Study Of The Potential Of Gliadins As Novel Carrier Materials For Drug Delivery Systems And Its Liver Targeting Capacity

Materials for targeting drug delivery system have now attracted attentions of many researchers in the area of pharmaceutics, and among them the study on nanoparticle vehicles is becoming one of the most hotspots for its merits. Recently, nanoparticles-based drug delivery systems has been reported all around the world, and mainly made from synthetic and natural polymers. The former includes polyvinyl alcohol (PVA), alkyl cyanoacrylate (ACA), poly-lactide (PLA), polylactide-co-polycolide (PLGA) et al; and the latter are commonly used as biodegradable materials such as albumin, gelatin and polysaccharides. Recently, the proteins of high molecular weight derived from plant present prominent behaviors in preparation of nanoparticles as drug targeting delivery system. The proteins from plant are pharmacological inactive and even no toxic and they can also be employed as vehicles to encapsulate numerous active compounds and they will be regarded as alternatives for synthetic polymers.The overall objective of the present study was to develop a new material for targetting drug delivery. In general, the dissertation was composed of five parts: The extraction and purification of the plant-derived-protein (Gliadins); Safty evaluation and biocompability of Gliadins; Formulation and optimization of gliadin nanoparticles; Preparation and characterization of Silibin loaded gliadin nanoparticle; The pharmacokinetic and biodistribution study of Silibin loaded gliadin nanoparticles.Firstly, the defatting techonology was investigated using the weight changes and the UV absorption of the solvents as the indexes and then the extraction parameters of gliadins were optimized according to the extraction rate. The SDS-PAGE was used to test the molecular weight of the protein composition and the gliadins purification process was decided. At the mean while, the solubility and the protein composition were also tested, the results showed that the gliadins obtained by the optimized techonology were a series of proteins with the molecular weight between 45-30kD and appeared as white crystal powder. It was absolutely dissolved in 70% alcohol with high solubility, but solubility decreased rapidly with the slight change of alcohol concentration.Secondly, the safety and biocompatibility of obtained Gliadins were preliminarily evaluated using ECV304 cell line as cell model, and MTT method was employed to detect the cytotoxicity of the material. The results showed that the gliadins solution and the aqueous extractions had no cytotoxicity of zero and one-level respectively. The cell grew well with the addition of material and no abnormal phenomenon was observed. The blood compatibility and haemolysis of the Gliadins were also tested. The haemolysis rate of this material was 2.11% and it exhibited very good blood compatibility. The acute toxicity experiment showed that the LD₅₀ of the material after iv administration to the mice was 96.7586 mg/ml (1.935g/kg) with a 95% confidence interval of 106.3950-87.9352 mg/mLThe Gliadins were then used to prepare nanoparticles and the preparation techonology were investigated. The methodology of the entrapment efficiency (EE%) and the drug loading (DL%) rate test assay were also evaluated. Three traditional Chinese medicines with anti-HBV activities were chosen as the model drug to prepare the drug loaded gliadin nanoparticles. The results suggested that the Gliadins nanoparticles can be fabricated by desolvation methods and hydrophobic inner core which provided spaces to the hydrophobic drugs formed. But as for hydrophilic drugs, which could only be absorbed on the surface of the particles and a very low EE% was found. And due to hydrophobic properties, the Silymarin loaded nanoparticles could achive a relative high EE% of 78.85%±7.53%, approximately.In order to elucidate the abilities of this new material as drug delivery system, Silybin was selected as model drug to complete the following experiments finally. The formulation of SN-GNPs was optimized using central composite design and response surface method. The effects of influence factors such as the ratio of drug to Gliadins, the concentration of emulsifier, the amount of stablizer were investigated. The overall desirability (OD) were produced and treated by a statistic analystical system to optimize the formulation. Under the optimal conditions, the mean diameter, EE%, DL% were 164.9nm, 68.95%, 4.79% respectively. The formulation of Lyophilyzation powder of SN-GNPs was optimized based on its appearance, color and dispersing properties. Finally, SN-GNP was freeze-dried with the mixture of 3% glucose and 2% Mannitol as cryoprotectants. The in vitro release kinetics showed that the release of SN-GNPs lyophilization powder could be well characterized by Weibull equation. Compared to the SN injection, it showed an absolutely sustained release in vitro.The Pharmacokinetics of SN-GNPs was studied in rats with the SN injection as a reference. Program DAS2.0 was employed to calculate the pharmacokinetic parameters. These results showed that the plasma drug concentration-time courses of SN-GNPs and SN injection were both conformed to two-compartment model. And SN-GNP can prolong the MRT compare to the SN injection, a slight sustained release of drugs was observed in vivo.Mice were employed in the body distribution studies after intravenous administration of SN-GNPs. The results showed that SN-GNPs distinctly changed the distribution of SN in vivo and gently increased the concentrations of Silibin in liver contrast to the SN injection after i.v. administration. All of this showed a potential liver targeting ability of the SN-GNPs.In conclusion, in the present studies, a new material Gliadins as vehicles for drug delivery system was developed. It is the first experiment in the world to evaluate its safety and biocompatibility of this material as the intravenous vehicles. And it's the first time to be used in preparation of traditional Chinese medicine loaded gliadin nanoparticles and evaluation of drug loading pattern. Finally, Gliadin nanoparticles achieved the potential of liver targeting of Silibin and which was verified by related investigations. All the above work has not been reported anywhere all around the world, a novel natural high molecular weight material for targeting drug delivery system was developed which enriched the research contents about this field and provide some useful information and reference for other reseachers. To some degree, this dissertation was practicle and creative.