX) spectroscopy of SPL-DMA-Ag was measured at 5 kV. Transmission electron microscopy (TEM) images of SPL-DMA-Ag were analyzed using an FEI Tecnai G2 F20 with acceleration voltages in the 100–120 kV range. Dynamic light scattering (DLS) was performed for the size distribution of silver nanoparticles present in the SPL-DMA-Ag hydrogel.

Results and Discussion

1H and 13C NMR Spectroscopies of SPL Polysaccharide

The 1H NMR and 13C NMR spectra of pure isolated polysaccharide Strychnos potatorum L. are presented in Figure S2. From the spectrum (Figure S2A), a broad peak was observed at δH 4.7 for solvent (D2O), and another anomeric signal at δH 4.5 was observed for the glycosidic bond in addition, the anomeric proton H2– H6 signals were observed at around δH 4.07, 3.82, 3.70, and 3.62. The methine peak of mannose polysaccharide and α- and β-anomeric protons were designated as β-d-Man and α-d-Gal, respectively. Mannose and galactopyranose of the polysaccharide were observed at δH 3.40 and 2.32, respectively. From the 1H NMR spectrum, the polysaccharide β-(1 → 4)-linked d-galctopyranose and β-mannopyranosyl, mannose, and galactose units are responsible for anomeric hydrogens that appear in different regions.[21,22]

The 13C NMR spectrum (Figure S2B) of the polysaccharide showed a chemical shift value of the anomeric region at δC 104.29 ppm associated with C-1-β-d-mannose and substituted α-d-galactose. The diagnostic peak was observed at 77.97 ppm attributed to the C-4-linkage of the mannose unit, and the resonance peak chemical shift values of β-d-mannose and galactose substituted peaks were attributed to C-5 and C-6, respectively, at δC 71.56, 73.25, and 74.26 ppm. These regions suggested chemical shift values attributed to broad-intensity peaks at δC 60.77, 65.49, and 69.86 ppm corresponding to galactose moiety units and at δC 40.84 ppm corresponding to the polysaccharide methine carbon atom. These values are conclusive for the linear structure of (1 → 4)-β-d-galactomannan of S. potatorum L polysaccharide.[18,21,22]

FTIR Spectroscopy Analysis

FTIR spectra (Figure ) of the SPL polysaccharide, SPL-DMA semi-IPN hydrogel, 5-FU loaded SPL-DMA semi-IPN hydrogel, 5-FU, DOX, CSLE, and CSLE-reduced SPL-DMA-Ag semi-IPN hydrogel were obtained to investigate the functional groups of polysaccharide, hydrogel formation, and the chemical stability of 5-FU/DOX in hydrogel and to identify functional groups of CSLE involved in the bioreduction of silver nanoparticles. In the spectrum of SPL polysaccharide (Figure A), the broad absorption peak at 3411.26 cm–1 was attributed to −OH stretching vibrations of the carbohydrate moiety, the peak at 1628.41 cm–1 to the carbonyl groups, and the peak at 1042.61 cm–1 to the C–O stretching vibrations in the galctopyranose ring. The peak at 878 cm–1 is attributed to the presence of α-d-glucose and those at 740.61 and 528.38 cm–1 to the presence of the polysaccharide glycoside linkage. In the spectrum of SPL-DMA semi-IPN hydrogel (Figure b), the broad absorption peak at 3411.06 cm–1 was attributed to −OH stretching vibrations of the polysaccharide. The peaks at 2924.38, and 2837.92 cm–1 are attributed to the C–H stretching vibrations of the hydrogel and the −CH3 group of DMAEMA. Also, 1669.52 and 1450.12 cm–1 peaks are attributed to the primary amide and the N–H stretching vibrations of DMAEMA and MBA. In the spectrum of 5-FU (Figure c), the peak at 1286 cm–1 is attributed to C–H bending vibration, peaks at 1231.71 and 1145.30 cm–1 are attributed to C–F bonds in 5-FU, and that at 809 cm–1 is attributed to the C–H out-of-plane deformation in 5-FU. Figure d illustrates the spectrum of 5-FU-loaded SPL-DMA semi-IPN hydrogel, which comprises all of the significant peaks of both hydrogel and drug. In the spectrum of DOX (Figure e), peaks at 3449 and 1625 cm–1 are attributed to −OH and −NH groups, respectively; peaks at 1576 and 1614 cm–1 are attributed to the aromatic double bond and primary amine of DOX, respectively, and the peak at 1210 cm–1 is attributed to the C–O–C asymmetric stretching vibrations of DOX. Figure f illustrates the spectrum of DOX-loaded SPL-DMA semi-IPN hydrogel, which comprises all of the significant peaks of both hydrogel and DOX. Hence, the drug (5-FU or DOX) encapsulated in the SPL-DMA hydrogel is structurally stable. In the spectrum shown in Figure g, major peaks are observed for CSLE at 3442.61, 2925.38, 2848.11, 1637.22, 1460.89, 1384.48, 1329.63, 1120.21, 1031.83, and 580.24 cm–1 attributed to −OH stretching, −C–H stretching of the methyl and amide groups, −C–H symmetrical twisting, sulphonates, −C–O single bond, −C–N stretching, and −C–S stretching vibrations. These functional groups may be responsible for the bioreduction of silver ions to silver nanoparticles (Figure h). Based on the FTIR results, the plausible schematic diagram of the hydrogel structure is presented in Figure .

Figure 1

FTIR spectra of S. potatorum polysaccharide (a), pure SPL-DMA hydrogel (b), pure 5-FU (c), 5-FU-loaded hydrogel (d), pure DOX (e), DOX-loaded hydrogel (f), the aqueous leaf extract of C. spinarum (g), and SPL-DMA hydrogel silver nanocomposites (h).

Figure 2

Plausible schematic diagram of SPL-DMA hydrogel fabrication and its applications.

UV–Vis Spectrophotometry Analysis

UV–visible spectroscopy is one of the simple and sensitive techniques used to monitor metal nanoparticle synthesis. Silver nanoparticles were synthesized by mixing known concentrations of AgNO3 and CSLE aqueous solutions to yield a dark brown solution within 4 h. To determine the bioreduction capacity of the CSLE, absorption spectra of CSLE and CSLE-Ag were obtained, which are presented in Figure S3. The spectrum of CSLE-Ag showed a maximum peak at the wavelength of around 430 nm, and no other specific peaks were identified between 300 and 600 nm, which results in the formation of silver nanoparticles without aggregation (silver clusters) by bioreduction of CSLE from silver ions, which facilitates the quantum mechanical phenomenon known as surface plasmon resonance. The same strategy was applied for the green synthesis of silver nanoparticles in the SPL-DMA hydrogel networks (as a nanoreactor), and the same characteristic strong peaks with a bathochromic shift were observed, which is responsible for the highly swelling nature of the hydrogel network. Here, semi-IPN hydrogels acted as templates for the synthesis of silver nanoparticles, due to the presence of nitrogen atoms of amide groups present in DMAEMA/MBA and oxygen atoms of hydroxyl/carboxyl functional groups present in the SPL polysaccharide. Hence, we believe that the present methodology (green) helps us to fabricate silver nanoparticle-embedded hydrogel networks, where silver nanoparticles are stabilized by the SPL polysaccharide with bioreduction by CSLE.

X-ray Diffraction (XRD) Analysis

XRD patterns of SPL polysaccharide, SPL-DMA semi-IPN hydrogel, 5-FU-loaded SPL-DMA semi-IPN hydrogel, DOX-loaded SPL-DMA semi-IPN hydrogel, 5-FU, DOX, and SPL-DMA-Ag nanocomposites are depicted in Figure . The XRD patterns in Figure A,B have no significant sharp peaks, indicating the amorphous nature of the SPL polysaccharide and SPL-DMA semi-IPN hydrogel. However, the XRD spectrum of pure 5-FU (Figure C) exhibits characteristic peaks observed at 2θ values of 25, 28, and 33° indicating the crystalline nature. The XRD spectrum of pure DOX (Figure D) exhibited peaks at 2θ = 18.30, 23.24, 26.12, 31.40, 35.25, and 41.19°, indicating the crystalline nature. In Figure E, the 5-FU-loaded SPL-DMA hydrogel exhibits low-intensity characteristic peaks observed at 2θ values of 19, 25, 28, and 30°. In Figure F, the DOX-loaded SPL-DMA hydrogel exhibits low-intensity characteristic peaks observed at 2θ values of 15.15, 17.85, 19.32, 22.14, and 24.18°. Hence, XRD patterns suggest that the dispersion of 5-FU and DOX in the SPL-DMA-5-FU hydrogel network is at the molecular level. The existence of Ag nanoparticles in the SPL-DMA hydrogel network was investigated through XRD peaks in Figure G observed at 2θ values of 27.64, 32.17, 38.32, 44.23, 64.23, and 77.46° for the planes 001, 111, 200, 220, and 311, respectively.[23,24]

Figure 3

XRD spectra of S. potatorum polysaccharide (A), pure SPL-DMA hydrogel (B), pure 5-FU (C), pure DOX (D), 5-FU-loaded hydrogel (E), DOX-loaded hydrogel (F), and SPL-DMA hydrogel silver nanocomposites (G).

Differential Scanning Calorimetry (DSC) and Thermogravimetry Analysis (TGA)

The thermal stability of SPL polysaccharide, SPL-DMA hydrogel, 5-FU, DOX, 5-FU-loaded hydrogel, and DOX-loaded hydrogel was characterized using DSC, and the curves are shown in Figure I. The pure polysaccharide (SPL) (Figure Ia) exhibits a broad endothermic peak at 69.43 °C, which might be due to bound and unbounded moisture evaporation or loss of water molecules from the polysaccharide unit. For the pure SPL-DMA hydrogel (Figure Ib), endothermic peaks were observed at 93.62, 248.57, and 359.94 °C due to the loss of absorbed moisture (water molecules) and decomposition of amide and carbonyl groups in polysaccharide units, respectively. 5-FU (Figure Ic) exhibits an endothermic peak at around 289.08 °C and another peak was observed at 343.21 °C, which indicates polymorphism and the melting point. DOX (Figure Id) exhibits endothermic peaks at 57.19 and 249.67 °C corresponding to its decomposition. The DOX-loaded hydrogels (Figure If) show endothermic peaks at around 83.59, 256.69, and 342.75 °C due to the loss of moisture content in the hydrogel network, carbonyl functional groups, and decomposition of polymer chain units, respectively. The 5-FU-loaded SPL-DMA hydrogel (Figure Ig) exhibited an endothermic peak at 83.59 °C due to the evaporation of moisture from the polymer backbone. Endothermic peaks were observed at 252.70 and 296.15 °C due to polymer transition and the loss of amide functional groups, respectively. The drugs DOX and 5-FU do not exhibit their original significant peaks in the drug-loaded hydrogel network. These results suggest that the drug may have a strong association and molecular-level distribution throughout the hydrogel network. The thermal degradation properties of SPL polysaccharide, pure SPL-DMA hydrogel, 5-FU-loaded hydrogel, DOX-loaded hydrogel, 5-FU, and DOX were investigated using TGA, and the thermograms are shown in Figure II. For the SPL polysaccharide (Figure IIa), a weight loss of 13.34% was observed in the region between 84.99 and 64.30 °C, which is due to the loss of absorbed water molecules. As seen in Figure IIb, the pure SPL-DMA hydrogel showed two steps of weight loss approximately; in the first step, we observed a 12.96% weight loss in the region between 137.23 and 272.94 °C due to dehydration of water molecules from the surface in the hydrogel network. In the second step, weight loss was of 16.72% was observed in the region between 353 and 416.29 °C, due to the decomposition of amide functional groups and polysaccharide side-chain units. As seen in Figure IIc, the pure 5-FU drug shows two steps of weight loss: in the first step, a 12.11% weight loss was found in the region of 133.47–225.33 °C due to the loss of amide chain, and in the second step, we observed a weight loss of 8.17% in the region of 401.59–473.54 °C due to the loss of mass and degradation of the 5-FU compound. As seen in Figure IId, the 5-FU composite SPL-DMA hydrogel weight loss was observed in two steps: the first-step weight loss was observed at 16.96% in the region of 150.81–306.64 °C due to polymer attributes in the bound and unbound water molecules and hydroxyl groups. The second-step weight loss was observed at 20.12% in the region of 371.41–452.79 °C. In Figure IIe, a weight loss of 5.3% was observed for pure DOX in the region of 156.46–214.20 °C due to degradation of the molecular mass of the DOX compound. As seen in Figure IIf, the DOX composite SPL-DMA hydrogel showed a two-step weight loss: in the first step, we observed a weight loss of 9.68% in the region of 122.86–249.82 °C, and in the second step, we observed a weight loss of around 19.59% in the region of 272.94–357.58 °C due to a loss of nitrogen-containing functional groups and decomposition of polysaccharide chain units. We observed high thermal stability for pure polysaccharide and SPL-DMA pure gel and the drug-loaded hydrogel network.

Figure 4

DSC thermograms of (I) SPL-DMA hydrogel (a), pure polysaccharide (b), pure SPL-DMA gel (c), pure 5-FU (d), pure DOX (e), 5-FU-loaded gel (f), and DOX-loaded gel and (II) TGA curves of SPL-DMA hydrogel (a), pure polysaccharide, (b) pure SPL-DMA gel, (c) pure 5-FU, (d) 5-FU-loaded gel, (e) DOX-loaded gel, and (f) pure DOX.

SEM, EDAX, TEM, and DLS Studies

Figure presents the SEM images of the pristine SPL-DMA hydrogel (A), drug-loaded SPL-DMA gel (B), SPL-DMA-Ag (C); EDAX spectra of SPL-DMA-Ag; TEM images of SPL-DMA-Ag (E-H); and DLS spectra of silver nanoparticles (I). Due to the presence of hydrophilic functionalities in the SPL-DMA hydrogel network, the surface morphology appears smooth, while the drug-loaded hydrogel exhibits a rough surface due to the surface-adhered crystallites of the drug. Rough surface morphology was also observed for the SPL-DMA-Ag composite hydrogel due to the formation of Ag particles not only inside the network but also on the surface. The presence of Ag nanoparticles is evidenced using EDAX, i.e., the appearance of a strong absorption band at around 3.2 keV. TEM images show a group of Ag nanoparticles and their sizes, i.e., 20 nm. The size of Ag nanoparticles is further evidenced by DLS.

Figure 5

FE-SEM images of SPL-DMA hydrogels: (a) pure hydrogels (b), drug-loaded gel (c), and SPL-DMA-Ag hydrogel (d); the SPL-DMA-Ag EDAX spectrum; and the TEM images of (e) the SAED pattern of SPL-DMA-Ag of (f) 2 nm, (g) 5 nm, and (h) 20 nm; and (i) DLS spectra of SPL-DMA-Ag.

Swelling Studies and Hydrogel Network Parameters

Swelling behavior is one of the essential characteristics of hydrogels,[25] and it mainly depends on the surrounding environment such as pH, temperature, and electric and magnetic fields. In the present study, SPL-DMA hydrogels are evaluated for swelling and deswelling kinetics and percentage of equilibrium swelling ratio (%ESR) at 32 °C. Figure S4 A shows the swelling kinetics; the hydrogels reached maximum swelling in 72 h, but maximum deswelling was observed in 10 h (Figure b). Overall, the %ESR is high for SPL-DMA-3 (3168) and low for SPL-DMA-4 (1811). High swelling behavior may be due to the presence of more PDMAEMA hydrophilic polymer chains in the hydrogel network. Figure S4C,D shows the %ESR of the hydrogels at various pH values (1–11) and temperatures (25–40 °C); the %ESR is more at a lower pH and a higher temperature. %ESR values of SPL-DMA hydrogels decrease in a low-to-high pH region (Figure S4C). This may be due to the presence of ionizable amino groups on the PDMAEMA polymer chains; at low pH values (1–3), these amino groups undergo electrostatic repulsion (protonation), and at high pH values (7–9), the hydrogels shrink (deprotonation).[2,26] The results of the temperature effect on %ESR are presented in Figure S4D. %ESR is increased on increasing the temperature from 25 to 40 °C. However, swelling does not increase much up to 30 °C, but a significant increase is observed at 40 °C.

Figure 7

Cell viability studies of SPL-DMA hydrogels against different cell lines, HeLa and 3T3-L1 cell. Cell viability after 24 h of incubation at various concentrations: untreated, 6.25, 12.5, 25, 50, and 100, respectively. (a) HeLa cells and (b) 3T3-L1 cell line treatment with hydrogels.

Based on the swelling data, various diffusion parameters such as the polymer–solvent interaction parameter (χ), molecular mass between cross-links (Mc), pore size (ξ), volume fraction (ϕ), and effective cross-linking density (υe) were calculated using the following equationsThe various network parameters χ, Mc, ξ, ϕ, and υe are represented in Table . Changes in feed composition and the amount of cross-linking agents have a significant impact on network characteristics. The overall SPL-DMA hydrogel χ values range from 0.52 to 0.53, indicating a satisfactory polymer–solvent interaction in the SPL-DMA hydrogel network. On decreasing the cross-linker density and increasing the DMAEMA concentration, both molecular mass between cross-links (Mc) and pore size (ξ) values increased. The effective cross-linking density (υ) values range between 0.18 and 0.37, and the observed (υe) values drop as the Mc values of the hydrogel network increase due to the presence of ionizable tertiary amino groups.[26,27] We observe the presence of hydrophilic functional groups −CONH and −OH in DMAEMA and protonation of amino groups in an acidic environment, which forms quaternary ammonium ions leading to network expansion.

Table 1

Feed Composition, Percentage of Equilibrium Swelling Ratio (%ESR), % Encapsulation Efficiency (%EE), and Network Parameters of SPL-DMA Hydrogels

									network parameters
sample code	SPL (2%) (mL)	Am (g)	DMAEMA (mL)	MBA 2% (w/v) (mL)	APS 10% (w/v) (mL)	% ESR	% EE of 5-FU	% EE of DOX	χ	Mc	ξ	ø	υe
SPL-DMA-1	5	0.5	0.5	1.0	1.0	2656	56.25 ± 1.3	39.01 ± 1.2	0.5310	30220	44.53	30.98	0.2773
SPL-DMA-2	5	0.5	1.0	1.0	1.0	2142	71.06 ± 1.4	49.25 ± 1.1	0.5286	40212	44.33	29.19	0.2249
SPL-DMA-3	5	0.5	1.5	0.5	1.0	3168	77.18 ± 0.8	61.28 ± 0.8	0.5360	48583	45.71	20.56	0.1877
SPL-DMA-4	5	0.5	0.5	2.0	1.0	1811	44.82 ± 0.8	32.14 ± 0.7	0.5238	11474	29.19	37.08	0.3283
SPL-DMA-5	5	0.5	0.5	1.0	1.0	2268	72.28 ± 1.3	56.78 ± 0.5	0.5281	24798	35.19	30.08	0.2421

Encapsulation and In Vitro Drug Release Studies

The encapsulation efficiency data of the SPL-DMA hydrogel network are shown in Table . 5-FU encapsulation efficiency of SPL-DMA hydrogels ranges between 44.82 ± 0.8 and 77.18 ± 0.8. DOX encapsulation efficiency of SPL-DMA hydrogels ranges between 32.14 ± 0.7 and 61.28 ± 0.8. The maximum % encapsulation efficiency is observed for SPL-DMA-3. The minimum % encapsulation efficiency was observed for SPL-DMA-4 formulation, these values were influenced by a high monomer concentration and a lower amount of cross-linking agent. The cross-linker variation decreases and swelling behavior increases, and all of the remaining formulations follow the same trend.

In vitro 5-FU delivery studies of SPL-DMA hydrogels were analyzed using a tablet dissolution tester in simulated gastric (pH 1.2) and intestinal (pH 7.4) environments at 37 °C. For SPL-DMA-3 formulation, 5-FU release studies were performed at pH 7.4 and 25 and 37 °C. The 5-FU and DOX release profiles are shown in Figure , and the drug release kinetics is plotted against the percentage of cumulative release versus time. These results are evaluated and discussed with respect to the monomer and concentration, as well as pH and temperature.

Figure 6

I. In vitro 5-FU release of the SPL-DMA hydrogels. (a) pH 1.2 and (b) pH 7.4 and (c) temperature effects at 25 and 37 °C. II. In vitro DOX release of the SPL-DMA hydrogels. (a) pH 1.2 and (b) pH 7.4 and (c) temperature effects at 25 and 37 °C.

The swelling kinetics drug release behavior of the SPL-DMA hydrogel controlled the drug release rate, and a higher amount of 5-FU was released at pH 1.2 rather than at pH 7.4 as displayed in Figure . The 5-FU drug release data of all formulations at pH 1.2 are shown in Figure A. The drug release rate of SPL-DMA -3 hydrogel formulation was higher than those of other formulations. The drug release behavior of all formulations of hydrogels was higher at pH 1.2 rather than at pH 7.4 because PDMAEMA concentration increased, and controlled leaching of drug molecules exhibit a higher swelling behavior at pH 1.2 as cationic amine groups of tertiary amine groups undergo protonation of the acidic region and the alkaline medium undergoes deprotonation of the hydrogel network. The alkaline medium of hydrogels easily ionizable of amine groups shrinking nature of SPL-DMA hydrogel network. The temperature-responsive behavior of SPL-DMA hydrogels is well accepted in the pharmaceutical application of biomaterials. The drug release and the mechanistic features of hydrogels change in the environment, especially at high temperatures. In vitro drug release studies were performed in media at pH 7.4 and 25 and 37 °C, as shown in Figure . The temperature effect of PDMAEMA near to physiological temperature lower critical solution temperature (LCST) around at 40–45 °C with respect to the environmental conditions. SPL-DMA-3 hydrogel 5-FU drug release temperature of 25 °C was observed at 2 h at 42.92%, 3 h at 62.48%, 86.80% at 5 h, 7 h at 94.40%, 12 h at 98.51%, and 24 h at 99.03%, and 37 °C 5-FU drug release temperature was observed at 2 h at 61.97%, 3 h at 65.73%, 5 h at 73.95%, 7 h at 78.62%, 12 h at 85.87%, 24 h at 90.87%, and 48 h at 96.18%. The drug release of SPL-DMA-3 formulation was higher at 25 °C (gel in a swollen state) than at 37 °C (in a collapsed state). The SPL-DMA-3 hydrogel drug released 85.87% in 12 h, but the release rate increased up to 96.18% in 48 h with a controlled release, as the cationic amine groups of tertiary amine groups undergo protonation easily at low temperature but higher-temperature DMAEMA deprotonation of the amine groups of the hydrogel is not easily ionizable due to the shrinking behavior of the SPL-DMA hydrogel network. The same results are observed for the DOX-loaded hydrogels (Figure ).

Figure 8

Antimicrobial studies of SPL-DMA-Ag hydrogels against (a) K. pneumoniae, (b) S. aureus, (c) P. aeruginosa, and (d) E. coli and (e) zone inhibition of SPL-DMA-Ag hydrogels.

The in vitro drug release of 5-FU and DOX from SPL-DMA hydrogels was analyzed using different kinetic models, i.e., zero-order, first-order, Higuchi, Hixson–Crowell, and Korsmeyer–Peppas models.where M and M∞ are the fractions of the drug released at a time t, k is the rate constant, and n is the drug release exponent.

All kinetic parameters with a correlation coefficient and rate constant are represented in Table . The in vitro drug release kinetics mechanism confirms that zero-order and Korsmeyer–Peppas models are better fitted compared to other kinetic models. The first case in the n < 0.5 polymer network follows Fickian diffusion, the second case, i.e., 0.5 < n < 0.89, corresponds to non-Fickian diffusion and an anomalous mechanism, and the third case, n = 1, completely follows non-Fickian and supercase II drug release kinetics. The drug release data analyzed to n values ranged between 0.35 and 0.50. n values indicate a completely Fickian diffusion transport mechanism and in relaxation of a polymer hydrogel network plays a vital role during the transport of drug molecules.[28,29]

Table 2

In vitro Drug Release of Various Kinetic Models for 5-FU and DOX Release Studies

sample code	zero-order		first-order		Higuchi		Hixson–Crowell		Korsmeyer–Peppas
									pH 1.2			pH 7.4
	R	K0	R	K1	R	Kh	R	Khc	k	n	r	k	n	r
5-FU
SPL-DMA-1	0.986	0.750	0.898	0.038	0.898	0.016	0.941	0.032	1.74	0.38	0.9825	7.47	0.49	0.9744
SPL-DMA-2	0.998	0.759	0.900	0.077	0.900	0.033	0.922	0.034	4.55	0.50	0.9423	6.76	0.52	0.9638
SPL-DMA-3	0.973	0.756	0.869	0.041	0.998	0.032	0.917	0.033	4.85	0.48	0.9774	7.37	0.50	0.9712
SPL-DMA-4	0.994	0.567	0.899	0.029	0.899	0.012	0.948	0.025	6.80	0.48	0.9401	8.25	0.47	0.9822
SPL-DMA-5	0.995	0.361	0.897	0.041	0.985	0.030	0.952	0.027	6.70	0.35	0.9691	2.31	0.47	0.9882
DOX
SPL-DMA-1	0.986	0.750	0.898	0.038	0.898	0.016	0.941	0.032	1.74	0.38	0.9825	7.47	0.49	0.9744
SPL-DMA-2	0.998	0.759	0.900	0.077	0.900	0.033	0.922	0.034	4.55	0.50	0.9423	6.76	0.52	0.9638
SPL-DMA-3	0.973	0.756	0.869	0.041	0.998	0.032	0.917	0.033	4.85	0.48	0.9774	7.37	0.50	0.9712
SPL-DMA-4	0.994	0.567	0.899	0.029	0.899	0.012	0.948	0.025	6.80	0.48	0.9401	8.25	0.47	0.9822
SPL-DMA-5	0.995	0.361	0.897	0.041	0.985	0.030	0.952	0.027	6.70	0.35	0.9691	2.31	0.47	0.9882

Antioxidant Activity Assay

The DPPH activity experiment demonstrates that SPL-DMA hydrogels have a discernible capability of radical scavenging, with the trend expected to vary with dose. The ability of the polymer to scavenge radicals is slightly lower than that of ascorbic acid. The reduction of DPPH is used to calculate the antioxidant activity of the SPL-DMA hydrogel. After 2 h, the DPPH radical scavenging activity was 60.73 ± 0.9%. Thongchai and co-workers have shown that natural polysaccharides have antioxidant properties.[30]

Cell Viability Assay of the SPL-DMA Hydrogel

Figure displays the dose-dependent cytotoxicity of pure SPL-DMA hydrogel, 5-FU-loaded SPL-DMA hydrogel, DOX-loaded SPL-DMA hydrogel, pure 5-FU, and pure DOX against HeLa and 3T3-L1 cells.

The IC50 value was determined using the linear regression equationHere, Y = 50, and M and C values were derived from the viability graph.

The IC50 values for pure ST hydrogel treated with HeLa and MT3L1 cells are 95.42 and 172. 66 μg/mL, respectively. The results of the anticancer activity of 5-FU on HeLa show high anticancer activity with an IC50 value of 14.88 μg/mL. The 5-FU-loaded hydrogel also shows good anticancer activity against HeLa cells with an IC50 value of 23.40 μg/mL. The results clearly demonstrated the anticancer activity of the 5-FU-loaded hydrogel with a 2.5-fold and two-fold reduction in anticancer activity as compared to pure 5-FU. It is expected that 5-FU delayed the release from hydrogel networks. In the case of DOX, the anticancer activity of HeLa is high with an IC50 value of 10.09 μg/mL. DOX-loaded hydrogels treated with HeLa cells show an IC50 value of 29.85 μg/mL. As expected, DOX-loaded hydrogels show high anticancer activity toward HeLa cells. However, the 5-FU- and DOX-loaded hydrogels are cytotoxic against 3T3-L1 cells with IC50 values of 84.21 μg/mL and 106.97 μg/mL, respectively, whereas pure hydrogel is not cytotoxic against 3T3-L1 cells (IC50 = 172.66 ug/mL). Overall, the 5-FU- and DOX-loaded hydrogels show high anticancer activity toward HeLa cells.[31,32]

Antimicrobial Activity Assay

Due to the stimuli-responsive behavior and importance of multifunctional hydrogels, they have been widely used in wound healing and tissue engineering applications.[33,34] In the present study, SPL-DMA-Ag hydrogel nanocomposites developed by a green method by reduction of silver ions into silver nanoparticles using an aqueous leaf extract of C. spinarum showed excellent antibacterial activity toward Gram-positive and Gram-negative bacteria, as shown in Figure . These nanocomposites showed significant zone inhibition against K. pneumonia ranging between 12 ± 0.9 and 14 ± 1.4 mm, Figure b shows S. aureus zone inhibition ranging between 12 ± 0.9 and 18 ± 1.2 mm, Figure c shows P. aeruginosa zone inhibition ranging between 10 ± 1.5 and 14 ± 1.3 mm, and Figure d shows E. coli zone inhibition ranging between 16 ± 0.3 and 18 ± 1.1 mm. Based on the above results, silver nanoparticles have a high surface-to-volume ratio, chemical stability, and controlled nanosize, which have a great impact on antimicrobial properties.[35] The positively charged silver ions are crucial for microbial activity through the electrostatic repulsion between positively charged silver ions and the negatively charged cell membrane of microorganisms.[36] The antibacterial mechanism of silver nanoparticles is mainly due to alteration of membrane permeability, modification of intracellular ATP levels and respiration, uncontrolled cellular transport, loss of ATP, and DNA replication. Silver nanoparticles also result in free radical generation and a release of silver ions that lead to cell death.[37] Also, the plates (Figure a–d) showed that the control ampicillin antibiotic drug’s significant zone inhibition of K. pneumonia was observed at 14 ± 0.4 mm, S. aureus at 22 ± 0.5 mm, and E. coli at 24 ± 1.3 mm, but P. aeruginosa did not show zone inhibition. From the study, the SPL-DMA-Ag hydrogel was observed to have strong and almost equal antimicrobial activity when compared with the standard drug “ampicillin”.

Conclusions

To confirm our hypothesis, we developed natural S. potatorum L. polysaccharide-based duel-responsive hydrogels with dimethylaminoethyl methacrylate by simple redox polymerization at ambient temperature. Also, these hydrogels were used as templates to fabricate silver nanocomposites using an aqueous leaf extract of C. spinarum (as a bioreducing agent). SPL-DMA hydrogels and their silver nanocomposites were characterized using UV–visible and FTIR spectroscopies, DSC, XRD, SEM, EDAX spectroscopy, TEM, and DLS. The dual-responsive behavior of the SPL-DMA hydrogel higher pH-equilibrium swelling in acidic regions and at low temperatures. Hydrogels exhibited a significant drug encapsulation efficiency and in vitro drug release characteristics of 5-fluorouracil and doxorubicin with stimuli-responsive behavior. Drug release data demonstrate that release and drug transport mechanisms solely followed Fickian diffusion. MTT assay shows that drug-loaded hydrogels show significant elimination of HeLa and 3T3-L1 cancer cells and hydrogels are compatible with cells. Another potential application of this study is the antimicrobial activity toward four different strains, namely, E. coli, K. pneumonia, Bacillus cereus, and S. aureus. The developed SPL-DMA hydrogels can load and deliver various other chemotherapeutic agents, are used as templates for metal nanoparticles, and have potential biomedical applications.