Efficient Delivery of Transducing Polymer Nanoparticles for Gene-Mediated Induction of Osteogenesis for Bone Regeneration

Abstract

Developing non-viral gene therapy vectors that both protect and functionally deliver nucleic acid cargoes will be vital if gene augmentation and editing strategies are to be effectively combined with advanced regenerative medicine approaches. Currently such methodologies utilize high concentrations of recombinant growth factors, which result in toxicity and off-target effects. Herein we demonstrate the use of modified cell penetrating peptides (CPPs), termed Glycosaminoglycan (GAG)-binding Enhanced Transduction (GET) peptides with plasmid DNA (pDNA) encapsulated poly (lacticco-glycolic acid) PLGA nanoparticles (pDNA-encapsulated PLGA NPs). In order to encapsulate the pDNA, it was first condensed with a cationic low molecular weight Poly L-Lysine (PLL) into 30-60 nm NPs followed by encapsulation in PLGA NPs by double emulsion; yielding encapsulation efficiencies (EE) of ∼30%. PLGA NPs complexed with GET peptides show enhanced intracellular delivery (up to sevenfold) and transfection efficiencies (up to five orders of magnitude). Moreover, the pDNA cargo has enhanced protection from nucleases (such as DNase I) promoting their translatability. As an example, we show these NPs efficiently deliver pBMP2 which can promote osteogenic differentiation in vitro. Gene delivery to human Mesenchymal Stromal Cells (hMSCs) inducing their osteogenic programming was confirmed by Alizarin red calcium staining and bone lineage specific gene expression (Q RT-PCR). By combining simplistic and FDA-approved PLGA polymer nanotechnology with the GET delivery system, therapeutic non-viral vectors could have significant impact in future cellular therapy and regenerative medicine applications.

Direct contact-mediated non-viral gene therapy using thermo-sensitive hydrogel-coated dressings

“…Importantly this is demonstrated for both reporter genes, and for therapeutic genes (VEGF, FigS. 16).…”

“…This technology exploits enhanced membrane-docking peptides which bind heparan sulfate glycosaminoglycans (GAGs), combined with a CPP. Functional quantities of many cargos were successfully delivered either in media [11][12][13][14] or from scaffolds [15], biomaterials [12,16] and hydrogels [17] to cells. Importantly nanoparticles (NPs) formed from GET peptides complexed with nucleic acids, such as plasmid (p)DNA have high transfection efficiency in vitro or in vivo [18][19][20][21].…”

Encapsulation of Large-Size Plasmids in PLGA Nanoparticles for Gene Editing: Comparison of Three Different Synthesis Methods

“…Moreover, these studies are usually carried out using medium to low molecular weight plasmids (typically around 5.5 kb but up to 7.3 kb), [ 35 , 42 , 64 , 65 , 66 ], which might be less sensitive to unfolding or sonication tearing than larger nucleic acid chains. In fact, to our knowledge, this is the first study validating ultrasound- or microfluidics-assisted double emulsion encapsulation of large pDNAs (9.4 kb), similar to those encoding the CRISPR/Cas gene editing system (which are typically about 9.10 kb in size) [ 2 ].…”

The Delivery and Activation of Growth Factors Using Nanomaterials for Bone Repair

“…To enhance the transfection efficiency of the plasmid, scientists used different modifications of the nanomaterials. Jalal and Dixon used glycosaminoglycan (GAG)-binding enhanced transduction (GET) peptides as the surface moiety of pBMP-2-encapsulated PLGA nanoparticles to enhance their cell penetration and achieve higher transfection efficiency by five orders of magnitude [ 159 ]. In addition to pBMP-2, the plasmid encoding VEGF, the most common GF that promotes angiogenesis, has been shown to promote bone repair.…”