Dendrimeric siRNA for Efficient Gene Silencing.

Programmable molecular self-assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second-generation dendrimers of siRNA can be effectively complexed with a low-molecular-weight, cationic polymer (poly(ß-amino ester), PBAE) to generate stable nanostructures about 160 nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility.

Optimization of polyethylene glycol shielding and mannose density on the lipid nanoparticles for efficient delivery to macrophages and spleens.

This study compared the effects of polyethylene glycol (PEG) shielding and mannose-conjugated ligands density on lipid nanoparticles (LNPs) for intracellular uptake to macrophages in vitro and accumulation in spleens in vivo. Fabricated phosphatidyl serine-incorporated LNPs (sLNPs) was physically decorated with mannose-conjugated DSPE-PEG (DPM) at different DPM/LNP molar ratios achieving the DPM density from 0 to 0.6 PEGs/nm2. We demonstrated that low PEG shielding sLNPs with mannose ligands (sLNP-DPMs) displayed superior uptake to macrophages (RAW 264.7 cells) compared with high PEG shielding sLNP-DPMs in vitro. However, high PEG shielding sLNP-DPMs showed significant spleen accumulation compared with low PEG shielding sLNP-DPMs in vivo after intravenous injection. In particular, high PEG shielding sLNPs coated with DSPE-methoxyPEG (DP) and DPM mixture at DP/DPM molar ratios of 5/5 exhibited greater accumulation in red pulp of spleens than naked sLNPs by 2.7-folds in vivo. These results suggested that the optimal PEG shielding and mannose densities per a particle might be different between in vitro cellular uptake to macrophages and in vivo spleen accumulation after systemic administration. Taken together, precision-tailored LNP-surface modifications achieved through optimization of PEG shielding and mannose density can greatly enhance accumulation of LNPs in red pulp of spleens, which could be applied for the delivery of nucleic acid-based drugs and vaccines to spleens in vivo.

Effects of Histidine Oligomers in Lipid Nanoparticles on siRNA Delivery.

In this study, histidine oligomer (oHis; 10mer)-incorporating LNPs (H10LNPs) are developed as a novel carrier for efficient siRNA delivery. Notably, the unmodified oHis (10mer) is greatly incorporated within LNPs through ionic interaction with siRNAs, which serves as an endosome escape enhancer. H10LNPs with a size of ≈65 nm demonstrate a significantly enhanced extent of endosomal escape, as evidenced by calcein assay and confocal microscopy images of intracellular fluorescence, surpassing conventional LNPs. Furthermore, the half inhibitory concentration (IC50) of the human endogenous globotriaosylceramide synthase (Gb3 synthase) gene in H10LNPs-treated cells exhibits a significant threefold decrease, compared to that in LNP-treated cells. Notably, H10LNPs maintain comparable biocompatibility and biodistribution both in vitro and in vivo. Considering that the fabricated siRNA H10LNPs exhibit excellent biocompatibility and superior gene silencing activity over conventional LNPs, these particles can be harnessed for the safe delivery of therapeutic siRNAs. Additionally, this study introduces promising, feasible, simple, and alternative formulation processes for integrating unmodified functional cationic peptides into LNPs to enhance the delivery efficiency of a wide range of nucleic acid-based drugs.

Engineered lipid nanoparticles enable therapeutic gene silencing of GTSE1 for the treatment of liver fibrosis.

Liver fibrosis is characterized by abnormal accumulation of extracellular matrix proteins, disrupting normal liver function. Despite its significant health impact, effective treatments remain limited. Here, we present the development of engineered lipid nanoparticles (LNPs) for targeted RNA therapeutic delivery in the liver. We investigated the therapeutic potential of modulating the G2 and S-phase expressed 1 (GTSE1) protein for treating liver fibrosis. Through screening, we identified P138Y LNP as a potent candidate with superior delivery efficiency and lower toxicity. Using these engineered LNPs, we successfully delivered siGTSE1 to hepatocytes, significantly reducing collagen accumulation and restoring liver function in a fibrosis animal model. Additionally, GTSE1 downregulation altered miRNA expression and upregulated hepatocyte nuclear factor 4 alpha (HNF4α). These findings suggest that therapeutic gene silencing of GTSE1 is a promising strategy for treating liver fibrosis by regenerating liver phenotypes and functions.

Innate immune responses against mRNA vaccine promote cellular immunity through IFN-ß at the injection site.

mRNA vaccines against SARS-CoV-2 have revolutionized vaccine development, but their immunological mechanisms are not fully understood. Here, we investigate injection site responses of mRNA vaccines by generating a comprehensive single-cell transcriptome profile upon lipid nanoparticle (LNP) or LNP-mRNA challenge in female BALB/c mice. We show that LNP-induced stromal pro-inflammatory responses and mRNA-elicited type I interferon responses dominate the initial injection site responses. By tracking the fate of delivered mRNA, we discover that injection site fibroblasts are highly enriched with the delivered mRNA and that they express IFN-ß specifically in response to the mRNA component, not to the LNP component of mRNA vaccines. Moreover, the mRNA-LNP, but not LNP alone, induces migratory dendritic cells highly expressing IFN-stimulated genes (mDC_ISGs) at the injection site and draining lymph nodes. When co-injected with LNP-subunit vaccine, IFN-ß induces mDC_ISGs at the injection site, and importantly, it substantially enhances antigen-specific cellular immune responses. Furthermore, blocking IFN-ß signaling at the injection site significantly decreases mRNA vaccine-induced cellular immune responses. Collectively, these data highlight the importance of injection site fibroblasts and IFN-ß signaling during early immune responses against the mRNA vaccine and provide detailed information on the initial chain of immune reactions elicited by mRNA vaccine injection.

Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics.

Several studies have utilized a lipid nanoparticle delivery system to enhance the effectiveness of mRNA therapeutics and vaccines. However, these nanoparticles are recognized as foreign materials by the body and stimulate innate immunity, which in turn impacts adaptive immunity. Therefore, it is crucial to understand the specific type of innate immune response triggered by lipid nanoparticles. This article provides an overview of the immunological response in the body, explores how lipid nanoparticles activate the innate immune system, and examines the adverse effects and immunogenicity-related development pathways associated with these nanoparticles. Finally, we highlight and explore strategies for regulating the immunogenicity of lipid nanoparticles.

Lipid nanoparticles (LNPs) for in vivo RNA delivery and their breakthrough technology for future applications.

RNA therapeutics show a significant breakthrough for the treatment of otherwise incurable diseases and genetic disorders by regulating disease-related gene expression. The successful development of COVID-19 mRNA vaccines further emphasizes the potential of RNA therapeutics in the prevention of infectious diseases as well as in the treatment of chronic diseases. However, the efficient delivery of RNA into cells remains a challenge, and nanoparticle delivery systems such as lipid nanoparticles (LNPs) are necessary to fully realize the potential of RNA therapeutics. While LNPs provide a highly efficient platform for the in vivo delivery of RNA by overcoming various biological barriers, several challenges remain to be resolved for further development and regulatory approval. These include a lack of targeted delivery to extrahepatic organs and a gradual loss of therapeutic potency with repeated doses. In this review, we highlight the fundamental aspects of LNPs and their uses in the development of novel RNA therapeutics. Recent advances in LNP-based therapeutics and preclinical/clinical studies are overviewed. Lastly, we discuss the current limitations of LNPs and introduce breakthrough technologies that might overcome these challenges in future applications.

In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

Surface PEGylation via native chemical ligation.

Native chemical ligation (NCL) is an emerging chemoselective chemistry that forms an amide bond by trans-thioesterification followed by intramolecular nucleophilic rearrangement between thioester and cysteine. The reaction is simple, occurs in a mild aqueous solution, and gives near-quantitative yields of a desired product. Since the first report in 1994, most studies involving the use of NCL have focused on the total synthesis of proteins to address fundamental questions pertaining to many aspects of protein science, such as folding, mirror images, and site-specific labeling of proteins, but applications of the NCL reaction for other areas remain largely unexplored. Herein, we present a facile strategy for surface immobilization of poly(ethylene glycol) (PEG) utilizing the NCL reaction. Surface immobilization of PEG (i.e., PEGylation) plays a key role in preventing nonspecific protein adsorption on surfaces, which is crucial in a wide variety of medical devices. Using cysteine-PEG and thioester-containing phosphonic acid conjugates, we achieved efficient surface PEGylation on titanium surfaces. Ellipsometry, goniometry, and X-ray photoelectron spectroscopy (XPS) unambiguously confirmed the presence of PEGs, which provided nonfouling effects of surfaces. This study indicates that the NCL reaction will be a useful toolkit for surface bioconjugation and functionalization.

The core composition of DNA block copolymer micelles dictates DNA hybridization properties, nuclease stabilities, and cellular uptake efficiencies.

Here, we report how the nature of the hydrophobic core affects the molecular interactions of DNA block copolymer assemblies. Three different amphiphilic DNA block copolymers, DNA-b-polystyrene (DNA-b-PS), DNA-b-poly(2-vinylpyridine) (DNA-b-P2VP), and DNA-b-poly(methyl acrylate) (DNA-b-PMA) were synthesized and assembled into spherical micelles composed of a hydrophobic polymer core and DNA corona. Interestingly, DNA block copolymer micelles having different hydrophobic cores exhibited markedly different molecular and biological interactions. DNA-b-PS exhibited higher melting temperature, sharper melting transition, higher stability to nuclease-catalyzed DNA degradation, and higher cellular uptake efficiency compared to DNA-b-P2VP and DNA-b-PMA. The investigation of the self-assembly behavior revealed a much higher aggregation number and DNA density for DNA-b-PS micelles, which explains the superior properties of DNA-b-PS. These results demonstrate that the type of the hydrophobic core polymer, which has been largely overlooked, has a profound impact on the molecular and biological interactions of the DNA shell.

Intracellular trafficking and unpacking of siRNA/quantum dot-PEI complexes modified with and without cell penetrating peptide: confocal and flow cytometric FRET analysis.

Cationic quantum dots (QDs) were utilized to complex small interfering RNA (siRNA) for studying intracellular trafficking, unpacking, and gene silencing. Positively charged polyethylenimine (PEI) was covalently conjugated on the surface of QDs to complex with cyanine dye labeled vascular endothelial growth factor siRNA (cy5-VEGF siRNA) for the formation of nanosized polyelectrolyte complexes (PEC). Fluorescence resonance energy transfer (FRET) was achieved between cy5-VEGF siRNA and PEI conjugated QDs (QD625) in the complex. From confocal microscopic analysis, intracellular uptake and release of siRNA from the PEC were visualized as a function of incubation time. The extent of cy5-siRNA release from the PEC was quantitatively evaluated by flow cytometric analysis. In addition, PEI conjugated QDs were further modified with a protein transduction domain (PTD) from human transcriptional factor, Hph-1. The two siRNA/QD-PEI complexes with and without Hph-1 have shown markedly different intracellular uptake behaviors and unpacking kinetics of cy5-siRNA. However, they exhibited similar extent of VEGF gene knockout regardless of Hph-1, but showed much higher gene silencing efficiency than siRNA/PEI complexes. The present study demonstrates that PEI conjugated QDs can be utilized as a useful siRNA carrier to analyze intracellular trafficking and unpacking pathway as well as to effectively silence a target gene.

Catechol-grafted poly(ethylene glycol) for PEGylation on versatile substrates.

We report on catechol-grafted poly(ethylene) glycol (PEG-g-catechol) for the preparation of nonfouling surfaces on versatile substrates including adhesion-resistant PTFE. PEG-g-catechol was prepared by the step-growth polymerization of PEO to which dopamine, a mussel-derived adhesive molecule, was conjugated. The immersion of substrates into an aqueous solution of PEG-g-catechol resulted in robust PEGylation on versatile surfaces of noble metals, oxides, and synthetic polymers. Surface PEGylation was unambiguously confirmed by various surface analytical tools such as ellipsometry, goniometry, infrared spectroscopy, and X-ray photoelectron spectroscopy. Contrary to existing PEG derivatives that are difficult-to-modify synthetic polymer surfaces, PEG-g-catechol can be considered to be a new class of PEGs for the facile surface PEGylation of various types of surfaces.

Controlled release of paclitaxel from heparinized metal stent fabricated by layer-by-layer assembly of polylysine and hyaluronic acid-g-poly(lactic-co-glycolic acid) micelles encapsulating paclitaxel.

Drug-eluting stent (DES) has been widely used for effective treatment of obstructive coronary artery disease, preventing the occurrence of restenosis that is mainly caused by hyper-proliferation of smooth muscle cells. Here, we demonstrate the immobilization of heparin on the metal surface via a bioinspired manner and subsequent build-up of a therapeutic layer-by-layer multilayer composed of paclitaxel (PTX) encapsulated poly(lactic-co-glycolic acid) grafted hyaluronic acid (HA-g-PLGA) micelles, heparin, and poly-L-lysine (PLL). It was hypothesized that the heparinized metallic surface would create a nonthrombogenic environment, while controlled release of PTX from the surface could induce antiproliferation of smooth muscle cells. For the surface immobilization of heparin on the surface of cobalt-chromium alloy (L605), dopamine-derivatized heparin was synthesized and anchored on the surface by a mussel-inspired adhesion mechanism. An amphiphilic graft copolymer of HA-g-PLGA was synthesized and utilized for the formation of anionic PTX loaded micelles. A PTX eluting multilayer composed of anionic HA-g-PLGA micelles, heparin, and PLL was self-assembled on the metal surface by a layer-by-layer fashion. The loading amount of PTX on the metal surface could be readily controlled with concomitantly achieving sustained release profiles of PTX over an extended period. The proliferation of human coronary artery smooth muscle cells was successfully arrested by controlled released PTX from the therapeutic multilayer coated on the metallic substrate.

A dynamic DNA nanostructure with switchable and size-selective molecular recognition properties.

Herein, we report a dynamic DNA nanostructure exhibiting switchable and size-selective molecular recognition properties. A DNA block copolymer, polystyrene-b-DNA (PS-b-DNA), and a thermo-responsive block copolymer, PS-b-poly(N-isopropylacrylamide) (PS-b-PNIPAM), were simultaneously assembled to form hybrid micelles composed of a PS core and a DNA/PNIPAM corona. PNIPAM strands did not significantly hinder the binding of molecular DNA for a broad range of PNIPAM lengths. On the other hand, they exerted significant steric hindrance for interactions with nanoscale species, which can be reversibly turned off by increasing the temperature above the lower critical solution temperature (LCST) of PNIPAM. Owing to the switchable and size-selective steric hindrance, the hybrid DNA micelles showed thermally controllable enzymatic degradation and cellular uptake. These results demonstrate that the binary self-assembly of two different responsive block copolymers is a promising approach to prepare dynamic nanostructures with controllable biological recognition properties.

Hyaluronic acid-paclitaxel conjugate micelles: synthesis, characterization, and antitumor activity.

Chemical conjugates of paclitaxel and hyaluronic acid (HA) were synthesized by utilizing a novel HA solubilization method in a single organic phase. Hydrophilic HA was completely dissolved in anhydrous DMSO with addition of poly(ethylene glycol) (PEG) by forming nanocomplexes. Paclitaxel was then chemically conjugated to HA in the DMSO phase via an ester linkage without modifying extremely hydrophilic HA. A series of HA-paclitaxel conjugates with different conjugation percentages were synthesized and characterized. HA-paclitaxel conjugates self-assembled in aqueous solution to form nanosized micellar aggregates, as characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). An intact form of paclitaxel was regenerated from HA-paclitaxel conjugate micelles at acidic pH conditions. HA-paclitaxel conjugate micelles exhibited more pronounced cytotoxic effect for HA receptor overexpressing cancer cells than for HA receptor deficient cells, suggesting that they can be potentially utilized as tumor-specific nanoparticulate therapeutic agents.

Shell cross-linked hyaluronic acid/polylysine layer-by-layer polyelectrolyte microcapsules prepared by removal of reducible hyaluronic acid microgel cores.

Shell cross-linked hollow polyelectrolyte microcapsules composed of hyaluronic acid (HA) and poly- l-lysine (PLL) were prepared by layer-by-layer (LBL) adsorption and subsequent core removal by a reductive agent. Disulfide cross-linked HA microgels were used as template core materials for the LBL deposition on the surface and removed by treatment of dithiothreitol at neutral pH condition. HA/PLL polyelectrolyte multilayers on the shell were chemically cross-linked via carbodiimide chemistry, and their physicochemical properties and drug release behaviors were investigated. Shell cross-linked HA/PLL polyelectrolyte microcapsules exhibited far enhanced physical stability against freeze-thaw cycles and acidic pH conditions compared to the un-cross-linked ones. The cross-linked HA/PLL multilayer shell also demonstrated pH responsive permeability, which became more permeable at low pH than at neutral pH. When bovine serum albumin (BSA), as a model protein drug, was loaded inside using the pH-dependent permeability, BSA release profiles from the microcapsules could be readily modulated by varying medium pH values or adding an HA digesting enzyme (hyaluronidase) in the incubation medium.

Hydrogel Based Biosensors for In Vitro Diagnostics of Biochemicals, Proteins, and Genes.

Hydrogel-based biosensors have drawn considerable attention due to their various advantages over conventional detection systems. Recent studies have shown that hydrogel biosensors can be excellent alternative systems to detect a wide range of biomolecules, including small biochemicals, pathogenic proteins, and disease specific genes. Due to the excellent physical properties of hydrogels such as the high water content and stimuli-responsive behavior of cross-linked network structures, this system can offer substantial improvement for the design of novel detection systems for various diagnostic applications. The other main advantage of hydrogels is the role of biomimetic three-dimensional (3D) matrix immobilizing enzymes and aptamers within the detection systems, which enhances their stability. This provides ideal reaction conditions for enzymes and aptamers to interact with substrates within the aqueous environment of the hydrogel. In this review, we have highlighted various novel detection approaches utilizing the outstanding properties of the hydrogel. This review summarizes the recent progress of hydrogel-based biosensors and discusses their future perspectives and clinical limitations to overcome.

Bioreducible-Cationic Poly(amido amine)s for Enhanced Gene Delivery and Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine.

PEGylation and HAylation via catechol: α-Amine-specific reaction at N-terminus of peptides and proteins.

UNLABELLED: The development of chemoselective, site-specific chemistries for proteins/peptides is essential for biochemistry, pharmaceutical chemistry, and other fields. In this work, we found that catechol, which has been extensively utilized as an adhesive molecule for material-independent surface chemistry and as a crosslinker in hydrogel preparation, specifically reacts with N-terminal α-amines, avoiding the ε-amine group in lysine. A conjugate of methoxy-poly(ethylene glycol)-catechol called mPEG-cat chemoselectively reacts with N-terminal amine groups at neutral pH resulting in site-specific PEGylation. To demonstrate the versatility of this catechol chemoselective reaction, we used four proteins (lysozyme, basic-fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), insulin, and erythropoietin (EPO)) as well as two peptides (hinge-3 and laminin-derived peptide (LDP)). All the tested macromolecules showed N-terminal site-specific modifications. Furthermore, we prepared another catechol grafted conjugate called hyaluronic acid-catechol (HA-cat) to demonstrate that this catechol-involved chemoselective chemistry is not specific for PEG conjugates. This new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for a variety of purposes. STATEMENT OF SIGNIFICANCE: Considering the fact that biological activities of proteins or peptides depend largely on their 3-dimensional conformation, the orientation-controllable reaction is very important for preserving the intrinsic functionality of them. In addition to PEG, many other bio-polymers such as oligonucleotides, antibodies, and oligosaccharides have been conjugated with proteins or peptides for various biomedical applications. Although several chemoselective conjugation chemistries have been reported, conjugation efficiencies are different depending on types of proteins or polymers, and thus there've been strong needs for the development of alternative strategy of chemoselective conjugation that can be applied for a variety of therapeutic proteins towards high biological activities. We are certain this new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for various purposes.

Self-assembled mirror DNA nanostructures for tumor-specific delivery of anticancer drugs.

Nanoparticle delivery systems have been extensively investigated for targeted delivery of anticancer drugs over the past decades. However, it is still a great challenge to overcome the drawbacks of conventional nanoparticle systems such as liposomes and micelles. Various novel nanomaterials consist of natural polymers are proposed to enhance the therapeutic efficacy of anticancer drugs. Among them, deoxyribonucleic acid (DNA) has received much attention as an emerging material for preparation of self-assembled nanostructures with precise control of size and shape for tailored uses. In this study, self-assembled mirror DNA tetrahedron nanostructures is developed for tumor-specific delivery of anticancer drugs. l-DNA, a mirror form of natural d-DNA, is utilized for resolving a poor serum stability of natural d-DNA. The mirror DNA nanostructures show identical thermodynamic properties to that of natural d-DNA, while possessing far enhanced serum stability. This unique characteristic results in a significant effect on the pharmacokinetics and biodistribution of DNA nanostructures. It is demonstrated that the mirror DNA nanostructures can deliver anticancer drugs selectively to tumors with enhanced cellular and tissue penetration. Furthermore, the mirror DNA nanostructures show greater anticancer effects as compared to that of conventional PEGylated liposomes. Our new approach provides an alternative strategy for tumor-specific delivery of anticancer drugs and highlights the promising potential of the mirror DNA nanostructures as a novel drug delivery platform.