Abstract
The objectives of this investigation were to investigate the effect of interaction of protein and polymers with varying end groups on the overall release profile of model proteins (Lysozyme, Bromelain and Bovine Serum Albumin (BSA)). |Lactide and/or glycolide-based polymers (intrinsic viscosity 0.15 to 0.22 d/L) differing in end groups (carboxylic acid or ester), were dissolved in an organic solvent mixture of various ratios of benzyl benzoate and benzyl alcohol. Polymer solutions were tested for injectability through 22 gauge needle. The model proteins were incorporated into the polymer solution by sonicating at 40W for 20 seconds. When polymer-protein solution was injected in releasing media (phosphate buffered saline, pH 7.4), it instantaneously formed a gel depot. Samples were withdrawn from the releasing media at specific time points and analyzed for the protein content as well as its conformational stability and biological activity. Lysozyme and Bromelain content was determined at 280 nm while BSA contents were determined at 277.8 nm by using a UV spectrophotometer. Conformational stability was determined by Fourier Transform Infra Red (FTIR) Spectroscopy and Differential Scanning Calorimeter (DSC). The peaks in the range of 1620-1695nm in FTIR spectrum were indicative of secondary structures of the model proteins. Enthalpy and mid-point of thermal transition in DSC thermogram were used as thermodynamic parameters indicating conformational stability. Biological activity was determined by enzyme activity assay using Micrococcus Lysodeikticus as substrate for Lysozyme, Tyrosinase for albumin, and Z-L-Lys-ONp hydrochloride for Bromelain. |Polymer concentrations up to 25% (w/w) were found injectable through 22 gauze needle. Polymers bearing carboxylic acid end group were not soluble in 100% BB which were well correlated with hydrophilic-lipophilic profiles of polymers and the solvent systems. Release studies indicated a lower burst release (< 15%) from formulations containing polymer with carboxylic acid end group than those containing ester end group (burst release > 25%). Polymers with acid end groups showed a faster rate of release than those with ester end groups in the second phase of the observed biphasic pattern of release. The amount of burst release and over all release profiles were explained on the basis of influence of end groups on the rate of gelation of the phase-sensitive smart polymer-based delivery systems. The polymers with free carboxylic end groups showed faster rate of gelation as compared to the polymer with ester end groups. The DSC thermograms of the protein solutions showed decreased conformational stability compared to the DSC thermograms of the protein formulations with time. The Tm of Lysozyme solution on day 84 was 26.94±0.096ºC and that of the Lysozyme formulations was > 47 ºC. The Tm of Bromelain solution on day 84 was 45.00±0.023 ºC and that of the Bromelain formulations was > 59 ºC. The Tm of Albumin solution on day 49 was 51.86±0.039 ºC and that of the Albumin formulations was > 54 ºC. This may be occurring due to the protective effect of the polymers on the protein formulations. FTIR data showed decrease in the area of secondary structures with time. The protein formulations however were found to have more stability as compared to the protein solutions. The corrected areas of secondary structures for Lysozyme solution on day 84 was 0.001 and for the Lysozyme formulations was >0.014. The corrected areas of secondary structures for Bromelain solution on day 49 was 0.0015 and for the Bromelain formulations was >0.042. The corrected areas of secondary structures for Albumin solution on day 49 was 0.04 and for the Albumin formulations was >0.2. Biological activity data showed that the protein formulations lost their biological activity with passage of time, but the biological activity of the protein formulations was more than that of protein solutions on any given day. FTIR, DSC, and enzyme activity data indicated significantly greater (p<0.05) conformational stability and activity in comparison to those of control samples.|It is concluded that the interaction of polymer end groups such as carboxylic acid with protein functional groups such as hydroxyl or amino can be used for reducing burst release and protecting conformational stability as well as biological activity of proteins incorporated in phase-sensitive smart polymer formulations.