A versatile engineered extracellular vesicle platform simultaneously targeting and eliminating senescent stromal cells and tumor cells to promote tumor regression.

Chemotherapy is an important therapeutic approach for malignant tumors for it triggers apoptosis of cancer cells. However, chemotherapy also induces senescence of stromal cells in the tumor microenvironment to promote tumor progression. Strategies aimed at killing tumor cells while simultaneously eliminating senescent stromal cells represent an effective approach to cancer treatment. Here, we developed an engineered Src-siRNA delivery system based on small extracellular vesicles (sEVs) to simultaneously eliminate senescent stromal cells and tumor cells for cancer therapy. The DSPE-PEG-modified urokinase plasminogen activator (uPA) peptide was anchored to the membranes of induced mesenchymal stem cell-derived sEVs (uPA-sEVs), and Src siRNA was loaded into the uPA-sEVs by electroporation (uPA-sEVs-siSrc). The engineered uPA-sEVs-siSrc retained the basic sEVs properties and protected against siSrc degradation. uPA peptide modification enhanced the sEVs with the ability to simultaneously target doxorubicin-induced senescent stromal cells and tumor cells. Src silencing by uPA-sEVs-siSrc induced apoptosis of both senescent stromal cells and tumor cells. The uPA-sEVs-siSrc displayed preferential tumor accumulation and effectively inhibited tumor growth in a tumor xenograft model. Furthermore, uPA-sEVs-siSrc in combination with doxorubicin significantly reduced the senescence burden and enhanced the therapeutic efficacy of chemotherapy. Taken together, uPA-sEVs-siSrc may serve as a promising therapy to kill two birds with one stone, not only killing tumor cells to achieve remarkable antitumor effect, but also eliminating senescent cells to enhance the efficacy of chemotherapeutic agent in tumor regression.

Identification of specific markers for human pluripotent stem cell-derived small extracellular vesicles.

Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs.

Injectable hypoxia-preconditioned cartilage progenitor cells-laden GelMA microspheres system for enhanced osteoarthritis treatment.

Osteoarthritis (OA) is the most common age-related degenerative joint disease mainly characterized by the destruction of articular cartilage. Owing to its native avascular property, intrinsic repair of articular cartilage is very limited. Thus, a chondrogenic microenvironment in the joint is essential to the preservation of healthy chondrocytes and OA treatment. Recently, cartilage progenitor cells (CPCs)-based therapy is emerging as a promising strategy to repair degenerated and damaged articular cartilage. In this study, injectable hypoxia-preconditioned three-dimensional (3D) cultured CPCs-laden gelatin methacryloyl (GelMA) microspheres (CGMs) were constructed and characterized. Compared to normoxia-pretreated 3D CPCs and two-dimensional (2D) cultured CPCs, hypoxia-preconditioned 3D cultured CPCs exhibited enhanced cartilage extracellular matrix (ECM) secretion and greater chondrogenic ability. In addition, hypoxia-preconditioned 3D cultured CPCs more effectively maintained cartilage matrix metabolism balance and attenuated articular cartilage degeneration in subacute and chronic rat OA models. Mechanistically, our results demonstrated hypoxia-preconditioned 3D cultured CPCs exerted chondro-protective effects by inhibiting inflammation and oxidative stress via NRF2/HO-1 pathway in vitro and in vivo. Together, through the 3D culture of CPCs using GelMA microspheres (GMs) under hypoxia environment, our results proposed an efficient articular cartilage regeneration strategy for OA treatment and could provide inspiration for other stem cells-based therapies.

Large extracellular vesicles secreted by human iPSC-derived MSCs ameliorate tendinopathy via regulating macrophage heterogeneity.

Tendinopathy is a common musculoskeletal disorder which results in chronic pain and reduced performance. The therapeutic effect of stem cell derived-small extracellular vesicles (sEVs) for tendinopathy has been validated in recent years. However, whether large extracellular vesicles (lEVs), another subset of extracellular vesicles, possesses the ability for the improvement of tendinopathy remains unknown. Here, we showed that lEVs secreted from iPSC-derived MSCs (iMSC-lEVs) significantly mitigated pain derived from tendinopathy in rats. Immunohistochemical analysis showed that iMSC-lEVs regulated the heterogeneity of infiltrated macrophages and several inflammatory cytokines in rat tendon tissue. Meanwhile, in vitro experiments revealed that the M1 pro-inflammatory macrophages were repolarized towards M2 anti-inflammatory macrophages by iMSC-lEVs, and this effect was mediated by regulating p38 MAPK pathway. Moreover, liquid chromatography-tandem mass spectrometry analysis identified 2208 proteins encapsulated in iMSC-lEVs, including 134 new-found proteins beyond current Vesiclepedia database. By bioinformatics and Western blot analyses, we showed that DUSP2 and DUSP3, the negative regulator of p38 phosphorylation, were enriched in iMSC-lEVs and could be transported to macrophages. Further, the immunomodulatory effect of iMSC-lEVs on macrophages was validated in explant tendon tissue from tendinopathy patients. Taken together, our results demonstrate that iMSC-lEVs could reduce inflammation in tendinopathy by regulating macrophage heterogeneity, which is mediated via the p38 MAPK pathway by delivery of DUSP2 and DUSP3, and might be a promising candidate for tendinopathy therapy.

Antibacterial Amino Acid-Based Poly(ionic liquid) Membranes: Effects of Chirality, Chemical Bonding Type, and Application for MRSA Skin Infections.

Bacteria induced infection remains a serious medical hazard to humans. Antibacterial polymeric materials, which can kill or inhibit bacteria by disrupting cell membranes, inhibiting certain enzymes, or interfering with the transcription or synthesis of DNA or RNA, have been applied to reduce or inhibit microbial drug resistance. Herein, amino acid-based ionic liquids (ILs) and poly(ionic liquid) (PIL) membranes were synthesized and used as antibacterial materials to treat skin wounds infected by methicillin-resistant Staphylococcus aureus (MRSA). The effects of chirality (D- or L-enantiomers) and chemical bonding (ionic or covalent) of the amino acid groups attached to the IL (or PIL) on antibacterial properties were investigated. Both the ILs and PIL membranes containing D-enantiomeric amino acid groups exhibited higher antibacterial activities compared with those containing L-enantiomeric amino acids. In addition, the ionically-bonded PIL membranes showed higher antibacterial activities than the corresponding covalently-bonded polymeric membranes. These results indicate that both the chirality and chemical bonding type of amino acid groups affect the antimicrobial activity of the PIL membranes. Additionally, the amino acid-based PIL membranes accelerated the wound-healing process, alleviated local tissue inflammation, and reduced the influence of bacteria on vital organs (liver and spleen) in MRSA-infected mouse models, demonstrating the potential applications for antimicrobial wound dressing.

Fast multipole boundary element method to calculate head-related transfer functions for a wide frequency range.

Head-related transfer functions (HRTFs) play an important role in spatial sound localization. The boundary element method (BEM) can be applied to calculate HRTFs from non-contact visual scans. Because of high computational complexity, HRTF simulations with BEM for the whole head and pinnae have only been performed for frequencies below 10 kHz. In this study, the fast multipole method (FMM) is coupled with BEM to simulate HRTFs for a wide frequency range. The basic approach of the FMM and its implementation are described. A mesh with over 70 000 elements was used to calculate HRTFs for one subject. With this mesh, the method allowed to calculate HRTFs for frequencies up to 35 kHz. Comparison to acoustically-measured HRTFs has been performed for frequencies up to 16 kHz, showing a good congruence below 7 kHz. Simulations with an additional shoulder mesh improved the congruence in the vertical direction. Reduction in the mesh size by 5% resulted in a substantially-worse representation of spectral cues. The effects of temperature and mesh perturbation were negligible. The FMM appears to be a promising approach for HRTF simulations. Further limitations and potential advantages of the FMM-coupled BEM are discussed.