Intravesical chemotherapy synergize with an immune adjuvant by a thermo-sensitive hydrogel system for bladder cancer

Abstract

Surgical resection remains the prefer option for bladder cancer treatment. However, the effectiveness of surgery is usually limited for the high recurrence rate and poor prognosis. Consequently, intravesical chemotherapy synergize with immunotherapy in situ is an attractive way to improve therapeutic effect. Herein, a combined strategy based on thermo-sensitive PLEL hydrogel drug delivery system was developed. GEM loaded PLEL hydrogel was intravesical instilled to kill tumor cells directly, then PLEL hydrogel incorporated with CpG was injected into both groins subcutaneously to promote immune responses synergize with GEM. The results demonstrated that drug loaded PLEL hydrogel had a sol-gel phase transition behavior in response to physiological temperature and presented sustained drug release, and the PLEL-assisted combination therapy could have better tumor suppression effect and stronger immunostimulating effect in vivo. Hence, this combined treatment with PLEL hydrogel system has great potential and suggests a clinically-relevant and valuable option for bladder cancer.

Keywords: Bladder cancer; Immunotherapy; Intravesical chemotherapy; Localized drug delivery; Thermo-responsive hydrogel.

Graphical abstract

Scheme 1

Schematic illustration of the PLEL-based combination strategy for bladder cancer. (A) The preparation of GEM/PLEL and CpG/PLEL hydrogels. (B) Chemo-immuno therapy to treat bladder cancer. Step 1: GEM/PLEL intravesical instillation. Step 2: CpG/PLEL subcutaneous injection in bilateral groins to boost tumor-specific immunity.

Fig. 1

Morphology and sol-gel phase transition behavior of GEM/PLEL (20 wt%) and CpG/PLEL (10 wt%) hydrogel. TEM images of PLEL micelles (A), GEM/PLEL micelles (B), CpG/PLEL micelles (C) at room temperature (0.1 wt%). Scale bar: 100 nm of (A) and (B), 200 nm of (C). (D) Reversible sol-gel phase transition process of GEM-loaded and CpG-loaded thermosensitive PLEL hydrogel. Changes in storage (G′) and loss modulus (G″) for GEM/PLEL hydrogel (20 wt%) (E) and CpG/PLEL hydrogel (10 wt%) (F) as a function of temperature.

Fig. 2

Drug release behavior from the PLEL hydrogel in vitro and retention of GEM in mice bladder. The cumulative released drug ratio of GEM from GEM/PLEL (20 wt%) (A) and CpG from CpG/PLEL (10 wt%) (B). (C) Representative images of extended release and retention of Cy5.5 as a substitute of GEM in the bladder from PLEL hydrogel. (D) Quantified bioluminescence intensity of Cy5.5. (E) In vivo cumulative release of Cy5.5 from PLEL hydrogel. Data are presented as mean ± sd (n = 5).

Fig. 3

In vitro cell viability and apoptosis and ICD of MB49 cells after treatment with different treatments. (A) Schematic illustration of Transwell co-culture system for a drug depot. (B) Cell viability of MB49 cells after incubating with blank PLEL hydrogel, free GEM and GEM/PLEL hydrogel. (C) Fluorescent morphology images of MB49 cells exposed to various treatments after live and dead cell staining. Scale bar: 100 μm. (D) Quantitative analysis of live/dead staining for each group. Representative flow cytometric analysis images (E) and quantification of expression levels (F) of CRT. (G) The ratio of extracellular ATP concentration to intracellular ATP concentration. Data are presented as mean ± sd (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4

In vivo anti-tumor efficiency and immune response of GEM/PLEL hydrogel on orthotopic bladder cancer model. (A) The process of intravesical instillation. (B) Schematic diagram of orthotopic bladder cancer model establishment and therapy schedule. Bioluminescence images (C) and quantified bioluminescence intensity (D) of mice treated with different formulations. Body weight (E) and survival curve (F) of mice in each group. The gray box represents the death of the mouse and the blue arrows denote time of administration. Data are presented as mean ± sd (n = 5). Quantification of surface markers on T cells (G–H), Treg cells (I–J), MDSCs (K–L) in spleen and tumor, and CRT (M) in tumor of Group 1–3. Data are presented as mean ± sd (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G1: Control, G2: 500 μg GEM/PBS, G3: 500 μg GEM/PLEL, G4: 1 mg GEM/PBS, G5: 1 mg GEM/PLEL, G6: 2 mg GEM/PBS, G7: 2 mg GEM/PLEL, G8: 4 mg GEM/PBS, G9: 4 mg GEM/PLEL).

Fig. 5

Efficacy of GEM/PLEL combined with different concentrations of CpG/PLEL. (A) Schematic diagram of therapy schedule. Bioluminescence images (B) and quantified bioluminescence intensity (C) of mice treated with different formulations. Body weight (D) and survival curve (E) of mice in each group. The gray box represents the death of the mouse. The blue arrows denote time of intravesical instillation of GEM/PLEL and the red arrow denotes the administration of CpG/PLEL or free CpG. Data are presented as mean ± sd (n = 5). (G1: Control, G2: GEM/PLEL, G3: (GEM + L CpG)/PLEL, G4: (GEM + M CpG)/PLEL, G5: (GEM + H CpG)/PLEL, G6: GEM/PLEL + H CpG).

Fig. 6

The related immune response after different treatment. (A) Representative flow cytometric analysis images of DCs. (B–E) Quantification of expression levels of CD11c, CD86, MHC I, MHC Ⅱ on the surface of DCs. (F–G) Representative flow cytometric analysis images of T cells and Treg cells in spleen. (H–I) Quantification of surface markers on T cells and Treg cells. Data are presented as mean ± sd (n = 3). The concentrations of IL-12p70, IL-6, IFN-γ, TNF at day 7 (J–M) and day 14 (N–Q) after injection of free CpG or CpG/PLEL. Data are presented as mean ± sd (n = 4–5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G1: Control, G2: GEM/PLEL, G3: (GEM + L CpG)/PLEL, G4: (GEM + M CpG)/PLEL, G5: (GEM + H CpG)/PLEL, G6: GEM/PLEL + H CpG).

Fig. 7

Anti-tumor efficacy of (GEM + M CpG)/PLEL treatment. Bioluminescence images (A) and quantified bioluminescence intensity (B) of mice in each group. Body weight (C) and survival curve (D) of mice in each group. The gray box represents the death of the mouse. The blue arrows denote time of intravesical instillation of GEM/PLEL and the red arrow denotes the administration of CpG/PLEL or free CpG. Data are presented as mean ± sd (n = 5). (G1: Control, G2: GEM/PLEL, G3: M CpG, G4: M CpG/PLEL, G5: GEM + M CpG, G6: (GEM + M CpG)/PLEL).

Fig. 8

The related immune response by combined therapy with PLEL hydrogel. (A–F) Quantification of expression levels of CD11c, CD86, MHC Ⅱ on the surface of DCs in tumor and lymph nodes. (G–J) Quantification of surface markers on T cells in tumor, spleen, lymph nodes and blood. Representative flow cytometric analysis images of B cells in tumor (K), Treg cells (L–M) and MDSCs (N–O) in tumor and spleen, CRT (P) in tumor. Quantification of expression levels of B cells in tumor (Q), Treg cells (R–S), MDSCs (T–U) in tumor and spleen, CRT (V) in tumor. Data are presented as mean ± sd (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G1: Control, G2: GEM/PLEL, G3: (G1: Control, G2: GEM/PLEL, G3: M CpG, G4: M CpG/PLEL, G5: GEM + M CpG, G6: (GEM + M CpG)/PLEL).