Jingyan Wei

A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy.

Targeted drug delivery vehicles with low immunogenicity and toxicity are needed for cancer therapy. Here we show that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, can deliver chemotherapeutics such as doxorubicin (Dox) to tumor tissue in BALB/c nude mice. To reduce immunogenicity and toxicity, mouse immature dendritic cells (imDCs) were used for exosome production. Tumor targeting was facilitated by engineering the imDCs to express a well-characterized exosomal membrane protein (Lamp2b) fused to αv integrin-specific iRGD peptide (CRGDKGPDC). Purified exosomes from imDCs were loaded with Dox via electroporation, with an encapsulation efficiency of up to 20%. iRGD exosomes showed highly efficient targeting and Dox delivery to αv integrin-positive breast cancer cells in vitro as demonstrated by confocal imaging and flow cytometry. Intravenously injected targeted exosomes delivered Dox specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity. Our results suggest that exosomes modified by targeting ligands can be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clinical applications.

Localized Electric Field of Plasmonic Nanoplatform Enhanced Photodynamic Tumor Therapy

Near-infrared plasmonic nanoparticles demonstrate great potential in disease theranostic applications. Herein a nanoplatform, composed of mesoporous silica-coated gold nanorods (AuNRs), is tailor-designed to optimize the photodynamic therapy (PDT) for tumor based on the plasmonic effect. The surface plasmon resonance of AuNRs was fine-tuned to overlap with the exciton absorption of indocyanine green (ICG), a near-infrared photodynamic dye with poor photostability and low quantum yield. Such overlap greatly increases the singlet oxygen yield of incorporated ICG by maximizing the local field enhancement, and protecting the ICG molecules against photodegradation by virtue of the high absorption cross section of the AuNRs. The silica shell strongly increased ICG payload with the additional benefit of enhancing ICG photostability by facilitating the formation of ICG aggregates. As-fabricated AuNR@SiO2-ICG nanoplatform enables trimodal imaging, near-infrared fluorescence from ICG, and two-photon luminescence/photoacoustic tomography from the AuNRs. The integrated strategy significantly improved photodynamic destruction of breast tumor cells and inhibited the growth of orthotopic breast tumors in mice, with mild laser irradiation, through a synergistic effect of PDT and photothermal therapy. Our study highlights the effect of local field enhancement in PDT and demonstrates the importance of systematic design of nanoplatform to greatly enhancing the antitumor efficacy.

Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows

Exosomes, molecular cargos secreted by almost all mammalian cells, are considered as promising biomarkers to identify many diseases including cancers. However, the small size of exosomes (30-200 nm) poses serious challenges in their isolation from complex media containing a variety of extracellular vesicles (EVs) of different sizes, especially in small sample volumes. Here we present a viscoelasticity-based microfluidic system to directly separate exosomes from cell culture media or serum in a continuous, size-dependent, and label-free manner. Using a small amount of biocompatible polymer as the additive in the media to control the viscoelastic forces exerted on EVs, we are able to achieve a high separation purity (>90%) and recovery (>80%) of exosomes. The proposed technique may serve as a versatile platform to facilitate exosome analyses in diverse biochemical applications.

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity.

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH‐S‐2,4‐dinitrophenyl t‐butyl ester (GSH‐S‐DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single‐chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv‐encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv‐B3) in Escherichia coli Rosetta. The scFv‐B3 was purified by Ni2+‐immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0–3.0 mg of proteins from 1 L culture. After the active site serines of scFv‐B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se‐scFv‐B3) with GPX activity of 1288 U/µmol. Copyright

Nanoparticle-mediated local depletion of tumour-associated platelets disrupts vascular barriers and augments drug accumulation in tumours

Limited intratumoural perfusion and nanoparticle retention remain major bottlenecks for the delivery of nanoparticle therapeutics into tumours. Here, we show that polymer–lipid–peptide nanoparticles delivering the antiplatelet antibody R300 and the chemotherapeutic agent doxorubicin can locally deplete tumour-associated platelets, thereby enhancing vascular permeability and augmenting the accumulation of the nanoparticles in tumours. R300 is specifically released in the tumour on cleavage of the lipid–peptide shell of the nanoparticles by matrix metalloprotease 2, which is commonly overexpressed in tumour vascular endothelia and stroma, thus facilitating vascular breaches that enhance tumour permeability. We also show that this strategy leads to substantial tumour regression and metastasis inhibition in mice.Polymer–lipid–peptide nanoparticles carrying an antiplatelet antibody and a chemotherapy drug deplete tumour-associated platelets to increase vascular permeability and augment the accumulation of the drug in tumours.

Improving GPX activity of selenium‐containing human single‐chain Fv antibody by site‐directed mutation based on the structural analysis

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni2+‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright

Unglycosylated recombinant human glutathione peroxidase 3 mutant from Escherichia coli is active as a monomer

Glutathione peroxidase 3 (GPx3) is a glycosylated member of GPx family and can catalyze the reaction of different types of peroxides with GSH to form their corresponding alcohols in vitro. The active center of GPx3 is selenocysteine (Sec), which is incorporated into proteins by a specific mechanism. In this study, we prepared a recombinant human GPx3 (rhGPx3) mutant with all Cys changed to Ser from a Cys auxotrophic strain of E. coli, BL21(DE3)cys. Although lacking post-translational modification, rhGPx3 mutant still retained the ability to reduce H2O2 and PLPC-OOH. Study on the quaternary structure suggested that rhGPx3 mutant existed as a monomer in solution, which is different from native tetrameric GPx3. Loss of the catalytic activity was considered to be attributed to both the absence of glycosylation and the failure of the tetramer. Further analysis was performed to compare the structures of rhGPx3 and GPx4 mutant, which were quite similar except for oligomerization loop. The differences of amino acid composition and electrostatic potentials on the oligomerization loop may affect the binding of large substrates to rhGPx3 mutant. This research provides an important foundation for biosynthesis of functionally selenium-containing GPx3 mutant in E.coli.

Expression and characterization of recombinant human phospholipid hydroperoxide glutathione peroxidase.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC1.11.1.12) is a selenoperoxidase that can directly reduce phospholipid and cholesterol hydroperoxides. The mature cytoplasmic GPx4 is a monomeric protein with molecular weight of 19.5 kDa. In this study, human GPx4 (hGPx4) gene was amplified from the complementary DNA library of human hepatoma cell line. Eukaryotic expression plasmid pSelExpress1‐leader‐GPx4 was constructed and transfected into the eukaryotic cells HEK293T. Expression of hGPx4 was detected by Western blotting, and the target protein was purified by immobilized metal affinity chromatography. The results of the activity and kinetics of the purified protein show that the obtained protein follows a “ping–pong” mechanism, which is similar to that of native cytosolic glutathione peroxidase (GPx1; EC1.11.1.9). This is the first time that hGPx4 could be expressed and purified from HEK293T cells, and this work will provide an important resource of hGPx4 for its functional study in vitro and in vivo.

Characterization of catalytic activity and structure of selenocysteine‐containing hGSTZ1c‐1c based on site‐directed mutagenesis and computational analysis

Human glutathione transferase zeta 1c‐1c (hGSTZ1c‐1c) is one of the glutathione transferase isoenzymes and considered to be a protein scaffold to imitate glutathione peroxidase (GPX) owing to the natural binding site of glutathione (GSH). In this report, several residues near GSH were mutated to selenocysteine (Sec) or cysteine (Cys) residues and the impacts of the substitutions on different activities were discussed. Mutations of Ser‐14 or/and Ser‐15 to Cys or Sec residues resulted in dramatic loss of catalytic activity of hGSTZ1c‐1c with chlorofluoroacetic acid as substrate, which indicated the importance of the hydroxyl groups in Ser‐14 and Ser‐15. And subsequent study by molecular modeling suggested that Ser‐15 was probably involved in catalysis, while Ser‐14 may play a crucial role in binding and orientation of GSH and possibly had a synergistic effect with Ser‐15 in catalysis. On the contrary, the result of converting Cys‐16 to Ser indicated its trivial role in catalysis. The investigations of the selenocysteine‐containing hGSTZ1c‐1c (seleno‐hGSTZ1c‐1c) and the mutant S17C implied that the substitutions of multi‐Sec for Cys residues at position 16, 137, and 205 could lead to subtle change in the structure of the protein molecule and concomitant change in catalytic activity as a direct result. This finding provides overwhelming evidence that the protein scaffold containing fewer cysteines should be chosen for imitating GPX using cysteine auxotrophic strain system to avoid unexpected structural changes.

A new human catalytic antibody Se-scFv-2D8 and its selenium-containing single domains with high GPX activity

Glutathione peroxidase (GPX) is a well‐known antioxidant selenoenzyme, which can catalyze the reduction of a variety of hydroperoxides and consequently protect cells and other biological tissues against oxidative damage. Many attempts have been made to mimic its function, and a human catalytic antibody Se‐scFv‐B3 with GPX activity has been prepared in our previous study. This time, a new clone 2D8 that bound specifically to the glutathione analog GSH‐S‐DNPBu was selected again by using the technology of phage display antibody library, and then scFv‐2D8 was successfully expressed in soluble form and purified using Ni2+‐immobilized metal affinity chromatography. After being converted into selenium‐containing scFv by chemically modification, it showed higher GPX activity than previous abzyme Se‐scFv‐B3. The heavy chain variable fragment of scFv‐2D8 was also prepared and converted into selenium‐containing protein using the same method. This selenium‐containing single‐domain antibody showed some GPX activity and, to the best of our knowledge, is the first human single‐domain abzyme with GPX activity, which lays a foundation for preparing GPX abzyme with human origin, lower molecular weight and higher activity. Copyright