Tomoya Suma

Odd-even effect of repeating aminoethylene units in the side chain of N-substituted polyaspartamides on gene transfection profiles

A series of the N-substituted polyaspartamides possessing repeating aminoethylene units in the side chain was prepared in this study to identify polyplexes with effective endosomal escape and low cytotoxicity. All cationic N-substituted polyaspartamides showed appreciably lower cytotoxicity than that of commercial transfection reagents. Interestingly, a distinctive odd-even effect of the repeating aminoethylene units in the polymer side chain on the efficiencies of endosomal escape and transfection to several cell lines was observed. The polyplexes from the polymers with an even number of repeating aminoethylene units (PA-Es) achieved an order of magnitude higher transfection efficiency, without marked cytotoxicity, than those of the polymers with an odd number of repeating aminoethylene units (PA-Os). This odd-even effect agreed well with the buffering capacity of these polymers as well as their capability to disrupt membrane integrity selectively at endosomal pH, leading to highly effective endosomal escape of the PA-E polyplexes. Furthermore, the formation of a polyvalent charged array with precise spacing between protonated amino groups in the polymer side chain was shown to be essential for effective disruption of the endosomal membrane, thus facilitating transport of the polyplex into the cytoplasm. These data provide useful knowledge for designing polycations to construct safe and efficient nonviral gene carriers.

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.

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/

Pancreatic cancer therapy by systemic administration of VEGF siRNA contained in calcium phosphate/charge-conversional polymer hybrid nanoparticles

Development of an efficient in vivo delivery vehicle of small interfering RNA (siRNA) is the key challenge for successful siRNA-based therapies. In this study, toward systemic delivery of siRNA to solid tumors, a smart polymer/calcium phosphate (CaP)/siRNA hybrid nanoparticle was prepared to feature biocompatibility, reversible stability and endosomal escape functionality using a pH sensitive block copolymer of poly(ethylene glycol) and charge-conversional polymer (PEG-CCP), of which anionic functional groups could be converted to cationic groups in an endosomal acidic condition for facilitated endosomal escape. Nanoparticles were confirmed to be approximately 100nm in size, narrowly dispersed and spherical. Also, the nanoparticle was highly tolerable in medium containing serum, while releasing the entrapped siRNA in a cytoplasm-mimicking ionic condition, presumably based on the equilibrium between CaP complexes and calcium ions. Further, the nanoparticle showed high gene silencing efficiency in cultured pancreatic cancer cells (BxPC3) without associated cytotoxicity. Ultimately, systemic administration of the nanoparticles carrying vascular endothelium growth factor (VEGF) siRNA led to the significant reduction in the subcutaneous BxPC3 tumor growth, well consistent with the enhanced accumulation of siRNA and the significant VEGF gene silencing (~68%) in the tumor. Thus, the hybrid nanoparticle was demonstrated to be a promising formulation toward siRNA-based cancer therapies.

Modulated Protonation of Side Chain Aminoethylene Repeats in N-Substituted Polyaspartamides Promotes mRNA Transfection

Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

Engineering low-fouling and pH-degradable capsules through the assembly of metal-phenolic networks.

Metal-phenolic coordination chemistry provides a simple and rapid way to fabricate ultrathin films. Here, we report a facile strategy for the preparation of low-fouling and pH-degradable metal-phenolic network (MPN) capsules using a synthetic polyphenol derivative, poly(ethylene glycol) (PEG)-polyphenol, as a building block. PEG-MPN capsules exhibit reduced nonspecific protein adsorption and cell association compared with tannic acid (TA)-MPN capsules. In addition, they show faster disassembly at a biologically relevant pH (5) than TA-MPN capsules (80% in 5 h vs 30% in 10 days). PEG-MPN capsules combine both the low fouling properties of PEG and the advantages of the MPN-driven assembly process (e.g., fast assembly and pH-degradability).

Metal–Phenolic Supramolecular Gelation

Materials assembled by coordination interactions between naturally abundant polyphenols and metals are of interest for a wide range of applications, including crystallization, catalysis, and drug delivery. Such an interest has led to the development of thin films with tunable, dynamic properties, however, creating bulk materials remains a challenge. Reported here is a class of metallogels formed by direct gelation between inexpensive, naturally abundant tannic acid and group(IV) metal ions. The metallogels exhibit diverse properties, including self-healing and transparency, and can be doped with various materials by in situ co-gelation. The robustness and flexibility, combined with the ease, low cost, and scalability of the coordination-driven assembly process make these metallogels potential candidates for chemical, biomedical, and environmental applications.

Boronate-Phenolic Network Capsules with Dual Response to Acidic pH and cis-Diols

Dual-responsive boronate-phenolic network (BPN) capsules are fabricated by the complexation of phenylborate and phenolic materials. The BPN capsules are stable in the presence of competing carbohydrates, but dissociate at acidic pH or in the presence of competing cis-diols at physiological pH. This engineered capsule system provides a platform for a wide range of biological and biomedical applications.

Rust‐Mediated Continuous Assembly of Metal–Phenolic Networks

The use of natural compounds for preparing hybrid molecular films-such as surface coatings made from metal-phenolic networks (MPNs)-is of interest in areas ranging from catalysis and separations to biomedicine. However, to date, the film growth of MPNs has been observed to proceed in discrete steps (≈10 nm per step) where the coordination-driven interfacial assembly ceases beyond a finite time (≈1 min). Here, it is demonstrated that the assembly process for MPNs can be modulated from discrete to continuous by utilizing solid-state reactants (i.e., rusted iron objects). Gallic acid etches iron from rust and produces chelate complexes in solution that continuously assemble at the interface of solid substrates dispersed in the system. The result is stable, continuous growth of MPN films. The presented double dynamic process-that is, etching and self-assembly-provides new insights into the chemistry of MPN assembly while enabling control over the MPN film thickness by simply varying the reaction time.

Engineered Metal-Phenolic Capsules Show Tunable Targeted Delivery to Cancer Cells

We engineered metal-phenolic capsules with both high targeting and low nonspecific cell binding properties. The capsules were prepared by coating phenolic-functionalized hyaluronic acid (HA) and poly(ethylene glycol) (PEG) on calcium carbonate templates, followed by cross-linking the phenolic groups with metal ions and removing the templates. The incorporation of HA significantly enhanced binding and association with a CD44 overexpressing (CD44+) cancer cell line, while the incorporation of PEG reduced nonspecific interactions with a CD44 minimal-expressing (CD44-) cell line. Moreover, high specific targeting to CD44+ cells can be balanced with low nonspecific binding to CD44- cells simply by using an optimized feed-ratio of HA and PEG to vary the content of HA and PEG incorporated into the capsules. Loading an anticancer drug (i.e., doxorubicin) into the obtained capsules resulted in significantly higher cytotoxicity to CD44+ cells but lower cytotoxicity to CD44- cells.