Delivery Of Small Interfering RNA Research Articles

Poly lactic-co-glycolic acid-modified Fe3O4 nanoclusters for small interfering RNA delivery

Objective To search for a safer and controlled released in vivo gene vector to replace previous viral vectors with the development of RNAi technology in gene therapy.Methods Poly lactic-coglycolic acid (PLGA)-modified Fe3 O4 nanoclusters loaded with small interfering RNA (siRNA) were synthesized via one-pot methods.The obtained siRNA nanoclusters were characterized by scanning election microscopy (SEM),TEM and XRD.Their abilities of protecting and releasing siRNA were determined by gel electrophoresis and Quant-iTTM PicogreenTM assay in vitro.Results A novel siRNA delivery vehicle of PLGA-modified magnetic nanoclusters was successfully synthesized and exhibited excellent abilities of protecting and releasing siRNA in vitro.Conclusion A low cytotoxicity,and highly dispersible and stable PLGA-modified Fe3O4 cluster nanocarrier for siRNA delivery was synthesized via a one-pot reaction.These as-prepared siRNA nanoclusters could encapsulate and protect large amounts of siRNA,and demonstrate favorable siRNA release kinetics in vitro. Key words: Nanomaterials; Magnetic materials; Small interfering RNA; Gene therapy

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.

Cationic Dendron-Bearing Lipids: Investigating Structure–Activity Relationships for Small Interfering RNA Delivery

A new family of cationic dendron-bearing lipids (CDLs) with poly(amidoamine) dendrons of first to third generation (named as A1, A2, and A3, respectively) was synthesized through a synthesis approach that permits facile variation of chemical structures. All CDLs could effectively bind small interfering RNA (siRNA) to form complexes confirmed by gel retardation analysis. In in vitro transfection experiments, A1/siRNA complexes exhibited significant gene silencing efficiency close to Lipofectamine 2000/siRNA complexes and much higher than A2/siRNA and A3/siRNA complexes. To reveal the underlying reason, we performed a series of experimental methods. The results suggested that the CDLs with smaller dendron sizes and higher proportion of hydrophobic segments could bind siRNA to form dendriplex aggregates with more compact structures and higher surface potentials. Therefore, they could be internalized via endocytosis more easily, which was believed to be the main reason for higher gene silencing efficiency. This paper provides an efficient CDL (A1) for siRNA delivery and indicates great potential for gene therapy.

Small‐interfering RNAs (siRNAs) as a promising tool for ocular therapy

RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments.

A novel peptide carrier for efficient targeting of antigens and nucleic acids to dendritic cells

Dendritic cells (DCs) initiate host immune responses by presenting captured antigens to naive T cells. Hence, DC-binding peptides may be used for antigen targeting to boost naive and memory immune responses. By biopanning peptide phage libraries on human monocyte-derived DCs, we identified novel DC-binding peptides. One of the selected phages, displaying the NW peptide (NWYLPWLGTNDW), bound DCs with high affinity, and its binding was inhibited by the corresponding synthetic peptide. Antigenic peptides or proteins conjugated to the NW peptide bound to DCs and were internalized without negative effects on DC phenotype and function. Ex vivo targeted delivery of CMV-pp65 peptides to DCs via the NW peptide increased T-cell responses in HLA-A2(+)/CMV(+) donors compared to untargeted peptides (P<0.001). Stimulation of CD45RO-depleted peripheral blood mononuclear cells from CMV(-) donors with the NW-pp65 fusion peptides expanded pp65-specific precursor T cells. Moreover, the NW peptide mediated small interfering RNA delivery to DCs, and a significant gene silencing was obtained. Collectively, the data reveal that proteins and nucleic acids can be directed to DCs through the NW peptide, enabling effective uptake and functional effects such as T-cell activation in the context of MHC class I and II molecules.

Enhanced cancer immunotherapy using nanoparticle-based modulation of SOCS1 genes in dendritic cells (P4242)

Abstract Although dendritic cells have important roles in cancer immunotherapy, their therapeutic efficiencies were limited due to the presence of immunosuppressive factors. Here, we report the fabrication and use of polymer nanoparticles for the simultaneous delivery of antigens and small interference RNA (siRNA) of immune suppressor genes into dendritic cells. The polymer nanoparticles containing ovalbumin (OVA, as a model antigen) and siRNA of suppressor of cytokine signaling 1 (SOCS1) were fabricated by double emulsion solvent evaporation method. The encapsulation of siRNA was about 57.6 % when OVA was present during the preparation, while that was only 2 % without the OVA. The polymer nanoparticles containing OVA and siRNA of SOCS1 were efficiently taken up by the dendritic cells and showed no detectable toxicity. The knockdown of SOCS1 genes in dendritic cells induced pro-inflammatory and anti-inflammatory cytokine expression. The polymer nanoparticles are expected to be potent nanotechnology platform for targeted delivery of siRNA and antigens into dendritic cells, and use as CD8+ T cells-based cancer immunotherapy.

Exploring polymeric micelles for improved delivery of anticancer agents: recent developments in preclinical studies.

As versatile drug delivery systems, polymeric micelles have demonstrated particular strength in solubilizing hydrophobic anticancer drugs while eliminating the use of toxic organic solvents and surfactants. However, the true promise of polymeric micelles as drug carriers for cancer therapy resides in their potential ability to preferentially elevate drug exposure in the tumor and achieve enhanced anticancer efficacy, which still remains to be fully exploited. Here, we review various micellar constructs that exhibit the enhanced permeation and retention effect in the tumor, the targeting ligands that potentiate the anticancer efficacy of micellar drugs, and the polyplex micelle systems suitable for the delivery of plasmid DNA and small interference RNA. Together, these preclinical studies in animal models help us further explore polymeric micelles as emerging drug carriers for targeted cancer therapy.

A novel dendritic nanocarrier of polyamidoamine-polyethylene glycol-cyclic RGD for &amp;ldquo;smart&amp;rdquo; small interfering RNA delivery and in vitro antitumor effects by human ether-&amp;agrave;-go-go-related gene silencing in anaplastic thyroid carcinoma cells

The application of RNA interference techniques is promising in gene therapeutic approaches, especially for cancers. To improve safety and efficiency of small interfering RNA (siRNA) delivery, a triblock dendritic nanocarrier, polyamidoamine-polyethylene glycol-cyclic RGD (PAMAM-PEG-cRGD), was developed and studied as an siRNA vector targeting the human ether-à-go-go-related gene (hERG) in human anaplastic thyroid carcinoma cells. Structure characterization, particle size, zeta potential, and gel retardation assay confirmed that complete triblock components were successfully synthesized with effective binding capacity of siRNA in this triblock nanocarrier. Cytotoxicity data indicated that conjugation of PEG significantly alleviated cytotoxicity when compared with unmodified PAMAM. PAMAM-PEG-cRGD exerted potent siRNA cellular internalization in which transfection efficiency measured by flow cytometry was up to 68% when the charge ratio (N/P ratio) was 3.5. Ligand-receptor affinity together with electrostatic interaction should be involved in the nano-siRNA endocytosis mechanism and we then proved that attachment of cRGD enhanced cellular uptake via RGD-integrin recognition. Gene silencing was evaluated by reverse transcription polymerase chain reaction and PAMAM-PEG-cRGD-siRNA complex downregulated the expression of hERG to 26.3% of the control value. Furthermore, gene knockdown of hERG elicited growth suppression as well as activated apoptosis by means of abolishing vascular endothelial growth factor secretion and triggering caspase-3 cascade in anaplastic thyroid carcinoma cells. Our study demonstrates that this novel triblock polymer, PAMAM-PEG-cRGD, exhibits negligible cytotoxicity, effective transfection, “smart” cancer targeting, and therefore is a promising siRNA nanocarrier.

Comparison of anti-EGFR-Fab&amp;rsquo; conjugated immunoliposomes modified with two different conjugation linkers for siRNA delivery in SMMC-7721 cells

BackgroundTargeted liposome-polycation-DNA complex (LPD), mainly conjugated with antibodies using functionalized PEG derivatives, is an effective nanovector for systemic delivery of small interference RNA (siRNA). However, there are few studies reporting the effect of different conjugation linkers on LPD for gene silencing. To clarify the influence of antibody conjugation linkers on LPD, we prepared two different immunoliposomes to deliver siRNA in which DSPE-PEG-COOH and DSPE-PEG-MAL, the commonly used PEG derivative linkers, were used to conjugate anti-EGFR Fab’ with the liposome.MethodsFirst, 600 μg of anti-EGFR Fab’ was conjugated with 28.35 μL of a micelle solution containing DSPE-PEG-MAL or DSPE-PEG-COOH, and then post inserted into the prepared LPD. Various liposome parameters, including particle size, zeta potential, stability, and encapsulation efficiency were evaluated, and the targeting ability and gene silencing activity of TLPD-FPC (DSPE-PEG-COOH conjugated with Fab’) was compared with that of TLPD-FPM (DSPE-PEG-MAL conjugated with Fab’) in SMMC-7721 hepatocellular carcinoma cells.ResultsThere was no significant difference in particle size between the two TLPDs, but the zeta potential was significantly different. Further, although there was no significant difference in siRNA encapsulation efficiency, cell viability, or serum stability between TLPD-FPM and TLPD-FPC, cellular uptake of TLPD-FPM was significantly greater than that of TLPD-FPC in EGFR-overexpressing SMMC-7721 cells. The luciferase gene silencing efficiency of TLPD-FPM was approximately three-fold high than that of TLPD-FPC.ConclusionDifferent conjugation linkers whereby antibodies are conjugated with LPD can affect the physicochemical properties of LPD and antibody conjugation efficiency, thus directly affecting the gene silencing effect of TLPD. Immunoliposomes prepared by DSPE-PEG-MAL conjugation with anti-EGFR Fab’ are more effective than TLPD containing DSPE-PEG-COOH in targeting hepatocellular carcinoma cells for siRNA delivery.

The Development of Ternary Nanoplexes for Efficient Small Interfering RNA Delivery

Targeted posttranscriptional gene silencing by RNA interference (RNAi) has garnered considerable interest as an attractive new class of drugs for several diseases, such as cancer. Chitosan and protamine are commonly used as a vehicle to deliver and protect small interfering RNA (siRNA), but the strong interaction still remains to be modulated for efficient siRNA uptake and silencing. Therefore, in this study, ternary nanoplexes containing chitosan and protamine were designed to substantially enhance the siRNA efficiency. Binary and ternary nanoplexes were prepared at different the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of siRNA (N/P) ratios and characterized in terms of size, zeta potential, morphology and serum stability. The silencing efficiencies and cytotoxicities of prepared nanoplexes were evaluated by enzyme-linked immunosorbent assay (ELISA) (for human vascular endothelial growth factor; hVEGF) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. The mean diameter of ternary nanoplexes ranged from 151 to 282 nm, depending on the weight ratio between polymers and siRNA. The gene silencing effect after transfection with ternary nanoplexes (chitosan/siRNA/protamine 83%) was significantly higher than that with binary nanoplexes (chitosan/siRNA 71% and protamine/siRNA 74%). Ternary nanoplexes showed the highest cellular uptake ability when compared with binary nanoplexes. Ternary nanoplexes did not induce substantial cytotoxicity. Serum stability and the lack of cytotoxicity of the nanoplexes provided advantages over other gene silencing studies. These results suggest ternary nanoplexes have the potential to be an effective siRNA carrier to study the gene silencing effect.

Nanoscale Small Interfering RNA Delivery Systems For Personalized Cancer Therapy

Summary: Nanoparticles represent particularly attractive delivery systems for small interfering RNA (siRNA) and may provide the foundation for rational design and formulation of RNAi-triggering nanomedicines. siRNA can be delivered with a therapeutic intent using lipid-based delivery platforms such as stable nucleic acid lipid particles (SNALP) with a lipid bilayer containing cationic as well as fusogenic lipids and a diffusible PEG-lipid coat, polymers, cationic complexes, recombinant fusion proteins, conjugates, or polyconjugates. Several investigators have reported preclinical or early clinical proof of concept studies demonstrating that systemic delivery of siRNA nanoparticles targeting specific gene transcripts can elicit biologic responses. Therapeutic nanoparticles containing siRNA targeting specific genes that contribute to the aggressiveness and/or radiochemotherapy resistance of cancer cells may facilitate a paradigm shift in modern cancer therapy.

Potent Gene Silencing In Vitro at Physiological pH Using Chitosan Polymers

Among non-viral cationic polymers, biodegradable chitosan has during the last decade become an attractive carrier for small interference RNA (siRNA) delivery. Currently, degradation of macromolecules in the lysosomes is assumed to be a major barrier for effective siRNA transfection. Hence, transfection protocols are focused toward endosomal release mechanisms. In this work, we have tested 3 novel chitosan polymers and their siRNA delivery properties in vitro. To obtain efficient gene silencing of our model gene, S100A4, various transfection parameters were investigated, such as pH, nitrogen/phosphate ratio, photochemical internalization (PCI), media for complex formation, and cell lines. Our results showed that 2 linear chitosan polymers demonstrated excellent siRNA gene silencing, better than Lipofectamine 2000. The silencing effect was achieved without PCI treatment, under physiological pH, and with no observable reduction in cell viability.

Unzipping and binding of small interfering RNA with single walled carbon nanotube: A platform for small interfering RNA delivery

In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, molecular dynamics (MD) simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, the complex is very stable which serves as one of the major mechanisms of siRNA delivery for biomedical applications. Since siRNA has to undergo unwinding process with the effect of RNA-induced silencing complex, our proposed delivery mechanism by SWCNT possesses potential advantages in achieving RNA interference.

A novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient siRNA delivery in breast cancer therapy: preparation and in vitro analysis

BackgroundSystemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs).MethodsAAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions.ResultsPEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection.ConclusionThe present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy.

Low molecular weight chitosan nanoparticulate system at low N:P ratio for nontoxic polynucleotide delivery

Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines.

Effective Plasmid DNA and Small Interfering RNA Delivery to Diseased Human Brain Microvascular Endothelial Cells

Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis.

Multifunctional fluorescent-magnetic polyethyleneimine functionalized Fe3O4–mesoporous silica yolk–shell nanocapsules for siRNA delivery

A facile, mild, environmentally friendly and reproducible strategy was used to fabricate the multifunctional fluorescent-magnetic polyethyleneimine functionalized Fe(3)O(4)-mesoporous silica yolk-shell nanocapsules for simultaneous fluorescent tracking and magnetically guided small interfering RNA delivery.

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.

Lipid-substituted polyethylenimine (PEI)-mediated therapeutic small interfering RNA delivery

Lipid-substituted polyethylenimine (PEI)-mediated therapeutic small interfering RNA delivery

Selective modification of HK peptides enhances siRNA silencing of tumor targets in vivo

Our research has focused on systemic delivery of small interference RNA (siRNA) by branched peptides composed of histidine and lysine. After studying several histidine-lysine (HK) peptides, one four-branched peptide, H3K(+H)4b, with a predominant repeating pattern of -HHHK-, was found to be an effective carrier of siRNA. Although the unmodified H3K(+H)4b carrier of siRNA targeting an oncogene was previously shown to have promise in a tumor-bearing mouse model, we sought to develop a more effective HK carrier of siRNA in this study. Our primary goal was to determine whether different ligand (cyclic RGD)-pegylation patterns on the H3K(+H)4b peptide affect siRNA delivery in vitro and in vivo. We compared the unmodified H3K(+H)4b with two modified H3K(+H)4b peptides for their ability to deliver siRNA in a tumor-bearing mouse model; one modified HK peptide, (RGD-PEG)(4)-H3K(+H)4b, had four cyclic RGD-polyethylene glycol (cRGD-PEG) conjugates per molecule, whereas the other peptide, (RGD-PEG)-H3K(+H)4b, had one cRGD-PEG per molecule. Although the modified HK peptides by themselves did not form stable nanoplexes with siRNA, combination of a highly charged unmodified HK peptide, H2K4b, with either of the modified HK peptides did form stable siRNA nanoparticles. For in vitro experiments with MDA-MB-435 cells that expressed luciferase (Luc), the H3K(+H)4b siRNA nanoplexes targeting Luc decreased its activity by 90% compared with negligible downregulation by the modified H3K(+H)4b nanoplexes (P<0.01). In contrast, the two modified H3K(+H)4b siRNA nanoplexes administered intravenously were more effective than the H3K(+H)4b nanoplexes in silencing Luc in a tumor xenograft model. The Luc activity in tumor lysates of mice administered H3K(+H)4b, (RGD-PEG)-H3K(+H)4b and (RGD-PEG)(4)-H3K(+H)4b nanoplexes decreased by 18, 35 and 75%, respectively. Thus, the siRNA nanoplex incorporating the highly modified peptide, (RGD-PEG)(4)-H3K(+H)4b, was the most effective at silencing its target in vivo (P<0.01). These studies demonstrate that selectively modified HK polymers are promising candidates for targeting oncogenes with siRNA.