A ß-Cyclodextrin-Based Nanoparticle with Very High Transfection Efficiency Unveils siRNA-Activated TLR3 Responses in Human Prostate Cancer Cells.

Synthetic double-stranded small interfering RNAs (siRNAs) mimic interference RNAs (RNAi) and can bind target mRNAs with a high degree of specificity, leading to selective knockdown of the proteins they encode. However, siRNAs are very labile and must be both protected and transported by nanoparticles to be efficiently delivered into cells. In this work, we used a Janus-type polycationic amphiphilic ß-cyclodextrin derivative to efficiently transfect siRNAs targeting mRNAs encoding mitogen-activated protein kinase (p42-MAPK) or Ras homolog enriched in brain (Rheb) into different cancer cell lines as well as astrocytes. We took advantage of this high transfection efficiency to simultaneously knock down p42-MAPK and Rheb to boost docetaxel (DTX)-mediated toxicity in two human prostate cancer cell lines (LNCaP and PC3). We found that double knockdown of p42-MAPK and Rheb increased DTX-toxicity in LNCaP but not in PC3 cells. However, we also observed the same effect when scramble siRNA was used, therefore pointing to an off-target effect. Indeed, we found that the siRNA we used in this work induced toll-like receptor 3 activation, leading to ß-interferon production and caspase activation. We believe that this mechanism could be very useful as a general strategy to elicit an immune response against prostate cancer cells.

Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system.

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(ß-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

Cyclodextrin-Based Nanostructure Efficiently Delivers siRNA to Glioblastoma Cells Preferentially via Macropinocytosis.

Small interfering ribonucleic acid (siRNA) has the potential to revolutionize therapeutics since it can knockdown very efficiently the target protein. It is starting to be widely used to interfere with cell infection by HIV. However, naked siRNAs are unable to get into the cell, requiring the use of carriers to protect them from degradation and transporting them across the cell membrane. There is no information about which is the most efficient endocytosis route for high siRNA transfection efficiency. One of the most promising carriers to efficiently deliver siRNA are cyclodextrin derivatives. We have used nanocomplexes composed of siRNA and a ß-cyclodextrin derivative, AMC6, with a very high transfection efficiency to selectively knockdown clathrin heavy chain, caveolin 1, and p21 Activated Kinase 1 to specifically block clathrin-mediated, caveolin-mediated and macropinocytosis endocytic pathways. The main objective was to identify whether there is a preferential endocytic pathway associated with high siRNA transfection efficiency. We have found that macropinocytosis is the preferential entry pathway for the nanoparticle and its associated siRNA cargo. However, blockade of macropinocytosis does not affect AMC6-mediated transfection efficiency, suggesting that macropinocytosis blockade can be functionally compensated by an increase in clathrin- and caveolin-mediated endocytosis.

Endocytosis: The Nanoparticle and Submicron Nanocompounds Gateway into the Cell.

Nanoparticles (NPs) and submicron particles are increasingly used as carriers for delivering therapeutic compounds to cells. Their entry into the cell represents the initial step in this delivery process, being most of the nanoparticles taken up by endocytosis, although other mechanisms can contribute to the uptake. To increase the delivery efficiency of therapeutic compounds by NPs and submicron particles is very relevant to understand the mechanisms involved in the uptake process. This review covers the proposed pathways involved in the cellular uptake of different NPs and submicron particles types as well as the role that some of the physicochemical nanoparticle characteristics play in the uptake pathway preferentially used by the nanoparticles to gain access and deliver their cargo inside the cell.

Evaluation of Amino-Functional Polyester Dendrimers Based on Bis-MPA as Nonviral Vectors for siRNA Delivery.

Herein, we present the first evaluation of cationic dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) as nonviral vectors for transfection of short interfering RNA (siRNA) in cell cultures. The study encompassed dendrimers of generation one to four (G1⁻G4), modified to bear 6⁻48 amino end-groups, where the G2⁻G4 proved to be capable of siRNA complexation and protection against RNase-mediated degradation. The dendrimers were nontoxic to astrocytes, glioma (C6), and glioblastoma (U87), while G3 and G4 exhibited concentration dependent toxicity towards primary neurons. The G2 showed no toxicity to primary neurons at any of the tested concentrations. Fluorescence microscopy experiments suggested that the dendrimers are highly efficient at endo-lysosomal escape since fluorescently labeled dendrimers were localized specifically in mitochondria, and diffuse cytosolic distribution of fluorescent siRNA complexed by dendrimers was observed. This is a desired feature for intracellular drug delivery, since the endocytic pathway otherwise transfers the drugs into lysosomes where they can be degraded without reaching their intended target. siRNA-transfection was successful in C6 and U87 cell lines using the G3 and G4 dendrimers followed by a decrease of approximately 20% of target protein p42-MAPK expression.

Molecular determinants for cyclo-oligosaccharide-based nanoparticle-mediated effective siRNA transfection.

AIM: To study the structural requirements that a cyclooligosaccharide-based nanoparticle must fulfill to be an efficient siRNA transfection vector. MATERIALS & METHODS: siRNA protection from degradation by RNAses, transfection efficiency and the thermodynamic parameters of the nanoparticle/siRNA interactions were studied on pairs of amphiphilic molecules using biochemical techniques and molecular dynamics. RESULTS: The lower the siRNA solvent accessible surface area in the presence of the nanoparticle, higher the protection from RNAse-mediated degradation in the corresponding nanocomplex; a moderate nanoparticle/siRNA binding energy value further facilitates reversible complexation and binding to the target cellular mRNA. CONCLUSION: The use, in advance, of these parameters will provide a useful indication of the potential of a molecular nanoparticle as siRNA transfecting vector.