| Ligand-receptor-mediated delivery systems have received major attention in the past few years because naturally occurring ligands and their receptors are not only able to achieve site-specific targeting to ligand-specific biosites, but they are also biodegradable, non-toxic and non-immunogenic. Transferrin (Tf) is a glycoprotein which transports a ferric ion in the body. A major pathway for cellular iron uptake is by internalization of the complex of iron-bound transferrin and the transferring receptor (TfR). Transferrin and TfR-mediated endocytosis is one of the best-characterized processes in cell biology. It was shown earlier that the number of TfR on the cell surface is increased significantly in tumor cells and other rapidly proliferating cells compared with slowly growing non-malignant cell populations. Tf is a stable, commercially available protein and has been used as a ligand-receptor-mediated delivery system for cancer chemotherapy.The conjugation of polyethylene glycol (PEG), PEGylation, is a procedure of growing interest for enhancing the therapeutic and biotechnological potential of peptides and proteins, and has been extensively used to modify various proteins. PEGylation has been considered a tool to enhance the drug delivery to neoplastic tissues by the passive enhanced permeation and retention mechanism.Recombinant human tumor necrosis factor alpha (rHuTNF-α, Mr= 17000), an anti-tumor cytokine, exhibits striking biological effects, such as direct cytotoxicity against various kinds of tumor cells, activation of immune anti-tumor response, and inducement of hemorrhagic necrosis of certain transplanted solid tumors. However, excessively frequent and high dose of rHuTNF-a are required for significant anti-tumor effects because of its short plasma half-life and it was found to have severe toxic side-effects. We have previously demonstrated that PEGylated rHuTNF-a had a longer plasma half-life, higher level accumulation in tumor tissues and better anti-tumor potency than unmodified one.The main goal for this study is to synthesize Tf-PEG- rHuTNF-a conjugates by binding Tf to PEG-rHuTNF-a, using heterofunctional NHS-PEG-MAL as a spacer. The Tf-PEG-rHuTNF-α conjugates may both maintain the advantages of PEGylation and achieve the function of active targeting tumor cells.Because rHuTNF-a is very expensive, in this study, P-lactoglobulin B (Mr= 18276, LG) was firstly selected as a model protein in order to explored the possibility of coupling Tf to PEGylation protein. On the basis of the successful synthesis of the Tf-PEG-LG conjugate, the Tf-PEG-rHuTNF-a conjugate was also prepared.The first step of synthesis was to prepare MAL-PEG-LG (or MAL-PEG-rHuTNF-a) via the formation of an amino bond between lysine amino groups of protein and the terminal N-hydroxylsuccinimide (NHS) group of NHS-PEG-MAL. Subsequently, the Tf-PEG-LG conjugates was synthesize by reacting the double bond of the maleimide (MAL) group at the other end terminus of MAL-PEG-LG (or MAL-PEG-rHuTNF-a) with thiolated human transferrin forming a stable thioether bond.LG and rHuTNF-awere modified with different molar ratio of NHS-PEG-MAL and sampled at different reaction time. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to determine the resulted PEGylated protein which the free protein and PEG-protein were completely separated by SDS-PAGE. The free protein band in SDS-gel was quantified by using the ImageMaster bio-imaging system, and then the degree of PEGylation were assessed. The degree of PEGylated LG and rHuTNF-a increased with increasing mass of PEG and reaction time.MAL-PEG-LG were separated and purified by gel filtration chromatography. The yield of purified MAL-PEG- LG was 51.6%, and the molar ratio of LG and PEG was 1:7.4. The Tf-PEG-LG conjugate was prepared by conjugating MAL-PEG-LG to thiolated Tf. After isolated by gel filtration chromatography, the Tf-PEG-LG conjugate was obtained at a molar ratio of 1:1.4 (LG/Tf). The total yield (base on LG) was 28.2%.MAL-PEG-rHuTNF-a was purified from the reation mixture by using cation-exchange perfusion chromatography and the yield was 85.7%. The molar ratio of rHuTNF-a and PEG was 1:4.8. The synthesis of Tf-PEG-rHuTNF-a conjugate was achieved at a molar ratio of 1:1.1 (rHuTNF-a/Tf). The total yield (based on rHuTNF-a) was 45.2%.To assess the specificity of the Tf conjugates binding, TfR expression was determined in K562, KB, S-l 80 and L02 cells by determining their maximum binding sites and affinity using the classic radioligand binding analysis. The results showed that K562, KB and S-l80 tumor cells expressed more TfR than human normal...
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