Light-Controlled Modulation of Gene Expression Using Polyamidoamine Formulations

Abstract

A promising method that offers both time- and site-specific delivery of macromolecules is photochemical internalization technology (PCI). Here, we have characterized various polyamidoamine (PAMAM) carriers [generation (G) 0-7], for light-directed delivery of nucleic acids in vitro by the use of PCI technology. A number of parameters for optimal delivery of nucleic acids into human cancer cells, that is, various light-doses, carrier-doses, and small interfering RNA (siRNA)/messenger RNA (mRNA) doses were investigated for either up- or down-regulation of enhanced green fluorescent protein (EGFP) gene expression. In summary, our results showed in an osteosarcoma cell line (OHS) [EGFP] model system the possibility for efficient light-directed siRNA silencing (>80% silencing) when using PAMAM G3 to G7 as carriers. Surprisingly, no EGFP mRNA up-regulation was detected either with or without PCI after EGFP mRNA/PAMAM (G0-G7) transfection in standard OHS cells. We have here identified properties for PAMAM formulations enabling light-directed siRNA delivery with the aim of developing a site-specific strategy for delivery of nucleic acids in vivo.

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research

“…For many types of delivery vehicles PCI seems to have the same positive effect across all the different nucleic acid cargos tested, but this is not always the case, e.g., for PAMAM dendrimers PCI seems to work well for siRNA, but not for mRNA delivery [167]. While one might expect the PCI effect on endosomal escape to be the same irrespective of the nucleic acid cargo, such difference may, e.g., be caused by different sensitivities of the cargo to degradation inside endocytic vesicles, e.g., making the timing of endosomal release an important parameter.…”

“…Further studies on using PCI for oligonucleotide mediated gene expression silencing have been performed with siRNA in combination with a variety of delivery vehicles. Thus, Bøe and co-workers showed good effects of PCI with siRNA with polymeric vehicles such as polyethylenimine [164], cyclodextrin-containing polymers [165], peptides [166] and polyamidoamine (PAMAM) carriers [167]; as well as with some, but not all of the tested lipid vehicles [164]. In case of PAMAM dendrimer complexed the authors tested several generations (0-7) of PAMAM dendrimers complexated with both a GFP siRNA and a GFP mRNA with regard to their PCI-mediated gene modulation efficiency in OHS-EGFP and OHS osteosarcoma cells respectively.…”

“…PAMAM dendrimers have also been successfully shown to carry splice switching oligonucleotides activated by PCI as seen by nuclear splice swithing activity [168]. For peptide vehicles, most of the poly(l-arginines) tested and some poly(l-lysines) showed a considerable PCI effect in s100A4 silencing in OHS cells, while poly(l-histidine) carriers were not so effective [167]. In contrast to the findings by Bøe et al [164], Oliveira and co-workers [169] also demonstrated good effects of PCI for lipofectamine mediated siRNA delivery, and most notably were able to show that this combination also could give strong downregulation of EGF receptor expression in vivo, leading to tumour growth retardation in an EGF dependent tumour model [170].…”

“…Since this vehicle has no endosomolytic properties in itself, in this case PCI could be used for turning mRNA expression on, against a background of virtually no expression without PCI. PCI-mediated mRNA delivery was also investigated with various PAMAM-based formulations, but while such formulations were effective for PCI-mediated siRNA delivery, they did not work with mRNA [167]. In vivo, PCI-mediated mRNA delivery using delivery vehicles have so far largely been unsuccessful, but very promising results have recently been obtained with PCI-mediated delivery of various types of "naked" mRNA molecules (Høgset et al, manuscript in preparation).…”

A combination hepatoma-targeted therapy based on nanotechnology: pHRE-Egr1-HSV-TK/131I-antiAFPMcAb-GCV/MFH

“…Although viral vectors have been proved efficacious, their small gene capacity, poor target specificity, self-immunogenicity, and especially serious biosafety concerns limit their further application. Despite avoiding the major security risks, non-viral vectors such as lipofection, polyamidoamine 26 , poly(ethyleneimine) 27 , and poly(amino acid) 28 are greatly inferior to viral vectors in transfection efficiency, and meaningful expression of target gene is hardly available in this system. Therefore, the gene delivery remains a challenge because of the lack of suitable vectors.…”