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.

Sulfated hyaluronic acid/collagen-based biomimetic hybrid nanofiber skin for diabetic wound healing: Development and preliminary evaluation.

Diabetic foot ulcers (DFUs) are one of the most serious and devastating complication of diabetes, manifesting as foot ulcers and impaired wound healing in patients with diabetes mellitus. To solve this problem, sulfated hyaluronic acid (SHA)/collagen-based nanofibrous biomimetic skins was developed and used to promote the diabetic wound healing and skin remodeling. First, SHA was successfully synthetized using chemical sulfation and incorporated into collagen (COL) matrix for preparing the SHA/COL hybrid nanofiber skins. The polyurethane (PU) was added into those hybrid scaffolds to make up the insufficient mechanical properties of SHA/COL nanofibers, the morphology, surface properties and degradation rate of hybrid nanofibers, as well as cell responses upon the nanofibrous scaffolds were studied to evaluate their potential for skin reconstruction. The results demonstrated that the SHA/COL, SHA/HA/COL hybrid nanofiber skins were stimulatory of cell behaviors, including a high proliferation rate and maintaining normal phenotypes of specific cells. Notably, SHA/COL and SHA/HA/COL hybrid nanofibers exhibited a significantly accelerated wound healing and a high skin remodeling effect in diabetic mice compared with the control group. Overall, SHA/COL-based hybrid scaffolds are promising candidates as biomimetic hybrid nanofiber skin for accelerating diabetic wound healing.

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.

Biochemical Characterization and Synthetic Application of WciN and Its Mutants From Streptococcus pneumoniae Serotype 6B.

The biochemical properties of α-1,3-galactosyltransferase WciN from Streptococcus pneumoniae serotype 6B were systemically characterized with the chemically synthesized Glcα-PP-(CH2)11-OPh as an acceptor substrate. The in vitro site-directed mutation of D38 and A150 residues of WciN was further investigated, and the enzymatic activities of those WciN mutants revealed that A150 residue was the pivotal residue responsible for nucleotide donor recognition and the single-site mutation could completely cause pneumococcus serotype switch. Using WciNA150P and WciNA150D mutants as useful tool enzymes, the disaccharides Galα1,3Glcα-PP-(CH2)11-OPh and Glcα1,3Glcα-PP-(CH2)11-OPh were successfully prepared in multi-milligram scale in high yields.

New injectable chitosan-hyaluronic acid based hydrogels for hemostasis and wound healing.

It is a challenge to develop hemostatic and wound dressings that are used for irregular shape and deep wound. Herein, a series of novel N-succinyl chitosan-oxidized hyaluronic acid based (NSC-OHA-based) hydrogels were fabricated, while calcium ions (Ca2+) and/or four-armed amine-terminated poly(ethylene glycol) (4-arm-PEG-NH2, labeled as PEG1) were introduced to regulate the mechanical behavior and bioactivities. We found all NSC-OHA-based hydrogels displayed self-healing and injectable performances. Besides, the addition of Ca2+ or PEG1 exhibited a positive effect on the adjustable mechanical behavior of hydrogels, providing the possibility to meet different mechanical requirements. Furthermore, Ca2+ or PEG1 significantly improved the biocompatibility, hemostasis and wound healing abilities of NSC-OHA hydrogel. Notably, compared with the commercial hemostatic agent (Arista™), hydrogels containing Ca2+ showed comparable hemostatic effects and significantly accelerated wound healing. Overall, the calcium-containing NSC-OHA hydrogels are promising for hemostasis and accelerating wound healing.

Hyaluronic acid oligosaccharide-collagen mineralized product and aligned nanofibers with enhanced vascularization properties in bone tissue engineering.

Considering the structural complexity of natural bone and the limitations of current treatment options, designing a biomimetic and functional tissue-engineered bone graft has been an urgent need for the replacement and regeneration of defected bone tissue. In light of the cell recruitment to the defect region, scaffold-guided bone tissue engineering has proven to be a viable strategy that is poised to deliver effective osseointegration and vascularization during bone remodeling. Herein, we provide an engineered bone scaffold based on aligned poly(lactic-co-glycolide) (PLGA) nanofibers incorporated with hyaluronic acid oligosaccharide-collagen mineralized microparticles (labeled oHA-Col/HAP) to guide the cell-specific orientation and osseointegration in bone healing. The aligned nanofibers were successfully prepared by a custom-made rotating mandrel with separating railings and HAs-Col/HAP mineralized microparticles were uniformly distributed in the composite scaffolds that acted as temporary templates for bone remodeling. The morphology, physicochemical properties and tensile strength of the scaffolds were characterized, the cell responses and in vivo biocompatibility and biodegradability of the scaffolds were also studied to evaluate the potential for bone tissue engineering. The experimental results illustrated that such anisotropic scaffolds loaded with oHA-Col/HAP microparticles mediated cell orderly arrangement conducive to the migration and recruitment of osseointegration-related cells and were stimulatory of cell proliferation. Those oHA-Col/HAP@PLGA scaffolds exhibited ideal biocompatibility and tissue regenerative capacity in vivo through a higher expression of vascularization-related genes. Overall, the novel engineered bone scaffold promises to serve as alternative candidates for bone tissue engineering applications.

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.

Fabrication and assessment of chondroitin sulfate-modified collagen nanofibers for small-diameter vascular tissue engineering applications.

Chondroitin sulfate (ChS) has shown promising results in promoting cell proliferation and antithrombogenic activity. To engineered develop a dual-function vascular scaffold with antithrombosis and endothelialization, ChS was tethered to collagen to accelerate the growth of endothelial cells and prevent platelet activation. First, ChS was used to conjugate with collagen to generate glycosylated products (ChS-COL) via reductive amination. Then, the fabricated ChS-COL conjugates were electrospun into nanofibers and their morphologies and physicochemical characteristics, cell-scaffold responses and platelet behaviors upon ChS-COL nanofibers were comprehensively characterized to evaluate their potential use for small-diameter vascular tissue-engineered scaffolds. The experimental results demonstrated that the ChS modified collagen electrospun nanofibers were stimulatory of endothelial cell behavior, alleviated thrombocyte activation and maintained an antithrombotic effect in vivo in 10-day post-transplantation. The ChS-COL scaffolds encouraged rapid endothelialization, thus probably ensuring the antithrombotic function in long-term implantation, suggesting their promise for small-diameter vascular tissue engineering applications.

Design and comprehensive assessment of a biomimetic tri-layer tubular scaffold via biodegradable polymers for vascular tissue engineering applications.

Considering the structural complexity of the native artery wall and the limitations of current treatment strategies, developing a biomimetic tri-layer tissue-engineered vascular graft is a major developmental direction of vascular tissue regeneration. Biodegradable polymers exhibit adequate mechanical characteristics and feasible operability, showing potential prospects in the construction of tissue engineering scaffold. Herein, we present a bio-inspired tri-layer tubular graft using biodegradable polymers to simulate natural vascular architecture. The inner layer made of polycaprolactone (PCL) nanofiber possesses high tensile strength and contributed to endothelial cell adhesion and proliferation. The middle layer consisted of poly(lactic-co-glycolide) (PLGA) with a three-dimensional porous structure is appropriate for vascular smooth muscle cells (SMCs) penetration. The polyurethane (PU) was selected to be the outer layer, aiming to hold the entire tubular structure, suggesting superior mechanical properties and ideal biocompatibility. Adhesion between independent layers is achieved by thermal crosslinking. The compliance, burst pressure and suture retention force of the tubular scaffold were 2.50 ± 1.60%, 2737.73 ± 583.41 mmHg and 13.06 ± 1.89 N, respectively. The in vivo study of subcutaneous implantation for 8 weeks demonstrated the biomimetic tri-layer vascular graft could maintain intimal integrity, cell infiltration, collagen deposition and scaffold biodegradation. Overall, the biomimetic tri-layer vascular graft promises to be a potential candidate for vascular replacement and regeneration.

Exploring the broad nucleotide triphosphate and sugar-1-phosphate specificity of thymidylyltransferase Cps23FL from Streptococcus pneumonia serotype 23F.

Glucose-1-phosphate thymidylyltransferase (Cps23FL) from Streptococcus pneumonia serotype 23F is the initial enzyme that catalyses the thymidylyl transfer reaction in prokaryotic deoxythymidine diphosphate-l-rhamnose (dTDP-Rha) biosynthetic pathway. In this study, the broad substrate specificity of Cps23FL towards six glucose-1-phosphates and nine nucleoside triphosphates as substrates was systematically explored, eventually providing access to nineteen sugar nucleotide analogs.

Improving in vitro biocompatibility on biomimetic mineralized collagen bone materials modified with hyaluronic acid oligosaccharide.

Hyaluronic acid (HA) has great potential in bone tissue engineering due to its favorable bioactivity and biocompatibility, especially hyaluronic acid oligosaccharides (oHAs) shows a promising result in endothelialization of blood vessel. To improve endothelialized effect and osteogenic performance of bone scaffold, we have created a biomimetic nanofiber network based on collagen modified with hyaluronic acid oligosaccharides (Col/oHAs) and its mineralized product. Biomimetically mineralized Col/oHAs based composite (Col/oHAs/HAP) was prepared via self-assembly at room temperature. The resultant composites were characterized by fourier transform infrared spectroscopy (FT-IR), X-Ray diffractometry (XRD), thermogravimetric analysis (TGA), scanning electron microscope (SEM) and transmission electron microscopy (TEM). They show some characteristics of natural bone both in composition and microstructure. The nanofiber was fabricated as a hybrid network which bionics extracellular matrix (ECM) and was prepared to culture artery endothelial cell (PIEC) and the mouse parietal bone cell (MC3T3-E1). Cells attached tightly to the nanofibers and infiltrated into the materials, forming an interconnected cell community. Moreover, the as-prepared nanofiber was found to noticeably enhance cells adhesion and proliferation and upregulate alkaline phosphatase activity (ALP) and osteocalcin (OCN) expression suggesting positive cellular responses. These results indicated that the Col/oHAs/HAP composite has a promising capacity to direct the osteogenic differentiation by providing an adaptable environment and can be expected as an excellent candidate for bone tissue engineering approaches with improved performance of promoting PIEC proliferation.

Hyaluronic acid oligosaccharide-modified collagen nanofibers as vascular tissue-engineered scaffold for promoting endothelial cell proliferation.

Hyaluronic acid oligosaccharides (oHAs) have shown promising results in promoting vascular endothelial cell (EC) proliferation and endothelialization. To engineered develop tissue scaffold for promoting EC proliferation and vessel endothelialization, different sizes of oHAs were prepared and grafted onto collagen to improve the biological properties of the synthesized materials, especially in angiogenesis. Firstly, oHAs were successfully prepared and conjugated with collagen to construct glycosylated collagens by reductive amination. The glycosylated collagens were then electrospun into various nanofibrous structures and their morphology and hemocompatibility, as well as EC responses on these nanofibrous scaffolds, were studied to evaluate the potential for vascular tissue engineering. The results showed that the nanofibrous scaffolds grafted by oHAs promoted EC proliferation whereas high molecular weight HA inhibited proliferation. The scaffolds had no detectable degree of hemolysis and coagulation, suggesting their promise as engineered vascular tissue scaffolds.

Biochemical studies of a ß-1,4-rhamnoslytransferase from Streptococcus pneumonia serotype 23F.

A new ß-rhamnoslytransferase Cps23FT from Streptococcus pneumonia serotype 23F was expressed and characterized. Its enzymatic activity and function were confirmed for the first time by utilizing enzymatically prepared dTDP-Rha and chemically synthesized Glcα-PP-(CH2)11-OPh as substrates. This reaction gave the desired disaccharide Rhaß-1,4-Glcα-PP-(CH2)11-OPh in a good isolated yield (67%), suggesting the potential of Cps23FT as a tool enzyme for the synthesis of complex oligosaccharides containing difficult ß-rhamnosyl linkages. Furthermore, site-directed mutagenesis of Cps23FT disclosed that its 271DKD273 motif was critical for the enzymatic activity and most likely the binding site for the required divalent metal cation.

Mechanical enhancement and in vitro biocompatibility of nanofibrous collagen-chitosan scaffolds for tissue engineering.

The collagen-chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen-chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen-chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen-chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young's modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen-chitosan nanofiber reinforced by TPU, both the break strength and the Young's modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen-chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.

One-pot four-enzyme synthesis of thymidinediphosphate-l-rhamnose.

A new, robust one-pot four-enzyme synthetic method was developed for thymidinediphosphate-l-rhamnose starting from d-glucose-1-phosphate. The enzymes, Glc-1-P thymidylyltransferase, dTDP-Glc-4,6-dehydratase, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase and dTDP-4-keto-Rha reductase were derived from Streptococcus pneumonia serotype 23F, expressed in Escherichia coli, and studied in detail to provide the first direct evidence for their functions.

Degradability of injectable calcium sulfate/mineralized collagen-based bone repair material and its effect on bone tissue regeneration.

The nHAC/CSH composite is an injectable bone repair material with controllable injectability and self-setting properties prepared by introducing calcium sulfate hemihydrate (CSH) into mineralized collagen (nHAC). When mixed with water, the nHAC/CSH composites can be transformed into mineralized collagen/calcium sulfate dihydrate (nHAC/CSD) composites. The nHAC/CSD composites have good biocompatibility and osteogenic capability. Considering that the degradation behavior of bone repair material is another important factor for its clinical applications, the degradability of nHAC/CSD composites was studied. The results showed that the degradation ratio of the nHAC/CSD composites with lower nHAC content increased with the L/S ratio increase of injectable materials, but the variety of L/S ratio had no significant effect on the degradation ratio of the nHAC/CSD composites with higher nHAC content. Increasing nHAC content in the composites could slow down the degradation of nHAC/CSD composite. Setting accelerator had no significant effect on the degradability of nHAC/CSD composites. In vivo histological analysis suggests that the degradation rate of materials can match the growth rate of new mandibular bone tissues in the implanted site of rabbit. The regulable degradability of materials resulting from the special prescriptions of injectable nHAC/CSH composites will further improve the workability of nHAC/CSD composites.

Improved workability of injectable calcium sulfate bone cement by regulation of self-setting properties.

Calcium sulfate hemihydrate (CSH) powder as an injectable bone cement was prepared by hydrothermal synthesis of calcium sulfate dihydrate (CSD). The prepared materials showed X-ray diffraction peaks corresponding to the CSH structure without any secondary phases, implying complete conversion from CSD phase to CSH phase. Thermogravimetric (TG) analyses showed the crystal water content of CSH was about 6.0% (wt.), which is near to the theoretic crystal water value of CSH. From scanning electron microscopy (SEM) micrographs, sheet crystal structure of CSD was observed to transform into rod-like crystal structure of CSH. Most interesting and important of all, CSD as setting accelerator was also introduced into CSH powder to regulate self-setting properties of injectable CSH paste, and thus the self-setting time of CSH paste can be regulated from near 30 min to less than 5 min by adding various amounts of setting accelerator. Because CSD is not only the reactant of preparing CSH but also the final solidified product of CSH, the setting accelerator has no significant effect on the other properties of materials, such as mechanical properties. In vitro biocompatibility and in vivo histology studies have demonstrated that the materials have good biocompatibility and good efficacy in bone regeneration. All these will further improve the workability of CSH in clinic applications.

Mechanical properties and in vitro bioactivity of injectable and self-setting calcium sulfate/nano-HA/collagen bone graft substitute.

Calcium sulfate hemihydrate (CSH) was introduced into the mineralized collagen (nHAC) to prepare an injectable and self-setting in situ bone graft substitute. The mechanical properties of materials, which are dependant on the L/S ratio, the content of nHAC and setting accelerator, were discussed based on the satisfying injectability and setting properties. It was found that the compressive strength and modulus of materials increased with the decrease of nHAC content and L/S ratio. CSD as setting accelerator hardly had an effect on the compressive properties of materials because it is not only the reactant of preparing CSH but also the final solidified product of CSH instead of a foreign body. Though the compressive properties of nHAC/CSD composites changed with the variety of nHAC content and L/S ratio, the compressive strength and modulus of the materials ranged from 2.0 to nearly 20.0 MPa and 100.0 to 800.0 MPa, respectively, which are similar to that of cancellous bone. In vitro cell behavior demonstrated that the composites could provide adequate environment for cell adhesion and proliferation. All these indicated that the nHAC/CSH composites were a potential scaffold for bone tissue engineering.

Injectable calcium sulfate/mineralized collagen-based bone repair materials with regulable self-setting properties.

An injectable and self-setting bone repair materials (nano-hydroxyapatite/collagen/calcium sulfate hemihydrate, nHAC/CSH) was developed in this study. The nano-hydroxyapatite/collagen (nHAC) composite, which is the mineralized fibril by self-assembly of nano-hydrocyapatite and collagen, has the same features as natural bone in both main hierarchical microstructure and composition. It is a bioactive osteoconductor due to its high level of biocompatibility and appropriate degradation rate. However, this material lacks handling characteristics because of its particle or solid-preformed block shape. Herein, calcium sulfate hemihydrate (CSH) was introduced into nHAC to prepare an injectable and self-setting in situ bone repair materials. The morphology of materials was observed using SEM. Most important and interesting of all, calcium sulfate dihydrate (CSD), which is not only the reactant of preparing CSH but also the final solidified product of CSH, was introduced into nHAC as setting accelerator to regulate self-setting properties of injectable nHAC/CSH composite, and thus the self-setting time of nHAC/CSH composite can be regulated from more than 100 min to about 30 min and even less than 20 min by adding various amount of setting accelerator. The compressive properties of bone graft substitute after final setting are similar to those of cancellous bone. CSD as an excellent setting accelerator has no significant effect on the mechanical property and degradability of bone repair materials. In vitro biocompatibility and in vivo histology studies demonstrated that the nHAC/CSH composite could provide more adequate stimulus for cell adhesion and proliferation, embodying favorable cell biocompatibility and a strong ability to accelerate bone formation. It can offer a satisfactory biological environment for growing new bone in the implants and for stimulating bone formation.