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Title: Controlling the rate of the release of proteins from microparticles for tissue engineering applications
Author: Qodratnama, Roozbeh
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2012
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Multifunctional tissue engineering scaffolds can be used to create a suitable niche for cells to grow and form neo-tissues. Microparticles with growth factorspecific release propelties and tissue-specific polymer degradation rates can serve as the building blocks that underlie the multifunctionality of these scaffolds. In this work, blending poly(DL-lactic acid-co-glycolic acid) - poly(ethylene glycol)poly( DL-lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymers with poly(DL-lactic acid-co-glycolic acid) (POL LGA) as a foundation polymer is introduced as an approach to accelerate lysozyme release from large PLGA microparticles. Here, the reproducible synthesis of composition ally different triblock copolymers is reported. Their reproducibility is controlled by controlling the lactic acid /poly(ethylene glycol) (LA/PEG) ratio. It is shown that a difference in LA/GA ratio and molecular weight of PEG can result in structurally different triblock copolymers with different hydrophobicity and different sol-gel transition temperatures. Large micropalticles (~ 200 I-lm in diameter) were fabricated from PLGA foundation polymer blended with PLGA-PEG-PLGA with morphology and size distribution closely similar to the control group (PLGA foundation polymer (~300 I-lm in diameter)). Blending PLGA foundation polymer with PLGA-PEGPLGA triblock copolymers reduced the glass transition temperature (Tg) and the decrease in Tg was correlated inversely with the mass oftriblock copolymer present in the polymer formulation. A comparison of the effect of two different triblock copolymers, namely PLGA-PEG 1500-PLGA (LA/GA on feed ratio of 2.5) and PLGA-PEG 1 OOO-PLGA LA/GA on feed ratio of 3) on lysozyme release rate and profile from these microparticles was made. It was shown that blending these triblock copolymers with PLGA 85: 15 as foundation polymer prior to microparticle manufacture significantly accelerated lysozyme release from these microparticles. It is demonstrated that the lysozyme release rate and the total release was propOltionate to the mass of triblock copolymer blended with foundation polymer - in the way that the higher the mass oftriblock copolymer in the polymer formulation the higher the release rate. At 37°C, microparticles with :1 polymer formulation consisting of PLGA 85: 15 without triblock copolymer (the control group) were shown to have a biphasic release profile consisting of a slow release phase (first week) and a prolonged lag phase. The slow release phase was composed of a burst release during which totally ~5% of entrapped protein (in first week) was released. No release was detectable after this release phase. Microparticles with polymer formulation consisting of PLGA85: 15/PLGAPEG- PLGA 90: 1 0 showed a tri -phasic release curve. After a burst release of 14- 17% in the first 24 hours, a slow or not detectable level of release was observed for 20-30 days. After this lag phase, another 20-25% was released over 10-20 days and the total release achieved by day 60 was 40-50% (of entrapped protein). Microparticles with polymer formulation consisting of PLGA85: 15/PLGA-PEGPLGA 70:30 showed a continuous release profile and totally released 70% of entrapped protein in the first 30 days and the release was undetectable after 30 days to the end. These results demonstrated that blending PLGA-PEG-PLGA triblock in PLGA 85:15 accelerated lysozyme release from microparticles. It was 11 found that the effect of PLGA-PEG 1 OOO-PLGA and PLGA-PEG 1500-PLGA on release rate and profile was not different. The environment surrounding microparticles and in a controlled release context, the incubation medium, can affect the release behaviour. In this work, release behaviour of FITC labelled lysozyme from microparticles fabricated from two different polymer formulations into a fibrin clot (3.5 mg/mL fibrinogen; physiological concentration) was compared with the release to PBS and fibrin gel with 50 mg/mL fibrinogen. Fibrin clot was used as a model of blood clot. It was shown that the release profile ofFITC labelled lysozyme in PBS and in fibrin gels are similar for a celtain formulation. However, the release rates were different in each incubation media. Very low release (2 .7-4.5% depending on the polymer formulation) was detectable in fibrin gel (50 mg/mL fibrinogen). The release rate was higher (3 .7-7% depending on the formulation) in fibrin clot (3.5 mg/mL fibrinogen). The highest release rates were detected in PBS (11 and 13%). Finally, the bioactivity of bone morphogenetic protein-2 (BMP-2) released from two compositionally different microparticles laden in fibrin clot (3.5 mg/mL), was examined by measuring its effects on cells over a 20 day period. It was revealed that BMP-2 released from microparticles can escape the fibrin gel to the culture medium. These BMP-2 molecules triggered osteogenetic cellular pathways. This was shown by detection of increased levels of alkaline phosphatase compared to a negative control. I In conclusion, it was shown for the first time that PLGA-PEG-PLGA triblock copolymers blended with PLGA as a foundation polymer can accelerate protein release from large microparticles. Generally large microparticles have very slow release rate and assemble into highly porous scaffolds.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available