Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth

The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for patholological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.     

Liposomal systems as viable drug delivery technology for skin cancer sites with an outlook on lipid-based delivery vehicles and diagnostic imaging inputs for skin conditions'

Skin cancer is among one of the most common human malignancies wide-spread world-over with mortality statistics rising continuously at an alarming rate. The increasing frequency of these malignancies has marked the need for adopting effective treatment plan coupled with better and site-specific delivery options for the desired therapeutic agent's availability at the affected site. The concurrent delivery approaches to cancerous tissues are under constant challenge and, as a result, are evolving and gaining advancements in terms of delivery modes, therapeutic agents and site-specificity of the therapeutics delivery. The lipid-based liposomal drug delivery is an attractive and emerging option, and which is meticulously shaping up beyond a threshold level to a promising, and viable route for the effective delivery of therapeutic agents and other required injuctions to the skin cancer. An update on liposomal delivery of chemotherapeutic agents, natural-origin compounds, photosensitizer, and DNA repair enzymes as well as other desirable and typical delivery modes employed in drug delivery and in the treatment of skin cancers is discussed in details. Moreover, liposomal delivery of nucleic acid-based therapeutics, i.e., small interfering RNA (siRNA), mRNA therapy, and RGD-linked liposomes are among the other promising novel technology under constant development. The current clinical applicability, viable clinical plans, future prospects including transport feasibility of delivery vesicles and imaging techniques in conjunction with the therapeutic agents is also discussed. The ongoing innovations in liposomal drug delivery technology for skin cancers hold promise for further development of the methodology for better, more effective and site-specific delivery as part of the better treatment plan by ensuring faster drug transport, better and full payload delivery with enough and required concentration of the dose.     

Opinion: miRNAs – The new wave of molecular cancer therapeutics

Cutting-edge advances in nanomedicine and the recent approval of two siRNA-based therapeutics by the Food and Drug Administration (FDA) has rekindled the interest in RNA interference (RNAi) as vehicles for the development of novel cancer therapeutics. In this perspective, we will briefly discuss how miRNAs are becoming the next-generation RNAi therapeutic, the advances in delivery vehicles for in vivo miRNA delivery, and where miRNA technology stands in terms of clinical translation.     

L1 retrotransposon-mediated stable gene silencing

RNA interference (RNAi) is widely used for functional studies and has been proposed as a potential therapeutic agent. Current RNAi systems are largely efficient, but have limitations including transient effect, the need for viral handling and potential insertional mutations. Here, we describe a simple L1 retrotransposon-based system for the delivery of small interfering RNA (siRNA) and stable silencing in human cells. This system demonstrated long-term siRNA expression and significant reduction in both exogenous and endogenous gene expression by up to 90%. Further characterization indicated that retrotransposition occurred in a controlled manner such that essentially only one RNAi-cassette was integrated into the host genome and was sufficient for strong interference. Our system provides a novel strategy for stable gene silencing that is easy and efficient, and it may have potential applications for ex vivo and in vivo molecular therapy.     

Quantification of siRNA using competitive qPCR

We have developed a PCR-based short interfering RNA (siRNA) quantification method based on competition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6 or enhanced green fluorescent protein genes. Having confirmed siRNA could not act as primer for amplicon generation, the lowest competing primer concentration yielding a linear relationship between template DNA amount (0.1–50 ng) and cycle of threshold (Ct) was determined (6.25 nM). Under these conditions addition of sequence-specific siRNA to the competitive quantitative PCR (cqPCR), resulted in a dose-dependent linear increase in Ct value. 2′-O-methyl ribose-modified siRNA retained an ability to inhibit template amplification in serum, unlike unmodified siRNAs that were susceptible to endonucleases. Mismatch-bearing or truncated siRNAs failed to inhibit template amplification confirming sequence specificity and an ability to discriminate between degraded and non-degraded siRNA sequences. Following delivery of E6 siRNA to C33-A cells using oligofectamine or His6 reducible polymers, siRNA uptake was quantified by cqPCR, revealing dose-dependent uptake. We anticipate that cqPCR will allow accurate determination of siRNA pharmacokinetics following in vivo delivery, greatly facilitating development of therapeutic siRNA delivery strategies.     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.     

A rapid, targeted, neuron-selective, in vivo knockdown following a single intracerebroventricular injection of a novel chemically modified siRNA in the adult rat brain

There has been a dramatic expansion of the literature on RNA interference and with it, increasing interest in the potential clinical utility of targeted inhibition of gene expression and associated protein knockdown. However, a critical factor limiting the experimental and therapeutic application of RNA interference is the ability to deliver small interfering RNAs (siRNAs), particularly in the central nervous system, without complications such as toxicity and inflammation. Here we show that a single intracerebroventricular injection of Accell siRNA, a new type of naked siRNA that has been modified chemically to allow for delivery in the absence of transfection reagents, even into differentiated cells such mature neurons, leads to neuron-specific protein knockdown in the adult rat brain. Following in vivo delivery, targeted Accell siRNAs were incorporated successfully into various types of mature neurons, but not glia, for 1 week in diverse brain regions (cortex, striatum, hippocampus, midbrain, and cerebellum) with an efficacy of delivery of approximately 97%. Immunohistochemical and Western blotting analyses revealed widespread, targeted inhibition of the expression of two well-known reference proteins, cyclophilin-B (38-68% knockdown) and glyceraldehyde 3-phosphate dehydrogenase (23-34% knockdown). These findings suggest that this novel procedure is likely to be useful in experimental investigations of neuropathophysiological mechanisms.     

Light-Activatable Gold Nanoshells for Drug Delivery Applications

Gold nanoshells (AuNSs) are currently being investigated as nanocarriers for drug delivery systems and have both diagnostic and therapeutic applications, including photothermal ablation, hyperthermia, drug delivery, and diagnostic imaging, particularly in oncology. AuNSs are valuable for their localized surface plasmon resonance, biocompatibility, low immunogenicity, and facile functionalization. AuNSs used for drug delivery can be spatially and temporally triggered to release controlled quantities of drugs inside the target cells when illuminated with a near-infrared (NIR) laser. Recently, many research groups have demonstrated that these AuNS complexes are able to deliver antitumor drugs (e.g., doxorubicin, paclitaxel, small interfering RNA, and single-stranded DNA) into cancer cells, which enhances the efficacy of treatment. AuNSs can also be functionalized with active targeting ligands such as antibodies, aptamers, and peptides to increase the particles’ specific binding to the desired targets. This article reviews the current research on NIR light-activatable AuNSs used as nanocarriers for drug delivery systems and cancer theranostics.     

A nanoformulation of siRNA and its role in cancer therapy: In vitro and in vivo evaluation

Overexpression of anti-apoptotic Bcl-2 is often observed in a wide variety of human cancers. It prevents the induction of apoptosis in neoplastic cells and contributes to resistance to chemotherapy. RNA interference has emerged as an efficient and selective technique for gene silencing. The potential to use small interfering RNA (siRNA) as a therapeutic agent for the treatment of cancer has elicited a great deal of interest. However, insufficient cellular uptake and poor stability have limited its therapeutic applications. The purpose of this study was to prepare chitosan nanoparticles via ionic gelation of chitosan by tripolyphosphate for effective delivery of siRNA to silence the anti-apoptotic Bcl-2 gene in neoplastic cells. Chitosan nanoparticles loaded with siRNA were in the size range 190 to 340 nm with a polydispersive index ranging from 0.04 to 0.2. They were able to completely bind with siRNA, provide protection against nuclease degradation, and enhance the transfection. Cell culture studies revealed that nanoparticles with entrapped siRNA could efficiently silence the antiapoptotic Bcl-2 gene. Studies on Swiss albino mice showed that siRNA could be effectively delivered through nanoparticles. There was significant decrease in the tumor volume. Blocking the expression of anti-apoptotic Bcl-2 can enhance the sensitivity of cancerous cells to anti-cancer drugs and the apoptosis rate. Therefore, nanoformulations with siRNA can be promoted as an adjuvant therapy in combination with anti-cancer drugs.     

Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.     

A novel delivery platform for therapeutic peptides

Although many peptides have therapeutic effects against diverse disease, their short half-lives in vivo hurdle their application as drug candidates. To extend the short elimination half-lives of therapeutic peptides, we developed a novel delivery platform for therapeutic peptides using an anti-hapten antibody and its corresponding hapten. We selected cotinine because it is non-toxic, has a well-studied metabolism, and is physiologically absent. We conjugated WKYMVm-NH₂, an anti-sepsis therapeutic peptide, to cotinine and showed that the conjugated peptide in complex with an anti-cotinine antibody has a significantly improved in vivo half-life while retaining its therapeutic efficacy. We suggest that this novel delivery platform for therapeutic peptides will be very useful to develop effective peptide therapeutics.     

Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer

Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared.     

Innovative Delivery of siRNA to Solid Tumors by Super Carbonate Apatite

RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.     

EGFP-EGF1-Conjugated PLGA Nanoparticles for Targeted Delivery of siRNA into Injured Brain Microvascular Endothelial Cells for Efficient RNA Interference

Injured endothelium is an important target for drug and/or gene therapy because brain microvascular endothelial cells (BMECs) play critical roles in various pathophysiological conditions. RNA-mediated gene silencing presents a new therapeutic approach for treating such diseases, but major challenge is to ensure minimal toxicity and target delivery of siRNA to injured BMECs. Injured BMECs overexpress tissue factor (TF), which the fusion protein EGFP-EGF1 could be targeted to. In this study, TNF alpha (TNF-α) was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro. The EGFP-EGF1-PLGA nanoparticles (ENPs) with loaded TF-siRNA were used as a new carrier for targeted delivery to the injured BMECs. The nanoparticles then produced intracellular RNA interference against TF. We compared ENP-based transfections with NP-mediated transfections, and our studies show that the ENP-based transfections result in a more efficient downregulation of TF. Our findings also show that the TF siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection while Lipofectamine-based transfections resulted in only 75% of the cells. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and for efficient RNA interference (RNAi). This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer.     

Identification of a novel cell-penetrating peptide from human phosphatidate phosphatase LPIN3

Biomolecules such as proteins, DNA, and RNA are macromolecules and can not cross the cell membrane. However, cell-penetrating peptide (CPP) has been shown to deliver therapeutic biomolecules successfully into cells. The various and widely used CPPs including TAT, VP22, and Antp are mostly non-human originated CPPs, and are limited by their potential toxicity and immunogenicity. We report here on a newly identified novel cell-penetrating sequence (LPIN; RRKRRRRRK) from the nuclear localization sequence (NLS) of human nuclear phosphatase, LPIN3. LPIN-EGFP recombinant protein was concentration- and time-dependently delivered into cells and localized to the nucleus as well as the cytoplasm. It penetrated the cell membrane by lipid raft-mediated endocytosis by binding to heparan sulfate proteoglycan. LPIN-EGFP was successfully delivered into primary mouse splenocytes in vitro and it could be delivered into various tissues including liver, kidney, and intestine in mice after intra-peritoneal injection. This research suggests that LPIN-CPP could be used in a drug delivery system to deliver therapeutic biomolecules including peptides, proteins, DNA, and RNA and without the limitations of non-human originated CPPs such as TAT-CPP.     

An intracellular delivery method for siRNA by an arginine-rich peptide

RNA interference has recently become a useful research tool for the studies of gene functions, regulations, and therapies. The double-stranded RNA is utilized to induce the sequence-specific gene silencing. To achieve this goal of specific gene silencing, a proper delivery system of siRNA is highly demanded. A number of approaches for delivering siRNA have been explored over the last few years. In the present study, we demonstrated a simple peptide-based siRNA delivery system in mammalian cells. A GC-EGFP cell line stably expressing enhanced green fluorescent protein was established from stable transfection of human gastric carcinoma cells. The synthetic nona-arginine peptide, an arginine-rich intracellular delivery peptide, or called protein transduction domain peptide, could noncovalently form stable complexes with EGFP siRNA and deliver these mixtures into cells. After entry, siRNA appeared to stay in perinuclear regions within cell, and ultimately fulfilled its targeted egfp gene silencing. These data were in consonance with that RNA-induced silencing complex components could be also localized to these perinuclear regions, creating a focal point for RNA interference factories. In the future, this non-toxic peptide may be proved to be a useful tool for the delivery of exogenous siRNA in RNA interference research.     

DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.