13 research outputs found
Polyamidoamine-Decorated Nanodiamonds as a Hybrid Gene Delivery Vector and siRNA Structural Characterization at the Charged Interfaces
Nanodiamonds have been discovered as a new exogenous
material source in biomedical applications. As a new potent form of
nanodiamond (ND), polyamidoamine-decorated nanodiamonds (PAMAM-NDs)
were prepared for E7 or E6 oncoprotein-suppressing siRNA gene delivery
for high risk human papillomavirus-induced cervical cancer, such as
types 16 and 18. It is critical to understand the physicochemical
properties of siRNA complexes immobilized on cationic solid ND surfaces
in the aspect of biomolecular structural and conformational changes,
as the new inert carbon material can be extended into the application
of a gene delivery vector. A spectral study of siRNA/PAMAM-ND complexes
using differential scanning calorimetry and circular dichroism spectroscopy
proved that the hydrogen bonding and electrostatic interactions between
siRNA and PAMAM-NDs decreased endothermic heat capacity. Moreover,
siRNA/PAMAM-ND complexes showed low cell cytotoxicity and significant
suppressing effects for forward target E6 and E7 oncogenic genes,
proving functional and therapeutic efficacy. The cellular uptake of
siRNA/PAMAM-ND complexes at 8 h was visualized by macropinocytes and
direct endosomal escape of the siRNA/PAMAM-ND complexes. It is presumed
that PAMAM-NDs provided a buffering cushion to adjust the pH and hard
mechanical stress to escape endosomes. siRNA/PAMAM-ND complexes provide
a potential organic/inorganic hybrid material source for gene delivery
carriers
LC/ESI MS/MS of trypsin/Glu-C digests of R27T and Rebif.
<p>(A) Extraction ion chromatogram at m/z 204 and 366 for product ion spectra at 21 min and 46 min, respectively. Amino acid sequencing of deglycosylated C3 and C13 peptides by LC/ESI MS/MS. (B) Deglycosylated C3 peptide MS spectrum and fragmentation information for R27T (Lot: 12104DS01 and 12103DS01) and Rebif. (C) MS spectrum of the deglycosylated C13 peptide and fragmentation information for R27T (Lot: 12104DS01 and 12103DS01) and Rebif.</p
Glycoengineering of Interferon-β 1a Improves Its Biophysical and Pharmacokinetic Properties
<div><p>The purpose of this study was to develop a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a) to improve its biophysical properties, such as aggregation, production and stability, and pharmacokinetic properties without jeopardizing its activity. To achieve this, we introduced additional glycosylation into rhIFN-β 1a via site-directed mutagenesis. Glycoengineering of rhIFN-β 1a resulted in a new molecular entity, termed R27T, which was defined as a rhIFN-β mutein with two N-glycosylation sites at 80<sup>th</sup> (original site) and at an additional 25<sup>th</sup> amino acid due to a mutation of Thr for Arg at position 27<sup>th</sup> of rhIFN-β 1a. Glycoengineering had no effect on rhIFN-β ligand-receptor binding, as no loss of specific activity was observed. R27T showed improved stability and had a reduced propensity for aggregation and an increased half-life. Therefore, hyperglycosylated rhIFN-β could be a biobetter version of rhIFN-β 1a with a potential for use as a drug against multiple sclerosis.</p></div
Size distribution of rhIFN-β 1a and R27T.
<p>The analysis of (A) rhIFN-β 1a and (B) R27T was performed at a scattering angle of 90° and distributed sizes, PDI, and Zeta averages were shown. (r.nm: radius in nanometers). (C) rhIFN-βs were quantified by CPE at each time up to 72 hr.</p
Construction and testing of rhIFN-β glycosylation analogs.
<p>(A) A schematic of the rhIFN-β protein. Boxes represented the locations of the five α-helices. Each vertical line represented a position with potential for an additional N-linked glycosylation site, as predicted by NetNGlyc. Introduced N-linked glycosylation consensus sequence sites were showed by diamonds(?). Purified samples were separated by (B) SDS-PAGE and (C) western blot analysis. (D) Analogs were subjected to N-glycanase digestion for the indicated times. (E) IEF analysis was performed over a pH range of 3–10.</p
<i>In vitro</i> activity.
<p>(A) Anti-viral, (B) anti-proliferative and (C) immunomodulatory activities of R27T (?), R27TΔGlyc (▪) and Rebif (▴).</p
Model of the N-glycosylated R27T/IFNAR2 complex.
<p>(A) front view and (B) side view. The complex structure oligosaccharides, R27T, and IFNAR2 were shown as orange, magenta, and cyan color, respectively. New hydrogen bonds between the oligosaccharide and complex were presented as red bold line in C (with IFNAR2) and D (R27T). Also, 27T and 80N in R27T protein were colored by yellow (A,B) and hydrogen-bonded residues in R27T and IFNAR2 were colored by green (C,D).</p
Mean arterial plasma concentration–time profiles for rhIFN-βs.
<p>(A) IV, (B) SC and (C) IM administration of R27T, R27TΔGlyc and Rebif at a dose of 1 MIU/kg in rats. Vertical bars indicated standard deviations.</p
Secondary structure ratios derived from the ATR-FTIR spectra of rhIFN-β 1a and R27T in solution.
<p>Secondary structure ratios derived from the ATR-FTIR spectra of rhIFN-β 1a and R27T in solution.</p
Sugar composition analysis of R27T and Rebif (mol/mol protein).
a<p>2 M TFA for neutral sugars at 100°C at 4 hr.</p>b<p>6 N HCl for amino sugars at 100°C at 4 hr.</p>c<p>0.1 N HCl for sialic acids(NANA+NGNA) at 80°C at 1 hr.</p