Optimization Of Preparation And Characteristics Of Ngf-PLGA Nanoparticles In Vitro

As the global population aging, there have more and more attention to central nervous system (Central nervous system, CNS) disease. Alzheimer's disease (Alzheimer's disease, AD) is a central nervous system degenerative diseases which function of cognitive and memory decline in clinical manifestations. The pathogenesis of AD is unclear, the treatment is very limited and the results would not be ideal. In current clinical mainly depend on the cholinesterase inhibitors, NMDA receptor antagonist and a variety of drugs to improve cerebral circulation. All treatments currently approved for AD have moderate efficacy in slowing the rate of cognitive decline in patients, and no efficacy in halting progression of the disease. Laboratory study found that nerve growth factor drugs can effectively prevent disease progression and improve symptoms of AD.To present nerve growth factor (Nerve growth factor, NGF) the most thorough study of as an example. NGF is a protein with high biological activity, it's receptor exists in various neurons of human central and peripheral nervous system. NGF's primary role is to nourish and protect nerve cells,restore damaged nerve cells and promote nerve growth. Animal studies have found that in treatment of Alzheimer's symptoms NGF also have a significantly improved results. Moreover, the isolated NGF has highly homologous in different species of animals,NGF in animal-derived also suitable for human to use. NGF will be the most promising drugs to treatment CNS disease.But NGF is a hydrophilic macromolecular protein, difficult to penetrate the blood-brain barrier, which is limited in clinical application. Though taken of administration of the drug delivery in cerebral ventricle in early clinical application was effective, but is bound to increase the patient's pain and risk of infection. Because of the short half-life of NGF, cerebrospinal fluid reflux and other factors, NGF is difficult to maintain the effective concentration in the brain, drug's utilization is very low. So to find a technology improving the permeability of blood-brain barrier for NGF and to increase the role of NGF in the brain is a persistence key for NGF to use in the clinical treatment.In recent years, with the develop of the nano-medical technology, there have been articles reporting that drugs can be brought by nano drug carriers to penetrate the blood-brain barrier and act in the brain, has brought new direction for NGF treatment CNS disease. In the 2005 FDA approved paclitaxel albumin nanoparticles injection suspension (paclitaxel, ABRAXANE) listed, to provide guarantee for the safety of nano-drug delivery. Therefore, we can envisage that NGF release nanoparticles can be prepared to solve the difficult problem that treatment of CNS diseases with NGF.In this thesis, nanoparticales were prepared by a double emulsification solvent evaporation method with poly(lactic-co-glycolic) acid (PLGA) as carrier material and methylene chloride (DCM) and acetone as organic solvent. Scanning electron microscopy (SEM) was used for the observation of nanoparticles. Mean diameter and particle size distribution were determined by photon correlation spectroscopy (PCS).The encapsulation efficiency of BSA or NGF in the nanoparticles was evaluated by BCA method or ELISA, which was also performed for the release of BSA or NGF from nanoparticles. Finally we established the preparation of nano drug carriers for our laboratory. Conclusion:1. Univariate analysis showed that since the size of nanoparticles increased as PVA concentration was also increased; With the larger time of sonication, the average particle size decreases; PLGA content increased, the average particle size increases; BSA concentration and volume of inner water phase had no significant effect on the average particle size.2. Orthogonal experimental design and variance analysis shows that PVA concentration and sonication time on the average particle size is not significant, but significantly affected on the entrapment efficiency. With the PVA concentration increased, the encapsulation efficiency increased; the time of sonication extended, the entrapment rate increases. However, the amount of PLGA on the particle size and entrapment rate has a significant impact. increasing the amount of particle size increased, the encapsulation efficiency increased. As prepare a smaller size order based on a higher entrapment rate nanoparticles, the final confirmation of the optimal prescription for the PVA concentration is 0.7%, extraction time is 20min, PLGA amount is 25mg.3. To optimize the prescription as a standard to prepared BSA-PLGA-NPs and the average diameter of a minimum was 219nm, the encapsulation efficiency was 44.7%. And to optimize the prescription preparation the NGF-PLGA-NPs of average diameter of a minimum was 243nm, encapsulation efficiency was 45.2%. Note that prepare protein peptide nano drug delivery systems with the material of PLGA, the average size less than 300nm, more than 40% encapsulation efficiency was. Also shows that with BSA as a model drug to explore the process of NGF nanoparticles experimental design is effective, and also has good repeatability.4. On studying the release mechanism of BSA-PLGA-NPs and NGF-PLGA-NPs in vitro find that both in vitro release into the medium release had burst release phase initial and slow release phases later. Burst release due to the surface of the protein adsorbed on the nanoparticles directly in the initial stage of dissolution and diffusion to the release medium. The release is due to in PLGA nanoparticles as protein within the framework of the gradual degradation and slow release. Prepared BSA-PLGA-NPs using the optimized conditions the release curve in the 2 to 28 days of with Higuchi equations as Q = 8.31t1 / 2 +43.60 (R2 = 0.982). The With the protein denaturation in NGF-PLGA-NPs, release time lasted only 14 days, 2 to 14 days of its release curves fitted with the Higuchi equation as Q = 9.24t1 / 2 +42.56 (R2 = 0.996)...