Fluorinated Oligoethylenimine Nanoassemblies for Efficient siRNA-Mediated Gene Silencing in Serum-Containing Media by Effective Endosomal Escape.

Efficient small interfering RNA (siRNA) delivery in the presence of serum is of crucial importance for effective gene therapy. Fluorinated vectors are considered to be attractive candidates for siRNA-mediated gene therapy because of their delivery efficacy in serum-containing media. However, the mechanisms driving the superior gene transfection behavior of fluorinated vectors are still not well-understood, and comprehensive investigations are warranted. Herein, we fabricated a library of perfluorooctanoyl fluoride-fluorinated (PFF-fluorinated) oligoethylenimines (f xOEIs, x is the PFF:OEI feeding ratio), which can readily form nanoassemblies (f xOEI NAs) capable of efficient siRNA delivery in cells cultured in medium both devoid of and supplemented with fetal bovine serum (FBS). The gene silencing test in serum-containing medium revealed that the f0.7OEI/siRNA NAs achieved a luciferase silencing of ∼88.4% in Luc-HeLa cells cultured in FBS-containing medium, which was almost 2-fold greater than the silencing efficacy of siRNA delivered by the commercially available vector Lipo 2000 (∼48.8%). High levels of apolipoprotein B silencing were also achieved by f0.7OEI/siRNA NAs in vivo. For an assessment of the underlying mechanisms of the efficacy of gene silencing of fluorinated vectors, two alkylated OEIs, aOEI-C8 and aOEI-C12, were fabricated as controls with similar molecular structure and hydrophobicity to that of f0.7OEI, respectively. In vitro investigations showed that the superior gene delivery exhibited by f0.7OEI NAs derived from the potent endosomal disruption capability of fluorinated vectors in the presence of serum, which was essentially attributed to the serum protein adsorption resistance of the f0.7OEI NAs. Therefore, this work provides an innovative approach to siRNA delivery as well as insights into fluorine-associated serum resistance.

Inhibition of proteases activity in intestine needs a sustainable acidic environment rather than a transient.

α-Chymotrypsin (α-CT) and trypsin are important components of the enzymatic barrier. They could degrade the therapeutic proteins and peptides, inhibit their activity consequently, and thereby reduce their oral bioavailability. Acidic agents, as one type of indirect protease inhibitors, have shown proof of concept in clinical trials. We report here the inactivated proteases due to acid influence can be reactivated immediately by environmental pH recovery regardless of how long the inactivation last. To keep the inactivation time of proteases for 4-5 h, we designed and prepared a sustained-release tablet containing citric acid (CA) which can effectively reduce the pH below 5.0 and maintain it for 5 h in the dissolution-reaction medium. The activity of α-CT and trypsin was quantified by analyzing the residual amount of their respective substrates BTEE and TAME. More than 80% of the substrates were survived in 5.0 h of incubation, whereas the common tablet inhibited the proteases activity for only two hours in the same experimental medium. It indicates that the sustained-release tablet loaded with CA can efficiently inhibit the α-CT and trypsin activity longer than the common tablet. The results will be beneficial for designing and formulating the peroral administration of peptide and protein drugs.

Transferrin-Dressed Virus-like Ternary Nanoparticles with Aggregation-Induced Emission for Targeted Delivery and Rapid Cytosolic Release of siRNA.

Viruses have evolved to be outstandingly efficient at gene delivery, but their use as vectors is limited by safety risks. Inspired by the structure of viruses, we constructed a virus-mimicking vector (denoted as TR4@siRNA@Tf NCs) with virus-like architecture and infection properties. Composed of a hydrophilic peptide, an aggregation-induced emission (AIE) luminogen, and a lipophilic tail, TR4 imitates the viral capsid and endows the vector with AIE properties as well as efficient siRNA compaction. The outer glycoprotein transferrin (Tf) mimics the viral envelope protein and endows the vector with reduced cytotoxicity as well as enhanced targeting capability. Because of the strong interaction between Tf and transferrin receptors on the cell surface, the Tf coating can accelerate the intracellular release of siRNA into the cytosol. Tf and TR4 are eventually cycled back to the cell membrane. Our results confirmed that the constructed siRNA@TR4@Tf NCs show a high siRNA silencing efficiency of 85% with significantly reduced cytotoxicity. These NCs have comparable transfection ability to natural viruses while avoiding the toxicity issues associated with typical nonviral vectors. Therefore, this proposed virus-like siRNA vector, which integrates the advantages of both viral and nonviral vectors, should find many potential applications in gene therapy.

Catalase-only nanoparticles prepared by shear alone: Characteristics, activity and stability evaluation.

Catalase is a promising therapeutic enzyme; however, it carries risks of inactivation and rapid degradation when it is used in practical bioprocess, such as delivery in vivo. To overcome the issue, we made catalase-only nanoparticles using shear stress alone at a moderate shear rate of 217s(-1) in a coaxial cylinder flow cell. Properties of nanoparticles, including particle size, polydispersity index and zeta potential, were characterized. The conformational changes of pre- and post-sheared catalase were determined using spectroscopy techniques. The results indicated that the conformational changes of catalase and reduction in α-helical content caused by shear alone were less significant than that by desolvation method. Catalase-only nanoparticles prepared by single shear retained over 90% of its initial activity when compared with the native catalase. Catalase nanoparticles lost only 20% of the activity when stored in phosphate buffer solution for 72h at 4°C, whereas native catalase lost 53% under the same condition. Especially, the activity of nanogranulated catalase was decreased only slightly in the simulated intestinal fluid containing α-chymotrypsin during 4h incubation at 37°C, implying that the catalase nanoparticle was more resistant to the degradation of proteases than native catalase molecules. Overall, catalase-only nanoparticles offered a great potential to stabilize enzymes for various pharmaceutical applications.

Two-photon polymerization microfabrication of hydrogels: an advanced 3D printing technology for tissue engineering and drug delivery.

3D printing technology has attracted much attention due to its high potential in scientific and industrial applications. As an outstanding 3D printing technology, two-photon polymerization (TPP) microfabrication has been applied in the fields of micro/nanophotonics, micro-electromechanical systems, microfluidics, biomedical implants and microdevices. In particular, TPP microfabrication is very useful in tissue engineering and drug delivery due to its powerful fabrication capability for precise microstructures with high spatial resolution on both the microscopic and the nanometric scale. The design and fabrication of 3D hydrogels widely used in tissue engineering and drug delivery has been an important research area of TPP microfabrication. The resolution is a key parameter for 3D hydrogels to simulate the native 3D environment in which the cells reside and the drug is controlled to release with optimal temporal and spatial distribution in vitro and in vivo. The resolution of 3D hydrogels largely depends on the efficiency of TPP initiators. In this paper, we will review the widely used photoresists, the development of TPP photoinitiators, the strategies for improving the resolution and the microfabrication of 3D hydrogels.

C2v symmetrical two-photon polymerization initiators with anthracene core: synthesis, optical and initiating properties.

A series of C(2v) symmetrical two-photon absorption compounds with anthracene core, 2,7-bis[2-(4-substituted phenyl)-vinyl]-9,10-dipentyloxyanthracenes designated as I, II and III (the substituted groups at the 4-position of phenyl of I, II and III were dimethylamino, methyl and cyano, respectively) were designed and synthesized as initiators in two-photon induced polymerization (TPIP). The anthracene ring was modified by linking vinylphenyl groups to the 2,7-position to extend conjugation system length and two pentyloxy groups to the 9,10-position to serve as electronic donors. Two-photon absorption cross section of I was around 300 GM, which was much larger than the 10 GM of II and 29 GM of III at 800 nm. I of 0.18% molar ratio in resin composed of methacrylic acid and dipentaerythritol hexaacrylate exhibited a dramatically low threshold of 0.64 mW compared with commercial photoinitiator benzil at a scanning speed 10 µm s(-1). Moreover, the threshold of photoinitiator I was only increased to 2.53 mW at a scanning speed of 1000 µm s(-1). The dependency of threshold on the concentration and exposure time was in accordance with theoretical calculation. Finally, a reasonable mechanism of the two-photon initiating process was proposed. This study provides good prospects for developing low threshold photoinitiator in TPIP.