A Simple Preparation Method of Gelatin Hydrogels Incorporating Cisplatin for Sustained Release

Abstract

The objective of this study was to develop a new preparation method for cisplatin (CDDP)-incorporated gelatin hydrogels without using chemical crosslinking nor a vacuum heating instrument for dehydrothermal crosslinking. By simply mixing CDDP and gelatin, CDDP-crosslinked gelatin hydrogels (CCGH) were prepared. CDDP functions as a crosslinking agent of gelatin to form the gelatin hydrogel. Simultaneously, CDDP is incorporated into the gelatin hydrogel as a controlled release carrier. CDDP's in vitro and in vivo anticancer efficacy after incorporation into CCGH was evaluated. In the in vitro system, the CDDP was released gradually due to CCGH degradation with an initial burst release of approximately 16%. CDDP metal-coordinated with the degraded fragment of gelatin was released from CCGH with maintaining the anticancer activity. After intraperitoneal administration of CCGH, CDDP was detected in the blood circulation while its toxicity was low. Following intraperitoneal administration of CCGH in a murine peritoneal dissemination model of human gastric cancer MKN45-Luc cell line, the survival time was significantly prolonged compared with free CDDP solution. It is concluded that CCGH prepared by the CDDP-based crosslinking of gelatin is an excellent sustained release system of CDDP to achieve superior anticancer effects with minimal side effects compared with free CDDP solution.

Keywords: cisplatin; crosslinking method; gastric cancer; gelatin hydrogel; peritoneal metastases; sustained release.

Figure 1

Preparation method for gelatin hydrogels incorporating CDDP for sustained CDDP release. (A) Two-step method reported previously. First, gelatin is crosslinked by chemical crosslinking using glutaraldehyde or dehydrothermal crosslinking, and then the CDDP is incorporated into it. (B) The newly developed one-step method. By crosslinking gelatin with CDDP, gelatin crosslinking and CDDP incorporation are performed simultaneously. The binding mechanism between CDDP and gelatin molecules can be a coordinate bond between platinum atoms and the carboxyl groups of gelatin side chains.

Figure 2

Characterization of CDDP-crosslinked gelatin hydrogels (CCGH). (A) A microscopic image of dried CCGH. Scale bar: 100 μm. (B) A microscopic image of CCGH dispersed in DDW. Scale bar: 100 μm. (C) Degradation profiles of CCGH. Collagenase D was added at the time indicated by the arrow (12 h). (n = 6). Data were represented as mean ± SD. (D) CDDP release profiles from CCGH. Collagenase D was added at the time indicated by the arrow (12 h). (n = 6). Data were represented as mean ± SD.

Figure 3

Characterization of CCGH prepared by varying the preparation conditions in vitro. (A–C) CDDP content per 1 mg CCGH (A), degradation profiles (B), and CDDP release profiles (C) of CCGH prepared by varying the stirring time of the gelatin aqueous solution and the CDDP aqueous solution at 40 °C (1 h; dotted line, 3 h; dashed line, 6 h; thick solid line, and 12 h; thin solid line) (n = 3). Data were represented as mean ± SD. Collagenase D was added at the time indicated by the arrow (12 h). (D–G) CDDP content per 1 mg CCGH (D), degradation profiles (E), CDDP release profiles (F), and recovery rate (G) of CCGH prepared by varying the standing time of the stirred solution at 4 °C (6 h; dotted line, 12 h; dashed line, 24 h; thick solid line, and 48 h; thin solid line). (n = 3). Data were represented as mean ± SD. Collagenase D was added at the time indicated by the arrow (12 h).

Figure 4

In vitro cytotoxicity of free CDDP solution and CCGH with and without degradation by collagenase. CCGH without degradation by collagenase (black circle), CCGH with degradation by collagenase (gray circle), and free CDDP solution (white triangle) (n = 4). The line means fitting curve.

Figure 5

In vivo retention evaluation of CDDP after intraperitoneal administration. (A) Serum CDDP concentration profiles after intraperitoneal administration of free CDDP solution, CCGH, and gelatin solution. CCGH (10 mg/kg; double dot-dash line), CCGH (30 mg/kg; double line), free CDDP solution (4 mg/kg; dot-dash line), and gelatin solution (thin solid line). (n = 5). Data were represented as mean ± SD. (B–F) Main pharmacokinetic parameters of CCGH (10, 30 mg/kg) and CDDP (4 mg/kg). (n = 5). (B) AUC, area under the serum concentration–time curve; (C) MRT, mean residence time; (D) t (1/2), elimination half-life; (E) CL, clearance; (F) Vd, distribution volume.

Figure 6

In vivo toxicity evaluation after treatment (day 1) in mice. (A) The profiles of body weight change. CCGH (10 mg/kg; double dot-dash line), CCGH (30 mg/kg; double line), free CDDP solution (2 mg/kg; dotted line), free CDDP solution (4 mg/kg; dot-dash line), and gelatin solution (thin solid line). (n = 5). Data were represented as mean ± SD. (B–F) Hematological examination data on day 10. (B) Serum concentrations of blood urea nitrogen (BUN). (C) Serum concentrations of creatinine (Cre). (D) Numbers of white blood cells (WBCs). (E) Numbers of red blood cells (RBCs). (F) Numbers of platelets. CCGH (10 mg/kg), CCGH (30 mg/kg), free CDDP solution (2 mg/kg), free CDDP solution (4 mg/kg), and gelatin solution. (n = 5).

Figure 6

In vivo toxicity evaluation after treatment (day 1) in mice. (A) The profiles of body weight change. CCGH (10 mg/kg; double dot-dash line), CCGH (30 mg/kg; double line), free CDDP solution (2 mg/kg; dotted line), free CDDP solution (4 mg/kg; dot-dash line), and gelatin solution (thin solid line). (n = 5). Data were represented as mean ± SD. (B–F) Hematological examination data on day 10. (B) Serum concentrations of blood urea nitrogen (BUN). (C) Serum concentrations of creatinine (Cre). (D) Numbers of white blood cells (WBCs). (E) Numbers of red blood cells (RBCs). (F) Numbers of platelets. CCGH (10 mg/kg), CCGH (30 mg/kg), free CDDP solution (2 mg/kg), free CDDP solution (4 mg/kg), and gelatin solution. (n = 5).

Figure 7

In vivo anticancer efficacy and toxicity evaluation after treatment in a mouse model with peritoneal metastases. (A) A schema of animal experiments. (B) Survival time profiles. CCGH (10 mg/kg; double dot-dash line), CCGH (20 mg/kg; double dashed line), CCGH (30 mg/kg; double line), free CDDP solution (2 mg/kg; dotted line), free CDDP solution (3 mg/kg; dashed line), free CDDP solution (4 mg/kg; dot-dash line), gelatin solution (thin solid line), and PBS (thick solid line). (n = 6). Drugs were injected at the time indicated by arrows (day 5, 12, and 19). (C) Body weight change profiles from the first injection (day 5) up to 7 days after the third injection (day 26). CCGH (10 mg/kg; double dot-dash line), CCGH (20 mg/kg; double dashed line), CCGH (30 mg/kg; double line), free CDDP solution (2 mg/kg; dotted line), free CDDP solution (3 mg/kg; dashed line), free CDDP solution (4 mg/kg; dot-dash line), gelatin solution (thin solid line), and PBS (thick solid line). (n = 6). Drugs were injected at the time indicated by arrows (day 5, 12, and 19).

Figure 7

In vivo anticancer efficacy and toxicity evaluation after treatment in a mouse model with peritoneal metastases. (A) A schema of animal experiments. (B) Survival time profiles. CCGH (10 mg/kg; double dot-dash line), CCGH (20 mg/kg; double dashed line), CCGH (30 mg/kg; double line), free CDDP solution (2 mg/kg; dotted line), free CDDP solution (3 mg/kg; dashed line), free CDDP solution (4 mg/kg; dot-dash line), gelatin solution (thin solid line), and PBS (thick solid line). (n = 6). Drugs were injected at the time indicated by arrows (day 5, 12, and 19). (C) Body weight change profiles from the first injection (day 5) up to 7 days after the third injection (day 26). CCGH (10 mg/kg; double dot-dash line), CCGH (20 mg/kg; double dashed line), CCGH (30 mg/kg; double line), free CDDP solution (2 mg/kg; dotted line), free CDDP solution (3 mg/kg; dashed line), free CDDP solution (4 mg/kg; dot-dash line), gelatin solution (thin solid line), and PBS (thick solid line). (n = 6). Drugs were injected at the time indicated by arrows (day 5, 12, and 19).