PLGA microspheres

Abstract Chemical modification of proteins may influence their formulation into and release from polymeric microspheres. Three chemical modifications of rat serum albumin (RSA) were effected on the amine groups of this protein: conjugation with a polyanion using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), intermolecular crosslinking using glutaraldehyde, and reductive alkylation using propyl aldehyde. The modified proteins had different physico-chemical properties as well as improved encapsulation efficiencies compared with native RSA microspheres. The microspheres were incubated at 37C for a period of over one month to investigate the influence of protein modification on the release profiles. Microsphere degradation accelerated from the 9th day of the release studies and this coincided with an increase in the release rates. The degradation rates of PLGA microspheres containing either native or crosslinked RSA were more rapid than those containing either heparin conjugated or propylated RSA. This was in agreement with the release data, since the release of the native and crosslinked RSA were more rapid than those of the other modified proteins. The release profiles of the RSA-heparin conjugates and the propylated RSA were approximately zero rather than first order between the 10th and 30th day of study. Chemical modification of protein may be a useful method to increase encapsulation efficiency and decrease release rates of proteins which are to be used in microsphere formulation of potent therapeutic proteins.

Discussion The RSA/heparin conjugation was heterogeneous since following activation of the heparin carboxyl groups several reactions may occur such as: conjugation of one RSA with one or more heparin; and conjugation of one heparin with more than one RSA. This is illustrated in figure 5. The RSA/glutaraldehyde reaction could be heterogeneous and result in intra- and intermolecular crosslinking RSA. The number of bands observed in the SDS-PAGE, and the shoulders observed on the peaks obtained using size exclusion chromatography confirmed the heterogeneous nature of these reactions. The reductive propylation reaction was relatively homogeneous as only one band was observed in SDS-PAGE and one peak in size exclusion chromatography.
The RSA-heparin conjugate and the crosslinked RSA had not only increased molecular weight, but also decreased hydrophilicity as a result of a reduction in the number of hydrophilic groups. The propylated RSA also had increased hydrophobicity. Consequently, these modified RSAs may have decreased partitioning into the aqueous phase during microsphere preparation which may explain the high encapsulation efficiencies of modified RSA compared to native RSA. Since there is no difference in apparent molecular weight between native and propylated protein, the hydrophobicity appears to have a dominant influence compared to molecular weight on the encapsulation efficiency. The encapsulation efficiency may also be improved as a result of interaction between the PLGA polymer and the modified proteins. For example, the propyl chain may interact with PLGA.
The slow release profiles of the modified proteins from PLGA microspheres correlated with the PLGA microsphere degradation rates as determined by GPC. Those microspheres containing the RSA-heparin conjugate and the propylated RSA had the slowest degradation rates. The RSA-heparin conjugate may have a buffering effect preventing significant pH reduction within the microspheres and therefore reducing acid catalyzed PLGA hydrolysis and consequently reducing release rates. The propylated RSA was the most hydrophobic of the modified proteins, according to the HPLC data, and consequently water uptake into these microspheres is likely to be slower, which would reduce the PLGA degradation rate. This is supported by the increased association constant and decreased dissociation constant of alkylated protein from the lipid bilayer coated optical waveguides (Michielin et al 1999). The release profile from the crosslinked RSA microspheres was similar to that of native RSA. A possible explanation of this is that the reduction in the buffer capacity of the crosslinked RSA, as a result of a reduction in the number of carboxyl and amino groups, may have the effect of offsetting the reduction in degradation rate expected as a consequence of the increased hydrophobicity of this molecule. The polydispersities of all the degraded PLGA microsphere samples were large compared with PLGA microspheres on the first day of the release studies. This may be due to auto-acceleration of PLGA degradation inside the microspheres by concentrated acid catalyzed hydrolysis and conservation of PLGA molecular weight at the surface of the microspheres as a consequence of dilution of acid in the aqueous media.
In conclusion, it appears that chemical modification of RSA can result in significantly increased encapsulation efficiency and in a reduction in release rates from PLGA microspheres. Modified RSA may be useful as a carrier protein to aid in the formulation of potent therapeutic proteins into PLGA microspheres.

By TAE-KYOUNG KIM AND DIANE J. BURGESS