Hiroyasu Takemoto

Polyion complex stability and gene silencing efficiency with a siRNA-grafted polymer delivery system

An siRNA-grafted polymer through disulfide linkage was prepared to improve the physicochemical properties and transfection efficacies of the polyion complexes (PICs) as a nanocarrier of siRNA. The siRNA-grafted polymer formed stable PICs due to its larger numbers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-grafted polymer showed a potent gene silencing effect without cytotoxicity or immunogenicity, demonstrating a promising feature of the siRNA-grafted polymer to construct the PIC-based nanocarrier for in vivo siRNA delivery.

Smart multilayered assembly for biocompatible siRNA delivery featuring dissolvable silica, endosome-disrupting polycation, and detachable PEG.

Multifunctional delivery systems of small interfering RNA (siRNA) are needed to overcome the intrinsic biological barriers toward efficient gene silencing in the cell cytoplasm. In this report, a smart multilayered assembly (SMA) was fabricated by a layer-by-layer method with polyionic materials. The SMA was designed to feature a siRNA-loaded core, a transiently core-stabilizing silica interlayer, an endosome-disrupting polycation interlayer, and a biocompatible poly(ethylene glycol) (PEG) shell with reductive environment-responsive detachability. The SMA was confirmed to be approximately 160 nm in size with narrow distribution and spherical morphology by DLS and TEM analyses. The PEG detachability of the SMA based on disulfide cleavage was also confirmed by the increase in both ζ-potential and size due to the exposure of the polycation interlayer and the compromised colloidal stability. The silica interlayer rendered the SMA highly tolerant to dissociation induced by anionic lipids, while after 24 h dialysis siRNA release from the SMA was clearly observed, presumably due to gradual dissolution of the silica interlayer based on the equilibrium shift to silicate ions. The entrapment ratio of siRNA delivered by the SMA within the endosome was significantly lower than that by nondisulfide control (NDC) without PEG detachability, suggesting the improved endosomal escape of SMA with the exposed, endosome-disrupting interlayer after PEG detachment. SMAs induced significantly higher gene silencing efficiency in various cultured cells, compared to NDC, without associated cytotoxicity. The systemic administration of SMAs for subcutaneous tumor-bearing mice achieved significant endogenous gene silencing in tumor tissue without hematological toxicity.

Precise Engineering of siRNA Delivery Vehicles to Tumors Using Polyion Complexes and Gold Nanoparticles

For systemic delivery of siRNA to solid tumors, a size-regulated and reversibly stabilized nanoarchitecture was constructed by using a 20 kDa siRNA-loaded unimer polyion complex (uPIC) and 20 nm gold nanoparticle (AuNP). The uPIC was selectively prepared by charge-matched polyionic complexation of a poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) copolymer bearing ∼40 positive charges (and thiol group at the ω-end) with a single siRNA bearing 40 negative charges. The thiol group at the ω-end of PEG-PLL further enabled successful conjugation of the uPICs onto the single AuNP through coordinate bonding, generating a nanoarchitecture (uPIC-AuNP) with a size of 38 nm and a narrow size distribution. In contrast, mixing thiolated PEG-PLLs and AuNPs produced a large aggregate in the absence of siRNA, suggesting the essential role of the preformed uPIC in the formation of nanoarchitecture. The smart uPIC-AuNPs were stable in serum-containing media and more resistant against heparin-induced counter polyanion exchange, compared to uPICs alone. On the other hand, the treatment of uPIC-AuNPs with an intracellular concentration of glutathione substantially compromised their stability and triggered the release of siRNA, demonstrating the reversible stability of these nanoarchitectures relative to thiol exchange and negatively charged AuNP surface. The uPIC-AuNPs efficiently delivered siRNA into cultured cancer cells, facilitating significant sequence-specific gene silencing without cytotoxicity. Systemically administered uPIC-AuNPs showed appreciably longer blood circulation time compared to controls, i.e., bare AuNPs and uPICs, indicating that the conjugation of uPICs onto AuNP was crucial for enhancing blood circulation time. Finally, the uPIC-AuNPs efficiently accumulated in a subcutaneously inoculated luciferase-expressing cervical cancer (HeLa-Luc) model and achieved significant luciferase gene silencing in the tumor tissue. These results demonstrate the strong potential of uPIC-AuNP nanoarchitectures for systemic siRNA delivery to solid tumors.

Systemic siRNA delivery to a spontaneous pancreatic tumor model in transgenic mice by PEGylated calcium phosphate hybrid micelles

Efficient systems for delivery of small interfering RNA (siRNA) are required for clinical application of RNA interference (RNAi) in cancer therapy. Herein, we developed a safe and efficient nanocarrier comprising poly(ethylene glycol)-block-charge-conversional polymer (PEG-CCP)/calcium phosphate (CaP) hybrid micelles for systemic delivery of siRNA and studied their efficacy in spontaneous bioluminescent pancreatic tumors from transgenic mice. PEG-CCP was engineered to provide the siRNA-loaded hybrid micelles with enhanced colloidal stability and biocompatibility due to the PEG capsule and with endosome-disrupting functionality due to the acidic pH-responsive CCP segment where the polyanionic structure could be converted to polycationic structure at acidic pH through cis-aconitic amide cleavage. The resulting hybrid micelles were confirmed to have a diameter of <50nm, with a narrow size distribution. Intravenously injected hybrid micelles significantly reduced the luciferase-based luminescent signal from the spontaneous pancreatic tumors in an siRNA sequence-specific manner. The gene silencing activity of the hybrid micelles correlated with their preferential tumor accumulation, as indicated by fluorescence imaging and histological analysis. Moreover, there were no significant changes in hematological parameters in mice treated with the hybrid micelles. These results demonstrate the great potential of the hybrid micelles as siRNA carriers for RNAi-based cancer therapy.

Acidic pH-responsive siRNA conjugate for reversible carrier stability and accelerated endosomal escape with reduced IFNα-associated immune response

Small interfering RNA (siRNA) has garnered much interest as a potential drug because of its strong gene-silencing activity. Toward the success in siRNA therapeutics, many strategies have been developed for efficient siRNA delivery into the cytosol of target cells. Among them, siRNA conjugates have arisen as one of the promising strategies in siRNA delivery, as siRNA can be readily conjugated to a functional molecule to acquire the ability of “programmed transfer” to the target sites. Indeed, several ligand molecules, such as lactose and RGD peptide, were conjugated with siRNA for site(or cell)-specific delivery. Furthermore, multimolecular siRNA conjugates enable stable polyion complex (PIC) formation because of the increased electrostatic interactions with polycations, leading to facilitated cellular uptake through charge neutralization of siRNA and also protection of siRNA from enzymatic degradations. However, those siRNA conjugates potentially stimulate immune responses through the activation of toll-like receptor 3 and/or protein kinase R, and thus they are desired to disintegrate into monomeric siRNAs (mono-siRNAs) in the cell for reduced immune responses. Meanwhile, considering that macromolecular drugs, including siRNA and its conjugates, would be taken up by cells through endocytosis and then delivered to the late endosome toward lysosomal degradation, siRNA needs to escape from the endosome into the cytosol for efficient gene silencing. Therefore, design of a smart siRNA conjugate for programmed endosomal escape and release of mono-siRNA is a great challenge for successful siRNA delivery. Herein, we developed a smart siRNA conjugate to fulfill the multifunctionality desired for enhanced siRNA delivery with reduced immunogenicity; that is, reversible PIC stability, endosomal escapability, and mono-siRNA releasability, based on a single chemical process. It is known that maleic acid amide (MAA) is relatively stable at extracellular neutral pH, while rapidly hydrolyzed at endosomal acidic pH. Thus, we utilized this MAA chemistry as an acid-labile anionic moiety for linking siRNA to an endosome-disrupting polycation and concurrently converting the cationic sites into a biologically inert anionic derivative. In design, the MAA-based conjugate is expected to improve the PIC stability through increased electrostatic interaction, while degrading the MAA moieties in the endosome for triggering three actions: 1) complex destabilization through unbalanced charges within PICs; 2) endosome disruption with the regenerated parent polycation; and 3) mono-siRNA release by MAA cleavage (Figure 1a). Figure 1b shows the chemical structure of siRNA-releasable/endosome-disrupting conjugate (REC), in which several siRNA molecules are grafted into the endosome-disrupting polymer side chains by the MAA linkage. The parent polycation is a polyaspartamide derivative with two repeating units of aminoethylene in each side chain (termed PAsp(DET)), which destabilizes the endosomal membrane integrity with the cationic diprotonated side chains to accelerate endosomal escape of the payload. A precursor polyanion was synthesized from PAsp(DET) to have a dibenzyl cyclooctyne (DBCO) group by MAA linkage as a conjugation site for siRNA. Then, an azidemodified siRNA (azide-siRNA) was reacted with the DBCO group in the polyanion side chains. Notably, the size exclusion chromatography (Supporting Information, Figure S5) confirmed that more than 95% of azide-siRNAs were conjugated to the polymer backbone utilizing a freeze–thaw treatment for the generation of a highly concentrated reactant phase. This successful conjugation at the quite high rate allows the use of the obtained conjugate without further purification. As a result, about 30 % of DBCO groups in the polymer side chains reacted with azide-siRNA; that is, about 5 siRNAs contained in the conjugate (Figure 1b). To investigate the [*] H. Takemoto, Dr. K. Kataoka Department of Materials Engineering, The University of Tokyo Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656 (Japan) E-mail: kataoka@bmw.t.u-tokyo.ac.jp Homepage: http://www.bmw.t.u-tokyo.ac.jp/

siRNA-Loaded Polyion Complex Micelle Decorated with Charge-Conversional Polymer Tuned to Undergo Stepwise Response to Intra-Tumoral and Intra-Endosomal pHs for Exerting Enhanced RNAi Efficacy

Small interfering RNA (siRNA) needs an efficient delivery vehicle to reach the cytoplasm of target cells for successful RNA interference (RNAi) therapy. This study aimed to develop an siRNA-loaded polyion complex (PIC) micelle equipped with a smart polymeric shell featuring tumor targetability and endosome escapability for enhanced RNAi activity in cancer cells. To this end, an acidic pH-responsive polypeptide was designed to exert a stepwise change in its charged state from negative to modestly positive and highly positive in response to slightly acidic environment of tumor (pH ∼6.7) and further lowered-pH condition of late endosomal compartments (pH ∼5.0), respectively, for selective binding to cancer cell surface and subsequent endosome disruption. This polypeptide, termed PAsp(DET-CDM/DBCO), was synthesized by introducing acid-labile carboxydimethyl maleate (CDM) and dibenzylcyclooctyne (DBCO) moieties into a polyaspartamide derivative bearing two-repeated aminoethylene side chains (PAsp(DET)). Then, PAsp(DET-CDM/DBCO) was installed on the surface of disulfide cross-linked PIC micelles prepared from cholesterol-modified siRNA (Chol-siRNA) and azide-poly(ethylene glycol)-b-poly[(3-mercaptopropylamidine)-L-lysine] (N3-PEG-b-PLys(MPA)) through the copper-free click reaction. Successful PAsp(DET-CDM/DBCO) coverage of PIC micelles was confirmed by a significant decrease in ζ-potential as well as a narrowly distributed size of 40 nm. The PAsp(DET-CDM/DBCO)-installed micelles significantly improved the gene-silencing efficiency in cultured lung cancer cells, compared with nonmodified control micelles, especially after incubation at pH 6.7. This improved silencing activity was nicely correlated with the facilitated cellular uptake of siRNA payloads at the acidic pH and the efficient endosomal escape. These results demonstrate that the acidic pH-responsive polypeptide shell is a promising design strategy for tumor-targeted siRNA delivery.

Enhanced transfection with silica-coated polyplexes loading plasmid DNA.

Silica-coating of positively charged polyplexes was demonstrated through silicic acid condensation to improve the polyplexes for enhanced complex stability and transfection efficiency. Silicic acid was efficiently condensed by polycations to form a silica network in the polyplex through electrostatic interaction and hydrogen bonding. The silica-coated (SC) polyplexes had an anionic surface charge of -20 mV and were 10-20 nm larger in size compared to the non-silica-coated control (+33.4 mV, 106 nm). Silica-coating significantly improved the polyplex stability against both dissociations by counter polyanion exchange and aggregation by salt. The silica network was dissolved to form silicic acid by removing free silicic acid based on the equilibrium, SiO(2) + 2H(2)O right arrow over left arrow Si(OH)(4). Indeed, dialysis of the SC polyplex solution against excess silica-free buffer permitted plasmid DNA release from the silica-coated polyplex, indicating the reversible nature of the silica-layer. The SC polyplex achieved significantly higher transfection efficiency without serious cytotoxicity compared to the polyplex without silica-coating. Detailed examinations of transfection using SC polyplexes revealed that the enhanced transfection efficiency was because of facilitated endosomal escape, possibly due to the protonation of the silica in acidic endosomal compartments. These findings demonstrate the utility of the silica-coating technique for polyplex-mediated gene delivery.

Targeted systemic delivery of siRNA to cervical cancer model using cyclic RGD-installed unimer polyion complex-assembled gold nanoparticles.

For systemic delivery of small interfering RNA (siRNA) to solid tumors, we developed an actively-targeted unimer polyion complex-assembled gold nanoparticle (uPIC-AuNP) by a two-step assembling process. First is the monodispersed uPIC formation from the single molecules of therapeutic siRNA and the block catiomer, cyclic RGD (cRGD) peptide-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (cRGD-PEG-PLL-LA). Second is the surface decoration of a 20nm-sized AuNP with uPICs. The cRGD-installed uPIC-AuNPs (cRGD-uPIC-AuNP) provided the targetability for selective binding to the cancer and cancer-related endothelial cellular surface, while regulating their size <50nm with a quite narrow distribution. The targeting efficacy of the cRGD-uPIC-AuNP was confirmed by in vitro cellular uptake in cultured cervical cancer (HeLa) cells and in vivo tumor accumulation in a subcutaneous HeLa model after systemic administration, compared with a non-targeted control uPIC-AuNP. Due to the targetability of the ligand, the cRGD-uPIC-AuNP achieved the significantly enhanced gene silencing ability in the subcutaneous HeLa tumor. Ultimately, the systemic delivery of siRNA targeted for papilloma virus-derived E6 oncogene by cRGD-uPIC-AuNP significantly inhibited the growth of subcutaneous HeLa tumor. This research demonstrates that the bottom-up construction of nanocarriers using monodispersed building blocks can be employed as delivery platforms for RNA interference-based cancer therapy.

Fine-Tuning of Charge-Conversion Polymer Structure for Efficient Endosomal Escape of siRNA-Loaded Calcium Phosphate Hybrid Micelles

For efficient delivery of siRNA into the cytoplasm, a smart block copolymer of poly(ethylene glycol) and charge-conversion polymer (PEG-CCP) is developed by introducing 2-propionic-3-methylmaleic (PMM) amide as an anionic protective group into side chains of an endosome-disrupting cationic polyaspartamide derivative. The PMM amide moiety is highly susceptible to acid hydrolysis, generating the parent cationic polyaspartamide derivative at endosomal acidic pH 5.5 more rapidly than a previously synthesized cis-aconitic (ACO) amide control. The PMM-based polymer is successfully integrated into a calcium phosphate (CaP) nanoparticle with siRNA, constructing PEGylated hybrid micelles (PMM micelles) having a sub-100 nm size at extracellular neutral pH 7.4. Ultimately, PMM micelles achieve the significantly higher gene silencing efficiency in cultured cancer cells, compared to ACO control micelles, probably due to the efficient endosomal escape of the PMM micelles. Thus, it is demonstrated that fine-tuning of acid-labile structures in CCP improves the delivery performance of siRNA-loaded nanocarriers.

Accelerated polymer-polymer click conjugation by freeze-thaw treatment

Herein, we report a unique technique to accelerate polymer-SNA conjugation based on copper-free click chemistry: gradual freeze-thawing of the reaction solution substantially increases the conjugation rate possibly because of the reactant concentration at the microenvironment scale. This technique was applied to the conjugation between a small interfering RNA (siRNA) and PEG in an aqueous buffer at/below room temperature.