School of Pharmacy Articles

Screening of siRNA Nanoparticles for Delivery to Airway Epithelial Cells Using High Content Analysis

Alan Hibbitts et al. — Wed, 02 May 2012 07:27:09 PDT

Background:

Delivery of siRNA to the lungs via inhalation offers a unique opportunity to develop novel methods of treating a range of poorly treated respiratory conditions. However progress has been greatly hindered by safety and delivery issues. This study developed a high-throughput method for screening novel nanotechnologies for pulmonary siRNA delivery

Methodology:

Following physico-chemical analysis, the ability of PEI-PEG/siRNA nanoparticles to facilitate siRNA delivery was determined using high content analysis (HCA) in Calu-3 cells. Results obtained from HCA were validated using confocal microscopy. Finally, cytotoxicity of the PEI-PEG/siRNA particles was analysed by HCA using the Cellomics® multiparamter cytotoxicity assay.

Conclusions:

PEI-PEG/siRNA nanoparticles facilitated increased siRNA uptake and luciferase knockdown in Calu-3 cells compared to PEI/siRNA.

The effect of liposome encapsulation on the pharmacokinetics of recombinant secretory leukocyte protease inhibitor (rSLPI) therapy after local delivery to a guinea pig asthma model.

Aileen Gibbons et al. — Wed, 02 May 2012 06:26:02 PDT

PURPOSE: Inhaled recombinant Secretory Leukocyte Protease Inhibitor (rSLPI) has shown potential for treatment of inflammatory lung conditions. Rapid inactivation of rSLPI by cathepsin L (Cat L) and rapid clearance from the lungs have limited clinical efficacy. Encapsulation of rSLPI within 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine]:Cholesterol liposomes (DOPS-rSLPI) protects rSLPI against Cat L inactivation in vitro. We aimed to determine the effect of liposomes on rSLPI pharmacokinetics and activity in vitro and after local delivery to the airways in vivo.

METHODS: Transport of DOPS-rSLPI and free-rSLPI across a polarised air-liquid epithelial monolayer was measured. An asthma guinea pig model was administered either DOPS-rSLPI liposomes or free-rSLPI by intratracheal instillation.

RESULTS: Apparent permeability (P(app)) of free-rSLPI was significantly higher at 4.9 x 10⁻⁶ cm/s than for DOPS-rSLPI, P(app) of 2.05 x 10⁻⁷ cm/s, confirmed by in vivo studies. Plasma rSLPI concentrations were highest in free-rSLPI-treated animals compared with those treated with DOPS-rSLPI; there also appeared to be a trend for higher intracellular rSLPI content in animals dosed with DOPS-rSLPI compared to free-rSLPI. Eosinophil influx was recorded as a measure of inflammation. Pre-dosing with either free-rSLPI or DOPS-rSLPI prevented inflammatory response to antigen challenge to levels comparable to control animals.

CONCLUSION: Encapsulation of rSLPI in DOPS:Chol liposomes improves stability, reduces clearance and increases residence time in the lungs after local delivery.

Incorporation of PLGA nanoparticles into porous chitosan-gelatin scaffolds: influence on the physical properties and cell behavior.

Vijay Kumar Nandagiri et al. — Wed, 16 Nov 2011 05:04:20 PST

Bone regeneration can be accelerated by localized delivery of appropriate growth factors/biomolecules. Localized delivery can be achieved by a 2-level system: (i) incorporation of biomolecules within biodegradable particulate carriers (nanoparticles), and (ii) inclusion of such particulate carriers (nanoparticles) into suitable porous scaffolds. In this study, freeze-dried porous chitosan-gelatin scaffolds (CH-G: 1:2 ratio by weight) were embedded with various amounts of poly(lactide-co-glycolide) (PLGA) nanoparticles, precisely 16.6%, 33.3% and 66.6% (respect to CH-G weight). Scaffolds loaded with PLGA nanoparticles were subjected to physico-mechanical and biological characterizations including morphological analysis, swelling and dissolution tests, mechanical compression tests and cell viability tests. Results showed that incorporation of PLGA nanoparticles into porous crosslinked CH-G scaffolds: (i) changed the micro-architecture of the scaffolds in terms of mean pore diameter and pore size distribution, (ii) reduced the dissolution degree of the scaffolds, and (iii) increased the compressive modulus. On the other hand, the water uptake behavior of CH-G scaffolds containing PLGA nanoparticles significantly decreased. The incorporation of PLGA nanoparticles did not affect the biocompatibility of CH-G scaffolds.

Effect of Microencapsulation Sheer Stress on the Structural Integrity and Biological Activity of a Model Monoclonal Antibody, Trastuzumab

Ritesh M. Pabari et al. — Mon, 14 Nov 2011 11:10:48 PST

The aim of the present study was to investigate the influence of process shearstressors on the stability of a model monoclonal antibody, trastuzumab. Trastuzumab, at concentrations of 0.4–4.0 mg/mL, was subjected to sonication, freeze-thaw, lyophilisation, spray drying and was encapsulated into micro- and nanoparticles. The stressed samples were analysed for structural integrity by gel electrophoresis, SDS-PAGE, and size exclusion chromatography (SEC), while the conformational integrity was analysed by circular dichroism (CD). Biological activity of the stressed trastuzumab was investigated by measuring the inhibition of cell proliferation of HER-2 expressing cell lines. Results show that trastuzumab was resistant to the process shear stresses applied and to microencapsulation processes. At the lowest concentration of 0.4 mg/mL, a low percent (<9%) of soluble/reversible aggregates may have been formed. No loss of structural integrity, conformation was observed and no significant change in the biological activity of trastuzumab was observed (ANOVA;p > 0.05). The results of this study conclude that

trastuzumab may be resistant to various processing stresses. These findings have important implications with respect to pharmaceutical processing of monoclonal antibodies.