siRNA Encapsulation Efficiency Research Articles

Preparation, Characterization, and In Vitro Evaluation of Lipidoid-Polymer Hybrid Nanoparticles for siRNA Delivery to the Cytosol.

RNA interference (RNAi) therapeutics are one of the most promising biological interventions in the efficient management of difficult-to-treat diseases. RNAi is mediated by small interfering RNA (siRNA), which induces specific and highly potent gene silencing. However, intracellular delivery of exogenous, chemically synthesized siRNA to the RNAi pathway in the cytosol remains a challenge, and is fully dependent on technologies that can facilitate cytosolic delivery without undesired side effects. One example is a novel delivery system referred to as lipidoid-polymer hybrid nanoparticles (LPNs), which we recently showed mediates highly efficient and safe gene silencing. Here we describe a double emulsion solvent evaporation method for the preparation of siRNA-loaded LPNs and methodologies employed for their physicochemical characterization and biological performance. A solution of siRNA in aqueous buffer is emulsified by sonication with an organic phase containing lipid and polymer into a primary emulsion. Subsequently, the primary emulsion is emulsified with a secondary water phase containing polyvinyl alcohol by sonication, and the organic phase is evaporated, eventually resulting in LPNs. The physicochemical characterization includes determination of (1) hydrodynamic particle size distribution, (2) zeta potential, (3) siRNA encapsulation efficiency, and (4) practical siRNA loading. The transfection experiments are conducted in a cell-based model system using enhanced green fluorescence protein as reporter. The gene silencing effect is also confirmed at the mRNA level by reverse transcription polymerase chain reaction (RT-PCR). The effect of the siRNA-loaded LPNs on cell viability is measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Preparation of Exosomes for siRNA Delivery to Cancer Cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 °C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 ± 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 ± 0.22 × 1012 particle/mL and 8.3 ± 1.7 × 1010 particle/µg, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

Novel lipid-polymer hybrid nanoparticles for siRNA delivery and IGF-1R gene silencing in breast cancer cells

BackgroundDelivery of siRNA has been one of the major challenges to application of siRNA therapeutics. Polymer/lipid hybrid nanoparticles (HNPs) combine the advantages of both polymeric and liposomal carriers with remarkable properties for efficient siRNA encapsulation and delivery. ObjectiveHerein, PLA-PEG-PLA/lipid hybrid nanoparticles were fabricated and utilized for siRNA encapsulation. Transfection efficiency of complex HNPs/siRNA was investigated in MCF7 breast cancer cells. MethodsPLA-PEG-PLA copolymer was synthesized by ring opening polymerization and its hybridization with cationic lipid was performed using sonication method. Quality controls and HNP cytotoxicity were determined using DLS, AFM, SEM, and MTT assay, respectively. Cellular uptake of HNP/siRNA complex was investigated by flow cytometry and fluorescence microscope. Silencing activity of HNPs/siRNA complexes was analyzed using semi-quantitative RT-PCR. ResultsFabricated HNPs exhibited improved biocompatibility compared to the cognate lipid nanoparticles and showed high siRNA encapsulation efficiency (80% ± 5). HNPs were capable of transferring of IGF-1R-siRNA into MCF7 cells, which resulted in expression down regulation of IGF-1R gene (70% ± 3). ConclusionThe excellent cyto-compatibility, high cellular uptake, and remarkable gene silencing ability made HNPs promising carriers for efficient siRNA delivery.

An enhanced RRM2 siRNA delivery to rheumatoid arthritis fibroblast-like synoviocytes through a liposome‑protamine-DNA-siRNA complex with cell permeable peptides.

Rheumatoid arthritis (RA) is considered to be a systemic autoimmune disease that induces systemic complications and progressive disability. It affects a large number of people. RA fibroblast-like synoviocytes (RA-FLS) promote the progression of RA through the secretion of proinflammatory cytokines and increasing invasiveness into the extracellular matrix. Therefore, targeting RA-FLS represents a potential approach for the treatment of RA. Ribonucleotide reductase M2 (RRM2), a critical protein for DNA synthesis and repair, may promote the proliferation of cells and inhibit cellular apoptosis. In previous studies it has been confirmed that the suppression of RRM2 markedly suppressed the proliferation of liver cancer cells. In the present study, a cell permeable peptide-conjugated liposome-polycation-DNA (LPD) complex loaded with RRM2 small interfering RNA (siRNA) (CCP-LPDR) was developed, aiming to increase the levels of apoptosis and inhibit the proliferation of RA-FLS. CCP-LPDR is a small-sized molecule (~130 nm) with high encapsulation efficiency of siRNA (>90%) and high stability. Furthermore, it was verified that CCP-LPDR markedly suppressed RRM2 gene and protein expression by ~80%. Notably, CCP-LPDR efficiently targeted RA-FLS, resulting in a marked decrease in the proliferation and increase in the level of apoptosis in RA-FLS. In addition, the levels of proinflammatory cytokines tumor necrosis factor-α and interleukin-6 were markedly decreased in RA-FLS following CCP-LPDR treatment. Therefore, CCP-LPDR may efficiently deliver RRM2 to RA-FLS and represent a potential treatment for RA.

Lipid nanoparticles with minimum burst release of TNF-α siRNA show strong activity against rheumatoid arthritis unresponsive to methotrexate

TNF-α siRNA has shown promising therapeutic benefits in animal models of rheumatoid arthritis. However, there continues to be a need for siRNA delivery systems that have high siRNA encapsulation efficiency and minimum burst release of TNF-α siRNA, and can target inflamed tissues after intravenous administration. Herein we report a novel acid-sensitive sheddable PEGylated solid-lipid nanoparticle formulation of TNF-α-siRNA, AS-TNF-α-siRNA-SLNs, prepared by incorporating lipophilized TNF-α-siRNA into solid-lipid nanoparticles composed of biocompatible lipids such as lecithin and cholesterol. The nanoparticles are approximately 120 nm in diameter, have a high siRNA encapsulation efficiency (>90%) and a minimum burst release of siRNA (<5%), and increase the deilvery of the siRNA in chronic inflammation sites in mouse models, including in a mouse model with collagen-induced arthritis. Importantly, in a mouse model of collagen antibody-induced arthritis that does not respond to methotrexate therapy, intravenous injection of the AS-TNF-α-siRNA-SLNs significantly reduced paw thickness, bone loss, and histopathological scores. These findings highlight the potential of using this novel siRNA nanoparticle formulation to effectively treat arthritis, potentially in patients who do not respond adequately to methotrexate.

The promoted delivery of RRM2 siRNA to vascular smooth muscle cells through liposome-polycation-DNA complex conjugated with cell penetrating peptides

Peripheral vascular disease (PVD) is a prevalent vascular disease that affect a large number of patients. The establishment of optimal treatments to mitigate the intimal hyperplasia (IH)-induced restenosis would help relieve the health burden of the PVD. Ribonucleotide reductase M2 (RRM2) is critical to cellular migration and proliferation. We have previously demonstrated that suppression of RRM2 expression could substantially inhibit hepatocellular carcinoma cell proliferation and migration. We hereby developed RRM2 small interfering RNA (siRNA)-loaded cell penetrating peptides-conjugated liposome-polycation-DNA complex (LPD) (RRM2-CLPD), aiming to inhibit the migration and proliferation of vascular smooth muscle cells (VSMCs) crucial for IH. RRM2-CLPD is of a small size (∼150 nm) and high siRNA encapsulation efficiency (∼90%). Further, we demonstrated that RRM2-CLPD could significantly inhibited RRM2 gene and protein expression by ∼80%. Notably, RRM2-CLPD was able to effectively bind to VSMCs, resulting in significant cellular proliferation and migration inhibition. Taken together, RRM2-CLPD represent a very promising treatment for IH.

Cosilencing Intestinal Transglutaminase-2 and Interleukin-15 Using Gelatin-Based Nanoparticles in an in Vitro Model of Celiac Disease.

In this study, we have developed a type B gelatin nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol-water solvent displacement method. Size, charge, and morphology of gelatin nanoparticles were evaluated using a Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA-containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15, and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an in vitro model of celiac disease by utilizing immunogenic α-gliadin peptide p31-43 in cultured J774A.1 cells. siRNA-containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217 ± 8.39 nm and -6.2 ± 0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3 ± 3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine-mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both TG2 and IL-15 genes, which corresponded to ∼200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle mediated knockdown of TG2 and IL-15 mRNA was tested in an in vitro model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31-43 stimulated J774A.1 cells upon either IL-15 or IL-15 + TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease.

Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications

BackgroundWe have previously defined mechanisms of intimal hyperplasia that could be targets for molecular therapeutics aimed at vascular pathology. However, biocompatible nanocarriers are needed for effective delivery. Cationic liposomes (CLPs) have been demonstrated as effective nanocarriers in vitro. However, in vivo success has been hampered by cytotoxicity. Recently, neutral PEGylated liposomes (PLPs) have been modified with cell-penetrating peptides (CPPs) to enhance cellular uptake. We aim to establish CPP-modified neutral liposomes as viable molecular nanocarriers in vascular smooth muscle cells. MethodsCLPs, PLPs, and CPP-modified PLPs (R8-PLPs) were assembled with short interfering RNA (siRNA) via ethanol injection. Characterization studies determined liposomal morphology, size, and charge. siRNA encapsulation efficiency was measured via RiboGreen assay. Vascular smooth muscle cells were exposed to equal lipid/siRNA across all groups. Rhodamine-labeled liposomes were used to quantify cell association via fluorometry, live/dead dual stain was used to measure cytotoxicity, and gene silencing was measured by quantitative polymerase chain reaction. ResultsR8-PLPs exhibited increased encapsulation efficiency equivalent to CLPs. PLPs and R8-PLP-5 mol% and R8-PLP-10 mol% had no cytotoxic effect. CLPs demonstrated significant cytotoxicity. R8-PLP-5 mol% and R8-PLP-10 mol% exhibited increased cell association versus PLPs. R8-PLP-10 mol% resulted in significant gene silencing, in a manner dependent on lipid-to-siRNA load capacity. ConclusionsThe negligible cytotoxicity and enhanced cellular association and gene silencing capacity exhibited by R8-PLPs reveal this class of liposomes as a candidate for future applications. Further modifications for optimizing R8-PLPs are still warranted to improve efficacy, and in vivo studies are needed for translational development. However, this could prove to be an optimal nanocarrier for vascular gene therapeutics.

Co-delivery of paclitaxel and anti-survivin siRNA via redox-sensitive oligopeptide liposomes for the synergistic treatment of breast cancer and metastasis

The overexpression of survivin in breast cancer cells is an important factor of paclitaxel (PTX) resistance in breast cancer. To overcome PTX resistance and improve the antitumor effect of PTX, we developed a novel liposome-based nanosystem (PTX/siRNA/SS-L), composed of a redox-sensitive cationic oligopeptide lipid (LHSSG2C14) with a proton sponge effect, natural soybean phosphatidylcholine (SPC), and cholesterol for co-delivery of PTX and anti-survivin siRNA, which could specifically downregulate survivin overexpression. PTX/siRNA/SS-L exhibited high encapsulation efficiency and rapid redox-responsive release of both PTX and siRNA. Moreover, in vitro studies on the 4T1 breast cancer cells revealed that PTX/siRNA/SS-L offered significant advantages over other experimental groups, such as higher cellular uptake, successful endolysosomal escape, reduced survivin expression, the lowest cell viability and wound healing rate, as well as the highest apoptosis rate. In particular, in vivo evaluation of 4T1 tumor-bearing mice showed that PTX/siRNA/SS-L had lower toxicity and induced a synergistic inhibitory effect on tumor growth and pulmonary metastasis. Collectively, the collaboration of anti-survivin siRNA and PTX via redox-sensitive oligopeptide liposomes provides a promising strategy for the treatment of breast cancer and metastasis.

In vitro and ex vivo delivery of tailored siRNA-nanoliposomes for E2F1 silencing as a potential therapy for colorectal cancer

Tailored developed nanoliposomes loaded with a siRNA against the transcription factor E2F1 (siE2F1), were produced and delivered to human colorectal adenocarcinoma cell lines and to intestinal human biopsies. siE2F1 loaded nanoliposomes were produced through a dedicated ultrasound assisted technique producing particles with about 40nm size (Small Unilamellar Vesicles, SUVs) and 100% siRNA encapsulation efficiency. Compared to other production methods, the one proposed here can easily produce particles in the nanometric scale by suitable ultrasonic duty cycle treatments. Furthermore, SUVs have a high degree of size homogeneity, a relevant feature for uniform delivery behaviour.siE2F1-loaded SUVs demonstrated a very low cytotoxicity in cells when compared to a commercial transfection agent. Moreover, SUVs loaded with siE2F1 were effective in the down regulation of the target in cultured colon carcinoma cells and in the consequent reduction of cell growth. Finally, a remarkable uptake and target silencing efficiencies were observed in cultured human biopsy of colonic mucosa. In conclusion, whereas further studies in more complex models are required, the siE2F1-SUVs generated have the potential to contribute to the development of novel effective inflammatory bowel diseases-associated colorectal cancer therapies for a future personalized medicine.

Distinctive polymer micelle designed for siRNA delivery and reversal of MDR1 gene-dependent multidrug resistance.

P-glycoprotein (P-gp) plays an importantrole in multidrug resistance (MDR), proved to be one of the major obstacles in cancer chemotherapy. Cationic polymers could specifically deliver siRNA to tumor cells and thus reverse MDR by the downregulation of P-gp. In this study, a triblock copolymer micelle was prepared based on the polymer of N-succinyl-chitosan-poly-l-lysine-palmitic acid (NSC-PLL-PA) to deliver siRNA-P-gp (siRNA-micelle) or doxorubicin (Dox-micelle). The resulting micelle exhibited an efficient binding ability for siRNA and high encapsulation efficiency for Dox, with an average particle size of ∼170nm. siRNA-micelle and Dox-micellewere instable at low pH, thereby enhancing tumor accumulation and intracellular release of the encapsulated siRNA and Dox. siRNA-micelle micelles could enhance the knockdown efficacy of siRNA by improving the transfection efficiency, downregulating P-gp expression, and passing the drug efflux transporters, thereby improving the therapeutic effects of Dox-micelle. However, P-gp could transfer from HepG2/ADM to HepG2 cells independent of the expression of mdr1, and the acquired resistance could permit tumor cells to survive and develop intrinsic P-gp-mediated resistance, thereby limiting the desired efficiency of chemotherapeutics. This study demonstrated the effectiveness of siRNA-micelle for tumor-targeted delivery, MDR reversal, and provided an effective strategy for the treatment of cancers that develop MDR. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2093-2106, 2017.

Synthesis and Characterization of Aptamer-Targeted SNALPs for the Delivery of siRNA.

Aptamers selected against cell surface receptors represent a unique set of ligands that can be used to target nanoparticles and other therapeutics to specific cell types. Here, we describe a method for using aptamers to deliver stable nucleic acid lipid particles (SNALPs) encapsulating small interfering RNA (siRNA) to cells in vitro. Using this method, we have demonstrated the ability of aptamer-conjugated SNALPs to achieve target-specific delivery and siRNA-mediated knockdown of a gene of interest. We also describe methods to characterize SNALP size, siRNA encapsulation efficiency, and aptamer conjugation efficiency.

One-step scalable preparation method for non-cationic liposomes with high siRNA content

Cationic liposomes (LPs) have been utilized for short interfering RNA (siRNA) delivery in vitro and in vivo owing to their high affinity for siRNA via electrostatic binding. However, both cytotoxicity and non-specific adsorption of cationic LPs in the body have prevented clinical siRNA applications. These situations have led to siRNA encapsulation in non-cationic LPs. We found that the instillation of neutral phospholipids dissolved in ethanol into aqueous solutions containing siRNA and CaCl2 resulted in high siRNA encapsulation (siRNA encapsulation efficiency: ∼80%; siRNA weight ratio: ∼10wt% of LPs). The products were monodispersed, ∼200nm, and negatively charged. Furthermore, when phospholipids with a high-phase transition temperature or cholesterol were used, the encapsulation efficiency and siRNA content remained high. Although anionic LPs could not encapsulate siRNAs using this method, the use of cholesterol-conjugated siRNA helped achieve substantial siRNA encapsulation in anionic LPs. These non-cationic siRNA-containing LPs did not show cytotoxicity in vitro, and could be formed with polyethylene glycol-conjugated phospholipids. When conjugated with targeting ligand, the non-cationic siRNA-containing LPs could suppress the expression of target gene in vitro. These data demonstrate that our preparation method would be suitable for large-scale LP production for systemic siRNA delivery.

Thermo-sensitive nanoparticles for triggered release of siRNA

Efficient delivery of small interfering RNA (siRNA) is crucially required for cancer gene therapy. Herein, a thermo-sensitive copolymer with a simple structure, poly (ethylene glycol) methyl ether acrylate-b-poly (N-isopropylacrylamide) (mPEG-b-PNIPAM) was developed. A novel kind of thermo-sensitive nanoparticles (DENPs) was constructed for the cold-shock triggered release of siRNA by double emulsion–solvent evaporation method using mPEG-b-PNIPAM and a cationic lipid, 3β [N-(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol [DC-Chol]. DENPs were observed by transmission electron microscopy and dynamical light scattering before and after ‘cold shock’ treatment. The encapsulation efficiency (EE) of siRNA in DENPs, which was measured by fluorescence spectrophotometer was 96.8% while it was significantly reduced to be 23.2% when DC-Chol was absent. DENPs/siRNA NPs exhibited a thermo-sensitive siRNA release character that the cumulatively released amount of siRNA from cold shock was approximately 2.2 folds higher after 7 days. In vitro luciferase silencing experiments indicated that DENPs showed potent gene silencing efficacy in HeLa-Luc cells (HeLa cells steadily expressed luciferase), which was further enhanced by a cold shock. Furthermore, MTT assay showed that cell viability with DENPs/siRNA up to 200 nM remained above 80%. We also observed that most of siRNA was accumulated in kidney mediated by DENPs instead of liver and spleen in vivo experiments. Thus, DENPs as a cold shock responsive quick release model for siRNA or hydrophilic macromolecules delivery provide a new way to nanocarrier design and clinic therapy.

Preparation and antitumor effect evaluation of composite microparticles co-loaded with siRNA and paclitaxel by a supercritical process.

The co-delivery of siRNA and therapeutic agents provides an effective method for cancer chemotherapy by avoiding drug resistance during the treatment. With a combination of ionic gelation and supercritical fluid technology, nanoparticle-embedded composite microparticles (CMPs) co-loaded with siRNA and paclitaxel (siRNA-PTX-CMPs) were successfully prepared. The results show that CMPs embedded with nanoparticles with a diameter of 50-100 nm exhibited a spherical shape and core-shell structure with a mean diameter of 323 nm. The encapsulation efficiency of siRNA in chitosan nanoparticles (CS NPs) was 96.97%. The drug load and encapsulation efficiency of PTX-loaded CMPs (5% dosage) were 1.40% and 27.95%, respectively; both these increased with an increase in dosage. It was found that no change had occurred in the functional groups of the components during the supercritical process, while the physical form of PTX had shifted to an amorphous state. In the cell experiments, the CMPs clustered around the nucleus after being taken up by the Bcap-37 cells. The results of the antitumor effect experiments revealed that the co-loaded siRNA-PTX-CMPs achieved a significantly better synergistic effect than single dosages, which indicated that the co-delivery system developed by the supercritical process could have potential in the application of cancer chemotherapy.

Nanolipoparticles-mediated MDR1 siRNA delivery: preparation, characterization and cellular uptake

Objective(s): Lipid-based nanoparticles (NLP) are PEGylated carriers composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The presence of PEG in the NLP formulation improves the particle pharmacokinetic behavior. The purpose of this study was to prepare and characterize NLPs containing MDR1 siRNA and evaluate their cytotoxicity and cellular uptake. MDR1 siRNA could be used in multidrug resistance reversal in cancer therapy.Materials and Methods: siRNAs were encapsulated into NLPs consisted of mPEG-DSPE/DOTAP/DOPE (10:50:40 molar ratio) by the detergent dialysis method. The particle diameters of NLPs and their surface charge were measured using dynamic light scattering. siRNA encapsulation efficiency was determined by an indirect method via filtration and free siRNA concentration determination. NLPs cytotoxicity was investigated by MTT assay. The ability of NLPs for siRNA delivery checked in two human cell lines (MCF-7/ADR and EPP85-181/RDB) by fluorescence microscopy and compared with oligofectamine.Results: NLPs containing MDR1 siRNA were prepared with the stable size of 80-90 nm and the zeta potential near to neutral. The siRNA encapsulation efficacy was more than 80%. These properties are suitable for in vivo siRNA delivery. NLPs cytotoxicity studies demonstrated they were non-toxic at the doses used. NLPs improved siRNA localization in both cell lines.Conclusion: NLPs containing MDR1 siRNA can be a good candidate for in vivo siRNA delivery studies.

PLGA microspheres encapsulating siRNA.

The therapeutic use of small interfering RNA (siRNA) represents a new and powerful approach to suppress the expression of pathologically genes. However, biopharmaceutical drawbacks, such as short half-life, poor cellular uptake, and unspecific distribution into the body, hamper the development of siRNA-based therapeutics. Poly(lactide-co-glycolide), (PLGA) microspheres can be a useful tool to overcome these issues. siRNA can be encapsulated into the PLGA microspheres, which protects the loaded nucleic acid against the enzymatic degradation. Moreover, PLGA microspheres can be injected directly into the action site, where the siRNA can be released in controlled manner, thus avoiding the need of frequent invasive administrations. The complete biodegradability of PLGA to monomers easily metabolized by the body, and its approval by FDA and EMA for parenteral administration, assure the safety of this copolymer and do not require the removal of the device after the complete drug release. In chapter, a basic protocol for the preparation of PLGA microspheres encapsulating siRNA is described. This protocol is based on a double emulsion/solvent evaporation technique, a well known and easy to reproduce method. This specific protocol has been developed to encapsulate a siRNA anti-TNFα in PLGA microspheres, and it has been designed and optimized to achieve high siRNA encapsulation efficiency and slow siRNA release in vitro. However, it can be extended also to other siRNA as well as other RNA or DNA-based oligonucleotides (miRNA, antisense, decoy, etc.). Depending on the applications, chemical modifications of the backbone and site-specific modification within the siRNA sequences could be required.

Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(d,l-lactide-co-glycolide acid) nanoparticles for intracellular delivery.

Calcium phosphate/poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles with a diameter below 200 nm, loaded with either nucleic acids or proteins, were synthesized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. The particles were stabilized by polyvinyl alcohol (PVA) and had a negative charge (zeta potential -26 mV). By the addition of calcium phosphate into the inner aqueous phase of the W1/O/W2-emulsion, the encapsulation efficiency of siRNA was increased to 37%, of DNA to 52%, and of bovine serum albumin to 78%, i.e. by a factor of 3 to 10 compared to PLGA nanoparticles without calcium phosphate. Total loadings of 8 μg siRNA, 5 μg DNA and 280 μg fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) per mg of PLGA nanoparticles were achieved by this method. The addition of an outer layer of either chitosan or polyethyleneimine (PEI) reversed the charge of the particles (zeta potential > +30 mV) and improved the cellular uptake as well as the endosomal escape of these particles as demonstrated by confocal laser scanning microscopy. Calcium phosphate-PLGA nanoparticles loaded with DNA encoding for enhanced green fluorescent protein (eGFP-DNA) showed a good transfection efficiency for epithelial cells (HeLa). Gene silencing with HeLa cells expressing eGFP gave knockdown efficiencies of 53% for anionic nanoparticles, of 68% for chitosan-coated cationic nanoparticles, and of 89% for polyethyleneimine-coated cationic nanoparticles.

Polymer nanoparticles for enhanced immune response: Combined delivery of tumor antigen and small interference RNA for immunosuppressive gene to dendritic cells

In this study, we report on polymer nanoparticles (NPs) that can induce an enhanced immune response in dendritic cell (DC)-based cancer immunotherapy by the combined delivery of tumor antigen and small interference RNA (siRNA) for the immunosuppressive gene to DCs. DCs are specialized antigen-presenting cells (APCs) that capture, process and present antigens and induce an antigen-specific cytotoxic T lymphocyte response. Because the suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of the APC-based immune response, the inhibition of SOCS1 gene expression is essential for DCs to enhance antigen-specific anti-tumor immunity. Multifunctional poly(lactide-co-glycolic acid) (PLGA) NPs that can deliver tumor antigen and siRNA for immunosuppressive SOCS1 genes to DCs simultaneously were fabricated by the emulsion solvent evaporation method. We have found that the encapsulation efficiency of small-sized and hydrophilic SOCS1 siRNA into hydrophobic PLGA matrix is drastically enhanced by the help of a tumor model antigen such as ovalbumin (OVA), and the encapsulation efficiency of siRNA in PLGA (SOCS1 siRNA only) NPs and PLGA (OVA/SOCS1 siRNA) NPs was ∼2% and 57.6%, respectively. PLGA (OVA/SOCS1 siRNA) NPs were efficiently taken up by bone-marrow-derived dendritic cells (BMDCs) and showed no detectable toxic effect. The knockdown of SOCS1 in BMDCs by PLGA (OVA/SOCS1 siRNA) NPs enhanced pro-inflammatory cytokine (tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-12 and IL-2) expression. Additionally, PLGA (OVA/SOCS1 siRNA) NP-treated BMDCs could elicit an immune response through cross-presentation in OVA-specific CD8 T cells that express IL-2 cytokine. Taken together, the combined delivery of NPs that can deliver both tumor antigen and immunosuppressive gene siRNA to BMDCs simultaneously could be a potent strategy to enhance immunotherapeutic effects in BMDC-based cancer therapy.

Enhanced endosomal/lysosomal escape by distearoyl phosphoethanolamine-polycarboxybetaine lipid for systemic delivery of siRNA

Cationic liposome based siRNA delivery system has improved the efficiencies of siRNA. However, cationic liposomes are prone to be rapidly cleared by the reticuloendothelial system (RES). Although modification of cationic liposomes with polyethylene glycol (PEG) could prolong circulation lifetime, PEG significantly inhibits siRNA entrapment efficiency, cellular uptake and endosomal/lysosomal escape process, resulting in low gene silencing efficiency of siRNA. In this study, we report the synthesis of zwitterionic polycarboxybetaine (PCB) based distearoyl phosphoethanolamine-polycarboxybetaine (DSPE-PCB) lipid for cationic liposome modification. The DSPE-PCB20 cationic liposome/siRNA complexes (lipoplexes) show an excellent stability in serum medium. The siRNA encapsulation efficiency of DSPE-PCB20 lipoplexes could reach 92% at N/P ratio of 20/1, but only 73% for DSPE-PEG lipoplexes. The zeta potential of DSPE-PCB20 lipoplexes is 8.19±0.53mV at pH 7.4, and increases to 24.6±0.87mV when the pH value is decreased to 4.5, which promotes the endosomal/lysosomal escape of siRNA. The DSPE-PCB20 modification could enhance the silencing efficiency of siRNA by approximately 20% over the DSPE-PEG 2000 lipoplexes at the same N/P ratio in vitro. Furthermore, DSPE-PCB20 lipoplexes could efficiently mediate the down-regulation of Apolipoprotein B (ApoB) mRNA in the liver and consequently decrease the total cholesterol in the serum in vivo, suggesting therapeutic potentials for siRNA delivery in hypercholesterolemia-related diseases.