| Backgroud:Bladder cancer is one of the most common tumors in the urinary system,with an estimated 80,500 newly diagnosed cases and 32,900 deaths in China in 2015.Currently,non-muscle invasive cancer(NMIBC)occupies approximately 80%of bladder tumors,which is confined to the inner lining of the bladder.Transurethral resection of bladder tumor(TURBT)is the gold standard treatment for NMIBC,with an 80%early success rate.However,50?70%of patients who undergo TURBT will relapse within five years.Moreover,20?30%of the recurrent cases progress to higher stages or grades,followed by metastasis of the tumor.Intravesical chemotherapy for NMIBC is a well-established adjuvant therapy for preventing recurrence and progression after TURBT.Drugs are directly administered to the bladder via catheter in a strategy called 'intravesical drug delivery'(IDD).IDD has many advantages.The bladder allows the catheter to be easily manipulated and accessed.Drugs are directly administered into the urinary bladder,resulting in greater exposure of the tissues to the drugs.Mitomycin C(MMC),the pharmacological actions of which include impairing DNA replication due to crosslinking with DNA chains and preventing protein synthesis,exerts its therapeutic activity against various cancers in a time-and dose-dependent manner.One of the important clinical applications of MMC is intravesical chemotherapy.However.the adjuvant therapy only reduces recurrence by 14?17%on average and has limited benefits against disease progression.reflecting the limitations of the conventional delivery method for MMC.Periodical discharge approximately every 2 h and the irritation caused by a high concentration of drugs reduce the exposure time,which is the crucial factor for the efficacy of MMC.Repeated instillation could lead to bladder fibrosis,infections,injury of the urinary tract and poor compliance.The inadequacy of conventional MMC delivery prompted the development of a new mode of administration.Several techniques have been investigated to optimize drug delivery in intravesical instillations.Co-instillation with hyaluronic acid,dimethyl sulfoxide or saponin is a chemical approach to enhance the permeability of the urothelium.Physical approaches,such as electromotive drug administration and hyperthermia,also focus on improving drug permeability.Some researchers also reported that photodynamic therapy had advantages in intravesical instillation.However,the disadvantages of the above approaches,such as limited effects,toxicity,diseconomy or inconvenience,restrict the application to intravesical instillation.However,the in situ forming depot(ISFD),nanoparticles and liposomes are good at improving drug bioavailability and patient compliance,and none of them have been studied for intravesical chemotherapy.ISFD is defined as a liquid formulation that transforms into a solid or semi-solid depot in situ after delivery into the body.Magnetic thermally induced gelling systems,one type of ISFDs comprising chitosan(CS),?-glycerophosphate(GP)and Fe3O4 magnetic nanoparticles(Fe3O4-MNPs),are promising candidates for the development of local drug delivery systems,reflecting the properties of targeted and sustained or controlled release of different agents.It was reported that insulin loaded in the thermosensitive gel was released in its native form over approximately 5 days in rats.The prolonged retention and targeting of doxorubicin for treating bladder cancer were also achieved after doxorubicin was loaded in magnetic targeted carriers.Thus,magnetic ISFDs can perhaps be used to optimize the effect of MMC as an intravesical chemotherapy.ObjectiveIn the present study,we designed a magnetic ISFD comprising CS,GP,Fe3O4-MNPs and MMC with prolonged retention of MMC in the bladder,and examined the sustained release,in vivo retention and anti-tumor activity of this delivery system for improving the effect of MMC as an intravesical chemotherapy.Methods1.In vitro studies1.1 MMC loaded Fe3O4-CS/GP gel system and magnetic detectionThe CS/GP solution was obtained as previously described.Briefly,the CS powder was dissolved in 0.1 M hydrochloric acid with magnetic stirring at room temperature.The GP powder was dissolved in distilled water.The two solutions were chilled in an ice bath;subsequently,the GP solution was added dropwise into CS solution under magnetic stirring at 4 ? to form a clear and homogeneous solution.Then,the Fe3O4-MNPs and MMC powder were successively added to the CS/GP solution under churning,followed by dispersal by ultrasound.A piece of Fe3O4-MMC-CS/GP gel sank in a beaker filled with distilled water.The magnetic property of Fe3O4-MMC-CS/GP gel was studied when a magnet was placed outside the beaker.1.2 Gelation time of Fe3O4-MMC-CS/GP and the influential factorsThe gelation time of the CS/GP and Fe304-MMC-CS/GP at 37 ? was determined using a classical method.A centrifuge tube loaded with the mixture solution was heated in a 37 ? water bath.At fixed time intervals,the tube was rotated upside down.The hydrogel formed when the sample remained stationary for 30 s,and this time point was recorded as the gelation time.The effect of temperature,Mw and the degree of deacetylation of CS on the gelation time were investigated.1.3 In vitro release studiesThe in vitro release of Fe3O4-MMC-CS/GP was examined using the dialysis method.Dialysis bags with molecular weight cut-of-f(MWCO)of 50 kDa were used to retain the Fe3O4-MMC-CS/GP gel and the released MMC was allowed to permeate into the medium.Initially,Fe3O4-MMC-CS/GP gel was loaded in a dialysis bag soaked in 20 ml of medium at 37 ?.Subsequently,the bag was tied and shaken in a horizontal shaker at 60 rpm and 37 ?.At fixed times the released medium was collected and replaced with 20 ml of fresh medium to mimic the urination discipline.The concentration of released MMC was determined using High Performance Liquid Chromatography(HPLC).The diffusion of the free MMC solutionwas analyzed as a control.The influence of the Fe3O4-MNPs,the Mw of CS and the initial amounts of MMC on release were observed.1.4 Scanning electron microscopy(SEM)and frozen section examinationThe surface morphology and structure of the Fe3O4-MMC-CS/GP gel were detected using SEM.The lyophilized Fe3O4-MMC-CS/GP gel was fixed onto adhesive carbon tapes,followed by gold coating.The sample was subsequently subjected to SEM analysis.A total of 0.1 ml of the Fe3O4-MMC-CS/GP mixture was instilled into 3 female rats.After recording the gelation time,the rats were sacrificed,and the bladder was harvested with the bladder neck tied for cryosectioning.followed by hematoxylin-eosin staining(HE staining).1.5 Swelling and degradation studiesThe weight of the lyophilized gel of CS/GP and Fe3O4-MMC-CS/GP(n=3)were recorded.Then the gels were immersed in 10 ml of isotonic phosphate buffered saline(PBS,pH 6.0)at 37 0C.At the setting time,the gels were taken out,removed the surface water using filter papers and recorded the wet weight.The swelling ratio was calculated.In vitro degradation studies were performed in two solutions:20 ml PBS(pH 6.0)and 20 ml PBS(pH 6.0)containing 4 mg/ml lysozyme.The dry gels(CS/GP and Fe3O4-MMC-CS/(GP,n=3)were placed in a shaking incubator at 37 ? and 60 rpm.At predetermined time points,samples were taken out,rinsed with distilled water,dried under vacuum and recorded the weight.Degradation solution was refreshed every day to ensure sustained enzyme activity.1.6 Cell viability assayT24 cells were seeded onto a 96-well plate and cultivated for 24 h to facilitate attachment to the surface.Then the growth medium was substituted with 100 ?l of DMEM.Group 1 was a control without treatment.Groups 2,3,4 and 5 were treated with a magnetic field,a magnetic field and Fe3O4-CS/GP,a magnetic field and MMC(100,50,and 25 pg/ml,respectively),and a magnetic field and Fe3O4-MMC-CS/GP,respectively.The Fe3O4-MMC-CS/GP gel was attracted to the sidewall using a magnetic field to avoid pressing the cells.DMEM was refreshed at 2,4,6,8,10,12 and 24 h to mimic the urination discipline.The gel and magnetic field were removed at 24 h and the CCK-8 solution was added.Subsequently,the cells were incubated for an additional 4 h at 37 ?.The optical density(OD)was measured using an absorbance microplate reader at a wavelength of 450 nm.1.7 Determination of apoptosis via flow cytometryT24 cells were seeded onto 6-well plates.The subgroups and treatments were the same as in Section 1.6,but only 25?g/ml MMC was used in this experiment.The cells were harvested with trypsin,washed twice with cold PBS.and resuspended in binding buffer.Subsequently,Annexin V-FITC and PI were added to the cell suspension and incubated for 15 min at room temperature in the dark.After incubation,400 ?l of binding buffer were added to the solution.The apoptotic cells were analyzed using a FACSDiva flow cytometer.2.In vivo studies2.1 In vivo evaluation of retentionThe retention study was performed using 27 female rats.After instillation,the rats were maintained in a magnetic field of 0.4 T and successively sacrificed at 2.4,6,8,10,12,24,48 and 72 h.Urine was discharged from bladders,and subsequently the bladder volume was maintained at approximately 0.3?0.5 ml using physiological saline.The bladders were submitted to cryosectioning,followed by HE staining.2.2 In vivo release studiesThe in vivo release studies were conducted using 6 female Wistar rats.All rats were prohibited from drinking water for 2 h before and after the instillation.0.1 ml of the Fe3O4-MMC-CS/GP mixture containing 0.1 mg of MMC was instilled through a 3F catheter in the experimental group,and the control group was instilled with 0.1 mg of free MMC solution.After instillation,the rats were transferred to metabolic cages under a magnetic field of 0.4 T for urine collection at 2,4,6,8,10,12,24,48 and 72 h.The collected urine was filtered through a 0.45 ?m membrane filter and stored at-80 ? until batch analysis via HPLC.The missing amounts of MMC were also calculated to evaluate the absorption of the bladder mucosa.2.3 In vivo anti-tumor activityThis study was conducted using 32 rats that were randomly assigned to one of the four treatment groups.In the tumor-induction phase,all rats were treated with 0.05%BBN to induce the autochthonous development of bladder tumors.After 10 weeks,all groups were treated six times with the intravesical instillation on a weekly basis.Each group was instilled with the following formulations:PBS,Fe3O4-CS/GP solution,MMC solution and Fe3O4-MMC-CS/GP mixture.After 20 weeks,all rats were sacrificed and subjected to necropsy.The bladders,ureters and kidneys were harvested from the rats and dissected for a macroscopic quantitative analysis.The volume and number of tumors in each group were recorded.2.4 Survival rateFrom the treatment phase,all rats were regularly assessed.The time of death.hematuria and weight loss were recorded.The graphs were generated with the number of live rats against time,and the Fe3O4-MMC-CS/GP was assessed in terms of the survival time of the rats.The mortality data were estimated using the Kaplan-Meier method.The statistical analysis was performed using the Mantel-Cox log-rank test,and P<0.05 was considered statistically significant.2.5 Immunohistochemistry studyThe bladder tumors of 4 groups were fixed in 4%paraformaldehyde for at least 24 h and subsequently embedded in paraffin for immunohistochemical staining of cleaved caspase-9 and cleaved caspase-3.Negative control sections were incubated without the primary antibody in citrate buffer.The digitized images were subsequently analyzed using Image-Pro Plus 6.0 and the average optical density(integrated optical density/area,IOD/Area)of the positive reactions was used to evaluate the expression.2.6 Statistical analysesAll determinations were performed in triplicate and the results are presented as the mean ± standard deviation(SD).Statistical significance was determined by one-way ANOVA,Student's t-test,or the Mantel-Cox log-rank test,as appropriate,using GraphPad Prism 5.Significance levels were defined as P<0.05(*),P<0.01(**)and P<0.001(***).Result1.1 The preparation of Fe3O4-MMC-CS/GP gel system and magnetic detectionMMC was loaded onto the Fe3O4-CS/GP successfully.The Fe3O4-MMC-CS/GP system was liquid at the room temperature,but it transformed into gel rapidly at 37 ? with homogeneous property.The clot of the gel was attracted to the sidewall by a magnet outside the beaker.And the gel still showed good magnetic ability with less amounts of Fe3O4-MNPs.1.2 Gelation time of Fe3O4-MMC-CS/GP and the influential factorsThe Mw and deacetylation degree of CS did affect the profile,showing an prolonged tendency with decreasing Mw and deacetylation.The transition was fast(3.5 min)at 37 ? using CS with 95%deacetylation.When replaced with CS with 85%deacetylation,the CS/GP solution did not gel at 37 ? in 30 min.but gelled rather slowly(10 min)at 45 ?.The gelation time of Fe3O4-MMC-CS/GP(95%deacetylation)was also 3.5 min,implying that neither the Fce3O4-MNPs nor MMC affected the gelation process.1.3 Release studies in vitroThe color of the medium containing MMC-CS/GP and Fe3O4-MMCCS/GP gradually changed to blue;however,the color of the MMC solution rapidly changed,and no blue color was observed by the naked eye after the first refreshment of medium.The release of MMC from the drug delivery system was gentle,with a weaker initial burst(75.33 ± 0.85 ?g/ml vs.98.44 ± 0.52 ?g/ml,decreased by 23.48%)and longer release time(approximately 10 h vs.2 h.increased by 4-fold)than the free MMC solution,indicating the sustained release of MMC from the delivery system.The Mw of CS,and the initial amounts of MMC had slight effect on the drug release,except Fe3O4-MNPs.1.4 Scanning electron microscopy(SEM)and frozen section examinationThe main characteristic was a loose and porous structure with a smooth matrix surface,whereas the external surface of the Fe3O4-MMC-CS/GP system was coarse and granular,reflecting the introduction of Fe3O4-MNPs and MMC,which adhered to or were embedded in the gel matrix.The loose and porous structure provided channels for drug delivery to the medium and therefore would be helpful for MMC release.The cryosections and HE staining of Fe3O4-MMC-CS/GP displayed a similar structure to the SEM images.The Fe3O4-MNPs and MMC were scattered throughout the delivery system,but the agglomeration of Fe3O4-MNPs was also observed.Themorphology and structure of the drug delivery system was well exhibited in the cryosections.1.5 Swelling and degradation studiesIt could be seen that the CS/GP and Fe3O4-MMC-CS/GP gel swelled fast and reached the equilibrium swelling after 1 h incubation in PBS,with swelling ratio of 255.19%± 16.02%vs.196.64%± 22.63%(P = 0.0216<0.05).This indicated the fast and good liquid absorption of delivery system which could be beneficial for dissolution,storage and sustained release of MMC.However,the swelling ratio of Fe3O4-MMC-CS/GP was significantly lower than CS/GP.reflecting the adherence and embedding of MMC and Fe3O4-MNPs in the delivery system,which occupied a part of space.Therefore,the swelling ratio of Fe3O4-MMC-CS/GP could be adjusted by regulating the amounts of MMC and Fe3O4-MNPs.The CS/GP and Fe3O4-MMC-CS/GP showed similar degradation curve in degradation medium,implying that neither the Fe3O4-MNPs nor MMC affected the degradation property.The Fe3O4-MMC-CS/GP in PBS degraded slowly with only 18.76%± 2.93%degradation rate after 10 days.When immersed in PBS with lysozyme,the delivery system degraded 75.93%±6.95%after 10 days reflecting the degraded effect of lysozyme to chitosan.In general,the degradation ratio of the new delivery system in harsh environment of the bladder was excellent.1.6 Cell viability assayAt the highest dose of MMC(100 ?g/ml),the OD values were not significantly different between groups 4 and 5,but were evidently lower than the first 3 groups,indicating that the magnet and Fe3O4-CS/GP had no detrimental effect on the efficacy of MMC and a large dose of MMC could kill almost all cells in 2 h.At moderate and small doses of MMC(50 and 25 pg/ml),the OD values of group 5 were less than group 4(0.537 ? 0.009 vs.0.323 ± 0.003,P=0.0007<0.001,and 1.727 ? 0.029 vs.0.626 ± 0.010,P 0.0003<0.001).The results supported the hypothesis that the delayed drug release behavior and extended exposure time resulted in the high mortality of the tumor cells.1.7 Fe3O4-MMC-CS/GP induces more apoptosis in tumor cellsFew apoptotic T24 cells were detected in the first 3 groups(1.17%± 1.07%,1.77%± 1.10%and 2.50%± 0.26%,respectively).The last 2 groups showed a larger percent of apoptotic cells(including early and late apoptotic cells,10.20%± 2.80%and 27.33%±4.51%,respectively)than the first 3 groups.Moreover,the number of apoptotic cells in group 5 were significantly increased by 167.94%compared with group 4(P 0.0124<0.05),suggesting that the sustained release of MMC from the Fe3O4-MMC-CS/GP system induced more powerful tumor cell apoptosis.2.1 In vivo evaluation of retentionThe Fe3O4-MMCCS/GP solution transformed into a gel on the bladder wall after instillation.Over time,the gel suffered from excretion,degradation and erosion,resulting in weight loss.The MMC was released either via immersion in urine or unpacking after the loss of the gel.The remaining magnetic gel and MMC were observed at 72 h after intravesical instillation.2.2 In vivo release studyThe release of MMC from Fe3O4-MMC-CS/GP lasted approximately 72 h(ti/2? 5.34 ± 1.67 h),which was increased by 17-fold compared with the free MMC solution(4 h,1t,= 0.25 ± 0.01 h).The free MMC solution had a strong initial burst release,but the concentration sharply decreased in 4 h.In contrast,the release of MMC from Fe3O4-MMC-CS/GP was slower and the decrease in the concentration was moderate and lasted for a longer time.Therefore,the sustained release resulted in a higher area under the curve(AUC)compared with the free MMC solution(418.70 ±12.79 vs.191.70 ± 57.45,increased by 118.41%,P 0.0182<0.05).Simultaneously,the prolonged effect of Fe3O4-MMC-CS/GP also enhanced the uptake of MMC through the bladder mucosa(21.25 ± 1.55 ?g vs.7.80±0.46 ?g,increased by 172.44%,P = 0.0012<0.01).2.3 Enhanced anti-tumor activity promoted by Fe3O4-MMC-CS/GPThe growth of the bladder tumors in groups 1 and 2 was exuberant;furthermore,ureter invasion accompanied by ipsilateral hydronephrosis appeared in these two groups.The tumors of groups 3 and 4 showed a marked reduction in volume compared with groups 1 and 2.Significant differences in the mean tumor volume per tumor were observed between groups 3 and 4(5.88 ± 0.65 mm3 vs.3.30 ± 0.56 mm3 P = 0.0083<0.01,inhibited by 43.88%)and fewer rats in group 4 had tumors(five of eight vs.seven of eight),but the number of tumors per rat with tumors was not significantly different(1.43 ± 0.30 vs.1.40±0.243,P>0.05).2.4 Survival rateThe survival rate is a definitive parameter in evaluating the antitumor efficacy of animal experiments upon tumor induction.It was obvious that group 4,which was treated with Fe3O4-MMC-CS/GP.had the highest survival rate and the most animals alive for the longest period among the 4 groups(P =0.0261<0.05).2.5 Enhanced apoptosis by Fe3O4-MMC-CS/GPThere was weak expression of cleaved caspase-3 in the bladder tumors of groups 1(12.97 ± 11.65)and 2(10.67 ± 6.13);however.groups 3(68.32 ± 14.31)and 4(159.64 ± 14.95)showed increased expression of cleaved caspase-3,and the bladder tumors of group 4 had significantly elevated levels compared with group 3(P<0.0001).The protein cleaved caspase-9,showed the same expression trend as cleaved caspase-3.These findings confirmed that the new delivery manner for MMC induced stronger apoptosis than the conventional intravesical instillation.ConclusionMMC was loaded into a biodegradable and injectable ISFD delivery system with thermosensitive and magnetic properties that showed a rapid transformation from a solution to a gel at 37 ? to improve the anti-tumor effect of MMC compared to the conventional intravesical instillation.More importantly,the delivery system displayed prolonged retention and sustained release of MMC in a magnetic field,with marked anti-tumor activity and enhanced tumor cell apoptosis in the bladder compared with the conventional intravesical instillation. |
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