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.

Graphical abstract

A combined strategy based on thermo-sensitive PLEL hydrogel drug delivery system for bladder cancer was developed. GEM loaded PLEL hydrogel was intravesical instilled firstly, then PLEL hydrogel incorporated with CpG was injected into both groins subcutaneously to promote immune responses. Results demonstrated that drug loaded PLEL hydrogel had a sol-gel phase transition behavior and presented sustained drug release, and the PLEL-assisted combination therapy could have better tumor suppression effect and stronger immunostimulating effect in vivo.

Highlights

• •The triblock polymer PDLLA-PEG-PDLLA (PLEL) is a thermos-sensitive hydrogel for localized drug delivery.
• •Drug loaded PLEL hydrogel achieves the sustained release of drug and extend retention time of Gemcitabine in bladder.
• •Intravesical chemotherapy with immunothrapy by thermo-sensitive PLEL system improves anti-tumor effect for bladder cancer.
• •Combined therapy strategy assisted by PLEL hydrogel promote related immune response to help kill bladder cancer cells.

1. Introduction

Bladder cancer (BC) is within the top 10 most common cancers worldwide, with estimated 573 000 new diagnoses and 212 000 deaths in 2020, which incidence and mortality rate are in a tendency to increase year by year [1,2]. Approximately 75% of newly diagnosed patients belongs to non-muscle-invasive bladder cancer (NMIBC) and 25% to muscle-invasive bladder cancer (MIBC) [3,4]. As one of the common malignant tumors of the urinary system, BC brings a large burden to patients due to repeated assessments and resections, high recurrence rate and poor prognosis [5], with about 70% of NMIBCs [4] and 50% of MIBCs recur within 2 years after surgery [6], and about 20% of NMIBCs progress to MIBC [7,8]. Transurethral resection of bladder tumor (TURBT) for NMIBC is a commonly used clinical treatment, but the recurrence occurs from 48% to 70%, with the rate of 7%–40% for the progression to MIBC. Therefore, intravesical instillation after TURBT has remarkably reduced the recurrence compared with TURBT alone [[9], [10], [11]], which can effectively increase the concentration of drugs, thereby improving the therapeutic efficacy and also reducing systemic toxic side effects.

As a commonly used chemotherapy drug for the treatment of cancer, Gemcitabine (GEM) has emerged as a feasible alternative to intravesical instillation for BC. It has shown that GEM can decrease the risk of tumor progression, with the similar efficacy to pyrirubicin and epibibraxin, and significantly reduces the recurrence of high-risk NMIBC compared with mitomycin C [[12], [13], [14]]. Moreover, GEM not only kills cancer cells directly, but also causes tumor immunogenic cell death (ICD) [[15], [16], [17]], which led to the exposure of calreticulin (CRT) and the release of adenosine triphosphate (ATP) [18]. It also mediates the immune effects associated with tumor immunotherapy, which increases the sensitivity of cancer cells to tumor-specific T cells, promotes the proliferation of cytotoxic T lymphocyte (CTL) [15,19,20], reduces the number of immunoregulatory Treg cells [15,21,22] and immune-suppressive myeloid-derived suppressor cells (MDSCs) [[22], [23], [24]]. However, GEM alone is not sufficient to elicit a strong immune response.

Several clinical researches indicated that the combination of immunotherapy with chemotherapeutic agents could improve the survival rate of patients with cancer [[25], [26], [27]]. Therefore, proper combination of intravesical instillation chemotherapywith immunotherapy can exert their respective advantages and synergistic anti-tumor immune efficacy [28]. As a toll-like receptor-9 (TLR9) agonist, unmethylated cytosine-phosphate-guanine oligonucleotide (CpG-ODN) is an immune-stimulating nucleic acid that has been widely used as an immune adjuvant that promotes antigen presentation in dendritic cells (DCs) [[29], [30], [31]], which increases the expression level of DC co-stimulatory molecules, thereby presenting antigens to tumor-specific T cells [15,19,21] and inducing the secretion of cytokines such as IFN-γ, IL-12, IL-6, and TNF [[32], [33], [34]]. Immune cytokines take an important part in anti-tumor immunity, IL-6 and IL-12p70, which is the activated form of IL-12, can activate CD8⁺ T cells and enhance its killing function, IFN-γ can up-regulate the expression of MHC Ⅰ and MHC Ⅱ in DCs, improve the differentiation of T cells, and directly inhibit tumor formation, TNF also can directly kill tumor cells [21,22]. Studies on the application of CpG in bladder cancer have shown that CpG could stimulate DCs maturation and production of related cytokines, playing an anti-tumor immunity effect, and CpG-ODN 1826 belongs to CpG-B, which is more likely optimal for anti-tumor response required T cells and B cells and results in tumor-specific immunity [[35], [36], [37], [38]]. In summary, CpG combined with GEM, which can produce an associated immune response, may have a powerful effect on cancer. CpG can be used to enhance the immunostimulating effect of GEM, in addition, GEM causes ICD to induce preliminary anti-tumor immunity and produces related immune effects, and reduces the immunosuppressive effect of the tumor immune microenvironment, enhancing the sensitivity of cancer cells to CpG-induced immune response. The combination of them produces direct killing effect on the one hand, but also improves the immune effect, better inhibits the occurrence and development of tumors on the other hand [22,23,[39], [40], [41], [42]].

However, because of the spontaneous excretion of the bladder, the urine flushing and the barrier of urothelium causing the difficulty for drug to penetrate, the drug concentration in the bladder will be low and the residence time will be short, resulting in poor treatment effect, which usually requires multiple administrations, causing inconvenience in clinical practical applications, and may leads to side effects such as bladder fibrosis and urinary tract infection [[43], [44], [45]]. In addition, toll-like receptors are pattern-aware receptors (PRRs) that are commonly expressed in vivo, therefore systemic administration of toll-like receptor agonists may causes serious immune-related adverse events, further, CpG also has poor stability [46,47], which results in limited use.

With the development of biomaterials, more and more drug delivery systems have been applied to intravesical instillation [44,48], such as polymers, liposomes, gelatin and other nanocarriers to increase local drug concentration, mucosal adhesion materials to improve adhesion, nanogels to enhance their adhesion and penetration, and thermosensitive hydrogels to develop in situ gel systems to improve the residence time of drugs in the bladder [[49], [50], [51], [52], [53]]. Moreover, various biological materials are used to deliver immune preparations [54]. Among them, the environmentally responsive intelligent hydrogel drug delivery system uses topical administration to slowly release immune adjuvants at the injection site, thereby activating the relevant immune response, reducing drug toxicity, and protecting drugs from degradation [55,56].

We designed a combination therapy strategy with thermo-sensitive hydrogel system based on the insufficiency of intravesical instilled GEM and the problem of systemic administration of CpG, as well as the advantages of hydrogel drug delivery systems (Scheme 1). The triblock polymer poly (D, L-lactide)-poly (ethylene glycol)-poly (D, L-lactide) (PDLLA-PEG-PDLLA, PLEL) has good reversible sol-gel phase transition behavior. Therefore, PLEL carried drugs can be injected into the body as the form of solution at room temperature, and the gel formed at body temperature can be used as a biological skeleton material to slowly release its loaded drugs, at the same time prevents the degradation of immune adjuvants. In addition, temperature-sensitive hydrogel PLEL has the advantages of biodegradability, good biocompatibility, and convenient synthesis [57,58]. We first used PLEL loaded GEM for intravesical instillation, which continuously and slowly released GEM in the bladder to kill tumor cells. After incorporating CpG 1826, which belongs to CpG-B, into PLEL and being injected subcutaneously into both groins, CpG released extendedly in vivo, causing related immune responses, thereby producing anti-tumor effects synergized with GEM. The first evaluation found that compared with free GEM, PLEL prolonged the residence time of GEM in the bladder, improved the therapeutic effect and reduced the toxic side effects of the drug. To further improve the treatment efficacy and reduce drug toxicity, we combined GEM/PLEL with CpG/PLEL. The results demonstrated that the PLEL-assisted combination therapy strategy had better tumor suppression effect and stronger immunostimulating effect, which promoted the maturation of DCs in lymph nodes, enhanced the immune response of CD8⁺ T cells and B cells, and reduced the proportion of immunosuppressive regulatory T (Treg) cells and MDSCs, while promoting the secretion of IL-6, IL-12p70, TNF and IFN-γ. In conclusion, the combination therapy strategy assisted by PLEL can effectively extend the residence time of GEM in the bladder while reduce the toxic side effects of chemotherapy drugs to improve the efficacy, and the combined immunotherapy further improves the treatment effect, which provides a new idea for the treatment of bladder cancer.

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.

2. Materials and methods

2.1. Materials and animals

Poly (ethylene glycol) (PEG, Mn = 1500), stannous octoate (Sn (Oct)2, 95%), Gemcitabine hydrochloride (99%) were obtained from Sigma-Aldrich (Saint Louis, USA). D, l-Lactide (D, L-LA) was bought from Daigang chemicals (Jinan, China). Penicillin-streptomycin, DMEM complete medium, and fetal bovine serum (FBS) were all obtained from Gibco (USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (methyl thiazolyltetrazolium) (MTT) were purchased from Sigma-Aldrich (USA), and Live & Dead Cytotoxicity Assay Kit were purchased from Jiangsu KGI Biotechnology Company (Nanjing, China). Cy5.5 was purchased from the Beijing Fanbo Biochemicals company (Beijing, China). D-luciferin potassium salt was purchased from Dalian Meilun Biotechnology Company (Dalian, China). CpG-ODN1826 (5ʹ- TCCATGACGTTCCTGACGTT-3ʹ) was obtained from Sangon Biotech (China). 7-AAD-PE, CD45-APC Cyanine7, CD3-FITC, CD4-PE, CD8a-PerCP Cy5.5, CD19-PE Cyanine7, CD20-FITC, CD25-PE Cyanine 7, FoxP3-AF647, Gr-1-APC, CD11b-PE, CD11c-APC, CD86-PE Cyanine7, MHC I-FITC, MHC II-PE, Transcription Factor Buffer Set and Mouse Inflammation CBA Kits were purchased from BD Pharmingen (USA). ATP assay kit were purchased from Beyotime. Calreticulin (D3E6) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) was purchased from Cell Signaling Technology. All other chemical agents were analytical pure grade and used without further purification.

Female C57BL/6 mice (6–8 weeks old) were purchased from GemPharmatech Company (China) and kept under a specific-pathogen-free condition with free access to standard food and water. All animal experiments were performed following the protocols approved by the Ethics Committee of the Animal Experimental Center of State Key Laboratory of Biotherapy of Sichuan University (Checking number: 20 210 409 028).

2.2. Preparation and characterization of PLEL, GEM/PLEL and CpG/PLEL hydrogel

2.2.1. Preparation of PLEL, GEM/PLEL and CpG/PLEL hydrogel

PLEL triblock copolymer was synthesized by ring-opening copolymerization of D, L-lactide with PEG, and characterized by nuclear magnetic resonance spectroscopy (¹H NMR, Varian 400 spectrometer, USA), fourier infrared spectroscopy (Bruker, INVENIO R, Germany) and gel permeation chromatography (GPC, Agilent 110 HPLC, USA) as described previously [58]. Then PLEL copolymer was dissolved in the phosphate buffered saline (PBS, pH = 8.0) at room temperature and stirred to gain the PLEL micelle solution. Finally, GEM was dissolved directly in the PLEL solution and CpG in PBS (pH = 7.0) mixed with PLEL copolymer solution at room temperature (T = 25 °C) to obtain GEM/PLEL (20 wt%) and CpG/PLEL (10 wt%) hydrogel. All drug-loaded samples were filtered by the 0.22 μm membrane for sterilization.

2.2.2. Morphology of PLEL, GEM/PLEL and CpG/PLEL micelles

The morphology of PLEL, GEM/PLEL and CpG/PLEL micelles was observed under a transmission electron microscope (TEM, Talos F200S G2, USA). Briefly, PLEL copolymer was dissolved in PBS (pH = 8.0) at room temperature and stirred to gain the PLEL micelle solution, then GEM was dissolved directly in PLEL solution and CpG in PBS mixed with PLEL copolymer solution at room temperature (T = 25 °C). Samples were kept in 4 °C before being examined, and diluted to 0.1 wt%. Then the samples were prepared by placing a drop of micelles suspension onto a copper grid covered with nitrocellulose, negatively stained with phosphotungsticacid, dried in air, and observed under TEM.

2.2.3. The thermosensitive sol-gel phase transition behavior of PLEL, GEM/PLEL and CpG/PLEL hydrogel

We carried out a test-tube-inversion method to investigate the thermosensitive sol-gel phase transition behavior of 20 wt% PLEL, 10 wt% PLEL, GEM/PLEL (20 wt%) and CpG/PLEL (10 wt%) hydrogel. Briefly, 2 mL samples were added to the vials and heated to 37 °C, then we also cooled them to 25 °C to observe whether sol phase were generated.

2.2.4. Dynamic rheological study of PLEL, GEM/PLEL and CpG/PLEL hydrogel

The dynamic rheological experiments of 20 wt% PLEL, 10 wt% PLEL, GEM/PLEL (20 wt%) and CpG/PLEL (10 wt%) were conducted using a rheometer (HAAKE Rheostress 6000, Thermo scientific, USA). Cold samples were stored in 4 °C before being examined. Changes in storage modulus (G′), loss modulus (G″) were measured as functions of temperature from 10 to 60 °C at a heating rate of 1 °C/min. The data were collected under a controlled stress of 1Pa and at a frequency of 1.0 Hz [58].

2.2.5. In vitro degradation of PLEL hydrogel

First, 2 mL of 10 wt% and 20 wt% PLEL solution were added into the tube respectively at 37 °C for 30 min to form stable hydrogel, then 8 mL PBS (pH = 7.4) was added. They were incubated in a 37 °C incubator whist shaking (100 rpm). The PBS was changed every 7 days to maintain its relatively constant pH environment, and pH of the solution was determined before each change, while the appearance of PLEL hydrogel were observed during degradation.

2.3. The retention behavior of PLEL in bladder

We first selected 15 wt%, 20 wt%, and 25 wt% PLEL hydrogel for the related experiments, and PLEL hydrogel was incorporated with soluble Prussian blue for convenient observation. Eight-week-old female C57BL6 mice were divided into three groups randomly. Mice were anesthetized with isoflurane and in supine position on a heated table. Second, bladder was emptied by palpating the lower abdomen above the bladder gently and washed by irrigating the bladder with 100 μL PBS twice. Third, a superficial 4/0 silk purse-string suture was placed around the urethral meatus, and a lubricated 24 G Jelco angiocatheter was passed through the urethra into the bladder. Fourth, mice of three groups were intravesical instilled with different concentrations of 15 wt%, 20 wt% and 25 wt% PLEL hydrogel by attaching a 1 mL syringe preloaded with PLEL hydrogel and injecting 75 μL into the bladder lumen. Then the suture was tied off as the angiocatheter was removed to temporarily obstruct the urethra. And released after 2 min. Finally, PLEL gels in the bladder was taken at specific time points for comparison.

Subsequently, we selected 20 wt% PLEL hydrogel for further investigation. Eight-week-old female C57BL6 mice were divided into four groups at random, instilled with 25 μL, 50 μL, 75 μL, 100 μL 20 wt% PLEL separately. Then we collected the gels in the bladder at specific time points. Further, three mice in each group were placed in metabolic cages to observe changes of urine output. Mice weight changes were also observed and mice were sacrificed on day 21 and major organs (heart, liver, spleen, lung and kidney) were collected for H&E staining.

2.4. Controlled release of GEM and CpG from PLEL hydrogel in vitro and retention of GEM in bladder

The release experiments of GEM and CpG from PLEL hydrogel were conducted in Transwell co-culture system. First, 1.5 mL pre-warmed PBS was placed on the lower layer, and 200 μL of GEM/PLEL (6.7 mg/mL, 20 wt% PLEL) were placed on the upper layer and equilibrated at 37 °C for 30 min to form stable hydrogel. Then they are incubated in a 37 °C incubator whist shaking (100 rpm). 500 μL of the lower layer PBS was harvested and replaced with pre-warmed PBS at specific time points. The study of CpG/PLEL (0.5 mg/mL, 10 wt% PLEL) was performed as the same way. Finally, the released GEM and CpG were determined by the high performance liquid chromatography (HPLC) and ultraviolet spectrophotometer respectively.

In order to know the release and retention of GEM from the GEM/PLEL hydrogel in bladder, we performed further exploration in vivo. A water-soluble fluorescence dye Cy5.5 was incorporated in PLEL hydrogel as a substitution for GEM. Cy5.5 was dissolved in different volumes of PBS and 20 wt% PLEL hydrogel and transurethral instilled into bladder. Then noninvasive intravital IVIS Lumina III imaging system was used to record the fluorescence intensity at different time points.

2.5. Cellular experiments

2.5.1. In vitro cytotoxicity assays of GEM

Mouse bladder cancer cell line MB49 was purchased from the American Type Culture Collection (ATCC, Rockville, USA), which was cultured in DMEM complete medium (supplemented with 10% FBS, 1% penicillin, and 1% streptomycin) and maintained at 37 °C with humidified 5% CO₂.

The MB49 cells were seeded into a 96-well plate with 2000 cells/well and incubated for 24 h, then culture medium with different concentrations of GEM were added and cultured for another 24 h, 48 h and 72 h. MTT assays were applied to assess the cellular viability, and the half maximal inhibitory concentration (IC₅₀) was calculated.

2.5.2. In vitro effect assays of GEM loaded PLEL hydrogel

We utilized a 24-well Transwell co-culture system to examine the cytotoxicity of GEM/PLEL (20 wt%) hydrogel on MB49 cells. MB49 cells were seeded in the lower chamber of the Transwell plate with 10 000 cells/well and cultivated for 24 h. Then the PLEL solution and GEM/PLEL hydrogel solution were kept at 37 °C for 30 min to form hydrogels and then placed in medium containing well plates for further co-culture. Further, PBS, free GEM solution (0.0060 μg/mL), PLEL hydrogel, GEM/PLEL hydrogel (0.0060 μg/mL, 20 wt%) were added to the Transwell upper chamber respectively. After co-culture for 24 h, 48 h and 72 h, cell viability was detected by the MTT method. Moreover, live dead staining experiment was applied to detect the antiproliferative effects of GEM/PLEL (20 wt%).

2.5.3. In vitro evaluation of immunogenic cell death (ICD) induced by GEM loaded PLEL hydrogel

MB49 cells were cultured and the PLEL hydrogel, free GEM solution, GEM/PLEL hydrogel groups were treated as the same method described above by 24-well Transwell co-culture system. After co-culture for 24 h, 48 h and 72 h, cells and culture medium were collected for determining the concentrations of ATP intracellularly and extracellularly by using enhanced ATP assay kit. To determine CRT exposure, cells were collected and incubated with Alexa 488-conjugated antibody for 1 h at room temperature. The CRT expression was then measured by flow cytometry.

2.6. In vivo evaluation of anti-tumor effect and immune response

2.6.1. Establish of orthotopic bladder cancer model

Six-week-old female C57BL6 mice were anesthetized with isoflurane and in supine position on a heated table. Then we made a horizontal incision on the lower abdomen above the pubic bone to find the bladder and compress the bladder to empty its contents. Third, we injected 5 × 10⁵ luciferase labeled mouse bladder cancer cell line MB49 (MB49-luc, purchased from ATCC, Rockville, USA) in 25 μL into the front wall of the bladder. Finally, we fell the bladder back and sutured the abdominal wall. The mice were injected penicillin intraperitoneally and kept warm on a heating pad until it awakens. The tumor fluorescence intensity was monitored by the noninvasive intravital IVIS Lumina III imaging system.

2.6.2. In vivo anti-tumor efficiency and immune response of GEM/PLEL hydrogel

After establishing the orthotopic bladder cancer model on day −1, mice were divided into nine groups (n = 5) randomly and intravesical instilled with different concentrations of GEM/PBS and GEM/PLEL hydrogel respectively on day 0 following the method mentioned above, and the time of temporarily obstructing the urethra is 20 min. The mice were treated once a week for 4 times totally (Fig. 4A and B). Tumor burden was monitored with IVIS. Body weight and survival of mice were also recorded. Tumors and spleens were collected 3 days later for flow cytometry analysis. Mice were sacrificed at day 90.

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).

2.6.3. In vivo evaluation of anti-tumor effect and immune response of GEM/PLEL combined with different concentrations of CpG/PLEL

With the aim to further achieve better therapeutic effect and safety and based primarily on the advantages of chemotherapy combined with immunotherapy, we chose to combine 500 μg GEM/PLEL (6.67 mg/mL, 20 wt%) with immunoadjuvant CpG 1826 to improve tumor inhibition effect and reduce drug toxicity. The tumor-bearing mice were grouped (n = 8) at random and treated according to Fig. 5A. Control group was not treated, GEM/PLEL group underwent intravesical instillation of GEM/PLEL once a week for 4 times, and other groups were injected in both groins 5 days after the first intravesical instillation, with 100 μL of low concentration of CpG/PLEL (L CpG/PLEL, 0.25 mg/mL, 10 wt%), medium concentration of CpG/PLEL (M CpG/PLEL, 0.5 mg/PLEL, 10 wt%), high concentration of CpG/PLEL (H CpG/PLEL, 1 mg/mL, 10 wt%) and high concentration of CpG solution (H CpG, 1 mg/mL). At the same time, we collected inguinal lymph nodes, spleens, blood 5 days later and serum at day 7 and 14 for flow cytometry analysis. Mice were sacrificed at day 90.

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).

2.6.4. In vivo evaluation of the PLEL based combined treatment strategy over the single-agent treatment

For subsequent analysis, we selected (GEM + M CpG)/PLEL (GEM/PLEL, 6.67 mg/mL, 20 wt%; CpG/PLEL, M CpG/PLEL, 0.5 mg/PLEL, 10 wt%) treatment group as the most suitable treatment strategy in this study to conduct subsequent evaluation of anti-tumor efficacy and immune responses. The tumor-bearing mice were divided into six groups (n = 8) and treated separately (Fig. 7A). Control group was not treated, GEM/PLEL group was intravesical instilled of GEM/PLEL once a week for 4 times, M CpG group and M CpG/PLEL group were injected with M CpG or M CpG/PLEL on day 5 inguinally, GEM + M CpG group and (GEM + M CpG)/PLEL group were injected with M CpG or M CpG/PLEL in both groins 5 days after the first intravesical instillation. Similarly, we collected tumors, inguinal lymph nodes, spleens, blood and serum to perform corresponding immunoassays and mice were sacrificed at day 90.

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).

2.6.5. Histology and immunohistochemistry analysis

Bladders of all groups were collected for H&E staining, and Ki-67 staining, TUNEL staining were performed to investigate the proliferation and apoptosis of cancer cells in tumor tissue on day 28. The main organs (heart, liver, spleen, lung and kidney) of (GEM + M CpG)/PLEL group were taken for HE staining.

2.7. Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 software and results were expressed as means ± S.D. Statistical significance was set at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

3. Results and discussion

3.1. Preparation and characterization of PLEL, GEM/PLEL and CpG/PLEL hydrogel

We synthesized the triblock PEG₁₅₀₀-PDLLA₁₅₀₀-PEG₁₅₀₀ copolymer, where 1500 represents the theoretical number average molecular weights (Mn) of PEG and PDLLA blocks. It was synthesized successfully according to the ¹H NMR spectrum and FT-IR spectrum and CPG spectrum, showing that the number average molecular weights (Mn) of PLEL was 4671, which was roughly consistent with the theoretical number average molecular weights (Fig. S1).

Amphiphilic polymer PLEL could self-assemble into core-shell-like micelles in water autonomously. It was previously found that PLEL micelles are dispersed in water at room temperature and they form a physical cross-linking points under the interaction of their hydrophobic nuclei when the temperature rises to 37 °C, which might contributes to the behavior of sol-gel phase transition [57,58]. PLEL hydrogels with concentrations ranging from 10 wt% to 25 wt% have suitable phase transition temperatures and are often selected for the preparation of drug delivery systems [59]. The morphology of PLEL, GEM/PLEL and CpG/PLEL micelles (0.1 wt%, with or without drugs) was observed under TEM, which showed the micelles were dispersed as individual spherical nanoparticles at room temperature, and the particle sizes of GEM/PLEL micelles was closed to PLEL micelles, but CpG/PLEL micelles was lager than them, as a result of the higher molecular weights of CpG than GEM. (Fig. 1A–C). As illustrated in Fig. 1D and Fig. S1, the obtained 20 wt% PLEL, 10 wt% PLEL, GEM/PLEL hydrogel (20 wt% PLEL) and CpG/PLEL hydrogel (10 wt% PLEL) were in sol state at 25 °C with good fluidity, and quickly changed to gel state when the temperature rose to 37 °C, with a reversible gel-sol transition upon cooling, indicating a good phase transition behavior. Furthermore, dynamic rheological experiments were performed to evaluate the changes of the storage modulus (Gʹ) and the loss modulus (G″) during the gelation process. When G' < G″, the material is sol phase; when G' > G″, the material is gel phase; and the intersection point of the G′ curve and the G″ curve is defined as the sol-gel phase transition temperature [60]. 20 wt% PLEL, 10 wt% PLEL, GEM/PLEL (20 wt% PLEL) and CpG/PLEL (10 wt% PLEL) showed similar rheological results. G″ was greater than G′ at room or low temperature, which exhibited the viscoelastic fluid characteristics, implying good flowability and injectability, while G′ was greater than G″ as the temperature increased to around 36 °C, which showed the viscoelastic solid characteristics, indicating the formation of a crosslinked physical hydrogel (Fig. 1E–F and Fig. S1). We also performed the degradation assay of PLEL hydrogel in vitro, which displayed the gross views of the remaining 10 wt% and 20 wt% PLEL hydrogel at 0 and 4 weeks (Fig. S1). During the period of degradation, the samples swelled gradually and the surface was eroded within the 4 weeks, and degradation rate of 10 wt% PLEL was quicker than 20 wt% PLEL, which showed flowing state earlier at around 10 days. Amounts of water-soluble products were dissolved into the buffer solution in 20 wt% PLEL hydrogel, and 10 wt% PLEL became free-flowing liquids (4 weeks). PH changes of buffers was also monitored (Fig. S1), which showed gradually decrease during the entire degradation process as a result of the generation of D, L -lactide and low-molecular-weight oligomer. However, the pH was still around 5.8 at 4 weeks, indicating a mild acidic effect during degradation, which may be attributed to the rapid diffusion of acidic degradation products and high water content in hydrogel. Notably, the pH of 10 wt% PLEL group was slightly higher than 20 wt% PLEL group, which may because the amount of PLEL polymer in 20 wt% PLEL was more than 10 wt%, making the production of D, L -lactide during degradation was higher than 10 wt% [58].

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.

As promising biomaterials, thermosensitive polymeric hydrogels have been extensively investigated for sustained drug delivery [61], such as natural hydrogels, commercially available triblock copolymers Pluronics or Poloxamers like poloxamer F127, PLLA/PEG copolymers and PCL/PEG copolymers, which have their own advantages for clinical use. But shortages about them limit the further application. For example, the degradation rate of natural hydrogels is too fast, studies have shown that poloxamer maintains its gel state in vivo for a short period of time and is only suitable for short-term sustained release of the drug, and it is non-biodegradable, potentially toxic [62], besides, the gel-sol transition property of PEG-PDLLA-PEG triblock copolymers make them not suitable for encapsulation of some drugs at higher temperature and the injection of hydrogel at higher temperature is uncomfortable for patients [63], moreover, the solution of the PCEC is unstable and gel is formed at room temperature and sol-gel transition is irreversible, which affects syringeability [58]. Compared with the thermosensitive hydrogels mentioned above and based on the results in this paper and our previous research [58,59], PLEL hydrogel has its advantages for drug delivery in vivo. First, synthesis is in one safe and facile step without using toxic coupling agent. Then, it exhibits reversible and sharp sol-gel transition between room temperature and body temperature without crystallization formed during the gelation and the sol phase is stable, which could be used as an injectable in-situ formed thermogel and is convenient to administer. Both in vitro and in vivo degradation assays demonstrated PLEL has suitable degradation rate and strong gel retention ability for longer-lasting drug delivery. Further, it has acceptable biocompatibility for biomedical applications.

3.2. The retention behavior of PLEL in bladder

Different concentrations of PLEL solutions were intravesical instilled at room temperature, which formed a gel state in bladder. Fig. S2 shown that 20 wt% and 25 wt% PLEL could retain in bladder for 72 h, while 15 wt% PLEL completely degraded at 24 h. Considering the difficulty of 25 wt% PLEL to pass through sterile membrane of 0.22 μM for sterilization, we chose 20 wt% PLEL for subsequent experiments. Retention of PLEL in bladder at different volumes would be slightly different, with PLEL at 100 μL and 75 μL remaining for up to 72 h, while PLEL at 50 μL and 25 μL showing complete degradation at 48 h (Fig. S2). All the results demonstrated that the higher the concentration and volume of PLEL, the longer its residence time in bladder, which may because the polymer micelle network of the hydrogel is denser and the mechanical strength is higher with the higher polymer concentration, and the larger volumes can form bigger hydrogel, which better maintains the integrity of hydrogel and are more resistant to urine flushing and mechanical force of bladder wall [64]. Moreover, from the potential risks of using PLEL hydrogel as a drug delivery system in bladder to cause the blockage of the urethra, we investigated the physiological changes of mice. Urine output of mice in the 75 μL PLEL and 100 μL PLEL groups was lower than that of other groups on day 2, but there was no difference afterwards. Mice in each group had normal body weight changes, and no abnormalities were observed in HE-stained sections (Fig. S2). Nevertheless, there still need more investigations of clinical use to improve the patient's compliance.

3.3. Controlled release of GEM and CpG from PLEL hydrogel in vitro and retention of GEM in bladder

The cumulative release rates of GEM at 24 h and 10 d were 57.7% and 90.8%, respectively, while CpG at 24 h and 96 h were 60.8% and 94.2%, separately, and both of them were released from PLEL on day 7 completely (Fig. 2A and B). These results suggested that GEM and CpG had similar drug release behavior, with a burst release on the first day and sustained release over the next few days, which is conducive to prolonging the retention time of GEM in bladder to improve therapeutic effect, and promoting continuously stimulation of CpG to body's immune system to exert a better immune effect. According to Fig. 2C–E, cumulative release rates of Cy5.5 solution dissolved in different volumes of PBS reached more than 90% at 1 h, while in 100 μL PLEL, 75 μL PLEL, 50 μL PLEL, and 25 μL PLEL were 44.3%, 51.6%, 75.1%, and 71.4%, respectively, and Cy5.5/100 μL PLEL group and Cy5.5/75 μL PLEL group still had fluorescence signals at 24 h, revealing that PLEL extended the residence time of GEM in bladder, and the drug stayed longer as the volume of PLEL was increased, which may due to the larger the volume of PLEL solution, the bigger the hydrogel form in the bladder, and the more resistant it is to urine flushing. Moreover, there still a little fluorescence in Cy5.5/100 μL PBS group and Cy5.5/75 μL PBS group, we speculated that there would be different degrees of absorption of each mouse after instilled for 20min, and there are obvious individual differences in mice urinating, besides, the larger volume caused the larger contact area between drugs and bladder wall, resulting in the greater possibility of its absorption. All the reasons may lead to some weak fluorescence signals in some mice in the PBS group, but from Fig. 2D–E, the retention time of Cy5.5/100 μL PLEL group and Cy5.5/75 μL PLEL group were higher than Cy5.5/100 μL PBS group and Cy5.5/75 μL PBS group significantly. In conclusion, considering the retention of PLEL in the bladder, safety of intravesical instillation, and release of drugs in bladder, we selected 20 wt% PLEL and 75 μL intravesical instilled PLEL for further analysis.

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).

3.4. In vitro anti-tumor efficiency and ICD induced by GEM loaded PLEL hydrogel

As a chemotherapy agent for the treatment of bladder cancer, we first tested the proliferation inhibitory effect of GEM on bladder cancer cells MB49. IC₅₀ values of GEM after incubating for 24 h, 48 h and 72 h were 0.34 μg/mL, 0.0060 μg/mL and 0.0054 μg/mL, respectively, and decreased by about 6 times from 24 h to 48 h (Fig. S3), indicating that anti-proliferative effect of GEM on bladder cancer cells increased with the extension of time within a certain period of time. Further, we selected GEM at a concentration of 0.0060 μg/mL and used a Transwell co-culture system to mimic the sustained drug release process (Fig. 3A) to investigate the anti-proliferative effect of GEM delivered hydrogel systems. As shown in Fig. 3B, blank hydrogel PLEL has no obvious cytotoxicity on MB49 cells, proliferation inhibitory effect of free GEM and GEM/PLEL on MB49 cells at 24 h was similar, and free GEM was slightly stronger than GEM/PLEL, which mainly is attributed to the slow release of GEM from PLEL. With the extension of time, cell viability of the GEM/PLEL group was significantly lower than that of free GEM group, which was the result of continuously released of GEM from PLEL to kill MB49 Cell. We also conducted live/dead cell staining experiment, which shown the consistent results (Fig. 3C and D), it could be seen that the proportion of viable cells induced by the drug-loaded PLEL hydrogel. Moreover, as illustrated in Fig. 3E–G and Fig. S3, GEM/PLEL induced notably secretion of CRT and ATP for a longer time, signifying an important role of PLEL hydrogel to improve the level of ICD caused by GEM.

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.

3.5. In vivo anti-tumor efficiency and immune response of GEM/PLEL hydrogel

We evaluated therapeutic effects of intravesical instillation of GEM/PLEL (20 wt% PLEL). Fig. 4C and D and Fig. S4 showed that fluorescence signal of tumor in control group was rapidly enhanced and mice died due to tumor overload at about day 30. Treatment efficacy was drug concentration dependent, the higher the concentration, the better the therapeutic effects. Further, bioluminescence intensity of GEM/PLEL groups were weaker than free GEM groups. In H&E staining study, bladder tumors in 500 μg GEM/PLEL group were significantly smaller than 500 μg GEM/PBS group, and bladder tumors in 1 mg GEM/PBS group and 1 mg GEM/PLEL group were smaller than other groups (Fig. S4). It also showed apoptosis and proliferation of bladder tumor cells in Group 1–4 in Fig. S4, which combined with the bioluminescence imaging results. All the mice with GEM/PLEL had higher body weight than the corresponding dose of free GEM groups, and mice lost weight to a certain extent after being instilled GEM with recovering after a period of time (Fig. 4E), which is caused by the weakness of mice after surgery on the one hand, and the adverse effect of GEM on the other hand. Moreover, survival time in GEM/PLEL groups was longer than free GEM groups, and it was obvious from 2 mg GEM/PBS group and 2 mg GEM/PLEL group that PLEL hydrogel played a role in drug sustained release, which reduced drug toxicity to some degree (Fig. 4F). Besides, there were death of mice in later treatment process of 1 mg GEM/PBS group and 1 mg GEM/PLEL group, which may be caused by multiple intravesical instillation under anesthesia and toxic side effects of GEM. Additionally, although therapeutic effect of 2 mg GEM/PLEL group and 4 mg GEM/PLEL group was better, it could be seen from physiological conditions of mice that the GEM instilled dose of 2 mg and 4 mg was too toxic. In conclusion, PLEL hydrogel prolonged the residence time of GEM in bladder, improved the treatment efficacy of GEM to effectively inhibited the occurrence and development of BC and reduced toxic side effect of GEM to some degree. Notably, the safety of 500 μg GEM/PLEL instilled therapy was better than 1 mg GEM/PLEL, while the effectiveness of 1 mg GEM/PLEL instilled treatment was better than 500 μg GEM/PLEL group.

Based on the above results, we chose 500 μg GEM/PBS and 500 μg GEM/PLEL to conduct related immunoassays. It showed that both the ratio of CD8⁺ T cells in tumor and spleen of 500 μg GEM/PLEL group was higher than other groups (Fig. 4G–H and Fig. S5), and CD4⁺ FoxP3⁺ CD25⁺ and CD11b⁺ GR-1⁺ were lower (Fig. 4I-L and Fig. S5), also the CRT⁺ in tumor increased remarkably (Fig. 4M and Fig. S5). All the results suggested that PLEL hydrogel elevated the related immune response of GEM effectively, to cause ICD, activate the proliferation of CD8⁺ T cells and reduces the number of Treg cells and MDSCs.

3.6. In vivo evaluation of anti-tumor effect and immune response of GEM/PLEL combined with different concentrations of CpG/PLEL

In order to further achieve better therapeutic effect and safety, we chose chemotherapy combined with immunotherapy to treat BC. As illustrated in Fig. 5B–C and Fig. S6, tumor fluorescence signal in (GEM + M CpG)/PLEL group, (GEM + H CpG)/PLEL group, and GEM/PLEL + H CpG group was significantly lower, and (GEM + M CpG)/PLEL group was close to GEM/PLEL + H CpG group, indicating that therapeutic effect of GEM/PLEL intravesical instillation combined with medium or high doses of CpG/PLEL was superior. H&E staining exhibited that mice bladder tumors of that 3 groups were significantly reduced (Fig. S6), and trend of apoptosis and proliferation of bladder tumor in Group 1–3 was consistent with the above results (Fig. S6). Body weight dropped by about 1–2 g after subcutaneous injection of free CpG or CpG/PLEL in the groin of mice, but it recovered after a few days, and except for control group, body weight of other groups were higher than GEM/PLEL group (Fig. 5D), which revealed that subcutaneous injection inguinally of CpG and CpG/PLEL would produce recoverable systemic adverse effects, and the adverse reactions of a single instillation of GEM/PLEL combined with a single injection of CpG and CpG/PLEL were milder than multiple intravesical instillation of GEM/PLEL. Survival period of (GEM + H CpG)/PLEL group was longer than GEM/PLEL + H CpG group, indicating that PLEL hydrogel loaded with CpG could reduce the drug toxicity of CpG to a certain extent (Fig. 5E). In summary, the use of PLEL hydrogel to carry CpG could reduce the drug toxicity of CpG, and the therapeutic strategy of intravesical instillation of GEM/PLEL combined with medium or high doses of CpG/PLEL could directly kill tumor cells.

Further, we detected the expression of relevant immune cell markers and cytokines. The ratio of CD11c⁺, CD11C⁺ CD86⁺, CD11c⁺ MHC Ⅰ⁺, CD11c⁺ MHC Ⅱ⁺ (Fig. 6A–E) in lymph nodes, CD3⁺, CD8⁺ CD4⁻, CD8⁺ CD4^-/CD4⁺ CD8⁻in lymph nodes and blood (Fig. S7), and CD8⁺ CD4⁻ in spleen (Fig. 6F, H) of (GEM + M CpG)/PLEL group, (GEM + H CpG)/PLEL group, GEM/PLEL + H CpG group increased remarkably. Besides, proportion of that 3 groups was reduced (Fig. 6G, I). CD8⁺ in spleen of GEM/PLEL group was different from Control group, CD4⁺ FoxP3⁺ CD25⁺ was also decreased by about 30% which may because GEM can stimulate CD8⁺ T cells activation and also inhibit the expression of Treg [15,65]. As illustrated in Fig. 6J-M, concentrations of IL-12p70, IL-6, IFN-γ, and TNF in (GEM + M CpG)/PLEL group, (GEM + H CpG)/PLEL group, and GEM/PLEL + H CpG group were all significantly increased on day 7, and the concentration of IFN-γ in GEM/PLEL group was higher than control group, which may because GEM can stimulate IFN-γ secretion [21]. On day 14, the concentrations of all cytokines decreased (Fig. 6N-Q), but concentrations in (GEM + M CpG)/PLEL group and (GEM + H CpG)/PLEL group were remarkably higher. All the experiment results demonstrated that GEM/PLEL combined with medium or high concentrations of CpG carried by PLEL could accordingly promote the proliferation and activation of DCs, improve the proportion of mature T lymphocytes, activate the proliferation and differentiation of CD8⁺ T cells, down-regulate the ratio of Treg cells, and stimulate the secretion of related cytokines for a longer time.

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).

3.7. In vivo evaluation of the PLEL based combined treatment strategy over the single-agent treatment

Based on the above work, we selected the (GEM + M CpG)/PLEL treatment group as the most suitable treatment strategy in this study, and carried out follow-up evaluations. Tumor fluorescence intensity in (GEM + M CpG)/PLEL group was lower than other groups, demonstrating that the effect of PLEL hydrogel adjuvant chemotherapy combined with immunotherapy excelled to chemotherapy or immunotherapy alone (Fig. 7A–B). Notably, bioluminescence intensity of GEM/PLEL group from day 11 to day 25 was higher than M CpG/PLEL group, indicating that directly killing tumor cells by GEM may have a certain role in cancer treatment compared with the delayed onset of immunotherapy. Besides, tumor inhibitory effect of M CpG/PLEL group was slightly better than free M CpG group, which suggested that PLEL hydrogel could improve the immune effect of CpG and tumor inhibitory ability. H&E staining showed that bladder tumors in (GEM + M CpG)/PLEL group were significantly reduced (Fig. S8), and the trend of the apoptosis and proliferation of the bladder tumor cells of Group 1–4 was consistent with the results above (Fig. S8). Mice lost body weight after each therapy but regained it few days later, and the survival period of mice in (GEM + M CpG)/PLEL group was longer than other groups (Fig. 7C and D). Moreover, H&E staining results shown the safety of GEM/PLEL combined with subcutaneous injection in the groin of M CpG/PLEL (Fig. S9).

The proportion of related immune markers of DCs in tumor (Fig. 8A–C, Fig. S10) and lymph nodes (Fig. 8D–F, Fig. S10), T cells in tumor (Fig. 8G, Fig. S11), spleen (Fig. 8H, Fig. S11), lymph nodes (Fig. 8I, Fig. S11) and blood (Fig. 8J, Fig. S11), B cells in tumors (Fig. 8K, Q) in (GEM + M CpG)/PLEL group increased notably, besides, related ratio of M CpG/PLEL group compared with M CpG group, and (GEM + M CpG)/PLEL group compared with GEM + M CpG group was also clearly elevated. Moreover, CD4⁺ FoxP3⁺ CD25⁺ ratio of tumor (Fig. 8L, R) and spleen (Fig. 8M, S), Gr-1⁺CD11b⁺ ratio of tumor (Fig. 8N, T) and spleen (Fig. 8O, U) in (GEM + M CpG)/PLEL group was reduced. Also, the CRT + ratio in tumor of (GEM + M CpG)/PLEL group increased remarkably (Fig. 8P, V). Moreover, as shown in Fig. S10, concentrations of IL-6, TNF and IFN-γ in serum of (GEM + M CpG)/PLEL group were higher on day 7, and the concentration of IL-12p70 in (GEM + M CpG)/PLEL group was higher than Group 1–4. The high concentration of IL-12p70 in GEM + M CpG group may be caused by the higher concentration of free CpG after administration, but there was no significant difference from (GEM + M CpG)/PLEL group. At day 14, concentrations of all cytokines decreased while the concentrations of cytokines in M CpG/PLEL group and (GEM + M CpG)/PLEL group were still higher. All above findings demonstrated that intravesical instillation of GEM/PLLE combined with M CpG/PLEL treatment could activate ICD, stimulate DC maturation, promote proliferation and differentiation of CD8⁺ T cells and B cells, reduce the proportion of Treg cells and MDSCs, and increase concentrations of IL-12p70, IL-6, IFN-γ, and TNF in mouse serum for a longer time.

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).

In a word, compared with surgery, which has high recurrence rate and declines life quality of patients [10], and systemic administration with potential toxic side effects [66,67], our study used intravesical instillation with GEM combined subcutaneous injection of immune adjuvant CpG in groins to effectively reduce the recurrence rate and possible toxic side effects caused by systemic administration of GEM and CpG, at the same time realized the slow release of drugs in vivo. Intravesical instillation is currently a common therapeutic strategy for BC, but there are still some problems such as short drug residence time, low penetration, low concentration of the drug in bladder, and inconvenience caused by the need for multiple administrations [43,44]. Some methods are designed for these problems, include intravesical radiofrequency-induced thermo-chemotherapy effect (RITE) [68], electromotive drug administration (EMDA) [69], the application of chemical agents [48] and hyaluronidase [70] to enhance the permeability of drugs, and various drug delivery systems to elevate drug residence time in bladder or improve the ability of drugs to penetrate bladder mucosa [44]. Compared with these pro-penetrating strategies or prodrug strategies like EMDA, RITE, which has the operational complexity and inconvenience, this research used temperature-sensitive hydrogel PLEL as drug delivery system to improve retention time of GEM in bladder, which is not only facile for administration, but also effectively shortens the blocking time when instilling drugs into bladder. Further, it avoids repeated intravesical instillation, which may cause urinary tract infections. Finally, compared with single intravesical instilled chemotherapy or immunotherapy, the combination therapeutic strategy of this study is based on the advantages of chemotherapy combined with immunotherapy, reducing the dose of GEM, while exerting the synergistic relevant anti-tumor immune effect of GEM and CpG to better achieve the purpose of killing cancer cells. This provides a new idea for the clinical treatment of bladder cancer by chemotherapy combined with immunotherapy based on the PLEL hydrogel system.

4. Conclusion

Overall, we adopted combined therapy strategy by incorporating intravesical chemotherapy with immunotherapy, using the advantages of killing the tumor cells directly and producing certain immune response to treat bladder cancer. We have developed a thermo-sensitive in situ formed PLEL hydrogel system to delivery GEM and CpG. The drug loaded PLEL hydrogel exhibited a quick and reversible sol-gel transition between room temperature and physiological temperature, with the characteristic of sustained drug release. Series studies demonstrated that the application of PLEL hydrogel as a vehicle for topical administration was of great help to prolong the residence time of local drug in bladder, enhance the ability of drugs to kill cancer cells, improve immunostimulating effect of immune adjuvants, and reduce adverse side effects. Consequently, the drug loaded PLEL hydrogel system showed great potential for intravesical instillation and immunotherapy for bladder cancer.

Ethics approval

All animal experiments were performed following the protocols approved by the Ethics Committee of the Animal Experimental Center of State Key Laboratory of Biotherapy of Sichuan University (Checking number: 20 210 409 028), and were carried out in compliance with all relevant ethical regulations.

CRediT authorship contribution statement

J. Liu: Conceptualization, Methodology, Investigation, Data curation, Software, Validation, Formal analysis, Writing - original draft. T.Y. Yang: Investigation. L.Q. Dai Methodology, Investigation. K. Shi Methodology. Y. Hao: Methodology. B.Y. Chu: Methodology. D.R. Hu: Methodology. Z.W. Bei: Investigation. L.P. Yuan: Investigation. M. Pan: Investigation. Z.Y. Qian: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no competing interests in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.