Rational Design and Development of a Soluble Mesoporous Carrier for the Solidification of a Preconcentrated Self-Nanoemulsion Formulation.

The solidification of self-preconcentrated nanoemulsion without changes in nanodroplet formation gains particular consideration due to the interaction between solidified carriers. This work aimed to develop mannitol mesoporous as a soluble carrier for supersaturated self-nanoemulsion (SSNE) using a design of experiment (DoE) approach. The mesoporous carrier was prepared by a spray-drying technique. The type of templating agent (TA) used to form a porous system, the amount of TA, and solid loading in the spray-drying process were studied. Several characterizations were performed for mannitol mesoporous formation, namely, powder X-ray diffraction, thermal analysis, scanning electron microscopy, and surface area analyzer. Solidification of SSNE incorporated into the mesoporous mannitol was carried out, followed by compaction behavior, flowability, and nanodroplet formation. The results revealed several process parameters for preparing the mesoporous mannitol, notably TA, which gained more significant consideration. Solid loading in the mesoporous preparation system reduced the surface area and pore size and did not affect solid SSNE flowability. The amount of TA increased the pore size and volume dramatically as well as the compactibility and flowability. Ammonium carbonate was the preferable TA for preparing the mesoporous carrier, particularly for the nanodroplet formulation process. In addition, synergistic and antagonistic interactions between factors were also observed. The optimized mesoporous carrier was applied for solidification, and there was no difference between SSNE and solid SSNE in the performance of nanodroplet formation.

Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product.

The aim of this research was to obtain recombinant human interferon alpha 2b (rhIFNalpha2b) from a synthetic open reading frame (ORF) overexpressed in Escherichia coli. For gene assembly, oligonucleotides were designed by Thermodynamically Balanced Inside Out (TBIO) method using the published synthetic codon optimized hIFNalpha2b ORF for high expression in E. coli. The synthetic ORF was assembled by a two-step Polymerase Chain Reaction (PCR) and cloned into a pGEM-T vector. The two-step PCR resulted in a DNA band of 522 base pairs (bp) corresponding to the size of hIFNalpha2b ORF. Fifteen recombinant pGEM-Ts were obtained and the sequencing results showed that the ORFs contained one to ten mutations with an error rate of 8.3 per kilo base. An ORF carrying one mutation was cloned into a pET32b vector and site-directed mutagenesis was performed to correct the mutation. The hIFNalpha2b ORF was overexpressed as a thioredoxin-his-tag fusion protein in E. coli BL21. The rhIFNalpha2b fusion protein was isolated from inclusion bodies (IB), renatured, and purified using Nickel columns, and all steps were monitored by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). A rhIFNalpha2b fusion protein of 37kDa in size was produced in high expression levels relative to total protein, renatured and purified from IB with a yield of 3.46mg/l without any further optimization. The purified rhIFNalpha2b was confirmed by peptide analysis with nano-LC-MS/MS2 mass spectrometry. Our current research demonstrates for the first time that by using the TBIO method a synthetic ORF encoding hIFNalpha2b gene can be expressed at high levels in E. coli.