| Objective:In1976, Bacillus Calmette-Gue’rin (BCG) was initially proposed to treat superficial bladder cancer by Morales and colleagues. Nowadays intravesical BCG instillations have proven to be the most successful adjuvant treatment for patients with non-muscle-invasive bladder cancer (NMIBC). However,30-50%of patients will fail initial BCG therapy. Some patients receiving intravesical BCG therapy may suffer severe adverse effects, leading to cessation of BCG therapy. To improve its antitumor effects and/or reduce the side effects, interesting approaches have been developed, such as genetically engineered BCG secreting relevant cytokines, administering various inflammatory cytokines and chemokines in addition to intravesical BCG and instillation of mycobacterial cell wall or its extracts as an alternative to BCG, etc.Responses to intravesical therapy are directly proportional to drug concentration rather than drug dose. The duration of BCG exposure is crucial for therapeutic response. However, the BCG exposure at the urothelium rarely lasts beyond the first voiding of urine after instillation, and often patients do not completely respond or the response is highly variable among patients. Strategies such as complete bladder emptying just before dose administration and restricted fluid intake have limited value. Inadequate conventional BCG delivery justifies the search for new vehicles to overcome the limitations inherent in intravesical route of BCG administration. Thermosensitive hydrogel based on chitosan (CS) and (3-glycerophosphate (GP) is currently a promising candidate. It has great potential in various applications, such as drug delivery, cell encapsulation and tissue engineering. Aqueous solutions of CS/GP form a free-flowing solution at room temperature and become a viscous hydrogel at body temperature. Upon incorporation of pharmaceutical agents, the hydrogel system could act as sustained drug release depot in situ. The therapeutic benefit of this system was demonstrated by delivering an anti-inflammatory drug in an interstitial cystitis rat model. In the light of the interesting application, we proposed this polymer could extend the residence time of BCG in bladder. Thereby increase in efficacy of BCG would be expected.Thermosensitive hydrogels form in bladder could still get washed out of the bladder during voiding, necessitating creative methods to make them resistant to excretion. An interesting study by Leakakos et al. attracted our attentions. Prolonged retention and targeting of doxorubin in swine bladder were achieved by instilling magnetic targeted carriers (MTC) composed of metallic iron and doxorubicin adsorbed onto activated carbon via an externally applied magnetic field to the bladder. Inspired by the experiment, we considered that magnetic particles could be utilized to prevent the thermosensitive hydrogels from being washed away during urine voiding. Fe3O4magnetic nanoparticle (Fe3O4-MNP) is a kind of magnetic ferric oxide and exhibits good superparamagnetic behavior. The nanoparticles are in preclinical studies as promising drug delivery formulations. When incorporated into thermosensitive hydrogel, they could respond to an external magnetic field and ensure attachment to the bladder wall.Thus, considering the features of formulations, a novel in situ gel system that contained Fe3O4-MNP and BCG dispersed within the thermosensitive hydrogel based on CS and GP for BCG instillation was developed. It was expected that the gel system would extend the duration of BCG in bladder and increase the antitumor efficacy. Motivated by these considerations, the present study was to investigate the feasibility of the gel system, characterize its bladder retention and evaluate the local immune activity.The aim of this study was to develop a magnetic thermosensitive hydrogel as intravesical BCG delivery system, which was formulated with chitosan (CS), β-glycerophosphate (GP) and Fe3O4magnetic nanoparticle (Fe3O4-MNP).Methods:1. Preparation of Fe3O4-BCG-CS/GP and Detection of its Characterization1.1Preparation of CS and GP solution (CS/GP) and CS/GP solution loaded with Fe3O4-MNP and BCGThe CS/GP solution was prepared as follow. Briefly, the CS powder was dissolved in0.1M hydrochloric acid under stirring for2h at room temperature. The insoluble particles in the chitosan solution were removed by filtration. The GP solution was prepared by dissolving the GP powder in distilled water. The two solutions were chilled at4℃for10min, followed by addition of the GP solution to the CS solution dropwise under stirring at4℃until a clear and homogeneous CS/GP solution was formed. The mixture for instillation was prepared by adding Fe3O4/MNPs and BCG powder to the CS/GP solution under stirring and then dispersing with ultrasound.1.2Gelation time of CS and GP solution and Fe3O4-BCG-CS/GP mixtureThe gelation time of CS/GP solution at37℃was determined by a typical method. The CS/GP solution was added to a centrifuge tube and heated in a water bath (37℃). At fixed time intervals, the tube was taken out and inverted. The sample that had not flowed for30s in the inverted tube was considered to form a hydrogel, and the time point was recorded as the gelation time. The effect of CS and GP concentration on gelation time was investigated.The resultant Fe3O4-BCG-CS/GP mixture was also incubated at37℃, and the gelation time was measured.1.3In vivo gelation and frozen section examination The gelation in vivo was investigated in3female Wistar rats. Before instillation, each rat was anesthetized through intraperitoneal injection and a shortened3F epidural anesthesia catheter was inserted through the urethra to the bladder lumen. The remaining urine was aspirated and then0.1ml Fe3O4-BCG-CS/GP mixture containing1mg of BCG was instilled via the catheter. The time to sacrifice depended on the gelation time determined before in vitro. The bladder neck was ligated and the whole bladder was removed for cryosection. The10μm thick sections were stained with hematoxylin-eosin staining (HE staining).1.4Digital microscope and scanning electron microscopy (SEM)Digital microscope and SEM was employed to display the surface morphology of the Fe3O4-BCG-CS/GP gel. The freeze-dried samples were first examined by digital microscope and then fixed on adhesive carbon tapes, and then gold coated with a Hitachi HUS-5GB sputter coater. The SEM images were obtained by using a Hitachi H-8010scanning system (an attachment of H-800transmission electron microscope) with the acceleration voltage of75to100kV.1.5In vivo evaluation of retentionThe retention of the Fe3O4-BCG-CS/GP gel in bladder was determined in36female rats. Water intake of rats was banned2h before and after instillation. The mixture was delivered into bladder according to Section1.3. All rats were maintained in a magnetic field of4k G The rats were sacrificed successively at determined time point after instillation (three rats at each time point). The bladders obtained from different time points were submitted to cryosection as described before. The sections were stained with HE staining, while adjacent frozen sections were stained with acid-fast staining, followed by HE staining, to display the BCG retained in the gel.2. Study of local immune activity2.1Study design The study comprised24female Wista rats. All rats were treated with0.05%BBN in drinking water for8weeks to induce the autochthonous development of bladder cancer. After10weeks,24rats were divided into four groups according to the treatment administered intravesically:group1, a control group given0.1ml phosphate-buffered saline (PBS); group2, a treatment group given lmg/O.lml BCG; group3, a CS/GP solution control group given0.1ml; group4, a treatment group given0.1ml Fe3O4/BCG-CS/GP mixture (containing1mg BCG). All groups received intravesical treatment weekly for six times.2.2Antitumor activityAfter20weeks, the rats were killed under anesthesia. At necropsy, the urinary bladders of rats were removed and inspected for grossly visible lesions. The number of tumors per rat and the volume of each tumor were recorded to calculate the incidence of tumors per group and the mean tumor volume per rat. A tumour was defined as a lesion of>0.5mm in diameter. Tumor volume was calculated by the following equation: V (mm3)=1/2×a×b2, where a is the longest diameter and b is the shortest diameter.2.3Urinary cytokines analysisAfter treating intravesically, rats were transferred to metabolic cages for urine collection. Urine was collected daily beginning at24h after the first therapeutic instillation, totally6times, and then collected once just before sacrificing. Each collection lasted for2h. Urine samples were collected in tubes on ice containing protease inhibitors, centrifuged and stored at-80℃until batch analysis via ELISA. Total amounts of urinary cytokines were evaluated by area under the curve (AUC). Mean AUC for urinary cytokines was calculated and normalized to baseline to measure immune response of rat bladder.2.4Immunohistochemistry studyThe bladders were fixed in4%paraformaldehyde for at least24h, embedded in paraffin, and submitted for immunohistochemistry. Sections were cut transversely through the mid-portion of the bladder. Infiltrating CD4+T cells were detected in the submucosa of bladder wall by immunohistochemistry with anti-CD4antibodies.2.5Statistical analysesAll determinations were made in triplicate and each result expressed as the mean±SEM. Statistical significance was determined either by one way ANOVA or by the Student t test as appropriate using Prism5. Statistical assessments were two-tailed and considered significant at P<0.05.Results:1. Characterization of CS/GP solution and Fe3O4-BCG-CS/GP mixtureBoth CS and GP concentration influenced the gelation time, which showed a decreased tendency with increase in CS and GP concentration. A CS/GP solution containing2%(w/v) of CS and10%(w/v) of GP and corresponding Fe3O4-BCG-CS/GP mixture containing0.6%(w/v) of Fe3O4-MNP and1%(w/v) of BCG was used in the following studies. The gelation time was relatively fast (6min) at37℃. In addition, the gelation time of Fe3O4/BCG-CS/GP mixture was also6min, indicating that Fe3O4-MNP and BCG did not have any effect on the thermogelling profile of the CS/GP gel.When put in saline, the gel was attracted in magnetic field and showed good magnetic response. Magnetic field gradient was achieved with a permanent magnet placed beside the beaker.2. Frozen section examination and SEMThe sol-gel transformation was also achieved in vivo. The results of frozen section examination showed that the gel revealed porous microstructures with different chamber diameters and distributions. The inner structure of the gel was well exhibited through the method of cryosection. The pores in the gel would provide pathways for medium flow and be beneficial to BCG release.Similar structure of porous network was observed through SEM micrographs. The inner surface of interconnected pores is rough, owing to the introduction of Fe3O4-MNP and BCG which are adhere to or embedded in the gel matrix.3. In vivo evaluation of retentionCryosection and staining was used to evaluate the effect of the gel and BCG on residence time in bladder. After intravesical administration, the gel formed in bladder and attached to the bladder wall. With the extension of intravesical exposure time, the excretion, degradation and erosion resulted in loss of the gel. The remaining gel matrix became attenuated and swelled, which could lead to release of entrapped BCG. The remanent gel and BCG could still be observed48h after instillation.4. Enhanced antitumor activity by Fe3O4-BCG-CS/GP gelAll rats completed the20-week protocol. Tumors from group1and group3showed large tumors, but group2and group4caused marked inhibition in bladder tumor growth. There were no statistical differences between group1and group3in both tumor volume and numbers. Significant differences were present between groups2and group4in mean tumor volume per rat but not in numbers of tumors per rat.5. High amounts of urinary cytokines induced by Fe3O4-BCG-CS/GP gelA significant increase in urinary cytokines was detected in group2and group4. Rats in group4had the highest AUC values of IL-2and IFN-y, which was indicative of enhanced immune response. The same cytokines could be detected in group2but at lower levels. In addition, concentrations of urinary IL-2and IFN-y a month after the last instillation were still higher in group4.6. Intense and lasting lymphocytic infiltration induced by Fe3O4-BCG-CS/GP gelCD4staining was performed to assess CD4+lymphocytic infiltration in submucosa. Rats in group1and group3contained few lymphocytes in submucosa of bladder, whereas rats in group2and group4showed multifocal lymphocytic infiltration. Rats of group4had significantly elevated levels of CD4+T cells infiltrating the submucosa when compared to rats of group2. These findings were consistent with the results of urinary cytokines analyses.Conclusion:This study firstly described the use of a magnetic thermosensitive CS/GP hydrogel as a suitable matrix for extended BCG delivery by intravesical route. The biodegradable and injectable thermosensitive gel showed a rapid sol-gel transformation both in vitro and in vivo at37℃and good superparamagnetic performance. Most importantly, this gel permitted an intravesical continuous release of BCG over the period of48h in the presence of magnetic field. Sustained delivery of BCG markedly increased the antitumor efficacy and induced a high local immunity in bladder. In conclusion, the magnetic chitosan hydrogel developed in this work can increase the immunogenic potency of BCG and has great potential for use as a promising intravesical BCG delivery system for superficial bladder cancer. |
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