CN116375805A

CN116375805A - Polypeptide with antibacterial and osteogenic activities, preparation and application thereof

Info

Publication number: CN116375805A
Application number: CN202310306245.6A
Authority: CN
Inventors: 姜文韬; 林正梅; 谢茁; 高现灵; 黄舒恒; 黄绮婷; 陈玲玲
Original assignee: ORAL SUBSIDIARY SUN YAT-SEN UNIVERSITY HOSPITAL
Current assignee: ORAL SUBSIDIARY SUN YAT-SEN UNIVERSITY HOSPITAL
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2023-07-04
Anticipated expiration: 2043-03-27
Also published as: CN116375805B

Abstract

The invention belongs to the technical field of biological medicines, and discloses a polypeptide with antibacterial and osteogenic activities, a preparation and application thereof. The invention designs a polypeptide with antibacterial and osteogenic activities, and the amino acid sequence of the polypeptide is shown as any one sequence of SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3. The invention provides a preparation containing the polypeptide or the derivative of the polypeptide, and the application of the polypeptide and the preparation in preparing medicines for treating jawbone infection or maxillofacial infectious bone defects. The polypeptide has small molecules and good effect of resisting maxillofacial infectious bone defect related bacteria; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. The invention solves the antibacterial problem and regeneration promoting problem of maxillofacial infectious bone defect at the same time, and has new design concept and good clinical application potential.

Description

Polypeptide with antibacterial and osteogenic activities, preparation and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a polypeptide with antibacterial and osteogenic activities, a preparation and application thereof.

Background

The maxillofacial infectious bone defect is one of the common diseases of the stomatology, is caused by various bacterial infections, such as the bone defect of the jawbone, the defect of the alveolar bone, the defect of periapical periodontitis, and the like, and is different from the common bone defect, the range of the bone defect can be continuously expanded due to the existence and the diffusion of the infection, and the local debridement combined with the implantation of bioactive materials is required to promote the healing of the bone defect and the application of antibiotics to control the infection in serious cases. Bone regeneration materials generally studied and used are focused on the induction regeneration of bone tissue, but lack the effect on infection control; the use of conventional antibiotics is also at risk of drug-resistant bacteria. Thus, there is a need for bioactive agents that have both antimicrobial and osteogenic properties. However, a large number of bioactive agents associated with bone defect repair have focused on promoting regeneration or anti-infection, and the usual agents are simply simple functional stacks of osteogenic material and antimicrobial components, and the usual bone defect repair materials do not have anti-infection effect, and antibiotics have the risk of causing drug resistance.

The antibacterial peptide is a polypeptide with antibacterial activity and derivatives thereof, is not easy to generate drug resistance, and can improve the antibacterial capability of the antibacterial peptide and enable the antibacterial peptide to have additional biological activity by modifying the sequence of the antibacterial peptide. Thus, engineering a polypeptide to have both antimicrobial and osteogenic activity is a potential means of achieving an "antimicrobial-osteogenic" dual-purpose formulation. However, the design of multifunctional polypeptides is difficult because the antimicrobial functional polypeptide fragment and the osteogenic functional polypeptide fragment may interfere with each other due to charge, hydrophilicity and hydrophobicity, structure, resulting in a decrease in antimicrobial and osteogenic capabilities. Few polypeptides claim to have antibacterial-osteogenic ability, but the effective concentration is high, the high concentration application is economical and the action efficiency is still further improved. Therefore, there is a need to develop a safe and effective polypeptide with the dual effects of antibacterial and osteogenesis so as to overcome the technical difficulty of the active preparation for infectious bone defect and apply the polypeptide to clinic.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a polypeptide with antibacterial and osteogenic activities, a preparation and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention designs a polypeptide with antibacterial and osteogenic activities, wherein the amino acid sequence of the polypeptide is shown as any one sequence of SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.

The polypeptide molecule with antibacterial and osteogenic activities is small, and the effect of resisting the bacteria related to maxillofacial infectious bone defects is good; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. The polypeptide provided by the invention simultaneously solves the antibacterial problem and regeneration promoting problem faced by maxillofacial infectious bone defect by a single molecule, and has new design concept and good clinical application potential.

In a second aspect, the invention provides a formulation having both antibacterial and osteogenic activity, said formulation comprising said polypeptide or a derivative of said polypeptide.

As a preferred embodiment of the formulation according to the invention, the derivative is an ester derivative.

As a preferred embodiment of the formulation according to the invention, the formulation further comprises a pharmaceutically acceptable salt.

Further, the pharmaceutically acceptable salt comprises at least one of hydrochloride, sulfate, acetate, mesylate, succinate, fumarate, citrate, malate, and organic amine salt.

As a preferred embodiment of the formulation according to the invention, the formulation further comprises a pharmaceutically acceptable carrier and/or adjuvant.

As a preferred embodiment of the formulation according to the invention, the formulation is a liquid formulation, a solid formulation or a semisolid formulation.

Further, the liquid preparation is a solution or an injection; the solid preparation is a tablet or a capsule; the semisolid preparation is ointment or gel.

In a third aspect, the invention applies the polypeptide and the preparation in preparing medicines for treating jawbone infection.

In a fourth aspect, the polypeptide and the preparation are applied to the preparation of drugs for maxillofacial infectious bone defects.

Compared with the prior art, the invention has the beneficial effects that:

the polypeptide molecule with antibacterial and osteogenic activities is small, and the effect of resisting the bacteria related to maxillofacial infectious bone defects is good; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. After the polypeptide is prepared into the antibacterial peptide medicine, compared with the traditional antibiotics, the antibacterial peptide medicine is difficult to cause drug resistance. The polypeptide provided by the invention simultaneously solves the antibacterial problem and regeneration promoting problem faced by maxillofacial infectious bone defect by a single molecule, and has new design concept and good clinical application potential.

Drawings

FIG. 1 is a helix pattern of polypeptides WL14, GK16, GL 14;

FIG. 2 is a mass spectrum of the prepared polypeptide WL 14;

FIG. 3 is a mass spectrum of the prepared polypeptide GK 16;

FIG. 4 is a mass spectrum of the prepared polypeptide GL 14;

FIG. 5 is a high performance liquid chromatogram of the prepared polypeptide WL 14;

FIG. 6 is a high performance liquid chromatogram of the prepared polypeptide GK 16;

FIG. 7 is a high performance liquid chromatogram of the prepared polypeptide GL 14;

FIG. 8 is a graph of the detection of hemolytic toxicity of polypeptide WL 14;

FIG. 9 is a graph showing the detection of hemolytic toxicity of the polypeptide GK 16;

FIG. 10 is a graph showing the detection of hemolytic toxicity of polypeptide GL 14;

FIG. 11 is the effect of polypeptide WL14 on BMSCs osteogenesis-related gene expression;

FIG. 12 is a graph showing the effect of polypeptide GK16 on BMSCs osteogenic related gene expression;

FIG. 13 shows the effect of polypeptide GL14 on BMSCs osteogenic related gene expression.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

Example 1: polypeptide with antibacterial and osteogenic activities

Designing a group of polypeptides with antibacterial and osteogenic activities, namely 'antibacterial osteogenic' polypeptides which are respectively marked as WL14, and the amino acid sequence of the polypeptides is WLKRWLRKLYKFGG (SEQ ID No. 1); GK16 with the amino acid sequence of GKLIWKLLRKAYKFGG-NH ₂ (SEQ ID No. 2); GL14, amino acid sequence GLKRWLRKLYKFGG-NH ₂ (SEQ ID No.3)。

A spiral wheel diagram of the polypeptides WL14, GK16, GL14 is shown in FIG. 1.

The preparation method of the polypeptide comprises the following steps:

(1) Fmoc-His (Trt) -Wang Resin is selected as Resin (carrier);

(2) The resin was fully swollen with Dichloromethane (DCM);

(3) The Fmoc-protecting group was removed with the appropriate concentration of DBLK (hexahydropyridine+DMF);

(4) Washing with N, N-Dimethylformamide (DMF) for several times to wash off DBLK;

(5) Weighing a proper condensing agent and an activator (HBTU, NMM) for coupling, wherein the second Fmoc-protected amino acid at the C terminal (Fomc-Leu-OH);

(6) The ninhydrin detection method ensures that the connection is complete;

(7) Washing with DMF for several times to remove residual residues and activator condensing agent;

(8) Coupling according to the amino acid sequences of the polypeptides WL14, GK16 and GL14, wherein the method refers to the steps (3) to (7);

(9) Removing the final Fmoc-protecting group by adopting the methods of the steps (3) and (4) after all amino acid connection is finished;

(10) Cracking with TFA cutting fluid, removing resin and amino acid protecting group to obtain crude product;

(11) Sending mass spectrum to confirm the correctness of the product;

(12) The crude product is sent to purification and separation, and the purity is improved.

Mass spectra of the prepared polypeptides WL14, GK16 and GL14 are shown in figures 2-4. The high performance liquid chromatograms of the prepared polypeptides WL14, GK16 and GL14 are shown in figures 5-7.

Example 2: determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of polypeptide

The minimum inhibitory concentration MIC and the minimum bactericidal concentration MBC are indicators of the antimicrobial activity of the drug, indicating the ability of the drug to inhibit and kill pathogenic microorganisms. The antibacterial ability of the polypeptides WL14, GK16 and GL14 prepared in example 1 on the common bacteria of maxillofacial infectious bone defects is detected by using a maxillofacial infectious bone defect common bacteria staphylococcus aureus (Staphylococcus aureus), methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus), periapical infection common bacteria enterococcus faecalis (Enterococcus faecalis) and candida Albicans (Canidia Albicans) through a microplate dilution method drug sensitivity test.

The method comprises the following specific steps:

(1) Picking the preserved single colony S.aureus and MRSA in 10mL LB liquid medium, and culturing E.faecalis in BHI medium at 37 ℃ by shaking table overnight; albicans were grown overnight in YPD medium at 35℃on a constant temperature shaker.

(2) Absorbing 20 μL of bacterial liquid in 10mL of corresponding culture medium, subculturing for 2 generations at 35 ℃ or 37 ℃ by a constant temperature shaking table, taking bacterial liquid in mid-log growth, diluting S.aureus and MRSA with MH culture medium, E.faecalis and C.albicans with RPMI 1640 culture medium containing 10% fetal bovine serum to 1×10 ⁶ CFU/mL was ready for use.

(3) The polypeptides WL14, GK16, GL14 were added to 96-well plates using a 2-fold dilution method, respectively, at 10. Mu.L per well. 90 mu L of bacterial liquid is added into each hole to ensure that the final concentration of the antibacterial peptide is 2 mu g/mL-128 mu g/mL. Sterile water served as a negative control.

(4) The 96-well plate is placed in a constant temperature culture tank at 35 ℃ or 37 ℃ for culture for 24 hours.

(5) MIC is the lowest antimicrobial peptide concentration of the plate-inside clearing wells.

(6) 50 mu L of bacterial liquid in the clarification hole is sucked, S.aureus and MRSA are coated on an LB agar plate, E.faecalis is coated on a BHI agar plate, C.albicans is coated on a YPD agar plate, and the culture is carried out for 24 hours at a constant temperature of 35 ℃ or 37 ℃. MBC is the lowest polypeptide concentration for sterile colony growth on plates.

As shown in Table 1, the polypeptides WL14, GK16 and GL14 have low MIC and MBC for S.aureus, MRSA, E.faecalis and C.albicans, and have good antibacterial capability between 8 mug/mL and 32 mug/mL.

TABLE 1 MIC and MBC of polypeptides WL14, GK16, GL14 vs S.aureus, MRSA, E.faecalis, C.albicans

WL14	MIC(μg/mL)	MBC(μg/mL)
			S.aureus	16.00±0.00	24.00±8.00
MRSA	16.00±0.00	24.00±8.00
			E.faecalis	32.00±0.00	32.00±0.00
C.albicans	16.00±0.00	16.00±0.00
			GK16	MIC(μg/mL)	MBC(μg/mL)
S.aureus	16.00±0.00	32.00±0.00
			MRSA	16.00±0.00	32.00±0.00
E.faecalis	32.00±0.00	32.00±0.00
			C.albicans	10.67±3.77	16.00±0.00
GL14	MIC(μg/mL)	MBC(μg/mL)
			S.aureus	32.00±0.00	32.00±0.00
MRSA	32.00±0.00	32.00±0.00
			E.faecalis	32.00±0.00	32.00±0.00
C.albicans	8.00±0.00	12.00±4.00

Note that: MIC and MBC are expressed as mean ± standard deviation of six independent replicates

Example 3: haemolytic toxicity of Polypeptides

The hemolytic test is an important index for commonly testing the safety of the antibacterial peptide, and the polypeptides WL14, GK16 and GL14 prepared in example 1 are taken for performing a hemolytic toxicity test to evaluate the biosafety.

The method comprises the following specific steps:

(1) Defibrinated sheep blood was centrifuged at 1000rpm for 10min, the supernatant was discarded, washed with PBS and centrifuged again to obtain red blood cells. Erythrocytes were resuspended with ten volumes of PBS to obtain a suspension of erythrocytes.

(2) The polypeptides WL14, GK16, GL14 were each added to the EP tube by a 2-fold dilution method, 100. Mu.L per tube. 900. Mu.L of the red blood cell suspension was added to each well to give a final concentration of the antimicrobial peptide of 8. Mu.g/mL to 128. Mu.g/mL. Another 100. Mu.L of 1% Triton X-100 was used as positive control and 100. Mu.L of PBS was used as negative control. Incubate at 37℃for 1h. After centrifugation at 1000rpm for 10min, the supernatant was taken and the OD was measured ₅₄₀ 。

Percent hemolysis = (OD _{540 processing group} -OD _{540 negative control group} )/(OD _540Triton _X-100 -OD _{540 negative control group} )×100％。

As shown in FIGS. 8-10, different lower case letters and Roman numerals indicate that there is a statistical difference, P<0.05。OD ₅₄₀ Can characterize the lytic action of the polypeptides on the mammal red blood cells, and the hemolytic toxicity of the three polypeptides for treating the mammal red blood cells for 1h is extremely weak at the concentration of 64 mug/mL or below, and is only below 4%; the three polypeptides only showed a significant hemolytic activity at a high concentration of 128. Mu.g/mL, with a hemolysis rate of WL14 of 20% and GL14 and GK16 of about 10%. The three polypeptides are proved to have good biological safety under the antibacterial concentration.

Example 4: effect of polypeptide on bone marrow mesenchymal Stem cell (BMSCs) osteogenic Gene expression

The effect of the polypeptides WL14, GK16 and GL14 prepared in example 1 on the expression of BMSCs bone formation related genes ALP, col-1, OCN, OPN, runx2 and BSP was examined, and the potential of the polypeptides to promote bone was evaluated.

The method comprises the following specific steps:

(1) BMSCs were inoculated in 12-well plates using DMEM/F-12 medium containing diabody, 10% fetal calf serum in CO ₂ In incubator (5% CO) ₂ Culturing at constant temperature of 95% air, 100% humidity and 37 ℃).

(2) Cultured adherent cells were changed daily for fresh medium containing the polypeptide. The polypeptides WL14, GK16 and GL14 are subjected to gradient dilution by using a culture medium, wherein the concentration range is 8-32 mug/mL of antibacterial concentration; the negative control group used a medium without antibacterial peptide. Cells were cultured for 14 days.

(3) Cells were harvested after 14 days and subjected to real-time quantitative PCR to measure the expression of osteogenic related genes. RNA rapid extraction kit (Yi Cunninghamia, shanghai, china) is used for separating and purifying RNA. RNA concentration and purity were measured using a NanoDrop 2000 (Thermo Scientific, USA). cDNA was synthesized using PrimeScript reverse transcription kit (TaKaRa, japan) containing gDNA Eraser. Using cDNA as template

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Green Master Mix (next holy, shanghai, china) in +.>

480-II (Roche, switzerland) was subjected to real-time quantitative PCR. GADPH is used as an internal reference, 2 ^-ΔΔCt And calculating the gene expression fold difference by a method.

As shown in fig. 11-13, different lowercase letters indicate that there was a statistical difference in gene expression for this group, P <0.05. Under the antibacterial concentration, the polypeptides WL14, GK16 and GL14 have obvious up-regulation on the expression of BMSCs osteogenesis related genes ALP, col-1, OCN, OPN, runx and BSP, which shows that the polypeptide has the antibacterial effect and simultaneously has the bone-promoting capability and the antibacterial-osteogenesis double effect.

In conclusion, the polypeptide molecule with antibacterial and osteogenic activities is small, and the effect of resisting maxillofacial infectious bone defect related bacteria is good; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. After the polypeptide is prepared into the antibacterial peptide medicine, compared with the traditional antibiotics, the antibacterial peptide medicine is difficult to cause drug resistance. The polypeptide provided by the invention simultaneously solves the antibacterial problem and regeneration promoting problem faced by maxillofacial infectious bone defect by a single molecule, and has new design concept and good clinical application potential.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A polypeptide with antibacterial and osteogenic activities is characterized in that the amino acid sequence of the polypeptide is shown as any one sequence of SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.

2. A formulation having both antibacterial and osteogenic activity, wherein the formulation comprises the polypeptide or derivative of the polypeptide of claim 1.

3. The formulation of claim 2, wherein the derivative is an ester derivative.

4. The formulation of claim 2, further comprising a pharmaceutically acceptable salt.

5. The formulation of claim 4, wherein the pharmaceutically acceptable salt comprises at least one of a hydrochloride, sulfate, acetate, mesylate, succinate, fumarate, citrate, malate, organic amine salt.

6. The formulation of claim 2, further comprising a pharmaceutically acceptable carrier and/or adjuvant.

7. The formulation of claim 2, wherein the formulation is a liquid formulation, a solid formulation, or a semi-solid formulation.

8. The formulation of claim 7, wherein the liquid formulation is a solution or an injection; the solid preparation is a tablet or a capsule; the semisolid preparation is ointment or gel.

9. Use of the polypeptide of claim 1, the formulation of any one of claims 2 to 8 for the manufacture of a medicament for treating a jawbone infection.

10. Use of the polypeptide of claim 1, the formulation of any one of claims 2 to 8 for the manufacture of a medicament for maxillofacial infectious bone defects.