Effect of polymer structure on micelles formed between siRNA and cationic block copolymer comprising thiols and amidines

Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.     

Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro

Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.     

Synthesis and in vitro characterization of an ABC triblock copolymer for siRNA delivery

The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential in therapy and in basic science. However, delivery systems capable of efficient and biocompatible delivery of siRNA to target cells are not yet satisfactory. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assemble with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), and a positively charged peptide (PEG-PPS-peptide). The diblock copolymer PEG(45)-PPS(5,10) was synthesized using anionic polymerization of propylene sulfide upon a PEG macroinitiator, and the peptide domain was coupled to the PPS terminus using a disulfide exchange reaction with an N-terminal cysteine residue on the peptide. The peptides were designed to interact electrostatically with siRNA, selecting the TAT peptide domain of HIV (RKKRRQRRR) and an oligolysine (Lys(9)). The resulting triblock copolymers were able to self-assemble with siRNA as demonstrated by dynamic light scattering and gel electrophoresis. Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic PPS block, achieving sizes ranging from 171 nm to 601 nm. Cell internalization and gene expression down-regulation studies showed that the triblock copolymers are able to transport siRNA inside the cell and mediate gene expression down-regulation, with the amount of internalization and gene transfer affected by charge ratio, PPS length, and the presence of serum. The proposed triblock was able to mediate gene expression down-regulation of GAPDH, achieving up to 90.5% +/- 0.02% down-regulation.     

Synthesis of folate-functionalized RAFT polymers for targeted siRNA delivery

Receptor-mediated, cell-specific delivery of siRNA enables silencing of target genes in specific tissues, opening the door to powerful therapeutic options for a multitude of diseases. However, the development of delivery systems capable of targeted and effective siRNA delivery typically requires multiple steps and the use of sophisticated, orthogonal chemistries. Previously, we developed diblock copolymers consisting of dimethaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-co-propylacrylic acid as potent siRNA delivery systems that protect siRNA from enzymatic degradation and enable its cytosolic delivery through pH-responsive, endosomolytic behavior. (1, 2) These architectures were polymerized using a living radical polymerization method, specifically reversible addition-fragmentation chain transfer (RAFT) polymerization, which employs a chain transfer agent (CTA) to modulate the rate of reaction, resulting in polymers with low polydispersity and telechelic chain ends reflecting the chemistry of the CTA. Here we describe the straightforward, facile synthesis of a folate receptor-targeted diblock copolymer siRNA delivery system because the folate receptor is an attractive target for tumor-selective therapies as a result of its overexpression in a number of cancers. Specifically, we detail the de novo synthesis of a folate-functionalized CTA, use the folate-CTA for controlled polymerizations of diblock copolymers, and demonstrate efficient, specific cellular folate receptor interaction and in vitro gene knockdown using the folate-functionalized polymer.     

The PEI-introduced CS shell/PMMA core nanoparticle for silencing the expression of E6/E7 oncogenes in human cervical cells

In this study, we examined the potential of cationic nanoparticle - polyethyleneimine-introduced chitosan shell/poly (methyl methacrylate) core nanoparticles (CS-PEI) for siRNA delivery. Initially, DNA delivery was performed to validate the capability of CS-PEI for gene delivery in the human cervical cancer cell line, SiHa. siRNA delivery were subsequently carried out to evaluate the silencing effect on targeted E6 and E7 oncogenes. Physicochemical properties including size, zeta potential and morphology of CS-PEI/DNA and CS-PEI/siRNA complexes, were analyzed. The surface charges and sizes of the complexes were observed at different N/P ratios. The hydrodynamic sizes of the CS-PEI/DNA and CS-PEI/siRNA were approximately 300-400 and 400-500nm, respectively. Complexes were positively charged depending on the amount of added CS-PEI. AFM images revealed the mono-dispersed and spherical shapes of the complexes. Gel retardation assay confirmed that CS-PEI nanoparticles completely formed complexes with DNA and siRNA at a N/P ratio of 1.6. For DNA transfection, CS-PEI provided the highest transfection result. Localization of siRNA delivered through CS-PEI was confirmed by differential interference contrast (DIC) confocal imaging. The silencing effect of siRNA specific to HPV 16 E6/E7 oncogene was examined at 18 and 24h post-transfection. The results demonstrated the capacity of CS-PEI to suppress the expression of HVP oncogenes.     

Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence disease-related genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal-lysosomal escape of siRNA delivery systems at the endosomal-lysosomal pH. The surfactants formed compact nanoparticles (160-260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40-88% silencing of luciferase expression with 100 nM siRNA and 35-75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.     

Nanoscale Diblock Copolymer Micelles: Characterizations and Estimation of the Effective Diffusion Coefficients of Biomolecules Release through Cylindrical Diffusion Model

Biomolecules have been widely investigated as potential therapeutics for various diseases. However their use is limited due to rapid degradation and poor cellular uptake in vitro and in vivo. To address this issue, we synthesized a new nano-carrier system comprising of cholic acid-polyethylenimine (CA-PEI) copolymer micelles, via carbodiimide-mediated coupling for the efficient delivery of small interfering ribonucleic acid (siRNA) and bovine serum albumin (BSA) as model protein. The mean particle size of siRNA- or BSA-loaded CA-PEI micelles ranged from 100–150 nm, with zeta potentials of +3-+11 mV, respectively. Atomic force, transmission electron and field emission scanning electron microscopy demonstrated that the micelles exhibited excellent spherical morphology. No significant morphology or size changes were observed in the CA-PEI micelles after siRNA and BSA loading. CA-PEI micelles exhibited sustained release profile, the effective diffusion coefficients were successfully estimated using a mathematically-derived cylindrical diffusion model and the release data of siRNA and BSA closely fitted into this model. High siRNA and BSA binding and loading efficiencies (95% and 70%, respectively) were observed for CA-PEI micelles. Stability studies demonstrated that siRNA and BSA integrity was maintained after loading and release. The CA-PEI micelles were non cytotoxic to V79 and DLD-1 cells, as shown by alamarBlue and LIVE/DEAD cell viability assays. RT-PCR study revealed that siRNA-loaded CA-PEI micelles suppressed the mRNA for ABCB1 gene. These results revealed the promising potential of CA-PEI micelles as a stable, safe, and versatile nano-carrier for siRNA and the model protein delivery.     

Vasopressin V2R-targeting peptide carrier mediates siRNA delivery into collecting duct cells

Internalization of receptor proteins after interacting with specific ligands has been proposed to facilitate siRNA delivery into the target cells via receptor-mediated siRNA transduction. In this study, we demonstrated a novel method of vasopressin V2 receptor (V2R)-mediated siRNA delivery against AQP2 in primary cultured inner medullary collecting duct (IMCD) cells of rat kidney. We synthesized the dDAVP conjugated with nine D-arginines (dDAVP-9r) as a peptide carrier for siRNA delivery. The structure of synthetic peptide carrier showed two regions (i.e., ligand domain to V2R (dDAVP) and siRNA carrying domain (nine D-arginine)) bisected with a spacer of four glycines. The results revealed that 1) synthesized dDAVP-9r peptides formed a stable polyplex with siRNA; 2) siRNA/dDAVP-9r polyplex could bind to the V2R of IMCD cells and induced AQP2 phosphorylation (Ser 256); 3) siRNA/dDAVP-9r polyplex was stable in response to the wide range of different osmolalities, pH levels, or to the RNases; 4) fluorescein-labeled siRNA was delivered into V2R-expressing MDCK and LLC-PK1 cells by siRNA/dDAVP-9r polyplex, but not into the V2R-negative Cos-7 cells; and 5) AQP2-siRNA/dDAVP-9r polyplex effectively delivered siRNA into the IMCD cells, resulting in the significant decrease of protein abundance of AQP2, but not AQP4. Therefore, for the first time to our knowledge, we demonstrated that V2R-mediated siRNA delivery could be exploited to deliver specific siRNA to regulate abnormal expression of target proteins in V2R-expressing kidney cells. The methods could be potentially used in vivo to regulate abnormal expression of proteins associated with disease conditions in the V2R-expressing kidney cells.     

Amphiphilic peptides with arginine and valine residues as siRNA carriers

An efficient and safe delivery carrier is required for the therapeutic application of siRNA. In this research, amphiphilic peptides with arginine and valine residues were evaluated as siRNA carriers. The peptides were composed of 1-4 arginine-blocks and 6 valine-blocks. In the aqueous solution, the arginine-valine peptides (RV peptides) formed micelles with hydrophobic cores comprised of a valine block and a cationic surface comprised of an arginine block. In a gel retardation assay, the RV peptides completely retarded siRNA at a 1:10 weight ratio (siRNA:peptide). A heparin competition assay suggested that the RV peptides formed more stable complexes with siRNA than they did with polyethylenimine (25 kDa, PEI25k). In an in vitro silencing assay, a dual luciferase expression (Renilla and firefly luciferases) vector, psiCHECK2, was co-transfected into human embryonic kidney 293 cells with Renilla-siRNA using the RV peptides. The specific silencing effect of Renilla luciferase was analyzed in reference to firefly luciferase. The results showed that the R3V6 peptide was more efficient than the R1V6, R2V6, and R4V6 peptides in silencing Renilla luciferase. In the flow cytometry and in vitro silencing studies, the R3V6 peptide delivered Renila-siRNA as efficiently as PEI25k. The siVEGF/R3V6 peptide also reduced endogenous vascular endothelial growth factor (VEGF) expression in CT27 cells as efficiently as PEI25k. A cytotoxicity assay showed that RV peptides did not cause any cytotoxicity. Therefore, RV peptides may be useful for the development of a safe and efficient delivery carrier of siRNA.     

SiRNA delivery systems based on neutral cross-linked dendrimers

A neutral cross-linked dendritic system is described for use of delivering small interfering RNA (siRNA) for targeted gene silencing. By replacing the terminal amines with hydrazide groups and N-acetylgalactosamine (GalNAc) ligands, cationic polyamidoamine (PAMAM) dendrimers were transformed into neutral glycosylated carriers for siRNA delivery. Mainly owing to the pH sensitivity and the proton-absorption capability of the tertiary amines within the interior branches, these PAMAM derivatives showing neutrality under physiological conditions (pH 7.4) formed complexes with siRNA through electrostatic interactions at pH 5. Cross-linking procedures via reactions with glutaraldehyde were established, and cytocompatible cross-linked systems loaded with siRNA obtained, with the particulate properties variable with the cross-linking condition and the GalNAc level in the dendritic carrier. In vitro cellular uptake and RNAi experiments showed that the cross-linked dendritic systems with an appropriate level of GalNAc composition effectively interacted with HepG2 cells and inhibited the expression of a luciferase reporter gene. Neutral cross-linked dendritic systems provide a new paradigm for designing siRNA delivery systems with biocompatibility and targeting capability.     

siRNA delivery using amphipathic cell-penetrating peptides into human hepatoma cells

Cell-penetrating peptides (CPPs) are an attractive tool for delivering membrane-impermeable compounds, including anionic biomacromolecules such as DNA and RNA, into living cells. Amphipathic helical peptides composed of hydrophobic amino acids and cationic amino acids are typical CPPs. In the current study, we designed amphipathic helical 12-mer peptides containing α,α-disubstituted α-amino acids (dAAs), which are known to stabilize peptide secondary structures. The dominant secondary structures of peptides in aqueous solution differed according to the introduced dAAs. Peptides containing hydrophobic dAAs and adopting a helical structure exhibited a good cell-penetrating ability. As an application of amphipathic helical peptides, small interfering RNA (siRNA) delivery into living human hepatoma cells was investigated. One of the peptides containing dAAs dipropylglycine formed stable complexes with siRNA at appropriate zeta-potential and size for intracellular siRNA delivery. This peptide showed effective RNA interference efficiency at short peptide length and low concentrations of peptide and siRNA. These findings will be helpful for the design of amphipathic helical CPPs as intracellular siRNA delivery.     

Application of an HIV gp41-derived peptide for enhanced intracellular trafficking of synthetic gene and siRNA delivery vehicles

Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.     

Hyaluronic acid-polyethyleneimine conjugate for target specific intracellular delivery of siRNA

A novel target specific small interfering RNA (siRNA) delivery system was successfully developed using polyethyleneimine (PEI)-hyaluronic acid (HA) conjugate. Anti-PGL3-Luc siRNA was used as a model system suppressing the PGL3-Luc gene expression. The siRNA/PEI-HA complex with an average size of ca. 21 nm appeared to be formed by electrostatic interaction between the negatively charged siRNA and the positively charged PEI of PEI-HA conjugate. The cytotoxicity of siRNA/PEI-HA complex to B16F1 cells was lower than that of siRNA/PEI complex according to the MTT assay. When B16F1 and HEK-293 cells were treated with fluorescein isothiocyanate (FITC) labeled siRNA/PEI-HA complex, B16F1 cells, with a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), showed higher green fluorescent intensity than HEK-293 cells because of the HA receptor mediated endocytosis of the complex. Accordingly, the PGL3-Luc gene silencing of anti-PGL3-Luc siRNA/PEI-HA complex was more efficient in B16F1 cells than in HEK-293 cells. In addition, the inhibited PGL3-Luc gene silencing effect in the presence of free HA in the transfection medium revealed that siRNA/HA-PEI complex was selectively taken up to B16F1 cells via HA receptor mediated endocytosis. All these results demonstrated that the intracellular delivery of anti-PGL3-Luc siRNA/PEI-HA complex could be facilitated by the HA receptor mediated endocytosis.     

Impact of tumor-specific targeting and dosing schedule on tumor growth inhibition after intravenous administration of siRNA-containing nanoparticles

This study addresses issues of relevance for siRNA nanoparticle delivery by investigating the functional impact of tumor-specific targeting and dosing schedule. The investigations are performed using an experimental system involving a syngeneic mouse cancer model and a theoretical system based on our previously described mathematical model of siRNA delivery and function. A/J mice bearing subcutaneous Neuro2A tumors approximately 100 mm(3) in size were treated by intravenous injection with siRNA-containing nanoparticles formed with cyclodextrin-containing polycations (CDP). Three consecutive daily doses of transferrin (Tf)-targeted nanoparticles carrying 2.5 mg/kg of two different siRNA sequences targeting ribonucleotide reductase subunit M2 (RRM2) slowed tumor growth, whereas non-targeted nanoparticles were significantly less effective when given at the same dose. Furthermore, administration of the three doses on consecutive days or every 3 days did not lead to statistically significant differences in tumor growth delay. Mathematical model calculations of siRNA-mediated target protein knockdown and tumor growth inhibition are used to elucidate possible mechanisms to explain the observed effects and to provide guidelines for designing more effective siRNA-based treatment regimens regardless of delivery methodology and tumor type.     

Monomolecular assembly of siRNA and poly(ethylene glycol)-peptide copolymers

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.     

Hyaluronic acid complexed to biodegradable poly L‐arginine for targeted delivery of siRNAs

Background

Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively‐charged HA and cationic poly L ‐arginine (PLR) for siRNA delivery systems.

Methods

Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA.

Results

Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP‐mediated delivery of siRNAs was highest in WM266.4 cells followed by B16F10 cells and COS‐7 cells, in parallel with CD44 surface densities of these cell lines. TC₅₀ values (i.e. the HP concentrations at which 50% of cells were viable after treatment) were used as indicators of cytotoxicity. HP101 showed TC₅₀ values that were 2‐fold and 23‐fold higher than those of HP110 and PLR, respectively. After delivery into cells, siRNA exerted target‐specific RNA interference effects on mRNA and protein levels. Three days after treatment of red fluorescent protein (RFP)‐expressing B16F10 cells with RFP‐specific siRNA complexed to HP101, cellular fluorescence signals were reduced. Intratumoral administration of RFP‐specific siRNA via HP101 delivery significantly reduced the expression of RFP in tumor tissues.

Conclusions

HP101 may function as a biocompatible polymeric carrier of siRNAs and have possible application to localized siRNA delivery in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Physicochemical and biological characterization of targeted, nucleic acid-containing nanoparticles

Nucleic acid-based therapeutics have the potential to provide potent and highly specific treatments for a variety of human ailments. However, systemic delivery continues to be a significant hurdle to success. Multifunctional nanoparticles are being investigated as systemic, nonviral delivery systems, and here, we describe the physicochemical and biological characterization of cyclodextrin-containing polycations (CDP) and their nanoparticles formed with nucleic acids including plasmid DNA (pDNA) and small interfering RNA (siRNA). These polycation/nucleic acid complexes can be tuned by formulation conditions to yield particles with sizes ranging from 60 to 150 nm, zeta potentials from 10 to 30 mV, and molecular weights from approximately 7 x 107 to 1 x 109 g mol-1 as determined by light scattering techniques. Inclusion complexes formed between adamantane (AD)-containing molecules and the beta-cyclodextrin molecules enable the modular attachment of poly(ethylene glycol) (AD-PEG) conjugates for steric stabilization and targeting ligands (AD-PEG-transferrin) for cell-specific targeting. A 70 nm particle can contain approximately 10 000 CDP polymer chains, approximately 2000 siRNA molecules, approximately 4000 AD-PEG5000 molecules, and approximately 100 AD-PEG5000-Tf molecules; this represents a significant payload of siRNA and a large ratio of siRNA to targeting ligand (20:1). The particles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application. Uptake of the nucleic acid-containing particles by HeLa cells is measured by flow cytometry and visualized by confocal microscopy. Competitive uptake experiments show that the transferrin-targeted particles display enhanced affinity for the transferrin receptor through avidity effects (multiligand binding). Functional efficacy of the delivered pDNA and siRNA is demonstrated through luciferase reporter protein expression and knockdown, respectively. The analysis of the CDP delivery vehicle provides insights that can be applied to the design of targeted nucleic acid delivery vehicles in general.     

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

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.

Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50?mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.

Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.

Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.     

Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing N-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000.     

Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA

To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to α_Vβ₃ integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the α_Vβ₃ integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0 μM PI/10 nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA.