Bacterial strains, culture condition, and materials
V. cholerae strains MTCC 3905 (reference strain), and Vibrio cholerae biofilm-forming rugose strain, HYR14 obtained from NICED, India, was used throughout this study12. The HYR14 strain was gifted by Prof. Thandavaraya Ramamurthy, National Institute of Cholera and Enteric Diseases, Kolkata, India. The strain was cultured in thiosulfate–citrate–bile salts–sucrose (TCBS) agar to confirm cell viability and propagated under standard growth conditions in Luria–Bertani (LB) broth at 37°C. An inoculum size of 0.2 (OD595) ≈ 105 cells was used for all the experiments13.
Cellulose acetate phthalate (MW 2534.12) was purchased from SRL, India. Sodium hydroxide pellets (MW 40 g/mol), potassium dihydrogen phosphate (MW 136.09 g/mol), and boric acid (MW 61.83 g/mol) were purchased from Merck Life Science, India. Pluronic F-127 was purchased from Sigma-Aldrich. Acetone was purchased from Pure Chemicals Co. India and Methanol from HiMedia, India. Minimal Essential Medium (MEM) with Earle salts, L-Glutamine, NEAA and sodium bicarbonate, Phosphate buffered saline (pH 7), trypsin, fetal bovine serum, antibiotic antimycotic solution (100X with 10,000 U Penicillin, 10mg Streptomycin and 25µg Amphotericin B per ml in 0.9% normal saline) was purchased from HiMedia, India. MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] for cell proliferation assay was purchased from HiMedia, Mumbai, India. Int 407 cells were procured from National Centre for Cell Science (NCCS), Pune, India. Bacteriological isolation media, growth media, and Giemsa stain were procured from HiMedia, India. Int 407 cell lines were grown using MEM with 10% FBS and 1% antibiotic and antimycotic solution at 37°C in a 5% CO2 incubator.
Methods
Nanoparticles synthesis and lyophilization.
The nanosuspension of 2Methoxy-4-vinyl phenol (2M4VP) with cellulose acetate phthalate (CAP) was prepared by a modified nanoprecipitation method. About 10 mg of CAP was dissolved in 1 mL of acetone and 10 mg of 2M4VP in 1 mL of methanol. Both the drug and the polymer preparation were mixed to obtain the organic phase. About 0.5% of Pluronic® F127 in distilled water was prepared to form the aqueous phase. The 2 mL of the organic phase was added dropwise into the aqueous media using a syringe and the dispersion was left under constant and continuous stirring at 700 RPM at room temperature until the organic phase completely evaporates. The colloidal aqueous solution containing the nano-precipitated particles of CAP-2M4VP was then freeze-dried by lyophilization14.
Material Characterization
The particle size distribution and stability of the CAP-2M4VP formulation were measured by the dynamic light scattering (DLS) and zeta potential using Zetasizer (NanoZs, Malvern Instruments, UK). The chemical interaction of drug and polymer present in the CAP-2M4VP particles was analyzed by Fourier-transform infrared spectroscopy (FTIR). The freeze-dried samples were placed on a hole of 1 mm on a stell gasket of 0.05 mm thickness on top of a diamond anvil cell. Then the sample was transferred to a stub and scanned from wavelength4000 cm−1 to 400 cm−1 15. The diffraction pattern of the CAP-2M4VP and 2M4VP were compared by X-ray diffractometer with Cu-Kα radiation at 30 mÅ of current and 40kV volatage (Rigaku, Japan). The analysis was performed by scanning the samples at 2θ from 0° to 100° 16. The variation in the thermal properties of the polymeric nanoparticles compared to the pure drug was studied using Thermogravimetry and Differential Scanning Calorimetry (NETZCH, Germany). About 5-10 mg of the samples were individually placed in an aluminum pan and heated at the rate of 10 °C/min up to 1000 °C and the result was represented as weight loss and heat flow against temperature. The surface morphology of the lyophilized CAP-2M4VP nanoparticles preparation and a pure sample of 2M4VP was captured after gold sputter-coating using Scanning Electron Microscope at 10kV (VEGA3, TESCAN Analytics, Czech Republic)
Entrapment efficiency
The percentage of 2M4VP entrapped within the nanoparticles was studied by centrifuging the CAP-2M4VP nanosuspension preparation at 12,000 rpm at 4 C for 20 mins. The total amount of 2M4VP entrapped within the CAP nanoparticles was determined by the estimation of unentrapped free drug present in the supernatant. The absorbance of the supernatant solution was measured at 268 nm. The percentage of entrapment efficiency was calculated by the following formula,17
Percentage Entrapment Efficiency = Total amount of drug - Un-entrapped drug X 100
The total amount of drug
Drug Content
The total amount of 2M4VP present within the CAP nanoparticles suspension was analyzed. About 1 mL of the nanosuspension was dissolved in 9 mL of methanol and left for 24 h at 4°C. The solution was sonicated and filtered and the amount of drug present was measured using a UV-Visible spectrophotometer at 268 nm using a respective blank 18,19.
In-vitro drug release studies
The percentage of drug release of 2M4VP encapsulated in CAP nanosuspension was studied in the presence of dissolution media - buffers at various pHs (1.2, 5.8, 7, 8.4, and 9). About 1 mg/mL of the CAP-2M4VP nanoparticles were loaded into a dialysis membrane (HiMedia, Mumbai, India; Pore size: up to 14 000 Daltons) and the ends were sealed. The membrane was then placed inside a screw cap bottle filled with 5 mL of dissolution media of specific pH. The set-up was placed under constant stirring at 200 RPM at 37°C. At regular time intervals, 5 mL of samples were removed using a syringe and were replaced with the 5 mL of the respective fresh buffers. The samples collected were analyzed at 268 nm using a UV-Visible spectrophotometer. The study was performed in triplicate and presented as the mean with standard deviation limits19.
Drug Release Kinetics
The cumulative percentage of drug release profiles obtained for pH 1.3, 5.8, 7.0, 8.0, and 9.0 was further analyzed by fitting the data into drug release kinetics models. The data from in-vitro drug release was fitted into zero-order, first-order, Higuchi, Korsmeyer-Peppas, Hixson-Crowell, Hopfenberg, Baker-Lonsdale, Makoid-Banakar, Weibull, and Gompertz kinetic models. The values of R2, Rate Constant (K), Sum of Squared Residual (SSR) and Release exponent (n) were identified for CAP-2M4VP nanoparticles at each pH included in this study 19.
Ex-vivo permeation assay
The permeation of CAP-2M4VP nanoparticles across excised goat intestinal mucosal membrane ex-vivo was performed using the Franz cell diffusion chamber. The Franz cell diffusion chamber comprises two compartments - an upper donor connected to a lower acceptor (receptor) compartment. The capacity of the acceptor compartment is approximately 15 mL and the available diffusion area is 1.8 cm2 in diameter. The acceptor compartment was filled with phosphate buffer pH 7 and a magnetic bead was placed inside. The 5x5 cm goat intestinal mucosal tissue was mounted on top of the acceptor compartment with the mucosal region facing towards the donor compartment with constant contact with the phosphate buffer pH 7.0 so that bubbles formation was prevented. The donor compartment was then placed on top of the intestinal tissue and both the compartment were held tightly in position with rubber clamps. The donor compartment was filled with phosphate buffer pH 7 containing 1 mg/mL of the CAP-2M4VP nanosuspension preparation. The opening of the donor compartment was covered with aluminium foil to prevent evaporation. The entire setup was placed on a magnetic stirrer at 50 RPM and continuously stirred at room temperature. About 500 μL of samples were collected from the receptor compartment at every 30 min interval for the first 2 hours and then at 1 h interval for 8 hours continuously. The cumulative amount of drug permeation was determined by a UV-Visible spectrophotometer at 268 nm. The volume withdrawn from the receptor compartment was replaced by the aliquoting same volume of the phosphate buffer into the donor compartment. The permeation data analysis was performed as per described by Ramyadevi and Rajan (2015)19. The results presented a mean + SD of experiments performed in triplicates.
Cell viability assay
The effect of the CAP-2M4VP nanoparticles on host cell viability was assayed using Int 407 cells, cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (w/v), and 1% L-glutamine using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The Int 407 cell lines were procured from National Centre for Cell Science (NCCS), Pune, India. About 1 × 104 Int 407 cells/well were seeded in a 96-well microtiter plate and incubated at 37°C in a CO2 incubator for 24 h. After the incubation period, the cells were treated with various concentrations (100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL) of CAP-2M4VP and incubated for another 24 h at 37°C in a CO2 incubator. After 24 h, 1 mg/ml of MTT was added, and the microtiter plate was incubated at 37°C for 3 h. Dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, and absorbance was read at 570 nm using a microplate reader spectrophotometer (Synergy H1)20.
Inhibition of growth
The effect of the CAP-2M4VP nanoparticles on the growth of V. cholerae MTCC 3905 and HYR14 was evaluated using a 96-well microtitre plate assay from concentrations ranging from 500 μg/mL to 0.06 μg/mL. V. cholerae MTCC 3905 and HYR14 strains were grown overnight in Luria – Bertani broth at 37◦C with shaking at 150 RPM. Briefly, overnight cultures of both the strains were diluted to 1:100 (OD595 = 0.2) using the fresh LB media. 100 μL of LB broth containing various concentrations of CAP-2M4VP from 500 μg/mL to 0.06 μg/mL was aliquoted in the microtitre plate wells. To this 10 μg/mL of overnight cultures of V. cholerae diluted to 1:100 (OD595 = 0.2) using the fresh LB, media were added. The plates were incubated at 37°C for 24 h statically. After 24 h, the growth was measured by reading the absorbance at 595nm using a microtitre plate reader (iMark, BIORAD, Japan). Experiments were performed in triplicates and the data are reported as mean with standard deviation21 .
Inhibition of biofilm formation
The ability of CAP-2M4VP to inhibit the biofilm formation of V. cholerae was evaluated by crystal violet assay using a 96-well microtitre plate. Briefly, overnight cultures of V. cholerae were diluted to 1:100 (OD595 = 0.2) using the fresh LB media. 100 μL of LB broth containing various concentrations of CAP-2M4VP from 250 μg/mL, 125 μg/mL, 62.50 μg/mL, 31.25 μg/mL, 15.63 μg/mL and 7.81 μg/mL were aliquoted into the wells. To these 10 μg/mL of overnight cultures of V. cholerae strains MTCC 3905 and HYR14 diluted to 1:100 (OD595 = 0.2) using the fresh LB media were added. The wells without CAP-2M4VP served as control and wells without inoculum served as blank. The plates were incubated at 37°C for 24 h statically. Biofilm quantification was performed using crystal violet assay as described previously. After 24h, the planktonic V. cholerae cells were removed by washing with PBS thrice. To the wells, 150 μL of 0.2% crystal violet stain was added and incubated for 20 mins for staining the adherent biofilm. After the incubation period, the unbound crystal violet stain was removed by washing the wells gently with PBS and the plates were air-dried. To this, 33% acetic acid was aliquoted to the wells to elute the crystal violet bound to the biofilm. The absorbance was read at 595nm using a microtiter plate reader (iMark, BIORAD, Japan). Experiments were performed in triplicates and the data are reported as mean with standard deviation. The lowest concentration that inhibited about 50% of V. cholerae biofilm formation at all the pHs (pH 6, 7, 8, and 9) was considered a minimum biofilm inhibitory concentration of 50 (MBIC50)22.
Fluorescent Microscopy of biofilm
The inhibition of biofilm formation by CAP-2M4VP against V. cholerae MTCC 3905 and HYR14 was observed using fluorescent microscopy. About 25 mL of LB media adjusted to pH 6, pH 7, pH 8, and pH 9 containing 31.25 μg/mL of CAP-2M4VP were aliquoted to 50 mL sterile crew capped tubes. One percent of the diluted (1:100 (OD595 = 0.2)) overnight culture of V. cholerae strains MTCC 3905 and HYR14 were added to the tubes. Sterile glass slides were placed inside the tubes and incubated at static conditions for 24h at 37°C. pH-adjusted media tubes without CAP-2M4VP served as control. After the incubation period, the slides were recovered from the tubes and rinsed with sterile 0.9% NaCl to remove planktonic cells and media. As per the manufacturer's protocol, the adherent biofilm was stained with BacLight Bacterial Viability Kit (ThermoFisher Scientific, USA). The images of biofilm were observed under a 40X objective using a fluorescent microscope (Nikon Eclipse Ts2)23.
Cell adhesion assay
Int 407 cells were grown up to 90% confluency using MEM supplemented with 10% FBS without antibiotics at 37°C in a 5% CO2 incubator. The cells were washed with phosphate-buffered saline at pH 7 and fresh media was added. The overnight culture of V. cholerae MTCC 3905 and HYR14 culture at a concentration of multiplicity of infection of 50 (MOI 50) was added along with 31.25 μg/mL of CAP-2M4V. The plates were incubated at 37°C in 5% CO2 for 1 hour. After the incubation period, the cells were washed with PBS to remove the non-adherent bacteria. The cells were lysed using 0.1% of Triton X-100 prepared in PBS. The CFU of the adherent bacteria was enumerated and presented as a percentage of bacteria adherent to Int 407 cells. The experiments were performed in triplicates (Letourneau et al., 2011). For imaging using light microscopy, the Int 407 cells were grown in Petri plates with sterile coverslips in MEM medium containing 10% FBS without antibiotics at 37°C in a 5% CO2 atmosphere. The infection procedure was followed and after the infection period, the coverslips were removed and fixed with methanol for 30 mins followed by staining with Giemsa stain for 20 mins. The stained cells were washed with PBS and air-dried. The bacteria adherent to Int 407 cells were observed at 40X and 100X magnification using a microscope24.
Gene expression analysis by quantitative RT-PCR
Gene expression analysis to understand the effect of CAP-2M4VP on the V. cholerae MTCC 3905 cells were investigated using quantitative real-time PCR. RNA was extracted from 24 h culture of V. cholerae MTCC 3905 cells treated with 31.25 μg/mL of CAP-2M4VP following the manufacturer’s guidelines of the HiMedia RNA Extraction kit (MB613) (HiMedia, India). The purity and integrity of the isolated RNA were analyzed using NanoDrop (Thermo Fisher Scientific, United States). The conversion of total RNA to cDNA was performed using the iScriptTM cDNA Synthesis kit following the manufacturer’s guidelines. The effect of CAP-2M4VP on the gene expression of MTCC 3905 was investigated using qRT-PCR using Real-Time PCR System (Applied Biosystems, USA). The primers and under the condition, as described previously. The relative gene expression was calculated using the 2–Δ Δ CT method25.
Statistical analysis
GraphPad Prism software version 8.0.2 (GraphPad Software Inc., San Diego, CA, United States) was used for statistical analysis of the results. The results were expressed as mean ± standard deviations (SD) and all the experiments were performed in triplicates. The significance was analyzed by Student t-test with p set at p ≤ 0.05.