Modeling interactions between Heparan sulfate and proteins based on the Heparan sulfate microarray analysis.

Heparan sulfate (HS), a sulfated polysaccharide abundant in the extracellular matrix, plays pivotal roles in various physiological and pathological processes by interacting with proteins. Investigating the binding selectivity of HS oligosaccharides to target proteins is essential, but the exhaustive inclusion of all possible oligosaccharides in microarray experiments is impractical. To address this challenge, we present a hybrid pipeline that integrates microarray and in silico techniques to design oligosaccharides with desired protein affinity. Using fibroblast growth factor 2 (FGF2) as a model protein, we assembled an in-house dataset of HS oligosaccharides on microarrays and developed two structural representations: a standard representation with all atoms explicit and a simplified representation with disaccharide units as "quasi-atoms." Predictive Quantitative Structure-Activity Relationship (QSAR) models for FGF2 affinity were developed using the Random Forest (RF) algorithm. The resulting models, considering the applicability domain, demonstrated high predictivity, with a correct classification rate of 0.81-0.80 and improved positive predictive values (PPV) up to 0.95. Virtual screening of 40 new oligosaccharides using the simplified model identified 15 computational hits, 11 of which were experimentally validated for high FGF2 affinity. This hybrid approach marks a significant step toward the targeted design of oligosaccharides with desired protein interactions, providing a foundation for broader applications in glycobiology.

Electrospray Nano-Micro Composite Sodium Alginate Microspheres with Shape-Adaptive, Antibacterial, and Angiogenic Abilities for Infected Wound Healing.

Nonhealing infectious wounds, characterized by bacterial colonization, wound microenvironment destruction, and shape complexity, present an intractable problem in clinical practice. Inspired by LEGOs, building-block toys that can be assembled into desired shapes, we proposed the use of electrospray nano-micro composite sodium alginate (SA) microspheres with antibacterial and angiogenic properties to fill irregularly shaped wounds instantly. Specifically, porous poly(lactic-co-glycolic acid) (PLGA) microspheres (MSs) encapsulating basic fibroblast growth factor (bFGF) were produced by a water-in-oil-in-water double-emulsion method. Then, bFGF@MSs were blended with the SA solution containing ZIF-8 nanoparticles. The resultant solution was electrosprayed to obtain nano-micro composite microspheres (bFGF@MS/ZIF-8@SAMSs). The composite MSs' size could be regulated by PLGA MS mass proportion and electrospray voltage. Moreover, bFGF, a potent angiogenic agent, and ZIF-8, bactericidal nanoparticles, were found to release from bFGF@MS/ZIF-8@SAMSs in a controlled and sustainable manner, which promoted cell proliferation, migration, and tube formation and killed bacteria. Through experimentation on rat models, bFGF@MS/ZIF-8@SAMSs were revealed to adapt to wound shapes and accelerate infected wound healing because of the synergistic effects of antibacterial and angiogenic abilities. In summation, this study developed a feasible approach to prepare bioactive nano-micro MSs as building blocks that can fill irregularly shaped infected wounds and improve healing.

All-in-one hydrogel patches with sprayed bFGF-loaded GelMA microspheres for infected wound healing studies.

The current wound healing process faces numerous challenges such as bacterial infection, inflammation and oxidative stress. However, wound dressings used to promote wound healing, are not well suited to meet the clinical needs. Hyaluronic acid (HA) not only has excellent water absorption and good biocompatibility but facilitates cell function and tissue regeneration. Dopamine, on the other hand, increases the overall viscosity of the hydrogel and possesses antioxidant property. Furthermore, chitosan exhibits outstanding performance in antimicrobial, anti-inflammatory and antioxidant activities. Basic fibroblast growth factor (bFGF) is conducive to cell proliferation and migration, vascular regeneration and wound healing. Hence, we designed an all-in-one hydrogel patch containing dopamine and chitosan framed by hyaluronic acid (HDC) with sprayed gelatin methacryloyl (GelMA) microspheres loaded with bFGF (HDC-bFGF). The hydrogel patch exhibits excellent adhesive, anti-inflammatory, antioxidant and antibacterial properties. In vitro experiments, the HDC-bFGF hydrogel patch not only showed significant inhibitory effect on RAW cell inflammation and Staphylococcus aureus (S. aureus) growth but also effectively scavenged free radicals, in addition to promoting the migration of 3 T3 cells. In the mice acute infected wound model, the HDC-bFGF hydrogel patch adhered to the wound surface greatly accelerated the healing process via its anti-inflammatory and antioxidant activities, bacterial inhibition and pro-vascularization effects. Therefore, the multifunctional HDC-bFGF hydrogel patch holds great promise for clinical application.

Integration of Zn2+, ATP, and bFGF to Nanodressing with Core-Shell Structure Fabricated by Emulsion Electrospinning for Wound Healing.

Basic fibroblast growth factor (bFGF) plays an important role in active wound repair. However, the existing dosage forms in clinical applications are mainly sprays and freeze-dried powders, which are prone to inactivation and cannot achieve a controlled release. In this study, a bioactive wound dressing named bFGF-ATP-Zn/polycaprolactone (PCL) nanodressing with a "core-shell" structure was fabricated by emulsion electrospinning, enabling the sustained release of bFGF. Based on the coordination and electrostatic interactions among bFGF, ATP, and Zn2+, as well as their synergistic effect on promoting wound healing, a bFGF-ATP-Zn ternary combination system was prepared with higher cell proliferation activity and used as the water phase for emulsion electrospinning. The bFGF-ATP-Zn/PCL nanodressing demonstrated improved mechanical properties, sustained release of bFGF, cytocompatibility, and hemocompatibility. It increased the proliferation activity of human dermal fibroblasts (HDFs) and enhanced collagen secretion by 1.39 and 3.45 times, respectively, while reducing the hemolysis rate to 3.13%. The application of the bFGF-ATP-Zn/PCL nanodressing in mouse full-thickness skin defect repair showed its ability to accelerate wound healing and reduce wound scarring within 14 days. These results provide a research basis for the development and application of this bioactive wound dressing product.

Expression, characterization and antivascular activity of amino acid sequence repeating collagen hexadecapeptide.

Type V collagen is an essential component of the extracellular matrix (ECM), and its remodeling releases specific protein fragments that can specifically inhibit endothelial cell responses such as proliferation, migration, and invasion. In this study, we have successfully constructed two engineered strains of Pichia pastoris capable of producing recombinant collagen through a new genetic engineering approach. Through high-density fermentation, the expression of 1605 protein and 1610 protein could reach 2.72 g/L and 4.36 g/L. With the increase of repetition times, the yield also increased. Bioactivity analysis showed that recombinant collagen could block the angiogenic effect of FGF-2 on endothelial cells by eliminating FGF-2-induced endothelial cell migration and invasion. Collectively, the recombinant proteins we successfully expressed have a wide range of potential for inhibiting angiogenesis in the biomaterials and biomedical fields.

Poly (Betulinic Acid) Nanoparticles Loaded with bFGF Improve Functional Recovery After Spinal Cord Injury.

Oxidative stress (OS) is one of the crucial molecular events of secondary spinal cord injury (SCI). Basic fibroblast growth factor (bFGF) is a multipotent cell growth factor with an anti-oxidant effect. However, bFGF has a short half-life in vivo, which limits its therapeutic application. Biodegradable polymers with excellent biocompatibility have been recently applied in SCI. The negative aspect is that polymers cannot provide a significant therapeutic effect. Betulinic acid (BA), a natural anti-inflammatory compound, has been polymerized into poly (betulinic acid) (PBA) to serve as a drug carrier for bFGF. This study explores the therapeutic effects and underlying molecular mechanisms of PBA nanoparticles (NPs) loaded with bFGF (PBA-bFGF NPs) in SCI. Results show that PBA-bFGF NPs produce remarkable biocompatibility in vivo and in vitro. The results also demonstrate that local delivery of PBA-bFGF NPs enhances motor function recovery, inhibits OS, mitigates neuroinflammation, and alleviates neuronal apoptosis following SCI. Furthermore, the results indicate that local delivery of PBA-bFGF NPs activates the nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling pathway following SCI. In summary, results suggest that local delivery of PBA-bFGF NPs delivers potential therapeutic advantages in the treatment and management of SCI.

Assessment of serum levels and placental bed tissue expression of IGF-1, bFGF, and PLGF in patients with placenta previa complicated with placenta accreta spectrum disorders.

OBJECTIVE: This study aims to detect the serum levels of IGF-1, bFGF, and PLGF and their expressions in placental bed tissues of patients with placenta previa complicated with PAS disorders. METHODS: This case and control study included 40 multiparous pregnant women with complete placenta previa between 34 weeks and 38 weeks of gestation and they were divided into two groups: 25 patients with PAS (case group) and 15 patients without PAS (control group). The venous blood samples were collected 2 h before the cesarean section, and the placental bed tissues were taken intraoperatively at the placental implantation site and then were histologically examined to evaluate the gravity of the myometrial invasion of the placenta. According to FIGO PAS increasing grading, the 25 patients were also divided into three groups: PAS grade I group, PAS grade II group, and PAS grade III group. The concentrations of IGF-1, bFGF, and PLGF in serum were measured using ELISA, and the mean ratio of the relative mRNA expression of each biomarker in placental bed tissues was calculated using qRT-PCR. The staining intensity and the positive cells were quantitatively measured and expressed as means by using Image J software for IHC analysis. RESULTS: IGF-1 had low serum levels and high placental bed expression in placenta previa patients with PAS disorders compared to those without PAS (all p < 0.0001). PLGF had high serum levels (p = 0.0200) and high placental bed expression (p < 0.0001) in placenta previa patients with PAS disorders compared to those without PAS. IGF-1 serum levels decreased up to PAS grade II (means were 24.3 ± 4.03, 21.98 ± 3.29, and 22.03 ± 7.31, respectively for PAS grade I, PAS grade II, PAS grade III groups, p = 0.0006). PLGF serum levels increased up to PAS grade II (means were 12.96 ± 2.74, 14.97 ± 2.56, and 14.89 ± 2.14, respectively for the three groups, p = 0.0392). However, IGF-1 and PLGF mRNA placental bed expression increased up to PAS grade III. The relative expression of mRNA means for the three groups was 3.194 ± 1.40, 3.509 ± 0.63, and 3.872 ± 0.70, respectively for IGF-1; and 2.784 ± 1.14, 2.810 ± 0.71, and 2.869 ± 0.48, respectively for PLGF (all p < 0.0001). Their IHC (immunohistochemical) staining also had increasing trends, but p > 0.05. bFGF was not significantly expressed in placenta previa with PAS disorders in most of the analysis sections (p > 0.05). CONCLUSIONS: Low serum levels and high expression in placental bed tissues of IGF-1, or high serum levels and high expression in placental bed tissues of PLGF, may differentiate placenta previa patients with FIGO PAS grade I and PAS grade II from those without PAS disorders. However, they could not significantly predict the degree of placental invasiveness in FIGO PAS grades II and III.

Improved myocardial performance in infarcted rat heart by injection of disulfide-cross-linked chitosan hydrogels loaded with basic fibroblast growth factor.

Myocardial infarction (MI) has been considered as the leading cause of cardiovascular-related deaths worldwide. Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor family that promotes angiogenesis after MI; however, it has poor clinical efficacy due to proteolytic degradation, low drug accumulation, and severe drug-induced side effects. In this study, an injectable disulfide-cross-linked chitosan hydrogel loaded with bFGF was prepared via a thiol-disulfide exchange reaction for MI treatment. The thiol-disulfide exchange reaction between pyridyl disulfide-modified carboxymethyl chitosan (CMCS-S-S-Py) and reduced BSA (rBSA) was carried out under physiological conditions (37 °C and pH 7.4). The mechanical properties of the disulfide-cross-linked chitosan hydrogel were evaluated based on the molar ratio of the pyridyl disulfide groups of CMCS-S-S-Py and the thiol groups of rBSA. The disulfide-cross-linked chitosan hydrogel showed good swelling performance, rapid glutathione-triggered degradation behavior and well-defined cell proliferation towards NIH 3T3 fibroblast cells. In the process of establishing a rat MI model, the squeezing heart method was used to make the operation more accurate and the mortality of rats was decreased by using a ventilator. The disulfide-cross-linked chitosan hydrogel loaded with bFGF (bFGF-hydrogel) was injected into a peri-infarcted area of cardiac tissue immediately following MI. Echocardiography demonstrated that the left ventricular functions were improved by the bFGF-hydrogel after 28 days of treatment. Histological results revealed that the hydrogel significantly reduced the fibrotic area of MI, and this was further improved by the bFGF-hydrogel treatment. TUNEL and immunohistochemical staining results showed that the bFGF-hydrogel had a more synergistic effect on antiapoptosis and proangiogenesis than using either bFGF or the hydrogel alone.

Spatiotemporal regulation of angiogenesis/osteogenesis emulating natural bone healing cascade for vascularized bone formation.

Engineering approaches for growth factor delivery have been considerably advanced for tissue regeneration, yet most of them fail to provide a complex combination of signals emulating a natural healing cascade, which substantially limits their clinical successes. Herein, we aimed to emulate the natural bone healing cascades by coupling the processes of angiogenesis and osteogenesis with a hybrid dual growth factor delivery system to achieve vascularized bone formation. Basic fibroblast growth factor (bFGF) was loaded into methacrylate gelatin (GelMA) to mimic angiogenic signalling during the inflammation and soft callus phases of the bone healing process, while bone morphogenetic protein-2 (BMP-2) was bound onto mineral coated microparticles (MCM) to mimics osteogenic signalling in the hard callus and bone remodelling phases. An Initial high concentration of bFGF accompanied by a sustainable release of BMP-2 and inorganic ions was realized to orchestrate well-coupled osteogenic and angiogenic effects for bone regeneration. In vitro experiments indicated that the hybrid hydrogel markedly enhanced the formation of vasculature in human umbilical vein endothelial cells (HUVECs), as well as the osteogenic differentiation of mesenchymal stem cells (BMSCs). In vivo results confirmed the optimal osteogenic performance of our F/G-B/M hydrogel, which was primarily attributed to the FGF-induced vascularization. This research presents a facile and potent alternative for treating bone defects by emulating natural cascades of bone healing.

Complexation of CXCL12, FGF-2 and VEGF with Heparin Modulates the Protein Release from Alginate Microbeads.

Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.

Technology transfer of a monitoring system to predict product concentration and purity of biopharmaceuticals in real-time during chromatographic separation.

Technological developments require the transfer to their location of application to make use of them. We describe the transfer of a real-time monitoring system for lab-scale preparative chromatography to two new sites where it will be used and developed further. Equivalent equipment was used. The capture of a biopharmaceutical model protein, human fibroblast growth factor 2 (FGF-2) was used to evaluate the system transfer. Predictive models for five quality attributes based on partial least squares regression were transferred. Six out of seven online sensors (UV/VIS, pH, conductivity, IR, RI, and MALS) showed comparable signals between the sites while one sensor (fluorescence) showed different signal profiles. A direct transfer of the models for real-time monitoring was not possible, mainly due to differences in sensor signals. Adaptation of the models was necessary. Then, among five prediction models, the prediction errors of the test run at the new sites were on average twice as high as at the training site (model-wise 0.9-5.7 times). Additionally, new prediction models for different products were trained at each new site. These allowed monitoring the critical quality attributes of two new biopharmaceutical products during their purification processes with mean relative deviations between 1% and 33%.

Selenium nanoparticles with various morphology for antiangiogenesis through bFGF-mediated P13K/AKT signaling pathways.

Selenium nanoparticles (Se NPs) have potential antitumor activity and immune properties. However, the mechanism between its antitumor activity and nanoparticle morphology has not been evaluated. Therefore, a simple method was used to synthesize three special shapes of Se NPs, which are fusiform, flower and spherical. Compared with fusiform selenium nanoparticles (Se NPs (S)) and flower-shaped selenium nanoparticles (Se NPs (F)), spherical selenium nanoparticles (Se NPs (B)) have better cell absorption effect and stronger antitumor activity. HRTEM showed that Se NPs (B) entered the nucleus through endocytosis and inhibited tumor angiogenesis by targeting basic fibroblast growth factor (bFGF). Se NPs (B) can competitively inhibit the binding of bFGF to fibroblast growth factor receptor through direct binding to bFGF, down-regulate the expression of bFGF in human umbilical vein endothelial cells (HUVEC), and significantly reduce the MAPK/Erk and P13K/AKT pathways activation of signaling molecules to regulate HUVEC cell migration and angiogenesis. These findings indicate that Se NPs have a special role in antitumor angiogenesis. This research provides useful information for the development of new strategies for effective drug delivery nanocarriers and therapeutic systems.

Half-life modeling of basic fibroblast growth factor released from growth factor-eluting polyelectrolyte multilayers.

Growth factor-eluting polymer systems have been widely reported to improve cell and tissue outcomes; however, measurements of actual growth factor concentration in cell culture conditions are limited. The problem is compounded by a lack of knowledge of growth factor half-lives, which impedes efforts to determine real-time growth factor concentrations. In this work, the half-life of basic fibroblast growth factor (FGF2) was determined using enzyme linked immunosorbent assay (ELISA). FGF2 release from polyelectrolyte multilayers (PEMs) was measured and the data was fit to a simple degradation model, allowing for the determination of FGF2 concentrations between 2 and 4 days of culture time. After the first hour, the FGF2 concentration for PEMs assembled at pH = 4 ranged from 2.67 ng/mL to 5.76 ng/mL, while for PEMs assembled at pH = 5, the concentration ranged from 0.62 ng/mL to 2.12 ng/mL. CRL-2352 fibroblasts were cultured on PEMs assembled at pH = 4 and pH = 5. After 2 days, the FGF2-eluting PEM conditions showed improved cell count and spreading. After 4 days, only the pH = 4 assembly condition had higher cells counts, while the PEM assembled at pH = 5 and PEM with no FGF2 showed increased spreading. Overall, the half-life model and cell culture study provide optimal concentration ranges for fibroblast proliferation and a framework for understanding how temporal FGF2 concentration may affect other cell types.

Discrete binding patterns of two heparin-reactive proteins, basic fibroblast growth factor and peptide p5R, in amyloid-laden and healthy mice.

Peptide p5R is a synthetic, polybasic, heparin-binding peptide that preferentially reacts with amyloid deposits in vivo and in tissue sections. Basic fibroblast growth factor (bFGF1) similarly interacts with heparin-like molecules, notably heparan sulfate proteoglycans (HSPG), in the extracellular matrix and on cell surfaces. The aim of this study was to compare the biodistribution of p5R and bFGF in healthy mice as well as those with systemic inflammation-associated amyloidosis (AA), which contains HSPG, by using SPECT/CT imaging, tissue biodistribution measurements and micro-autoradiography. Although both proteins are known to bind heparan sulfate, their biodistribution was remarkably different in the healthy and diseased animals. Imaging revealed uptake of both radiolabeled proteins in the liver, spleen, and kidneys of mice with amyloidosis; however, 125I-bFGF, but not 125I-p5R, was observed in normal tissue at sites of HSPG expression, including the hepatic and splenic sinusoids and renal glomerulae. Microautoradiography demonstrated that while p5R bound exclusively to amyloid deposits in the spleen and liver of AA mice, bFGF had a broader binding pattern. Consequently, even though bFGF and p5R both interact with heparan sulfate moieties, p5R binding was restricted to HSPG in amyloid deposits and did not bind HSPG in healthy tissues, whereas bFGF preferentially reacted with HSPG in normal tissue. The data suggest that peptide p5R selectively binds HSPG in amyloid and that the HSPG in healthy tissue, recognized by bFGF, is not targeted by the peptide.

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation.

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.

Heparin-mediated electrostatic immobilization of bFGF via functional polymer films for enhanced self-renewal of human neural stem cells.

Preserving the self-renewal capability of undifferentiated human neural stem cells (hNSCs) is one of the crucial prerequisites for efficient hNSC-based regenerative medicine. Considering that basic fibroblast growth factor (bFGF) is one of the key contributing factors in maintaining the self-renewal property of hNSCs, the bioactivity and stability of bFGF in the hNSC culture should be regulated carefully. In this study, we developed a functional polymer film of poly(glycidyl methacrylate (GMA)-co-N,N-dimethylaminoethyl methacrylate (DMAEMA)) (coGD, or p(GMA-co-DMAEMA)) via initiated chemical vapor deposition (iCVD), which facilitated a stable, electrostatic adsorption of heparin and subsequent immobilization of bFGF. The bFGF-immobilized coGD surface substantially enhanced the proliferation rate and neurosphere forming ability of hNSCs compared to tissue culture plate (TCP). The expression of the stemness markers of hNSCs such as NESTIN and SOX-2 was also upregulated prominently on the coGD surface. Also, the hNSCs cultured on the coGD surface showed enhanced neurogenesis upon spontaneous differentiation. The immobilized bFGF on the coGD surface stimulated the expression of bFGF receptors and subsequently activated the mitogen-activated protein kinase (MAPK) pathway, attributed to the increase in self-renewal property of hNSCs. Our results indicate that the coGD surface allowed in situ heparin-mediated bFGF immobilization, which served as a robust platform to generate hNSC neurospheres with enhanced self-renewal and differentiation capabilities and thereby will prompt an advance in the field of therapeutics of neurodegenerative diseases.

bFGF and collagen matrix hydrogel attenuates burn wound inflammation through activation of ERK and TRK pathway.

Burn injuries are most challenging to manage since it causes loss of the integrity of large portions of the skin leading to major disability or even death. Over the years, hydrogels are considered as a significant delivery system for wound treatment because of several advantages over other conventional formulations. We hypothesized that the bFGF-collagen-AgSD incorporated hydrogel formulation can accelerate the rate of burn healing in animal model and would promote fibroblast cell proliferation. Neovascularization and re-epithelialization is a hall mark of burn wound healing. In the present study, histopathological investigation and scanning electron microscopy of skin tissue of Wistar rats showed almost complete epithelialisation after 16 days in the treatment group. The developed hydrogel showed significantly accelerated wound closure compared with a standard and control group. The faster wound closure resulted from increased re-epithelialization and granulation tissue formation because of the presence of collagen and growth factor. Expressions of proteins such as TrkA, p- TrkA, ERK1/2, p-ERK1/2, NF-kß, and p-NF-kß involved in nerve growth factor (NGF) signalling pathway were analysed by western blot. All the findings obtained from this study indicated that the hydrogel can be considered as a promising delivery system against second degree burn by faster healing.

Generation and functional characterization of a single-chain variable fragment (scFv) of the anti-FGF2 3F12E7 monoclonal antibody.

Single-chain variable fragments (scFvs) are small-sized artificial constructs composed of the immunoglobulin heavy and light chain variable regions connected by a peptide linker. We have previously described an anti-fibroblast growth factor 2 (FGF2) immunoglobulin G (IgG) monoclonal antibody (mAb), named 3F12E7, with notable antitumor potential revealed by preclinical assays. FGF2 is a known angiogenesis-associated molecule implicated in tumor progression. In this report, we describe a recombinant scFv format for the 3F12E7 mAb. The results demonstrate that the generated 3F12E7 scFv, although prone to aggregation, comprises an active anti-FGF2 product that contains monomers and small oligomers. Functionally, the 3F12E7 scFv preparations specifically recognize FGF2 and inhibit tumor growth similar to the corresponding full-length IgG counterpart in an experimental model. In silico molecular analysis provided insights into the aggregation propensity and the antigen-recognition by scFv units. Antigen-binding determinants were predicted outside the most aggregation-prone hotspots. Overall, our experimental and prediction dataset describes an scFv scaffold for the 3F12E7 mAb and also provides insights to further engineer non-aggregated anti-FGF2 scFv-based tools for therapeutic and research purposes.

bFGF-chitosan scaffolds effectively repair 20 mm sciatic nerve defects in adult rats.

The repair of peripheral nerve injury is still a great challenge in clinic. Autologous nerve transplantation is the gold standard for the treatment of long-distance peripheral nerve defects, but this method remains associated with high morbidity of the donor site and lack of matching donor. In this study, a novel chitosan scaffold (CS) loaded with control-released basic fibroblast growth factor (bFGF) was used to repair 20 mm sciatic nerve defects in adult rat. The ultrastructure of bFGF-CS was observed by scanning electron microscope. The tensile tester and nano-indentation were used to evaluate its mechanical properties. Cholera toxin B-subunit (CTB) tracing, sciatic nerve function index, electromyography, immunofluorescence staining of regenerated nerve and motor endplate were used to evaluate the regeneration of sciatic nerve in rats. The results showed that the structure and mechanical properties of bFGF-CS was beneficial to the regeneration of sciatic nerve. At 12 weeks after operation, bFGF-CS facilitated sciatic nerve regeneration in rat. CTB successfully crossed the sciatic nerve defect area to reach the cell body of sciatic nerve. The motor endplate was reconstructed, thus promoting the behavioral recovery. These findings suggest that the bFGF-CS provides an effective means of repairing 20 mm sciatic nerve defects and shows great potential for clinical application.

Construction of heparan sulfate microarray for investigating the binding of specific saccharide sequences to proteins.

Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships.