Patent ID: 12187770

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention will be further described below in conjunction with embodiments. The description of the following embodiments is only used to help understand the specific implementation of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made to the present invention without departing from the essence of the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention, and the protection scope of the present invention is subject to the claims.

Synthesis Example 1

CKIPKASSVPTELSAISMLYLGPGGDWIVAC (SEQ ID NO:1) Solid-Phase Polypeptide Synthesis

The specific steps were as follows:1. Resin swelling0.8 g of 2-Chlorotrityl Chloride Resin with a substitution degree of 0.35 mmol/g was weighed and placed into a reaction tube with addition of DCM (concentration: 15 ml/g) under shaking for 30 min.2. Linking a first amino acidSolvent DCM was removed through a sand core suction filter, with addition of 3-fold excess of Fmoc-Cys(Trt)-OH amino acid in molar, and then 8-fold excess of DIEA (N,N-Diisopropylethylamine) in molar were added, followed by adding a small amount of DMF to dissolve under shaking for 1 h finally. They were washed alternately with DMF and DCM for 6 times3. Deprotectiona 15 ml solution of 20% piperidine in DMF (15 ml/g) was added for 5 min, which was removed followed by adding a 15 ml solution of 20% piperidine in DMF (15 ml/g) for 15 min.4. testingThe piperidine solution was removed by suction filtration to obtain the resin, from which dozens of resins were took and washed with ethanol for 3 times, with addition of one drops of ninhydrin, KCN, and phenol solutions separately, followed by heating at 105° C.-110° C. for 5 min, and color change to dark blue indicated a positive reaction.5. WashingWashing twice with DMF (10 ml/g), twice with methanol (10 ml/g), and twice with DMF (10 ml/g).6. CondensationThree-fold excess of a protected amino acid (Fmoc-Leu-OH) and three-fold excess of HBTU, both dissolved in a small amount of DMF, were added into a reaction tube, followed by adding ten-fold excess of DIEA to react for 40 min.7. WashingWashing once with DMF (10 ml/g), twice with methanol (10 ml/g), and twice with DMF (10 ml/g);8. Steps 2 to 7 were repeated to link amino acids in the sequence from right to left.9. testingWashing twice with DMF (10 ml/g), twice with DCM (10 ml/g), twice with DMF (10 ml/g), followed by draining for 10 min. Ninhydrin test was negative.10. WashingWashing 3 times with methanol (10 ml/g).11. Polypeptide cleavage from resinPreparing a cleavage solution (10/g): 94.5% TFA, 2.5% water, 2.5% EDT, and 1% TISResin was placed into a flask or a centrifuge tube based on a ratio of the resin and the cleavage solution at 10 ml/g under constant temperature shaking for 120 min.12. Blow drying and washingThe cleavage solution was blow dried with nitrogen as much as possible, and then was subjected to chromatography with ether, followed by washing with ether for six times, and then drying by volatilization at room temperature. The crude peptide sequence was obtained.13. Oxidative cyclization10 mg of the crude produce were cyclized by adding 100 ml of 5% DMF solution to oxidize sulfhydryl groups.14. Purifying the polypeptide by HPLC, with the specific steps of:1) 200 mg of crude peptides were placed into a container. They were dissolved with 2-5 ml of 50% acetonitrile in water. They could be sonicated slightly for 2 min.2) The dissolved solution was filtered with a 0.45 μm filter membrane.3) Analysis: 3 μl of the crude product was analyzed by analytical HPLC. The mobile phase was water and acetonitrile conducting a gradient elution for 30 min, and equilibrating HPLC firstly at an initial gradient of 95% water and 5% acetonitrile to a terminal ratio of 5% water and 95% acetonitrile for 5 min, and followed by injection.4) Preparation: the dissolved sample was made ready for injection. Preparative HPLC was equilibrated for 10 min at an initial gradient of 95% water and 5% acetonitrile to a terminal gradient of 25% water and 75% acetonitrile for 40 min. The sample from the detector was collected.5) identification: taking the collected sample for purification and MS identification.15. Finally, the purified solution was lyophilized to obtain the finished product.16. The powdered peptide was in a sealed package and stored at −20° C.

Synthesis Example 2

CKIPKASSVPTELSAISMLYLGPGGDWIVAC (SEQ ID NO:1) Solid-Phase Polypeptide Synthesis

The specific steps were as follows:1. Resin swelling0.9 g of 2-Chlorotrityl Chloride Resin with a substitution degree of 0.3 mmol/g was weighed and placed into a reaction tube with addition of DCM (concentration: 18 ml/g) under shaking for 30 min.2. Linking a first amino acidSolvent was removed through a sand core suction filter, with addition of 3-fold excess of Fmoc-Cys(Trt)-OH amino acid in molar, and then 10-fold excess of DIEA in molar were added, followed by adding a small amount of DMF to dissolve under shaking for 1 h finally. They were washed alternately with DMF and DCM for 6 times3. Deprotectiona solution 18 ml of 20% piperidine in DMF (15 ml/g) was added for 5 min, which was removed followed by adding a 15 ml solution of 20% piperidine in DMF (15 ml/g) for 15 min.4. testingThe piperidine solution was removed by suction filtration to obtain the resin, from which 15 g of resins were took and washed with ethanol for 3 times, with addition of one drops each of ninhydrin, KCN, and phenol solutions, followed by heating at 105° C.-110° C. for 5 min, and color change to dark blue indicated a positive reaction.5. WashingWashing twice with DMF (10 ml/g), twice with methanol (10 ml/g), and twice with DMF (10 ml/g).6. CondensationThree-fold excess of a protected amino acid (Fmoc-Leu-OH) and three-fold excess of HBTU, both dissolved in a small amount of DMF, were added into a reaction tube, followed by adding ten-fold excess of DIEA. The reaction was lasted for 40 min.7. WashingWashing once with DMF (10 ml/g), twice with methanol (10 ml/g), and twice with DMF (10 ml/g).8. Steps 2 to 7 were repeated to link amino acids in the sequence from right to left.9. testingWashing twice with DMF (10 ml/g), twice with DCM (10 ml/g), twice with DMF (10 ml/g), followed by draining for 10 min. Ninhydrin test was negative.10. WashingWashing 3 times with methanol (10 ml/g).11. Polypeptide cleavage from resinPreparing a cleavage solution (10 ml): 94.5% TFA, 2.5% water, 2.5% EDT, and 1% TISResin was placed into a flask or a centrifuge tube based on a ratio of the resin and the cleavage solution at 1 g:10 ml under constant temperature shaking for 120 min.12. Blow drying and washingThe cleavage solution was blow dried with nitrogen as much as possible, and then was subjected to chromatography with ether, followed by washing with ether for seven times, and then drying by volatilization at room temperature. The crude peptide sequence was obtained.13. oxidative cyclization10 mg of the crude produce were cyclized by adding 100 ml of 5% DMF solution to oxidize sulfhydryl groups.14. Purifying of the polypeptide by HPLCThe specific steps were as follows:1, 200 mg of crude peptides were placed into a container. They were dissolved with 4 ml of 50% acetonitrile in water. They could be sonicated slightly for 2 min.2, The dissolved solution was filtered with a 0.45 μm filter membrane.3, Analysis: 3 μl of the crude product was analyzed by analytical HPLC. The mobile phase was water and acetonitrile conducting a gradient elution for 30 min, and equilibrating HPLC firstly at an initial gradient of 95% water and 5% acetonitrile to a terminal ratio of 5% water and 95% acetonitrile for 5 min, and followed by injection.4, Preparation: the dissolved sample was made ready for injection. Preparative HPLC was equilibrated for 10 min at an initial gradient of 95% water and 5% acetonitrile to a terminal gradient of 25% water and 75% acetonitrile for 40 min. The sample from the detector was collected.5, Identification: the collected sample was took for purification and MS identification.15. Finally, the purified solution was lyophilized to obtain the finished product.16. The powdered peptide was in a sealed package and stored at −20° C.

FIGS.1to5showed schematic diagrams of docking between the cyclized polypeptide from modified BMP2 with BMP type I and VEGF type 2 receptors. The cyclized polypeptide from BMP2 had high affinity for BMP type I receptor and VEGF type 2 receptor simultaneously, which enabled the cyclized polypeptide from BMP2 to rapidly induce receptor complex formation and mediate its intracellular signaling pathway. Wherein,FIG.1: a structure diagram of a cyclized polypeptide form BMP2;FIG.2: BMP I type receptor;FIG.3: VEGF type 2 receptor;FIG.4: surface interaction in an optimal structural diagram of docking between a cyclized polypeptide from BMP2 and BMP type I receptor molecule, wherein blue represented the positive surface area, red represented the negative surface area, and green represented the hydrophobic surface area;FIG.5: a optimal structural diagram of docking between a cyclized polypeptide from BMP2 and VEGF type 2 receptor molecule.

Example 3

Promoting the Repair of Cranial Defects in Rats by Dripping a Cyclized Polypeptide from BMP2 Alone Study

The specific operation was as follows: SD rats were anesthetized with 3% pentobarbital sodium by intraperitoneal injection, prone on the operating table, fixed limbs, shaved the parietal bones, and covered with 1% iodine disinfection drape. The skin fascia periosteum was cut in layers, and the skull parietal bone was exposed, then the skull parietal bone was cut with a 1 cm trephine using an implanter at 600 rpm, until it turned into a blue-purple ring near the skull. The bone fragment from one end was pried with a stripper, and 8 mm circular bone fragment was removed by a vascular forcep.

The cyclized polypeptide from modified BMP2 of the present invention (CKIPKASSVPTELSAISMLYLGPGGDWIVAC) (SEQ ID NO:1) (50 ng/mL), and the modified linear polypeptide of the present invention (KIPKASSVPTELSAISMLYLGPGGDWIVA) (SEQ ID NO:4) (50 ng/mL), a simple cyclized peptide at a concentration of 50 ng/mL (CKIPKASSVPTELSAISMLYLC) (SEQ ID NO: 2), 50 ng/mL of a simple linear polypeptide (KIPKASSVPTELSAISMLYL) (SEQ ID NO: 3), BMP2 protein (50 ng/mL) and equivalent saline (control) were respectively dripped on the medical collagen membrane, then the membrane was implanted into the skull defect site, and the periosteum and skin were sutured in layers and disinfected with iodine. Within 3 days after surgery, rats were injected daily with gentamicin for preventation infection and carprofen for analgesia, and they were fed normally in separate cages. Four weeks later, samples were taken for Micro-CT scanning, and the software was used to analyze the indicators of new bone formation.

As shown inFIGS.8A-8C, there was a round standard bone defect with a diameter of 8 mm in Wistar rat skull. The equivalent polypeptide or protein was loaded on the medical collagen membrane and then implanted into the bone defect area. After 4 weeks, Microfil perfusion and Micro-CT scan were performed to calculate the results of new bone and vascular reconstruction. (L-BMP2: a linear peptide (SEQ ID NO: 3); C-BMP2: a cyclized polypeptide (SEQ ID NO: 2); M-L-BMP2: a modified linear peptide (SEQ ID NO: 4); M-C-BMP2: a modified cyclized polypeptide (SEQ ID NO: 1); the last group: BMP2 protein, set as a control group).

The data statistics chart on the left showed that the volume ratio of new bone in the modified circlized peptide group was significantly higher than that of other groups with statistically difference (FIG.8B). From the data inFIG.8B, it could be seen that, overall, the effect of the cyclized polypeptide was greater than that of the linear peptide, but the modified cyclized polypeptide of the present invention had the best effect, which was significantly different from other treatment, and it had better effect than the protein. The figure on the right showed that the area of new blood vessels in the modified cyclized polypeptide group was higher than that of other groups (FIG.8C), which was significantly different from the BMP2 protein group.

Example 4

The Collagen Sponge Loaded with a Cyclized Polypeptide from BMP2 is Used to Promote the Repair of the Limit Bone Defect of the Rat Skull

The cyclized polypeptide from unmodified BMP2, modified linear polypeptide, simple cyclized polypeptide, simple linear polypeptide, BMP2 protein and equivalent saline were dissolved in normal saline, respectively, and they were evenly drop on the collagen sponge (the diameter of 0.5 cm, the thickness of 1 mm, the volume of 50 μl, each peptide or protein had equivalent concentration). After lyophilization, they were sealed and stored at low temperature. The pre-prepared sponge was implanted into the skull defect. After 4 weeks of sampling, MicroCT showed that compared with the blank control group, equivalent cyclized polypeptide from BMP2 had a significant effect on promoting new bone formation with the amount of new bone nearly twice that of the blank group.

Example 5

Effect of a Linear Peptide and a Cyclized Polypeptide on Human Osteoblast Cell Line (hFOB1.19), respectively.

5.1 Osteogenesis-Related Protein Expression Detection

a) hFOB1.19, BMSC cell ALP activity detection and staining: ALP activity was detected using LabAssay™ ALP colorimetric assay kit, and the total protein concentration was measured by BCA method for calibration. After 7 days of culture, the cells were fixed for alkaline phosphatase staining. The specific operation of activity detection was as follows:{circle around (1)} The medium in the well plate was discarded and the plate was washed three times with PBS.{circle around (2)} Cells were lysed by adding cell lysate, and were crushed by ultrasound to promote the release of cell protein.{circle around (3)} The supernatant was pipetted after centrifugation for 15 min (4° C., 15000 rpm) with addition of ALP detection buffer, followed by incubating at 37° C. for 30 min.{circle around (4)} Stop solution was added to stop the reaction, and the absorbance at 405 nm wavelength was measured. ALP vitality was calculated.{circle around (5)} Total cell protein was quantified by BCA method and the ALP activity was corrected.

The specific method of ALP staining was as follows:{circle around (1)} The original medium was discarded and washed 3 times with PBS.{circle around (2)} Cells were fixed with 4% paraformaldehyde for 20 min;{circle around (3)} Washing 3-5 times with PBS, 3-5 min each time;{circle around (4)} BCIP/NBT staining working solution was formulated according to instructions of the kit;{circle around (5)} After the last washing, the washing solution was removed, followed by adding 500 ul BCIP/NBT dyeing working solution to each well to ensure that the sample could be fully covered;{circle around (6)} After incubation at 37° C. in an incubator in the dark for 30 min, the BCIP/NBT staining working solution was removed, and washed 1-2 times with PBS to stop the color reaction.b) Cell secretion type I collagen staining detection: after culture for 14 days, cells were washed 2 times with PBS after removal of medium, followed by washing 2 times with deionized water after fixation, and then stained with the kit, and dried and took pictures after washing.
5.2 Outer Matrix Mineralization Detectiona) Extracellular matrix Alizarin Red staining: after cell inoculation and culture, cells were washed 2 times with PBS, followed by washing 2 times with deionized water after fixation, and stained with Alizarin red solution, then washed with deionized water until calcium nodules becoming red to remove excess dye solution, followed by drying and observation.b) Correlation area analysis: photographs and image data analysis: each group of cell culture wells was took picture by NIS-Elements F2.20 (Nikon Eclipse 80i), and the area of red-stained calcified nodules in each culture well was measured by Image-Pro Plus 6.0 software.

The above was only the general treatment process for the cells, and the specific concentration was shown inFIG.6.

Result:

As shown inFIG.6, the linear peptide and cyclized polypeptide of the present invention were used to detect osteogenic indicators by acting on the human osteoblast cell line (hFOB1.19): inFIG.6, (A) indicates that ALP staining after culture for 7 days showed that the osteogenic effect of the cyclized polypeptide was significantly better than that of the linear peptide, and the effect of 50 ng/ml cyclized peptide was better than that of protein BMP2. (B) indicated that after induction culture in the mineralization medium for 28 days, Alizarin Red staining showed that the area of mineralized nodules induced by equivalent concentration of cyclized polypeptide was significantly higher than that of equivalent protein BMP2. (C) indicated that the quantitative detection of ALP after culture for 7 days revealed that the amount of ALP induced by 50 ng/ml cyclized peptide was significantly higher than that of protein BMP2. (D) indicated that the 28-day Alizarin Red quantitative analysis showed that the amount of mineralized nodules induced by 50 ng/ml cyclic peptide was 2.6 times higher than that induced by equivalent protein BMP2. (LP: a modified linear peptide of the present invention; CP: a modified cyclized polypeptide of the present invention; last group: BMP2 protein.*p<0.05; **p<0.01; ***p<0.001 had a significant difference; n.s.: no difference).

Example 6

Four Peptides and Proteins Act on HUVEC and BMSC, Respectively

The Specific Operation Steps were as Follows: Osteogenesis Related Index Test was as FOB Cell Tube Formation Test

a) On the first day, the matrigel was placed in a refrigerator at 4° C. overnight.b) On the next day, the matrigel was centrifuged for a few minutes after freezing and thawing.c) Operation was carried out on ice. Matrigel was mixed with a pre-cooled pipette tip in the pre-cooled EP tube, and aliquoted into 500 μl.d) The 96-well plate was pre-cooled in advance, with addition of 50 μl of matrigel to each well with avoiding air bubbles. It was placed at 37° C. in an incubator for 45 min.e) When the HUVEC cells were 70-80% confluence, they were digested and resuspended in DMEM containing 10% FBS. 50 μl of the resuspension solution was added to each well at a concentration of 3×10000 cells per well, with three duplicate wells.f) After incubation at 37° C. in an incubator, blood vessel formation could be seen after four hours.
Result:

As shown inFIG.7, the four peptides and proteins acted on HUVEC and BMSC respectively to detect angiogenesis and osteogenic indicators: (A) Cell tubular morphology observation after 3 hours showed that the modified cyclized polypeptide had the best effect. (B) The result of extracellular matrix mineralization after 14 days showed that the new mineralized nodules of modified cyclized polypeptide were significantly higher than those of other groups. (C) The statistical analysis of tube forming junction points showed that the number of nodes induced by the modified cyclized polypeptide was four times that of the BMP2 protein group. (D) The statistical analysis of the area of mineralized nodules showed that the modified cyclized polypeptide had a more obvious promotion effect on the formation of mineralized nodules than BMP2 protein. (L-BMP2: a linear peptide (SEQ ID NO: 3); C-BMP2: a cyclized polypeptide (SEQ ID NO: 2); M-L-BMP2: a modified linear peptide (SEQ ID NO: 4); M-C-BMP2: a modified cyclized polypeptide (SEQ ID NO: 1); the last group: BMP2 protein. Each treatment had equivalent concentration. p<0.05; **p<0.01; ***p<0.001 had a significant difference; n. s: no difference).

In the absence of any elements or limitations specifically disclosed herein, the invention shown and described herein can be realized. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It is therefore to be understood that, although the present invention has been particularly disclosed by various embodiments and optional features, modifications and variations of the concepts herein described may be resorted to by a person skilled in the art, and that such modifications and variations are considered to fall within the scope of the present invention as defined by the appended claims.

The contents of the articles, patents, patent applications, and all other documents and electronically available information described or described herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants hereby incorporate into this application any and all materials and information retained from any such article, patent, patent application or other document.