Characterization of Multilayered Nanoparticles Encapsulated in Yeast Cell Wall Particles for DNA Delivery

Abstract

Nonviral gene delivery technologies have been developed using layer-by-layer self-assembly of nanomaterials held together by electrostatic interactions in order to provide nanoparticulate materials that protect and deliver DNA to cells. Here we report a new DNA delivery technology based on the in situ layer-by-layer synthesis of DNA nanoparticles caged within hollow yeast cell wall particles (YCWP). YCWP provide protection and facilitate oral and systemic receptor-targeted delivery of DNA payloads to phagocytic cells. The nanoparticles inside YCWP consist of a core of tRNA/polyethylenimine (PEI) followed by a DNA layer that is finally coated with a protective outer layer of PEI. Using fluorescein and rhodamine labeling of tRNA, PEI, and DNA, the layer-by-layer formation of the nanoparticles was visualized by fluorescent microscopy and quantitated by fluorescence spectroscopy and flow cytometry. Optimal conditions (tRNA:YCWP, PEI:YCWP ratios and DNA load levels) to synthesize YCWP encapsulated nanoparticles were determined from these results. The high in vitro transfection efficiency of this encapsulated DNA delivery technology was demonstrated by the transfection of NIH3T3-D1 cells with YCWP-tRNA/PEI/gWizGFP/PEI formulations containing low amounts of the plasmid gWizGFP per particle to maximally express green fluorescent protein (GFP).

Delivery strategies to enhance oral vaccination against enteric infections

“…GPs can be efficiently loaded with antigen (N90%), target M cells for uptake and also exhibit an intrinsic immunomodulatory capacity [210,211]. β-glucans are fungal PAMPs, signalling through receptors such as dectin-1 and complement receptor 3 expressed on DCs, monocytes and neutrophils [209,212].…”

Recent advances in oral vaccine development

“…93,94,[98][99][100][101][102][103] Based on the in situ layer-by-layer synthesis through electrostatic interactions, the group of Soto initially engineered yeast cell wall particles for DNA delivery. 104 This encapsulation strategy enables a high nucleic acid capacity and delivery, in addition to the oral bioavailability of β-glucan particles and their inherent β-glucan receptor targeting capacity toward APCs. However, a disadvantage associated with the sequential adsorption of polymers in particular of polyethylenimine (PEI) is that high concentration ranges are cytotoxic.…”

Controlled-release approaches towards the chemotherapy of tuberculosis

“…44 The use of GPs for delivery of macromolecules, such as DNA, siRNA, and proteins, has been successfully established both in vitro and in vivo. 45,46 Soto et al have successfully encapsulated the TB antibiotic rifampin in glucan particles by making use of an alginate or chitosan hydrogel to close the GP pores and prolong the drug release. 44 The void holes of the GPs permit the efficient absorption and encapsulation of drugs.…”