磁性温敏凝胶作为卡介苗载体在膀胱肿瘤灌注治疗中的应用研究
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摘要
研究背景
膀胱癌是泌尿系统最常见的恶性肿瘤之一,发病率较高。膀胱癌包括尿路上皮癌、鳞状细胞癌和腺细胞癌,其中尿路上皮癌最为常见,虽然理论上经尿道膀胱肿瘤电切术可完全切除非肌层浸润性膀胱癌,但术后复发率可高达67%。因此中国、欧洲及美国的泌尿外科指南建议所有非肌层浸润性膀胱癌患者术后均应进行辅助性膀胱灌注治疗。卡介苗(Bacillus Calmette-Guerin, BCG)是膀胱灌注常用的一种生物制剂,为一种减毒活菌,具有一定的抗原性、致敏性和残余毒性。经过多年的实践,BCG己被公认为最有效的预防膀胱肿瘤复发的免疫制剂,适用于表浅的、非肌层浸润性膀胱肿瘤的术后灌注治疗。
BCG膀胱灌注虽然疗效确切,但其不良反应发生率较高,限制了其临床应用。且有研究报道,30～50%的膀胱肿瘤病人BCG灌注失败,因无法耐受副作用、对BCG治疗反应差、肿瘤进展或复发等,最终停用BCG灌注治疗。增加BCG的灌注剂量是增强BCG治疗反应的最直接方法,但矛盾的是BCG灌注的剂量越大,虽然抗肿瘤效果明显,但副作用随之增加,如何提高BCG的疗效同时降低其不良反应,成为研究的热点。
由于人周期性的排尿,药物灌注后很快随尿液排出体外,其与膀胱粘膜的作用时间较短,通常只有1～2小时。药物膀胱内灌注后,在较短的时间段内,病人对膀胱内药物的反应个体差异较大,部分病人甚至没有反应。膀胱内药物灌注的效果主要与膀胱内药物浓度及持续时间相关,而非药物灌注剂量。因此,BCG灌注后在膀胱内的持续作用时间对治疗效果至关重要。
基于壳聚糖(CS)及p-甘油磷酸钠(β-glycerophosphate, GP)的温敏凝胶是一种生物材料,在室温条件下,CS和GP的混合溶液呈液态,加热至37℃体温条件下,即转变为CS/GP凝胶。CS凝胶在药物载体、细胞包裹及组织工程等方面得到广泛应用,是优良且极具发展前途的医用缓释体系。因此我们设想,CS/GP温敏凝胶可以装载BCG进行膀胱灌注,以期延长BCG在膀胱内的滞留时间,提高BCG的疗效。
CS/GP温敏凝胶载体进入膀胱后依然会随尿液排出体外。我们设想利用磁性颗粒及外加磁场防止凝胶载体随尿液排出。Fe304磁性纳米粒(Fe3O4magnetic nanoparticle, Fe3O4-MNP)是一种纳米级铁氧体,具有磁响应性好,在液体中可均匀分散,且在生物体内可被降解,无毒副作用等特性,作为磁性材料在生物领域中得到广泛应用。因此我们将Fe3O4-MNP包装进入CS/GP温敏凝胶内,在外加磁场条件下,利用Fe3O4-MNP所提供的磁牵引力,实现凝胶载体在膀胱内的贴壁粘附。
研究目的
本课题中我们以CS/GP凝胶为基质,设计了一种搭载BCG、Fe3O4-MNP的载体复合物(Fe3O4-BCG-CS/GP),在常温条件下为液体,进入体内后可转变为凝胶,用以进行BCG的膀胱灌注,以期延长BCG在膀胱内的作用时间,并最终增强BCG的抗膀胱肿瘤作用。
方法
1. Fe3O4-BCG-CS/GP凝胶载体系统的构建、表征检测及体内应用可行性评估
1.1Fe3O4-BCG-CS/GP载体复合物的制备
根据已有文献报道及我们的前期实验,制备方法如下：
1)将脱乙酰度为95%的CS溶于0.1mol/L的稀盐酸中；
2)室温下持续充分搅拌2小时；
3)将CS溶液中不溶性颗粒过滤；
4)将p-GP粉末溶于蒸馏水中；
5)将CS溶液及β-GP溶液在4℃C冰箱中冷藏10分钟；
6)持续搅拌的状态下将p-GP溶液逐滴加入CS溶液中,直至形成均一的溶液；
7)然后将一定量的BCG及Fe3O4-MNP加入混合溶液中,采用机械搅拌、超声分散的方法混合均匀；
1.2CS/GP溶液及Fe3O4-BCG-CS/GP复合物溶液的体外凝胶化时间测定
不同浓度配比的CS/GP溶液及Fe3O4-BCG-CS/GP载体复合物凝胶化时间的测定按照如下经典方法进行：
将溶液加入离心管中；37℃水浴锅中加热；按照一定的时间间隔,从水浴锅中取出离心管,倒置观察；倒置30s样品不流动,即可认为凝胶化完成；记录不同CS和GP配比的CS/GP溶液凝胶化时间,Fe3O4-BCG-CS/GP载体复合物溶液凝胶化时间同理测定。
1.3大鼠体内凝胶化时间测定及冰冻切片检查
利用大鼠测定体内凝胶化时间。灌注前,进行腹腔注射麻醉,取3Fr硬膜外导管自制大鼠导尿管。用空针抽出大鼠膀胱内尿液。将0.1ml Fe3O4-BCG-CS/GP载体复合物溶液注入大鼠膀胱内。根据步骤2中测定的凝胶化时间,处死大鼠,取出膀胱,观察凝胶化完成情况。然后对大鼠膀胱进行冰冻切片检查,观察复合物凝胶后的形态。
1.4数码显微镜及扫描电镜检查
我们采用数码显微镜及扫描电镜来观察Fe3O4-BCG-CS/GP复合物凝胶的表面形态。Fe3O4-BCG-CS/GP复合物溶液在37℃水浴中完成凝胶化。取样品少量冷冻干燥。放置于数码显微镜下观察摄片。喷镀贵金属银,增强导电性,处理完成后在扫描电镜下观察复合物凝胶的表面形态。
1.5Fe3O4-BCG-CS/GP复合物凝胶大鼠膀胱内滞留时间测定
雌性’Wistar大鼠36只用以测定Fe3O4-BCG-CS/GP复合物凝胶在膀胱内的滞留时间。大鼠膀胱灌注前2小时禁饮。麻醉后进行膀胱灌注,所有大鼠处于4kG外加磁场中饲养。灌注后每4小时处死大鼠3只；取膀胱标本立即进行冰冻切片检查,HE染色观察膀胱内残留凝胶形态,抗酸染色及复染HE染色观察凝胶中剩余BCG的形态。
2. Fe3O4-BCG-CS/GP凝胶载体系统抗膀胱肿瘤作用的研究
2.1实验设计
本部分实验使用24只雌性Wistar大鼠。根据以往成熟的实验设计,采用BBN诱发大鼠膀胱肿瘤原位模型后进行抗肿瘤实验。BBN加入每日饮水中,浓度为0.05%,共喂养8周。10周后,24只大鼠根据灌注药物的不同随机分为4组：
第一组(Group1):为对照组,给予0.1ml的PBS灌注；
第二组(Group2):给予1mg/0.1ml的BCG进行膀胱灌注；
第三组(Group3):CS/GP溶液灌注组,每次灌注0.1ml；
第四组(Group4):给予Fe3O4-BCG-CS/GP复合物溶液0.1ml,含1mg BCG。四组大鼠每周灌注一次,共6次。实验的1-8周为肿瘤诱导期,11～16周为治疗期。四组大鼠均饲养于强度为4kG的磁场环境中。
2.2抗肿瘤效果验证
20周后麻醉下处死所有大鼠,收集大鼠膀胱组织观察。详细记录每只大鼠的肿瘤数目及每个肿瘤的体积,计算每组大鼠的肿瘤发生率及每只大鼠的平均肿瘤体积。直径超过0.5mm的肿物拟定为肿瘤,列入统计中。肿瘤体积按如下公式计算：
V (mm3)=1/2XaXb2(a是肿瘤的最大直径,b为肿瘤的最短直径)。
2.3尿液细胞因子分析
膀胱灌注后,将4组大鼠转移至代谢笼内收集尿液。第一次膀胱灌注后24小时收集尿液,每日收集1次,共6次。按实验计划,在处死大鼠前收集尿液1次。每次收集的时间约2小时。收集尿液的离心管内含蛋白酶抑制剂,收集时放置于冰块上。收集完成后离心、储存于-80℃冰箱中待检。本实验采用双抗体夹心ELISA法来检测大鼠尿液中IL-2和IFN-γ的浓度,描绘出其变化曲线。通过计算曲线下面积(area under the curve, AUC)来估算大鼠接受单次膀胱灌注后尿液中细胞因子分泌的总量,据此评估大鼠膀胱内免疫反应的强弱。
2.4免疫组化检查
大鼠膀胱灌注BCG后引起膀胱内CD4+的淋巴细胞浸润,其在BCG抗膀胱肿瘤的作用机制中起重要作用。处死所有大鼠后,收集大鼠膀胱组织进行免疫组化检查。我们采用CD4抗体免疫组化染色的方法检测膀胱粘膜中浸润的CD4+淋巴细胞。
2.5统计分析
所有数据读取等检测步骤重复进行三遍,指标进行统计学处理,采用Prism5统计软件进行分析,计量数值以x±s表示,根据需要选用配对资料的t检验或单因素方差分析进行统计分析,选用双侧检验。P<0.05为差异有统计学意义。
结果
1. Fe3O4-BCG-CS/GP凝胶载体系统的构建、表征检测及体内应用可行性评估
实验所制备的CS/GP溶液无色半透明状,性质均一,在体外加热至37℃C的条件下,CS/GP溶液成功完成凝胶化,凝胶化后溶液失去流动性。倾倒离心管,凝胶固定不流动。Fe3O4-BCG-CS/GP复合物成功完成凝胶化,样品呈灰黑色,性质均一。实验发现,CS及GP的浓度皆可影响凝胶化时间。由于复合物凝胶中Fe3O4-MNP的存在,凝胶保留了一定的磁响应性。经冰冻切片、HE染色后观察,Fe3O4-BCG-CS/GP载体复合物亦在大鼠膀胱内完成凝胶化。由于CS为有机物,冰冻切片法较好地保留了复合物凝胶在膀胱内的形态,显示Fe3O4-BCG-CS/GP复合物凝胶载体呈网格状的微结构,大小、直径不一。数码显微镜及电镜下可观察到复合物凝胶的表面同样呈现大小不同的网格状结构,与冰冻切片所示结构相似。在磁场作用下,复合物凝胶在膀胱内贴附到膀胱壁表面。随着复合物凝胶在膀胱内作用时间的延长,其在粘膜表面的降解增多、随尿液排出的损耗增大以及尿液的溶蚀作用,其浓度和密度也逐渐降低,凝胶网格溶胀扩大,网格壁变薄。经过48小时,我们仍可在膀胱内检测到少量凝胶存留,因此我们可以认为,磁性凝胶作为BCG载体,可以在外加磁场的作用下,在膀胱内滞留时间长达48个小时。
2. Fe3O4-BCG-CS/GP凝胶载体系统抗膀胱肿瘤作用的研究
在抗大鼠膀胱肿瘤的研究中,大鼠灌注Fe3O4-BCG-CS/GP复合物后,所有24只大鼠均按计划完成20周的实验过程。在第20周末,处死大鼠,取大鼠膀胱组织进行检查。Fe3O4-BCG-CS/GP复合物与普通BCG都显示出明显抗肿瘤诱导作用。Fe3O4-BCG-CS/GP复合物组大鼠的平均肿瘤体积较BCG组大鼠明显减小,但在肿瘤数目方面的差异没有统计学意义。Fe3O4-BCG-CS/GP复合物灌注后大鼠尿液中IL-2及IFN-γ浓度较普通BCG组明显升高,且分泌总量较大。通过CD4染色,Fe3O4-BCG-CS/GP复合物灌注的大鼠膀胱粘膜下有更多CD4+淋巴细胞浸润。
结论
本实验中成功制备了以CS/GP温敏凝胶为载体、以Fe3O4-MNP为磁性牵引物、搭载BCG的Fe3O4-BCG-CS/GP凝胶载体系统,经验证可在体内、体外成功完成凝胶化,保留了较好的磁响应性。凝胶载体呈网格状,BCG等搭载物在凝胶载体中相对独立,利于BCG的释放。Fe3O4-BCG-CS/GP凝胶载体系统在外加磁场作用下,可在大鼠膀胱内延长BCG的作用时间达48小时；动物实验显示,与普通BCG相比,同等剂量条件下Fe3O4-BCG-CS/GP凝胶载体所搭载的BCG可刺激膀胱分泌更多细胞因子,募集大量炎性细胞浸润,在膀胱内产生更强的粘膜免疫反应,其抗膀胱肿瘤作用优于普通BCG。我们认为以CS/GP温敏凝胶为载体搭载Fe3O4-MNP、BCG后构建的Fe3O4-BCG-CS/GP凝胶载体系统,增强了BCG的免疫活性及抗肿瘤效应,为BCG的应用提供了一种新的思路,有望成为用于膀胱癌术后灌注预防膀胱癌复发的更好的免疫制剂。
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 Characterization
1.1Preparation of CS and GP solution (CS/GP) and CS/GP solution loaded with Fe3O4-MNP and BCG
The 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 mixture
The 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 retention
The 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 activity
2.1Study 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 activity
After20weeks, 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 analysis
After 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 study
The 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 analyses
All 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 mixture
Both 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 SEM
The 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 retention
Cryosection 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 gel
All 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 gel
A 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 gel
CD4staining 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.
膀胱癌是泌尿系统最常见的恶性肿瘤之一,发病率较高。膀胱癌包括尿路上皮癌、鳞状细胞癌和腺细胞癌,其中尿路上皮癌最为常见,虽然理论上经尿道膀胱肿瘤电切术可完全切除非肌层浸润性膀胱癌,但术后复发率可高达67%。因此中国、欧洲及美国的泌尿外科指南建议所有非肌层浸润性膀胱癌患者术后均应进行辅助性膀胱灌注治疗。卡介苗(Bacillus Calmette-Guerin, BCG)是膀胱灌注常用的一种生物制剂,为一种减毒活菌,具有一定的抗原性、致敏性和残余毒性。经过多年的实践,BCG己被公认为最有效的预防膀胱肿瘤复发的免疫制剂,适用于表浅的、非肌层浸润性膀胱肿瘤的术后灌注治疗。
BCG膀胱灌注虽然疗效确切,但其不良反应发生率较高,限制了其临床应用。且有研究报道,30～50%的膀胱肿瘤病人BCG灌注失败,因无法耐受副作用、对BCG治疗反应差、肿瘤进展或复发等,最终停用BCG灌注治疗。增加BCG的灌注剂量是增强BCG治疗反应的最直接方法,但矛盾的是BCG灌注的剂量越大,虽然抗肿瘤效果明显,但副作用随之增加,如何提高BCG的疗效同时降低其不良反应,成为研究的热点。
由于人周期性的排尿,药物灌注后很快随尿液排出体外,其与膀胱粘膜的作用时间较短,通常只有1～2小时。药物膀胱内灌注后,在较短的时间段内,病人对膀胱内药物的反应个体差异较大,部分病人甚至没有反应。膀胱内药物灌注的效果主要与膀胱内药物浓度及持续时间相关,而非药物灌注剂量。因此,BCG灌注后在膀胱内的持续作用时间对治疗效果至关重要。
基于壳聚糖(CS)及p-甘油磷酸钠(β-glycerophosphate, GP)的温敏凝胶是一种生物材料,在室温条件下,CS和GP的混合溶液呈液态,加热至37℃体温条件下,即转变为CS/GP凝胶。CS凝胶在药物载体、细胞包裹及组织工程等方面得到广泛应用,是优良且极具发展前途的医用缓释体系。因此我们设想,CS/GP温敏凝胶可以装载BCG进行膀胱灌注,以期延长BCG在膀胱内的滞留时间,提高BCG的疗效。
CS/GP温敏凝胶载体进入膀胱后依然会随尿液排出体外。我们设想利用磁性颗粒及外加磁场防止凝胶载体随尿液排出。Fe304磁性纳米粒(Fe3O4magnetic nanoparticle, Fe3O4-MNP)是一种纳米级铁氧体,具有磁响应性好,在液体中可均匀分散,且在生物体内可被降解,无毒副作用等特性,作为磁性材料在生物领域中得到广泛应用。因此我们将Fe3O4-MNP包装进入CS/GP温敏凝胶内,在外加磁场条件下,利用Fe3O4-MNP所提供的磁牵引力,实现凝胶载体在膀胱内的贴壁粘附。
研究目的
本课题中我们以CS/GP凝胶为基质,设计了一种搭载BCG、Fe3O4-MNP的载体复合物(Fe3O4-BCG-CS/GP),在常温条件下为液体,进入体内后可转变为凝胶,用以进行BCG的膀胱灌注,以期延长BCG在膀胱内的作用时间,并最终增强BCG的抗膀胱肿瘤作用。
方法
1. Fe3O4-BCG-CS/GP凝胶载体系统的构建、表征检测及体内应用可行性评估
1.1Fe3O4-BCG-CS/GP载体复合物的制备
根据已有文献报道及我们的前期实验,制备方法如下：
1)将脱乙酰度为95%的CS溶于0.1mol/L的稀盐酸中；
2)室温下持续充分搅拌2小时；
3)将CS溶液中不溶性颗粒过滤；
4)将p-GP粉末溶于蒸馏水中；
5)将CS溶液及β-GP溶液在4℃C冰箱中冷藏10分钟；
6)持续搅拌的状态下将p-GP溶液逐滴加入CS溶液中,直至形成均一的溶液；
7)然后将一定量的BCG及Fe3O4-MNP加入混合溶液中,采用机械搅拌、超声分散的方法混合均匀；
1.2CS/GP溶液及Fe3O4-BCG-CS/GP复合物溶液的体外凝胶化时间测定
不同浓度配比的CS/GP溶液及Fe3O4-BCG-CS/GP载体复合物凝胶化时间的测定按照如下经典方法进行：
将溶液加入离心管中；37℃水浴锅中加热；按照一定的时间间隔,从水浴锅中取出离心管,倒置观察；倒置30s样品不流动,即可认为凝胶化完成；记录不同CS和GP配比的CS/GP溶液凝胶化时间,Fe3O4-BCG-CS/GP载体复合物溶液凝胶化时间同理测定。
1.3大鼠体内凝胶化时间测定及冰冻切片检查
利用大鼠测定体内凝胶化时间。灌注前,进行腹腔注射麻醉,取3Fr硬膜外导管自制大鼠导尿管。用空针抽出大鼠膀胱内尿液。将0.1ml Fe3O4-BCG-CS/GP载体复合物溶液注入大鼠膀胱内。根据步骤2中测定的凝胶化时间,处死大鼠,取出膀胱,观察凝胶化完成情况。然后对大鼠膀胱进行冰冻切片检查,观察复合物凝胶后的形态。
1.4数码显微镜及扫描电镜检查
我们采用数码显微镜及扫描电镜来观察Fe3O4-BCG-CS/GP复合物凝胶的表面形态。Fe3O4-BCG-CS/GP复合物溶液在37℃水浴中完成凝胶化。取样品少量冷冻干燥。放置于数码显微镜下观察摄片。喷镀贵金属银,增强导电性,处理完成后在扫描电镜下观察复合物凝胶的表面形态。
1.5Fe3O4-BCG-CS/GP复合物凝胶大鼠膀胱内滞留时间测定
雌性’Wistar大鼠36只用以测定Fe3O4-BCG-CS/GP复合物凝胶在膀胱内的滞留时间。大鼠膀胱灌注前2小时禁饮。麻醉后进行膀胱灌注,所有大鼠处于4kG外加磁场中饲养。灌注后每4小时处死大鼠3只；取膀胱标本立即进行冰冻切片检查,HE染色观察膀胱内残留凝胶形态,抗酸染色及复染HE染色观察凝胶中剩余BCG的形态。
2. Fe3O4-BCG-CS/GP凝胶载体系统抗膀胱肿瘤作用的研究
2.1实验设计
本部分实验使用24只雌性Wistar大鼠。根据以往成熟的实验设计,采用BBN诱发大鼠膀胱肿瘤原位模型后进行抗肿瘤实验。BBN加入每日饮水中,浓度为0.05%,共喂养8周。10周后,24只大鼠根据灌注药物的不同随机分为4组：
第一组(Group1):为对照组,给予0.1ml的PBS灌注；
第二组(Group2):给予1mg/0.1ml的BCG进行膀胱灌注；
第三组(Group3):CS/GP溶液灌注组,每次灌注0.1ml；
第四组(Group4):给予Fe3O4-BCG-CS/GP复合物溶液0.1ml,含1mg BCG。四组大鼠每周灌注一次,共6次。实验的1-8周为肿瘤诱导期,11～16周为治疗期。四组大鼠均饲养于强度为4kG的磁场环境中。
2.2抗肿瘤效果验证
20周后麻醉下处死所有大鼠,收集大鼠膀胱组织观察。详细记录每只大鼠的肿瘤数目及每个肿瘤的体积,计算每组大鼠的肿瘤发生率及每只大鼠的平均肿瘤体积。直径超过0.5mm的肿物拟定为肿瘤,列入统计中。肿瘤体积按如下公式计算：
V (mm3)=1/2XaXb2(a是肿瘤的最大直径,b为肿瘤的最短直径)。
2.3尿液细胞因子分析
膀胱灌注后,将4组大鼠转移至代谢笼内收集尿液。第一次膀胱灌注后24小时收集尿液,每日收集1次,共6次。按实验计划,在处死大鼠前收集尿液1次。每次收集的时间约2小时。收集尿液的离心管内含蛋白酶抑制剂,收集时放置于冰块上。收集完成后离心、储存于-80℃冰箱中待检。本实验采用双抗体夹心ELISA法来检测大鼠尿液中IL-2和IFN-γ的浓度,描绘出其变化曲线。通过计算曲线下面积(area under the curve, AUC)来估算大鼠接受单次膀胱灌注后尿液中细胞因子分泌的总量,据此评估大鼠膀胱内免疫反应的强弱。
2.4免疫组化检查
大鼠膀胱灌注BCG后引起膀胱内CD4+的淋巴细胞浸润,其在BCG抗膀胱肿瘤的作用机制中起重要作用。处死所有大鼠后,收集大鼠膀胱组织进行免疫组化检查。我们采用CD4抗体免疫组化染色的方法检测膀胱粘膜中浸润的CD4+淋巴细胞。
2.5统计分析
所有数据读取等检测步骤重复进行三遍,指标进行统计学处理,采用Prism5统计软件进行分析,计量数值以x±s表示,根据需要选用配对资料的t检验或单因素方差分析进行统计分析,选用双侧检验。P<0.05为差异有统计学意义。
结果
1. Fe3O4-BCG-CS/GP凝胶载体系统的构建、表征检测及体内应用可行性评估
实验所制备的CS/GP溶液无色半透明状,性质均一,在体外加热至37℃C的条件下,CS/GP溶液成功完成凝胶化,凝胶化后溶液失去流动性。倾倒离心管,凝胶固定不流动。Fe3O4-BCG-CS/GP复合物成功完成凝胶化,样品呈灰黑色,性质均一。实验发现,CS及GP的浓度皆可影响凝胶化时间。由于复合物凝胶中Fe3O4-MNP的存在,凝胶保留了一定的磁响应性。经冰冻切片、HE染色后观察,Fe3O4-BCG-CS/GP载体复合物亦在大鼠膀胱内完成凝胶化。由于CS为有机物,冰冻切片法较好地保留了复合物凝胶在膀胱内的形态,显示Fe3O4-BCG-CS/GP复合物凝胶载体呈网格状的微结构,大小、直径不一。数码显微镜及电镜下可观察到复合物凝胶的表面同样呈现大小不同的网格状结构,与冰冻切片所示结构相似。在磁场作用下,复合物凝胶在膀胱内贴附到膀胱壁表面。随着复合物凝胶在膀胱内作用时间的延长,其在粘膜表面的降解增多、随尿液排出的损耗增大以及尿液的溶蚀作用,其浓度和密度也逐渐降低,凝胶网格溶胀扩大,网格壁变薄。经过48小时,我们仍可在膀胱内检测到少量凝胶存留,因此我们可以认为,磁性凝胶作为BCG载体,可以在外加磁场的作用下,在膀胱内滞留时间长达48个小时。
2. Fe3O4-BCG-CS/GP凝胶载体系统抗膀胱肿瘤作用的研究
在抗大鼠膀胱肿瘤的研究中,大鼠灌注Fe3O4-BCG-CS/GP复合物后,所有24只大鼠均按计划完成20周的实验过程。在第20周末,处死大鼠,取大鼠膀胱组织进行检查。Fe3O4-BCG-CS/GP复合物与普通BCG都显示出明显抗肿瘤诱导作用。Fe3O4-BCG-CS/GP复合物组大鼠的平均肿瘤体积较BCG组大鼠明显减小,但在肿瘤数目方面的差异没有统计学意义。Fe3O4-BCG-CS/GP复合物灌注后大鼠尿液中IL-2及IFN-γ浓度较普通BCG组明显升高,且分泌总量较大。通过CD4染色,Fe3O4-BCG-CS/GP复合物灌注的大鼠膀胱粘膜下有更多CD4+淋巴细胞浸润。
结论
本实验中成功制备了以CS/GP温敏凝胶为载体、以Fe3O4-MNP为磁性牵引物、搭载BCG的Fe3O4-BCG-CS/GP凝胶载体系统,经验证可在体内、体外成功完成凝胶化,保留了较好的磁响应性。凝胶载体呈网格状,BCG等搭载物在凝胶载体中相对独立,利于BCG的释放。Fe3O4-BCG-CS/GP凝胶载体系统在外加磁场作用下,可在大鼠膀胱内延长BCG的作用时间达48小时；动物实验显示,与普通BCG相比,同等剂量条件下Fe3O4-BCG-CS/GP凝胶载体所搭载的BCG可刺激膀胱分泌更多细胞因子,募集大量炎性细胞浸润,在膀胱内产生更强的粘膜免疫反应,其抗膀胱肿瘤作用优于普通BCG。我们认为以CS/GP温敏凝胶为载体搭载Fe3O4-MNP、BCG后构建的Fe3O4-BCG-CS/GP凝胶载体系统,增强了BCG的免疫活性及抗肿瘤效应,为BCG的应用提供了一种新的思路,有望成为用于膀胱癌术后灌注预防膀胱癌复发的更好的免疫制剂。
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 Characterization
1.1Preparation of CS and GP solution (CS/GP) and CS/GP solution loaded with Fe3O4-MNP and BCG
The 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 mixture
The 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 retention
The 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 activity
2.1Study 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 activity
After20weeks, 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 analysis
After 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 study
The 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 analyses
All 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 mixture
Both 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 SEM
The 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 retention
Cryosection 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 gel
All 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 gel
A 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 gel
CD4staining 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.
引文
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