Metformin-Loaded Chitosan Hydrogels Suppress Bladder Tumor Growth in an Orthotopic Mouse Model via Intravesical Administration

Abstract

Our previous study found that the intravesical perfusion of metformin has excellent inhibitory effects against bladder cancer (BC). However, this administration route allows the drug to be diluted and excreted in urine. Therefore, increasing the adhesion of metformin to the bladder mucosal layer may prolong the retention time and increase the pharmacological activity. It is well known that chitosan (Cs) has a strong adhesion to the bladder mucosal layer. Thus, this study established a novel formulation of metformin to enhance its antitumor activity by extending its retention time. In this research, we prepared Cs freeze-dried powder and investigated the effect of metformin-loaded chitosan hydrogels (MLCH) in vitro and in vivo. The results showed that MLCH had a strong inhibitory effect against proliferation and colony formation in vitro. The reduction in BC weight and the expression of tumor biomarkers in orthotopic mice showed the robust antitumor activity of MLCH via intravesical administration in vivo. The non-toxic profile of MLCH was observed as well, using histological examinations. Mechanistically, MLCH showed stronger functional activation of the AMPKα/mTOR signaling pathway compared with metformin alone. These findings aim to make this novel formulation an efficient candidate for managing BC via intravesical administration.

Keywords: bladder cancer; chitosan hydrogels; intravesical administration; metformin; orthotopic model.

Figure 1

Physical characteristics of Cs hydrogels. (A) Concentration-dependent transmittance changes in the Cs hydrochloride solution at a wavelength of 600 nm. Data are presented as the mean ± standard deviation of three repetitions. (B) After placing it in a vacuum at low temperature, the clear solution (left) of regular Cs powder dissolved in 1% hydrochloric acid (v/v) became a uniform of lyophilized Cs powder (right). (C) Lyophilized Cs powder could be dissolved in Met physiological saline to form even MLCH (right) whereas regular Cs powder could not (left). (D) Micro-structural differences between the regular and lyophilized powder. The images were taken using an inverted microscope. (E) Results of the Tyndall effect assay on MLCH and Met. (F) Infrared spectra of MLCH, Met, and Cs hydrogels. (G) ¹H NMR spectra of MLCH, Met, and Cs hydrogels. The solvent used for ¹H NMR spectra is D₂O. (H) The prepared Cs lyophilized powder showed a loose and porous three dimensional network structure so it has a high metformin loading capacity. MLCH, metformin-loaded Cs hydrogel; Met, metformin.

Figure 2

Characterization of MLCH. (A) The UV spectra of MLCH showed the characteristic absorption peak of Met at 233 nm. (B) After freeze-drying, MLCH had a pH of ~5, which satisfied the requirements for bladder perfusion. (C) MLCH had a positive ζ potential, which is beneficial for adhesion to the bladder mucosa. MLCH, metformin-loaded Cs hydrogel.

Figure 3

Different concentrations of Cs hydrogels and Met synergistically inhibited bladder cancer growth in vitro. (A) MB49 cells were treated with Cs hydrogel for 2 h. Then, the cells were cultured for 48 h after removal of the Cs hydrogels. Images were taken using an inverted microscope (Red arrows indicate that Cs shell adhered to the cells). (B,C) MB49 cells were treated with Cs or MLCH for 2 h. Then, the cells were cultured for 48 h after removal of the Cs or MLCH, the viability of MB49 cells was assessed by MTT assay. (D,E) MB49 cells were treated with Cs or MLCH for 2 h (twice in 1 week) and then cultured with a complete medium; the viability of MB49 cells was assessed by colony formation assays. (F,G) Quantification of the colony formation assays. The wells were scanned at a wavelength of 550 nm. Data are presented as the mean ± standard deviation of five independent experiments. * p < 0.05, ** p < 0.01, ns = not significant. MLCH, metformin-loaded chitosan hydrogel; Ctrl, control; Cs, chitosan alone; Met, metformin alone.

Figure 4

MLCH inhibits BC growth in vivo when administered intravesically. (A) Bioluminescent images of the mouse orthotopic implantation model following the different treatments. (B) Total weight of mice during the entire procedure. (C) Images of the explanted bladder tissues. (D) Weights of the mouse bladders, including those of mice that died before the end of the experiment. Data are presented as the mean ± standard deviation of five independent experiments. ** p < 0.01, *** p < 0.001, ns = not significant. BC, bladder cancer; MLCH, metformin-loaded chitosan hydrogel; Ctrl, control; Cs, chitosan alone; Met, metformin alone.

Figure 5

Inhibitory effect of MLCH on AMPKα signaling pathways in bladder cancer. (A) Western blot analysis of p-AMPKα, p-mTOR, p-P70s6k, and p-4Ebp1 protein expression in bladder tissues from the mice in the five different groups. (B) Relative levels of p-AMPKα, mTOR, P70s6k, and 4Ebp1 are shown as the mean ± the standard error of the mean. * p < 0.05, *** p < 0.001. (C,D) Histological sections of the bladder tissues were subjected to hematoxylin and eosin staining or were used for immunohistochemistry analysis of Ki67 expression to confirm the presence or absence of tumors. MLCH, metformin-loaded chitosan hydrogel; Ctrl, control; Cs, chitosan alone; Met, metformin alone; p-, phosphor; P70s6k, ribosomal protein S6 kinase; 4Ebp1, eukaryotic translation initiation factor binding protein 1.

Figure 6

Pathological examination of the vital organs. (A) Pathological examination of the vital organs in the five groups of mice. Hematoxylin and eosin staining were used for pathological examination of tissue sections of the heart, lung, liver, and kidneys. (B) Observation of the inner wall of the bladder showed Cs was retained for ≥24 h. The bladder tissue was assessed using hematoxylin and eosin staining following the administration of MLCH for 24 h to observe the retention of Cs. MLCH, metformin-loaded chitosan hydrogel; Ctrl, control; Cs, chitosan alone; Met, metformin alone.

Figure 7

Scheme of the MLCH delivery system and mechanism of action. (A) MLCH exerts a beneficial therapeutic effect in an orthotopic mouse model of bladder cancer via extending the metformin retention time. (B) MLCH activates AMPKα and down-regulates the mTOR signal pathway, thereby inhibiting P70s6k and 4Ebp1 phosphorylation to block the tumor cell growth.