siRNA Biodistribution Research Articles

Unprotonatable and ROS-Sensitive Nanocarrier for NIR Spatially Activated siRNA Therapy with Synergistic Drug Effect.

Although small interfering RNA (siRNA) therapy has achieved great progress, unwanted gene inhibition in normal tissues severely limits its extensive clinical applications due to uncontrolled siRNA biodistribution. Herein, a spatially controlled siRNA activation strategy is developed to achieve tumor-specific siRNA therapy without gene inhibition in the normal tissues. The quaternary ammonium moieties are conjugated to amphiphilic copolymers via reactive oxygen species (ROS)-sensitive thioketal (TK) linkers for co-delivery of siRNA and photosensitizer chlorin e6 (Ce6), showing excellent siRNA complexation capacity and near infrared (NIR)-controlled siRNA release. In the normal tissue, siRNAs are trapped and degraded in the endo-lysosomes due to the unprotonatable property of quaternary ammonium moiety, showing the siRNA activity "off" state. When NIR irradiation is spatially applied to the tumor tissue, the NIR irradiation/Ce6-induced ROS trigger siRNA endo-lysosomal escape and cytosolic release through the photochemical internalization effect and cleavage of TK bonds, respectively, showing the siRNA activity "on" state. The siRNA-mediated glutathione peroxidase 4 gene inhibition enhances ROS accumulation. The synergistic antitumor activity of Ce6 photodynamic therapy and gene inhibition is confirmed in vivo. Spatially controlled tumor-specific siRNA activation and co-delivery with Ce6 using unprotonatable and ROS-sensitive cationic nanocarriers provide a feasible strategy for tumor-specific siRNA therapy with synergistic drug effects.

Small Interfering RNA (siRNA) of Mini circle DNA Transfection (McD) is Encapsulated by Gold Nanoparticles from Folate’s Receptors of Gold Conjugation as Curative Therapy for Non-Small’s Cell of Lung Cancer

Background: Lung cancer is one of the non-communicable diseases that have an increasing number of events that each year with a mortality rate of 18.4% and an incidence of 11.6%, occupies the top position based on GLOBOCAN data in 2018. One of management in the NSCLC is currently in the form of surgery and adjuvant therapy such as chemotherapy, radiotherapy and target therapy. However, there were weaknesses and limitations in care for its patients therefore development of curative therapy for NSCLC’s patients were needed. The aim of this review is to learn and investigate about the potential of siRNA that is transfected into McD and folate receptors alpha-conjugated gold nanoparticle encapsulation as a therapeutic opportunity that could be developed as a treatment in management of NSCLC.Method: The writing method used in this review article was the study method of literature. The used data comes from 45 of relevant literature sources and was arranged systematically in accordance with the topic of the problem discussed, along with the inclusion and exclusion criteria.Result: This treatment could increase gene transfection, siRNA biodistribution in organs, reduced KRAS, Bcl-2, and VEGF expression. Moreover, siRNA had entered phase III clinical trials and FOLR1 antibodies were in phase II of clinical trials.Conclusion: Utilization of siRNA specifically was designed for three genes such for KRAS, VEGF and Bcl-2 which were three genes that played a role in the pathogenesis of NSCLC, could be the right modality choice for treatment of NSCLC.

Effects of PEG anchors in PEGylated siRNA lipoplexes on invitro gene‑silencing effects and siRNA biodistribution in mice.

Polyethylene glycol (PEG)-modifications (PEGylations) of cationic liposome/small interfering RNA complexes (siRNA lipoplexes) can enhance their systemic stability. The present study determined the effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene-silencing effects and siRNA biodistribution after intravenous injection. Three types of dialkyl or trialkyl cationic lipids were used in the current study for the preparation of cationic liposomes. Additionally, various PEGylated siRNA lipoplexes that contained PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG), PEG-cholesterol (PEG-Chol) and PEG-chondroitin sulfate conjugate (PEG-CS) were prepared. The results revealed that PEGylation of siRNA lipoplexes with PEG-DSPE strongly decreased gene-silencing effects in cells. In contrast, those with PEG-DSG, PEG-Chol and PEG-CS did not largely decrease gene-silencing effects. However, regardless of the PEG-derivative type, PEGylation of siRNA lipoplexes decreased their agglutination with erythrocytes. Furthermore, intravenous injection of PEGylated siRNA lipoplexes markedly decreased the accumulation of siRNA in the lungs, regardless of the type of PEG-derivative. However, non-PEGylated siRNA lipoplexes accumulated mainly in the lungs regardless of the siRNA lipoplex cationic lipid type. The results indicated that PEGylation of siRNA lipoplexes with PEG-DSG, PEG-Chol and PEG-CS may improve systemic stability without losing transfection activity by PEGylation.

Carbonate Apatite and Hydroxyapatite Formulated with Minimal Ingredients to Deliver SiRNA into Breast Cancer Cells In Vitro and In Vivo.

Introduction: Cancer is one of the top-ranked noncommunicable diseases causing deaths to nine million people and affecting almost double worldwide in 2018. Tremendous advancement in surgery, chemotherapy, radiation and targeted immunotherapy have improved the rate of cure and disease-free survival. As genetic mutations vary in different cancers, potential of customized treatment to silence the problem gene/s at the translational level is being explored too. Yet delivering therapeutics at the required dosage only to the affected cells without affecting the healthy ones, is a big hurdle to be overcome. Scientists worldwide have been working to invent a smart drug delivery system for targeted delivery of therapeutics to tumor tissues only. As part of such an effort, few organic nanocarriers went to clinical trials, while inorganic nanoparticles (NPs) are still in development stage despite their many customizable properties. Carbonate apatite (CA), a pH sensitive nanocarrier has emerged as an efficient delivery system for drugs, plasmids and siRNAs in preclinical models of breast and colon cancers. Like hydroxyapatite (HA) which serves as a classical tool for delivery of genetic materials such as siRNA and plasmid, CA is an apatite-based synthetic carrier. We developed simplified methods of formulating CA-in-DMEM and a DMEM-mimicking buffer and HA in a HEPES-buffered solution and characterized them in terms of size, stability, protein corona (PC) composition, cytotoxicity, siRNA delivery efficiency in breast cancer cells and siRNA biodistribution profile in a mouse model of breast cancer. Methods: Particle growth was analyzed via spectrophotometry and light microscopy, size was measured via dynamic light scattering and scanning electron microscopy and confirmation of functional groups in apatite structures was made by FT-IR. siRNA-binding was analyzed via spectrophotometry. Stability of the formulation solutions/buffers was tested over various time points and at different temperatures to determine their compatibility in the context of practical usage. Cellular uptake was studied via fluorescence microscopy. MTT assay was performed to measure the cytotoxicity of the NPs. Liquid chromatography—mass spectrometry was carried out to analyze the PC formed around all three different NPs in serum-containing media. To explore biodistribution of all the formulations, fluorescence-labeled siRNA-loaded NPs were administered intravenously prior to analysis of fluorescence intensity in the collected organs and tumors of the treated mice. Results: The size of NPs in 10% serum-containing media was dramatically different where CA-in-DMB and HA were much larger than CA-in-DMEM. Effect of media was notable on the PC composition of all three NPs. All three NPs bound albumin and some common protease inhibitors involved in bone metabolism due to their compositional similarity to our bone materials. Moreover, CA also bound heme-binding proteins and opsonins. Unlike CA, HA bound different kinds of keratins. Difference in PC constitution was likely to influence accumulation of NPs in various organs including those of reticuloendothelial system, such as liver and spleen and the tumor. We found 10 times more tumor accumulation of CA-in-DMB than CA-in-DMEM, which could be due to more stable siRNA-binding and distinct PC composition of the former. Conclusion: As a nanocarrier CA is more efficient than HA for siRNA delivery to the tumor. CA prepared in a buffer containing only the mere constituents was potentially more efficient than classical CA prepared in DMEM, owing to the exclusion of interference attributed by the inorganic ions and organic molecules present in DMEM.

Optimized combination of cationic lipids and neutral helper lipids in cationic liposomes for siRNA delivery into the lung by intravenous injection of siRNA lipoplexes

In this study, we examined the effect of neutral helper lipids in cationic liposomes on gene silencing efficacy in the lungs after intravenous injection of cationic liposomes/siRNA complexes (siRNA lipoplexes) into mice. We used 6 kinds of dialkyl or trialkyl cationic lipids as cationic lipids and prepared cationic liposomes composed of each cationic lipid with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), DOPE/cholesterol (Chol), or Chol. Regardless of the type of cationic lipid in the cationic liposomes, the increase in Chol content in the formulation decreased the in vitro gene silencing efficacy, and it increased siRNA accumulation in the lungs after intravenous injection of siRNA lipoplexes. However, gene silencing efficacies in the lungs were not greatly affected by the type of neutral helper lipid in cationic liposomes composed of cationic lipids with saturated dialkyl chains (C16–C18), although they were increased by the inclusion of Chol into formulations of cationic liposomes composed of cationic lipids with unsaturated dialkyl chains (C18). These findings indicated that siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of siRNA lipoplexes were strongly affected by combination of a cationic lipid and neutral helper lipid in cationic liposomes.

Effect of cationic lipid in cationic liposomes on siRNA delivery into the lung by intravenous injection of cationic lipoplex

Cationic liposomes composed of dialkyl cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can efficiently deliver siRNA to the lungs following the intravenous injection of cationic liposome/siRNA complexes (lipoplexes). In this study, we examined the effect of cationic lipid of cationic liposomes on siRNA delivery to the lungs after intravenous injection. We used six kinds of cationic cholesterol derivatives and 11 kinds of dialkyl or trialkyl cationic lipids as cationic lipids, and prepared 17 kinds of cationic liposomes composed of a cationic lipid and 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) for evaluation of siRNA biodistribution and in vivo gene silencing effects. Among cationic liposomes, those composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18), 2-((1,5-bis(octadecyloxy)-1,5-dioxopentan-2-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-3-18D), 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), or cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) with DOPE exhibited high accumulation of siRNA in the lung and significant suppression of Tie2 mRNA expression after the intravenous injection of cationic lipoplexes with Tie2 siRNA. Furthermore, DC-1-16/DOPE and DC-1-18/DOPE lipoplexes with protein kinase N3 (PKN3) siRNA could suppress the tumour growth when intravenously injected into mice with lung LLC metastasis. These findings indicate that the siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of cationic lipoplexes were strongly affected by the type of cationic lipid of cationic liposomes.

Effect of chondroitin sulfate on siRNA biodistribution and gene silencing effect in mice after injection of siRNA lipoplexes

Previously, we found that intravenous injection of chondroitin sulfate (CS), followed by intravenous injection of siRNA/cationic liposome complexes (siRNA lipoplexes) could deliver siRNAs to the liver and suppress expression of target genes. Here, we examined the effect of injection order of CS and siRNA lipoplexes on the biodistribution of siRNA and gene silencing in the liver after sequential injection. When siRNA lipoplexes were injected intravenously into mice, the siRNA largely accumulated in the lungs. However, injection of siRNA lipoplexes, followed by injection of CS, reduced siRNA accumulation in the lungs and increased it in the liver. In addition, agglutinates of erythrocytes caused by the addition of siRNA lipoplexes were re-dispersed by the addition of CS, indicating that the agglutinates accumulating in the lungs by injection of siRNA lipoplexes were broken up by CS injection. However, injection of apolipoprotein B (ApoB) siRNA lipoplexes, followed by injection of CS did not suppress ApoB mRNA levels in the liver. From there results on sequential injection, the injection order of CS and siRNA lipoplexes was important for gene silencing effects in the liver, although the sequential injection could deliver siRNA efficiently into the liver regardless of the injection order of CS and siRNA lipoplexes.

Small interfering RNA delivery into the liver by cationic cholesterol derivative-based liposomes

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50 mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.

Hydrophobic interactions between polymeric carrier and palmitic acid-conjugated siRNA improve PEGylated polyplex stability and enhance in vivo pharmacokinetics and tumor gene silencing

Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core relative to the analogous polyplexes formed with unmodified siRNA, si-NPs. Hydrophobized siPA loaded into the NPs at a lower charge ratio (N+:P−) relative to unmodified siRNA, and siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (∼40% at 24 h after one treatment and ∼60% at 48 h after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and tumor gene silencing relative to conventional formulations that are stabilized solely by electrostatic interactions.

Nanoparticle-based nonviral delivery of siRNA in zebrafish heart: gene silencing efficiency and siRNA biodistribution

Event Abstract Back to Event Nanoparticle-based nonviral delivery of siRNA in zebrafish heart: gene silencing efficiency and siRNA biodistribution Fang Wang1, Lu Gao2, Liuyi Meng1, Jingwei Xiong2 and Ying Luo1 1 Peking university, Biomedical Engineering, China 2 Peking University, Institute of Molecular Medicine, China Introduction: Non-viral nanomaterials hold promise for use as siRNA delivery vectors for treating cardiac diseases. However, the delivery mechanisms of nanomaterials in heart have not been elucidated due to the difficulty to track and image nanoparticles in vivo. Here, we aim to investigate the efficiency, kinetics and distribution of siRNA delivered locally to zebrafish heart through nanomaterials. In particular, the cellular distribution of siRNA was studied via flk:GFP and cmcl2:GFP transgenic zebrafish models in which endothelial cells and cardiomyocytes express GFP, respectively. Materials and Methods: A neutral polyethelenimine (PEI)-based material, PEI-HYD-RGD[1], and a dendrimer-based system, f-PAM4-PEG-R9, were synthesized through coupling reactions[2]. The materials were formulated with siRNA targeting Raldh2 (a retinoic acid-synthesizing enzyme) to form two types of nanoparticles, which were injected intrapleurally to zebrafish models with amputated hearts. The gene silencing effect was evaluated by qPCR. The uptake kinetics and the distribution of CY5-labled siRNA were analyzed via confocal microscopy of cryo-sectioned heart tissues. Results and Discussion: Both delivery systems showed gene silencing effects in zebrafish heart: The expression of Raldh2 was efficiently downregulated by about 50% and 80% via the PEI-HYD-RGD and f-PAM4-PEG-R9 vectors, respectively, compared to the models injected with DPBS only (Fig.1). In the f-PAM4-PEG-R9 system, the uptake of siNC-CY5 by the injured heart tissue began at 3 h after injection, reached the highest level in heart at 6 h and decreased at 24 h (Fig 2A-C). In the transgenic models, nanoparticle mainly located in the injured areas. In the PEI-HYD-RGD-medicated delivery, more nanoparticles were found associated with the cardiomyocytes (Fig 2E) than the endothelial cells (Fig 2D). The results indicate that nanoparticles may preferentially release therapeutic siRNA locally in injured heart tissues and induce differential distribution of siRNA in different cell types. Conclusions: Zebrafish provides a useful model to study the pharmacological mechanisms of siRNA in heart tissue. Nanoparticle-based system can effectively deliver siRNA in heart and may be further studied for optimal gene-silencing efficiency and siRNA distribution in vivo to develop new therapeutics for cardiac diseases.

Indium-Labeling of siRNA for Small Animal SPECT Imaging.

Ever since the discovery of RNA interference (RNAi), therapeutic delivery of siRNA has attracted a lot of interest. However, due to the nature and structure of siRNA, a carrier is needed for any mode of systemic treatment. Furthermore, specific imaging techniques are required to trace where the deposition of the siRNA occurs throughout the body after treatment. Tracking in vivo siRNA biodistribution allows understanding and interpreting therapeutics effects and side effects. A great advantage of noninvasive imaging techniques such as SPECT imaging is that several time points can be assessed in the same subject. Thus, the time course of biodistribution or metabolic processes can be followed. Therefore, we have described an approach to modify siRNA with a DTPA (Diethylene Triamine Pentaacetic Acid) chelator in order to utilize an indium labeled siRNA for SPECT imaging. Here, we explain the details of the labeling and purification procedures.

Invited Presentation: Single-Walled Carbon Nanotubes for Delivery of siRNA: Producing a Pharmaceutically Stable and Effective Drug Product

siRNA has the potential to be a selective targeted cancer therapy but currently lacks an effective delivery mechanism. Ensysce has been exploring the use of single-walled carbon nanotubes (SWCNT) as delivery vehicles for siRNA and has extensively examined the safety, and in vitro and in vivo efficacy of this platform. We have determined that siRNA/SWCNT complexes are well tolerated, and can be administered on a multi-weekly intravenous (i.v.) dosing schedule over extended periods of time. Our work has explored formulation and processing to provide a drug product with a good pharmacokinetic half-life allowing distribution of the SWCNT to desired tissues in animals and that provides suitable pharmaceutic properties for use in a clinical setting. Complexes with various siRNA payloads have been evaluated in animal models with human tumor xenografts. Antitumor activity has been demonstrated with siRNA/SWCNT complexes targeting one or two growth driving proteins. Preclinical development has begun and a product has been selected to move through IND enabling toxicology. SWCNT offer a novel approach to safely and effectively deliver oligonucleotides, and by manipulating formulation of the complex we can alter the siRNA biodistribution to tissues of interest with resulting target inhibition for disease control.

Delivery of Therapeutic siRNA to the Lung Endothelium via Novel Lipoplex Formulation DACC

Posttranscriptional gene silencing by RNA interference can be therapeutically exploited to inhibit pathophysiological gene expression. However, in contrast to the established effectiveness of RNAi in vitro, safe and effective delivery of siRNAs to specific organs and cell types in vivo remains the major hurdle. Here, we report the development and in vivo characterization of a novel siRNA delivery system (DACC lipoplex) suitable for modulating target gene expression specifically in the lung vasculature. Systemic administration of DACC in mice delivered siRNA cargo functionally to the lung pulmonary endothelium. A single dose of DACC lipoplexes administered by bolus injection or by infusion was sufficient to specifically silence genes expressed in pulmonary endothelial cells such as CD31, Tie-2, VE-cadherin, or BMP-R2. When tested in a mouse model for lung cancer, repeated treatment with DACC/siRNA(CD31) reduced formation of lung metastases and increased life span in a mouse model of experimental lung metastasis.

Development of Pre-Clinical Models for Evaluating the Therapeutic Potential of Candidate siRNA Targeting STAT6

Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification = 0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular toxicity or reduced efficacy. We have established a robust, sensitive method for determining siRNA bio-distribution in vivo, and developed a nasal model to aid evaluation. Further work is warranted.

Balancing Cationic and Hydrophobic Content of PEGylated siRNA Polyplexes Enhances Endosome Escape, Stability, Blood Circulation Time, and Bioactivity in Vivo

A family of pH-responsive diblock polymers composed of poly[(ethylene glycol)-b-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)], PEG-(DMAEMA-co-BMA), was reversible addition-fragmentation chain transfer (RAFT) synthesized with 0-75 mol % BMA in the second polymer block. The relative mole % of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and in vivo bioactivity. The polymer with 50:50 mol % of DMAEMA:BMA (polymer "50 B") in the RAFT-polymerized block efficiently condensed siRNA into 100 nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape. In vitro delivery of siRNA with polymer 50 B produced up to 94% protein-level knockdown of the model gene luciferase. The PEG corona of the NPs blocked nonspecific interactions with constituents of human whole blood, and the relative hydrophobicity of polymer 50 B increased NP stability in the presence of human serum or the polyanion heparin. When injected intravenously, 50 B NPs enhanced blood circulation half-life 3-fold relative to more standard PEG-DMAEMA (0 B) NPs (p < 0.05), due to improved stability and a reduced rate of renal clearance. The 50 B NPs enhanced siRNA biodistribution to the liver and other organs and significantly increased gene silencing in the liver, kidneys, and spleen relative to the benchmark polymer 0 B (p < 0.05). These collective findings validate the functional significance of tuning the balance of cationic and hydrophobic content of polyplex NPs utilized for systemic siRNA delivery in vivo.

Gene Silencing via RNAi and siRNA Quantification in Tumor Tissue Using MEND, a Liposomal siRNA Delivery System

Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5' RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability.

Abstract 5621: Single-walled carbon nanotubes for delivery of siRNA: Antitumor efficacy comparing formulated complexes in EGFR overexpressing A431 xenografts.

Abstract siRNA has the potential to be a selective targeted cancer therapy but currently lacks an adequate delivery mechanism. Ensysce has been exploring the use of single-walled carbon nanotubes (SWCNT) as delivery vehicles for siRNA and has previously reported on the safety, in vitro and in vivo efficacy of this platform. We have determined that siRNA/SWCNT complexes are well tolerated, and can be administered in a multi-weekly intravenous (i.v.) dosing schedule over extended periods of time. The pharmacokinetics and distribution of the SWCNT in animals have been examined. The goal of the current study was to determine an optimal formulation of siRNA/SWCNT that provides maximal tumor accumulation and antitumor efficacy. siRNA/SWCNT complexes were formulated in aqueous solutions with PEG-lipid analogues and the circulation and tissue distribution were evaluated. All solutions prepared were examined by atomic force microscopy (AFM), dynamic light scattering (DLS) and for z-potential. siRNA payloads were quantified and siRNA/SWCNT complexes were examined for stability in serum and systemic efficacy. Solutions of siRNA/SWCNT formulated complexes were administered i.v. to C57Bl6 mice or to nu/nu mice bearing MiaPaCa human pancreatic or A431 human epidermoid carcinoma tumors. Mice were sacrificed at various times after i.v. administration. Blood and tissues were collected and SWCNT concentrations were determined by measuring near infrared fluorescence using NS2 Nanospectralizer. The presence and persistence of SWCNT in the tissues was determined. Animals bearing A431 xenografts were treated with SWCNT/siEGFR/PEG-lipid analogue complexes and tumor growth control was compared to that produced by erlotinib. Data to be presented will include details of the analytical characterization of the SWCNT, circulating half-life (t1/2) and tissue concentrations versus time of administration. Tumor and tissue SWCNT content were found to depend on the t1/2 of the formulation of preparations that were delivered. Microscopic examination of tumors indicated that SWCNT presence in tumor increased with the increasing t1/2. The siRNA target knockdown in tumor xenografts of up to 90% was observed with complexes comprised of a number of siRNA payloads including siKRAS, siEGFR and siTRX. Antitumor activity has also been demonstrated with complexes targeting one or two growth driving proteins. Formulations providing t1/2 of 13 hr for the circulating SWCNT were evaluated with siEGFR against A431 EGFR overexpressing cells, with the optimal formulation showing tumor growth delay of 16 to 20 days versus the free siRNA treated control. In summary, SWCNT offer a novel approach to deliver oligonucleotides safely and effectively and formulation of the complexes can offer a means to control the siRNA biodistribution. Citation Format: D. Lynn Kirkpatrick, Olga Gliko, Michelle Weiss, Iris Owusu, Anton Naumov. Single-walled carbon nanotubes for delivery of siRNA: Antitumor efficacy comparing formulated complexes in EGFR overexpressing A431 xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5621. doi:10.1158/1538-7445.AM2013-5621

&lt;i&gt;In Vivo&lt;/i&gt; Imaging of Liposomal Small Interfering RNA (siRNA) Trafficking by Positron Emission Tomography

In the development of nucleic acid medicines such as small interfering RNA (siRNA) drugs, one problem is how to study the pharmacokinetics and pharmacodynamics, since the precise in vivo behavior of siRNA is hard to detect. In this research, to establish a highly sensitive detection system of siRNA biodistribution in the whole body, the technology of positron imaging was applied. First, a one-step synthetic method in which double-stranded siRNA was directly labeled by a positron emitter, (18)F, was developed. By using [(18)F]-labeled siRNA ([(18)F]-siRNA), the complex of siRNA and polycation liposomes (PCL) containing dicetylphosphate tetraethylenepentamine (TEPA-PCL) was prepared. Then, the biodistribution of the siRNA after intravenous administration to mice was analyzed by planar positron imaging system (PPIS). As a result, whereas naked [(18)F]-siRNA was immediately excreted in mouse bladder after administration, the complex with cationic liposome (CL) was trapped in the lungs. Furthermore, [(18)F]-siRNA carried with PEGylated CL (PL) was distributed throughout the body, suggesting that it circulated in the bloodstream for an extended period of time. Additionally, PET imaging revealed more detailed biodistribution of the siRNA than in vivo imaging system (IVIS) because PET imaging is not affected by the depth variation of target tissues. On the other hand, to induce high accumulation of siRNAs against c-myc, MDM2, and VEGF in tumor tissue, a tumor-targeting probe, RGD peptide, was grafted at the top of PEG chain in PEGylated TEPA-PCL and the effect of the complex on experimental lung metastasis of B16 melanoma was examined. The complex suppressed the progression of tumor. We believe that the positron imaging data would support the development of siRNA agent for clinical use.

Abstract 2888: Lipid nanoparticle-mediated delivery of siRNA to extrahepatic tumors

Abstract While lipid nanoparticles (LNPs) proved to be effective in delivering siRNA to the liver and hepatocellular carcinoma (HCC) via systemic administration, they showed inferior or no efficacy in the extrahepatic tumor models. To understand the underlying mechanism for LNP activity in delivering siRNA to extrahepatic tumors, we determined siRNA biodistribution and gene-silencing activity of the LNP-siRNA nanoparticles containing different percentages of a polyethylene glycol (PEG) lipid, distinct PEG lipids with a short (C-14) or long (C-18) alkyl chain, or an identical composition with different particle sizes in mouse subcutaneous tumor xenograft models derived from either human HCC (Hep3B) or colon cancer (HT29 or HCT116) cells. We show that the LNPs containing C-18 DSA PEG exhibited a higher retention of siRNA in both Hep3B and HT29 tumors than those with C-14 DMA PEG following an intravenous administration. Intriguingly, LNPs containing 2% of DSA or DMA PEG resulted in similar levels (∼50%) of target knockdown in Hep3B tumors while those containing 6% of DSA or DMA PEG caused no target silencing. Also, none of these LNPs reduced target expression in HT29 tumors despite the retention of siRNA in tumor tissues. When LNPs with an identical composition (2% DMG PEG) but different particle sizes (80 nm vs. 120 nm) were tested, we observed a comparable and moderate suppression of target expression in Hep3B tumors, but no target knockdown in HCT116 tumors. This is despite the fact that the expression of low density lipid receptor, which is shown to be involved in mediating LNP efficacy, is detected in HT29 and HCT116 tumors. Finally, intratumor injection of LNPs only induced minimal target silencing in subcutaneous tumors while provoking significant target knockdown in the liver. Together, enhanced tumor retention of siRNA might not be readily translated to target silencing activity in tumors. Other factors such as the efficiency of transfecting tumor cells and the passive targeting effect through opsonization could impact on LNP efficacy in extrahepatic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2888. doi:1538-7445.AM2012-2888

Delivery of siRNA to the Mouse Lung via a Functionalized Lipopolyamine

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.