Prolonged Hypocalcemic Effect by Pulmonary Delivery of Calcitonin Loaded Poly(Methyl Vinyl Ether Maleic Acid) Bioadhesive Nanoparticles

The purpose of the present study was to design a pulmonary controlled release system of salmon calcitonin (sCT). Therefore, poly(methyl vinyl ether maleic acid) [P(MVEMA)] nanoparticles were prepared by ionic cross-linking method using Fe2+and Zn2+ions. Physicochemical properties of nanoparticles were studiedin vitro. The stability of sCT in the optimized nanoparticles was studied by electrophoretic gel method. Plasma calcium levels until 48 h were determined in rats as pulmonary-free sCT solution or nanoparticles (25 μg·kg−1),ivsolution of sCT (5 μg·kg−1), and pulmonary blank nanoparticles. The drug remained stable during fabrication and tests on nanoparticles. The optimized nanoparticles showed proper physicochemical properties. Normalized reduction of plasma calcium levels was at least 2.76 times higher in pulmonary sCT nanoparticles compared to free solution. The duration of hypocalcemic effect of pulmonary sCT nanoparticles was 24 h, while it was just 1 h for theivsolution. There was not any significant difference between normalized blood calcium levels reduction in pulmonary drug solution andivinjection. Pharmacological activity of nanoparticles after pulmonary delivery was 65% of theivroute. Pulmonary delivery of P(MVEMA) nanoparticles of sCT enhanced and prolonged the hypocalcemic effect of the drug significantly.

Expression and Identification of Mutated Osteoprotegerin in Culture Cells and Larvae of Silkworm

The mutated osteoprotegerin (OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-OPG-372 was screened and identified by Southern blotting. The recombinant human OPG-372 was expressed in cultured cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that a 42 kD recombinant protein was expressed in BmN cells and a 46 kD one in larvae respectively. The bioactivities of the recombinant proteins were determined by hypocalcemic effect assay in the mice. The results showed that the recombinant proteins had a significant hypocalcemic effect on mice sera.