Self-assembling Peptide Nanofiber Hydrogel Research Articles

A self-assembling peptide nanofiber hydrogel for biomaterials with rapid stimulation response to naturally positively charged group substances

Peptides as unique supramolecular nanofibrous materials are favored choices to fabricate hydrogel for artificial extracellular matrices. Herein, we devised an amphiphilic polypeptide that could be motivated by multiple substances with positively charged group at physiological temperature and pH to assemble supramolecular nanofiber hydrogels since the electrostatic repulsion between monomers was attenuated. The hydrogelation phenomenon and spectroscopy were compatible with molecular dynamics simulations (MDs) results for revealing the assembly mechanism of this peptide, confirming that the behavior of assembling and the typical secondary structure of the assemblies was β-sheet. The impact of peptide concentration on the mechanical properties of hydrogels were described using rheology. We found that the peptide formed elastic gels from 0.3 wt% onwards in practical use and that these recovered by themselves after shear thinning that indicated they were injectable hydrogels. The successful establishment of cell spheres with high cell viability during 15 days culture in 3D space demonstrated the nanofiber hydrogels had potential in cell model construction and microtissue culture. Overall, the superior biologically friendly gel-forming conditions and excellent biological properties of this supramolecular assembly peptide material have invigorated the progress of biomedical fields such as tissue regeneration based on in vitro 3D culture.

A novel octa-arginine-modified injectable self-assembling peptide hydrogel for multidrug-resistant cancer therapy

Surgery combined with systemic chemotherapy is currently the main modality of cancer treatment. However, the development of cancer multidrug resistance and inevitable surgical residual lesions lead to high rates of cancer recurrence and mortality. Meanwhile, systemic drug toxic side effects and surgical bleeding complications seriously reduce the quality of life of patients. To avoid cancer multidrug resistance and comprehensively optimize cancer treatment, this work provides a novel injectable cell penetrating peptide octa-arginine (R8)-modified RADA16 (RR) self-assembling peptide nanofiber hydrogel as an anticancer drug carrier for multidrug-resistant cancer therapy. In RR hydrogel, highly compatible RADA16 functions as the β-sheet-based self-assembling backbone which tightly adheres to cancer tissue, prevents postoperative bleeding, and sustainedly releases anticancer drugs for long-term effects. The introduced R8 motifs endow RR hydrogel with the capacity to selectively kill cancer cells and reverse cancer multidrug resistance, which minimally impact normal tissue cells. Specifically, R8 motifs on the hydrogel scaffold selectively interfere with cancer cell membranes, increase drug uptake and penetration into cancer cells and tissues by enhancing multiple internalization pathways, and reduce drug efflux by inhibiting P-gp and BCRP multidrug-resistant transporters. Therefore, this study provides a multifunctional hydrogel material with strong potential for multidrug-resistant cancer clinical translation.

Repair of critical-sized rat cranial defects with RADA16-W9 self-assembled peptide hydrogel

Bone defects are common in orthopaedics and there is an urgent need to explore effective bone repair materials with osteoinductive activity. Peptide self-assembled nanomaterials have a fibrous structure like that of the extracellular matrix and are ideal bionic scaffold materials. In this study, a short peptide WP9QY (W9) with strong osteoinductive effect was tagged to a self-assembled peptide RADA16 molecule through solid phase synthesis to design a RADA16-W9 peptide gel scaffold. A rat cranial defect was used as a research model to explore the effect of this peptide material on the repair of bone defects in vivo. The structure characteristic of the functional self-assembling peptide nanofiber hydrogel scaffold RADA16-W9 was evaluated by atomic force microscopy (AFM). Then adipose stem cells (ASCs) were isolated from Sprague-Dawley (SD) rat and cultured. the cellular compatibility of scaffold was evaluated through Live/Dead assay. Furthermore, we explore the effects of hydrogels in vivo with the critical-sized mouse calvarial defect model. Micro-CT analysis showed that the RADA16-W9 group had higher levels of bone volume/total volume (BV/TV) (P < 0.05)，Trabecular number(TB.N) (P < 0.05)，bone mineral density (BMD)(P < 0.05) and trabecular thickness (Tb. Th) (P < 0.05) compared with the RADA16 and PBS groups. Hematoxylin and eosin (H&E) staining showed that RADA16-W9 group had the highest bone regeneration level. Histochemical staining showed significantly higher expression levels of osteogenic factors such as alkaline phosphatase (ALP) and osteocalcin (OCN) in the RADA16-W9 group than in the other two groups (P < 0.05). Reverse transcription polymerase chain reaction (RT-PCR) quantification showed higher mRNA expression levels of osteogenic-related genes ALP, Runt-related transcription factor 2(Runx2), OCN, Osteopontin (OPN) in the RADA16-W9 group than in the RADA16 and PBS groups (P < 0.05). The live/dead staining results showed that RADA16-W9 is not toxic to rASCs and has good biocompatibility. In vivo experiments show that it accelerates the process of bone reconstruction, significantly promoting bone regeneration and can be used to develop a molecular drug for bone defect repair.

Repairing sciatic nerve injury with self-assembling peptide nanofiber scaffold-containing chitosan conduit

BackgroundAn increasing number of nerve guide scaffolds have been used to replace the “gold-standard” autologous nerve graft for repairing peripheral nerve defects, but nerve regeneration is usually far from complete.MethodsHere, we designed and prepared two functionalized self-assembling peptides (SAP) with the IKVAV and KLT sequences, which were derived from the combination of laminin and VEGF, respectively. Their mixtures were also obtained to combine the effects of neuroprotective and neurotrophic and proangiogenic factors.ResultsThe beneficial effect of peptide gels on nerve regeneration was evaluated in vitro using Schwann cells (SCs). As a useful intraluminal filling, a three-dimensional (3D) functionalized self-assembling peptide (SAP) nanofiber hydrogel was formed in the hollow lumen of chitosan conduits under physiological conditions. In vivo, the combination of the two functionalized SAP gels containing a chitosan nerve conduit significantly accelerated nerve healing and enhanced morphological repair.ConclusionBased on the current findings, the combined application of two functionalized SAP gels with chitosan nerve conduit is a promising therapy for the engineering of peripheral nerve regeneration.

Stem cell-homing hydrogel-based miR-29b-5p delivery promotes cartilage regeneration by suppressing senescence in an osteoarthritis rat model.

Osteoarthritis (OA) is a common joint disease characterized by progressive loss of cartilage and reduction in lubricating synovial fluid, which lacks effective treatments currently. Here, we propose a hydrogel-based miRNA delivery strategy to rejuvenate impaired cartilage by creating a regenerative microenvironment to mitigate chondrocyte senescence that mainly contributes to cartilage breakdown during OA development. An aging-related miRNA, miR-29b-5p, was first found to be markedly down-regulated in OA cartilage, and their up-regulation suppressed the expression of matrix metalloproteinases and senescence-associated genes (P16INK4a/P21) via ten-eleven-translocation enzyme 1 (TET1). An injectable bioactive self-assembling peptide nanofiber hydrogel was applied to deliver agomir-29b-5p, which was functionalized by conjugating a stem cell–homing peptide SKPPGTSS for endogenous synovial stem cell recruitment simultaneously. Sustained miR-29b-5p delivery and recruitment of synovial stem cells and their subsequent differentiation into chondrocytes led to successful cartilage repair and chondrocyte rejuvenation. This strategy enables miRNA-based therapeutic modality to become a viable alternative for surgery in OA treatment.

Sa12b-Modified Functional Self-Assembling Peptide Hydrogel Enhances the Biological Activity of Nucleus Pulposus Mesenchymal Stem Cells by Inhibiting Acid-Sensing Ion Channels

Various hydrogels have been studied for nucleus pulposus regeneration. However, they failed to overcome the changes in the acidic environment during intervertebral disc degeneration. Therefore, a new functionalized peptide RAD/SA1 was designed by conjugating Sa12b, an inhibitor of acid-sensing ion channels, onto the C-terminus of RADA16-I. Then, the material characteristics and biocompatibility of RAD/SA1, and the bioactivities and mechanisms of degenerated human nucleus pulposus mesenchymal stem cells (hNPMSCs) were evaluated. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that RAD/SA1 self-assembling into three-dimensional (3D) nanofiber hydrogel scaffolds under acidic conditions. Analysis of the hNPMSCs cultured in the 3D scaffolds revealed that both RADA16-I and RAD/SA1 exhibited reliable attachment and extremely low cytotoxicity, which were verified by SEM and cytotoxicity assays, respectively. The results also showed that RAD/SA1 increased the proliferation of hNPMSCs compared to that in culture plates and pure RADA16-I. Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting demonstrated that the expression of collagen I was downregulated, while collagen II, aggrecan, and SOX-9 were upregulated. Furthermore, Ca2+ concentration measurement and western blotting showed that RAD/SA1 inhibited the expression of p-ERK through Ca2+-dependent p-ERK signaling pathways. Therefore, the functional self-assembling peptide nanofiber hydrogel designed with the short motif of Sa12b could be used as an excellent scaffold for nucleus pulposus tissue engineering. Moreover, RAD/SA1 exhibits great potential applications in the regeneration of mildly degenerated nucleus pulposus.

Paracrine study of adipose tissue-derived mesenchymal stem cells (ADMSCs) in a self-assembling nano-polypeptide hydrogel environment

Abstract To research the paracrine role of adipose tissue-derived mesenchymal stem cells (ADMSCs) in promoting angiogenesis under the three-dimensional culture conditions consisting of a functionalized self-assembling peptide nanofiber hydrogel. ADMSCs were isolated, extracted, and then identified. Three kinds of peptides (RADAI-16, RGD, and KLT) were prepared, and a functionalized self-assembling peptide nanofiber hydrogel was produced by mixing RADAI-16, RGD, and KLT in a volume ratio 2:1:1. AFM was used to observe RADAI-16, RGD, KLT, and the functionalized self-assembling peptide nanofiber hydrogel. Then, ADMSCs were cultured under three-dimensional conditions consisting of the peptide nanofiber hydrogel, and AFM was used to observe cell migration. The ADMSCs in the common culture group (37°C, 5% CO2 cell culture box) and hypoxic culture group (37°C, 10% CO2, and 1% O2 hypoxic culture box) acted as controls. ADMSCs were three-dimensionally cultured in situ for 1 day, and then the concentrations of HGF and VEGF in the supernatant were determined by ELISA. Cells were extracted from the peptide nanofiber hydrogel, and HO-1 expression was detected by western blotting. ADMSCs have high expression levels of CD29, CD90, and CDl05 and low expression levels of CD34 and CD45. In addition, they can differentiate into adipocytes and osteocytes. The diameters of the fibers of RADAI-16, RGD, KLT, and the functionalized self-assembling peptide hydrogel are 17.34 ± 1.82, 15.50 ± 1.41, 13.77 ± 1.18, and 20.26 ± 1.25 nm, respectively. AFM indicated that cells in the functionalized self-assembling peptide nanofiber hydrogel migrated farther than those in RADAI-16. The concentrations of HGF under common, hypoxic, and three-dimensional culture conditions were 47.31 ± 6.75, 247.86 ± 17.59, and 297.25 ± 17.95 pg/mL, respectively, while the concentrations of VEGF were 218.30 ± 3.03, 267.13 ± 4.27, and 289.14 ± 3.11 pg/mL, respectively. Both HGF and VEGF were expressed more in the presence of the functionalized self-assembling peptide nanofiber hydrogel than in its absence (P &lt; 0.05). Using western blotting, ADMSCs cultured under hypoxic and three-dimensional conditions were found to have high expression levels of HO-1. Culturing ADMSCs under three-dimensional conditions consisting of functionalized self-assembling peptide nanofiber hydrogels can promote their paracrine role in angiogenesis, such as HGF and VEGF, and hypoxia is one of the important elements.

Injectable self-assembling peptide nanofiber hydrogel as a bioactive 3D platform to promote chronic wound tissue regeneration

Chronic wounds remain a worldwide clinical challenge, and bioactive materials that can promote skin regeneration are required. Self-assembling peptide (SAP) hydrogels have shown great potential in tissue repair, but their regenerative efficacy and possible mechanism in chronic wound healing are unclear. Here, we report an SAP (KGH) that enhances extracellular matrix (ECM) remodeling and angiogenesis, thereby promoting chronic wound healing in diabetic mice. In vivo, the KGH hydrogel was retained in wounds up to 7 days after injection, and it was effective in speeding up wound closure by ∼20% compared to the control groups and enhancing angiogenesis (e.g., VEGFA, CD31+ capillaries), cell proliferation (e.g., PCNA+ cells), formation of granulation tissue (e.g., α-SMA), and ECM deposition/remodeling (e.g., collagen I, fibronectin). In vitro, the KGH hydrogel created a 3D microenvironment for skin cells, maintained the sustained growth of cell spheroids, and increased the secretion of ECM proteins (e.g., laminin) and growth factors (e.g., PDGFB, VEGFA, and TGF-β) in skin keratinocytes compared to the conventional 2D culture. Mechanistically, the KGH hydrogel might promote wound tissue regeneration by activating the Rho/ROCK and TGF-β/MEK/MAPK pathways. As a type of designed material, SAP can be further re-engineered with biological motifs, therapeutic reagents, or stem cells to enhance skin regeneration. This study highlights that SAP hydrogels are a promising material platform for advanced chronic wound healing and might have translational potential in future clinical applications. Statement of significanceChronic wounds are a common and serious health issue worldwide, and bioactive dressing materials are required to address this issue. SAP hydrogels have shown certain tissue repair potential, but their regenerative efficacy and underlying mechanism in chronic wound healing remain elusive. Herein, we report that SAP hydrogels create a native 3D microenvironment that can remarkably stimulate angiogenesis and ECM remodeling in diabetic wounds. Mechanistically, the SAP hydrogel promoted ECM proteins and GFs secretion in skin cells through the activation of the Rho/ROCK and TGF-ß/MEK/MAPK pathways. Additionally, SAP can be readily engineered with various bioactive motifs or therapeutic drugs/cells. This work highlights SAP hydrogels as a promising biomaterial platform for chronic wound healing and the regeneration of many other tissues.

Crosstalk between PC12 cells and endothelial cells in an artificial neurovascular niche constructed by a dual-functionalized self-assembling peptide nanofiber hydrogel

Crosstalk between PC12 cells and endothelial cells in an artificial neurovascular niche constructed by a dual-functionalized self-assembling peptide nanofiber hydrogel

Proangiogenic peptide nanofiber hydrogel/3D printed scaffold for dermal regeneration

Dermal substitutes have been widely used as therapeutic options in skin damage to regenerate the skin. Rapid vascularization of a dermal substitute can provide channels for nutritional support, excretion and create an interventional pathway for the immune system during the early wound healing stages. Therefore it is important to evaluate the vascularization property of dermal substitutes in the process of dermal reconstruction. In this study, a proangiogenic self-assembling peptide nanofiber hydrogel (SLg) was synthesized and combined with UV-cured gelatin (GelMA) to prepare a series of three-dimensional (3D) printed scaffolds. Interpenetrating polymer networks (IPNs) comprising interwoven gelatin and peptide were constructed during 3D printing process, resulting in advancement in absorbency and elasticity. Moreover, the 3D printed GelMA/SLg scaffolds exhibited controlled porosity, pore size and connected pore properties. These properties provided the appropriate microenvironment for cellular migration, proliferation and were conducive for the growth of blood vessels leading to early angiogenesis. An in vivo study found that 3D printed GelMA/SLg scaffolds containing 20% peptide hydrogel exhibited the best collagenous fibrous structure, fastest repair cycles in revascularization and dermal regeneration. These results imply that the proangiogenic scaffold promotes early rapid vascularization and tissue healing, and is a promising therapeutic option for high-quality regenerative repair of skin damage.

Proangiogenic Peptide Nanofiber Hydrogel/3D-Printed Scaffold to Promote Dermal Regeneration

Dermal substitutes are widely used for wound repair and show a good advantage in skin regeneration. Vascularization is an important index for evaluating dermal materials. Rapid vascularization can provide channels for nutritional support and excretion and create an intervention pathway for the immune system in the early stage of wound healing. In this study, we synthesized a proangiogenic self-assembling peptide nanofiber hydrogel and combined it with UV-cured gelatin to form a series of 3D-printed scaffolds. As the percentage of peptide increased, the microstructure of the scaffolds transformed to nanoscale, and the water absorption rate and weight loss ratio in phosphate-buffered saline (PBS) buffer also increased. Mouse fibroblasts (L929) and human umbilical vein endothelial cells (HUVECs) adhered and proliferated on 3D-printed scaffolds, and a vascular model test showed that many connected tubes were present in the HUVECs after 8 h of culture with material extract, indicating the promising proangiogenic capability of these 3D-printed scaffolds. An in vivo study demonstrated that the 3D-printed scaffold combined with 20% peptide hydrogel had the best collagenous fiber structure and fastest repair cycles in dermis regeneration. The results indicate that our proangiogenic scaffold constitutes a promising therapeutic option for high-quality skin regeneration.

Self-assembling peptide hydrogels functionalized with LN- and BDNF- mimicking epitopes synergistically enhance peripheral nerve regeneration.

The regenerative capacity of the peripheral nervous system is closely related to the role that Schwann cells (SCs) play in construction of the basement membrane containing multiple extracellular matrix proteins and secretion of neurotrophic factors, including laminin (LN) and brain-derived neurotrophic factor (BDNF). Here, we developed a self-assembling peptide (SAP) nanofiber hydrogel based on self-assembling backbone Ac-(RADA)4-NH2 (RAD) dual-functionalized with laminin-derived motif IKVAV (IKV) and a BDNF-mimetic peptide epitope RGIDKRHWNSQ (RGI) for peripheral nerve regeneration, with the hydrogel providing a three-dimensional (3D) microenvironment for SCs and neurites.Methods: Circular dichroism (CD), atomic force microscopy (AFM), and scanning electron microscopy (SEM) were used to characterize the secondary structures, microscopic structures, and morphologies of self-assembling nanofiber hydrogels. Then the SC adhesion, myelination and neurotrophin secretion were evaluated on the hydrogels. Finally, the SAP hydrogels were injected into hollow chitosan tubes to bridge a 10-mm-long sciatic nerve defect in rats, and in vivo gene expression at 1 week, axonal regeneration, target muscular re-innervation, and functional recovery at 12 weeks were assessed.Results: The bioactive peptide motifs were covalently linked to the C-terminal of the self-assembling peptide and the functionalized peptides could form well-defined nanofibrous hydrogels capable of providing a 3D microenvironment similar to native extracellular matrix. SCs displayed improved cell adhesion on hydrogels with both IKV and RGI, accompanied by increased cell spreading and elongation relative to other groups. RSCs cultured on hydrogels with IKV and RGI showed enhanced gene expression of NGF, BDNF, CNTF, PMP22 and NRP2, and decreased gene expression of NCAM compared with those cultured on other three groups after a 7-day incubation. Additionally, the secretion of NGF, BDNF, and CNTF of RSCs was significantly improved on dual-functionalized peptide hydrogels after 3 days. At 1 week after implantation, the expressions of neurotrophin and myelin-related genes in the nerve grafts in SAP and Autograft groups were higher than that in Hollow group, and the expression of S100 in groups containing both IKV and RGI was significantly higher than that in groups containing either IKV or RGI hydrogels, suggesting enhanced SC proliferation. The morphometric parameters of the regenerated nerves, their electrophysiological performance, the innervated muscle weight and remodeling of muscle fibers, and motor function showed that RAD/IKV/RGI and RAD/IKV-GG-RGI hydrogels could markedly improve axonal regeneration with enhanced re-myelination and motor functional recovery through the synergetic effect of IKV and RGI functional motifs.Conclusions: We found that the dual-functionalized SAP hydrogels promoted RSC adhesion, myelination, and neurotrophin secretion in vitro and successfully bridged a 10-mm gap representing a sciatic nerve defect in rats in vivo. The results demonstrated the synergistic effect of IKVAV and RGI on axonal regrowth and function recovery after peripheral nerve injury.

Injectable extracellular vesicle-released self-assembling peptide nanofiber hydrogel as an enhanced cell-free therapy for tissue regeneration

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great potential for tissue repair, but their therapeutic capacity is limited by rapid clearance and short half-life. Herein, we purposed a hydrogel-based slow release strategy to enhance the therapeutic potency of EVs. A matrix metalloproteinase-2 (MMP2) sensitive self-assembling peptide (KMP2) hydrogel was used for the local delivery of MSC-EVs. The structure and controlled release properties of the KMP2 hydrogel were analyzed. The effects of the EV-loaded KMP2 hydrogel (KMP2-EVs) on cell apoptosis, inflammation and angiogenesis were evaluated in mice with renal ischemia-reperfusion (I/R) injury. In vitro, KMP2 formed a cross-linked nanofiber hydrogel to encapsulate MSC-EVs. KMP2 showed greater degradation and EV release in response to MMP2. The released EVs had similar structures and bioactivities as fresh, isolated EVs. In vivo, I/R mice treated with KMP2-EVs showed improved renal function by reducing tubular cell apoptosis, pro-inflammatory cytokine expression, and macrophage infiltration than mice receiving either EVs or KMP2. Moreover, KMP2-EVs showed better efficacy on promoting endothelial cell proliferation and angiogenesis than KMP2 or EVs alone, which subsequently decreased chronic renal fibrosis in I/R mice. This study highlighted that the EV-released KMP2 hydrogel is a promising cell-free therapy for tissue repair.

Synergistic effects of dual-presenting VEGF- and BDNF-mimetic peptide epitopes from self-assembling peptide hydrogels on peripheral nerve regeneration.

The crosstalk between vascularization and nerve regeneration in the peripheral nervous system has recently been suggested to play an important role in the treatment of peripheral nerve injury. Regenerative strategies via synergistic delivery of multiple biochemical cues have received growing attention, especially the combination of pro-angiogenic factors and neurotrophic factors. Here we developed a self-assembling peptide nanofiber hydrogel dual-functionalized with vascular endothelial growth factor (VEGF)- and brain-derived neurotrophic factor (BDNF)-mimetic peptide epitopes for peripheral nerve reconstruction. It could simultaneously present VEGF- and BDNF-mimetic peptide epitopes and provides a three-dimensional (3D) neurovascular microenvironment for endothelial cell and neural cell growth. In vitro cellular experiments showed that the functionalized peptide hydrogel scaffold effectively promoted the pro-myelination of Schwann cell, as well as the adhesion and proliferation of endothelial cell compared with scaffolds presenting VEGF- or BDNF-mimetic peptide epitope alone. When implanted in a rat model to bridge a critical-size sciatic nerve gap in vivo, the functionalized peptide hydrogel significantly improved the number of newly formed blood vessels, the density of regenerating axons, the morphometric analysis of the regenerated muscles and the electrophysiological findings, indicating the synergistic effect of the two bioactive motifs on peripheral nerve regeneration. Collectively, constructing an artificial neurovascular microenvironment in the lesion area by using the functionalized self-assembling peptide nanofiber hydrogel may have a great potential for promoting nerve tissue engineering and regeneration in other tissues.

Chemotherapeutic Delivery from a Self-Assembling Peptide Nanofiber Hydrogel for the Management of Glioblastoma.

Localized chemotherapy has gained significant impetus for the management of malignant brain tumors. In the present study, we appraised the versatility of an in-situ gel forming self-assembling peptide, ac-(RADA)4-CONH2, as a biocompatible delivery depot of the chemotherapeutic drug doxorubicin (DOX) and the anticancer agent curcumin (CUR), respectively. The morphology and mechanical properties of ac-(RADA)4-CONH2 were assessed with scanning electron microscopy (SEM) and rheological studies. The in vitro drug release from ac-(RADA)4-CONH2 was monitored in phosphate-buffered saline pH7.4. Distribution of the fluorescent actives within the peptide matrix was visualized with confocal laser scanning microscopy (CLSM). The in vitro biological performance of the ac-(RADA)4-CONH2-DOX and ac-(RADA)4-CONH2-CUR was evaluated on the human glioblastoma U-87 MG cell line. SEM studies revealed that the ac-(RADA)4-CONH2 hydrogel contains an entangled nanofiber network. Rheology studies showed that the more hydrophobic CUR resulted in a stiffer hydrogel compared with ac-(RADA)4-CONH2 and ac-(RADA)4-CONH2-DOX, due to the interaction of CUR with the hydrophobic domains of the peptide nanofibers as confirmed by CLSM. In vitro release studies showed a complete DOX release from ac-(RADA)4-CONH2 within 4days and a prolonged release for ac-(RADA)4-CONH2-CUR over 20days. An increased cellular uptake and a higher cytotoxic effect were observed for ac-(RADA)4-CONH2-DOX, compared with DOX solution. Higher levels of early apoptosis were observed for the cells treated with the ac-(RADA)4-CONH2-CUR, compared to CUR solution. The current findings highlight the potential utility of the in-situ depot forming ac-(RADA)4-CONH2 hydrogel for the local delivery of both water soluble and insoluble chemotherapeutic drugs.

Bioactive Self-Assembling Peptide Hydrogels Functionalized with Brain-Derived Neurotrophic Factor and Nerve Growth Factor Mimicking Peptides Synergistically Promote Peripheral Nerve Regeneration.

Various artificial materials have been fabricated as alternatives to autologous nerve grafts in peripheral nerve regeneration, and these afford positive recovery effects without the disadvantages of the gold standard. In this study, we prepared a three-dimensional functionalized self-assembling peptide nanofiber hydrogel containing two neurotrophic peptides (CTDIKGKCTGACDGKQC and RGIDKRHWNSQ derived from nerve growth factor and brain-derived neurotrophic factor, respectively) that reflected the structure and properties of the neural extracellular matrix. The material was used to promote axonal regrowth and functional recovery. Scanning electron microscopy revealed a three-dimensional porous matrix within the hydrogel. Circular dichroism spectroscopy and atomic force microscopy confirmed that the peptides displayed a β-sheet structure and self-assembled into long nanofibers. Rheology measurements and atomic force microscopy indicated that the elasticity of the peptide hydrogels was close to that of the nerve tissue matrix. In vitro work with Schwann cells and dorsal root ganglia showed that the hydrogels exhibited good cell compatibility. Furthermore, the hydrogel containing CTDIKGKCTGACDGKQC and RGIDKRHWNSQ promoted the neurite outgrowth of PC12 cells significantly compared to non-functionalized peptide. In vivo, the hydrogels were placed into chitosan tubes and used to bridge 10 mm long sciatic nerve defects in rats. We found that the combination of CTDIKGKCTGACDGKQC and RGIDKRHWNSQ accelerated axonal regeneration and afforded good functional recovery, suggesting that they synergistically facilitate peripheral nerve regeneration.

A neurotrophic peptide-functionalized self-assembling peptide nanofiber hydrogel enhances rat sciatic nerve regeneration

Nerve guidance conduit (NGC) is a potential alternative to autologous nerve for peripheral nerve regeneration. A promising therapeutic strategy is to modify the nerve guidance conduit intraluminal microenvironment using physical and/or chemical guidance cues. In this study, a neurotrophic peptide-functionalized self-assembling peptide nanofiber hydrogel that could promote PC12 cell adhesion, proliferation, and neuronal differentiation in vitro was prefilled in the lumen of a hollow chitosan tube (hCST) to accelerate axonal regeneration in a rat sciatic nerve defect model. The functionalized self-assembling peptide was developed by introducing a neurotrophic peptide (RGI, RGIDKRHWNSQ) derived from brain-derived neurotrophic factor (BDNF) to the C-terminus of the self-assembling peptide RADA16-I (Ac-(RADA)4-CONH2). Morphological, histological, electrophysiological, and functional analyses demonstrated that the RGI-functionalized, self-assembling, peptide nanofiber hydrogel RAD/RGI could produce a neurotrophic microenvironment that markedly improved axonal regeneration with enhanced re-myelination and motor functional recovery.

Paracrine of human adipose-derived mesenchymal stem cells in three-dimensional culture containing functionized self assembling peptide nanofiber hydrogel

Objective To study the paracrine of human adipose-derived mesenchymal stem cells (ADMSCs) in three-dimensional culture containing functionized self assembling peptide nanofiber hydrogel and the influencing factors. Methods ADMSCs were isolated and extracted. Three kinds of peptides (RADAI-16, RGD, KLT) were prepared and functionized self assembling peptide nanofiber hydrogel was made by mixing RADAI-16, RGD and KLT with a volume ratio of 2∶1∶1. Under AFM, the morphology of RADAI-16, RGD, KLT and functionized self assembling peptide nanofiber hydrogel was observed (n=4). ADMSCs were cultured under the three-dimensional condition containing peptide nanofiber hydrogel. ADMSCs were subjected to in situ three-dimensional cultivation for 1 day, then the concentrations of hepatocyte growth factor (HGF, n=3) and vascular endothelial growth factor (VEGF, n=3) were determined in the supernatatn by enzyme linked immunosorbent assay (ELISA). The cells were extracted in peptide nanofiber hydrogel and the expression of heme oxygenase-1 (HO-1, n=3) was detected by Western blotting. The cultured ADMSCs in hypoxic condition and normal circumstance served as controls. Results ADMSCs showed long spindle shape and grew in a whirlpool shape. The diameter of RADAI-16, RGD, KLT and functionized self assembling peptide nanofiber hydrogel was (17.34±1.82), (15.50±1.41), (13.77±1.18) and (20.26±1.25) nm, respectively. The concentration of HGF under three conditions was (47.31±6.75), (247.86±17.59), and (297.25±17.95) ng/L, respectively, and that of VEGF was (218.30±3.03), (267.13±4.27) and (289.14±3.11) ng/L, respectively. Both HGF and VEGF were paracrined more in condition of functionized self assembling peptide nanofiber hydrogel (P=0.000). Western blotting results showed that the expression of HO-1 in the three-dimensional culture group was about 2 times (P=0.000) higher than that in the ordinary culture group, and was 1.2 times higher than that in the hypoxia culture group (P=0.033). Conclusion The three-dimensional culture mediated by the functionized self assembling peptide nanofiber hydrogel can promote the paracrine of ADMSCs, such as HGF and VEGF, and hypoxia is one of the important elements. Key words: Adipose derived Mesenchymal stem cells; Three-dimensional culture; Functionized self assembling peptide nanofiber hydrogel; Paracrine; Hypoxia

Self-Assembling Peptide Nanofiber Hydrogels for Controlled Ocular Delivery of Timolol Maleate.

The self-assembling peptides Ac-(RADA)4-CONH2 and Ac-(IEIK)3I-CONH2, which form hydrogels in physiological conditions, were evaluated as carriers for ocular delivery of the β-blocker timolol maleate. Electron microscopy studies revealed that hydrogels contain nanofibers, whereas rheological studies showed that the Ac-(IEIK)3I-CONH2 self-assembles in a stiffer hydrogel compared with the Ac-(RADA)4-CONH2 peptide. The in vitro release and ex vivo permeation studies demonstrated controlled release and transport of the drug through the cornea, which depended on the self-assembling peptide sequence. In vivo studies in rabbits showed significant increase in the area under the concentration-time curve (AUC) after administration of the drug through the Ac-(RADA)4-CONH2 hydrogel compared to drug solution, whereas a sustained reduction of intraocular pressure for up to 24 h after instillation was achieved for both drug-loaded hydrogels. Histological studies revealed good ocular tolerability upon application of the formulations, suggesting that self-assembling peptide hydrogels are promising systems for sustained ocular drug delivery.

Designer D-form self-assembling peptide scaffolds promote the proliferation and migration of rat bone marrow-derived mesenchymal stem cells.

Self-assembling peptide (SAP) nanofiber hydrogel scaffolds have become increasingly important in tissue engineering due to their outstanding bioactivity and biodegradability. However, there is an initial concern on their long-term clinical use, since SAPs made of L-form amino acid sequences are sensitive to enzymatic degradation. In this study, we present a designer SAP, D-RADA16, made of all D-amino acid. We investigated the nanofiber morphology of D-RADA16, its potential for the culture of bone marrow-derived mesenchymal stem cells (BMSCs), and the proteolytic resistance of the biomaterial. The results revealed that D-RADA16 exhibited stable β-sheets and formed interwoven nanofiber scaffolds in water. D-RADA16 and L-RADA16 hydrogel scaffolds were both found to promote the proliferation and migration of rat BMSCs in the 3D cell culture microenvironment. Furthermore, the D-RADA16 scaffolds exhibited a higher proteolytic resistance against proteinase K than the L-RADA16 scaffolds. These observations indicate that D-RADA16 hydrogel scaffolds have excellent bioactivity, biocompatibility and biostability, and thus may serve as promising candidates for long-term application in vivo.