Anionic and cationic dextran hydrogels for post-loading and release of proteins.
In this study, post-loading of proteins in and release from chemically crosslinked dextran hydrogels exploiting reversible electrostatic interactions was investigated. Methacrylated dextran (Dex-MA) was co-polymerized with methacrylic acid (MA) or dimethylaminoethyl methacrylate (DMAEMA) to form negatively and positively charged hydrogels, respectively. Incubation of negatively charged hydrogels in a low ionic strength (10 mM HEPES, pH 7.4) solution of cytochrome C (isoelectric point (pI) 10.2) led to quantitative absorption of the protein in the hydrogel. BSA (pI 4.8) and myoglobin (pI 7.2) were post-loaded into positively charged gels at neutral pH and negatively charged gels at pH 5, respectively. Loading efficiency and protein distribution in the gels were dependent on network charge (maximum loading efficiency at 100-150 μmol charged monomer/g gel) and crosslink density (higher and more homogenous loading at lower crosslink density) and on the ionic strength during loading (lower but more homogenous loading at higher ionic strength). Diffusion controlled release of the loaded protein was triggered by incubation of the hydrogel in HEPES buffered saline (HBS) pH 7.4. The amount of released cytochrome C in HBS varied from 94% to 70% from gels containing 60 and 150 MA/g, respectively. Importantly, quantitative release was obtained in 1 M NaCl, indicating that post-loading led to neither the formation of insoluble protein aggregates nor irreversible immobilization of the protein in the matrix. ESI-MS analysis of the released cytochrome C revealed that post-loading did not result in oxidation of the protein, as opposed to loading during preparation of the gels. In conclusion, this paper shows that post-loading of proteins in dextran hydrogels and release exploiting reversible charge interactions can be applied for efficient loading of proteins that are negative, positive or neutral at physiological pH. Importantly, our data demonstrate that using this loading method no chemical modification to the protein occurred.
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
Joris P
JP
Schillemans
Ellen
E
Verheyen
Arjan
A
Barendregt
Wim E
WE
Hennink
Cornelus F
CF
Van Nostrum
D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000008:administration & dosage*; D003911:Dextrans / Q000737:chemistry*; D006736:Horses; D020100:Hydrogels / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D008689:Methacrylates / Q000737:chemistry*; D009211:Myoglobin / Q000008:administration & dosage*; D009994:Osmolar Concentration; D012710:Serum Albumin, Bovine / Q000008:administration & dosage*
D003911:Dextrans; D020100:Hydrogels; D007477:Ions; D008689:Methacrylates; D009211:Myoglobin; C514458:methacrylated dextran; D012710:Serum Albumin, Bovine; D045304:Cytochromes c