Supramolecular bispecific self-assembly polypeptide for promoting endothelial cell repair and preparation method, application and medicine thereof
Through supramolecular bispecific self-assembly polypeptide BsSA targeting blood-brain barrier endothelial cells and activate the IGF-1R/IR signaling pathway, the problems of insufficient blood-brain barrier targeting and poor endothelial cell repair in the prior art are solved, and significant TBI treatment effect is achieved.
Patent Information
- Application Number
- CN202510070509.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-04-18
AI Technical Summary
In the treatment of traumatic brain injury (TBI), it is difficult to effectively target the blood-brain barrier and promote endothelial cell repair, resulting in inefficiency.
A supramolecular bispecific self-assembled polypeptide BsSA is used, which is obtained by self-assembly of SA-INS and SA-IGF polypeptides. By targeting the IGF-1R/IR of endothelial cells of blood-brain barrier, it activates downstream signaling pathways and promotes endothelial cell proliferation and survival.
BsSA significantly improves the blood-brain barrier targeting ability and bioavailability, significantly alleviates endothelial lesions and neuromemory defects after TBI, and prolongs survival. Its therapeutic effect is far better than that of IGF-1 protein.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to a supramolecular bispecific self-assembling polypeptide for promoting endothelial cell repair, a preparation method thereof, an application and a medicine thereof. Background Art
[0002] Traumatic brain injury (TBI) is characterized by high mortality and disability rates. Its prominent pathological feature is the primary damage of the blood-brain barrier (BBB), especially the cerebral microvessels, which are the main components of the BBB, are vulnerable to external damage. Therefore, drugs that can enhance the repair of damaged blood vessels may promote the recovery of traumatic brain injury. There are currently no TBI-related clinical trials specifically targeting angiogenesis. A variety of natural proteins have been shown to have therapeutic effects on TBI, but the current therapeutic proteins that can treat vascular injury after brain trauma have certain limitations, including short half-life, poor brain targeting ability, low bioavailability, high cost, and low immunogenicity. One of the key factors leading to poor efficacy is insufficient blood-brain barrier targeting and poor recovery of endothelial cell function. Strategies that can enhance targeting of the blood-brain barrier and protect endothelial cells are essential for improving the outcomes of TBI treatment. Summary of the invention
[0003] In order to solve the above problems, the present invention provides a supramolecular bispecific self-assembling polypeptide that promotes endothelial cell repair, and a preparation method, application and medicine thereof. The present invention proposes a new method to solve the challenges associated with blood-brain barrier targeting and promoting endothelial cell repair in TBI treatment. The present invention introduces a supramolecular bispecific self-assembling polypeptide (BsSA) that can promote endothelial cell repair, which uses the potential of targeted binding to IGF-1R / IR of blood-brain barrier endothelial cells to activate downstream signaling pathways to promote endothelial cell proliferation / survival, thereby effectively treating TBI.
[0004] In order to achieve the above object, the present invention provides the following technical solutions:
[0005] The present invention provides a supramolecular bispecific self-assembling polypeptide for promoting endothelial cell repair, which is obtained by self-assembly of a SA-INS polypeptide and a SA-IGF polypeptide;
[0006] The amino acid sequence of the SA-INS polypeptide is Nap-GFFY;
[0007] The amino acid sequence of the SA-IGF polypeptide is Nap-FFG-GYGSSSRRAPQT;
[0008] The configurations of amino acids in the SA-INS polypeptide and the SA-IGF polypeptide are both L configurations, and Nap is 2-naphthylacetic acid.
[0009] Preferably, the SA-INS polypeptide and SA-IGF polypeptide are both synthesized by Fmoc-solid phase synthesis.
[0010] Preferably, the Fmoc-solid phase synthesis method comprises the following steps:
[0011] (1) The C-terminus of the Fmoc-amino acid is bound to the resin;
[0012] (2) Removal and washing of Fmoc protecting group;
[0013] (3) the C-terminus of the next Fmoc-amino acid is coupled to the N-terminus of the amino acid or peptide on the resin and washed;
[0014] (4) Repeat steps (2) to (3), finally add 2-naphthyl acetic acid for coupling, and wash;
[0015] (5) removing the peptide derivative from the resin to obtain a crude product;
[0016] (6) Purify the crude product using high performance liquid chromatography.
[0017] The present invention also provides a method for preparing the supramolecular bispecific self-assembling polypeptide described in the above technical solution, comprising the following steps:
[0018] 1) dissolving the SA-INS polypeptide and the SA-IGF polypeptide in a solvent, adjusting the pH value to 7.4, to obtain a polypeptide solution;
[0019] 2) heat-treating and cooling the polypeptide solution obtained in step 1) to obtain a supramolecular bispecific self-assembling polypeptide.
[0020] Preferably, the concentrations of the SA-INS polypeptide and the SA-IGF polypeptide in the polypeptide solution in step 1) are both 3 mM.
[0021] Preferably, the solvent in step 1) is physiological saline; a 1M sodium carbonate solution is used to adjust the pH value;
[0022] The conditions of the heat treatment in step 2) include: heating to boiling and then cooling; the cooling is natural cooling.
[0023] The present invention also provides the use of the supramolecular bispecific self-assembling polypeptide described in the above technical solution in the preparation of a drug for treating traumatic brain injury.
[0024] The present invention also provides the use of the supramolecular bispecific self-assembling polypeptide described in the above technical solution in the preparation of a drug for promoting endothelial cell proliferation and migration.
[0025] The present invention also provides the use of the supramolecular bispecific self-assembling polypeptide described in the above technical solution in the preparation of a drug for promoting the repair of endothelial cell damage.
[0026] The present invention also provides a medicine for treating traumatic brain injury, which contains the supramolecular bispecific self-assembling polypeptide described in the above technical solution.
[0027] Beneficial effects:
[0028] BsSA was constructed using a simple co-assembly strategy, combining two self-assembling peptides: SA-INS, which targets and binds to the insulin receptor (IR) and SA-IGF, which targets the insulin-like growth factor 1 receptor (IGF1R). This unique combination enables BsSA to selectively bind to blood-brain barrier endothelial cells, promoting their proliferation and survival to treat TBI.
[0029] After administration through the tail vein, BsSA self-assembles and interacts with blood-brain barrier endothelial cells by binding to IGF1R / IR. BsSA has superior blood-brain barrier targeting ability and bioavailability. By activating IGF-1R / IR and downstream AKT / GSK3β / eNOS signaling pathways, it induces cell survival / proliferation to repair endothelial cells damaged by the blood-brain barrier and thus treats traumatic brain injury. Therefore, BsSA treatment can significantly alleviate endothelial lesions, neurological memory deficits and prolong survival after TBI. Its therapeutic effect is far superior to IGF-1 protein treatment, overcoming the limitations of insufficient targeting and poor therapeutic effect.
[0030] Beneficial properties and advantages: BsSA design offers several significant properties and advantages in the field of TBI treatment. First, it is able to bind IGF1R / IR to target blood-brain barrier endothelial cells and promote the proliferation / survival of damaged endothelial cells through the AKT / GSK3β / eNOS signaling pathway. This approach significantly improves the therapeutic effect of TBI by overcoming the limitations of insufficient targeting and poor tissue retention of natural proteins.
[0031] Secondly, BsSA demonstrated the transformation behavior of self-assembling peptides. After the single-molecule peptides self-assemble into nanostructures, their in vivo stability is greatly improved; the supramolecular structure formed by multiple peptides interacts with cell membrane receptors through multivalent binding and other effects, which is more conducive to improving the binding affinity with cells and exerting their effects. This transformation process helps the peptides target, enrich and retain damaged blood-brain barrier endothelial cells, further improving the therapeutic effect of TBI.
[0032] Conclusion: In conclusion, supramolecular bispecific self-assembling peptides BsSA that promote endothelial cell repair represent a major advance in the field of TBI treatment. By addressing the limitations of insufficient blood-brain barrier targeting of natural proteins and poor restoration of endothelial cell function, this new approach has the potential to significantly improve the treatment of TBI. The unique properties and advantages of BsSA represent a new strategy for self-assembling peptides to treat endothelial lesions induced by traumatic brain injury and provide valuable insights into other neurological diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0034] Figure 1 The structural formula and HPLC mass spectrum of the polypeptide of Example 1;
[0035] Figure 2 The structural formula and HPLC mass spectrum of the polypeptide of Example 2;
[0036] Figure 3 Optical images of solutions configured in Examples 1, 2, and 3;
[0037] Figure 4 The polypeptide microscopic morphology of the polypeptides of Examples 1, 2, and 3;
[0038] Figure 5 The secondary structure diagram of the polypeptides of Examples 1, 2, and 3 is shown in FIG.
[0039] Figure 6 This is a diagram showing that the polypeptide in Experimental Example 4 promotes endothelial cell proliferation and migration;
[0040] Figure 7 This is a diagram showing that the polypeptide promotes endothelial cell damage and repair in Experimental Example 5;
[0041] Figure 8 This is an immunoblot image of the protein expression related to the pathway of the mechanism by which the polypeptides of Examples 1, 2, and 3 in Experimental Example 6 promote endothelial cell proliferation / survival;
[0042] Fig. 9 In vivo imaging of the aggregation ability of the Cy5.5-labeled peptide in the brain of normal mice and TBI mice in Experimental Example 7
[0043] Fig.10 This is a statistical chart of the mortality rate of mice during treatment in Experimental Example 8;
[0044] Fig.11 This is a behavioral test diagram of mice during treatment in Experimental Example 8;
[0045] Fig.12This is a graph showing the blood perfusion of mice treated with TBI and polypeptides in Experimental Example 8;
[0046] Fig.13 These are MRI images of mice treated with TBI and polypeptides in Experimental Example 8. DETAILED DESCRIPTION
[0047] The present invention provides a supramolecular bispecific self-assembling polypeptide for promoting endothelial cell repair, which is obtained by self-assembly of a SA-INS polypeptide and a SA-IGF polypeptide; the amino acid sequence of the SA-INS polypeptide is Nap-GFFY; the amino acid sequence of the SA-IGF polypeptide is Nap-FFG-GYGSSSRRAPQT; the configurations of the amino acids in the SA-INS polypeptide and the SA-IGF polypeptide are both L configurations, and Nap is 2-naphthylacetic acid.
[0048] In the present invention, the SA-INS polypeptide and SA-IGF polypeptide are preferably synthesized by Fmoc-solid phase synthesis. In the present invention, the Fmoc-solid phase synthesis method preferably includes the following steps: (1) the C-terminus of the Fmoc-amino acid is bound to the resin; (2) the Fmoc protecting group is removed and washed; (3) the C-terminus of the next Fmoc-amino acid is coupled to the amino acid or the N-terminus of the polypeptide on the resin, and washed; (4) steps (2) to (3) are repeated, and finally 2-naphthaleneacetic acid is added for coupling and washed; (5) the polypeptide derivative is cut off from the resin to obtain a crude product; (6) the crude product is purified by high performance liquid chromatography. The present invention does not specifically limit other condition parameters of the Fmoc-solid phase synthesis method, and those skilled in the art can follow the routine.
[0049] The present invention also provides a method for preparing the supramolecular bispecific self-assembling polypeptide described in the above technical solution, comprising the following steps:
[0050] 1) dissolving the SA-INS polypeptide and the SA-IGF polypeptide in a solvent, adjusting the pH value to 7.4, to obtain a polypeptide solution;
[0051] 2) heat-treating and cooling the polypeptide solution obtained in step 1) to obtain a supramolecular bispecific self-assembling polypeptide.
[0052] In the present invention, the concentration of SA-INS polypeptide and SA-IGF polypeptide in the polypeptide solution is preferably 3 mM. In the present invention, the solvent is preferably physiological saline. In the present invention, a 1 M sodium carbonate solution is preferably used to adjust the pH value. In the present invention, the heat treatment conditions preferably include: heating to boiling and then cooling; the cooling is natural cooling.
[0053] The present invention also provides the use of the supramolecular bispecific self-assembling polypeptide described in the above technical solution in the preparation of a drug for treating traumatic brain injury.
[0054] The present invention also provides the use of the supramolecular bispecific self-assembling polypeptide described in the above technical solution in the preparation of a drug for promoting endothelial cell proliferation and migration.
[0055] The present invention also provides the use of the supramolecular bispecific self-assembling polypeptide described in the above technical solution in the preparation of a drug for promoting the repair of endothelial cell damage.
[0056] The present invention also provides a drug for treating traumatic brain injury, comprising the supramolecular bispecific self-assembling polypeptide of the above technical solution. In the present invention, the dosage form of the drug is preferably an injection, and the content of the supramolecular bispecific self-assembling polypeptide in the injection and the preparation method are not particularly limited in the present invention, and can be conventionally set by those skilled in the art.
[0057] In the embodiments of the present invention, the sources of the preparations involved are as follows:
[0058] 2-Cl-Trt resin: purchased from Jier Biochemical (Shanghai) Co., Ltd., with a substitution rate of 1.158 mmol / g.
[0059] Amino acids: purchased from Gill Biochemical (Shanghai) Co., Ltd., with a purity of 98%.
[0060] N,N-Diisopropylethylamine (DIEPA): purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a purity of 99%.
[0061] 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU): purchased from Tianjin Xiensi Biochemical Technology Co., Ltd. with a purity of 98%.
[0062] Trifluoroacetic acid (TFA): purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a purity of 99%.
[0063] Triisopropylsilane (TIS): purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a purity of 99%.
[0064] Anhydrous dichloromethane (DCM): purchased from Tianjin Chemical Reagent Company.
[0065] N,N-Dimethylformamide (DMF): purchased from Tianjin Chemical Reagent Company.
[0066] Chromatographic grade methanol was purchased from Tianjin Concord Technology Co., Ltd.
[0067] 2-Naphthylacetic acid: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0068] Hoechst 33342 stain: purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.
[0069] EDU staining kit: purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.
[0070] DCFH-DA: provided by Sigma-Aldrich.
[0071] Cy5.5: provided by Thermofisher Scientific.
[0072] Piperidine: purchased from Tianjin Chemical Reagent Company.
[0073] Cell culture medium (EBM2): provided by LONZA, Switzerland.
[0074] Fetal bovine serum: provided by Thermofisher Scientific, sterile.
[0075] C57 / BL6 mice: 8 weeks old, male, purchased from Weitonglihua Biotechnology Co., Ltd.
[0076] The devices involved in the embodiments of the present invention are as follows:
[0077] High performance liquid chromatography (HPLC): manufactured by Lumtech, Germany.
[0078] High performance liquid chromatography-mass spectrometry: manufactured by Shimadzu, Japan, model LC-MS2020.
[0079] Electronic balance: manufactured by Sarturious, Germany, model BS124S.
[0080] Transmission electron microscope (TEM): Tecnai G2 F20 system.
[0081] Freeze dryer: manufactured by Beijing Yatai Kelong, model LGJ-1-50.
[0082] Laser confocal microscope: manufactured by Leica, Germany, model is TCS SP8.
[0083] Circular dichroism spectrometer: manufactured by BioLogic, France, model MOS-450.
[0084] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0085] Examples 1 and 2
[0086] The synthesis of the polypeptide Nap-GFFY (SA-INS) of Example 1 and the polypeptide Nap-FFG-GYGSSSRRAPQT (SA-IGF) of Example 2 is as follows:
[0087] The peptide was synthesized by the classic Fmoc-short peptide solid phase synthesis method. The obtained polypeptide is referred to as SA-INS or SA-IGF. The specific steps are as follows:
[0088] Place 0.5 mmol of 2-Cl-Trt resin in a solid phase synthesis tube, add 15 mL of DCM, and shake on a shaker for 10 minutes to swell the 2-Cl-Trt resin;
[0089] Use an ear bulb to squeeze DCM out of the solid phase synthesizer containing 2-Cl-Trt resin;
[0090] Dissolve 0.75 mmol of Fmoc-protected amino acid in 10 mL of DCM, add 1.5 mmol of DIEPA, mix well, add to the solid phase synthesizer, and react at room temperature for 1 hour;
[0091] Carry out the blocking step: first remove the reaction solution in the solid phase synthesis tube, then wash with 10 mL DCM, wash for 1 minute each time, wash 3 times in total, then add 16 mL of a solution of DCM: DIEPA: methanol in a ratio of 17:1:2, and react at room temperature for 15 minutes;
[0092] Remove the reaction solution from the solid phase synthesis tube, wash with DCM, 10 mL each time, 1 minute, 3 times in total, then wash with DMF, 10 mL each time, 1 minute, 3 times in total. Add 15 mL of DMF containing 20% piperidine (volume ratio), react for 30 minutes, then wash with DMF, 10 mL each time, 1 minute, 5 times in total.
[0093] Weigh 1 mmol of the second Fmoc-protected amino acid, 1 mmol of HATU, and 2 mmol of DIEPA, dissolve them in 10 mL of DMF, add the dissolved amino acid solution into the solid phase synthesis tube, and react at room temperature for 1 hour.
[0094] Repeat steps 5) and 6) to add amino acids and end-capping group 2-Nap in sequence, then wash with DMF 5 times and dichloromethane 5 times to prepare for the next reaction.
[0095] Prepare 10 mL of cutting solution with a ratio of 95% TFA, 2.5% TIS, and 2.5% HO. 2O (volume ratio), added to a solid phase synthesizer, and reacted at room temperature for 1 hour. The product was cut from the 2-Cl-Trt resin, and the solvent was removed by a rotary evaporator to obtain a crude product. Subsequently, it was separated and purified by HPLC to obtain the polypeptides of Examples 1 and 2. After LC-MS detection, the results are shown in Figure 1 and Figure 2 The structural formula of the polypeptides in Examples 1 and 2 is as follows: Figure 1 and Figure 2 shown.
[0096] Experimental Example 1
[0097] Solution preparation
[0098] Configuration of Examples 1 (SA-INS) and 2 (SA-IGF):
[0099] 1) The lyophilized SA-INS and SA-IGF peptides were dissolved in physiological saline to a final concentration of 3 mM.
[0100] 2) Add 1M Na 2 CO 3 The final pH of the solution was adjusted to 7.4.
[0101] 3) The solution is heated to an appropriate temperature (heated to boiling) and then cooled to ensure that the peptide is fully dissolved.
[0102] 4) In subsequent experiments, the prepared peptide solution was appropriately diluted as needed to obtain the desired concentration.
[0103] Example 3
[0104] Configuration of (BsSA):
[0105] 1) The lyophilized SA-INS and SA-IGF peptides were dissolved in physiological saline to a final concentration of 3 mM.
[0106] 2) Add 1M Na 2 CO 3 The final pH of the solution was adjusted to 7.4.
[0107] 3) The solution is heated to an appropriate temperature (heated to boiling) and then cooled to ensure that the peptide is fully dissolved.
[0108] 4) In subsequent experiments, the prepared peptide solution was appropriately diluted as needed to obtain the desired concentration.
[0109] Experimental Example 2
[0110] Transmission electron microscopy experiment
[0111] According to the preparation method described in Experimental Example 1, solutions of Examples 1 (polypeptide concentration is 3mM), 2 (polypeptide concentration is 3mM), and 3 (polypeptide concentration is 3mM) were prepared, and electron microscopy samples were prepared. First, 10μL of polypeptide self-assembly solution was dripped onto the copper mesh, left for one minute, and then filter paper was used to remove excess solution from the edge. Next, 10μL of phosphotungstic acid was dripped onto the copper mesh and left for one minute for negative staining, and filter paper was used again to remove excess dye from the edge. The treated copper mesh was placed in a drying oven, and after it was completely dried, it was photographed using an electron transmission microscope. See the results. Figure 4 .
[0112] Figure 4 It was shown that Example 1 (SA-INS) and Example 2 (SA-IGF) formed nanofibers respectively. The co-assembled BsSA (Example 3) formed nanofibers with thicker diameters than Example 1 (SA-INS) and Example 2 (SA-IGF), and the occurrence of self-assembly was further confirmed by fluorescence resonance energy transfer experiments.
[0113] Experimental Example 3
[0114] Secondary structure determination by circular dichroism
[0115] For CD signal detection, the sample was diluted to a concentration of 50 μM according to the preparation method described in Experimental Example 1. Then, 200 μL of the diluted sample was added to a quartz cell with a diameter of 0.1 cm. The CD spectrum was recorded in the range of 190-260 nm using a circular dichroism spectrometer (MOS-450, BioLogic). Figure 5 shown.
[0116] SA-INS showed two characteristic peaks at 193 and 203 nm, respectively, tending to form a β-sheet conformation. SA-IGF showed two characteristic peaks at 189 and 207 nm, respectively, tending to form a β-sheet conformation. In addition, BsSA showed two characteristic peaks at 192 and 205 nm, respectively, indicating that it tends to form a β-sheet conformation.
[0117] Experimental Example 4
[0118] Study on the Proliferation and Migration Ability of BsSA on Endothelial Cells by EdU Staining and Scratch Test
[0119] EDU staining: 5-ethyl-2′-deoxyuridine (EdU) staining was used to detect the proliferation rate of HUVECs. Briefly, HUVECs were seeded in 12-well plates and cultured overnight. Then, SA-INS (10nM), SA-IGF (10nM), BsSA (10nM), or IGF1 protein (10nM) was added to EBM2 medium without R3-IGF-1 and incubated for 48h. Subsequently, EdU working solution was added to the culture medium and cultured at 37°C for 2h. Afterwards, the cells were fixed with 4% paraformaldehyde (PFA), the membrane was permeated with 0.3% TritonX-100, and then reacted with click buffer for 30 minutes. Hoechst nucleus staining. Confocal laser scanning microscopy (CLSM) imaging to evaluate EdU + Cells. Figure 6 As shown in A, it can be observed that the BsSA group in Example 3 has the best ability to promote endothelial cell proliferation compared with other groups.
[0120] Scratch test: Place a wound healing 2-well plug (ibidi, 81176) in a 24-well plate and seed HUVECs in the plug. After the cells were cultured overnight, remove the plug and wash the cells with PBS. SA-INS (10nM), SA-IGF (10nM), BsSA (10nM) and IGF1 protein (10nM) were added to EBM2 medium without R3-IGF-1 and incubated with HUVECs for 24h. The images at 0 and 24h were analyzed using ImageJ software. Figure 6 As shown in B, it can be observed that the BsSA group in Example 3 significantly promoted endothelial cell migration compared with the other groups.
[0121] Experimental Example 5
[0122] Antioxidative stress ability of BsSA
[0123] DCFH-DA probe was used to evaluate the level of ROS in endothelial cells. HUVECs were plated in 6-well plates and cultured, and pretreated with SA-INS (10 nM), SA-IGF (10 nM), BsSA (10 nM) and IGF1 (10 nM) for 24 h. DCFH-DA was added to the cells and incubated at 37 °C and 5% CO 2 Afterwards, the cells were washed three times with PBS. 2 O 2 After 2 h of treatment, flow cytometry was used to detect the ROS generation of HUVECs in each group. Figure 7 As shown, H 2 O 2 Treatments showed elevated ROS levels, and the ROS level of endothelial cells in the BsSA group of Example 3 was the lowest compared to the other treatments.
[0124] Experimental Example 6
[0125] Mechanistic pathways of BsSA promoting endothelial cell proliferation / survival
[0126] In order to explore the mechanism of BsSA promoting endothelial cell proliferation / survival in Example 3, the expression levels of related proteins were detected by Western blot. SA-INS (10nM), SA-IGF (10nM), BsSA (10nM) and IGF1 (10nM) were cultured in EBM2 without R3-IGF-1 for 48h. Cell proteins were extracted and WB was used to detect the levels of p-IGF1R / IR, p-AKT, p-eNOS and p-GSK3β. The results are shown in Figure 3. Figure 8 As shown, the above proteins in the endothelial cells of the BsSA group in Example 3 showed stronger expression levels than those in other groups.
[0127] Experimental Example 7
[0128] BsSA aggregation ability in the brain
[0129] Using a small animal in vivo imaging system, the ability of Examples 2 and 3 and IGF1 protein to aggregate in the brain was evaluated. Based on the dosage calculation of the cyanine dye Cy5.5-NHS, the polypeptide self-assembly solution of the example was prepared, with a dosage of 1 μM kg -1 The drug was administered by tail vein injection with a volume of 150 μL. The time after drug administration was marked as 0 hours (0h). In vivo imaging was performed at different time points (0h, 4h, 8h, 24h). Fig. 9 shown.
[0130] The accumulation of BsSA and SA-IGF in the brain of sham-operated mice reached a peak at 4 h after injection, and the fluorescence intensity of BsSA was approximately 1.8 times that of SA-IGF. At all observation time points (0, 4, 8, and 24 h), BsSA showed significantly greater brain accumulation compared with SA-IGF and IGF-1 ( Fig. 9 In TBI mice, BBB disruption resulted in significantly increased brain fluorescence accumulation of SA-IGF, BsSA, and IGF1 compared with sham-operated mice. The temporal distribution of BsSA and SA-IGF was consistent with that of sham-operated mice ( Fig. 9 In contrast, IGF1 reached its peak accumulation at 24 h after injection, probably due to its larger molecular weight, which may hinder its passage through the damaged blood-brain barrier.
[0131] Experimental Example 8
[0132] To evaluate the therapeutic effect of BsSA on TBI brain injury. To evaluate the survival rate of mice, to evaluate the memory ability of mice using the water maze test, to evaluate the improvement of brain blood flow in mice using the laser speckle test, and to evaluate the brain injury volume and edema degree of mice using MRI.
[0133] TBI model was established in mice. The mice underwent craniectomy one day before model establishment. Anesthesia was performed by intraperitoneal injection of tribromoethanol (0.2 mL / 10 g). Subsequently, the head of the mouse was fixed on a stereotaxic frame, and an incision was made in the midline of the scalp to expose the bottom layer of the skull. A dental drill was used to perform a craniotomy on the right parietal bone, located between the junction of the amniotic membrane and the medulla, and between the lateral skull and the sagittal suture. A needle cap was placed around the craniotomy site, and the bottom was fixed with reinforced glass ionomer adhesive. Sterile saline was injected into the central area and sealed with a sterile venous cap to prevent the dura mater from being exposed to the environment. After the mice were anesthetized and revived, a hydraulic impact device (MODEU01-B, Newsun, USA) was used to set the hydraulic impact pressure to 200-220 kPa for impact. The mice in the control group only underwent scalp incision and bone hole grinding, and no hydraulic impact was performed. The experiment was performed by tail vein injection. The mice were injected with peptide derivatives (1 μM kg -1 ) and PBS for 3 consecutive days, a total of 3 doses, and the survival of mice in each group was monitored. Fig.10 As shown, the survival rate of mice in the BsSA group was the highest compared with the mice in other groups.
[0134] The memory ability of mice was evaluated by water maze experiment. Ten mice were randomly selected from each group for water maze experiment. The positioning navigation experiment started on the 15th day after modeling and continued for 5 days. The mice were put into the water facing the pool wall in random order until the mice found and climbed onto the platform. The computer monitored and recorded the time (latency), total distance and swimming speed required for the mice from entering the water to climbing onto the platform. If the mouse did not find the platform within 90 seconds, it was towed to the platform and stayed for 30 seconds. At this time, the latency was recorded as 90 seconds. Statistical analysis was performed between the groups. The spatial exploration experiment was carried out on the second day after the positioning navigation experiment. The underwater platform was removed, and each group of mice swam freely in the water tank for 90 seconds. The time spent in the correct quadrant and the number of times the platform position was crossed were calculated. In addition, the swimming speed was recorded to evaluate motor skills. The data of the spatial learning and memory test were recorded and analyzed by a video tracking system (EthoVision XT 16). The results are shown in Figure 2. Fig.11 As shown, mice in the BsSA group showed stronger memory ability.
[0135] Laser speckle experiment was used to evaluate the improvement of brain blood flow in mice. After the mice were anesthetized, a straight incision was made in the middle of the top of the head, extending forward to the nose and then to the neck to fully expose the parietal bone. The mice were fixed in a prone position on a Rayward mouse stereotaxic instrument. After removing the periosteum with a cotton swab, hair and other interfering impurities were removed under the CCD camera to obtain satisfactory detection results. A thin layer of sterile glycerin was applied on the top of the head to avoid changes in the detection value caused by evaporation of water on the skull surface. The mice were placed under a HR laser speckle blood flow dynamic imager to observe the cerebral blood flow. The experiment was conducted at room temperature, and the body temperature of the mice was kept constant at about 37 degrees during the experiment. The basic indicators of the HR laser speckle blood flow dynamic imager were as follows: the monitoring distance between the cortex of the C57 mouse head and the CCD camera was set to 10 cm; the indicator laser parameter was 660 nm, the maximum output power was 1 mW, and the mouse head was evenly irradiated at 90 degrees above the mouse skull; the monitoring laser parameter was 785 nm, the maximum output power was 1 mW, and the CCD camera of the microscope collected the data, with a resolution better than 7.5 um / pixel; the average monitoring area was 2.1×2.8 cm; the area of the region of interest was set at 3-5 mm 2 The sampling density is medium density; the image acquisition speed is 5 images / second; only one frame of color photo is taken at the beginning. The data collected by the CCD camera is used to calculate the brain perfusion (perfusion unit, PU) through the computer's internal dedicated software, and finally converted into a blood flow map to display the perfusion amount. The results are as follows Fig.12 As shown, BsSA treatment significantly improved brain blood flow in brain-injured mice.
[0136] MRI was used to evaluate the brain injury volume and edema of mice. MRI scans were performed using a 9.4-TBrukerBioSpec 94 / 30USR (Brucke, Karlsruhe, Germany) 3 days after FPI. The acquired T2, T2*map, and contrast-enhanced T1 (CE-T1) images were analyzed independently by two trained neurologists. T2 images were acquired using a fast spin echo pulse sequence with the following parameters: TR = 2802.4 msec, TE = 33 msec, resolution = 78 × 78 μm 2 , the slice thickness was 0.5 mm. In this study, a multi-echo gradient echo pulse sequence was used to obtain T2*map, and the parameters were as follows: TR = 947.4 msec; TE = 3.5, 8.5, 13.5, 18.5, 23.5, 28.5, 33.5, 38.5 msec; FA = 50°; matrix size = 256 × 256; FOV = 20 × 20 mm 2 ; resolution = 78 × 78 μm 2; Slice thickness was 0.5 mm. Five minutes after intraperitoneal injection of 46.9 mg of gadopentetate dimeglumine (Magnevist; Bayer, Guangzhou, China), CE-T1 examination was performed to detect contrast enhancement of brain lesions caused by blood-brain barrier (BBB) disruption: TR = 200 msec, TE = 3 msec, resolution = 78 × 78 μm 2 The slice thickness was 0.5 mm. The lesion volume was assessed 3 days after injury in T2 and CE-T1 scans and delineated along the borders of the high-intensity signal areas. The measurements were made using 3D Slicer (version 5.6.2). Fig.13 As shown, BsSA treatment significantly reduced brain lesion volume, significantly improved blood-brain barrier leakage, and alleviated brain edema in TBI mice compared with other treatments.
[0137] The present invention proposes an innovative method to treat TBI-induced endothelial lesions by targeting the blood-brain barrier and protecting endothelial cells using supramolecular bispecific self-assembling peptide BsSA that can promote endothelial cell repair. We found that BsSA has superior blood-brain barrier targeting ability and bioavailability. By activating the IGF-1R / IR and downstream AKT / GSK3β / eNOS signaling pathways, endothelial cell survival / proliferation is induced to repair endothelial cells damaged by the blood-brain barrier, thereby treating traumatic brain injury. Therefore, BsSA treatment can significantly alleviate endothelial lesions, neurological memory deficits and prolong survival after TBI, and its therapeutic effect is far superior to IGF-1 protein treatment. This peptide is expected to become an important breakthrough in the field of TBI treatment, significantly improve patient prognosis, and provide new ideas for the treatment of neurological injury-like diseases. Self-assembling peptides represent a new strategy for treating endothelial lesions induced by traumatic brain injury and provide valuable insights into other neurological diseases.
[0138] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A supramolecular bispecific self-assembling polypeptide for promoting endothelial cell repair, characterized in that: It is obtained by self-assembly of SA-INS polypeptide and SA-IGF polypeptide; The amino acid sequence of the SA-INS polypeptide is Nap-GFFY; The amino acid sequence of the SA-IGF polypeptide is Nap-FFG-GYGSSSRRAPQT; The configurations of amino acids in the SA-INS polypeptide and the SA-IGF polypeptide are both L configurations, and Nap is 2-naphthylacetic acid.
2. The supramolecular bispecific self-assembling polypeptide according to claim 1, characterized in that: The SA-INS polypeptide and SA-IGF polypeptide are both synthesized by Fmoc-solid phase synthesis method.
3. The supramolecular bispecific self-assembling polypeptide according to claim 2, characterized in that: The Fmoc-solid phase synthesis method comprises the following steps: (1) The C-terminus of the Fmoc-amino acid is bound to the resin; (2) Removal and washing of Fmoc protecting group; (3) the C-terminus of the next Fmoc-amino acid is coupled to the N-terminus of the amino acid or peptide on the resin and washed; (4) Repeat steps (2) to (3), finally add 2-naphthyl acetic acid for coupling, and wash; (5) removing the peptide derivative from the resin to obtain a crude product; (6) Purify the crude product using high performance liquid chromatography.
4. The method for preparing the supramolecular bispecific self-assembling polypeptide according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) dissolving the SA-INS polypeptide and the SA-IGF polypeptide in a solvent, adjusting the pH value to 7.4, to obtain a polypeptide solution; 2) heat-treating and cooling the polypeptide solution obtained in step 1) to obtain a supramolecular bispecific self-assembling polypeptide.
5. The preparation method according to claim 4, characterized in that: In the step 1), the concentrations of the SA-INS polypeptide and the SA-IGF polypeptide in the polypeptide solution are both 3 mM.
6. The preparation method according to claim 4, characterized in that: The solvent in step 1) is physiological saline; a 1M sodium carbonate solution is used to adjust the pH value; The conditions of the heat treatment in step 2) include: heating to boiling and then cooling; the cooling is natural cooling.
7. Use of the supramolecular bispecific self-assembling polypeptide according to any one of claims 1 to 3 in the preparation of a drug for treating traumatic brain injury.
8. Use of the supramolecular bispecific self-assembling polypeptide according to any one of claims 1 to 3 in the preparation of a drug for promoting endothelial cell proliferation and migration.
9. Use of the supramolecular bispecific self-assembling polypeptide according to any one of claims 1 to 3 in the preparation of a drug for promoting the repair of endothelial cell damage.
10. A drug for treating traumatic brain injury, characterized in that: A supramolecular bispecific self-assembling polypeptide comprising any one of claims 1 to 3.