Convection-Enhanced Delivery of Alkaloid - Loaded Maghemite Nanoparticles against 9L - Gliomass Cell Line

Materials

Isolated piperideine alkaloid, glycine, divinyl sulfone (DVS), triethylamine (TEA), dimethyl sulfoxide (DMSO), FeCl2,NaNO3, potassium persulfate, 1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo NHS), NaOH, gelatin (from porcine skin), human serum albumin (HSA), polyethylene glycol (PEG, MW 400), PEG (MW 400) methacrylate (PEGMA), PEG dimethacrylate (PEGDMA), amino terminated PEGMA (NH2PEGMA) and Salts for buffers were purchased from Sigma Lab. Pvt. Ltd. (Mumbai, India). Formaldehyde was purchased from Meru Chem Pvt. Ltd. (Mumbai India).

Collection, Cultivation and Isolation of Piperideine Alkaloid

Papaya leaves were collected from the botanical garden of Columbia group of institutions; subsequently leaves were authenticated by Dr. SP Rao (Professor, Department of Pharmacognosy, Columbia Institute of Pharmacy, and Raipur). Furthermore 5 kg air-dried leaves were crushed as well as powdered by mixer grinder. There after soaked in 6 liter of 89 % ethanol, 10 % water and 1 % alcohol acetate for 2 days. Additionally the extract was decanted and the extraction repeated. The combined extracts were evaporated at 60°C in a rotary evaporator and converted into thick syrup which was shaken with 1 liter of water containing 20 ml alcohol acetate. The mixture was extracted with ethanol and the aq. phase was separated, adjusted to pH 11 with K2CO3 and then extracted thoroughly with ethanol. Whatsmore ethanol extract was washed with water, extracted with 5% hydrochloric acid and the acid extract was again adjusted to pH 11 and extracted with ethanol [8]. This ethanol, after drying over dry magnesium sulphate was stored to a dark brown ‘crude extract’ of papaya leaf alkaloids, from which piperidine precipitated collected after chilling. Isolated piperidine was confirmed by Dragendorff’s, Mayer’s and Hager’s test [9].

Preparation of γ-Fe2O3 NPs

γ-Fe2O3 NPs were narrow size of distribution prepared by nucleation as per the controlled growth of iron oxide thin films onto the gelatin/iron oxide nuclei, as earlier described. Subsequently, γ-Fe2O3 NPs were prepared in the range of 20.03±2.5 nm diameter. Along with addition of FeCl2 aqueous solution (20mmol/10mL in 0.1 N HCl) to 40 mL of an aqueous solution containing 120mg of porcine gelatin, followed by the addition of a sodium nitrate solution (15mmol/10mL in water). After a reaction time of 10 minutes, a sodium hydroxide aqueous solution (1 N) was added until a pH of 9.5 was obtained. This procedure was repeated four more times. During the entire procedure, the aqueous suspension was agitated at 60°C and kept in an inert environment [10]. The formed γ-Fe2O3 NPs were then washed from excess reagents with water using magnetic columns.

Conjugation of Human Serum Albumin (HSA) onto γ-Fe2O3 NPs: 5mg of HSA was added to 25mL of the γ-Fe2O3 NPs. There after NPs dispersed in water (1 mg/0.5mL). Mixture was then shaken at pH 9.5 and 60°C for 24 hours, and then cooled at room temperature. The physically coated NPs obtained as γ-Fe2O3 - HAS NPs which is further washed from excess HSA with phosphate-buffered saline by magnetic columns [11]. Polymerization of Polyethylene Glycol Acrylate Derivatives on the Surface of the γ-Fe2O3 NPs: Polymerization of polyethylene glycol acrylate monomers on the surface of γ-Fe2O3 NPs was accomplished by mixing 5mL of the γ-Fe2O3 NPs dispersed in a bicarbonate buffer (0.1 M, pH 8.1, 2.0 mg/mL) with 0.35mL of polyethylene glycol γ-Fe2O3 acrylate (PEGMA), 0.05mL of PEG dimethacrylate (PEGDMA), 0.1mL of NH2PEGMA, and 24mg of potassium persulfate dissolved in 25mL of a bicarbonate buffer (0.1M, pH 8.1). For the polymerization process, the above mixture was shaken at 75°C for 3 hours. The formed PEGM NPs were then washed from excess monomers by magnetic columns [12]. Physical Conjugation of Piperidine Alkaloids to the HSA-Coated NPs: Piperidine alkaloids was physically conjugated to the HSA coated NPs, γ-Fe2O3 - HSA, by adding 1mg of piperidine alkaloid dissolved in 0.5mL of dimethyl sulphoxide (DMSO) to 10mL of the nanoparticle dispersion in physiological buffer solution (PBS) (0.5mg/0.5mL). The reaction mixture was then shaken at room temperature for 18 hours [13]. The obtained γ-Fe2O3- HAS – piperidine alkaloid NPs were washed from excess piperidine alkaloid extract with PBS (0.1 M, pH 7.3) by magnetic columns. Higher concentrations of piperidine were obtained by increasing piperidine alkaloid concentration [14]. Piperidine Alkaloid Conjugation to the HSA – Coated and PEG – Coated γ-Fe2O3 NPs: Piperidine alkaloids were covalently conjugated to the HSA-coated γ-Fe2O3 NPs, In a typical experiment, 5mg of piperidine alkaloids were dissolved in 2.5mL of DMSO was added to 2.5mg of 1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide (EDC) and 5mg sulfo-NHS dissolved in 2.5mL of water. The mixture was then shaken at room temperature for 30 minutes. Furthermore 20ml of the HSA-coated NPs were then added and the mixture was shaken at room temperature for 1 hour, after which the NPs were washed from excess reagents by magnetic columns. In a similar way piperidine alkaloid was conjugated to the terminal NH2 groups of the γ-Fe2O3 - PEG NPs. Higher concentrations of piperidine alkaloids were obtained by increasing the initial concentration of piperidine [15]. Evaluation of the Bound HSA Concentration: HSA bound to the γ-Fe2O3 NPs were evaluated by measuring the unbound proteins using Bradford assay 33 & 34 as well as data’s were compared from the predetermined concentration. Subsequently the average values were calculated by repeating measurement at least three times for each sample. The binding yield was calculated by multiplying the ratio of the concentration of the bound piperidine alkaloid to the initial concentration by 100. Evaluation of the Bound Piperidine Alkaloid Concentration: The concentration of piperidine bound to the γ-Fe2O3 NPs was indirectly determined by measuring the absorbance of the unbound drug at 303 nm. The binding yield was calculated piperidine concentration.

Development of 9L-Gliomass Cell Culture

9L glio-sarcoma cells were used in existing work which reveals more foliate receptor inducing effect were maintained at room temperature as well as 5% of carbon monoxide in Dulbecco’s Modified Eagle’s Medium (DMEM) (Biological Industries, Pune, India) enriched with 10% Fetal Calf Serum (FCS) and 1% penicillin/streptomycin and subcultured twice a week. The cells were plated at a density of 6X104 per well in 24 well plates for the cell-kill experiment.