The aim of this investigation is preparation of Mitomycin-C encapsulated
with chitosan nanoparticles synthesis using ionic gelation technique for intravesical controlled drug delivery systems. This study was conducted in vitro. Cumulative amount of drug released from the nanoparticles was calculated. Mitomycin-C release studies were examined for different pH values. During the drug loading and release studies, initial amount of drug was changed (i.e., 0.5, 1.25 and 2.5 mg) to get different release profiles and the release studies were repeated (n = 6). The loading efficiencies of Mitomycin-C with three different initial concentrations 0.5mg/ml, 1.25 mg/ml and 2.5 mg/ml into chitosan nanoparticles were 54.5%, 47.1% and 36.4%, respectively. For different pH values, the cumulative releases of Mitomycin-C from chitosan nanoparticles were 47% and 53% for pH 6.0 and 7.4, respectively (p < 0.01). For different drug doses, the cumulative releases of Mitomycin-C (MMC) from Chitosan nanoparticles were 44%, 53% and 65% for 0.5 mg/mL, 1.25 mg/mL and 2.5 mg/mL respectively (p < 0.01). The anticancer activity of Mitomycin-C loaded chitosan nanoparticles was measured in T24 bladder cancer cell line in vitro, and the results revealed that the 2.5 MMC coated Chitosan nanoparticles had better tumor cells decline activity. From this investigation, we conclude that the drug encapsulated synthesized chitosan nanoparticles possess a high ability to be used as pH and dose responsive drug delivery system. This systematic investigation demonstrates a promising future for the intravesical installation in treatment of the superficial bladder cancer.
Bladder carcinoma is one of the most predominant malignancies of the urinary tract. Most bladder tumors are superficial at the diagnostics stage, since the symptoms are nonspecific and may be linked with many other conditions. Many urothelial cell carcinomas diagnoses are not invasive, since they go no deeper than the superficial layer (mucosa) of the bladder [1] . These tumors not only can be treated by surgical resection but also adjuvant intravesical installation of Bacille Calmette Guerine (BCG) and other chemotherapeutic agents including Mitomycin-C (MMC), Epirubicin, Doxorubicin and Adriamycin [2] [3] [4] [5] . Additionally, Mitomycin-C, Epirubicin and Doxorubicin can be used for a single, immediate, post-operative intravesical instillation after resection of the tumor [6] [7] . MMC is frequently used as an intravesical agent. However, the optimal dose usage and installation frequency of the MMC is somewhat controversial [8] . At present, some methods such as microwave-induced hyperthermia or electromotive drug administration, have been known to increase efficacy of MMC, however, the optimal usage of MMC has not been described [9] . It is further known that MMC should be administrated for an extended period with repeated dose administration to prevent of further tumor recurrences and progression. The optimal MMC molecule demonstrates certain characteristics, such as long-acting, reduced administration of repeated dose, reduced side-effect and low dose requirement, however there is no known ideal MMC molecule.
Drug delivery systems employ specific technologies that focus mainly on targeting release or delivery of drugs or any substance that is of medicinal importance. As an example, controlled drug release systems using anti-neoplastic drugs are currently being investigated by several researchers. The nanomaterial landscape is immense since well-characterized polymers, lipids, peptides and proteins, sugars, and surfactants that can be engineered into novel Nano formulation platforms. Liposomes, dendrimers, and nanogels have been used for both controlled drug delivery and cell growth scaffolds and are presented in many nanomedicines and will appear, to some extent, also in the nanomedicines in the future [10] [11] . Using advanced nanomaterials, such as functionalized quantum dots (QDs), nanoparticles can pave the way towards improved nano-bio systems with minimal resources, reduced health hazard and environmental friendly impacts [12] [13] . It has been further reported that major challenges in drug delivery, with synthesized drug delivery carriers’ molecules, are their accumulations in the targeted cells, targeting a specific tissue with nuclear uptake and endosomal transfer [13] . Additionally, many other materials of therapeutic relevance, can cure cancer and other related diseases are under studies for targeted and time-release drug delivery. Some of these substances under current investigations include xanthan, gelatine, albumin and phospholipids [14] .
Polymeric nanocarriers have paramount important in the field of science and have revealed many applications in the field of nanotechnology as well development in drug delivery systems. Nanocarriers have some specific properties which include size and shape, surface characteristics and chemical constituents that differs significantly with other polymers [15] [16] . Chitosan are highly basic polysaccharides that occur naturally with some specific properties which include the formation of polyoxysalt, formation of film in solution, optical structural properties and ability to chelate metal ions. Chitosan nanoparticles also have similar but more optimized properties than the normal chitosan solution, as revealed in a study that compared insulin chitosan solution and its nanoparticles, in which the nanoparticles showed more effective treatment in lowering the blood glucose level, more optical characteristics and its presence in the physiological environment is also less [17] . Chitosan nanoparticles was synthesized using a novel technique based on ionic gelation using sodium tripolyphosphate (TPP) as cross-linking agent TPP and the particles are known to have a different morphology and surface characteristic [18] . In this method, the TPP solution flows through the pores of a micro engineered membrane into the chitosan solution put in a stirred cell. The ionic cross-linking method is the most common among physical cross-linking techniques since the preparation procedure is simple, does not involve the use of organic solvent or high temperature, and no chemical interaction is involved. The advantages make this method efficient and safe for production of thermosensitive therapeutic agents such as proteins, peptides, hormones, and vaccines loaded into chitosan particulate systems. Assembly and formation of the particles is achieved by ionic cross- linking between chitosan and one of its derivatives, being cationic in nature, and either negatively charged macromolecules or anionic cross-linking agents. In some studies, the controlled drug delivery systems including MMC, Epirubicin and Doxorubicin has been performed by the synthesis with different nanogel and nanoparticles and the efficacy of drugs in the tissue can be increased [19] [20] . The muco-ad- hesive properties of these systems are an important factor in their retention and action in drug delivery. We aimed to synthesis a pH sensitive drug delivery system that will reduce the intravesical administration frequency with an enhanced efficacy of installation.
2. Experimental2.1. Materials
Low viscous chitosan from shrimp shells [mol. wt. 50,000 - 190,000 Da (based on viscosity)] was purchased from Sigma Aldrich (USA). Glacial acetic acid (Glacial/27225/USA) solution was employed as a solvent for chitosan. Sodium tripolyphosphate (Alfa-Aesar 013440/USA) was used as a cross-linker. Phosphate Buffer Saline (PBS) was purchased from Fluka (Switzerland). Spectra/Por Dialysis membrane bag was used in vitro drug release studies (molecular weight cut-off was 12 - 14 kDa, Cole Parmer Ind.). MMC was obtained from Roche Diagnostics GmbH (Mannheim, Germany). All reagents were of analytical grade and used without further purification. Bladder cancer cell line T24 was sourced from the American Type Culture Collection (Manassas, VA). Dulbecco modified Eagle’s medium (DMEM), 1% penicillin/streptomycin, trypsin-EDTA and 10% fetal bovine serum (FBS), were purchased from Thermo Fisher Scientific (MA, USA). MTT (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide (MTT) kit was obtained from Sigma-Aldrich.
2.2. The Synthesis of Chitosan Nanoparticles
Chitosan nanoparticles were synthesized by using ionic gelation technique [18] . In a typical procedure, low viscous chitosan (% 0.5 w/v) was dissolved in 1% Glacial acetic acid solution. The pH of the prepared chitosan solution was adjusted to 4.5 by drop wise addition of 10 N NaOH. After pH adjusting, 4 mL of tripolyphosphate (TPP) (0.25% w/v) solution was dropped into 12 mL of chitosan solution. The resulting solution was mechanically stirred for 1 hr. at room temperature. The chitosan nanoparticles thus formed were recovered by centrifugation at 12.000 rpm for 30 min and was subsequently washed three times followed by freeze drying for further studies.
2.3. The Characterization of Chitosan Nanoparticles
Dynamic Light Scattering (DLS)
After centrifugation, the supernatant was decanted and the nanoparticles were suspended in 2 ml of distilled water using ultra sonication. The charge of the nanoparticle and their mean size were measured by using Dynamic Light Scattering (Malvern Instruments, Model 3000 HSA, England). The nanoparticle zeta potential was determined in distilled water. Analyses were performed in triplicate at room temperature.
Atomic Force Microscopy (AFM)
Morphological evaluations of chitosan nanoparticles were realized with an Atomic Force Microscope (AFM) (Nano magnetics, Turkey) that was used to obtain AFM images of nanoparticles. Samples were prepared by pouring the solutions on a mica surface. 20 μL solution was dried at room temperature before analysis.
Fourier Transform Infrared Spectroscopy (FTIR)
Physicochemical characteristics of free drug MMC, chitosan nanoparticles and MMC encapsulated chitosan nanoparticles were analyzed by Shimadzu IR Prestige 21 model FT-IR spectroscopy.
2.4. MMC Loading Studies
MCC is compatible with anionic agents. Different concentrations of MMC were dissolved in TPP solution (0.25% w/v) and then TPP solution was dropped into the chitosan solution. The recovery of the chitosan nanoparticles was done by centrifugation at 12,000 rpm for 30 min.; then it was washed with distilled water several times and lyophilized.
Drug Loading Efficiency (DLE)
In other to determine the quantity of drug loaded, separation of nanoparticle from the aqueous suspension was done by centrifugation at 12.000 rpm for 30 min. The amount of free MMC concentration in the supernatant solution was determined by measuring of absorbance values. (Thermo Scientific, Nanodrop 1000) at a wavelength of 358 nm. DLE were calculated according to the following equation.
MMC was also confirmed by the FTIR studies.
2.5. In Vitro Release Studies
In vitro release analysis was carried out in a double-cell system mimicking the urination system including a dialysis membrane bag (molecular weight cut-off was 12 - 14 kDa) between the cells. 5 ml of phosphate buffer solution (pH = 7.4) was put into one side and drug loaded lyophilized nanoparticles as well as specific quantity of MMC were suspended in 5 ml PBS and put into another cell. During the drug loading and release studies, the initial amount of MMC was changed (i.e., 0.5, 1.25 and 2.5 mg) to get different release profiles. The nanoparticle samples were placed in a water bath with mild agitation and were set at a temperature of 37˚C. At particular time intervals of 30, 60, 90, 120, 150, 180, 240, 300, 360 min etc.; 100 μL released medium was taken out from the nanoparticle- free cell and the same volume fresh PBS solution was added. The content of released MMC was detected using Ultraviolet Spectrometer (Thermo Scientific, Nanodrop 1000) with a wavelength of 358 nm (n = 6). Cumulative amount of drug (in %) released from the nanoparticles was calculated. The amount of MMC in the solution was calculated by creating a calibration curve with known amounts of drug concentrations. All of the release studies were repeated (n = 6). Additionally, MMC release studies have examined for different pH values. The pH of PBS solution was adjusted to 6 and 7.4 (n = 6). During the pH experiments, the nanoparticles were loaded with 1.25 mg MMC.
2.6. In Vitro Cytotoxicity Studies
The standard MTT assay was performed to determine bare NPs and drug loaded nanoparticle toxicity. T24 cells were cultured in DMEM medium with additional 1% penicillin/streptomycin mixture and 10% FBS. Briefly, T24 cells were seeded onto a 96-well plate at a density of 1 × 104 cells/well per well. After 24 h, the medium was removed and the cells were treated with a range of concentrations at 0.5, 1.25 and 2.50 mg/ml of MMC, bare NPs and MMC/CS nanoparticles. The untreated cells were accepted as a control. After 24 h incubation, 200 µL of 3 mg/ml MTT solution was added to each well and the plate was incubated at 37˚C, 5% CO2 for 4 h. DMEM was refreshed at 2, 4, 6, 8, 10, 12 and 24 h to mimic the urination discipline. The absorbance was measured using an absorbance micro plate reader (Thermo Fisher Scientific, 1510, Finland) at a wavelength of 450 nm. The viability of the cell was deducted by adopting the following equation:
where AT is the Absorbance of treated cells and AU is Absorbance of untreated cells. The cell viability graph is drawn using the mean absorbance of three independent replicates.
2.7. Statistical Analysis
All measurements were calculated as the mean ± standard deviation (SD). One- way analysis of variance (ANOVA) was adopted in the analysis of the results with the Student-Newman-Keuls multiple comparisons or t-test when comparing the differences between the means of two groups at the same time point. The statistical significance was benched at <0.05 probability.
3. Results and Discussion3.1. Size Distribution of CS and MMC/CS Nanoparticles
In this study, chitosan nanoparticles syntheses were carried out through ionic gelation technique and MMC was loaded successfully to these nanoparticles. Morphological evaluation showed that the obtained nanoparticles were sphere like in shape with a narrow size distribution range at around 140 nm as shown in dynamic light scattering output and the polydispersity (Q) was 0.370. It is further shown that the obtained nanoparticles were sphere like in shape with a narrow size distribution range at around 140 nm as shown in dynamic light scattering output. The size of chitosan nanoparticles was smaller than 200 nm, which due to the chitosan nanoparticle size, allows for targeting and accumulation in cancerous tissues [21] . Such a range of nanoparticles may accumulate more accurately in tumor due to the enhanced permeability and retention (EPR) effect, which is often regarded as “passive” targeting [22] .
As shown in Table 1, polydispersity indices of chitosan nanoparticles (CS) and MMC encapsulated chitosan nanoparticles (MMC/CS) were lower than 0.4, indicating the size distributions of nanoparticles were narrow and formulations of nanoparticle were mono dispersed in size distribution. Compared to the size results of nanoparticles and drug carrying nanoparticles, the encapsulation of MMC moderately increased the size of the nanoparticles as the ionic interaction that exist among the chitosan TPP can be affected by drug loading. Drug anions can engage the CS cations due to the hydrophilicity of MMC in aqueous solution. This on the other hand can hamper the CS and TPP interaction. This may
Z-average zeta-potential and particle size of the drug encapsulated CS nanoparticles
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