Efficient siRNA Delivery System Research Articles

Nontoxic, Biodegradable Hyperbranched Poly(β-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(β-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer

BackgroundSmall interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. MethodsPEG12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. ResultsOur results showed that PEG12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. ConclusionIn conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

ErbB3-Targeting Oncolytic Adenovirus Causes Potent Tumor Suppression by Induction of Apoptosis in Cancer Cells.

Cancer is a multifactorial and deadly disease. Despite major advancements in cancer therapy in the last two decades, cancer incidence is on the rise and disease prognosis still remains poor. Furthermore, molecular mechanisms of cancer invasiveness, metastasis, and drug resistance remain largely elusive. Targeted cancer therapy involving the silencing of specific cancer-enriched proteins by small interfering RNA (siRNA) offers a powerful tool. However, its application in clinic is limited by the short half-life of siRNA and warrants the development of efficient and stable siRNA delivery systems. Oncolytic adenovirus-mediated therapy offers an attractive alternative to the chemical drugs that often suffer from innate and acquired drug resistance. In continuation to our reports on the development of oncolytic adenovirus-mediated delivery of shRNA, we report here the replication-incompetent (dAd/shErbB3) and replication-competent (oAd/shErbB3) oncolytic adenovirus systems that caused efficient and persistent targeting of ErbB3. We demonstrate that the E1A coded by oAd/shErbB, in contrast to dAd/shErbB, caused downregulation of ErbB2 and ErbB3, yielding stronger downregulation of the ErbB3-oncogenic signaling axis in in vitro models of lung and breast cancer. These results were validated by in vivo antitumor efficacy of dAd/shErbB3 and oAd/shErbB3.

Enzyme-sensitive nanoparticles, smart TAT and cetuximab conjugated immunoliposomes to overcome multidrug resistance in breast cancer cells

Due to recent advances in the field of small molecule-based drugs, designing an efficient siRNA delivery system seems essential. Here, modified sets of lipids conjugated with cell-penetrating TAT peptide, MMP2 enzyme-sensitive moiety, and cetuximab antibodies against the EGF receptor were synthesized, purified and verified on HPLC, TLC, SEM, and DLS analyses. Different cellular and molecular experiments were designed to evaluate the transfection efficiency, targeting properties, and functions, including cytotoxicity assay, resensitization assessments, flow cytometry-based uptake assay, BCRP silencing efficiency, real-time PCR, and western blotting. The final targeted liposomes represented an average diameter of 160 nm; zeta-potential and siRNA encapsulation rates were respectively around −28.9 ± 3.16 mV and 88.3 ± 0.9 w/w. The siBCRP carried by the TAT+Cetuximab+ liposome led to an increase in the tumoricidal effect of mitoxantrone by a reduction in IC50 value by 4-fold (*** P < 0.001). Flow cytometry results showed that the cellular uptake rate of final immunoliposomes was significantly higher than the naked liposomes (*** P < 0.001). The Targeted siRNA encapsulating liposomes decreased BCRP transcript and protein levels in MCF7-MX cells by 0.24 and 0.2-fold after 48 h, respectively. Due to the silencing results of the BCRP by the encapsulated siRNA and the inhibitory effects of cetuximab on the EGFR, this formulation could widely be utilized as a carrier for tumor-directed gene delivery.

Bioengineered Protein Nanocage by Small Heat Shock Proteins Delivering mTERT siRNA for Enhanced Colorectal Cancer Suppression.

The efficient delivery of small interfering RNA (siRNA) for target gene silencing holds great promise for cancer therapy. Protein nanocages have attracted considerable attention as ideal drug delivery systems because of their material-derived advantages and unique structural properties. However, most studies about siRNA delivery have not indicated the real role of protein nanocages in inhibiting tumor growth in vivo. Herein, we fabricated an efficient siRNA delivery system using a small heat shock protein (Hsp) nanocage decorated with Arg-Gly-Asp (RGD) and the transactivator of transcription (Tat) peptide. Hsp-Tat-RGD NC showed good cellular uptake and lysosomal escape in colorectal cancer cells. In addition, the nanocage could efficiently transfect siRNA into the cytoplasmic area of CT26 cells. Hsp-Tat-RGD NC delivering telomerase reverse transcriptase (TERT)-targeting siRNA could significantly downregulate TERT protein expression and trigger tumor cell apoptosis in vitro. More importantly, Hsp-Tat-RGD/siTERT complexes nearly completely inhibited the tumor growth after five times of treatment in mice bearing CT26 xenograft. Our results demonstrate the great potential of the Tat/RGD-decorated Hsp nanocage as a promising siRNA delivery platform for cancer therapy.

Self-assembled tetrahedral framework nucleic acid mediates tumor-associated macrophage reprogramming and restores antitumor immunity

There is increasing interest in depleting or repolarizing tumor-associated macrophages (TAMs) to generate a proinflammatory effect. However, TAMs usually display an immunosuppressive M2-like phenotype in the tumor microenvironment. Apparently, developing a macrophage-targeting delivery system with immunomodulatory agents is urgent. In this study, an efficient siRNA and CpG ODNs delivery system (CpG-siRNA-tFNA) was prepared with nucleic acid stepwise self-assembled. The tFNA composed of CpG ODNs and siRNA showed a higher stability and an enhanced cellular uptake efficiency. Moreover, the CpG-siRNA-tFNA effectively reprogrammed TAMs toward M1 phenotype polarization with increased proinflammatory cytokine secretion and NF-κB signal pathway activation, which triggers dramatic antitumor immune responses. Additionally, the CpG-siRNA-tFNA exhibited superior antitumor efficacy in a breast cancer xenograft mouse model without obvious systemic side effects. Taken together, CpG-siRNA-tFNA displayed greatly antitumor effect by facilitating TAM polarization toward M1 phenotypes in favor of immunotherapy. Hence, we have developed an efficient therapeutic strategy with immunomodulatory agents for clinical applications.

Cyclic Peptide-Gadolinium Nanocomplexes as siRNA Delivery Tools.

We have recently reported that a cyclic peptide containing five tryptophan, five arginine, and one cysteine amino acids [(WR)5C], was able to produce peptide-capped gadolinium nanoparticles, [(WR)5C]-GdNPs, in the range of 240 to 260 nm upon mixing with an aqueous solution of GdCl3. Herein, we report [(WR)5C]-GdNPs as an efficient siRNA delivery system. The peptide-based gadolinium nanoparticles (50 µM) did not exhibit significant cytotoxicity (~93% cell viability at 50 µM) in human leukemia T lymphoblast cells (CCRF-CEM) and triple-negative breast cancer cells (MDA-MB-231) after 48 h. Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptakes of Alexa-488-labeled siRNA were found to be enhanced by more than 10 folds in the presence of [(WR)5C]-GdNPs compared with siRNA alone in CCRF-CEM and MDA-MB-231 cells after 6 h of incubation at 37 °C. The gene silencing efficacy of the nanoparticles was determined via the western blot technique using an over-expressed gene, STAT-3 protein, in MDA-MB-231 cells. The results showed ~62% reduction of STAT-3 was observed in MDA-MB-231 with [(WR)5C]-GdNPs at N/P 40. The integrity of the cellular membrane of CCRF-CEM cells was found to be intact when incubated with [(WR)5C]-Gd nanoparticles (50 µM) for 2 h. Confocal microscopy reveals higher internalization of siRNA in MDA-MB-231 cells using [(WR)5C]-GdNPs at N/P 40. These results provided insight about the use of the [(WR)5C]-GdNPs complex as a potent intracellular siRNA transporter that could be a nontoxic choice to be used as a transfection agent for nucleic-acid-based therapeutics.

Layer by Layer Assembled Chitosan-Coated Gold Nanoparticles for Enhanced siRNA Delivery and Silencing.

Delivery of small interfering RNA (siRNA) provides one of the most powerful strategies for downregulation of therapeutic targets. Despite the widely explored capabilities of this strategy, intracellular delivery is hindered by a lack of carriers that have high stability, low toxicity and high transfection efficiency. Here we propose a layer by layer (LBL) self-assembly method to fabricate chitosan-coated gold nanoparticles (CS-AuNPs) as a more stable and efficient siRNA delivery system. Direct reduction of HAuCl4 in the presence of chitosan led to the formation of positively charged CS-AuNPs, which were subsequently modified with a layer of siRNA cargo molecules and a final chitosan layer to protect the siRNA and to have a net positive charge for good interaction with cells. Cytotoxicity, uptake, and downregulation of enhanced Green Fluorescent Protein (eGFP) in H1299-eGFP lung epithelial cells indicated that LBL-CS-AuNPs provided excellent protection of siRNA against enzymatic degradation, ensured good uptake in cells by endocytosis, facilitated endosomal escape of siRNA, and improved the overall silencing effect in comparison with commercial transfection reagents Lipofectamine and jetPEI®. Therefore, this work shows that LBL assembled CS-AuNPs are promising nanocarriers for enhanced intracellular siRNA delivery and silencing.

Simple FRET Electrophoresis Method for Precise and Dynamic Evaluation of Serum siRNA Stability.

Small interfering RNAs (siRNAs) are potent therapeutic molecules, but despite recent progress, their administration in vivo remains challenging due to their low stability in the bloodstream. Thus, techniques for investigating the stability of siRNA are fundamental for the development of efficient siRNA delivery systems. We designed a simple FRET electrophoresis method to dynamically evaluate serum siRNA stability in parallel with its interaction with the serum components. Each strand of the siRNA was labeled with the fluorophore carboxyfluorescein (FAM) at the 5'-end and the quencher carboxytetramethylrhodamine (TAMRA) at the 3'-end. After incubation in serum, molecular stability was proportional to the FRET efficiency that could be quantified in-gel by ImageJ analysis. Compared to the usual gel-shift and other plate-based FRET assays, this method is more sensitive and allows investigation of the stability of serum siRNA and siRNA-based nanoparticles, as well as the extrapolation of degradation kinetics in parallel with interaction analysis.

Size Matters: Arginine-Derived Peptides Targeting the PSMA Receptor Can Efficiently Complex but Not Transfect siRNA

Oligoarginine sequences conjugated to a short cancer-targeting peptide (CTP) selective for the prostate-specific membrane antigen (PSMA) receptor was developed for selective small interfering RNA (siRNA) delivery to a human metastatic/castration-resistant prostate cancer (PCa) cell line, which expresses PSMA on the surface. The PSMA-Rn (n = 6 and 9) peptides were synthesized by solid-phase peptide synthesis, characterized by liquid chromatography-mass spectrometry (LC-MS) and condensed with glucose-regulated protein (GRP)-silencing siRNAs. Native gels showed formation of stable CTP:siRNA ionic complexes. Furthermore, siRNA release was effected by heparin competition, supporting the peptides’ capabilities to act as condensing and releasing agents. However, dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies revealed large anionic complexes that were prone to aggregation and limited cell uptake for RNAi activity. Taken together, these data support the notion that the development of efficient peptide-based siRNA delivery systems is in part contingent on the formulation of discrete nanoparticles that can effectively condense and release siRNA in cells.

Delivery of Survivin siRNA Using Cationic Diphenylalanine Vesicles

Small interfering RNA(siRNA) has been proved to be a powerful tool for silencing target gene in cells, raising the possibility that siRNA can be employed as a therapy for treating cancers and other genetic diseases. However, siRNA transfection has the limitation due to the difficulty in the delivery of siRNA to target cells and tissues. To explore an efficient biocompatible siRNA delivery system, cationic diphenylalanine vesicles(CDPVs) were constructed to transfer survivin siRNA to human ovarian cancer cells. The morphology of CDPVs was characterized by scanning electron microscopy(SEM) and the distribution of survivin siRNA was characterized by confocal laser scanning microscopy, which reveal that diphenylalanine and the survivin siRNA were successfully co-delivered. After co-incubation for 48 h, the CDPVs/siRNA exhibited enhanced tumor cell growth inhabitation and apoptosis inducted in human SK-OV-3 ovarian carcinoma cells. Overall, CDPVs is an efficient siRNA delivery system and has a promising prospect for cancer therapy.

Tumor-specific delivery of therapeutic siRNAs by anti-EGFR immunonanoparticles.

BackgroundEfficient target-specific siRNA delivery has always been a primary concern in the field of siRNA clinical application.PurposeIn this study, four different types of anti-epidermal growth factor receptor (EGFR) antibody-conjugated immunonanoparticles were prepared and tested for cancer cell-targeted therapeutic siRNA delivery.Materials and methodsThe prepared nanoparticles encapsulating siRNAs were character-ized by gel retardation and particle analysis using a Zetasizer. In vitro transfection and reduction of target genes, vimentin and JAK3, were determined using quantitative reverse transcription polymerase chain reaction. In vivo tumor targeting and antitumoral efficacies of the nanoparticles were evaluated in mice carrying tumors.ResultsAmong these immunonanoparticles, anti-EGFR immunolipoplexes and immunoviroplexes exhibited remarkable cell binding and siRNA delivery to EGFR-expressing tumor cells compared to immunoliposomes and immunovirosomes. Especially, the anti-EGFR immunoviroplexes exhibited the most efficient siRNA transfection to target tumor cells. Therefore, antitumoral vimentin and Janus kinase-3 siRNAs were loaded in the anti-EGFR immunolipoplexes and immunoviroplexes, which were tested in mice carrying SK-OV-3 tumor xenografts. In fact, the therapeutic siRNAs were efficiently delivered to the tumor tissues by both delivery vehicles, resulting in significant inhibition of tumor growth. Moreover, administration of doxorubicin in combination with anti-EGFR immunoviroplexes resulted in remarkable and synergistic tumor growth inhibition.ConclusionThis study provides experimental proof that cancer cell-targeted immunoviroplexes are an efficient siRNA delivery system for cancer therapy. Moreover, this study also suggests that a combination of conventional chemotherapy and tumor-directed anticancer siRNA therapy would be a better modality for cancer treatment.

Enhancing Gene-Knockdown Efficiency of Poly(N-isopropylacrylamide) Nanogels.

Polo-like-kinase 1 (PLK1), which is a serine–threonine protein kinase overexpressed in cancer cells, is known to regulate tumor growth and have recently gathered attention as a target gene for RNA interference because of the poor bioavailability and nonspecificity of the available inhibitors. However, the lower transfection efficiency of siRNA and its poor stability in biological mileu necessitate the need of efficient siRNA delivery systems. Here, we report efficacious polymeric nanoparticles for the delivery of PLK1 siRNA in mammalian cancer cells. N-Isopropylacrylamide (NIPAm) and N-isopropylmethacrylamide-co-NIPAm nanogels were synthesized and modified using poly-ε-lysine. Furthermore, their ability to induce gene silencing was investigated by flow cytometry and real-time polymerase chain reaction, and the silencing efficiency observed was related to the polymer composition and its effect on the gene loading and protection ability and the endosomal escape capability. This study attempts to leverage the understanding of the cell–material interaction, thus, addressing the bottlenecks of siRNA delivery for enhancing the efficacy of the poly(N-isopropylacrylamide)-based delivery vehicle.

Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible Fluorinated Peptide Dendrimers for Cancer Therapy.

RNA interference (RNAi) has a great promise in treating various acquired and hereditary diseases. However, it remains highly desirable to develop new delivery system to circumvent complex extra- and intracellular barriers for successful clinical translation. Here, we report on a versatile polymeric vector, bioreducible fluorinated peptide dendrimers (BFPD), for efficient and safe small interfering RNA (siRNA) delivery. In virtue of skillfully integrating all of the unique advantages of reversible cross-linking, fluorination, and peptide dendrimers, this novel vector can surmount almost all extra- and intracellular barriers associated with local siRNA delivery through highly improved physiological stability and serum resistance, significantly increased intratumoral enrichment, cellular internalization, successful facilitation of endosomal escape, and cytosolic siRNA release. BFPD polyplexes, carrying small interfering vascular endothelial growth factor (siVEGF), demonstrated excellent VEGF silencing efficacy (∼65%) and a strong capability for inhibiting HeLa cell proliferation. More importantly, these polyplexes showed superior performance in long-term enrichment in the tumor sites and had a high level of tumor growth inhibition. Furthermore, these polyplexes not only exhibited excellent in vivo antitumor efficacy but also demonstrated superior biocompatibility, compared with LPF2000, both in vivo and in vitro. These findings indicate that BFPD is an efficient and safe siRNA delivery system and has remarkable potential for RNAi-based cancer treatment.

Conjugates of HA2 with octaarginine-grafted HPMA copolymer offer effective siRNA delivery and gene silencing in cancer cells

The key for successful gene silencing is to design a safe and efficient siRNA delivery system for the transfer of therapeutic nucleic acids into the target cells. Here, we describe the design of hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer displaying multiple copies of octaarginine (R8) and its use in promoting the effective delivery of small interfering RNA (siRNA) molecules intracellularly. Fluorescein-5-isothiocyanate (FITC)-labeled HPMA copolymer-bound R8 (P-R8-FITC) was synthesized with increasing R8 molar ratios (4–9.5mol-%) to define the optimal R8 content that allowed the polymer to serve both as a siRNA-binding domain and as an intracellular transduction moiety mediating improved cellular delivery. A subunit of the influenza virus hemagglutinin (HA2), known for its ability to disrupt endosomal membranes, was further conjugated to P-R8-FITC copolymer to promote endosomal escape. Of the different P-(R8)-FITC conjugates considered, only that polymer containing the highest mol-% of R8 (P-(R8)9.5-FITC) was able to encapsulate siRNA molecules into nano-sized polyion complexes (PICs) presenting positive surface charge, low in vitro cytotoxicity, and high serum stability. P-(R8)9.5-FITC/cy5-siRNA complexes can efficiently deliver siRNA molecules into cells, while naked siRNA or siRNA encapsulated within polymers with lower R8mol-% were unable to transfect the same cells. Conjugation of HA2 fusogenic peptide to P-(R8)-FITC significantly decreased the oncogenic RAC1 mRNA levels in cancer cells. This indicates that P-(R8)-(HA2)-FITC can deliver siRNA into target cells, and that the siRNA can reach the perinuclear region where it interacts with the RNA-induced silencing complex.

Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol–Schiff base–polyethylene glycol (Chol–SIB–PEG)-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol–SIB–PEG was successfully synthesized and confirmed with FTIR and 1H-NMR. An Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application.

N-Isopropylacrylamide Modified Polyethylenimines as Effective siRNA Carriers for Cancer Therapy.

N-isopropylacrylamide modified PEI (PEN) was synthesized via Michael addition and was developed as an efficient siRNA delivery system both in vitro and in vivo. PEN showed significant enhanced cytocompatibility compared with commercial PEI-25k. The complexation of PEN with siRNA was studied by gel retardation, particle size and zeta potential measurement. The in vitro transfection ability of PEN was measured by qRT-PCR assay, and achieved obviously enhanced gene silencing efficiency compared with PEI-25k. The confocal imaging and flow cytometric analysis further validated its excellent intracellular trafficking ability. For antitumor treatment experiment, PEN mediated siVEGF showed obviously therapeutic effects for the treatment of CT26 tumor. Therefore, the present study demonstrated a useful strategy for constructing efficient siRNA delivery vehicles for antitumor therapy.

Targeted delivery of siRNA to activated T cells via transferrin-polyethylenimine (Tf-PEI) as a potential therapy of asthma

Asthma is a worldwide health problem. Activated T cells (ATCs) in the lung, particularly T helper 2 cells (Th2), are strongly associated with inducing airway inflammatory responses and chemoattraction of inflammatory cells in asthma. Small interfering RNA (siRNA) as a promising anti-sense molecule can specifically silence inflammation related genes in ATCs, however, lack of safe and efficient siRNA delivery systems limits the application of siRNA as a therapeutic molecule in asthma. Here, we designed a novel pulmonary delivery system of siRNA, transferrin-polyethylenimine (Tf-PEI), to selectively deliver siRNA to ATCs in the lung. Tf-PEI polyplexes demonstrated optimal physicochemical properties such as size, distribution, zeta-potential, and siRNA condensation efficiency. Moreover, in vitro studies showed significantly enhanced cellular uptake and gene knockdown mediated by Tf-PEI polyplexes in human primary ATCs. Biodistribution of polyplexes in a murine asthmatic model confirmed that Tf-PEI polyplexes can efficiently and selectively deliver siRNA to ATCs. In conclusion, the present work proves the feasibility to target ATCs in asthma via Tf receptor. This strategy could potentially be used to design an efficient siRNA delivery system for asthma therapy.

Receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient delivery system for MRTF silencing in conjunctival fibrosis

There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye.

Receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent a novel and efficient siRNA delivery system to prevent conjunctival fibrosis

PurposeGlaucoma is the leading cause of irreversible blindness worldwide and fibrosis is the main cause of failure of glaucoma surgery. We have previously described how the Myocardin‐related transcription factor/Serum response factor (MRTF/SRF) pathway is intricately linked to all the key pathways in ocular fibrosis. Our aim was to develop a novel liposome‐peptide‐siRNA (LYR) nanoparticle as an efficient delivery system for MRTF siRNA in conjunctival fibrosis.MethodsThe LYR nanoparticles were characterised with regard to particle size and zeta potential. Real‐time qPCR and western blotting were used to compare the silencing efficiency in human Tenon's fibroblasts using different MRTF siRNA concentrations, targeting peptides, and liposomes. The cytotoxicity of the LYR nanoparticles was assessed using the MTT cell assay. Three‐dimensional fibroblast‐populated collagen matrices were also used as a functional assay to measure contraction in vitro.ResultsAll LYR nanoparticles were strongly cationic with sizes around 100 nm and PDIs &lt; 0.4. The LYR nanoparticles efficiently silenced the MRTF gene by 76% and 84% using 50 nM and 100 nM siRNA respectively. The MRTF gene was also efficiently silenced by 76% and 75% using the targeting peptides Y and ME27 respectively. The MRTF protein expression was significantly decreased by the LYR nanoparticles. The non‐PEGylated liposome formulations showed higher silencing efficiency than the cationic PEGylated formulations. The MRTF nanoparticles were also not cytotoxic at 50 nM siRNA concentration and prevented matrix contraction after a single transfection treatment.ConclusionsThis is the first study to show that receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent an efficient and safe siRNA delivery system that could be used to prevent fibrosis after glaucoma surgery.