CN113633823B - Functional self-assembly nano polypeptide hydrogel, preparation method, application and preparation - Google Patents
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- CN113633823B CN113633823B CN202110813402.3A CN202110813402A CN113633823B CN 113633823 B CN113633823 B CN 113633823B CN 202110813402 A CN202110813402 A CN 202110813402A CN 113633823 B CN113633823 B CN 113633823B
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Abstract
The invention discloses a functional self-assembly nano-polypeptide hydrogel, a preparation method, an application and a preparation, wherein the functional self-assembly nano-polypeptide hydrogel comprises Ca 2+ Conjugated binding peptides and cross-linking peptides responsive to coagulation factor 13, wherein the molar ratio of binding peptides to cross-linking peptides is 1:1-10; the invention forms nano-fiber by self-assembly of cross-linking peptide (CRP) and binding peptide (CBP) in Ca 2+ The second stage of self-assembly is driven to weave into a mesh network to form NBA, the NBA can rapidly and effectively stop bleeding of a rat liver scratch model, and meanwhile inflammation around a wound is effectively reduced, and wound healing is promoted.
Description
Technical Field
The invention belongs to the field of vascular tissue engineering, and particularly relates to a functional self-assembled nano-polypeptide hydrogel, a preparation method, an application and a preparation.
Background
It is estimated that 580 million people die worldwide each year from severe trauma, corresponding to approximately 16000 deaths per day. According to the World Health Organization (WHO) statistics, road traffic accidents, suicide, murder and war are the major causes of trauma, in which case the liver is the most commonly damaged internal organ, causing at least 10% mortality. Uncontrolled and post-traumatic massive liver bleeding remains a major and potentially preventable cause of mortality, and even if rescued in a timely manner, these patients suffer from life-threatening coagulopathies. Whether in battlefield or daily rescue, the core principle of liver trauma emergency is the rapid and effective hemostasis. In addition, bleeding inevitably occurs in common liver surgery including partial liver resection and liver transplantation, and is closely related to the postoperative effect. While much clinical effort has been made to manage and prevent excessive blood loss during liver surgery, surgery-complicated bleeding can still occur in individual patients, particularly those who undergo extensive superficial resection of the liver. More importantly, the body's own blood clotting cannot stop bleeding in time to prevent a large amount of sustained blood loss, and thus timely hemostasis using hemostatic agents is of interest for both first aid trauma and surgical procedures involving the liver.
For this reason, two broad classes of hemostatic materials have emerged: 1) Combat Gauze, quikClot and WundStat, and 2) inorganic materials represented by polymer materials, hemCo and Celox, and the like. Inorganic materials generally promote the formation of blood clots and catalyze the conversion of cellulose to fibrin, resulting in good hemostasis. The polymeric hemostatic material always serves as a physical barrier, having the function of preventing bleeding. While these two materials have had many successes in hemostasis in trauma emergencies and surgical procedures involving the liver, significant challenges remain in rapidly and effectively controlling bleeding, especially puncture wounds that are not compressible for hemostasis and large wounds that are not suturable. In addition, commercial hemostatic agents often have side effects, including secondary damage from in vitro reactions of inorganic products and bio-toxicity from degradation of polymeric products. Therefore, the development of biocompatible hemostatic materials without side effects is urgently needed.
To solve these problems, self-assembling peptide hydrogels are uniquely advantageous as hemostatic materials because of their inherent biocompatibility and safety. Driven by noncovalent weak interactions such as hydrophobic interaction, electrostatic interaction, and hydrogen bonding, self-assembling peptides (such as SPG-178, EAK16, SLAc, RADA16, etc.) can gel at bleeding sites, significantly reducing blood loss. This self-assembly, driven by weak interaction, is a double-edged sword: the ability to self-assemble into specific network nanostructures is one of the greatest benefits of these peptides, but the weak interoccludal interactions make them fragile and unsuitable for blocking bleeding from arterial vessels, such as hepatic arteries, with high pressure. Therefore, it remains a challenge to develop peptide-derived hemostatic materials with mechanical properties to rapidly and effectively control bleeding.
Disclosure of Invention
The invention aims to provide a functional self-assembly nano-polypeptide hydrogel, a preparation method, an application and a preparation, and aims to solve the problem that the existing gel and hemostatic material cannot deal with large wound surfaces and high-pressure vascular hemorrhage.
The invention adopts the following technical scheme: a functional self-assembled nano-polypeptide hydrogel contains Ca 2+ A conjugated binding peptide and a cross-linking peptide that is responsive to coagulation factor 13, wherein the binding peptide and cross-linking peptide are in a molar ratio of 1:1-10.
Further, the molar ratio of binding peptide to cross-linking peptide was 3:1.
Further, the sequence of the cross-linking peptide is: (RADA) 4 -GGQQLK, the sequence of the binding peptide is: (RADA) 4 -GSVLGYIQIR。
A preparation method of functional self-assembly nano-polypeptide hydrogel comprises the following steps: mixing the water solution of the binding peptide and the cross-linking peptide into a polypeptide mixed solution according to the molar ratio of 3:1, completely dissolving and uniformly mixing the polypeptide by ultrasonic treatment, and inducing the polypeptide mixed solution to self-assemble at room temperature to form hydrogel.
The functional self-assembled nano polypeptide hydrogel is used for preparing tissue engineering materials.
Further, the tissue engineering material is an artificial blood vessel tissue material.
An artificial blood vessel obtained by three-dimensional culture of ex vivo mammalian-derived vascular endothelial cells in the functional self-assembled nano-polypeptide hydrogel of any one of claims 1 to 3, the ex vivo vascular endothelial cells being taken from lung, skin, aorta or umbilical vessels of a mammal.
A functional self-assembling nano-polypeptide formulation comprising the functional self-assembling nano-polypeptide of any one of claims 1-3.
Furthermore, the dosage form of the functional self-assembly nano-polypeptide preparation comprises powder or liquid preparation, and the functional self-assembly nano-polypeptide preparation also comprises pharmaceutically acceptable carriers and/or auxiliary materials.
The application of the functional self-assembly nano polypeptide preparation comprises the following steps: preparing the hemostatic material.
The invention has the beneficial effects that: the functional self-assembly nano polypeptide hydrogel (NBA) quickly and effectively stops bleeding through a three-stage self-assembly strategy of two functional peptides. NBA is first constructed by two-stage self-assembly from cross-linking peptide (CRP) and binding peptide (CBP) to form nanofibers at Ca 2+ Driven by the driving force, the second stage self-assembly is woven into a mesh network, and then the third stage self-assembly is carried out under the catalysis of blood coagulation factor thirteen (FXIIIA), so that a compact physical barrier is formed to prevent bleeding. According to the expectation, NBA can rapidly and effectively stop bleeding of a rat liver scratch model, and meanwhile, inflammation around a wound is effectively reduced, and wound healing is promoted. More importantly, NBA has excellent hemostatic effect on unstitched hemorrhage caused by 1/3 liver defect of rats and incompressible hemorrhage caused by penetrating wounds, the polypeptide of the invention has no toxic or side effect at all, the safety of the hemostatic material is greatly increased, the bionic three-stage self-assembly strategy provides a clinically potential peptide-based treatment method for fatal liver hemorrhage, and the research and development of the self-assembly peptide hydrogel hemostatic are repeated, so that the peptide hydrogel hemostatic has wide potential application prospect.
Drawings
FIG. 1 shows the design and characteristics of NBA; (A) Schematic diagram of three-stage self-assembly strategy for two functionalized polypeptides CRP and CBP; (B) And (C) LC-MS characterization for the synthesis of CBP (B) and CRP (C); (D) The nano-fibers are self-assembled in PBS buffer solution at the first stage to form a transmission electron microscope image; (E) Is CBP and Ca 2+ Isothermal Titration Calorimetry (ITC) determination of affinity, in PBS buffer at pH7.4, at 25 ℃ to determine its binding affinity; (F) And (G) is Ca 2+ Driving the second stage self-assembled TEM image (F) and SEM image (G); (H) And (I) is a transmission electron microscope image (H) and a scanning electron microscope image (I) after the blood coagulation factor XIIIA (FXIIIa) catalyzes the second-stage self-assembly; (J) is the degree of gelatinization quantified as the absorbance at 600 nm; (K) Showing C-reactive protein/CBP peptide hydrogel pairs for representative photography 2+ The reaction with FXIIIa is gelatinized;
FIG. 2 shows the biological function of NBA; (A) The scanning electron microscope image of NBA on FXIIIa, fibrinogen and blood combined action shows that NBA has great potential in the aspect of weaving a compact physical barrier consisting of polypeptide and fibrin; (B) The whole blood coagulation evaluation of NBA and control groups is shown for representative photographs; (C) For in vitro kinetic whole blood coagulation evaluation of NBA and control groups, the absorbance of the blood solution was measured at a wavelength of 600nm and the half coagulation time (BCI) was fitted with a logarithmic equation 50 ) (D) is a representative picture of rat liver hemostasis after a sagittal plane cut wound with the length of 1cm and the depth of 0.5cm is made; (E) And (F) hemostasis time (E) and blood loss (F) (mean ± SD, n = 6/group) for rat liver scratch model, P value, P was calculated by ANOV method<0.001，p<0.0001，p<0.05，p<0.01，p<0.05，p<0.0001；
FIG. 3 is a representation that NBA is effective in reducing inflammatory response around the wound and promoting wound healing; (A) Volcano plots of wound tissue gene differential expression after NBA treatment versus PBS treatment (n = 3); (B) And (C) is the result of Gene Set Enrichment Analysis (GSEA) of inflammatory response (B) and IL6-JAK-STAT3 signal pathway (C) after NBA treatment; (D) Is FIBRILLAR TM V-olcano profile of wound tissue differentially expressed genes (n = 3) after treatment and PBS treatment (n = 3); (E) And (F) is para-FIBRILAR TM The inflammatory response of the treatment (E) and the GSEA outcome of the IL6-JAK-STAT3 signaling pathway (F); (G) The content of IL-6, MCP-1, NF-kB and TNF alpha in wound tissues is detected by an ELISA method; (H) Representative H for hepatic wounds at day 14 post treatment&E, imaging;
FIG. 4 shows that NBA has good hemostatic effects on both rat liver defect lethal suturable hemorrhage model and wound penetration incompressible hemorrhage model; (A) Establishing a rat liver hemostasis diagram for a rat liver 1/3 defect lethal suturable bleeding model; (B) And (C) hemostasis time (B) and blood loss (C) for rat liver scratch model (mean ± standard deviation, n = 6/group); (D) Blood cell counts and liver biochemical determinations for day 14 post-treatment; (E) Representative HE images of liver wounds at day 14 post treatment; (F) Is a liver hemostasis diagram of a rat model with penetrating injury and non-compressible hemorrhage; (G) And (H) hemostasis time (G) and blood loss (H) for rat liver scratch model (mean ± SD, n = 6/group); (I) blood cell counts on day 3 post treatment; (J) P values were calculated using ANOVA for representative HE images of liver wounds at day 14 post-treatment. p <0.001, p < -0.0001, p < -0.05, p < -0.01, p < -0.05, p < -0.0001;
fig. 5 is a graph of CRP: scanning electron microscope images of NBA composed of CBP;
FIG. 6 is a HRTEM (Transmission Electron microscopy) image and calcium elemental analysis of NBA;
FIG. 7 is the time-dependent cytotoxic activity of NBA of 0.075%, 0.050%, 0.025%, and 0% on (A) AML12 (murine hepatocytes) and (B) HUVEC (human umbilical vein endothelial cells) human umbilical vein endothelial cells;
FIG. 8 is a representative histological HE stain image of a cut of heart, liver, spleen, lung, and kidney tissue after liver scoring in rats;
FIG. 9 is a representative histological HE stain image of heart, liver, spleen, lung, and kidney tissue sections from rats in a lethal suturable hemorrhage model of 1/3 defects in rat liver.
Detailed Description
The invention is described in detail below with reference to the drawings and the detailed description.
The present invention contemplates a three-stage self-assembly strategy for developing a peptide-derived hemostatic material that is capable of treating non-compressible puncture wounds and non-suturable large wounds. In this case, two custom-designed peptides (RADA) 4 GGQQLK (cross-linking peptide, CRP) and (RADA) 4 -GSVLGYIQIR(Ca 2+ Binding peptide, CBP) was efficiently synthesized by Fmoc chemistry (fig. 1A). In the first stage of self-assembly, (RADA) 4 The motif may confer the ability of CRP and CBP to self-assemble into nanofibers in combination. The functional motif VLGYIQIR in the flanking CBP around the self-assembled nanofiber is able to interact with Ca with a coordination number of 3 2+ In combination, the second stage of self-assembly into a mesh-like network called nano-wound patches (NBA) is driven (fig. 1A). As for the third stage of self-assembly, another motif around the nanofibers (QQLK in CRP) can pass through the coagulation cascade (FIG. 1A)The Factor XIIIA (FXIIIA) catalyzed ethyl transfer reactions further cross each other and to cellulose. Thus, when NBAs encounter blood, they can form a physical barrier including polypeptides and cellulose in situ at the site of bleeding. More importantly, NBA is a viscous liquid before encountering blood, facilitating adherence to puncture and large wounds. As expected, NBA rapidly and effectively controls bleeding in rat liver scarification, penetrating wound non-compression bleeding models, and rat liver defect lethal non-suturable bleeding models, while maintaining good safety profile. The bionic three-stage self-assembly strategy provides a clinically potential peptide-based treatment method for fatal hepatic hemorrhage, and revives the effort of developing self-assembly peptide hydrogel as a hemostatic agent, so that the bionic three-stage self-assembly strategy has wide potential application prospects.
The invention discloses a functional self-assembly nano-polypeptide hydrogel which comprises Ca 2+ A binding peptide and a cross-linking peptide responsive to coagulation factor 13FXIIIa, wherein the molar ratio of the binding peptide to the cross-linking peptide is 1:1-10, preferably 3:1, and the sequence of the binding peptide is: (RADA) 4 -GSVLGYIQIR, said cross-linked peptide having the sequence: (RADA) 4 -GGQQLK。
The invention also discloses a preparation method of the functional self-assembly nano-polypeptide hydrogel, which comprises the following steps: mixing the water solution of the binding peptide and the cross-linking peptide into a polypeptide mixed solution according to the molar ratio of 3:1, completely dissolving and uniformly mixing the polypeptide by ultrasonic treatment, and inducing the polypeptide mixed solution to self-assemble at room temperature to form hydrogel.
The invention also discloses the application of the functional self-assembly nano polypeptide hydrogel in preparing tissue engineering materials, wherein the tissue engineering materials are artificial vascular tissue materials.
The invention also discloses an artificial blood vessel, which is obtained by three-dimensionally culturing the isolated vascular endothelial cells derived from mammals in the functional self-assembly nano polypeptide hydrogel, wherein the isolated vascular endothelial cells are taken from the lungs, the skin, the aorta vessels or the umbilical vessels of the mammals.
The invention also discloses a functional self-assembly nano-polypeptide preparation, which comprises the functional self-assembly nano-polypeptide, the dosage form of the functional self-assembly nano-polypeptide preparation comprises powder or liquid preparation, the functional self-assembly nano-polypeptide preparation further comprises pharmaceutically acceptable carriers and/or auxiliary materials, and the invention also discloses an application of the functional self-assembly nano-polypeptide preparation: preparing the hemostatic material.
Materials and methods
All synthetic peptides were from CS Bio (Shanghai) Ltd. All other chemicals used in this study were purchased from Sigma-Aldrich unless otherwise noted. Acetonitrile and water (HPLC grade) were purchased from Fisher Science Ltd. All products were used as received without further purification. All data are expressed as mean ± standard deviation (s.d.). Statistical significance analysis was performed using the t-test to determine the significance of the two individual data, with P <0.05 as significant.
Preparation and characterization of NBA
(RADA) 4-GGQLK (cross-linked peptide, CRP) and (RADA) 4-GSVLGYIQIR (Ca) were synthesized on appropriate resins using the HBTU/HOBt method using the HBTU activation/DIEA in situ neutralization protocol developed for FMOC-chemistry SPPS, using a CS bio 336X full-automatic polypeptide synthesizer 2+ Binding peptide, CBP). The crude product was found to contain 88% TFA, 5% phenol, 5%H 2 O and 2% of TIPS, cleaved, deprotected in cocktail reagent, precipitated with cold ether, and purified to homogeneity by preparative C18 reverse phase High Performance Liquid Chromatography (HPLC). The molecular weight was determined by electrospray ionization mass spectrometry (ESI-MS).
For preparing hydrogel, the (RADA) 4-GGQLK cross-linking peptide and (RADA) 4-GSVLGYIQIR binding peptide were combined with 0.1mg/mL CaCl 2 In PBS solution (3): 1 mass ratio. During the crosslinking, the hydrogels were soaked in FXIIIa (Solarbio, G8661 in PBS) at 37 ℃ to prepare concentrations of 0-1.25%, 2.5%, 5%, 7.5% and 15% (w/v), respectively, to explore a suitable concentration. The absorbance of the hydrogel at 600nm was measured with a microplate reader (Epoch, biotek).
To study the interaction of NBA with blood, NBA was added to whole blood and left at 37 ℃ for 5min. The whole blood was centrifuged at 120g for 10min to obtain Platelet Rich Plasma (PRP). Then, PRP was dropped on NBA, left at 37 ℃ for 1h, and finally washed 3 times with PBS to remove physically attached blood cells and platelets. These samples were fixed with 2.5% glutaraldehyde in PBS at 4 ℃ for 3h, and then dehydrated twice in succession with 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol for further morphological observation, as shown in FIG. 1.
CRP and CBP are synthesized by FMOC chemical solid phase peptide synthesis (SPSS), purified by preparative High Performance Liquid Chromatography (HPLC), and collected after freeze-drying. As shown in fig. 2B and 2C, two polypeptides were successfully synthesized with correct molecular weight and purity over 95% by liquid chromatography-mass spectrometry (LC-MS) analysis. Notably, the yields of CRP and CBP were both greater than 75%, promoting their potential for use.
The microstructure of the hydrogel was observed using a Hitachi S-4800 scanning electron microscope. The lyophilized samples were further observed by spraying an 8nm platinum layer on the dried hydrogel scaffolds. And the elemental (Ca) profile was recorded. The microstructure of the hydrogel was observed by transmission electron microscopy (Tecnai G2 SPIRIT, FEI). Method for preparing transmission electron microscope sample, 10 μ L of polypeptide solution is dropped on copper net coated with carbon film, and dyed with 2% phosphotungstic acid solution.
To investigate the biological function of NBA, scanning Electron Microscopy (SEM) was used to observe the ultrastructural changes of NBA after incubation of FXIIa with fibrinogen and blood. As shown in fig. 2A, fibrinogen further increased the compaction of NBA, probably due to FXIIIa catalyzed co-crosslinking between fibrinogen and NBA. In addition, increased compactability was also observed after incubation of NBA with FXIIIa and blood (fig. 2A). These results indicate that NBA has great potential in weaving a tight physical barrier in situ at the hemorrhage site complexed by polypeptide and fibrin.
As shown in the transmission electron microscope image, the dissolution of CRP and CBP in PBS buffer (ph 7.4) triggered the first stage self-assembly into sparse-wired nanofibers (fig. 1D). Furthermore, as designed, CBP has the function of binding calcium ions, which is the driving force for secondary self-assembly.
Isothermal titration experiments were performed using a PEAQ-ITC microcalorimeter (MicroCal) to examine the interaction of the hydrogel with calcium ions. The sample was dissolved in deionized water. The syringe was filled with 200. Mu.l of calcium chloride (100. Mu.M) and the cells were loaded with 260. Mu.l of (RADA) 4-GSVLGYIQIR (10. Mu.M). The titration protocol included 19 injections of 2. Mu.l, separated by 90 seconds.
Isothermal titration calorimetry showed CBP and Ca 2+ The binding affinity of (2.14. Mu.M) and the number of binding sites was 0.356 (FIG. 1E), indicating that one CBP can bind to 3 Ca 2+ And (4) effectively combining. As a result, this feature induces cross-linking between nanofibers, organized into a network called NBA (fig. 1F and G). To optimize this network structure, the ratio of CBP and CRP was screened. As shown in the SEM image in fig. 5, the optimal ratio is 3:1 (CBP: CRP) is most favorable for the conformation of this mesh network (FIG. 5). Notably, the overlap of TEM images with in situ Ca elemental analysis further supports this result, where Ca can be found at the intersection of nanofibers (fig. 6). In addition, further cross-linking between the QQLK motifs in CRP will drive the third stage of self-assembly. As expected, FXIIIa could catalyze this reaction and weave the mesh network into more compact nanofiber tangles (fig. 1H and I). Furthermore, gel analysis again demonstrated the self-assembly of this tertiary polypeptide in the presence of Ca 2+ And FXIIIa, the absorbance at 600nm corresponding to the most dense gel is highest, in sharp contrast to the lowest absorbance of the polypeptide only condition (FIGS. 1J and K). Notably, the 5% peptide density is a critical concentration at which NBA remains mobile before FXIIIa catalysis and is completely transferred to the solid gel after catalysis (fig. 1K). Thus, this concentration is used for subsequent functional tests.
Whole blood coagulation assay
A50. Mu.L volume (5%w/v) of hydrogel was placed in a flat bottom petri dish. Then, 50. Mu.L of calcium-containing whole blood solution (0.2M calcium chloride, 20mM in blood) was slowly dropped on the surface of the hydrogel after preheating at 37 ℃. The plates containing the blood samples were incubated at 37 ℃ for 0s, 30s, 60s, 90s, 120s, 150s, 180s and 210s, respectively. At the corresponding time point, 5ml of deionized water was gently added to release free blood without disturbing the clot. An equivalent amount (2.5 mg) of hemostatic powder (Fu Hetai), oxidized regenerated cellulose (Easy Peel), gelatin sponge (Xiang En) was treated and analyzed in the same manner as the gel. The absorbance of the supernatant was measured at 542nm using a microplate reader (Epoch, biotek). 4 replicates were performed. The coagulation index (BCI) of the material was quantified as follows:
wherein Ir represents the absorbance of the sample, ir represents the absorbance of 50. Mu.L of calcium-containing whole blood in 5mL of deionized water, and Ic represents the absorbance of deionized water.
The in vitro clotting properties of NBA were evaluated by the dynamic whole blood clotting assay. Is prepared from commercially available gelatin hemostatic sponge (gelatin sponge), chitosan hemostatic powder (chitosan) and hemostatic fiber yarn (FIBRILAR) TM ) Is a positive control. In this test, a lower transmission of the hemoglobin solution indicates a higher coagulation rate. As shown in FIG. 2B, NBA and FIBRILAR TM Clarification appeared after 60 seconds of incubation with blood, indicating that they had very good clotting properties. The absorbance of each group of blood solutions at a wavelength of 600nm was measured and the semi-clotting time (BCI) was fitted with a logarithmic equation 50 ) To quantify the clotting ability. NBA, gelfoam, chitosan and FIBRILAR as shown in FIG. 2C TM Can obviously shorten the semi-coagulation time of blood. Importantly, BCI of NBA 50 The shortest value, amazing 2.0s, and FIBRILAR TM Having sub-level coagulation ability, BCI 50 21.7s, therefore FIBRILLAR was chosen TM As a positive control for in vivo testing.
Hemostasis for hepatic hemorrhage model
The experimental procedure was approved by the fourth department of medical ethics committee of the university of military medical science. SD rats (male,. About.200 g) were randomly grouped. Isofluoroether (R510-22, VETEASY) was used for anesthesia at an induction level of 3% and a maintenance level of 2%. The median incision of the abdomen was followed, and the left lobe of the liver was revealed. Three liver injury models are established in the invention. In the first model, a partial (approximately 1/3) left lobe (puncture) was excised. In the second model, two perpendicular wounds, 3cm long and 5mm deep, were made on exposed livers (puncture wounds). In the third model, a near full-thickness wound of 4mm in diameter was made with biopsy punch (a bleeding wound that could not be sutured). The experimental group was applied to the wound surface 10s after injury with a 5% (w/v) hydrogel or oxidized regenerated cellulose hemostat. The wounds of the control group were not treated. Bleeding time and amount were recorded separately. After surgery, rats were sutured and placed independently. The rats without any treatment were blank. All animals receiving human care and study were in compliance with institutional animal care and use committee guidelines.
The hemostatic properties of NBA were further evaluated using a rat model of severe liver injury, with a 1cm long, 0.5cm deep cross sagittal incision simulating a severe wound that could not be sutured. As shown in FIG. 2D, severe liver damage resulting in rapid massive hemorrhage was achieved by 5% NBA or adjuvant FIBRILAR 15s after hemorrhage TM And (6) treating. Unexpectedly, in the NBA-treated liver, the hydrogel rapidly conformed to the lesion and immediately gelled in situ. More importantly, NBA hemostasis, FIBRILAR, occurs in less than 50 seconds TM The hemostasis time of (a) was 120 seconds, whereas that of the PBS control group exceeded 220 seconds (fig. 2E). In addition, with FIBRILAR TM NBA statistically significantly reduced blood loss compared to the PBS treatment group (Ctrl) (fig. 2F). These results indicate that NBA has good hemostatic capabilities in vivo.
Transcriptome sequencing
Liver tissues of the puncture wound experimental group, the control group and the blank group were collected 3 days after the molding and the treatment, respectively. Tissue RNA was extracted using TRIzol (Invitgen) as required by the instructions. RNA-Seq library set
Ultra TM RNA Library Prep Kit for
(E7530L, new England Biolabs) constructionAnd sequenced on the NovaSeq6000 platform (2X 150 bp).
Due to the rapid hemostatic action and good biocompatibility of NBA, it is speculated that NBA can effectively alleviate inflammatory reactions around the wound, which is very beneficial for the healing of the wound. To confirm this, 1cm was isolated from the injured liver after 3d of hemostatic treatment 3 The wound surface tissue of (1). RNA sequencing analysis showed that NBA elicited a 920 differential changes in gene expression compared to the PBS control group (fig. 3A), and a consistent and reproducible abundance of inflammatory regulatory gene expression signatures could be found by Gene Set Enrichment Analysis (GSEA) (fig. 3B and C). Specifically, NBA caused a significant down-regulation of the inflammatory response pathway (fig. 3B) and the IL6-JAK-STAT3 signaling pathway (fig. 3C). In sharp contrast, the FIBRILLAR is used TM Comparison with PBS control, although only 162 differentially expressed genes were found (FIG. 3D), FIBRILLAR TM The inflammatory response (FIG. 3E) and the IL6-JAK-STAT3 signaling pathway (FIG. 3F) were statistically significantly upregulated, possibly due to its limited biocompatibility. To further validate these results, enzyme-linked immunosorbent assays were performed on 4 inflammatory factors in wound tissue: the quantitative determination of IL-6, MCP-1, NF-kB and TNF alpha is carried out, and the result supports that NBA can effectively reduce the inflammatory reaction of wound tissues. The results were that NBA treatment promoted wound healing, which was confirmed by wound liver pathology sections at day 14 post-treatment (fig. 3H), which were further supported by normalized liver function indices (fig. 4D) and full blood counts (fig. 4D). In addition, no pathological changes were observed in the heart, spleen, lung, and kidney of the mice after treatment with NBA (fig. 8 and 9), supporting the safety of NBA. Overall, NBA is excellent in hemostatic therapy and anti-inflammatory, showing great potential as a rapid, efficient and safe liver hemostatic material.
Blood routine examination and blood biochemistry
To assess regeneration of the liver, tissue specimens were collected 2 weeks after molding and treatment and fixed with 4% paraformaldehyde. Paraffin-embedded tissue sections were 40- μm thick and stained with hematoxylin-eosin. All sections were taken by microscope (IX 53, olympus, japan).
Blood routine and blood biochemical indices were collected from the inferior vena cava of rats in experimental groups (scratch and puncture), control group and blank group 2 weeks after molding and treatment, respectively. To separate the serum, the whole blood sample was centrifuged at 6000g for 10min. The blood routine examination was performed using an automatic blood cell analyzer (BC-2800VET, mindray). The determination of the biochemical parameters of the blood was carried out using the corresponding kit (Kayto) according to the instructions of a fully automatic biochemical analyzer (Chemray 800).
Prior to functional testing, the in vitro acute toxicity of NBA was first investigated by testing its activity after incubation with hepatocytes (AML 12 cells) and vascular endothelial cells (HUVEC cells). As shown in fig. 7, no cytotoxicity was found during the 7 day incubation, indicating that NBA is safe and biocompatible.
To further challenge the hemostatic capability, a rat liver defect lethal suturable hemorrhage model and a penetrating wound non-compressible hemorrhage model are established. As shown in FIG. 4A, more than 1/3 of the liver was excised and more than half of the whole blood would be lost after injury (FIGS. 4B and 4C). 15s after hemorrhage, pressurizing the wound surface in control group, and treating the other two groups with NBA or FIBRILAR TM And (5) treating the wound surface. Without leaving the material, it is combined with compressing the wound surface group or FIBRILLAR TM Compared with the treatment group, NBA significantly shortens the hemostasis time. At the same time, NBA treatment reduced bleeding volume by half compared to compression (fig. 4C). Moreover, NBA also promoted wound healing and liver function recovery (fig. 4D and 4E), while maintaining good safety (fig. 9).
The small and deep penetrating wounds caused by small calibre weapons and improvised explosive devices are incompressible and conventional hemostatic agents are also difficult to stop bleeding. As shown in FIG. 4F, a 50mm diameter penetrating wound resulted in severe bleeding, but FIBRILAR TM There was no hemostatic effect on major bleeding (fig. 4G and H). In sharp contrast, NBA passed rapidly through the wound and immediately gelled in situ to stop bleeding (supplement video clip and fig. 4F). As a result, NBA treatment reduced bleeding time by half (fig. 4G) and bleeding volume by half (fig. 4H). Furthermore, a significant drop in White Blood Cells (WBCs) and neutrophils 3 days after treatment indicates a reduction in inflammatory response following NBA treatment (fig. 4I). In addition, NBA also promoted wound healing (fig. 4J), eachInflammatory reaction was not observed in the organs (FIG. 8).
Enzyme linked immunosorbent assay
After the model is made and 3 days after the treatment, serum samples are respectively collected from an experimental group (the bleeding wound surface is not sutured), a control group and a blank group, and enzyme-linked immunosorbent assay is carried out. ELISA assays for TNF α, NF-KB, IL-6, MCP-1, TGF α, EGF and HGF were performed according to the manufacturer's instructions.
Cell survival rate
AML12 and HUVEC cell lines were purchased from Chinese Shanghai Zhongji cell banks at 37 ℃ under CO 2 Culturing in an incubator with a concentration of 5% at a rate of 1000 cells per well, changing to 0.075% NBA, 0.05% NBA, 0.025% NBA, 0.001mg/mLCaCl 2 (corresponding medium) and fresh medium. And the corresponding culture medium is changed every day for 7 days. Cell viability was measured by the CCK8 (Dojindo) method at 0, 1, 2, 3, 5, and 7d, respectively.
The invention quickly and effectively stanchs through a three-stage self-assembly strategy of two functionalized polypeptides. In the first two steps of self-assembly, CRP and CBP are assembled into nanofibers first, then in Ca 2+ Are woven into a mesh network. This NBA is a viscous liquid before it encounters blood, and thus facilitates coverage and conformance of perforated and large wounds. Eventually, the wound will activate factor XIIIA (FXIIIa) to catalyze NBA to self-assemble in the third phase, forming a dense physical barrier to effectively stop bleeding. Without the material, NBA can rapidly prevent rat liver scratch bleeding, and simultaneously effectively reduce inflammation around the wound surface and promote wound surface healing. More importantly, NBA has good hemostatic effect on hemorrhage which can not be sutured in 1/3 liver defect of rat and non-compressible hemorrhage of penetrating wound, and simultaneously maintains good safety. The bionic three-stage self-assembly strategy provides a polypeptide treatment method with clinical potential for fatal liver bleeding, and the research and development of the self-assembly peptide hydrogel hemostatic are repeated, so that the bionic three-stage self-assembly strategy has wide potential application prospects.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (8)
1. A functional self-assembled nano-polypeptide hydrogel is characterized by comprising Ca 2+ Conjugated binding peptides and cross-linking peptides responsive to coagulation factor 13, wherein the molar ratio of the binding peptides to the cross-linking peptides is 3:1; the sequence of the cross-linking peptide is: (RADA) 4 -GGQQLK, the sequence of the binding peptide being: (RADA) 4 -GSVLGYIQIR。
2. A method of making the functional self-assembling nano-polypeptide hydrogel of claim 1, the method comprising the steps of: (RADA) 4-GGQLK cross-linking peptide and (RADA) 4-GSVLGYIQIR binding peptide were combined as 3:1 mass ratio, dissolving and mixing to obtain a polypeptide mixed solution, carrying out ultrasonic treatment to completely dissolve and uniformly mix the polypeptide, and inducing the polypeptide mixed solution to self-assemble at room temperature to form hydrogel.
3. Use of the functional self-assembled nano-polypeptide hydrogel of claim 1 in the preparation of tissue engineering materials.
4. Use according to claim 3, characterized in that the tissue engineering material is an artificial vascular tissue material.
5. An artificial blood vessel obtained by three-dimensionally culturing ex-vivo blood vessel endothelial cells derived from a lung, skin, aorta or umbilical vessel of a mammal in the functional self-assembled nano-polypeptide hydrogel according to claim 1.
6. A functional self-assembling nano-polypeptide preparation, wherein the functional self-assembling nano-polypeptide preparation comprises the functional self-assembling nano-polypeptide of claim 1;
the functional self-assembly nano polypeptide is as follows: comprises with Ca 2+ Conjugated binding peptides and cross-linking peptides responsive to coagulation factor 13, wherein the molar ratio of the binding peptides to the cross-linking peptides is 3:1; the sequence of the cross-linking peptide is: (RADA) 4 -GGQQLK, the sequence of the binding peptide being: (RADA) 4 -GSVLGYIQIR。
7. The functional self-assembling nano-polypeptide preparation according to claim 6, wherein the dosage form of the functional self-assembling nano-polypeptide preparation comprises powder or liquid preparation, and the functional self-assembling nano-polypeptide preparation further comprises pharmaceutically acceptable carriers and/or excipients.
8. Use of the functional self-assembling nano-polypeptide formulation of claim 1 or the functional self-assembling nano-polypeptide formulation of claim 6 or 7: preparing the hemostatic material.
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