Self-Assembled Cationic ß-Cyclodextrin Nanostructures for siRNA Delivery.

Functionalized cyclodextrin molecules assemble into a wide variety of superstructures in solution, which are of interest for drug delivery and other nanomaterial and biomaterial applications. Here we use a combined simulation and experimental approach to probe the coassembly of siRNA and cationic cyclodextrin (c-CD) derivatives into a highly stable gene delivery nanostructure. The c-CD form supramolecular structures via interdigitation of their aliphatic tails, analogous to the formation of lipid bilayers and micelles. The native conformation of siRNA is preserved by the encapsulating c-CD superstructure in an extensive hydrogen-bonding network between the positively charged side arms of c-CD and the negatively charged siRNA backbone. The stability of the complexation is confirmed using isothermal titration calorimetry, and the experimental/simulation codesign methodology opens new avenues for creation of highly engineerable gene delivery vectors.

Cyclodextrin-siRNA conjugates as versatile gene silencing agents.

Functional siRNAs (luciferase and PLK1) have been conjugated to ß-cyclodextrin and the ability of the conjugates to retain gene knockdown activity has been assessed by delivery to cancer cell lines using various formulations. Initially two formulations used complexation with polycations, namely Lipofectamine 2000 and an amphiphilic polycationic cyclodextrin. Gene knockdown results for human glioblastoma cells (U87) and prostate cancer cells (PC3, DU145) showed that conjugation to the cyclodextrin did not reduce gene silencing by the RNA. A third mode of delivery involved formation of targeted nanoparticles in which the conjugate was first complexed with adamantyl-PEG-ligands (targeting ligand RVG peptide or dianisamide) by adamantyl inclusion in the cyclodextrin cavities of the conjugates, followed by charge neutralisation with the cationic polymer chitosan. Enhanced knockdown was achieved by these ligand-targeted formulations. In summary, while this study illustrated the gene silencing efficacy of a simple cyclodextrin-siRNA conjugate it is envisaged that future studies will explore the use of conjugates with a modified cyclodextrin which would be self-delivering. Detailed data such as stability, lysosomal escape etc. will then be reported for each conjugate, since this will be appropriate for conjugates which are intended to exploit, rather than merely demonstrate, the concept. The present paper was intended to demonstrate the viability and generality of this novel concept.

Folate-targeted amphiphilic cyclodextrin nanoparticles incorporating a fusogenic peptide deliver therapeutic siRNA and inhibit the invasive capacity of 3D prostate cancer tumours.

The main barrier to the development of an effective RNA interference (RNAi) therapy is the lack of a suitable delivery vector. Modified cyclodextrins have emerged in recent years for the delivery of siRNA. In the present study, a folate-targeted amphiphilic cyclodextrin was formulated using DSPE-PEG5000-folate to target prostate cancer cells. The fusogenic peptide GALA was included in the formulation to aid in the endosomal release of siRNA. Targeted nanoparticles were less than 200nm in size with a neutral surface charge. The complexes were able to bind siRNA and protect it from serum nucleases. Incubation with excess free folate resulted in a significant decrease in the uptake of targeted nanoparticles in LNCaP and PC3 cells, both of which have been reported to have differing pathways of folate uptake. There was a significant reduction in the therapeutic targets, ZEB1 and NRP1 at mRNA and protein level following treatment with targeted complexes. In preliminary functional assays using 3D spheroids, treatment of PC3 tumours with targeted complexes with ZEB1 and NRP1 siRNA resulted in more compact colonies relative to the untargeted controls and inhibited infiltration into the Matrigel™ layer.

Antibody-Targeted Cyclodextrin-Based Nanoparticles for siRNA Delivery in the Treatment of Acute Myeloid Leukemia: Physicochemical Characteristics, in Vitro Mechanistic Studies, and ex Vivo Patient Derived Therapeutic Efficacy.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with high relapse rates. It is known that leukemia stem cells (LSCs), a very small subpopulation of the total number of leukemic cells, maintain the leukemia phenotype (∼80-90% of AML remain the same as at first diagnosis), display chemotherapy resistance, and contribute to disease regeneration. Therefore, targeting LSCs could control the relapse of AML. Small interfering RNA (siRNA), an effector of the RNA interference (RNAi) pathway, can selectively downregulate any gene implicated in the pathology of disease, presenting great potential for treatment of AML. In this study an antibody targeted cyclodextrin-based nanoparticle (NP) (CD.DSPE-PEG-Fab) was developed for siRNA delivery specifically to AML LSCs. The targeted CD.siRNA.DSPE-PEG-Fab formulation, where Fab specifically targets the IL-3 receptor α-chain (IL-3Rα, also known as CD123, a cell surface antigen for human AML LSCs), achieved antigen-mediated cellular uptake in KG1 cells (an AML leukemia stem and progenitor cell line). Efficient delivery of bromodomain-containing protein 4 (BRD4) siRNA using the targeted formulation resulted in downregulation of the corresponding mRNA and protein in KG1 cells and in ex vivo primary AML patient derived samples. The resulting silencing of BRD4 induced myeloid differentiation and triggered leukemia apoptosis. In addition, a synergistic therapeutic effect was detected when administered in combination with the chemotherapeutic, cytarabine (Ara-C). These results indicate the clinical potential of the antibody-tagged cyclodextrin NP for targeted delivery of therapeutic siRNA in the treatment of AML.

Formulation and Evaluation of Anisamide-Targeted Amphiphilic Cyclodextrin Nanoparticles To Promote Therapeutic Gene Silencing in a 3D Prostate Cancer Bone Metastases Model.

In recent years, RNA interference (RNAi) has emerged as a potential therapeutic offering the opportunity to treat a wide range of diseases, including prostate cancer. Modified cyclodextrins have emerged as effective gene delivery vectors in a range of disease models. The main objective of the current study was to formulate anisamide-targeted cyclodextrin nanoparticles to interact with the sigma receptor (overexpressed on the surface of prostate cancer cells). The inclusion of octaarginine in the nanoparticle optimized uptake and endosomal release of siRNA in two different prostate cancer cell lines (PC3 and DU145 cells). Resulting nanoparticles were less than 200 nm in size with a cationic surface charge (∼+20 mV). In sigma receptor-positive cell lines, the uptake of anisamide-targeted nanoparticles was reduced in the presence of the sigma receptor competitive ligand, haloperidol. When cells were transfected in 2D, the levels of PLK1 mRNA knockdown elicited by targeted versus untargeted nanoparticles tended to be greater but the differences were not statistically different. In contrast, when cells were grown on 3D scaffolds, recapitulating bone metastasis, targeted formulations showed significantly higher levels of PLK1 mRNA knockdown (46% for PC3 and 37% for DU145, p < 0.05). To our knowledge, this is the first time that a targeted cyclodextrin has been used to transfect prostate cancer cells in a 3D model of bone metastasis.

Nanoparticle-mediated siRNA delivery assessed in a 3D co-culture model simulating prostate cancer bone metastasis.

siRNA has emerged as a potential therapeutic for the treatment of prostate cancer but effective delivery remains a major barrier to its clinical application. This study aimed to develop and characterise a 3D in vitro co-culture model to simulate prostate cancer bone metastasis and to assess the ability of the model to investigate nanoparticle-mediated siRNA delivery and gene knockdown. PC3 or LNCaP prostate cancer cells were co-cultured with hFOB 1.19 osteoblast cells in 2D on plastic tissue culture plates and in 3D on collagen scaffolds mimicking the bone microenvironment. To characterise the co-culture model, cell proliferation, enzyme secretion and the utility of two different gene delivery vectors to mediate siRNA uptake and gene knockdown were assessed. Cell proliferation was reduced by∼50% by day 7 in the co-culture system relative to monoculture (PC3 and LNCaP co-cultures, in 2D and 3D) and an enhanced level of MMP9 (a marker of bone metastasis) was secreted into the media (1.2-4-fold increase depending on the co-culture system). A cationic cyclodextrin gene delivery vector proved significantly less toxic in the co-culture system relative to the commercially available vector Lipofectamine 2000(®). In addition, knockdown of both the GAPDH gene (minimum 15%) and RelA subunit of the NF-κB transcription factor (minimum 20%) was achieved in 2D and 3D cell co-cultures. Results indicate that the prostate cancer-osteoblast in vitro co-culture model was more physiologically relevant vs the monoculture. This model has the potential to help improve the design and efficacy of gene delivery formulations, to more accurately predict in vivo performance and, therefore, to reduce the risk of product failure in late-stage clinical development.

Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications.

Folate-targeted amphiphilic cyclodextrin.siRNA nanoparticles for prostate cancer therapy exhibit PSMA mediated uptake, therapeutic gene silencing in vitro and prolonged circulation in vivo.

In this study, a folate targeted cyclodextrin (CD) nanoparticle was prepared by co-formulating CD.siRNA complexes with DSPE-PEG5000-folate to target the prostate specific membrane antigen (PSMA). Targeted formulations showed increased uptake, relative to untargeted controls, in two prostate cancer cell lines expressing PSMA (VCaP and LNCaP). Competitive uptake studies, using excess folate, significantly reduced uptake of targeted nanoparticles in PSMA positive cell lines (P<0.001). Relative to untreated controls, folate-targeted nanoparticles significantly reduced the levels of RelA mRNA in VCaP and LNCaP cells by 44% and 22% respectively (P<0.001). In contrast there was no significant reduction in RelA mRNA in these cell lines by untargeted complexes. Pharmacokinetic (PK) data indicated that the incorporation of PEG into the formulation increased the circulation time of siRNA 8-fold. This study highlights the ability of incorporating a folate ligand into CD.siRNA nanoparticles to allow for targeted delivery of siRNA to prostate cancer cells via the PSMA.

A novel, anisamide-targeted cyclodextrin nanoformulation for siRNA delivery to prostate cancer cells expressing the sigma-1 receptor.

Prostate cancer is a leading cause of cancer-related death in men and RNA interference (RNAi) has emerged as a potential therapeutic option. However, the absence of a safe and specific delivery vector remains a major obstacle to the clinical application of RNAi. Cyclodextrin derivatives are known to be efficient delivery systems with low toxicity in a variety of cell types. In this study, a cationic cyclodextrin derivative was synthesized to complex siRNA. The nanoparticle was then further modified by exploiting the ability of the ß-cyclodextrin cavity to form an inclusion complex with the hydrophobic molecule adamantane. PEGylated adamantane derivatives were synthesized with and without the anisamide-targeting ligand on the terminal end of the PEG chain. Anisamide is known to bind specifically to the sigma receptor which is overexpressed on the surface of prostate cancer cells. The resulting nanocomplexes were slightly cationic and less than 300 nm in size. They successfully protected siRNA from serum-induced nuclease degradation and were non-toxic to prostate cancer cells. In addition, the targeted nanoparticles mediated high levels of siRNA cellular uptake and corresponding PLK1 gene knockdown in prostate cancer cells in vitro. To our knowledge, this is the first time that the ability of cyclodextrins to form inclusion complexes with adamantane derivatives has been exploited for the targeted delivery of siRNA to prostate cancer cells via the sigma receptor.

The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics.

Prostate cancer bone metastases are a leading cause of cancer-related death in men with current treatments offering only marginally improved rates of survival. Advances in the understanding of the genetic basis of prostate cancer provide the opportunity to develop gene-based medicines capable of treating metastatic disease. The aim of this work was to establish a 3D cell culture model of prostate cancer bone metastasis using collagen-based scaffolds, to characterise this model, and to assess the potential of the model to evaluate delivery of gene therapeutics designed to target bone metastases. Two prostate cancer cell lines (PC3 and LNCaP) were cultured in 2D standard culture and compared to 3D cell growth on three different collagen-based scaffolds (collagen and composites of collagen containing either glycosaminoglycan or nanohydroxyapatite). The 3D model was characterised for cell proliferation, viability and for matrix metalloproteinase (MMP) enzyme and Prostate Specific Antigen (PSA) secretion. Chemosensitivity to docetaxel treatment was assessed in 2D in comparison to 3D. Nanoparticles (NPs) containing siRNA formulated using a modified cyclodextrin were delivered to the cells on the scaffolds and gene silencing was quantified. Both prostate cancer cell lines actively infiltrated and proliferated on the scaffolds. Cell culture in 3D resulted in reduced levels of MMP1 and MMP9 secretion in PC3 cells. In contrast, LNCaP cells grown in 3D secreted elevated levels of PSA, particularly on the scaffold composed of collagen and glycosaminoglycans. Both cell lines grown in 3D displayed increased resistance to docetaxel treatment. The cyclodextrin.siRNA nanoparticles achieved cellular uptake and knocked down the endogenous GAPDH gene in the 3D model. In conclusion, development of a novel 3D cell culture model of prostate cancer bone metastasis has been initiated resulting, for the first time, in the successful delivery of gene therapeutics in a 3D in vitro model. Further enhancement of this model will help elucidate the pathogenesis of prostate cancer and also accelerate the design of effective therapies which can penetrate into the bone microenvironment for prostate cancer therapy.

Synthesis and characterization of rabies virus glycoprotein-tagged amphiphilic cyclodextrins for siRNA delivery in human glioblastoma cells: in vitro analysis.

In man brain cancer is an aggressive, malignant form of tumour, it is highly infiltrative in nature, is associated with cellular heterogeneity and affects cerebral hemispheres of the brain. Current drug therapies are inadequate and an unmet clinical need exists to develop new improved therapeutics. The ability to silence genes associated with disease progression by using short interfering RNA (siRNA) presents the potential to develop safe and effective therapies. In this work, in order to protect the siRNA from degradation, promote cell specific uptake and enhance gene silencing efficiency, a PEGylated cyclodextrin (CD)-based nanoparticle, tagged with a CNS-targeting peptide derived from the rabies virus glycoprotein (RVG) was formulated and characterized. The modified cyclodextrin derivatives were synthesized and co-formulated to form nanoparticles containing siRNA which were analysed for size, surface charge, stability, cellular uptake and gene-knockdown in brain cancer cells. The results identified an optimised co-formulation prototype at a molar ratio of 1:1.5:0.5 (cationic cyclodextrin:PEGylated cyclodextrin:RVG-tagged PEGylated cyclodextrin) with a size of 281 ± 39.72 nm, a surface charge of 26.73 ± 3 mV, with efficient cellular uptake and a 27% gene-knockdown ability. This CD-based formulation represents a potential nanocomplex for systemic delivery of siRNA targeting brain cancer.

PEGylated cyclodextrins as novel siRNA nanosystems: correlations between polyethylene glycol length and nanoparticle stability.

Silencing disease-related genes in the central nervous system (CNS) using short interfering RNA (siRNA) holds great promise for treating neurological disorders. Yet, delivery of RNAi therapeutics to the brain poses major challenges to non-viral systems, especially when considering systemic administration. Cationic nanoparticles have been widely investigated for siRNA delivery, but the tendency of these to aggregate in physiological environments limits their intravenous application. Thus, strategies to increase the stability of nanoparticles have been developed. Here, we investigated the ability of modified cationic amphiphilic or PEGylated amphiphilic cyclodextrins (CD) to formulate stable CD.siRNA nanoparticles. To this end, we describe a simple method for post-modification of pre-formed cationic CD.siRNA nanoparticles at their surface using PEGylated CDs of different PEG lengths. PEGylated CD.siRNA nanoparticles presented reduced surface charges and increased stability in physiological salt conditions. Stability of PEGylated CD.siRNA nanoparticles in vitro increased with both PEG length and PEG density at the surface. Furthermore, in a comparative pharmacokinetic study, increased systemic exposure and reduced clearance were achieved with CD-formulations when compared to naked siRNAs. However, no significant differences were observed among non-PEGylated and PEGylated CD.siRNAs suggesting that longer PEG lengths might be required for improving stability in vivo.

Biophysical and structural characterisation of nucleic acid complexes with modified cyclodextrins using circular dichroism.

Modified cyclodextrins (CDs) have shown great promise as non-viral gene and siRNA delivery vectors in a range of in vitro and in vivo studies. In the current study, structural and biophysical characterisation of selected CDs was carried out to enhance our understanding of their interaction with nucleic acids. The methods used for such characterisation were dynamic light scattering, zeta potential measurements and circular dichroism. Variations in the chemistries of individual CDs and in the type of formulation were shown to affect key properties of complexes such as size, surface charge and nucleic acid conformation. Furthermore, the effects of temperature and pH on the conformation of nucleic acids were investigated. pH studies were intended to mimic the conditions encountered by cationic complexes during endocytosis. Circular dichroism studies revealed that changes occurred in DNA and siRNA conformation upon complexation with CDs and when exposed to increasing temperature and decreasing pH. Overall, siRNA appeared to be more susceptible to conformational changes although complexation of siRNA with CDs tended to have a stabilising effect.

Differential nanotoxicological and neuroinflammatory liabilities of non-viral vectors for RNA interference in the central nervous system.

Progression of RNA interference-based gene silencing technologies for the treatment of disorders of the central nervous system (CNS) depends on the availability of efficient non-toxic nanocarriers. Despite advances in the field of nanotechnology undesired and non-specific interactions with different brain-cell types occur and are poorly investigated. To this end, we studied the cytotoxic and neuroinflammatory effects of widely-used transfection reagents and modified amphiphilic ß-cyclodextrins (CDs). All non-viral vectors formed positively charged nanoparticles with distinctive physicochemical properties. Differential and significant cytotoxic effects were observed among commercially available cationic vectors, whereas CDs induced limited disruptions of cellular membrane integrity and mitochondrial dehydrogenase activity. Interestingly, murine derived BV2 microglia cells and a rat striatal in vitro model of Huntington's disease (ST14A-HTT120Q) were more susceptible to toxicity than human U87 astroglioma cells. BV2 microglia presented significant increases in cytokine, toll-like receptor 2 and cyclooxygenase-2 gene expression after transfection with selected commercial vectors but not with CD.siRNA nanoparticles. Non-viral siRNA nanoparticles formulated with G6 polyamidoamine (PAMAM) also significantly increased cytokine gene expression in the brain following injections into the mouse striatum. Together our data identify modified CDs as nanosystems that enable siRNA delivery to the brain with low levels of cytotoxicity and immunological activation.

Gastrointestinal gene delivery by cyclodextrins--in vitro quantification of extracellular barriers.

Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers. Stability and transfection efficiency of CD·DNA complexes were assessed post incubation in simulated gastric and intestinal fluids, bile salts and mucin, or with intestinal enzymes to represent extracellular barriers to intestinal gene delivery. Stability was determined by gel electrophoresis and transfection was measured by luciferase expression in intestinal epithelial cells (Caco-2). Transfection efficiency of CD·DNA complexes was enhanced after incubation in bile salts but was reduced after incubation in gastric and intestinal fluids and mucin. CD·DNA complexes were stable after incubation with pancreatic enzymes and with a model lower intestinal enzyme. Furthermore, the CD protected pDNA from degradation by DNase. In summary, physiologically relevant in vitro models were established and used to quantify the barriers posed by the intestinal extracellular environment to gene delivery. This systematic assessment identified the advantages and limitations of the CD vector and facilitated the proposal of formulation strategies to overcome these barriers.

Cationic and PEGylated Amphiphilic Cyclodextrins: Co-Formulation Opportunities for Neuronal Sirna Delivery.

Optimising non-viral vectors for neuronal siRNA delivery presents a significant challenge. Here, we investigate a co-formulation, consisting of two amphiphilic cyclodextrins (CDs), one cationic and the other PEGylated, which were blended together for siRNA delivery to a neuronal cell culture model. Co-formulated CD-siRNA complexes were characterised in terms of size, charge and morphology. Stability in salt and serum was also examined. Uptake was determined by flow cytometry and toxicity was measured by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Incorporation of a PEGylated CD in the formulation had significant effects on the physical and biological properties of CD.siRNA complexes. Co-formulated complexes exhibited a lower surface charge and greater stability in a high salt environment. However, the inclusion of the PEGylated CD also dramatically reduced gene silencing efficiency due to its effects on neuronal uptake. The co-formulation strategy for cationic and PEGylated CDs improved the stability of the CD.siRNA delivery systems, although knockdown efficiency was impaired. Future work will focus on the addition of targeting ligands to the co-formulated complexes to restore transfection capabilities.

Nanostructures of cationic amphiphilic cyclodextrin complexes with DNA.

Complexes of cationic amphiphilic cyclodextrins heptakis[2-(ω-amino-oligo(ethylene glycol))-6-deoxy-6-hexadecylthio]-ß-cyclodextrin and heptakis[2-(ω-amino-oligo(ethylene glycol))-6-deoxy-6-dodecylthio]-ß-cyclodextrin with DNA were examined by small-angle X-ray scattering and dynamic as well as electrophoretic light scattering. The first cyclodextrin forms bilayer vesicles in water, which, in the presence of calf thymus DNA, transform to a multilamellar complex. In this complex, the DNA lies between the two polar layers of the cyclodextrin's protonated amino groups in alternation with the lipidic bilayers. The cyclodextrin with shorter lipid chains, in contrast, forms micelles in water, and electrostatic clustering of these about DNA does not affect their intrinsic structure. These results are relevant to the potential of such cyclodextrins in therapeutic gene delivery, showing that their self-assembly modes in isolation influence their complex formation with DNA and possibly their efficiency in promoting cell transfection.

Self-assembling modified ß-cyclodextrin nanoparticles as neuronal siRNA delivery vectors: focus on Huntington's disease.

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a toxic Huntingtin (HTT) protein. The use of short interfering RNAs (siRNAs) to silence the mutant protein is one of the most promising therapeutic strategies under investigation. The biggest caveat to siRNA-based approaches is the lack of efficient and nontoxic delivery vectors for siRNA delivery to the central nervous system. In this study, we investigated the potential of modified amphiphilic ß-cyclodextrins (CDs), oligosaccharide-based molecules, as novel siRNA neuronal carriers. We show that CDs formed nanosize particles which were stable in artificial cerebrospinal fluid. Moreover, these complexes were able to reduce the expression of the HTT gene in rat striatal cells (ST14A-HTT120Q) and in human HD primary fibroblasts. Only limited toxicity was observed with CD·siRNA nanoparticles in any of the in vitro models used. Sustained knockdown effects were observed in the striatum of the R6/2 mouse model of HD after single direct injections of CD·siRNA nanoparticles. Repeated brain injections of CD·siRNA complexes resulted in selective alleviation of motor deficits in this mouse model. Together these data support the utility of modified ß-CDs as efficient and safe siRNA delivery vectors for RNAi-based therapies for neuropsychiatric and neurodegenerative disorders.

In vitro investigations of the efficacy of cyclodextrin-siRNA complexes modified with lipid-PEG-Octaarginine: towards a formulation strategy for non-viral neuronal siRNA delivery.

PURPOSE: Development of RNA interference based therapeutics for neurological and neurodegenerative diseases is hindered by a lack of non-viral vectors with suitable properties for systemic administration. Amphiphilic and cationic cyclodextrins (CD) offer potential for neuronal siRNA delivery. We aimed to improve our CD-based siRNA formulation through incorporation of a polyethyleneglycol (PEG) shielding layer and a cell penetrating peptide, octaarginine (R8). METHODS: CD.siRNA complexes were modified by addition of an R8-PEG-lipid conjugate. Physical properties including size, charge and stability were assessed. Flow cytometry was used to determine uptake levels in a neuronal cell model. Knockdown of an exogenous gene and an endogenous housekeeping gene were used to assess gene silencing abilities. RESULTS: CD.siRNA complexes modified with R8-PEG-lipid exhibited a lower surface charge and greater stability to a salt-containing environment. Neuronal uptake was increased and significant reductions in the levels of two target genes were achieved with the new formulation. However, the PEG layer was not sufficient to protect against serum-induced aggregation. CONCLUSIONS: The R8-PEG-lipid-CD.siRNA formulation displayed enhanced salt-stability due to the PEG component, while the R8 component facilitated transfection of neuronal cells and efficient gene silencing. Further improvements will be investigated in the future in order to optimise stability in serum and enhance neuronal specificity.

Anisamide-targeted cyclodextrin nanoparticles for siRNA delivery to prostate tumours in mice.

A hepta-guanidino-ß-cyclodextrin (G-CD), its hepta-PEG conjugate (G-CD-PEG), and the corresponding anisamide-terminated PEG conjugate (G-CD-PEG-AA) have been synthesised and compared as delivery vectors for siRNA to prostate cancer cells and tumours in vivo. The G-CD-PEG-AA.siRNA formulations (in which anisamide targets the sigma receptor), but not the non-targeted formulations, induced prostate cell-specific internalisation of siRNA resulting in approximately 80% knockdown in vitro of the reporter gene, luciferase. Following intravenous administration of the anisamide-targeted formulation in a mouse prostate tumour model significant tumour inactivation with corresponding reductions in the level of vascular endothelial growth factor (VEGF) mRNA were achieved, without demonstrating enhanced toxicity. This data imply significant potential for anisamide-conjugated cyclodextrin vectors for targeted delivery of therapeutic siRNAs in the treatment of prostate cancer.