A Hydrogel-Based Epirubicin Delivery System for Intravesical Chemotherapy

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Article A Hydrogel-Based Epirubicin Delivery System for Intravesical Chemotherapy

Ching-Wen Liu 1, Yu-Tse Wu 1,*,†, Kai-Jen Lin 2, Tsan-Jung Yu 3, Yu-Liang Kuo 4 and Li-Ching Chang 5,6,*,†

1 School of Pharmacy, Kaohsiung Medical University, No.100, Shih-Chuan 1st Road, Sanmin District, Kaohsiung 807, Taiwan; [email protected] 2 Department of Pathology, E-Da Hospital, I-Shou University, No.1, Yida Road, Yanchao District, Kaohsiung 824, Taiwan; [email protected] 3 Department of Urology, E-Da Hospital, I-Shou University, No.1, Yida Road, Yanchao District, Kaohsiung 824, Taiwan; [email protected] 4 Department of Medical Imaging and Radiological Sciences, Chung Shan Medical University, No.110, Sec. 1, Jianguo North Rd., South District., Taichung 402, Taiwan; [email protected] 5 Department of Occupational Therapy, I-Shou University, No.8, Yida Road, Yanchao District, Kaohsiung 824, Taiwan 6 Department of Pharmacy, E-Da Hospital, I-Shou University, No.1, Yida Road, Yanchao District, Kaohsiung 824, Taiwan * Correspondence: [email protected] (Y.-T.W.); [email protected] (L.-C.C.); Tel.: +886-7-3121101 (Y.-T.W.); +886-7-6151100 (L.-C.C.) † These authors contributed equally to this work.

Academic Editor: Derek J. McPhee Received: 20 March 2016; Accepted: 26 May 2016; Published: 1 June 2016

Abstract: This study aimed to examine the efficacy of epirubicin-loaded gelatin hydrogel (EPI-H) in the treatment of superficial urothelium carcinoma. Hydrogel was prepared by Schiff base-crosslinking of gelatin with glutaraldehyde. EPI-H exhibited high entrapment efficiency (59.87% ˘ 0.51%). EPI-H also increased epirubicin accumulation in AY-27 cells when compared with the effect of aqueous solutions of epirubicin (EPI-AQ); respective epirubicin-positive cell counts were 69.0% ˘ 7.6% and 38.3% ˘ 5.8%. EPI-H also exhibited greater cytotoxicity against AY-27 cells than that of EPI-AQ; IC50 values were 13.1 ˘ 1.1 and 7.5 ˘ 0.3 µg/mL, respectively. Cystometrograms showed that EPI-H reduced peak micturition, threshold pressures, and micturition duration, and that it increased bladder compliance more so than EPI-AQ. EPI-H enhanced epirubicin penetration into basal cells of urothelium in vivo, whereas EPI-AQ did so only to the umbrella cells. EPI-H inhibited tumor growth upon intravesical instillation to tumor-bearing bladder of F344 rats, inducing higher levels of caspase-3 expression than that observed with EPI-AQ treatment; the number of caspase-3 positive cells in treated urothelium carcinoma was 13.9% ˘ 4.0% (EPI-AQ) and 34.1% ˘ 1.0%, (EPI-H). EPI-H has value as an improved means to administer epirubicin in intravesical instillation treatments for bladder cancer.

Keywords: epirubicin; hydrogel; intravesical; bladder cancer

1. Introduction The standard-of-care therapy for non-muscle-invading bladder cancer (NMIBC) is transurethral resection of bladder tumor (TURBT) followed immediately by a single intravesical dose of a chemotherapeutic agent (e.g., epirubicin) or immunotherapeutic agent (e.g., BCG) to reduce recurrence and progression to a muscle-invasive disease [1]. Intravesical delivery provides regional therapy in which drugs are directly administered into the bladder through a urethral catheter [2]. Intravesical

Molecules 2016, 21, 712; doi:10.3390/molecules21060712 www.mdpi.com/journal/molecules Molecules 2016, 21, 712 2 of 16 therapy has advantages in allowing more targeted delivery of therapeutic agents to tumor cells located in the bladder epithelium and in reducing the side effects associated with systemic drug absorption. Overall, this enables a broader therapeutic window. However, two major physiological hurdles restrict intravesical efficacy, and this reduced efficacy can result in a high cancer recurrence rate. The first hurdle is limited dwell time of a drug in the bladder (2 h) due to washout during voiding. The second hurdle is the limited uptake of drug into the urothelial cells (normal or malignant) due to the unique permeability barrier properties of the urothelium [3]. The current formulation of epirubicin to be instilled in the bladder is a lyophilized powder that is dissolved in normal saline. This formulation is typically administered in episodes of 2-h durations. Irritation to the urothelium, urine washout, and watertight urothelium barriers prevent this aqueous epirubicin solution from achieving sustained exposure to the urothelium. The 1- and 2-year cancer recurrence-free rates after initial TURBT are 25% and 12.5%, respectively, and the recurrence-free rates when epirubicin treatment is applied after TUR are 58.5% and 38.6%, respectively [4]. Given the high recurrence rates, we speculate that a scaffold for optimizing epirubicin release to the luminal bladder may improve drug efficacy. Strategies for improving intravesical therapy include physical and chemical approaches to increasing both drug penetration and drug residence time in the bladder [5,6]. We previously constructed poly(ethyl-2-cyanoacrylate) (PECA) epirubicin-loaded nanoparticles for enhancing intravesical delivery of epirubicin [7]. We have since then developed gelatin-based muco-adhesive nanocomposites as intravesical delivery scaffolds for gene therapy applications [8]. Gelatin-based hydrogels provide good viscosity for transduction into the bladder through a urethral tube. They also exhibit urothelium adhesion, thus enhancing urothelial penetration of drugs associated with the hydrogel. Here, the present study evaluated the feasibility of using hydrogel to provide an intravesical scaffold for applications of epirubicin in the chemotherapy of NMIBC. We are the first to propose the application of epirubicin-loaded gelatin hydrogel carriers for intravesical usage and the first to study its feasibility in therapies targeting bladder tumors. Prior to the biological applications described in this study, we fully characterized the appearance of epirubicin-loaded 15% gelatin hydrogel (denoted as EPI-H). This characterization included an evaluation of fine structure by scanning electron microscopy (SEM). The cytotoxicity of epirubicin-loaded gelatin against the AY-27 bladder cancer cell line was determined in vitro. The potential for enhanced apoptosis after EPI-H treatment was evaluated by means of double-labeling with FITC-tagged annexin V and propidium iodide. Apoptosis was also assessed through the cleaved/pro caspase-3 ratio as determined by western blotting. The adhesion of EPI-H to F344 rat bladder urothelium and penetration of epirubicin through the urothelium were assessed using fluorescent microscopy; histological features of the urothelium were subsequently inspected. Urodynamic parameters assessed after administration of EPI-H included peak micturition pressure, threshold pressure, micturition duration, and bladder compliance. Finally, the efficacy of EPI-H against bladder tumors formed by AY-27 cells in F344 rats was assessed.

2. Results

2.1. Characterization of EPI-H We first characterized the impact of gelatin type (A75 or A175) on hydrogel yield, hydration ratio, size, viscosity, and torque (Table1). For 15% gelatin type A75 and A175 hydrogel, mean yields were 89.7% and 98.0%, mean hydration ratios were 86.8% and 87.2%, mean particle sizes were 183.6 and 124.6 nm, mean viscosities were 32.2 and 78.7 Pa.s., and mean torques were 56.7% and 67.0%, respectively. The thermosensitivities of 15% gelatin A75 and A175 hydrogel were examined by visual inspection (fluidity and turbid) at 25 ˝C and 37 ˝C (Figure1A). The 15% gelatin A175 hydrogel showed high viscosity at 25 ˝C and became fluid at 37 ˝C. The influence of temperature changes on gelatin A175 hydrogel viscosity values is shown in Figure1B. This optimal fluidity made it easy to Molecules 2016, 21, 712 3 of 16

Moleculesviscosity,2016, 21 and, 712 thermosensitive fluidity, was selected as the carrier for epirubicin in the biological3 of 16 evaluations conducted in this study. The entrapment efficiency of epirubicin (0.25 mg/mL) in 15% gelatin A175 was 59.87% ± 0.51%. administer, providing good ﬂow, coverage, and adhesion to the urothelium in the bladder cavity. Thus, The SEM photographs showed epirubicin as a fine irregular crystal structure (Figure 1C). The the 15%lyophilized gelatin A17515% gelatin (denoted A175 as hydrogel H), which showed afforded fibrous satisfactory details (Figure yield rate,1D). When hydration epirubicin ratio, was viscosity, and thermosensitiveloaded into the hydrogel, ﬂuidity, SEM was showed selected the as epirubicin the carrier to be for partially epirubicin dispersed, in the and biological that it formed evaluations a conducteddense and in this conglomerate study. structure (Figure 1E).

25 °C 37 °C 120 A75 A175 A75 A175 100 80

Viscosity (Pa.s) 20 Gel Sol

22 24 26 28 30 32 34 36 38 Temperature (°C)(。C ) (A) (B)

Figure 1. (A) Visual inspection of gelatin hydrogel at 25 °C or 37 °C; (B) Viscosity (Pa.s) vs. temperature Figure 1. (A) Visual inspection of gelatin hydrogel at 25 ˝C or 37 ˝C; (B) Viscosity (Pa.s) vs. temperature for gelatin A175 hydrogel. Scanning electron microscopy (SEM) images showing microstructures of (C) for gelatin A175 hydrogel. Scanning electron microscopy (SEM) images showing microstructures of (C) epirubicin; (D) gelatin A175 hydrogel (H); and (E) epirubicin-loaded hydrogel (EPI-H). Magnification: epirubicin; (D) gelatin A175 hydrogel (H); and (E) epirubicin-loaded hydrogel (EPI-H). Magnification: (b,d) ×5000, (c) ×1000. (b,d) ˆ5000, (c) ˆ1000. Table 1. Characterization of gelatin hydrogel. Table 1. Characterization of gelatin hydrogel. Gelatin Yield (%) Hydration Ratio (%) Size (nm) Viscosity (Pa.s) Torque (%) A75 89.7 ± 21.0 86.8 ± 0.0 183.6 ± 4.6 32.2 ± 1.3 56.7 ± 6.6 Gelatin Yield (%) Hydration Ratio (%) Size (nm) Viscosity (Pa.s) Torque (%) A175 98.0 ± 24.9 87.2 ± 0.1 124.6 ± 0.9 78.7 ± 3.0 67.0 ± 4.5 A75 89.7 ˘ 21.0 86.8 ˘ 0.0 183.6 ˘ 4.6 32.2 ˘ 1.3 56.7 ˘ 6.6 2.2.A175 In Vitro Epirubicin 98.0 ˘ 24.9 Accumulation 87.2 and˘ Cytotoxicity0.1 124.6 ˘ 0.9 78.7 ˘ 3.0 67.0 ˘ 4.5 Epirubicin (0.25 mg/mL) was dissolved in normal saline (EPI-AQ) or dispersed in hydrogel The(EPI-H). entrapment Treatment efficiency with EPI-H of epirubicinallowed greater (0.25 epir mg/mL)ubicin inaccumulation 15% gelatin in A175 AY-27 was cells 59.87% than that˘ 0.51%. Theallowed SEM photographs by treatment with showed EPI-AQ epirubicin (Figure 2A). as The a epirubicin-positive fine irregular crystalcell counts structure from EPI-AQ (Figure and1 C). The lyophilizedEPI-H treatments 15% of gelatin AY-27 A175were 38.3% hydrogel ± 5.8% showed and 69.0% fibrous ± 7.6%, details respectively (Figure (Figure1D). 2B). When As shown epirubicin was loadedin Figure into 2C, the the hydrogel, half-maximal SEM inhibitory showed concentrations the epirubicin (IC to50 be values) partially of hydrogel dispersed, (H), and EPI-AQ, that itand formed a denseEPI-H and in conglomerate AY-27 cells were structure 196.2 ± 0.7, (Figure 13.11 ±E). 1.1, and 7.5 ± 0.3 μg/mL, respectively. EPI-H exhibited significantly increased cytotoxicity against AY-27 cells when compared with the effect of EPI-AQ 2.2. In(p Vitro= 0.013). Epirubicin The lackAccumulation of cytotoxicity andassociated Cytotoxicity with the hydrogel (H) confirmed that it could be safely used in intravesical applications. Epirubicin (0.25 mg/mL) was dissolved in normal saline (EPI-AQ) or dispersed in hydrogel (EPI-H). Treatment with EPI-H allowed greater epirubicin accumulation in AY-27 cells than that allowed by treatment with EPI-AQ (Figure2A). The epirubicin-positive cell counts from EPI-AQ and EPI-H treatments of AY-27 were 38.3% ˘ 5.8% and 69.0% ˘ 7.6%, respectively (Figure2B). As shown in Figure2C, the half-maximal inhibitory concentrations (IC 50 values) of hydrogel (H), EPI-AQ, and EPI-H in AY-27 cells were 196.2 ˘ 0.7, 13.1 ˘ 1.1, and 7.5 ˘ 0.3 µg/mL, respectively. EPI-H exhibited significantly increased cytotoxicity against AY-27 cells when compared with the effect of Molecules 2016, 21, 712 4 of 16

EPI-AQ (p = 0.013). The lack of cytotoxicity associated with the hydrogel (H) conﬁrmed that it could be safelyMolecules used 2016 in, intravesical21, 712 applications. 4 of 16

EPI-AQ EPI-H

Fluorescence Fluorescence

30.0 μm 30.0 μm

Phase-contrast Phase-contrast

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* 80 80

60 60

40 40 Survial (%) 20 H EPI-AQ EPI-H 20 EPI positivecells (%) 0 0 1.2 2.5 5 10 20 0 EPI-AQ EPI-H Concentration (μg/mL) (B) (C)

Figure 2. Epirubicin accumulated in AY-27 cells and induced cytotoxicity. (A) Fluorescent images of Figure 2. Epirubicin accumulated in AY-27 cells and induced cytotoxicity. (A) Fluorescent images of epirubicin (red) and DAPI (nuclei, blue), with phase-contrast images of AY-27 cells after EPI-AQ and epirubicin (red) and DAPI (nuclei, blue), with phase-contrast images of AY-27 cells after EPI-AQ and EPI-H treatment for 18 h. (magnification, ×400) (B) The statistics of epirubicin-positive cells in the ˆ EPI-HEPI-AQ treatment and forEPI-H 18 h.trea (magniﬁcation,tment AY-27 cells; 400)(C) The (B) cytotoxicity The statistics of AY-27 of epirubicin-positive cells with H, EPI-AQ, cells and in the EPI-AQEPI-H and EPI-Htreatment treatment for 18 h AY-27 prior cells;to the (MTSC) The assay. cytotoxicity All results of are AY-27 expressed cells with as the H, mean EPI-AQ, ± standard and EPI-H treatmenterror for of four 18 h independent prior to the experiments. MTS assay. * Allp < 0.05 results in cases are expressedof EPI-H versus as the EPI-AQ. mean ˘ standard error of four independent experiments. * p < 0.05 in cases of EPI-H versus EPI-AQ. 2.3. Enhanced Apoptosis by Caspase-3 Pathway 2.3. EnhancedThe Apoptosis apoptotic byeffect Caspase-3 of EPI-H Pathway was examined by Annexin V-FITC (AV) and propidium iodide (PI) double staining, and by fluorescence-activated cell sorting (FACS). AV-positive/PI-negative cells The apoptotic effect of EPI-H was examined by Annexin V-FITC (AV) and propidium iodide were scored as early apoptotic, and AV-positive/PI-positive cells were scored as late apoptotic cells. (PI) double staining, and by ﬂuorescence-activated cell sorting (FACS). AV-positive/PI-negative cells As shown in Figure 3A,C, the percentages of AY-27 cells in the early/late apoptotic stage observed were scoredafter control, as early EPI-AQ, apoptotic, and EPI-H and AV-positive/PI-positivetreatments were 0.0%/0.0%, cells6.4%/2.5%, were scored12.4%/2.1%, as late respectively. apoptotic In cells. As shownother in words, Figure EPI-H3A,C, treatment the percentages induced higher of AY-27 numb cellsers of in AY-27 the early/late cells to enter apoptotic early apoptosis. stage observed after control, EPI-AQ, and EPI-H treatments were 0.0%/0.0%, 6.4%/2.5%, 12.4%/2.1%, respectively. In other words, EPI-H treatment induced higher numbers of AY-27 cells to enter early apoptosis.

Molecules 2016, 21, 712 5 of 16 Molecules 2016, 21, 712 5 of 16 PI

AV (A) (B)(C) EPI-H Control EPI-AQ pro-caspase-3

cleavaged-caspase-3

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3.0

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0.0 Control EPI-AQ EPI-H

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3.0

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0.0 Control EPI-AQ EPI-H (E)

Figure 3. EPI-H enhanced apoptosis in AY-27 cells through activation of caspase 3 pathway. (A) AY-27 Figure 3. EPI-H enhanced apoptosis in AY-27 cells through activation of caspase 3 pathway. (A) AY-27 cells were treated with (B) EPI-AQ and (C) EPI-H then double-stained with Annexin V-FITC (AV) and cells were treated with (B) EPI-AQ and (C) EPI-H then double-stained with Annexin V-FITC (AV) and PI followed by flow cytometry analysis. Data are presented as a percentage of the cell population; PI followed by flow cytometry analysis. Data are presented as a percentage of the cell population; (D) As shown by western blot analysis EPI-AQ and EPI-H induce conversion of pro-caspase 3 to D ( ) Ascleaved-caspase-3 shown by western in AY-27 blot analysiscells. (E) EPI-AQHistogram and showing EPI-H the induce ratios conversion of cleaved-/pro-caspase-3 of pro-caspase in 3 to cleaved-caspase-3control, EPI-AQ-, in AY-27 and EPI-H-treated cells. (E) Histogram cells. Results showing are expressed the ratios as of the cleaved-/pro-caspase-3 mean ± SD. * p < 0.05 in in cases control, EPI-AQ-,of EPI-AQ and EPI-H-treated and EPI-H vs. cells.control. Results + p < 0.05 are for expressed EPI-H vs. as EPI-AQ. the mean ˘ SD. * p < 0.05 in cases of EPI-AQ and EPI-H vs. control. + p < 0.05 for EPI-H vs. EPI-AQ. Pro- and cleaved-caspase-3 proteins were measured by western blotting to assess apoptotic Pro-rates andFigure cleaved-caspase-3 3D shows that EPI-H proteins treatment were measuredcaused a reduction by western in pro-caspase-3 blotting to assess levels apoptoticand a rates Figure3D shows that EPI-H treatment caused a reduction in pro-caspase-3 levels and a concomitant increase in levels of cleaved-caspase-3 in Ay-27 cells. Figure3E shows that mean ratios of Molecules 2016, 21, 712 6 of 16 Molecules 2016, 21, 712 6 of 16 cleaved-/pro-caspase-3concomitant increase levelsin levels associated of cleaved-caspase-3 with control, in EPI-AQ,Ay-27 cells. and Figure EPI-H 3E treatments shows that weremean 0.27,ratios 1.13, and 3.83,of cleaved-/pro-caspase-3 respectively. These resultslevels associated indicated with that thecontrol, enhanced EPI-AQ, apoptosis and EPI-H induced treatments by EPI-H were in0.27, AY-27 cells1.13, occurred and 3.83, through respectively. the caspase These activation results indicated pathway. that the enhanced apoptosis induced by EPI-H in AY-27 cells occurred through the caspase activation pathway. 2.4. Urodynamic Parameters 2.4. Urodynamic Parameters Intravesical instillation of epirubicin to patients causes severe bladder irritation and induces early voiding. Thus,Intravesical understanding instillation theof epirubicin urodynamic to effectspatients of causes EPI-H severe is crucial bladder for assessingirritation itsand potential induces for early voiding. Thus, understanding the urodynamic effects of EPI-H is crucial for assessing its future clinical application. CMG profiles following intravesical instillation of normal saline (NS), H, potential for future clinical application. CMG profiles following intravesical instillation of normal EPI-AQ, and EPI-H in rats was recorded (Figure4A). The peak micturition pressures in NS, H, EPI-AQ, saline (NS), H, EPI-AQ, and EPI-H in rats was recorded (Figure 4A). The peak micturition pressures and EPI-H treatment groups were 21.2 ˘ 0.3, 18.7 ˘ 0.1, 51.0 ˘ 1.8, and 31.1 ˘ 1.7 cmH O, respectively in NS, H, EPI-AQ, and EPI-H treatment groups were 21.2 ± 0.3, 18.7 ± 0.1, 51.0 ± 1.8, and2 31.1 ± 1.7 (Figure4B). EPI-AQ elicited the highest peak micturition pressure, and EPI-H induced less bladder cmH2O, respectively (Figure 4B). EPI-AQ elicited the highest peak micturition pressure, and EPI-H hyperactivity.induced less The bladder threshold hyperactivity. pressures The observed threshold in NS,pressures H, EPI-AQ, observed and in EPI-H NS, H, treatment EPI-AQ, and groups EPI-H were 1.6 ˘treatment0.1, 1.8 ˘ groups0.4, 5.3 were˘ 0.4, 1.6 ± and 0.1,2.0 1.8 ˘± 0.4,0.5 cmH5.3 ± 20.4,O, respectivelyand 2.0 ± 0.5 cmH (Figure2O, 4respectivelyC). EPI-H treatment(Figure 4C). also significantlyEPI-H treatment reduced also threshold significantly pressure reduced when thresh comparedold pressure with thewhen treatment compared effect with of the EPI-AQ. treatment effectBladder of EPI-AQ. capacity was measured as the amount of saline infused into the bladder at the time micturitionBladder commenced. capacity Thewas infusionmeasured durations as the amount observed of saline in the infused NS, H, into EPI-AQ, the bladder and EPI-H at the treatment time groupsmicturition were 445.0 commenced.˘ 5.0, 313.3 The ˘infusion10.6, 309.2duration˘ 4.7,s observed and 214.0 in the˘ 3.0NS, s, H, respectively EPI-AQ, and (Figure EPI-H4D). The bladdertreatment compliances groups were of 445.0 the NS,± 5.0, H, 313.3 EPI-AQ, ± 10.6, and 309.2 EPI-H ± 4.7, treatment and 214.0 groups ± 3.0 s, wererespectively 44.4 ˘ (Figure2.6, 40.4 4D).˘ 12.1, The bladder compliances of the NS, H, EPI-AQ, and EPI-H treatment groups were 44.4 ± 2.6, 40.4 ± 12.1, 13.4 ˘ 1.2, and 36.5 ˘ 9.1 µL/cmH2O, respectively (Figure4E). EPI-AQ treatment appreciably reduced 13.4 ± 1.2, and 36.5 ± 9.1 μL/cmH2O, respectively (Figure 4E). EPI-AQ treatment appreciably reduced bladder compliance (p < 0.05) when compared with the treatment effects of NS and H. In contrast, EPI-H bladder compliance (p < 0.05) when compared with the treatment effects of NS and H. In contrast, treatment elicited bladder compliance results similar to that observed with the NS and H treatments. EPI-H treatment elicited bladder compliance results similar to that observed with the NS and H Overall,treatments. these observations Overall, these demonstratedobservations demonstrated that EPI-H that infusion EPI-H diminishesinfusion diminishes epirubicin-induced epirubicin-induced bladder hyperactivitybladder hyperactivity by reducing by micturition reducing micturition peak and peak threshold and threshold pressure, pressure, lessening lessening bladder bladder capacity, and increasingcapacity, and bladder increasing compliance. bladder compliance.

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Figure 4. Cont. Molecules 2016, 21, 712 7 of 16 Molecules 2016, 21, 712 7 of 16

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20 + # 60 O) 102 50 l/cmH Bladder complianceBladder ( μ 040 NS H EPI-AQ EPI-H 30 (E) 20 + # FigureFigure 4.4.Effects Effects of of intravesical intravesical epirubicin epirubicin10 on on voiding voiding function function in in the the rat. rat. (A )(A Representative) Representative tracings tracings of

of cystometrograms (CMG) recorded complianceBladder ( 0 from F344 rats following intravesical instillation of normal saline cystometrograms (CMG) recorded from F344NS rats H following EPI-AQ EPI-H intravesical instillation of normal saline (NS), H, EPI-AQ, and EPI-H. Urodynamic parameters after treatments were (B) peak micturition (NS), H, EPI-AQ, and EPI-H. Urodynamic parameters(E) after treatments were (B) peak micturition pressure (cmH2O); (C) threshold pressure (cmH2O); (D) micturition duration (s); and (E) bladder pressure (cmH2O); (C) threshold pressure (cmH2O); (D) micturition duration (s); and (E) bladder Figure 4. Effects of intravesical epirubicin on voiding+ function in the rat. (A) Representative# tracings compliancecompliance (µμL/cmH2O).O). Data Data are are mean mean ± S.D.˘ S.D. p <+ 0.05p < 0.05compared compared with NS. with p NS. < 0.05# p compared< 0.05 compared with H. of cystometrograms2 (CMG) recorded from F344 rats following intravesical instillation of normal saline * p < 0.05 compared with EPI-AQ. with H.(NS), * p < H, 0.05 EPI-AQ, compared and EPI-H. with EPI-AQ.Urodynamic parameters after treatments were (B) peak micturition

pressure (cmH2O); (C) threshold pressure (cmH2O); (D) micturition duration (s); and (E) bladder 2.5. In Situ Urothelium Penetration + # 2.5. In Situ Urotheliumcompliance (μL/cmH Penetration2O). Data are mean ± S.D. p < 0.05 compared with NS. p < 0.05 compared with H. * p < 0.05 compared with EPI-AQ. EPI-HEPI-H andand EPI-AQEPI-AQ werewere eacheach intravesicallyintravesically instilledinstilledinto into ratrat bladderbladder andand epirubicinepirubicin penetrationpenetration intointo the the2.5. urothelium urothelium In Situ Urothelium associated associated Penetration with with each each treatment treatmen wast was measured. measured. Epirubicin Epirubicin excites excites and and emits emits as red as underred under ﬂuorescence fluorescenceEPI-H and microscopy. EPI-AQ microscopy. were As each shown intravesically As shown in Figure instilin 5Fi,led EPI-AQ,gure into 5,rat EPI-AQ, whichbladder isand which the epirubicin form is usedthe penetration form in the used reported in the clinicalreported intravesicalinto clinical the urothelium intravesical instillations, associated instillations, was withpresent each treatmen was at extremelypresentt was measured. at low extremely levels Epirubicin in low the excites superﬁciallevels and in emits the plaque as superficial layer red under fluorescence microscopy. As shown in Figure 5, EPI-AQ, which is the form used in the ofplaque the bladder. layer of EPI-H the bladder. forms a EPI-H dense forms coating a (indense red) coating on the (in urothelial red) on surface, the urothelial which indicatedsurface, which that indicatedreported that clinicalmore epirubicinintravesical instillations,penetrates was the present plaques at extremelyand that low the levels epirubicin in the superficial is distributed to more epirubicinplaque layer penetrates of the bladder. the plaques EPI-H forms and thata dense the coating epirubicin (in red) is distributedon the urothelial to umbrella surface, which cells, Tb cells, umbrella cells, Tb cells, and basal cells above the lamina propria. and basalindicated cells above that more the laminaepirubicin propria. penetrates the plaques and that the epirubicin is distributed to umbrella cells, Tb cells, andEPI-AQ basal cells above the lamina propria. EPI-H EPI-AQ EPI-H

U U I I B B S S Fluorescence Fluorescence Fluorescence Fluorescence

U I B

H&E U S I B

H&E S

Figure 5. EPI-H enhanced penetration of epirubicin to urothelium. Fluorescence images of urothelium Figure 5.showedEPI-H epirubicin enhanced (red) penetration and DAPI (nuclei, of epirubicin blue) af toter urothelium.intravesical EPI-AQ Fluorescence or EPI-H imagesinstillation of urotheliumin showedF344 epirubicin rats. Histopathology (red) and DAPI sections (nuclei, were blue) stained after by intravesical hematoxylin/eosin EPI-AQ (H or&E). EPI-H Umbrella instillation cells, in F344 rats.Figure Histopathology intermediate5. EPI-H enhanced cells, sections basal penetration cells, were and stained subepithelial of epirubicin by hematoxylin/eosin connective to urothelium. tissue of the (H&E). Fluorescence urothelium Umbrella are images respectively cells, of intermediate urothelium indicated by U, I, B, and S. (magnification, ×200) cells,showed basal epirubicin cells, and (red) subepithelial and DAPI connective (nuclei, tissueblue) af ofter the intravesical urothelium EPI-AQ are respectively or EPI-H indicated instillation by U,in

I,F344 B, and rats. S. (magniﬁcation,Histopathologyˆ 200)sections were stained by hematoxylin/eosin (H&E). Umbrella cells, intermediate cells, basal cells, and subepithelial connective tissue of the urothelium are respectively indicated by U, I, B, and S. (magnification, ×200)

Molecules 2016, 21, 712 8 of 16 Molecules 2016, 21, 712 8 of 16

Histological assessment following following a a 2-h instillation with EPI-AQ revealed that the urothelium of the urinary bladder was intact, with rounded surf surfacesaces of the umbrella cells, and showed slight submucosal edemaedema andand inflammation. inflammation With. With instillation instillation of EPI-H,of EPI-H, the the umbrella umbrella and and intermediate intermediate cells cellsof the of urothelium the urothelium exhibited exhibited a mild a mild loss of loss integrity of integrity at focal at areasfocal areas where where the epithelium the epithelium showed showed some somesubtle subtle denudation. denudation. EPI-H EPI-H showed showed detrimental detrimental effects effects with with signs signs of cell of damage,cell damage, inflammation inflammation and andepithelium epithelium denudation. denudation. Histological Histological staining staining revealed revealed no remarkableno remarkable microscopic microscopic features features in the in thesuburothelial suburothelial stroma. stroma. Figure 66 presentspresents thethe tissuetissue concentration-depthconcentration-depth profileprofile ofof EPI-AQEPI-AQ andand EPI-HEPI-H inin thethe pigpig bladderbladder wall following the Franz diffusion assay. The concentrationsconcentrations of epirubicin we werere normalized according to the weight of wet tissue.

100

EPI-H 80 EPI-AQ

60 * g of EPI/mg of tissue) tissue) of g of EPI/mg

μ 40 ** 20 ** *** Tissue levels ( levels Tissue 0 200 400 600 800 1000 Tissue Depth (μm)

Figure 6. Tissue level-depth proﬁlesprofiles of epirubicinepirubicin inin bladderbladder tissuetissue followingfollowing exposureexposure toto 22 mg/mLmg/mL EPI-AQ or EPI-H formulations. Tissues Tissues were incubated for 2 h and sectioned by cryotome. cryotome. Values Values are means ±˘ SEMSEM ( (nn == 3). 3). * *pp << 0.05, 0.05, ** ** p p< <0.01 0.01 and and *** *** p p< <0.001 0.001 in in cases cases of of EPI-AQ EPI-AQ vs.vs. EPI-H.EPI-H.

The mean concentrations of epirubicin (μg/mg tissue) at EPI-H/EPI-AQ groups at a depth from The mean concentrations of epirubicin (µg/mg tissue) at EPI-H/EPI-AQ groups at a depth from the apical urothelium were 81.4/77.9 (at 200 μm), 55.8/50.5 (at 400 μm, p < 0.05), 39.9/20.1 (at 600 μm, the apical urothelium were 81.4/77.9 (at 200 µm), 55.8/50.5 (at 400 µm, p < 0.05), 39.9/20.1 (at 600 µm, p < 0.01), 32.7/7.3 (at 800 μm, p < 0.01), and 16.5/1.8 (at 1000 μm, p < 0.01), respectively. Tissue p < 0.01), 32.7/7.3 (at 800 µm, p < 0.01), and 16.5/1.8 (at 1000 µm, p < 0.01), respectively. Tissue concentrations of epirubicin decreased as the tissue depth from the urothelium increased. EPI-H concentrations of epirubicin decreased as the tissue depth from the urothelium increased. EPI-H released more epirubicin into urothelium over that of EPI-AQ. released more epirubicin into urothelium over that of EPI-AQ.

2.6. In In Vivo Vivo Antitumor Efficacy Efficacy The potential anti-cancer activity activity of of EPI-H EPI-H ag againstainst AY-27 AY-27 bladder tumors in F344 rats was evaluated. NormalNormal (control)(control) rat rat bladder bladder showed showed an an intact intact urothelium, urothelium, including including surface surface umbrella umbrella cells cellswith with scalloped scalloped appearance appearance and fuzzy and fuzzy superficial superficial cytoplasm cytoplasm (Figure (Figure7A). AY-27 7A). administrationAY-27 administration caused causedbladder bladder tumor invasion tumor invasion from the from urothelium the urothelium to the muscle to the layer muscle (T2 layer stage). (T2 The stage). tumor The cells tumor exhibited cells exhibitedan increase an in increase the nuclear-to-cytoplasmic in the nuclear-to-cytoplasmic (N/C) ratio (N/C) with ratio markedly with markedly increased increased variation variation in nuclear in nuclearsize and size shape, and and shape, mitotic and figures. mitotic Thefigures. hydrogel The hydrogel treatment treatment group (H) group also showed (H) also similar showed histologic similar histologicfeatures associated features associated with bladder with tumor bladder including tumor including marked nuclear marked pleomorphism. nuclear pleomorphism. In the EPI-AQ In the EPI-AQtreatment treatment group, low group, to moderate low to moderate dysplasia dysplasi and concentrationa and concentration of inflammatory of inflammatory cells were cells evident were evident(T1 stage). (T1 Intravesical stage). Intravesical instillation instillation of EPI-H however of EPI-H restricted however tumor restricted growth totumor the urothelium growth to layer, the urotheliumprevented progress layer, prevented to advanced progress stages, to and advanced elicited stages, necrosis and of tumorelicited cells. necrosis EPI-H of showed tumor cells. detrimental EPI-H showedeffects with detrimental signs of effects cell damage, with signs inflammation of cell damage, and epitheliuminflammation denudation. and epithelium Themicro-features denudation. The of micro-featuresthe EPI-H treatment of the groupEPI-H showedtreatment marked group inflammatoryshowed marked cell inflammatory infiltration (e.g., cell lymphocytes infiltration (e.g., and lymphocytesneutrophils) inand the neutrophils) tumor. Several in the tumor tumor. nests Several were tumor apparent nests with were cell apparent debris with confined cell inside.debris confinedThese observations inside. These demonstrated observations the anti-cancerdemonstrated activity the ofanti-cancer EPI-H treatment activity against of EPI-H bladder treatment tumors. against bladder tumors. Levels of caspase-3 proteins were assessed by immunofluorescence microscopy of paraffin-embedded AY-27 tumor sections of F344 rats as a means to evaluate apoptosis (Figure 7B).

Molecules 2016, 21, 712 9 of 16

EPI-H treatment induced higher caspase-3 levels than that induced by EPI-AQ treatment. These results confirmed that the increased apoptosis induced by EPI-H in AY-27 cells occurred through the caspase-3 activation pathway. The percentage of caspase-3-positive cells in the urothelium carcinoma (UC), hydrogel (H), EPI-AQ, and EPI-H groups were 3.1% ± 1.0%, 1.8% ± 0.9%, 13.9% ± 4.0%, and

Molecules34.1% ± 20161.0%,, 21 ,respectively 712 (Figure 6C). These results were consistent with the in vitro study results9 of 16 shown in Figure 3.

Normal urothelium UC H

EPI-AQ EPI-H EPI-H (magnified view)

(A) UC H

EPI-AQ EPI-H

(B)

Figure 7. Cont. Molecules 2016, 21, 712 10 of 16 Molecules 2016, 21, 712 10 of 16

40 *

10 caspase-3 positive cells (%) cells caspase-3 positive 0 UC H EPI-AQ EPI-H

(C)

Figure 7. EPI-H enhanced antitumor effica efficacycy in F344/AY-27 rat model. ( A)) Histopathologic Histopathologic findings findings with H&E staining showed normal normal urothelium; urothelium; ur urotheliumothelium carcinoma carcinoma (UC), (UC), and and UC UC treated treated with with H, H, EPI-AQ, EPI-H, EPI-H, and and the enlarged image of yellow box in EPI-H. The yellow arrowheads indicated apoptotic nuclei. ( (BB)) Immunofluorescence Immunofluorescence of of bladder bladder section section showed showed caspase-3 caspase-3 (green) (green) and and DAPI (nuclei, blue) in UC, H, EPI-AQ, andand EPI-H groups. (magnification,(magnification, 200200×)ˆ)( (CC)) Quantitative Quantitative analysis analysis of of caspase-3caspase-3 in in immunofluorescence immunofluorescence images. images. Average Average of the positively stained stained area area was evaluated evaluated from from threethree randomly selected selected observation observation fields fields in in each bladder section. * * p < 0.001, 0.001, compared with the EPI-AQ group.

3. Discussion Levels of caspase-3 proteins were assessed by immunofluorescence microscopy of paraffin-embeddedThis study examined AY-27 the tumor optimal sections epirubicin of F344 deliv ratsery as asystem means for to evaluateintravesic apoptosisal therapy (Figure of bladder7B). cancerEPI-H treatmentas a means induced to improve higher drug caspase-3 efficacy levels and thanto reduce that induced adverse by effects EPI-AQ in a treatment. rat tumor These model. results The responseconfirmed rate that for the intravesical increased drug apoptosis instillation induced is approximately by EPI-H in 30% AY-27 when cells tumors occurred are present, through and the approximatelycaspase-3 activation 70% pathway.after tumor The surgery, percentage and of caspase-3-positiveit differs according cells to inthe the agent urothelium used [9]. carcinoma Major limitations(UC), hydrogel of intravesical (H), EPI-AQ, chemotherapeutic and EPI-H groups drugs wereare attributable 3.1% ˘ 1.0%, to low 1.8% bladder˘ 0.9%, residency 13.9% and˘ 4.0%, low penetrationand 34.1% ˘ into1.0%, the respectively urothelium. (Figure This7C). study These show resultsed, were to our consistent knowledge, with the forin the vitro firststudy time results the feasibilityshown in Figureof gelatin3. hydrogel carriers as a means to administer intravesical epirubicin to treat more effectively superficial bladder cancer. 3. DiscussionThe advantage of intravesical chemotherapy is its restriction to the bladder and insignificant systemicThis drug study absorption examined and the distribution. optimal epirubicin The plasma delivery uptake system of any for drug intravesical administered therapy intravesically of bladder iscancer dependent as a means on its tomolecular improve weight drug efficacy(MW), and and to to a reducelesser degree, adverse on effects its lipid in solubility. a rat tumor Systemic model. absorptionThe response from rate the for bladder intravesical of drugs drug with instillation MW > is 300 approximately is minimal [10]. 30% whenAbsorption tumors of areepirubicin present, (MWand approximately 342) instilled into 70% the after bladder tumor is generally surgery, andslight it, and differs plasma according levels do to thenot agentexceed used 0.5 ng/mL [9]. Major [11]. Inlimitations this study, of intravesical intravesical instillation chemotherapeutic of EPI-H drugs resulted are in attributable more epirubicin to low retention bladder residencyin the urothelium and low thanpenetration that achieved into the by urothelium. EPI-AQ instillation This study showed,in vivo in to an our orthotopic knowledge, rat for model. the first Using time the feasibilityhydrogel carrier,of gelatin more hydrogel epirubicin carriers penetrated as a means plaques to administer and distributed intravesical to umbrella epirubicin cells, to intermediate treat more effectivelycells, and basalsuperficial cells. Epirubicin bladder cancer. reached the lamina propria but did not significantly penetrate muscle or bladder serosa.The These advantage experiments of intravesical indicated chemotherapy that gelatin hy isdrogel its restriction carriers toare the suitable bladder for and applications insignificant in superficialsystemic drug urothelium absorption cancer, and distribution.a health problem The plasma providing uptake a major of any indication drug administered for intravesical intravesically (rather thanis dependent systemic) on chemotherapy. its molecular weight The efficacy (MW), andof toEPI-H a lesser was degree, also observed on its lipid through solubility. histological Systemic measurements.absorption from EPI-H the bladder induced of tumor drugs cell with necrosis MW > and 300 isprevented minimal tumor [10]. Absorptionprogressionof inepirubicin a 2-week instillation(MW 342) instilled protocol. into the bladder is generally slight, and plasma levels do not exceed 0.5 ng/mL [11]. In thisGelatin study, transforms intravesical from instillation gel to solution of EPI-H (sol) resulted by raising in morethe temperature. epirubicin retention The reversible in the melting urothelium and congelationthan that achieved of gelatin, by EPI-AQnamely, instillation solation andin vivogelationin an, orthotopictake place ratby model.heating Usingand cooling, the hydrogel respectively. carrier, Gelatedmore epirubicin gelatin penetratedcontains three plaques straight and distributed chain molecules to umbrella that cells,combine intermediate to form cells,complicated and basal three cells. dimensionalEpirubicin reached structures. the lamina Heating propria of gelatin but did causes not significantly the disintegration penetrate of musclethese three-dimensional or bladder serosa. structures followed by the dispersion of the chain molecules [12,13]. In present study, there is gel-sol

Molecules 2016, 21, 712 11 of 16

These experiments indicated that gelatin hydrogel carriers are suitable for applications in superficial urothelium cancer, a health problem providing a major indication for intravesical (rather than systemic) chemotherapy. The efficacy of EPI-H was also observed through histological measurements. EPI-H induced tumor cell necrosis and prevented tumor progression in a 2-week instillation protocol. Gelatin transforms from gel to solution (sol) by raising the temperature. The reversible melting and congelation of gelatin, namely, solation and gelation, take place by heating and cooling, respectively. Gelated gelatin contains three straight chain molecules that combine to form complicated three dimensional structures. Heating of gelatin causes the disintegration of these three-dimensional structures followed by the dispersion of the chain molecules [12,13]. In present study, there is gel-sol transition at 25 ˝C. The aim of present hydrogel is applied for intravesical chemotherapy, thus, solution status in body temperature provide suitable fluidity to cover and adherent urothelium. The loading efficiency of gelatin varies according to preparation methods and drug characteristics. A loading efficiency of 29%–32% was obtained by means of a water/oil emulsion method for methotrexate in gelatin nanoparticles [14]. A 15%–19% loading efficiency was reported for chloroquine phosphate hydrogel using a similar protocol [15]. The gelatin hydrogels produced in the present study were covalently crosslinked by glutaraldehyde, which served to increase stability and mechanical properties [16]. Glutaraldehyde is indispensable for tizanidine hydrochloride-loading; loading is 13.8% with glutaraldehyde-based preparation but only 1.1% without glutaraldehyde-mediated crosslinking [17]. Loading efficiency is also influenced by physiochemical properties of the drugs and of the gelatin concentration. In the present study, the epirubicin loading efficiency in 5% and 15% gelatin was 47.40% and 57.87%, respectively. The major adverse effects of epirubicin instillation are bladder irritation, hematuria, pyuria, atrophic bladder, and hemorrhagic cystitis [18]. Weekly instillations of epirubicin were reported to induce changes in vesical volume and compliance, although only the change in bladder compliance was associated with statistical significance [11]. Decreased bladder compliance is probably due to direct superficial epithelial damage and cell loss, leading to mucosal irritation and submucosal edema. In humans, chemical cystitis after intravesical chemotherapy manifests primarily as urinary frequency, urgency, and/or bladder pain. In this study, we determined whether functional bladder damage occurred after intravesical chemotherapy with EPI-H. Higher levels of epirubicin entering the urothelium would seem to inevitably lead increase the risk of chemical cystitis. In comparison with EPI-AQ treatment, the use of EPI-H resulted in lower micturition duration, indicating decrease bladder capacity. Fortunately, the bladder compliance was significantly higher in the EPI-H treatment group when compared with that of the EPI-AQ treatment group. Flow cytometry analysis showed that EPI-H treatment doubled the number of AY-27 cells entering the early stages of apoptosis. We determined that this was related to an increased cleaved-/pro-caspase-3 ratio in the treated AY-27 cells. Caspase-3 expression in the AY-27 rat bladder cancer cell line is known to be associated with the apoptotic response [19]. Caspase-3 is also involved in bladder cancer apoptosis when cells are exposed to epirubicin [20]. Apoptosis failure is a crucial step in the initiation and progression of cancer. This information can be used clinically to select chemotherapeutic drugs that act in a hormone-dependent or independent manner. For therapeutic intravesical chemotherapy, bladder cancer cells are more sensitive to EPI-H than to the currently used EPI-AQ. Intravesical EPI-H instillation exhibited an anti-cancer effect on a urothelium carcinoma that had been orthotopically implanted into a rat model. An orthotopic bladder tumor model is indispensable in studies of therapeutic effects. AY-27 rat bladder cancer cells transplanted orthotopically into Fischer F344 female rats exhibit features of transitional cell carcinoma (TCC) [21]. Patchy carcinoma in situ can be detected histologically at 12–13 days after inoculation, and the condition progresses to papillary tumor or invasive disease thereafter. An NMIBC study indicated that the 2-year recurrence-free survival rate following epirubicin treatment is 55% [22]. Intravesical epirubicin in the NMIBC study resulted in a recurrence rate of 36%–62%. There were 14–38 months in median time to recurrence, Molecules 2016, 21, 712 12 of 16 and the 5-year cumulative recurrence rate was 58%–69% [1]. The presently developed EPI-H system has an improved intravesical chemotherapeutic ability to prevent massive tumor progression and invasion, and it could improve the numbers described above.

4. Materials and Methods

4.1. Materials Epirubicin was obtained from Calbiochem (La Jolla, CA, USA). Gelatin, glycine, propidium iodide (PI), and glutaraldehyde were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All organic solvents were of HPLC grade. All cell culture media and reagents were from Gibco BRL (Grand Island, NY, USA) or Hyclone (Logan, UT, USA). The annexin V-FITC kit was purchased from Calbiochem. Fischer F344 rats were purchased from the National Laboratory Animal Center (NLAC) (Taipei, Taiwan). Animal protocols were approved by the Animal Ethics Committee of I-Shou University.

4.2. HPLC Analysis of Epirubicin Epirubicin was analyzed by HPLC with fluorescence detection (FLD) [7]. Briefly, HPLC-FLD analyses were carried out using a model L7100 solvent delivery system (Hitachi, Tokyo, Japan). An AcclaimR120 (150 mm ˆ 4.6 mm ID) (Dionex, Surrey, UK) column was used for separation of EPI. The mobile phase consisted of acetonitrile/0.1 M orthophosphoric acid/triethylamine/water at volumetric ratios 27:3:0.07:70 with an apparent pH of 2.8. The flow rate of the mobile phase was 1.0 mL/min. Doxorubicin was used as an internal standard. The column outlet stream was monitored at λex-474 nm and λem-551 nm. Linearity was found to be in the range of 0.2–40 µg/mL with a coefficient of determination (R2) of 0.9999. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.07 µg/mL, respectively.

4.3. Preparation of Gelatin Hydrogel Hydrogel was prepared according to previous methods [8] using A type gelatin (Sigma) with 75 or 175 bloom numbers. An aqueous solution of 5% (w/v) gelatin (1 mL) with 0.8 µg/mL of glutaraldehyde was left at 4 ˝C overnight for gelation and cross-linking action. The cross-linked gelatin hydrogel was immersed in 50 mM glycine aqueous solution under agitation for 1 h to block the residual aldehyde groups of glutaraldehyde, followed by washing twice in double-distilled water for 1 h. The resulting hydrogel was freeze-dried for 48 h. Freeze-dried gelatin was dissolved in 1 mL of normal saline to form 15% (w/v) hydrogel, and the solution was heated to 37 ˝C.

4.4. Characterization of Hydrogel After allowing the freeze-dried hydrogel to swell for 24 h at 37 ˝C in normal saline, the weight of swollen hydrogel (Ws) was measured. The swollen hydrogel was dried in a vacuum drying oven at 60 ˝C for 6 h, and then the weight of vacuum-dried hydrogel (Wd) was measured. The hydration ratio was calculated using the following equation: [(Ws ´ Wd)/Ws] ˆ 100. The viscosity of hydrogel was determined using a Carri-Med CSL2 100 rheometer (TA Instruments, New Castle, DE, USA). Particle size distribution and mean diameter were determined using an N5 Submicron Particle Size Analyzer (Beckman, Hialeah, FL, USA). The surface morphology of freeze-dried hydrogel was determined using a ﬁeld emission scanning electron microscope (FE-SEM; JEOL JSM-5600 LV, Tokyo, Japan). Hydrogel was frozen in liquid nitrogen prior to freeze-drying to maintain the existing morphology. Sectioned gels were mounted on metal holders and vacuum-coated with a gold layer prior to SEM examination. The ﬂuidity of the hydrogel was visually monitored at 25 ˝C and 37 ˝C[8].

4.5. Entrapment Efﬁciency of EPI-H Epirubicin was distributed in 15% (w/v) hydrogels for the following physicochemical properties and bioactivity evaluation. To determine the entrapment efﬁciency, EPI-H hydrogel (0.1 g) was Molecules 2016, 21, 712 13 of 16 dissolved in 10 mL of normal saline, centrifuged and the epirubicin content of supernatant was determined by the aforementioned HPLC method:

actual epirubicin concentration entrapmentefﬁciencyp%q “ ˆ 100 theoretical epirubicin concentration

4.6. Cell Culture and Cytotoxic Assay The rat bladder cancer cell line AY-27 (a kind gift from Professor R. Moore, University of Alberta, Edmonton, AB, Canada) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine ˝ serum (FBS), 2% L-glutamine, and 0.2% penicillin/streptomycin at 37 C with 5% CO2 [21]. Cell viability was determined using the CellTiter 96 Aqueous nonradioactive cell proliferation assay according to the manufacturer’s instructions (Promega, Madison, WI, USA) [23]. AY-27 cells (1 ˆ 104) were seeded in 96-well plates and then treated with epirubicin-loaded hydrogels for 18 h. Cell-growth inhibition potency was expressed as IC50 values, deﬁned as the concentration of the drug necessary to inhibit the growth of cells by 50% in 18 h. The percentage of surviving cells was deﬁned as (mean absorbance of treated wells/mean absorbance of untreated wells) ˆ 100%. Data are the means ˘ S.D. of four experiments.

4.7. Flow Cytometry with Annexin V-FITC and PI Staining AY-27 cells were pretreated with EPI-H or EPI-AQ (as final concentration of epirubicin 0.25 µg/mL) for 18 h. AY-27 cells were harvested, washed with ice-cold phosphate buffered saline (PBS), and stained with Annexin V-FITC (AV)/PI reagent as described previously [24]. Apoptosis rates were quantified by means of fluorescence-activated cell sorting flow cytometry (BD Biosciences, Ann Arbor, MI, USA). AV-positive/PI-negative cells were scored as early apoptotic, and AV-positive/PI-positive cells were counted as late apoptotic cells.

4.8. Western Blotting AY-27 cells were pretreated with EPI-H or EPI-AQ (as final concentration of epirubicin 0.25 (µg/mL) for 18 h. AY-27 cells were washed twice in cold PBS, and lysed in TBS containing 1 mM EDTA, 1 mM DTT, 0.2% Triton, 0.1% SDS, and the complete protease inhibitors mixture (Roche, Indianapolis, IN, USA). Proteins were subjected to SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Boehringer Mannheim, Indianapolis, IN, USA). Membranes were probed with antibodies specific for caspase-3 (Cell Signaling, Danvers, MA, USA), cleaved caspase-3 (Cell Signaling), or actin (Merck Millipore, Darmstadt, Germany), and incubated with the HRP-conjugated anti-rabbit Ig or anti-mouse Ig Ab. Signals were revealed with the ECL kit (Merck Millipore), and visualized by autoradiography. Increased levels of cleaved-caspase-3 (17 kDa) with a concomitant decrease in levels pro-caspase-3 (32 kDa) is regarded as an indicator of apoptosis.

4.9. Cystometrogram (CMG) Analysis The CMGs were carried out according to previously described methods [25,26]. Fischer F344 rats (7 weeks) were anesthetized with Zoletil-50 (1 mg/kg). Before the beginning of each CMG, the bladder was emptied, and a urethral catheter was inserted to fill the bladder and to measure bladder pressure. The catheter was connected via a T-tube to a syringe pump (KDS250, KD Scientific Corp., MA, USA) and to a pressure transducer and amplifier (ML866 and ML224, PowerLab, AD Instruments, Colorado Springs, CO, USA). Data were recorded on a chart recorder and digitized for computer data collection (Labchart 7, AD Instruments, Colorado Springs, CO, USA). The bladder was then infused with 500 µL normal saline (as control), and epirubicin-loaded hydrogel at a steady rate (0.07 mL/min), during which time the pressure was measured in-line through the catheter. A voiding contraction was defined as an increase in bladder pressure that resulted in urine loss. CMG was recorded until the bladder pressure was stable, and at least 5 filling/voiding cycles were measured on each rat before drug Molecules 2016, 21, 712 14 of 16 administration, and used as baseline values. CMG parameters recorded for each animal included peak micturition pressure, threshold pressure, duration of non-voiding contractions (without urine leakage during bladder infusion), and bladder compliance. Peak micturition pressure was the maximum pressure during micturition as observed in CMG. Threshold pressure was the intravesical pressure immediately prior the initiation of micturition. Bladder compliance was measured by infused volume (µL)/threshold pressure (∆cmH2O).

4.10. Ethics Statement All in-life rat studies were performed under Association for Assessment and Accreditation of Laboratory Animal Care-approved conditions and approved by the I-Shou University Institutional Animal Care and Use Committee (Approval numbers IACUC-ISU-102025). Animals were sacriﬁced by carbon dioxide asphyxiation.

4.11. In Vivo Urothelium Permeability and Histologic Analysis Female F344 rats (7 weeks) were treated with EPI-H or EPI-AQ (500 µL/180 g b.w.) for 2 h via urethral catheter. After administration, rat urinary bladder tissue pieces with flat surfaces were sectioned by CM 1900 cryostat (Leica, Amtzell Germany) and observed by fluorescence microscope (Eclipse 4000, Nikon, Tokyo, Japan). The remaining rat bladder tissue was fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for histological analysis [27].

4.12. Determination of Epirubicin in the Bladder Tissues Fat removed, freshly excised porcine bladders were opened and cut into pieces measuring approximately 3 ˆ 3 cm in a shallow bath of pH 7.4 PBS buffer. Bladder sections were mounted onto a Franz diffusion cell apparatus, such that the luminal side of the bladder wall faced upwards to contact the drug solution as previously described. The tissue sections were not stretched and measured approximately 2–3 mm thick. Receptor chambers contained 6 mL of 37 ˝C PBS buffer (pH 7.4). The donor chambers contained 0.5 mL of EPI-H and EPI-AQ (all 2 mg/mL drug). The diffusion cells were maintained at 37 ˝C for 2 h. The drug contact areas of the tissue samples were then cut out and rapidly frozen with liquid nitrogen on a bed of dry ice. Tissues were then sectioned into 200 µm thick samples by cryostat (Leica CM 1900). Epirubicin concentrations in the tissue were determined by HPLC [7].

4.13. Bladder Tumor Implantation, Intravesical Drug Instillation and Histology Analysis In situ urothelium cancer was induced by intravesical instillation of the rat AY-27 bladder tumor cell line [21]. F344 rats were anaesthetized, and the bladder was catheterized via urethral catheter. To facilitate tumor seeding, the bladder mucosa was conditioned with 0.4 mL of 0.1 N hydrochloric acid (HCl) for 15 s and neutralized with 0.4 mL of 0.1 N potassium hydroxide (KOH) for 15 s. The bladder was then drained and flushed with sterile PBS. Immediately after bladder conditioning, the AY-27 cells ((1 ˆ 107) were instilled and left indwelling for at least 1 h. The rats were turned 90˝ every 15 min to facilitate whole bladder exposure to the tumor cell suspension. After 1 h, the catheter was removed and the rats were allowed to void the suspension spontaneously. After tumor implantation (on 1st day), F344 rat bladders were intravesically instilled with EPI-H on the 8th, 10th, and 12th days. All of the rats were killed on the 14th day. After killing, bladders were excised, fixed in 4% formalin overnight, dehydrated, and then embedded in paraffin. The embedded tissues were cut into 5-µm slices and stained with hematoxylin and eosin (H&E) for histological analysis.

4.14. Urothelium Caspase-3 Immunofluorescent Staining Paraffin-embedded tumor sections were subjected to immunofluorescence analysis. Briefly, sections were deparaffinized, rehydrated, subjected to heat-induced antigen retrieval, and quenched. Molecules 2016, 21, 712 15 of 16

Sections were blocked with Antibody Diluent (Dako, Carpinteria, CA, USA) and incubated with a caspase-3 antibody (1:500) (Cell Signaling). Sections were then washed and incubated with anti-rabbit IgG and counterstained with mounting media containing DAPI and analyzed using a microscope (Eclipse 4000, Nikon). The number of caspase-3+ cells in the bladder sections was counted in 3 randomly selected ﬁelds (ˆ200), and the average of the 3 ﬁelds was calculated [28].

4.15. Statistical Analysis The data are presented as mean ˘ standard deviation (SD). Data were analyzed using the student t-test and one-way analysis of variance (ANOVA). Statistical signiﬁcance was determined at the level of p < 0.05.

5. Conclusions Gelatin hydrogels are effective carriers of epirubicin for intravesical chemotherapy of superficial urothelium cancer. The strong bio-adhesive property of gelatin hydrogel minimizes flushing away by urine, thereby enhancing antitumor efficacy of loaded drugs. Hydrogel delivery of epirubicin represents an attractive strategy for intravesical drug development.

Acknowledgments: We acknowledge the financial supports of Ministry of Science and Technology, R.O.C. (MOST 102-2314-B-214-003-MY3, MOST 103-2320-B-214-003, MOST 104-2320-B-214-002). Author Contributions: Y.-T.W. and L.-C.C. conceived and designed the experiments. C.-W.L. performed the experiments and wrote the manuscript. K.-J.L., T.-J.Y. and Y.-L.K. assisted with the experiments. Conflicts of Interest: The authors have no conflicts of interest to declare.

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Sample Availability: Sample of epirubicin-loaded gelatin hydrogel is available from the authors.

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