Preparation Of Biodegradable And Thermosensitive Hydrogels Of Different Sizes And Studies On Post-fabrication Encapsulation Of Protein Drugs Via Microgels

With the fast growth of biotechnology, more and more biologically active peptides and protein drugs are produced. The clinical applications of these protein drugs, however, still face many obstacles because of their low stability, short circulatory and large molecular size. The classic drug delivery systems (DDSs) for small molecular drugs are not suitable for protein drugs. Therefore, it is a challenging topic to develop novel controlled release systems in which proteins and peptides are stable, absorbable and have a long half-life time. This topic is an interdisciplinary one among pharmaceutics, biology, and material science.Hydrogel is a good candidate delivery carrier for protein drugs. The properties of hydrogels, such as size, morphology, intelligence and biodegradability, need to meet the practical requirements, and the preparation techniques are rather important. The formulation conditions are vital to pharmaceuticals, which are directly related to the uniform of products. So, the postdealing technique is also very important.This Ph. D thesis focuses on a potential protein drug delivery system based on synthetic hydrogels. The work mainly includes synthesis of biodegradable and thermosensitive hydrogel of different size, and preliminary studies on formulations of post-fabrication encapsulation of protein drugs via microgel. The innovations of this Ph.D thesis are listed as fillows:1. A kind of thermosensitive and biodegradable chemically-crosslinked nanogels was synthesized and a macroscopic physically-crosslinked hydrogels was prepared based on the nanogels as building block. A triblock copolymer, poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEG-PPG-PEG) was end-capped by acryloyl groups using a biodegradable oligolactide as spacer, and such biodegradable amphiphilic macromers could form micelles in water. A nanogel was prepared via polymerizing macromers in a micelle, and a macroscopic physical gelation was found upon heating a concentrated aqueous nanogel suspension. Such a sol-gel transition with a chemically-crosslinked nanogel as the building block was thermoreversible. This kind of hydrogel affords a promising injectable biomaterial, meanwhile, this research reveals new physics of the thermogelling mechanism of amphiphilic block copolymers.2. The measurement methods of drug loading amount of protein post-fabrication encapsulation into microgels were investigated systematically. Our group previorusly designed and synthesized a kind of thermosensitive and biodegradable microgels. In the research of this thesis, the microgels were prepared by inversion suspension polymerization from the macromer described above. By modulating the formulation conditions, the microgels of different sizes were obtained, and the properties such as thermosensitity and biodegradation of microgels were investigated. We also made preliminary studies on post-fabrication encapsulation of protein drugs via microgels. An improvement in the course of the present Ph. D thesis is the examinations of meaurment approachs of loading amount. Five methods were used in this thesis and the results are as follows. Method 1: after equilibrium in protein solutions, the solutions were filtrated and the protein mass in the remaining solutions was measured, Thus the protein mass entrapped into the microgels were obtained, and the drug loading amount were calculated. The error in this method is large. Method 2: after equilibrium in protein solutions, the protein concentration in the remaining solution was measured and the mass of proteins was calculated, which was minus by the total protein mass and the protein mass entrapped into the microgels was obtained, then drug loading amount was calculated. The value in this method is larger than the other methods. Method 3: after equilibrium in protein solutions, microgels were separated and dried, then controlled released till the complete biodegradation of microgels, the protein cumulative release amounts at the end point are regards as the total amounts of protein in microgels, and then drug loading amount can be calculated. Method 4: after equilibrium in protein solutions, microgels were separated and dried, then degraded in PBS without taking out sample during the time, the protein concentrations were measured and drug loading amount was calculated. Methods 5: after equilibrium in protein solutions, microgels are separated and dried, then degraded in NaOH solution (containing 5% SDS), the protein concentrations were measured and drug loading amount was calculated. The results of the latter three methods have no significant difference, and thus all these three methods can be used to characterize the drug loading amount. Meanwhile, we can conclude that in our DDS, the proteins were released completely, which embodied the advantages of our post-fabrication encapsulation technology on the other hand.3. The effects of drying approaches on physico-chemical properties of the microgels and the post-fabrication encapsulation of a protein were investigated. Before the loading of protein drugs, the microgels were dried by freeze-drying or acetone drying. The results demonstrated that the drying processes had a significant influence on morphology, swelling ratio and thermosensitivity of microgels. As a model protein drug, bovine serum albumin (BSA) was loaded by immersing the dried microgels into the BSA solution. After such a post-fabrication encapsulation, the loaded microgels were dried by one of three approaches, freeze-drying, vacuum-drying at room temperature, and slow drying in a 4℃refrigerator at normal pressure. The effects of drying approaches, both before and after drug loading, on the release behaviors were examined. The results indicated that although the drying processes after drug loading did not influence the release behaviors to a large extent, the drying approaches of microgels before drug loading had significant effects on both protein loading into the hydrogels and protein release out of the hydrogels. This paper thus demonstrates that the very popular freeze-drying is not suitable for our hydrogel system and drying approaches in preparation of a hydrogel should be paid much attention when this kind of wet materials is aimed to be used as a drug carrier.