Quantum dot assisted long-term intracellular trafficking and development of safe and efficient non-viral vector

A number of non-viral DNA delivery systems and transfection protocols are under investigation. However, these show limited transfection efficiency and some are toxic to cells. The research described herein was aimed at developing novel methods to investigate potential roadblocks to the trafficking of non-viral DNA delivery systems and to develop novel and safer non-viral delivery systems. ^ As a novel approach to assessing intracellular DNA stability, plasmid DNA was dual labeled with rhodamine and fluorescein at two different sites. Gel-electrophoresis and time-lapse confocal microscopy confirmed visualization of DNA as a yellow color due to the close proximity of two fluorophores and separation of colors in the event of strand break. Thus color separation is a probable indication of DNA degradation inside cells. ^ To overcome photo-instability limitations of organic fluorophores, quantum dots (QDs) conjugated plasmid DNA was utilized, for long-term single cell tracking studies. Plasmid DNA-QD labeling via peptide nucleic acid linkers was facilitated by encapsulating cadmium selenide/zinc sulphide QDs in maleimide functionalized PEG lipids. QD-DNA labeling was confirmed by atomic force microscopy and gel electrophoresis. Transfection efficiencies of QD-DNA conjugates were comparable to unconjugated plasmid DNA demonstrating no loss in DNA functionality. Cellular uptake, cytoplasmic and nuclear distribution of QD-DNA conjugates were evaluated using confocal microscopy. QD-DNA conjugates were non-toxic as revealed by cytotoxicity assay and provide a unique tool for investigation of intracellular trafficking. ^ As an approach to develop a safe and efficient DNA delivery system, anionic liposomal systems formed by complexing 1,2-dioleoyl-sn-glycero-3-phospho-glycerol/1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (anionic/zwitterionic lipid) liposomes with plasmid DNA and Ca2+ were developed and optimized for high transfection efficiency and low cytotoxicity. Particle size analysis, gel electrophoresis, transmission electron microscopy and confocal studies assisted in characterization of optimized formulations. Maximum transfection efficiency occurred at 15-20 mM Ca2+ and 20/80 (anionic/zwitterionic lipid) and at high lipid/DNA ratios, 15–20 μg/0.8 μg. The transfection efficiency and cell viability of anionic lipoplexes in serum were ∼78% and ∼93%, respectively, which was comparable to cationic lipoplexes, which had transfection efficiency and cell viability values of ∼68% and ∼35%, respectively. Thus anionic lipoplexes appear to be suitable candidates for DNA delivery.

Non-Aqueous Suspensions of Antibodies are Much Less Viscous Than Equally Concentrated Aqueous Solutions

Purpose: The aim of this study was to markedly lower the viscosities of highly concentrated protein, in particular antibody, formulations. An effective approach elaborated herein for γ-globulin and a monoclonal antibody is to replace aqueous solutions with equimolar suspensions in neat organic solvents. Methods: Viscosities of aqueous solutions and non-aqueous suspensions of the model protein bovine γ-globulin and a murine monoclonal antibody were examined under a variety of experimental conditions. In addition, protein particle sizes were measured using dynamic light scattering and light microscopy. Results: Concentrated suspensions of amorphous γ-globulin powders (up to 300 mg/mL, composed of multi-micron-sized particles) in absolute ethanol and a number of other organic solvents were found to have viscosities up to 38 times lower than the corresponding aqueous solutions. Monoclonal antibody follows the same general trend. Additionally, the higher the protein concentration and lower the temperature, the greater the viscosity benefit of a suspension over a solution. Conclusions: The viscosities of concentrated γ-globulin and monoclonal antibody suspensions in organic solvents are drastically reduced compared to the corresponding aqueous solutions; the magnitude of this reduction depends on the solvent, particularly its hydrogen-bonding properties.Sanofi Aventis (Firm)Massachusetts Institute of Technology. Biophysical Instrumentation Facility (Study of Complex Macromolecular Systems (NSF-0070319))Massachusetts Institute of Technology. Biophysical Instrumentation Facility (Study of Complex Macromolecular Systems (NIH GM68762 instrumentation grant

Morphologically-Directed Raman Spectroscopy as an Analytical Method for Subvisible Particle Characterization in Therapeutic Protein Product Quality

Abstract Subvisible particles (SVPs) are a critical quality attribute of injectable therapeutic proteins (TPs) that needs to be controlled due to potential risks associated with drug product quality. The current compendial methods routinely used to analyze SVPs for lot release provide information on particle size and count. However, chemical identification of individual particles is also important to address root-cause analysis. Herein, we introduce Morphologically-Directed Raman Spectroscopy (MDRS) for SVP characterization of TPs. The following particles were used for method development: (1) polystyrene microspheres, a traditional standard used in industry; (2) photolithographic (SU-8); and (3) ethylene tetrafluoroethylene (ETFE) particles, candidate reference materials developed by NIST. In our study, MDRS rendered high-resolution images for the ETFE particles (> 90%) ranging from 19 to 100 μm in size, covering most of SVP range, and generated comparable morphology data to flow imaging microscopy. Our method was applied to characterize particles formed in stressed TPs and was able to chemically identify individual particles using Raman spectroscopy. MDRS was able to compare morphology and transparency properties of proteinaceous particles with reference materials. The data suggests MDRS may complement the current TPs SVP analysis system and product quality characterization workflow throughout development and commercial lifecycle