Cytoplasmic dynein associated peptide-linked nanoparticles for non-viral vector applications

Abstract

Event Abstract Back to Event Cytoplasmic dynein associated peptide-linked nanoparticles for non-viral vector applications Ercia Carbone1, Komal Rajpura1, Ho Man Kan1, Tao Jiang1 and Wai Hong Lo1 1 University of Connecticut Health Center, Institute for Regenerative Engineering, United States Statement of Purpose: Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NP) hold great promise for gene delivery applications. Non-viral polymeric nanoscale vectors offer several advantages over viral vectors[1]. However, most non-viral vectors suffer from significantly lower transfection efficiencies than viral vectors. This is because during their way to the nuclei of cells, non-viral vectors must overcome a series of diffusional and enzymatic barriers [1]. Interestingly, it has been shown that a number of viruses exploit microtubules by interacting with cytoplasmic dynein motor protein (a motor protein responsible for retrograde movement along microtubules) in order to enter the nucleus of the host cell. Cytoplasmic dynein is a large, multisubunit molecular motor that translocates various cargoes toward the nucleus. The consensus peptide sequence “K/R-X-T-Q-T” has been shown to be important for interacting with one of the dynein subunits [2]. Here, we develop dynein associated peptide-linked polymeric nanoparticles for non-viral vector applications. It is hypothesized that the novel dynein peptide-linked nanoparticles will hijack the endogenous dynein motor system intracellularly, and consequently move along microtubules toward the nucleus (-ve end of microtubule) (Fig.1). Methods: Dynein-associated peptide “LysGluThrGlnThrCys (KETQTC)” and the control peptide “LysGluThrAlaThrCys (KETATC)” were synthesized and labeled with FITC commercially at the N-terminus of the peptides (LifeTein, Inc). Note that the additional Cys residue is to facilitate the conjugation of the Asp to PLGA NPs; Maleimide-PEG-PLGA and Methoxy-PEG-PLGA were purchased from Polyscitech. This NP was fabricated using a water-in-oil-in-water (W/O/W) double emulsion technique using a mixture of 1:9 (Maleimide-PEG-PLGA : Methoxy-PEG-PLGA) [3]. The particle size distribution and zeta potential measurements of the NPs were determined by dynamic laser scattering. Conjugation of the peptides to the NP was performed as previously described [3]. Equal amounts of peptide-linked NPs or control peptide-linked NPs were mixed with purified dynein LC8 protein (1 mg) (ProSpec) for 1 hour at 4°C. The complex, i.e. peptide-linked NPs bound to LC8, was precipitated by centrifugation. The precipitated NPs were washed 3x with PBS and subsequently boiled with 2x sample buffer (Bio-rad). The proteins were resolved by SDS-PAGE (Bio-rad) and subsequently analyzed by immunoblot analysis with anti-LC8 antibody (Millipore). Human MG63 osteoblasts (ATCC) were cultured on coverslips with dynein peptide-linked NPs or control peptide-linked NPs for various time points. After removing the NPs and washing the coverslips 4x with PBS, the cells were fixed with 4% PFA (Santa Cruz). Nuclei were stained with popdium iodide (PI) nucleic acid stain. Cells were then mounted by a mounting solution, and fluorescent signals on the cells were observed via confocal microscopy (Zeiss LSM ConfoCor 3). Results: The average diameter of the NPs is 143.8 nm and the zeta potential is -26.43 mV. Biochemical binding analysis showed that purified recombinant dynein LC8 protein efficiently binds to KETQT-NPs. In contrast, LC8 protein does not bind well to the control peptide (KETAT)-NPs (Fig.2A). We further demonstrated that the KETQT-NPs can interact with endogenous dynein LC8 protein in MG63 cell lysates (Fig. 2B). To investigate the localization of the peptide-NPs in cells, we incubated the NPs with cells for various time points. Figure 2C shows that the FITC signals from KETQT-NPs (green) are colocalized with cells’ nuclei (red), whereas the FITC signals from the control NPs do not. Conclusions: Taken together, our data demonstrated that the KETQT-NPs can bind to dynein subunit (LC8) in vitro and in vivo, and these NPs can be enriched in nuclei region when they were incubated with cell culture. Future research including live-cell imaging will be used to trace the movement of the NPs in cells. Our dynein associated NPs may have a significant impact in non-viral vector development for gene therapy applications. This work was supported by funding from the State of Connecticut Stem Cell Research Foundation.