Magnetic Multiwalled Carbon Nanotubes With Controlled Release Of Epirubicin:An Intravesical Instillation System For Bladder Cancer

Backgroud:Urothelial bladder cancer(UBC)is a common malignancy with a significant impact on public health.UBC is the 7th most common cancer in men and the 17th most common in women worldwide.Approximately 75%of newly diagnosed UBCs are non-muscle invasive bladder cancer(NMIBC).NMIBC has a high rate of recurrence and progression despite transurethral resection of the bladder tumor(TURBT).After initial treatment,up to 70%of NMIBC patients experience disease recurrence.NMIBC has the highest recurrence rate among solid tumors and is the most expensive cancer over the lifetime of the patient due to its high frequency of recurrenceNMIBC recurs because of topical occult micrometastases in the bladder.The conventional systemic administration of drugs is very ineffective in bladder diseases due to the poorly vascularized urothelium;hence only a small fraction of the drug actually reaches the affected site.Thus,'intravesical drug delivery' has been described whereby a drug is directly instilled into the bladder via a catheter to attain high local concentrations with minimal systemic effects.The limitations of traditional intravesical instillation treatment are primarily due to insufficient perfusion of anticancer agents and limited residence time of the chemotherapy drugs in the bladder.A long duration of exposure is crucial for good therapeutic response.However,instilled drug solutions are periodically diluted with urine and are washed out of the bladder during voiding.This leads to repeated drug infusions.These repeated instillations improve drug exposure,but they lower compliance.Therefore,strategies for improving intravesical instillation therapy after TURBT in patients with NMIBC are an important research topic to reduce the clinical and economic burden of NMIBC.Concurrently,magnetic nanoparticles(MNPs)with good superparamagnetic behavior have attracted much attention due to their inherent materials properties and huge potential in cancer diagnosis and treatment.Our previous studies have demonstrated that thermosensitive hydrogel systems including Fe3O4 magnetic nanoparticles prolonged the retention time of chemotherapy drugs in bladders with external magnets.However,these systems have limited impact on disease sites.First,most drugs in hydrogel systems would separate from bladder mucosa via the loose structure of the cross-linking.Second,the hydrogel may swell and block the vesicoureteric junction.Third,temperature-dependent preparation of hydrogel systems would restrict clinical applications.We describe a novel magnetic carboxylated multiwalled carbon nanotubes(mMWCNTs)-based system with nanoscale characteristics that solves these issues CNTs have a large surface area and are conjugated easily to chemotherapy drugs including single walled CNTs(SWCNTs)or MWCNTs.MWCNTs have lower biological toxicity than SWCNTs.The strong acid-carboxylated MWCNTs(MWCNTs-COOH)not only mitigate toxicity but also increase the dispersibility and increase the efficiency of loaded drugs.The most common intravesical chemotherapeutic agents for bladder cancer are pirarubicin,epirubicin,doxorubicin,hydroxycamptothecine,mitomycin C and gemcitabine.For patients with NMIBC,early intravesical instillation with epirubicin(EPI)after TURBT is an effective method.And the MWCNTs-COOH can efficiently load EPI via supramolecular ?-? stacking because of its large surface area and hydrogen bonding.Thus,we proposed to prepare mMWCNTs-based delivery system for EPI intravesical instillation with external magnets.This scheme is flexible because the magnet controls drug deliveryObjectiveThe aim of this study was to report an epirubicin(EPI)-loaded magnetic multiwalled carbon nanotubes(mMWCNTs-EPI)system for intravesical instillation in place of the current formulation.The mMWCNTs-EPI system was formulated with carboxylated MWCNTs,Fe3O4 magnetic nanoparticles,and EPI.The features and anti-tumor activity of mMWCNTs-EPI system were investigatedMethodsPreparation of mMWCNTsThe preparation of mMWCNTs is illustrated in Scheme 1A.The Fe3O4 were attached to a chemically modified MWCNTs-COOH surface producing magnetic multiwalled carbon nanotubes(mMWCNTs).First,750 mg MWCNTs were dispersed in 300 mL solution of mixed acid(sulfuric acid:nitric acid = 1:3)by 80 kHz sonication with a KQ-500DE digital control ultrasonic cleaner(Kunshan Ultrasonic Instruments Co.,Ltd,China)for 30 min and then heated to 95? for 4 h.The MWCNTs were transformed into MWCNTs-COOH with mixed acid treatment The MWCNTs-COOH were subsequently dried in a drying oven.Next,100 mg dried MWCNTs-COOH were dispersed in double distilled water,and then 42.8 mg FeCl2·4H2O and 116.8 mg FeCl3·6H2O were added(the feed molar ratio of Fe2+:Fe3+ is 1:2).The weight ratio of MWCNTs-COOH to Fe3O4 is 2:1,After sonication for 5 min,the solution was purged with Ar2 for 0.5 h.The reaction system was then heated to 60?,and ammonium hydroxide was added.The reaction system was kept at 60? for 2 h and then 90? for 0.5 h.Finally,the resulting mMWCNTs were washed 6 times with 200 mL double distilled water and dried in an oven.The dried mMWCNTs were laid out thinly in an ultra-clean stage(Suzhou Bolante Laboratory System Engineering Co.,Shenzhen,China),and bacteria/spore-free mMWCNTs were produced by ultraviolet radiation for 6 h.Characterization of mMWCNTsThe TEM images were obtained on JEM-1011(JEOL,Japan)transmission electron microscope with an acceleration voltage of 60 kV.The dried samples were fixed on adhesive carbon tapes and then gold coated with a Hitachi E-1010 ion sputter coater(Tokyo,Japan).The FESEM images were collected by a Hitachi SU-70 SEM system(Tokyo,Japan)at an accelerating voltage of 30-100 kV.The zeta potential measurements of the mMWCNTs were confirmed by the DelsaTM Nano C Particle Analyzer(Beckman Coulter,Brea,CA,USA).The FTIR spectra was determined on an ALPHA FTIR Spectrometer(Bruker,Billerica,MA,USA)The wave number ranges from 4000 to 400 cm-1,and the resolution is 4 cm-1.The crystalline structure of mMWCNTs was considered by the X'Pert3 Powder diffractometer(PANalytical,Almelo,Netherlands).The magnetization evaluation was carried out with MicroMagTM Model 2900 AGM System(Lake Shore Cryotronics,Westerville,OH,USA)according to the manufacturer's instruction at room temperature.Cell cultureThe T24 cells were grown in McCoy's 5A medium supplemented with 10%fetal bovine serum(FBS)and 5637 cells were grown in Roswell Park Memorial Institute-1640(RPMI-1640)medium supplemented with 10%FBS.All cells were grown at 37? in an atmosphere containing 5%CO2 and 95%air and were harvested when they were in the logarithmic growth phase.Cell viability assayThe cytotoxicity of mMWCNTs against human bladder cancer cells were evaluated with CCK-8 assays.The 5637 and T24 cells were seeded onto a 96-well plate at a density of 5 × 103 cells/well in 100 ?L of culture medium.After attached to the surface of 96-well plate,the cells were treated with 0,0.625,1.25,2.5,5,10,20 and 40 ?g/mL mMWCNTs for 72 h.The cells were subsequently treated with a CCK-8 solution and incubated for an additional 4 h.The optical density(OD)was measured using an absorbance microplate reader(Thermo Fisher Scientific,Waltham,MA,Finland)at 450 nm.The CCK-8 assays were performed to test the anti-tumor activity of mMWCNTs-EPI against human bladder cancer cells.Group 1 was a control without treatment.Groups 2-4 were treated 24 h with the following formulations:magnetic field and 40 ?g/mL mMWCNTs;2 ?g/mL EPI;magnetic field and mMWCNTs-EPI(containing 2 ?g/mL EPI),respectively.The culture medium was refreshed at 2,4,6,8,10,12 and 24 h to mimic urination.The CCK-8 solution was added in accordance with the protocol described above.Cell morphology testTo visualize the influence of mMWCNTs on cell F-actin cytoskeleton arrangement,the cells were incubated with fluorescein isothiocyanate-labeled phalloidin.5637 and T24 cells were divided into control group and experimental group,respectively.Experimental groups were treated with 40 ?g/mL mMWCNTs for 72 h.Control groups were treated with phosphate buffered saline(PBS)for the same time.The cells were fixed with methanol for 10 min and stained with fluorescein isothiocyanate-labeled phalloidin for 1 h at room temperature.Nuclei were counterstained with DAPI.The images were captured using a fluorescence microscope(Olympus,Tokyo,Japan).In vivo toxicity of mMWCNTs studiesThe toxicity of mMWCNTs in vivo was determined in 12 female rats.Six rats were maintained in the magnetic field with 3200 Gauss magnets fixed near their bladders.Then,2.5 mg/1 mL mMWCNTs were instilled intravesically every 3 days for 1 month.Another 6 rats were fed as the control.Blood samples were collected after 1 month for analysis of blood serum chemistry.Serum samples were obtained from blood by centrifugation(3000 rpm for 10 min).Alanine aminotransferase(ALT),aspartate aminotransferase(AST),serum creatinine(Scr)and blood urea nitrogen(BUN)were determined by fully automatic biochemical analyzer(Chemray 240;Rayto Life and Analytical Sciences Co.,Shenzhen,China).The hearts,livers,spleens,lungs,kidneys,and brains were excised at the time of euthanization to detect the systemic toxicity of mMWCNTs.The tissues were fixed with 4%paraformaldehyde,embedded in paraffin and serially sectioned.The 4-?m-thick sections were subjected to hematoxylin-eosin(H&E)stainingLoading and release studiesThe EPI was dissolved in double distilled water at a concentration of 1 mg/mL.The same mass of mMWCNTs were then mixed into EPI solution by sonication for 30 min.The mMWCNTs-EPI were produced and then purified via external magnets.Briefly,the suspension of mMWCNTs-EPI and free EPI was placed on a magnetic rack(Solarbio Life Sciences,Beijing,China)for 10 min.The mMWCNTs-EPI were then held tightly to the flask wall by magnetic forces.The free EPI aspirated and measured on a Shimadzu SCL-lOAvp Series High Performance Liquid Chromatography(HPLC)system(Japan)coupled with UV-detector set at 480 nm and C18 analytical column(150 × 4.6 mm,5 ?m particle size)(Beckman Coulter,Inc.,USA).The binary mobile phase was sodium dihydrogen phosphate(68%)and acetonitrile(32%)delivered at 1.0 mL/min.The loading efficiency of EPI was calculated using following formula:Loading efficiency =(Weight of total EPI-free EPI)/(Weight of total EPI)x 100%The mMWNTs-EPI were then dispersed in PBS(pH = 4?6?8)on a shaking table.Every 2 h,the released solution was collected with external magnets and PBS was refreshed(mimicking physiological condition of urine).The EPI release ratio from mMWCNTs-EPI was determined according to the change in concentration.The areas under the concentration-time curves(AUC)values for EPI were calculated.In vivo evaluation of retentionThe retention time of mMWCNTs-EPI in bladders were determined in 15 female rats with a 3200 Gauss magnetic field.The rats were sacrificed at 12,24,48,72 and 96 h after mMWCNTs-EPI instillation(three rats at each time point).The bladders excised at different time points were fixed with 4%paraformaldehyde,embedded in paraffin,and serially sectioned.The 4-?m thick sections underwent hematoxylin-eosin(H&E)stainingQuantitative detection of apoptosis in vitroTo quantitatively evaluate the cell status,5637 and T24 cells were seeded onto 6-well plates at a density of 1 X 105 cells/well.Group 1 was a control without treatment.Groups 2-4 were treated for 24 h with the following formulations:magnetic field and 40 ?g/mL mMWCNTs;2 ?g/mL EPI;magnetic field and mMWCNTs-EPI(containing 2 ?g/mL EPI).Culture medium was refreshed at 2,4,6,8,10,12 and 24 h to mimic urination.The cells were collected and subjected to annexin V and propidium iodide(PI)staining using a FITC Annexin V Apoptosis Detection Kit following the manufacturer's protocol.Apoptotic cells were then analyzed by a FACSDiva flow cytometer(BD Biosciences,San Jose,CA,USA)Quantitative detection of proliferation in vitroTo examine proliferation of 5637 and T24 cells,the cells were seeded onto 24-well plates at a density of 5 × 104 cells/well.The groups were treated as described in cell morphology test and quantitative detection of apoptosis in vitro.All groups were treated with EdU 2 h and then incubated with the Cell-Light EdU Apollo567 InVitro Imaging Kit at room temperature.Nuclei were counterstained with DAPI.Images were captured using a fluorescence microscope,and digital histomorphometric analysis was performed with Image J software.The resulting data were expressed as ratio of labeled nuclei to total nuclei.For each section,three high-power fields were detected and each measurement was performed in triplicate.Chemically-induced bladder cancer model in ratsAn N-methyl-N-nitrosoure(MNU)-induced rat bladder tumor model described previously was used with some modifications.Female Wistar rats were anesthetized with intraperitoneal injections of 3%pentobarbital(30 mg/kg).Then MNU was instilled intravesically via a shortened 3F epidural anesthesia catheters within 45 min of preparation after the bladders were drained.The instillation was performed every other week for a total of 4 doses(8 weeks).Anti-tumor activity in vivoThe study comprised 30 female Wistar rats.All the 30 rats were divided randomly into 5 groups.Group 1 was administered a normal diet as control.Other groups were induced bladder cancer model as described above.After 8 weeks,groups 2-5 received intravesical instillation with the following formulations:0.1 mL PBS;0.25 mg/0.1 mL mMWCNTs;0.1 mg/0.1 mL EPI;0.1 mL mMWCNTs-EPI(containing 0.1 mg EPI,0.25 mg mMWCNTs),respectively.They received intravesical instillation on a weekly basis,6 times.Groups 3 and 5 were maintained in the 3200 Gauss magnetic field.All rats were euthanized and subjected to necropsy(one in the mMWCNTs group was killed by an unintended anesthetic overdose).The bladders,ureters,and kidneys of all groups were obtained at the end of the treatment.The total tumor volume per rat and the volume per tumor in each group were recorded.A tumor was defined as a lesion of>0.5 mm in diameter.Tumor volume was calculated by the following equation:V(mm3)= 1/2 ×(length)×(width).Immunohistochemistry studyThe bladders of all groups were fixed in 4%paraformaldehyde for at least 24 h and subsequently embedded in paraffin for immunohistochemical staining of Bcl-2,Bax,Cleaved caspase-3,and immunofluorescence staining of Ki67.Negative control sections were incubated without the primary antibody.The digitized images were subsequently analyzed using Image-Pro Plus 6.0,and the average optical density(integrated optical density/area,IOD/Area)positive reactions was used to evaluate the expression of Bcl-2,Bax,Cleaved caspase-3.The ratio of Ki67+ cells was analyzed with Image J software.Statistical analysisAll statistical analyses were conducted with SPSS 20.0 software(SPSS Inc.USA).Data were expressed as mean± SD for continuous variables.Continuous data were compared among the groups via a one-way ANOVA.The Tukey's test was used to analyze multiple comparisons between groups.A two-tailed P<0.05 indicated statistical significanceResultsCharacterization of mMWCNTsThe results displayed the TEM and FESEM images of mMWCNTs prepared in the conditions described above.The length of raw MWCNTs is 10-30 ?m;the length of MWCNTs-COOH cut by mixed acid became 200-1000 nm.A close observation revealed that the MWCNTs-COOH were wrapped tightly with nano-Fe3O4 on the surface.The zeta potential of the mMWCNTs suspensions was-47.31± 0.16(mV),which showed good stability in aqueous solution.The FTIR spectra of MWCNTs-COOH and mMWCNTs showed several peaks at 3434 cm-1,1626 cm-1,1384 cm-1,and 1045 cm-1.These peaks were ascribed to O-H bonds,C=O groups,C-OH and C-O stretching vibrations,respectively.The peak at 582 cm-1 of mMWCNTs was specified to metal-oxygen.The X-ray diffraction(XRD)confirmed the metal-oxygen bonds in the mMWCNTs.According to the JCPDS file No.190629 for the magnetite,six diffraction peaks of mMWCNTs at 20 = 30.16°,35.58°,43.22?,53.70°,57.22° and 62.87°could be attributed to the(220),(311),(400),(422),(511)and(440)crystal planes of cubic Fe3O4,respectively.Furthermore,the main features of X-ray diffraction pattern of CNTs is a graphite-like peak(002).The diffraction peaks of mMWCNTs at 20 = 26.02°could be attributed to the(002)peak of CNTs,which is not obvious.This suggested that mMWCNTs were wrapped by Fe3O4 nanoparticles.The magnetic hysteresis curve of mMWCNTs was determined by the alternating gradient magnetometer.The mMWCNTs have superparamagnetic behavior,and the maximum saturation magnetization value is 19.13 emu/g.The residual magnetization and coercivity were both zero.The magnetization curve showed an invertible“S" shape.There was no hysteresis in the sample.The macroscopic magnetic parameter of mMWCNTs was measured via the 1 mg/mL mMWCNTs suspension.When an external magnet was applied,the mMWCNTs separated from the suspension and were rapidly attracted to the magnet to clear the suspensions.However,the mMWCNTs returned to suspensions after gentle shaking Only a trace of mMWCNTs sediments were observed after storage for 15 days indicating excellent aqueous stability.Toxicity of mMWCNTsWhen treated with different concentrations of mMWCNTs,the mMWCNTs showed little toxicity against 5637 and T24 cells.The ratio of EdU-labeled cells was calculated to examine the effect of mMWCNTs on cell proliferation.There was no difference between 40 ?g/mL mMWCNTs groups and control groups.The f-actin staining was predominantly found in the cortical structures around the cell periphery with a few thin stress fibers located within cell body.Alignment of F-actin fibers increased in all periods of mitosis.There were no obvious morphological changes or re-organization of F-actin cytoskeleton in either group.The toxicity of mMWCNTs in vivo was determined in 12 female rats.During the experiment,there was no mortality or systemic serum biochemical toxicity induced by mMWCNTs.Neither mMWCNTs agglomerates nor any visible signs of toxicity(e.g.,inflammatory cells or histopathological changes)were found in major organs.There were no abnormal behavioral changes including diarrhea,vomiting,anorexia,or lethargy.There was no significant difference in body weight between the experimental group and the control group.Sustained EPI releasing and prolonged retention in rat bladderThe loading procedure for mMWCNTs with EPI solutions resulted in a loading percentage of 40.4 ± 9.6%.The results show that the release of EPI from mMWCNTs-EPI was slower,and the decrease in the concentration was moderate and lasted longer than free EPI.The sustained release of EPI from the mMWCNTs-EPI resulted in the AUC nearly tripling.The mMWCNTs-EPI were stable in rat bladder after 12h with external magnets.The amount of mMWCNTs-EPI and the mMWCNTs-EPI-covered surface areas along the urothelium were reduced with time.There were some remnants until 96h.In vitro anti-tumor activityThe mMWCNTs-EPI showed more significant cytotoxicity to 5637 and T24 cells than free EPI when the culture medium was refreshed every two hours.The flow cytometry results demonstrated that free EPI and mMWCNTs-EPI groups showed higher apoptotic ratios than control and mMWCNTs groups.Versus free EPI,the apoptotic ratios in the mMWCNTs-EPI groups were significantly increased.Significantly lower ratios of EdU-labeled cells per high-power field(magnification×200)were observed in mMWCNTs-EPI-treated cells relative to free EPI groups Statistical analysis showed that mMWCNTs-EPI groups demonstrated significantly less proliferation than free EPI groups.In vivo anti-tumor activityRepresentative bladder tumors from the five groups are shown in the article.The administration of PBS and mMWCNTs caused no markedly different reduction in the growth of bladder tumors—neither the total tumor volume per rat nor volume of each tumor.In terms of efficacy,the mMWCNTs-EPI group achieved better efficacy than free EPI for inhibition of tumor volume.In addition,one unilateral hydronephrosis appeared in both the PBS and mMWCNTs groups;one bilateral hydronephrosis appeared in the PBS group.The hydronephrosis was caused by ureter or vesicoureteric junction invasion.Enhanced apoptosis and inhibited proliferation by mMWCNTs-EPIThe expression of Bax and Cleaved caspase-3 was weak while the Bcl-2 increased in PBS and mMWCNTs groups;the EPI and mMWCNTs-EPI groups had contrasting expression patterns compared with PBS and mMWCNTs groups.The data also showed that the mMWCNTs-EPI group had higher expression of Bax and Cleaved caspase-3 and lower expression of Bcl-2 than the EPI group.The immunofluorescence staining results indicated that Ki67+ cells were nearly negative in the control group but obviously increased in PBS and mMWCNTs groups.After receiving intravesical instillation therapy with EPI or mMWCNTs-EPI,the ratios of Ki67+ cells were significantly decreased.In addition,the mMWCNTs-EPI group showed more inhibition in Ki67 expression than the EPI group.ConclusionThis study successfully developed a safe magnetic adhesion system for the urothelium.The results showed that the mMWCNTs-EPI system effectively extended the duration of EPI in intravesical instillation under external magnets.The mMWCNTs-EPI system enhanced EPI cytotoxicity and inhibited cell proliferation in bladder cancer—both in vitro and in vivo.The mMWCNTs-EPI system can enhance the therapeutic effects and decrease side effects of bladder cancer by utilizing the synergistic effects of magnetic retention and chemotherapy.