Chemicals: Isoniazid, rifampicin, pyrazinamide and dexamethasone, PEG-20000, polyvinyl alcohol, and dextran-70000 were obtained from Sigma [St. Louis, USA], n-butyl cyanoacrylate was obtained from Reevax Pharmaceuticals, Bangalore, India. Oleic albumin dextrose catalase [OADC] enrichment was of Difco-Becton-Dickinson [USA]. EDTA, FBS, MEM was obtained from Invitrogen Corporation [Gibco], New York, USA. Acetonitrile, methanol, potassium dihydrogen phosphate and ammonium acetate were of HPLC grade from Sigma [St. Louis, USA].
Cells and Animals: C6 glioma cell line was obtained from National Centre of Cell Sciences [NCCS], Pune. Swiss albino mice of either sex [3-4 weeks, 20-25g] were procured from the Central Animal House of Post Graduate Institute of Medical Education and Research, Chandigarh and were provided standard pellet diet and water ad libitum. Swiss albino mice of either sex (3-4 weeks old, 20-25g) were obtained from the Central Animal House of Post Graduate Institute of Medical Education and Research, Chandigarh. The animals were housed in cages or animal isolators in case of infected animals (Nuaire Instruments, NU 605-600E, Series 6) and provided with standard pellet diet (Hindustan Lever Ltd, Mumbai) and water ad libitum. The plan of work was approved by the Institute Animal Ethics Committee and all the animal experiments were carried out according to the ethical guidelines (Institute Animal
Ethics Committee no 224)
Preparation of polybutylcyanoacrylate nanoparticles encapsulating antitubercular drugs and dexamethasone: Nanoparticles encapsulating Isoniazid, Rifampicin, Pyrazinamide and Dexamethasone were prepared as described with few modifications [16]. n-butylcyanoacrylate monomer was added to a mixture of 1% dextran and pluronic F-68 (pH 2.2) under constant stirring. Each drug was added to the stirring mixture for encapsulation. The mixture was stirred for 3h and pH was neutralized using 0.1N NaOH. The mixture was further stirred for 1h and filtered through a G1 glass filter to remove the agglomerates. Nanoparticles were obtained by centrifuging filtrate at 20,000 rpm for 1h. Pellet was washed with water and lyophilized with 3% Mannitol as a cryoprotectant. Nanoparticles were coated with2% solution of PEG-2000 and polysorbate-80.Successful polymerization of n-butylcyanoacrylate monomer to Polybutylcyanoacrylate and coating of PBCA nanoparticles was confirmed by FTIR.
Characterization of PBCA nanoparticles containing drugs: The drug-loaded PBCA nanoparticles were analyzed for particle size and polydispersity index using Zetasizer, Malvern Corp., USA.
Drug analysis: HPLC was used for the analysis of drugs. Parameters for HPLC analysis of each drug are depicted in Table 1.
Drug entrapment efficiency: Amount of drug in nanoparticle was estimated by calculating the difference between the amount of drug in the supernatant and the total amount of drug used for the preparation of nanoparticles. The free drug in the pooled supernatant was determined by HPLC.
Percentage drug entrapment efficiency was calculated as:
Drug loading capacity: Drug loading capacity was expressed as the amount of drug entrapped per gram of polymer.
Surface Morphology: Transmission electron microscopy (TEM) was done to determine the surface morphology of nanoparticles.
Ex vivo studies: Uptake of fluorescent PBCA nanoparticles containing rhodamine was studied in C6 glioma cells. Rhodamine nanoparticles were prepared using anionic polymerization and coated with either polysorbate-80 or mixture of PEG-20000 + P-80. Cells were grown in MEM, 10% FBS under standard conditions in the CO2 incubator and were plated in 24 well plates and allowed to attach for 24h. Cells were treated with rhodamine containing nanoparticle suspension for different time intervals. After 4 and 8h, cells were fixed and observed under fluorescent microscope. For FACS, cells were deadhered and the cell suspension was immediately analyzed on flow cytometer at 488nm excitation and 585 nm emission [17].
Brain uptake In vivo studies: A comparative study was carried out to study the delivery of nanoparticles to the brain using oral vs intravenous routes of administration. Rhodamine-loaded PBCA nanoparticles were given orally or intravenously to mice and delivery of rhodamine to mice brain was evaluated by fluorescent microscopy. Mice were divided into 6 groups on the basis of route of administration and type of rhodamine formulation: Group I: free Rhodamine was given intravenously, Group II: free Rhodamine was given orally, Group III: Rhodamine-loaded PBCA uncoated nanoparticles were given intravenously, Group IV: Coated Rhodamine-loaded PBCA nanoparticles were given intravenously, Group V: Uncoated Rhodamine-loaded PBCA nanoparticles given orally, Group VI: Coated Rhodamine-loaded PBCA nanoparticles were given orally. Animals were sacrificed at 24, 48, 72, and 96h and cryo sections of brain tissue were prepared for rhodamine detection using fluorescent microscopy[18].
Pharmacokinetic analysis:
Blood/tissue sampling: For pharmacokinetic analysis, in healthy mice (5-6) mice per time point, blood samples (100 µL)were collected from the saphenous vein with Microvette capillary blood collection tubes treated with Lithium Heparin(Sarstedt, France). Blood was drawn at 0.25h, 0.5h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h 72h, 96h, 120h, 144h, 168h, 192h, 216h, 240h after single drug dose administration. Saline solution (100 µL) was administered subcutaneously to compensate for the lost blood volume. Samples were centrifuged at 2000g for 5min at 4oC to separate the plasma. Tissues were obtained from mice after sacrifice at day1,4,8 and 12 to check the drug levels in various tissues. Lung, spleen and brain tissues were isolated and homogenized in saline (100mg/ml) for drug analysis.
Drug administration and dosage: Free drugs, drug-loaded PBCA nanoparticles and coated drug-loaded PBCA nanoparticles were administered orally to different groups of mice [6 to10 mice per group].Group I: Free Isoniazid, Group II: Uncoated Isoniazid nanoparticles, Group III: Coated Isoniazid nanoparticles, Group IV: Free Rifampicin, GroupV: Uncoated Rifampicin nanoparticles, Group VI: Coated Rifampicin nanoparticles, Group VII: Free Pyrazinamide, Group VIII: Uncoated Pyrazinamide nanoparticles, Group IX: Coated Pyrazinamide nanoparticles, Group X: Dexamethasone, Group XI: Dexamethasone nanoparticles, Group XII: Coated Dexamethasone nanoparticles. Drug dosage was determined according to adult human therapeutic dose(Isoniazid-25mg/kg, Rifampicin-10mg/kg, Pyrazinamide-150mg/kg, Dexamethasone -0.2mg/kg).
Drug estimation plasma/tissue by HPLC and pharmacokinetic analysis. Tissue homogenates and plasma samples were deproteinized and then protein-free filtrates were analyzed by HPLC. Data were analyzed for Pharmacokinetic and pharmacodynamic parameters[19].
Pharmacokinetic analysis was performed using the online version of PKSolver (an add-in program for pharmacokinetic and pharmacodynamic data analysis Microsoft Excel). The pharmacokinetic parameters evaluated comprised: AUC, AUMC , Cmax (the maximum serum concentration that a drug achieves in a specified compartment or test area of the body after the single dose of the drug has been administrated),t1/2 or elimination half-life( the time taken for the plasma concentration to fall by half its original value), kele, (first-order rate constant for plasma clearance)
Therapeutic efficacy of PBCA nanoparticles encapsulating antitubercular drugs and dexamethasone against mice model of tuberculous meningitis
A murine model of tuberculous meningitis was established as described earlier[20] Briefly, mice were anesthetized and placed in a stereotactic device. To inoculate the infection, a burr hole was made with a 26 gauge needle at the inoculation site [0mm × plane, 3.5mm y plane and 2mm z plane from the bregma] after midline sagittal incision. Bacillary dose [105 CFU/50µl] was injected using Hamilton syringe and scalp incision was stitched with sterile suture. To monitor the development of infection, mice were sacrificed 4 weeks after infection and tissues were prepared for histopathological examination and CFU enumeration. Chemotherapy was initiated to evaluate the therapeutic effects of free drugs vs drugs delivered via the nanoparticle drug delivery system. Free drugs were administered daily while the encapsulated drugs were administered every 8thday. The encapsulated drugs were used both in PEG+P-80 coated form as well as in uncoated form in order to evaluate the effects of PEG+P-80 coating on the chemotherapeutic potential of the PBCA nanoparticles. The chemotherapy was given for 4 and 8 weeks. At each time interval, the mice from each treatment group and untreated control group were sacrificed and tissues were analyzed for CFU count, histopathology
Four weeks post-infection, mice were subjected to drug dosage administration as described below:
Group I: Untreated Controls
Group II: Free antitubercular drugs (Isoniazid+ Rifampicin+ Pyrazinamide) administered orally
Group III: Uncoated encapsulated antitubercular drugs (Isoniazid+ Rifampicin + Pyrazinamide), orally
Group IV: Coated encapsulated antitubercular drugs (Isoniazid+ Rifampicin + Pyrazinamide), orally
After 4 or 8 weeks of chemotherapy, mice from each group were sacrificed under sodium pentothal anesthesia. Brain, lungs, and spleen were isolated, half of each organ was homogenized and half preserved for histopathological studies.
CFU enumeration: Organ homogenates were plated on Middlebrook 7H11 agar plates to determine bacterial load in each organ. Plates were incubated at 37oC for 21days to obtain the bacterial count.
Histopathological examination: Tissues were fixed in 10% buffered formalin for 24h and processed for histopathological examination.
Statistical analysis: The pharmacokinetic or pharmacodynamic data analysis was done using ANOVA followed by multiple comparisons using post hoc test and Bonferroni correction. For CFU data comparison among various groups, ANOVA was used followed by student’s ‘t’-test to compare treatment groups with each other and untreated control. Data were analyzed using MS Excel and SPSS 16 software.