Preparation And Pharmacodynamic Study Of Functionalized Glycol Chitosan/PLGA/TP5Nanocarriers For Oral Delivery

Thymopoietin oligo-peptides are biologically active fragments of thymushormone thymopoietinⅡ, which includs thymopentin (TP5), thymocartin (TP4) andthymotrinan (TP3). All of them possess similar physiological activities asthymopoietinⅡ, and could be used as bidirectional immunomodulatory agents to dealwith immune-related diseases. Currently, only TP5is clinically approved forintramuscular or subcutaneous injection. Oral administration as one of alternatives forinjection has better compliance in patients. However, as for other peptide therapeutics,poor intestinal stability and low permeability are two key barriers to be overcome fororal delivery of TP5.In order to protect thymopoietin oligo-peptides, two enzyme inhibitors, Bestatinand EDTA, were selected to perform glycol chitosan (GCS) modification, and theresultant modified GCS were further evaluated for their enzyme inhibitory activity.Moreover, they were used as coating materials of drμg-loaded PLGA nanoparticles,and the preliminary pharmacodynamics of functionalized nanoparticles was studied invivo by oral administration to mice.First, GCS-Bestatin was synthesized throμgh covalent conjμgation, followed byfurther purification. On the other hands, GCS-gly-Bestatin was also synthesized, andamong glycine residue was used as a linker. The results of FTIR and fluorescentspectroscopy showed that both GCS-Bestatin and GCS-gly-Bestatin were successfullyconjμgated mainly throμgh forming amide bonds, and that few ester bonds existed inGCS-gly-Bestatin conjμgate. It was also found that the weight percent of Bestatin inGCS-gly-Bestatin was as two-fold high as that in GCS-Bestatin. Moreover,GCS-EDTA also was synthesized, and the results of FTIR confirmed that the covalentattachment of EDTA to GCS was achieved by the formation of amide bonds betweenthe carboxylic acid group of EDTA and amino groups of GCS. Second, the enzyme inhibitory activity of the resultant conjμgates was evaluated,and molecular interaction mechanism was also illustrated. The results of in vitroinhibitory experiments revealed that both GCS-Bestatin and GCS-gly-Bestatin couldefficiently inhibit aminopeptidase N activity and the former possessed more potentialthan the later in inhibitory efficiency under the same conditions. Further investigationindicated that the space hindrance and the loss of one pair of hydrogen bonds,resulting from the covalent conjμgation of GCS with Bestatin, did not prevent theinteraction between the leucyl residue of Bestatin and the hydrophobic pocket S1’ ofaminopeptidase N. In addition, it was found that the synthesized GCS-EDTA couldcompetitively chelate Ca2+from calcein, sμggesting that the resultant conjμgate stillhad the metal ion-chelating ability.Third, TP5-loaded PLGA nanoparticles were prepared using w/o/w emulsionevaporation method, and formulations were optimized based on the evaluation ofparticle size and encapsulation efficiency (EE). The selected preparation parametersare as following：40%sonication powder for50s,3%(w/v) PVA in outer aqueoussolution,130mg/mL PLGA, and50μl internal aqueous solution. The resultant PLGAnanoparticles were further coated with GCS, GCS-Bestatin, and GCS-EDTA,respectively. The experimental results showed that the coating had no significanteffect on either particle size or zeta potential of PLGA nanoparticles. The particle sizeis around250nm, and the EE is about30%. The coating with GCS and GCS-Bestatinled to a significant change in zeta potential of PLGA nanoparticles, from negative topositive. In comparison, PLGA nanoparticles coated with GCS-EDTA still possesseda negative zeta potential. The results of TEM experiment revealed that thenanoparticles in all groups are spherical and displayed a narrow size distribution. Theresults of in vivo studies with immunosuppressed mice showed that the ratios ofCD4+/CD8+in groups administrated with GCS-Bestatin and GCS-EDTA-coatedPLGA nanoparticles were higher than that of TP5solution group. Thepharmcodynamics will be further studied with the improved immunosuppressedmodel mice. In conclusion, this study indicated that the covalent conjμgation of Bestatin andEDTA to GCS could render GCS enzyme inhibitory activity, and that TP5-loadedPLGA nanoparticles coated with functionalized GCS may be used as potential carriersfor TP5oral delivery.