DNA Nanoparticles Protocols

Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery

Weijun Li and Francis C. Szoka, Jr.1

Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California at San Francisco, California 94143-0446, USA

1Corresponding author (szoka@cgl.ucsf.edu)

INTRODUCTION

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo.

Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery

Li Shyh-Dar1, Li Song2, and Huang Leaf1,3

1 Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599, USA
2 Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pennsylvania 15213, USA

3Corresponding author (leafh@pitt.edu)

INTRODUCTION

Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. The LPD nanoparticles contain a highly condensed DNA core surrounded by lipid bilayers with an average size of ~100 nm. The nanoparticle complex is injected into mice, and expression of the transfected DNA is monitored with an appropriate assay.

PEI Nanoparticles for Targeted Gene Delivery

Frank Alexis, Jieming Zeng, and Wang Shu1,2

Institute of Bioengineering and Nanotechnology, Singapore 138669
1Department of Biological Sciences, National University of Singapore, Singapore 117543

2Corresponding author (swang@ibn.a-star.edu.sg)

INTRODUCTION

This protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing uptake by nontarget cells. We describe here methods for theconjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection using these complexes. The conjugation step involves the use of the succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC), a heterobifunctional cross-linker, to form a stable bond between PEI and peptides containing thiol groups.

Preparation of Gold Nanoparticle–DNA Conjugates

T. Andrew Taton1
1University of Minnesota, Minneapolis, Minnesota

Publication Name:

Current Protocols in Nucleic Acid Chemistry

Unit Number: UNIT 12.2

DOI: 10.1002/0471142700.nc1202s09

Online Posting Date: August, 2002

ABSTRACT

This unit describes the preparation of conjugates between nanometer-scale gold particles and synthetic oligonucleotides. Oligonucleotide-functionalized gold nanoparticles are finding increased use in both the construction of complex, tailored nanostructures and the optimization of DNA sequence analysis. The protocols in this unit outline the synthesis, purification, and characterization of nanoparticle-DNA conjugates for applications in nanotechnology and biotechnology. Separate procedures are presented for nanoparticles functionalized with just one or a few oligonucleotide strands and for nanoparticles functionalized with a dense layer of oligonucleotide strands. The different physical and chemical properties of these two types of conjugates are discussed, as are their stability and utility in different environments.

Chitosan-plasmid DNA nanoparticles used for contraceptive vaccine.

Publication: Immunotherapy Weekly

Publish date: July 21, 2004

2004 JUL 21 - (NewsRx.com & NewsRx.net) -- Researchers have prepared chitosan-plasmid DNA nanoparticles encoding zona pellucida glycoprotein-3 alpha and characterized its expression in the mouse.

"In the present study, the porcine zona pellucida (ZP)-3alpha eukaryotic expression vector pVAX1-pZP3alpha was constructed by genetic recombinant technology, then the recombinant plasmid was encapsulated in nanoparticles with chitosan, and the imaging of chitosan/pVAX1-pZP3alpha nanoparticles by Atomic Force Microscope (AFM) was processed. Feeding mouse with those microencapsulation by gastric larvae, and after five days, detecting its expression in mouse intestine by RT-PCR …