Poly (N-Vinylcaprolactam-Grafted-Sodium Alginate) Based Injectable pH/Thermo Responsive In Situ Forming Depot Hydrogels for Prolonged Controlled Anticancer Drug Delivery; In Vitro, In Vivo Characterization and Toxicity Evaluation

Abstract

This study was aimed to develop novel in situ forming gels based on N-vinylcaprolactam, sodium alginate, and N,N-methylenebisacrylamide. The in situ Poly (NVRCL-g-NaAlg) gels were developed using the cold and free radical polymerization method. The structure formation, thermal stability, and porous nature of gels was confirmed by FTIR, NMR, DSC, TGA, and SEM. The tunable gelation temperature was evaluated by tube titling and rheological analysis. Optical transmittance showed that all formulations demonstrated phase transition around 33 °C. The swelling and release profile showed that gels offered maximum swelling and controlled 5-FU release at 25 °C and pH (7.4), owing to a relaxed state. Porosity and mesh size showed an effect on swelling and drug release. The in vitro degradation profile demonstrated a controlled degradation rate. An MTT assay confirmed that formulations are safe tested against Vero cells. In vitro cytotoxicity showed that 5-FU loaded gels have controlled cytotoxic potential against HeLa and MCF-7 cells (IC₅₀ = 39.91 µg/mL and 46.82 µg/mL) compared to free 5-FU (IC₅₀ = 50.52 µg/mL and 53.58 µg/mL). Histopathological study demonstrated no harmful effects of gels on major organs. The in vivo bioavailability in rabbits showed a controlled release in gel form (C_max, 1433.59 ± 45.09 ng/mL) compared to a free drug (C_max, 2263.31 ± 13.36 ng/mL) after the subcutaneous injection.

Keywords: 5-FU in situ depot; MTT assay; N-(vinylcaprolactam); anticancer drugs; chemical grafting; hydrogels; pharmacokinetics; rheology; sodium alginate.

Figure 1

¹H-NMR spectrum analysis of (A) pure N-vinylcaprolactam, (B) sodium alginate, and (C) Poly (NVCL-g-NaAlg) sample.

Figure 1

¹H-NMR spectrum analysis of (A) pure N-vinylcaprolactam, (B) sodium alginate, and (C) Poly (NVCL-g-NaAlg) sample.

Figure 2

FTIR spectra’s of pure 5-FU, pure NVCL, pure NaAlg, and bare chemically grafted Poly (NVCL-g-NaAlg) in situ depot gel sample.

Figure 3

DSC analysis of pure NVCL, pure NaAlg, and bare chemically grafted Poly (NVCL-g-NaAlg) in situ depot gel sample.

Figure 4

Thermogravimetric analysis of pure NVCL, pure NaAlg, and bare chemically grafted Poly (NVCL-g-NaAlg) in situ depot gel sample.

Figure 5

SEM analysis of chemically grafted thermoresponsive Poly (NVCL-g-NaAlg) in situ depot hydrogels. Surface morphology ×100 (A), ×200 (B), Cross-sectional morphology ×200 (C), ×250 (D), ×300 (E), ×500 (F), ×800 (G), and ×1000 (H).

Figure 6

Sol-gel state transition of chemically grafted Poly (NVCL-g-NaAlg) depot gels.

Figure 7

Oscillatory sweep tests and corresponding time data for depot gel formulations under strain = 1% and frequency = 1Hz (A–C). Change of G′ and G″ over 25 °C to 40 °C with increasing temperature for chemically grafted Poly (NVCL-g-NaAlg) depot gels (D). Frequency sweep test of thermoresponsive chemically grafted Poly (NVCL-g-NaAlg) depot gels under controlled strain of 1% (E). Effect of increasing shear rate (0.1–10 s⁻¹) on viscosity of gel formulations for 10 min at 25 °C (F). All the experiments were conducted in triplicates (n = 3).

Figure 8

Temperature dependence and effect of increasing polymeric and MBA contents on optical transmittances of thermoresponsive chemically grafted Poly (NVCL-g-NaAlg) depot gels (A) Effect of polymeric and crosslinking agent concentrations on percent crosslinking of the chemically grafted Poly (NVCL-g-NaAlg) depot gels (B). The data were analyzed for statistical significance with one-way ANOVA. The data were found statistically significant with p-value of <0.01. In vitro degradation profile of chemically grafted Poly (NVCL-g-NaAlg) depot gels at 37 °C (C). All the experiments were conducted in triplicates (n = 3).

Figure 9

The response of chemically grafted Poly (NVCL-g-NaAlg) in situ depot gels to swelling media of variable pH values at constant temperature i.e., 25 °C. A significant difference was seen in data at pH = 7.4 and 1.2. The data were analyzed with one-way ANOVA and found significant with p-value of <0.001 (A) Response of chemically grafted Poly (NVCL-g-NaAlg) in situ thermoresponsive depot gels to equilibrium swelling at variable pH and temperature programs (B) Effect of variable crosslinking agent concentrations (MBA) on equilibrium swelling ratio as function of time (C) Effect of MBA contents on ESR of chemically grafted Poly (NVCL-g-NaAlg) in situ depot gels at different pH and temperature programs (D). Heating and cooling cycles (swelling-deswelling-reswelling kinetics) of chemically grafted Poly (NVCL-g-NaAlg) depot gels (E) The data indicate the mean of (n = 3) individual experiments.

Figure 10

The response of thermoresponsive Poly (NVCL-g-NaAlg) depot gels to in vitro cumulative 5-FU release at variable pH and temperature programs. A significant difference was seen in drug release data at pH = 7.4 and 1.2. The data were analyzed with one-way ANOVA and found significant with p-value of <0.001 (A). Effect of increasing NaAlg concentrations in feed composition ratio of thermoresponsive Poly (NVCL-g-NaAlg) in situ depot gels on cumulative 5-FU release with time in PBS (pH = 7.4) at 25 °C (B), 37 °C (C), and in acidic buffer solution (pH = 1.2) (D). Cumulative 5-FU release in buffer solutions of variable pH values at different temperature programs with variable NaAlg contents (E). The data indicate the mean ± SD of (n = 3) individual experiments.

Figure 11

Effect of variable MBA contents on cumulative 5-FU release from Poly (NVCL-g-NaAlg) in situ depot gels with time in DW at 25 °C (A), in pH 7.4 (PBS, 5 mM) at 25 °C (B), and in PBS (pH = 7.4) at 37 °C (C). Cumulative 5-FU release in buffer solutions of variable pH values at different temperature programs with variable MBA contents (D). A significant decrease in drug release data was found with increasing MBA contents. The data were tested for statistical significance with one-way ANOVA and found significant with p-value of <0.1. The data show the mean ± SD of (n = 3).

Figure 11

Effect of variable MBA contents on cumulative 5-FU release from Poly (NVCL-g-NaAlg) in situ depot gels with time in DW at 25 °C (A), in pH 7.4 (PBS, 5 mM) at 25 °C (B), and in PBS (pH = 7.4) at 37 °C (C). Cumulative 5-FU release in buffer solutions of variable pH values at different temperature programs with variable MBA contents (D). A significant decrease in drug release data was found with increasing MBA contents. The data were tested for statistical significance with one-way ANOVA and found significant with p-value of <0.1. The data show the mean ± SD of (n = 3).

Figure 12

Effect of NVCL contents on cumulative 5-FU release from Poly (NVCL-g-NaAlg) in situ depot gels with time in pH 7.4 (PBS, 5 mM) at 25 °C (A), pH 7.4 (PBS, 5 mM) at 37 °C (B), and in acidic buffer solution (pH = 1.2) (C). Cumulative 5-FU release in buffer solutions of variable pH values at different temperature programs with variable NVCL contents (D). The data indicate the mean ± SD of (n = 3).

Figure 12

Effect of NVCL contents on cumulative 5-FU release from Poly (NVCL-g-NaAlg) in situ depot gels with time in pH 7.4 (PBS, 5 mM) at 25 °C (A), pH 7.4 (PBS, 5 mM) at 37 °C (B), and in acidic buffer solution (pH = 1.2) (C). Cumulative 5-FU release in buffer solutions of variable pH values at different temperature programs with variable NVCL contents (D). The data indicate the mean ± SD of (n = 3).

Figure 13

In vitro cytocompatibility sketch of bare thermoresponsive chemically grafted Poly (NVCL-g-NaAlg) in situ depot gels against Vero cell lines using MTT assay (A). Cytotoxicity evaluation of chemically grafted Poly (NVCL-g-NaAlg) in situ depot gels against Human cervical (HeLa) cancer cell lines (B). Cytotoxicity evaluation of chemically grafted Poly (NVCL-g-NaAlg) in situ depot gels against MCF-7 cancer cell lines (C). Data reported show mean ± SD of (n = 3). The data were tested with one-way ANOVA and found significant with p-value of <0.01.

Figure 14

Plasma drug concentration-time plots of 5-FU administered through the subcutaneous route in free pure solution form (A) and loaded in depot gel form (B).

Figure 14

Plasma drug concentration-time plots of 5-FU administered through the subcutaneous route in free pure solution form (A) and loaded in depot gel form (B).

Figure 15

Histopathological micrographs of different rabbit organs of control, pure free 5-FU treated, and 5-FU loaded injectable gel groups after the subcutaneous administration.