Construction of a Targeting Nanoparticle of 3',3″-Bis-Peptide-siRNA Conjugate/Mixed Lipid with Postinserted DSPE-PEG2000-cRGD.

The cyclic Arg-Gly-Asp (cRGD) peptides are widely used as tumor-targeting ligands due to their specific binding ability to integrin αvß3, which is overexpressed on the surface of various cancer cells and the endothelial cells of new blood vessels within tumor tissues. In this paper, the postinsertion strategy of DSPE-PEG2000-cRGD has been applied to the nanoparticles of 3',3″-bis-peptide-siRNA (pp-siRNA) encapsulated by gemini-like cationic lipid (CLD) and neutral cytosin-1-yl lipid (DNCA) from our lab. It was confirmed that the nanoparticles of pp-siRNA/CLD/DNCA/DSPE-PEG2000-cRGD (PCNR) were able to specifically target tumor cells with highly expressed integrin αvß3; moreover, it efficiently downregulated the levels of BRAF mRNA and the BRAF protein and inhibited cell proliferation in A375 cells, in comparison with the nontargeted nanocomplex of pp-siRNA/CLD/DNCA/cRAD (PCNA). The uptake pathways of PCNR are mostly dependent on CvME-mediated endocytosis and macropinocytosis in A375 cells, which could bypass lysosome or quickly lead to the lysosomal escape to reduce siRNA degradation. Finally, the biodistribution study showed that PCNR exhibited a high ability to accumulate in tumor tissues. These results suggest that the nanocomplex of PCNR is promising to be highly effective in the treatment of melanomas including their mutation.

Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion.

With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

Site-Specific PEGylated Adeno-Associated Viruses with Increased Serum Stability and Reduced Immunogenicity.

Adeno-associated virus (AAV) is one of the most extensively studied and utilized viral vectors in clinical gene transfer research. However, the serum instability and immunogenicity of AAV vectors significantly limit their application. Here, we endeavored to overcome these limitations by developing a straightforward approach for site-specific PEGylation of AAV via genetic code expansion. This technique includes incorporation of the azide moiety into the AAV capsid protein followed by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs) through click chemistry. Using this approach, only the chosen site(s) was consistently PEGylated under mild conditions, preventing nonselective conjugation. Upon a series of in vitro examinations, AAVs conjugated with 20-kD PEG at sites Q325+1, S452+1, and R585+1 showed a 1.7- to 2.4-fold stability improvement in pooled human serum and a nearly twofold reduction in antibody recognition. Subsequent animal research on Sprague Dawley rats displayed a promising 20% reduction in antibody inducement and a higher virus titer in the blood. Together, our data demonstrate successful protection of an AAV vector from antibody neutralization and blood clearance, thereby increasing the efficiency of therapeutic gene delivery.

Freeze-resistant, rapidly polymerizable, ionic conductive hydrogel induced by Deep Eutectic Solvent (DES) after lignocellulose pretreatment for flexible sensors.

In this paper, ternary DES (choline chloride, glycerol, Lewis acid) was used to pretreat lignocellulose, and the DES solution with dissolved lignin was utilized as the medium of hydrogel to prepare DES-based polyacrylic acid/polyvinyl alcohol (PAA/PVA) double network hydrogels with great mechanical properties, self-adhesion, and high electrochemical sensitivity. The entanglement of PAA with PVA chains, the covalent linkage between Al3+ and PAA chains and the metal phenol network (MPN) formed by Al3+ and lignin improved the mechanical properties of the hydrogels, enabling the prepared hydrogels to achieve a tensile strain of 400 % and an elongation at break of 150 kPa. Secondly, the introduction of DES solution endowed the hydrogel with excellent electrical sensing ability and anti-freezing property, so that the hydrogel still maintains good flexibility and ionic conductivity at -20 °C. It was also found that the above hydrogel can achieve a high gauge factor of 4.19 as a flexible sensor, which provides scientific ideas for the application of the pretreated DES solution in the field of flexible wearable.

Utilization of lignin upon successive fractionation and esterification in polylactic acid (PLA)/lignin biocomposite.

The study presents the preparation of novel biocomposites based on different lignin fractions and polylactic acid (PLA). Based on the improvement of PLA mechanical properties, we proposed a process to assemble a series of lignin samples and PLA matrix using melt blending to investigate the effects on PLA properties from the perspective of the structure and molecular weight for lignin. The lignin was extracted from pine residue using deep eutectic solvent (DES) and was subjected to fractionation with ethanol and acetone as well as esterification modification with succinic anhydride (SAn). The treated lignin samples were used as additives, which not only improved the mechanical properties of PLA on the basis of retaining its thermal stability, but also granted excellent antimicrobial and biocompatibility properties. The results showed that the molecular weight of lignin was negatively correlated with the phenolic hydroxyl content, and the mechanical properties of the composites were also significantly affected by different molecular weights of lignin, indicating that the inhomogeneity of lignin affects its value-added utilization. The enhanced mechanical properties, antibacterial effect, and steady biocompatibility provide potential possibilities for lignin-based composites in biomedical applications.

Culturing rhodotorula glutinis in fermentation-friendly deep eutectic solvent extraction liquor of lignin for producing microbial lipid.

Currently, deep eutectic solvents (DES) have attracted increasing attention due to their excellent performance in delignification. However, few studies focused on the treatment of DES waste liquid after extraction of lignin. In this work, the fermentation-friendly DES comprised of glycerol, choline chloride (ChCl) and acetic acid (AA) was applied for delignification of lignocellulose. Subsequently, the extraction effects of different DES were investigated, and the DES extraction liquor was used for lipid production. Results shows ChCl made little difference to lipid synthesis, while excessive AA exerted inhibitory effect on the growth of cells. Following pretreatment, the delignification exceeded 63%. When the DES liquid obtained after lignin extraction was used to produce lipid, the delay period was obvious, while the lipid yield and content were unaffected. Not only is the DES prepared in this study effective in delignification of lignocellulose, it is also applicable as raw material to produce lipid.

Recent Progress in Aptamer Discoveries and Modifications for Therapeutic Applications.

Aptamers are oligonucleotide sequences with a length of about 25-80 bases which have abilities to bind to specific target molecules that rival those of monoclonal antibodies. They are attracting great attention in diverse clinical translations on account of their various advantages, including prolonged storage life, little batch-to-batch differences, very low immunogenicity, and feasibility of chemical modifications for enhancing stability, prolonging the half-life in serum, and targeted delivery. In this Review, we demonstrate the emerging aptamer discovery technologies in developing advanced techniques for producing aptamers with high performance consistently and efficiently as well as requiring less cost and resources but offering a great chance of success. Further, the diverse modifications of aptamers for therapeutic applications including therapeutic agents, aptamer-drug conjugates, and targeted delivery materials are comprehensively summarized.

A new synthetic access to bicyclic polyhydroxylated alkaloid analogues from pyranosides.

A facile, versatile and stereoselective synthesis of bicyclic polyhydroxylated alkaloids as castanospermine analogues is described. The synthetic route started from methyl pyranosides. The key steps involved a high-yielding expeditious one-pot tandem reaction from alkenes to N-substituted delta-lactams. The delta-lactams were stereoselectively vinylated to give the dienes, which were followed by the ring-closing metathesis to produce the cyclized products. The functional group transformations of the resulting bicyclic compounds furnished diverse polyhydroxylated alkaloids in good yields.

Precise and combinatorial PEGylation generates a low-immunogenic and stable form of human growth hormone.

In this study, we aimed to develop a safe and stable form of human growth hormone (hGH) and to refine PEGylation methods for therapeutic proteins via genetic code expansion. Through this precise approach, a series of polyethylene glycol (PEG) moieties and sites were combined in various ways. Additionally, the effects of combinatorial PEGylation on the biological, pharmacological, and immunogenic properties of hGH in vitro and vivo were analyzed. Our results showed that combinatorial PEGylation at Y35, G131, and K145 significantly reduced immunogenicity and improved pharmacokinetic (PK) profiles compared with mono-PEGylation, while retaining biological activity. Upon re-examination of the pharmacodynamics in hypophysectomized rats, multi-PEGylated hGH was found to be much more stable than mono-PEGylated hGH. Thus, this method for combinatorial, precise PEGylation may facilitate the development of next-generation, long-acting hGH with low immunogenicity.

Development of next generation of therapeutic IFN-α2b via genetic code expansion.

With the aim to overcome the heterogeneity associated with marketed IFN-α2b PEGylates and optimize the size of the PEG moiety and the site of PEGylation, we develop a viable and facile platform through genetic code expansion for PEGylation of IFN-α2b at any chosen site(s). This approach includes site-specific incorporation of an azide-bearing amino acid into IFN-α2b followed by orthogonal and stoichiometric conjugation of a variety of PEGs via a copper-free click reaction. By this approach, only the chosen site(s) within IFN-α2b is consistently PEGylated under mild conditions, leading to a single and homogenous conjugate. Furthermore, it makes the structure-activity relationship study of IFN-α2b possible by which the opposite effects of PEGylation on the biological and pharmacological properties are optimized. Upon re-examination of the PEGylated IFN-α2b isomers carrying different sizes of PEG at different sites, we find mono-PEGylates at H34, A74 and E107 with a 20-, 10- and 10-kDa PEG moiety, respectively, have both higher biological activities and better PK profiles than others. These might represent the direction for development of the next generation of PEGylated IFN-α2b.

Nucleolin-targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas.

BRAF gene mutation is found in more than 60% of malignant melanomas, which are difficult to treat. In this study, a new tumor-targeting liposome was developed to deliver anti-BRAF siRNA (siBraf) for the treatment of melanomas. Nucleolin is overexpressed on the surface of cancer cells. AS1411, an aptamer showing specific binding to nucleolin, was conjugated to PEGylated cationic liposome as the targeting probe ASLP (AS1411-PEG-liposome). The ASLP/siRNA complex was formed through electrostatic interaction between ASLP and siRNA. The binding of AS1411 to the surface of PEGylated liposomes was confirmed by gel electrophoresis and capillary electrophoresis. Real-time PCR and Western blot analysis showed that ASLP/siBraf exhibited strong silencing activity of BRAF gene. The much higher accumulation of the siRNA in tumor cells comparing with normal cells indicated that ASLP displayed excellent tumor-targeting capability. Notably, ASLP/siBraf showed significant silencing activity in A375 tumor xenograft mice and inhibited the melanoma growth. These results suggested that the new nucleolin-targeted siRNA delivery system by AS1411 may have the potential for the treatment of melanoma.

Polymer-assisted structural modification on nucleosides and nucleotides.

Nucleosides and their analogues play important roles in biological research and clinical therapeutics. Polymer-assisted structural modifications of nucleosides and nucleotides enable parallel and rapid construction of nucleoside library. For some nucleosides, higher chemical selectivity and regioselectivity can be achieved using solid-phase synthesis when compared to classic solution-phase synthesis.