Ionizable liposomal siRNA therapeutics enables potent and persistent treatment of Hepatitis B.

Small interfering RNA (siRNA) constitutes a promising therapeutic modality supporting the potential functional cure of hepatitis B. A novel ionizable lipidoid nanoparticle (RBP131) and a state-of-the-art lyophilization technology were developed in this study, enabling to deliver siRNA targeting apolipoprotein B (APOB) into the hepatocytes with an ED50 of 0.05 mg/kg after intravenous injection. In addition, according to the requirements of Investigational New Drug (IND) application, a potent siRNA targeting hepatitis B virus (HBV) was selected and encapsulated with RBP131 to fabricate a therapeutic formulation termed RB-HBV008. Efficacy investigations in transient and transgenic mouse models revealed that the expressions of viral RNAs and antigens (HBsAg and HBeAg), as well as viral DNA, were repressed, dose-dependently and time-dependently at multilog decreasing amplitude, in both circulation and liver tissue. In contrast, entecavir (ETV), the first-line clinically-employed nucleoside analog drug, barely recused the antigen expression, although it triggered as high as 3.50 log reduction of viral DNA, in line with clinical observations. Moreover, the toxicity profiles suggested satisfactory safety outcomes with ten times the therapeutic window. Therefore, this study provides an effective nucleic acid delivery system and a promising RNAi agent for the treatment of hepatitis B.

Core Role of Hydrophobic Core of Polymeric Nanomicelle in Endosomal Escape of siRNA.

Efficient endosomal escape is the most essential but challenging issue for siRNA drug development. Herein, a series of quaternary ammonium-based amphiphilic triblock polymers harnessing an elaborately tailored pH-sensitive hydrophobic core were synthesized and screened. Upon incubating in an endosomal pH environment (pH 6.5-6.8), mPEG45-P(DPA50-co-DMAEMA56)-PT53 (PDDT, the optimized polymer) nanomicelles (PDDT-Ms) and PDDT-Ms/siRNA polyplexes rapidly disassembled, leading to promoted cytosolic release of internalized siRNA and enhanced silencing activity evident from comprehensive analysis of the colocalization and gene silencing using a lysosomotropic agent (chloroquine) and an endosomal trafficking inhibitor (bafilomycin A1). In addition, PDDT-Ms/siPLK1 dramatically repressed tumor growth in both HepG2-xenograft and highly malignant patient-derived xenograft models. PDDT-Ms-armed siPD-L1 efficiently blocked the interaction of PD-L1 and PD-1 and restored immunological surveillance in CT-26-xenograft murine model. PDDT-Ms/siRNA exhibited ideal safety profiles in these assays. This study provides guidelines for rational design and optimization of block polymers for efficient endosomal escape of internalized siRNA and cancer therapy.

Harnessing pH-Sensitive Polycation Vehicles for the Efficient siRNA Delivery.

pH-sensitive hydrophobic segments have been certificated to facilitate siRNA delivery efficiency of amphiphilic polycation vehicles. However, optimal design concepts for these vehicles remain unclear. Herein, by studying the library of amphiphilic polycations mPEG-PAMA50-P(DEAx-r-D5Ay) (EAE5x/y), we concluded a multifactor matching concept (pKa values, "proton buffering capacities" (BCs), and critical micelle concentrations (CMCs)) for polycation vehicles to improve siRNA delivery efficiency in vitro and in vivo. We identified that the stronger BCs in a pH 5.5-7.4 subset induced by EAE548/29 (pKa = 6.79) and EAE539/37 (pKa = 6.20) are effective for siRNA delivery in vitro. Further, the stronger BCs occurred in a narrow subset of pH 5.5-6.5 and the lower CMC attributed to higher siRNA delivery capacity of EAE539/37 in vivo than EAE548/29 after intravenous administration and subcutaneous injection. More importantly, 87.2% gene knockdown efficacy was achieved by EAE539/37 via subcutaneous injection, which might be useful for an mRNA vaccine adjuvant. Furthermore, EAE539/37 also successfully delivered siRRM2 to tumor via intravenous administration and received highly efficient antitumor activity. Taken together, the suitable pKa values, strong BCs occurred in pH 5.5-6.5, and low CMCs were probably the potential solution for designing efficient polycationic vehicles for siRNA delivery.

Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle.

mRNA is a novel class of therapeutic modality that holds great promise in vaccination, protein replacement therapy, cancer immunotherapy, immune cell engineering etc. However, optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application. Here we present an ionizable lipid nanoparticle (iLNP) based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs. mRNAs encoding luciferase or erythropoietin (EPO) were prepared by in vitro transcription and formulated with proposed iLNP, to form iLP171/mRNA formulations. It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes. The maximum amount of protein expression was found at 6 h post-administration. The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA. Moreover, no toxicity or immunogenicity was observed for these mRNA formulations. Therefore, our study provides a useful and promising platform for mRNA therapeutic development.

Potent and rapid reversal of the von Willebrand factor inhibitor aptamer BT200.

BACKGROUND: BT200, a pegylated form of the aptamer BT100, inhibits binding of von Willebrand factor (VWF) to platelet glycoprotein GPIb, preventing arterial thrombosis in cynomolgus monkeys. It is being developed for secondary prevention of arterial thrombosis such as stroke or myocardial infarction. Inhibition of thrombogenesis by BT200 is expected to provide a therapeutic benefit. However, there may be unexpected bleeding (eg, incidental trauma) in which a reversal agent is required. To address this need, BT101, a complementary aptamer, has been developed to specifically inhibit BT100 and BT200 function. OBJECTIVES: To characterize the effects of BT101 both in vitro and in vivo. METHODS: The direct interaction between BT101 and the core aptamer BT100 was evaluated using polyacrylamide gel electrophoresis. The binding of BT200 to purified human VWF and inhibition of VWF activity was further characterized using enzyme-linked immunosorbent assay. VWF-dependent platelet function was measured by the platelet function analyzer and aggregometry in whole blood. In addition, both the in vivo pharmacokinetic profile of BT101 as well as its ability to reverse BT200 activity, were evaluated in cynomolgus monkeys. RESULTS: BT101 bound to the core aptamer BT100 at a 1:1 ratio, inhibited BT200 binding to purified human VWF, and reversed BT200-induced inhibition of both VWF activity and VWF-dependent platelet function in vitro. After intravenous injection to monkeys, BT101 reversed BT200-induced effects on VWF activity and platelet function within minutes, without causing any adverse effects. CONCLUSIONS: The results of this study demonstrate that BT101 is an effective reversal agent for BT200.

The development and characterization of a long acting anti-thrombotic von Willebrand factor (VWF) aptamer.

BACKGROUND: Thrombus formation involves coagulation proteins and platelets. The latter, referred to as platelet-mediated thrombogenesis, is predominant in arterial circulation. Platelet thrombogenesis follows vascular injury when extracellular von Willebrand factor (VWF) binds via its A3 domain to exposed collagen, and the free VWF A1 domain binds to platelet glycoprotein Ib (GPIb). OBJECTIVES: To characterize the antiplatelet/antithrombotic activity of the pegylated VWF antagonist aptamer BT200 and identify the aptamer VWF binding site. METHODS: BT100 is an optimized aptamer synthesized by solid-phase chemistry and pegylated (BT200) by standard conjugation chemistry. The affinity of BT200 for purified human VWF was evaluated as was VWF inhibition in monkey and human plasma. Efficacy of BT200 was assessed in the monkey FeCl3 femoral artery thrombosis model. RESULTS: BT200 bound human VWF at an EC50 of 5.0 nmol/L and inhibited VWF A1 domain activity in monkey and human plasma with mean IC50 values of 183 and 70 nmol/L. BT200 administration to cynomolgus monkeys caused a time-dependent and dose-dependent effect on VWF A1 domain activity and inhibited platelet function as measured by collagen adenosine diphosphate closure time in the platelet function analyzer. BT200 demonstrated a bioavailability of ≥77% and exhibited a half-life of >100 hours after subcutaneous injection. The treatment effectively prevented arterial occlusion in an FeCl3 -induced thrombosis model in monkeys. CONCLUSIONS: BT200 has shown promising inhibition of human VWF in vitro and prevented arterial occlusion in non-human primates. These data including a long half-life after subcutaneous injections provide a strong rationale for ongoing clinical development of BT200.

An oligonucleotide synthesizer based on a microreactor chip and an inkjet printer.

Synthetic oligonucleotides (oligos) are important tools in the fields of molecular biology and genetic engineering. For applications requiring a large number of oligos with high concentration, it is critical to perform high throughput oligo synthesis and achieve high yield of each oligo. This study reports a microreactor chip for oligo synthesis. By incorporating silica beads in the microreactors, the surface area of the solid substrate for oligo synthesis increases significantly in each microreactor. These beads are fixed in the microreactors to withstand the flushing step in oligo synthesis. Compared to conventional synthesis methods, this design is able to avoid protocols to hold the beads and integrate more microreactors on a chip. An inkjet printer is utilized to deliver chemical reagents in the microreactors. To evaluate the feasibility of oligo synthesis using this proof-of-concept synthesizer, an oligo with six nucleotide units is successfully synthesized.

The study of relationships between pKa value and siRNA delivery efficiency based on tri-block copolymers.

Tri-block copolymers have exhibited great potentials in small interfering RNA (siRNA) therapeutics. To reveal structure-activity relationships, we here synthesized a series of tri-block copolymers with different hydrophobic segments, PEG-PAMA-P(C6Ax-C7Ay-DPAz-DBAm) (EAAS) and PEG-PDAMAEMA-P(C6Ax-C7Ay-DPAz-DBAm) (EDAS), termed from EAASa to EAASh and EDASa to EDASh, with pKa ranging from 5.2 to 7.0. Our data showed that the better gene silencing efficiency was located in pKa of 5.8-6.2, which was contributed from higher endosomal escape observed with confocal images and hemolysis assay. EAASc, the leader polymer, showed excellent gene knockdown at w/w ratio of 14.5 on HepG2 (89.94%), MDA-MB-231 (92.45%), 293A (83.06%), and Hela cells (80.27%), all better than lipofectamine 2000. Besides, EAASc mediated effective gene silencing in tumor when performed peritumoral injection. This work found out that polymers with pKa ranging from 5.8 to 6.2 were efficient in siRNA delivery, which provided an optimization strategy for siRNA delivery systems, especially for tri-block copolymers.

Efficient delivery of nucleic acid molecules into skin by combined use of microneedle roller and flexible interdigitated electroporation array.

Rationale: Delivery of nucleic acid molecules into skin remains a main obstacle for various types of gene therapy or vaccine applications. Here we propose a novel electroporation approach via combined use of a microneedle roller and a flexible interdigitated electroporation array (FIEA) for efficient delivery of DNA and siRNA into mouse skin. Methods: Using micromachining technology, closely spaced gold electrodes were made on a pliable parylene substrate to form a patch-like electroporation array, which enabled close surface contact between the skin and electrodes. Pre-penetration of the skin with a microneedle roller resulted in the formation of microchannels in the skin, which played a role as liquid electrodes in the skin and provided a uniform and deep electric field in the tissue when pulse stimulation was applied by FIEA. Results: Using this proposed method, gene (RFP) expression and siRNA transfection were successfully achieved in normal mice skin. Anti-SCD1 siRNA electroporated via this method mediated significant gene silencing in the skin. Moreover, electroporation assisted by the microneedle roller showed significant advantages over treatment with FIEA alone. This allowed nucleic acid transportation at low voltage, with ideal safety outcomes. Principal conclusions: Hence, the proposed electroporation approach in this study constitutes a novel way for delivering siRNA and DNA, and even other nucleic acid molecules, to mouse skin in vivo, potentially supporting clinical application in the treatment of skin diseases or intradermal/subcutaneous vaccination.

AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism.

Type 2 diabetes mellitus (T2DM) is a common metabolic disease influenced by both genetic and environmental factors. In this study, we performed an in-house genotyping and meta-analysis study using three independent GWAS datasets of T2DM and found that rs3743121, located 1 kb downstream of AQR, was a novel susceptibility SNP associated with T2DM. The risk allele C of rs3743121 was correlated with the increased expression of AQR in white blood cells, similar to that observed in T2DM models. The knockdown of AQR in HepG2 facilitated the glucose uptake, decreased the expression level of PCK2, increased the phosphorylation of GSK-3ß, and restored the insulin sensitivity. Furthermore, the suppression of AQR inhibited the mTOR pathway and the protein ubiquitination process. Our study suggests that AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism.

Parametric optimization of electric field strength for cancer electrochemotherapy on a chip-based model.

Electrochemotherapy (ECT), as one of the very few available treatments for cutaneous and subcutaneous tumors when surgery and radiotherapy are no longer available, requires applying a proper electric field to the tumor to realize electroporation-mediated cytotoxic drug delivery. It is impossible to exhaust all possible electrical parameters on patients to realize the optimal tradeoff between tumor suppression and adverse effects. To address this issue, this study provides a feasible solution by developing a four-leaf micro-electrode chip (F-MEC) in which the electric field was specially designed by linear distribution to cover all possible electric field strengths for ECT. Methods: We developed a F-MEC that provides a linearly varied electric field and a capacity for in situ observation of cell status. By culturing tumor cells on the F-MEC surface and in situ monitoring the cell responses to ECT drugs, the optimal electric field strength for any given cell type could be rapidly and accurately calculated in a few, or even only one, simple assay. Results: Using this chip, we monitored MCF-7 and A315 cell responses to ECT and determined the optimum ECT voltage. More importantly, we successfully verified that the in vitro determined voltage coincided with the optimal value for in vivo ECT in mice. Conclusion: In this proof-of-concept study, the in vivo tumor suppression assays proved that the optimal parameters acquired from in vitro F-MEC assay could be used for in vivo ECT.

Site-Specific Modification Using the 2'-Methoxyethyl Group Improves the Specificity and Activity of siRNAs.

Rapid progress has been made toward small interfering RNA (siRNA)-based therapy for human disorders, but rationally optimizing siRNAs for high specificity and potent silencing remains a challenge. In this study, we explored the effect of chemical modification at the cleavage site of siRNAs. We found that modifications at positions 9 and 10 markedly reduced the silencing potency of the unmodified strand of siRNAs but were well tolerated by the modified strand. Intriguingly, addition of the 2'-methoxyethyl (MOE) group at the cleavage site improved both the specificity and silencing activity of siRNAs by facilitating the oriented RNA-induced silencing complex (RISC) loading of the modified strand. Furthermore, we combined MOE modifications at positions 9 and 10 of one strand together with 2'-O-methylation (OMe) at position 14 of the other strand and found a synergistic effect that improved the specificity of siRNAs. The surprisingly beneficial effect of the combined modification was validated using siRNA-targeting endogenous gene intercellular adhesion molecule 1 (ICAM1). We found that the combined modifications eliminated its off-target effects. In conclusion, we established effective strategies to optimize siRNAs using site-specific MOE modifications. The findings may allow the creation of superior siRNAs for therapy in terms of activity and specificity.

Elaboration on the Distribution of Hydrophobic Segments in the Chains of Amphiphilic Cationic Polymers for Small Interfering RNA Delivery.

Hydrophobization of cationic polymers, as an efficient strategy, had been widely developed in the structure of cationic polymer micelles to improve the delivery efficiency of nucleic acids. However, the distribution of hydrophobic segments in the polymer chains is rarely considered. Here, we have elaborated three types of hydrophobized polyethylene glycol (PEG)-blocked cationic polymers with different distributions of the hydrophobic segments in the polymer chains PEG-PAM-PDP (E-A-D), PEG-PDP-PAM (E-D-A), and PEG-P(AM/DP) (E-(A/D)), which were synthesized by reversible addition-fragmentation chain transfer polymerization of methoxy PEG, cationic monomer aminoethyl methacrylate, and pH-sensitive hydrophobic monomer 2-diisopropylaminoethyl methacrylate, respectively. In aqueous solution, all of the three copolymers, E-A-D, E-D-A, and E-(A/D), were able to spontaneously form nanosized micelles (100-150 nm) (ME-A-D, ME-D-A, and ME-(A/D)) and well-incorporated small interfering RNA (siRNA) into complex micelles (CMs). The effect of distributions of the hydrophobic segments on siRNA delivery had been evaluated in vitro and in vivo. Compared with ME-D-A and ME-(A/D), ME-A-D showed the best siRNA binding capacity to form stable ME-A-D/siRNA CMs less than 100 nm, mediated the best gene-silencing efficiency and inhibition effect of tumor cell growth in vitro, and showed better liver gene-silencing effect in vivo. In the case of ME-(A/D) with a random distribution of cationic and hydrophobic segments, a gene-silencing efficiency higher than Lipo2000 but lesser than ME-A-D and ME-D-A was obtained. As the mole ratio of positive and negative charges increased, ME-D-A/siRNA and ME-A-D/siRNA showed similar performances in size, zeta potential, cell uptake, and gene silencing, but ME-(A/D)/siRNA showed reversed performances. In addition, ME-A-D as the best siRNA carrier was evaluated in the tumor tissue in the xenograft murine model and showed good anticancer capacity. Obviously, the distribution of the hydrophobic segments in the amphiphilic cationic polymer chains should be seriously considered in the design of siRNA vectors.

The pH-Triggered Triblock Nanocarrier Enabled Highly Efficient siRNA Delivery for Cancer Therapy.

Small interfering RNA (siRNA) therapies have been hampered by lack of delivery systems in the past decades. Nowadays, a few promising vehicles for siRNA delivery have been developed and it is gradually revealed that enhancing siRNA release from endosomes into cytosol is a very important factor for successful delivery. Here, we designed a novel pH-sensitive nanomicelle, PEG-PTTMA-P(GMA-S-DMA) (PTMS), for siRNA delivery. Owing to rapid hydrolysis in acidic environment, PTMS NPs underwent hydrophobic-to-hydrophilic transition in endosomes that enabled combination of proton sponge effect and raised osmotic pressure in endosomes, resulting in vigorous release of siRNAs from endosomes into cytosol. In vitro results demonstrated that PTMS/siRNA complexes exhibited excellent gene silencing effects in several cell lines. Their gene silencing efficiency could reach ~91%, ~87% and ~90% at the N/P ratio of 50/1 in MDA-MB-231, A549 and Hela cells respectively, which were better than that obtained with Lipofectamine 2000. The highly efficient gene silencing was then proven from enhanced siRNA endosomal release, which is mainly attributed to pH-triggered degradation of polymer and acid-accelerated siRNA release. In vivo experiments indicated that NPs/siRNA formulation rapidly accumulated in tumor sites after i.v. injection. Tumor growth was effectively inhibited and ~45% gene knockdown efficacy was determined at the siRRM2 dose of 1mg/kg. Meanwhile, no significant toxicity was observed during the whole treatment. We also found that PTMS/siRNA formulations could lead to significant gene silencing effects in liver (~63%) and skin (~80%) when injected by i.v. and s.c., respectively. This research work gives a rational strategy to optimize siRNA delivery systems for tumor treatments.

Target-Recognition Mechanism and Specificity of RNA Activation.

Small activating RNA (saRNA)-mediated gene activation has opened a new avenue for upregulating the expression of target genes by promoting endogenous transcription, a phenomenon known as RNA activation (RNAa). RNAa is distinct from the established RNAi mechanistic framework, although AGO2 is required by both. The precise mechanism of RNAa is currently disputable and has become a bottleneck in the development of this new technology. saRNA may achieve activation of target genes by directly binding to DNA targets in promoter, or interacting with antisense transcripts transcribed from overlapping promoter sequences, or by silencing other genes. In this chapter, we focused on recent development in our understanding of the target-recognition mechanism in RNAa. Conflicting results on saRNA targets are also discussed. Despite that the target mechanism of RNAa is more complex than expected and not completely understood so far, independent lines of evidence have suggested that saRNAs work by an "on-site" mechanism by binding to target genomic DNA in a "seed-region"-dependent manner. Finally, "off-target" effects of saRNA are observed and should be carefully controlled in designing experiments for and interpreting results from RNAa-related studies.

Balancing Biocompatibility, Internalization and Pharmacokinetics of Polycations/siRNA by Structuring the Weak Negative Charged Ternary Complexes with Hyaluronic Acid.

Polycations are generally used to work as delivery vector to develop siRNA-based therapy for gene-related diseases. The contradiction between inevitable toxicity, internalization, and pharmacokinetics of polycations/siRNA is a critical challenge for polycations and impedes their further application. Herein, we synthesized the ECMD polycations and constructed ECMD/siRNA/HA complexes with slight negative charge to address the above mentioned issue. We found that equipping with HA could effectively shield the positive charge and dramatically improve cell viability. Moreover, the ternary complexes with slight negative charge exhibited similar cellular uptake efficiency and knockdown efficiency compared with ECMD/siRNA binary complexes because of CD44 protein-mediated endocytosis. Pharmacokinetics experiment and in vivo distribution elucidated that the ternary complexes with negative charge could help to prolong the circulation time of siRNA in blood and affect the organs distribution after i.v. injection. In addition, with time going by, the accumulation amount of siRNA loaded by the ternary complexes was much more in tumor compared with the binary complexes. Therefore, we believed that building the complexes was a feasible method to further develop polycationic vectors for siRNA delivery.

pH-Sensitive Nanomicelles for High-Efficiency siRNA Delivery in Vitro and in Vivo: An Insight into the Design of Polycations with Robust Cytosolic Release.

The extremely low efficient cytosolic release of the internalized siRNA has emerged recently as a central issue for siRNA delivery, while there is a lack of guidelines to facilitate the cytosolic release of internalized siRNA. To address these concerns, we studied the contribution of the pH-sensitive inner core on handling the cytosolic release of siRNA delivered by a series of PG-P(DPAx-co-DMAEMAy)-PCB amphiphilic polycation nanomicelles (GDDC-Ms) with extremely low internalization (<1/4 of lipofactamine 2000 (Lipo2000)). Significantly, just by varying the mole ratio of DPA and DMAEMA to adjust the initial disassembly pH (pHdis) of the core near to 6.8, GDDC4-Ms/siRNA could get nearly 98.8% silencing efficiency at w/w = 12 with 50 nM siRNA and ∼78% silencing efficiency at w/w = 30 with a very low dose of 5 nM siRNA in HepG-2 cell lines, while Lipo2000 only got 65.7% with 50 nM siRNA. Furthermore, ∼98.4% silencing efficiency was also realized in the hard-to-transfect human acute monoblastic leukemia cell line U937 by GDDC4-Ms/siRNA (at w/w = 15, 50 nM siRNA), in the inefficient case for Lipo2000. Additionally, the high silencing efficiency (∼80%) in skin tissue in vivo was discovered. Undoubtedly, the robust potential of GDDC4-Ms in handling the cytosolic release paves a simple but efficient new way for the design of the nonviral siRNA vector.

Pharmacokinetic Behaviors of Intravenously Administered siRNA in Glandular Tissues.

The pharmacokinetics of small interfering RNAs (siRNAs) is a pivotal issue for siRNA-based drug development. In this study, we comprehensively investigated the behavior of siRNAs in vivo in various tissues and demonstrated that intravenously-injected naked siRNA accumulated remarkably in the submandibular gland, bulbourethral gland, and pancreas, with a respective half-life of ~22.7, ~45.6, and ~30.3 h. This was further confirmed by gel separation of tissue homogenates and/or supernatants. In vivo imaging and cryosectioning suggested that delivery carriers significantly influence the distribution and elimination profiles of siRNA. Gene-silencing assays revealed that neither naked nor liposome-formulated siRNA resulted in gene knockdown in the submandibular and bulbourethral glands after systemic administration, suggesting that these glands function as drug reservoirs that enable slow siRNA release into the circulation. But robust gene-silencing was achieved by local injection of liposome-encapsulated siRNA into the submandibular gland. Our results enhance understanding of the pharmacokinetic properties of siRNAs and we believe that they will facilitate the development of siRNA therapy, especially for the submandibular gland.

Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing.

Self-assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self-assembly remains challenging. In this work, through a combination of experimental and computational approaches, the self-assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high-generation dendrimers is reported. It is demonstrated that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self-assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA and achieve effective gene silencing both in cells - including the highly refractory human hematopoietic CD34(+) stem cells - and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self-assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self-assembling nanosystems for complex and functional applications.

Inter-molecular ß-sheet structure facilitates lung-targeting siRNA delivery.

Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-ß, which was able to form inter-molecular ß-sheet structures. Results showed that K-ß peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications.