Decorating Delivery Vehicles Using Hyaluronic Acid Oligosaccharides Enables Active Targeting Toward Cancer and Minimizes Adverse Effect of Chemotherapeutics.

The major drawback of conventional chemotherapeutic treatment is the non-specificity or inability to ascertain and target cancerous cells directly. In this study, an active targeting strategy that is poised to carry the anticancer agents to the desired sites for therapeutic action while avoiding toxicity to normal organs is provided. The active targeting of delivery vehicles is achieved by ligand-receptor interactions, in particular the specific binding between hyaluronic acid oligosaccharides (oHAs) and CD44 receptors. This study first prepares oHAs by the size-exclusion chromatography and utilizes them to decorate chitosan (CTS) as basic materials (oHAs-CTS) for drug delivery, then fabricates oHAs-CTS into micro/nanoscale carriers to encapsulate agents for cancer chemotherapy. The oHAs-CTS micro/nanocarriers exhibit high drug encapsulation efficiency (58-87%), and the drug releases present a sustained behavior. Notably, oHAs-CTS delivery vehicles display an enhanced active targeting toward cancers and alleviate the cytotoxic effects on normal cells. Additionally, in vivo results show that drug-laden oHAs-CTS nanocarriers demonstrate a significant inhibitory effect on 4 T1 tumors without any toxicity to the major organs. Taken together, the findings highlight the potential of oHAs-CTS micro/nanospheres as delivery vehicles with enhanced active targeted capability toward cancers and minimized adverse effects of chemotherapeutic agents for cancer treatment.

Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae.

Hyaluronic acid (HA) is a well-known functional marine polysaccharide. The utilization and derivative development of HA are of great interest. Hyaluronan lyase has wide application prospects in the production of HA oligosaccharides and lower molecular weight HA. In this study, a strain of Enterobacter asburiae CGJ001 with high hyaluronan lyase activity was screened from industrial wastewater. This strain exhibited an impressive enzyme activity of 40,576 U/mL after being incubated for 14 h. Whole genome sequencing analysis revealed that E. asburiae CGJ001 contained a cluster of genes involved in HA degradation, transport, and metabolism. A newly identified enzyme responsible for glycosaminoglycan degradation was designated as HylEP0006. A strain of E. coli BL21(DE3)/pET-22b(+)-hylEP0006 was successfully constructed. HylEP0006 exhibited optimal degradation at 40 °C and pH 7.0, showing a high activity of 950,168.3 U/mg. HylEP0006 showed specific activity against HA. The minimum degradation fragment of HylEP0006 was hyaluronan tetrasaccharides, and HylEP0006 could efficiently degrade HA into unsaturated disaccharides (HA2), with HA2 as the final product. These characteristics indicate that HylEP0006 has a potential application prospect for the extraction and utilization of hyaluronic acid.

Construction of enzyme-laden vascular scaffolds based on hyaluronic acid oligosaccharides-modified collagen nanofibers for antithrombosis and in-situ endothelialization of tissue-engineered blood vessels.

The current use of synthetic grafts often yields low patency in the reconstruction of small-diameter blood vessels owing to the deposition of thrombi and imperfect coverage of the endothelium on the graft lumen. Therefore, the design of vascular scaffolds with antithrombotic performance and endothelialization is greatly required. Herein, we developed an enzyme-laden scaffold based on hyaluronic acid oligosaccharides-modified collagen nanofibers (labeled HA-COL) to improve the anti-platelet capacity and endothelialization of vascular grafts. In this study, HA-COL nanofibers not only encouraged the endothelialization of vascular scaffolds, but acted as an antiplatelet enzyme-laden platform. Apyrase (Apy) and 5'-nucleotidase (5'-NT) were covalently grafted onto the nanofibers, which in turn converted the platelet-sensitive substance: adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and adenosine, thereby, improving the antithrombotic performance of the scaffolds. Notably, the catalytic end-product: adenosine would work in coordination with HA-COL to synergistically enhance the endothelialization of the vascular scaffolds. The results demonstrated that the enzyme-laden scaffolds maintained catalytic performance, reduced platelet adhesion and aggregation, and guaranteed higher patency after 1-month in situ transplantation. Moreover, these scaffolds showed optimal cytocompatibility, tissue compatibility, scaffold biodegradability and tissue regenerative capability during in vivo implantation. Overall, these engineered vascular scaffolds demonstrated their capacity for endothelialization and antithrombotic performance, suggesting their potential for small-diameter vascular tissue engineering applications. STATEMENT OF SIGNIFICANCE: Considering the critical problems in small-diameter vascular reconstruction, the enzyme-laden vascular scaffolds were prepared for improving in-situ endothelialization and antithrombotic performances of artificial blood vessels. The electrospun HA-COL nanofibers were used as the main matrix materials, which provided favorable structural templates for the regeneration of vasculature and functioned as a platform for the loading of enzymes. The enzyme-laden scaffolds with the biomimetic cascading reaction would convert ADP into adenosine, thereby, decreasing the sensitivity of platelets and improving the antithrombotic performance of tissue-engineered blood vessels (TEBVs). The nanofibrous scaffolds exhibited optimal cytocompatibility, tissue compatibility and regenerative capability, working together with catalytic products of dual-enzyme reaction that would synergistically contribute to TEBVs endothelialization. This study provides a new method for the improvement of in-situ endothelialization of small-diameter TEBVs while qualified with antithrombotic performance.

Hyaluronic acid oligosaccharides modified mineralized collagen and chitosan with enhanced osteoinductive properties for bone tissue engineering.

In this study, we prepared a biomimetic hyaluronic acid oligosaccharides (oHAs)-based composite scaffold to develop a bone tissue-engineered scaffold for stimulating osteogenesis and endothelialization. The functional oHAs products were firstly synthesized, namely collagen/hyaluronic acid oligosaccharides/hydroxyapatite (Col/oHAs/HAP), chitosan/hyaluronic acid oligosaccharides (CTS/oHAs), and then uniformly distributed in poly (lactic-co-glycolic acid) (PLGA) solution followed by freeze-drying to obtain three-dimensional interconnected scaffolds as temporary templates for bone regeneration. The morphology, physicochemical properties, compressive strength, and degradation behavior of the fabricated scaffolds, as well as in vitro cell responses seeded on these scaffolds and in vivo biocompatibility, were investigated to evaluate the potential for bone tissue engineering. The results indicated that the oHAs-based scaffolds can promote the attachment of endothelial cells, facilitate the osteogenic differentiation of MC3T3-E1 and BMSCs, and have ideal biocompatibility and tissue regenerative capacity, suggesting their potential to serve as alternative candidates for bone tissue engineering applications.

Hyaluronic Acid Oligosaccharides Improve Myocardial Function Reconstruction and Angiogenesis against Myocardial Infarction by Regulation of Macrophages.

Myocardial infarction (MI) is identified as one of the major causes of mortality and disability worldwide. For severe myocardial infarction, even advanced forms of clinical intervention often lead to unsatisfactory therapeutic results. Thus, alternative strategies for MI treatment are still desirable. Previously studies reported the capacity of degradative fragment of h-HA (high molecular weight hyaluronic acid), hyaluronan oligosaccharides (<10 disaccharides units, o-HA), for wound healing by influence on angiogenesis, inspiring us to study its potential for myocardial functional recovery against MI. However, there are few reports about o-HA in MI therapy. Methods: In our study, we synthesized o-HA with 6~10 disaccharides (4-5 kDa) by enzymatic degradation and investigated its therapeutic effects on MI. Results: We found that o-HA could reduce infarct size and apoptosis in MI region, also promote myocardial angiogenesis and myocardial function reconstruction in MI mouse model. Furthermore, our results also indicated that o-HA in cardiac improved polarization of M2 type macrophage, removed the inflammatory response caused by neutrophil for accelerating myocardial function reconstruction in vivo. The transcriptomic analyses revealed that o-HA could activate expression of chemokines Ccl2 and Cxcl5 for promoting macrophage polarization and stimulate MAPK and JAK/STAT signaling pathway for compensatory response of myocardial function. Conclusion: Collectively, our results suggested o-HA with 6~10 disaccharides might be a potential agent for reconstruction of cardiac function against MI.