Lipoprotein-based Nanoplatform For Cellular Sirna Delivery

Although small interfering RNA (siRNA) can silence the expression of disease-related genes, delivery of these highly charged molecules is challenging. Delivery approaches for siRNA are actively being pursued and improved strategies are required for non-toxic and efficient delivery for gene knockdown. Lipoproteins (LDL and HDL) are natural and endogenous nanoparticles that have a rich history as delivery vehicles, while lipoprotein mimetics are novel nanocarriers that can be clinically translationable. This study examined LDL nanoparticles and HDL mimicking peptide-phospholipid scaffold (HPPS) as nanocarriers for siRNA delivery and overcome the siRNA delivery problems, including targeting delivery and cytosolic release.1) We used purified LDL nanoparticles as carriers for functional siRNA delivery. When siRNA was covalently conjugated to cholesterol, over 25 chol-siRNAs could be incorporated onto each LDL without changing nanoparticle morphology. The resulting LDL-chol-siRNAs nanoparticles were selectively taken up into cells via LDL receptor mediated endocytosis, resulting in enhanced gene silencing compared to free chol-siRNA (39% gene knock down versus 0% knock down at 100 nM). However, silencing efficiency was limited by the receptor-mediated entrapment of the LDL-chol-siRNA nanoparticles in endosomes. Photochemical internalization demonstrated that endolysosome disruption strategies significantly enhance LDL-mediated gene silencing.2) We introduce the cytosolic release of Low density lipoprotein nanoparticles (LNPs) loaded cargos using photochemical internalization (PCI) approach. Three types of fluorescent dyes mimicking the therapeutics were loaded onto LNPs via various approaches, including intercalation in the phospholipids monolayer exterior (surface loading), conjugation to the amino acids of apoB-100 protein (protein labeling) or reconstitution into the lipid core of LDL (core loading). Fluorescence imaging demonstrated the cellular uptake of cargo-loaded LDL nanoparticles (CLNPs) was through LDL receptor (LDLR) mediated endocytosis in the lung cancer A549 cells. When the PCI was performed, the measurement of fluorescence changing folds before and post irradiation showed a varying degree of cargo release, a large amount of DiI release, parts of FITC release and seldom Fluo-BOA release. Thus, PCI is well-suited to improve LDL mediated siRNA delivery efficiency. Together, this study demonstrated the effectiveness of cytosolic release of LDL loaded cargos was loading methods dependent, as well as providing insight into the design of drug release using PCI.3) We report a novel approach to efficiently deliver siRNA into the cytosol of cancer cells via the scavenger receptor class B typeâ… (SRB1) pathway. This was accomplished by using a HDL-mimicking peptide-phospholipid scaffold (HPPS) as a nanocarrier and cholesterol-modified siRNA targeting bcl-2 gene (chol-si-bcl-2) as the siRNA payload. Eight chol-si-bcl-2 molecules were stably incorporated into each HPPS particle while maintaining its structural integrity and homogenity. Bcl-2 protein expression was silenced by the resulting HPPS-chol-si-bcl-2 in SRB1 over-expressing cells but not in SRB1 low-expressing cells. Direct cytosolic transport of HPPS-chol-si-bcl-2 was confirmed by confocal imaging and subcellular fractionation assay. Compared with chol-si-bcl-2 alone, HPPS-chol-si-bcl-2 down-regulated Bcl-2 expression by 4.8-fold and increased apoptosis by 2.5-fold. The direct cytosolic delivery mechanism and biocompatibility of HPPS are uniquely suited for the delivery of intracellular active RNAi therapeutics.4) We use a fluorescence imaging approach to show that HPPS conjugated to epidermal growth factor (EGF-HPPS) are able to specifically recognize and target lung cancer cells expressing a GFP-labeled epidermal growth factor receptor (EGFR-GFP). This study provides a framework for evaluation and validation of specific nanoparticle targeting ligands using fluorescence imaging. Here we investigated the use of lipoproteins and lipoprotein mimetics as carriers for siRNA delivery. By the use of PCI and the specific delivery mechanism of SRB1, the cytosolic delivery of siRNA was achieved for LDL and HPPS, respectively, making them useful tools for the cytosolic delivery of siRNAs, as well as providing potential opportunities for the gene therapy using siRNAs. Using a dual fluorescent imaging technique, we validated the specific targeting ability of EGF-HPPS, which would dramatically extend the application of HPPS in cancer therapy. In addition, it provided a useful technique for the validation of the influence of unintended targets on the desired ligand-receptor interaction.