AU2020102985A4 - Dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, polypeptide derivative and salt thereof, and application - Google Patents
Dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, polypeptide derivative and salt thereof, and application Download PDFInfo
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- AU2020102985A4 AU2020102985A4 AU2020102985A AU2020102985A AU2020102985A4 AU 2020102985 A4 AU2020102985 A4 AU 2020102985A4 AU 2020102985 A AU2020102985 A AU 2020102985A AU 2020102985 A AU2020102985 A AU 2020102985A AU 2020102985 A4 AU2020102985 A4 AU 2020102985A4
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- polypeptide
- caries
- bacteria
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- resisting
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Cosmetics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a dual-effect anti-caries polypeptide with functions of resisting to
cariogenic bacteria and promoting remineralization, a polypeptide derivative or a polypeptide
medicinal salt. The polypeptide contains an amino acid sequence a and an amino acid sequence
b, wherein the amino acid sequence a is Xn, X is S, T, Y, K, R, N or Q, and n is a number of
amino acid repetition times and an integer of 5-8; and the amino acid sequence b is an
antibacterial peptide with an alpha double helix structure, positive charges and 8-12 amino
acids, and hydrophilic and hydrophobic amino acids appear alternately in the sequence. The
dual-effect polypeptide with functions of resisting to cariogenic bacteria and promoting
remineralization disclosed by the invention can inhibit growth of Streptococcus mutans, inhibit
formation of a Streptococcus mutans biofilm, reduce a metabolic capacity of the formed
Streptococcus mutans biofilm, and kill living bacteria in the formed Streptococcus mutans
biofilm; the polypeptide can promote remineralization of demineralized enamel and reduce a
caries depth and mineral losses of demineralized enamel; and the polypeptide has good stability
and no obvious cytotoxicity.
-1/5
3.0
2.5-L11
2.0
1.5
1.0- ---
0.5
0.0
0 1 2 4 8 16 32 64 128256512
SH18(idl)
Figure 1
Description
-1/5
3.0
2.5-L11 2.0
1.5
1.0- ---
0.5
0.0 0 1 2 4 8 16 32 64 128256512 SH18(idl)
Figure 1
Dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, polypeptide derivative and salt thereof, and application
The invention belongs to the field of biomedicines, in particular to a dual-effect anti caries polypeptide with functions of resisting to cariogenic bacteria and promoting remineralization, a polypeptide derivative, a polypeptide medicinal salt and application thereof.
Caries is a kind of disease that causes chronic progressive destruction of hard tissues of tooth under the action of multiple factors mainly bacteria. It is also the most common oral disease of human beings and widely exists all over the world. The World Health Organization lists it as one of the three major diseases endangering human health. As verified by a lot of evidence, the existence of bacteria is a prerequisite for occurrence of caries. The direct cause of dental caries is the metabolism of carbohydrates by oral cariogenic bacteria, which can lead to acid generation, and then demineralization of hard tissues of teeth. However, development of caries is a process of repeated alternation and dynamic changes of demineralization and remineralization. Artificial methods can promote remineralization of demineralized hard tissues, restore the hardness, and terminate or eliminate early caries. Therefore, early prevention and treatment of dental caries mainly involves two important directions: inhibiting cariogenic bacteria and promoting remineralization of hard tissues of teeth.
In the aspect of inhibiting cariogenic bacteria, anti-microbial agents such as chlorhexidine and tetracycline can directly act on bacteria to prevent caries, but long-term use of them can easily cause oral flora imbalance, causing bacteria to produce drug resistance and generating obvious toxic and side effects. Traditional Chinese medicines such as catechu, rhubarb, scutellaria baicalensis Georgi, apiary, areca nut, pseudo-ginseng, tea polyphenol, etc. have been proved to have the effects of interfering with bacterial metabolism and inhibiting plaque biofilm formation or the like, but there are problems such as liquid medicine dyeing of teeth and generation of drug resistance. Immune caries prevention has certain effects, but there are problems concerning enhancement of immunogenicity and safety in human application. In addition, the problems affecting the ecology of oral flora also need to be solved urgently.
In the aspect of promoting remineralization of tooth hard tissues, the remineralization effects of amorphous calcium phosphate, xylitol and sorbitol that replace sugar, traditional Chinese medicine Chinese gall and Cynanchum wilfordii, nano hydroxyapatite, casein phosphopeptide, trace elements, olive oil, resin, etc. have been reported successively, but the conclusions have not been unified at present because the effects are not obvious or the experimental results are different. In recent years, based on the biomineralization function of enamel matrix proteins, the synthesis of artificial hydroxyapatite from enamel matrix proteins in vitro has been successful. Chen et al. simulated the enamel mineralization process and used enamel matrix protein to control the synthesis of nanorod-shaped hydroxyapatite, which is very similar to natural enamel in chemical composition and crystal size. Yamagish et al. formed fluorapatites with an enamel structure on the enamel surface. These fluorapatites are densely arranged, parallel arranged and perpendicular to the enamel surface. Domestic scholars Wang Zhiwei et al. placed enamel in a phosphate agar-calcium acetate solution system containing SD rat enamel matrix proteins for 7 days. The results showed that crystal banded structure appeared on the enamel surface added with enamel matrix proteins. All these experimental results all suggest that enamel matrix protein has strong potential to induce enamel remineralization, but it has not become a mature product to be directly used for caries prevention.
An ideal anti-caries preparation should have the functions of resisting to cariogenic bacteria and promoting remineralization of hard tissues of teeth at the same time, so as to improve the prevention and treatment effect of caries from two aspects at the same time. Fluorine is a common "dual-effect" anti-caries agent, which effectively reduces the incidence of caries. However, the widespread use of fluorine also increases the incidence of dental fluorosis and skeletal fluorosis. Therefore, it is of great significance to find safe and effective dual-effect anti-caries preparations.
With the development of molecular biology, polypeptides can be flexibly modified according to needs. They are safe and effective, and have become the focus of dental caries prevention and control research. The researchers of the invention have obtained certain research results in the in-depth research on antibacterial functional polypeptides and remineralization functional polypeptides in the early stage. The patents CN201310354537.3 and CN201310355804.9 provide polypeptides with enamel remineralization induction effect, and the tests prove that the effect is equivalent to that of fluoride. In addition, the patent application CN201510207973.7 provides a small molecule antibacterial polypeptide with a stable structure, a good bactericidal effect and a wide range. However, these polypeptides only have a single function of resisting to cariogenic bacteria or promoting remineralization promoting. Patients with dual requirements for resistance to bacteria and remineralization need to use two drugs at the same time. On the one hand, the drug use burden of patients is increased, leading to poor compliance. On the other hand, the two drugs may affect each other and reduce their respective curative effects. Therefore, the research and development of polypeptides with "dual effects" of resisting to bacterial and promoting remineralization dual-effect are of a high clinical practical value in caries prevention and treatment based on both the two directions of preventing cariogenic bacteria and promoting remineralization.
A first objective of the invention is to: summarize the existing research results aiming at the above research background, innovatively put forward the idea of using protein splicing through rigorous scientific reasoning, and construct a dual-function anti caries polypeptide with functions of resisting to cariogenic bacteria and promoting remineralization, a polypeptide derivative or a polypeptide medicinal salt. On the one hand, the polypeptide can reduce cariogenic bacteria and caries occurrence; and on the other hand, the polypeptide can promote remineralization of demineralized enamel (early caries) and treat early caries, thereby generating better effects of caries prevention and treatment.
A second objective of the invention is to provide a pharmaceutical composition and a preparation thereof containing the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt.
A third objective of the invention is to provide application of the dual-effect anti caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt in preparation of anti-caries drugs.
The above objectives of the invention are realized by the following technical scheme:
A dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, a polypeptide derivative or a polypeptide medicinal salt, wherein the polypeptide comprise an amino acid sequence a: Xn, X is S, T, Y, K, R, N or Q, and n is an integer selected from 5 to 8 for the number of amino acid repetitions; and
An amino acid sequence b: b is an antibacterial peptide with an alpha double helix structure, positive charges, 8-12 amino acids, and alternate occurrence of hydrophilic and hydrophobic amino acids in the sequence.
Preferably, the amino acid sequence a is located at the N-terminal of the polypeptide. According to the research of the present inventors, when the sequence is reversed, the antibacterial activity of the polypeptide is significantly reduced.
According to some embodiments of the present invention, the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, wherein the amino acid sequence a is Xn, X is S or T, and n is 5 or 6.
According to some embodiments of the present invention, the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, wherein the amino acid sequence a is SSSSSS, SSSSS or TTTTTT.
According to some embodiments of the present invention, the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, wherein the amino acid sequence b is GLLWHLLHHLLH, LLRRLLRRLLRR or LLKKLLLKKLLKK, or a polypeptide having 80% or more identity to them.
According to some embodiments of the invention, the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, wherein the polypeptide comprises one of the following amino acid sequences, preferably SEQ ID NO. 1:
SEQIDNO.1: SSSSSSGLLWHLLHHLLH;
SEQ ID NO.2: SSSSSSLLRRLLRRLLRR;
SEQ ID NO.3: SSSSSSLLKKLLKKLLKK;
SEQ ID NO.4: SSSSSGLLWHLLHHLLH;
SEQ ID NO.5: SSSSSLLRRLLRRLLRR;
SEQ ID NO.6: SSSSSLLKKLLKKLLKK;
SEQ ID NO.7: TTTTTTGLLWHLLHHLLH;
SEQ ID NO.8: TTTTTTLLRRLLRRLLRR;
SEQ ID NO.9: TTTTTTLLKKLLKKLLKKo
According to some embodiments of the present invention, the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, wherein the polypeptide derivative is a modifier of the C-terminal amidation of the polypeptide, such as the C terminal amidst of SEQ ID NO. 1-9.
According to some embodiments of the present invention, the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, wherein the salt includes, but is not limited to, hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate, organic amine salt, and the like.
The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt of the present invention can be prepared by referring to the patents CN201310354537.3 and CN201310355804.9, or can also be prepared according to the embodiments of the present invention.
A preparation method according to an embodiment of the invention comprises the following steps:
1. Taking Rink-Amide-Am Resin as the resin (carrier);
2. Fully swelling the resin with DCM;
3. Removing a Fmoc-protective group with an appropriate concentration of DBLK (hexahydropyridine + DMF);
4. Carrying out washing for several times with DMF to remove DBLK;
5. Weighing suitable condensing agents and activators (HBTU, NMM) and the first Fmoc-protected amino acid (Fomc-His (trt)-OH) at the C terminal for condensation;
6. Using a ninhydrin detection method to ensure complete connection;
7. Carrying out cleaning with DMF for several times to remove various residual residues and an activator condensing agent;
8. Carrying out condensation according to the amino acid sequence of the polypeptide, wherein the method can be refers to in Step 3-7;
9. After connection of all amino acids, removing the last Fmoc-protecting group by the method of Step 3 and 4;
10. Carrying out cracking with a TFA cutting solution to remove resin and amino acid protective groups, and obtaining a crude product;
11. Using spectrometry to confirm that the product is correct (a molecular weight conforms to theoretical value); and
12. Treating the crude product with purification and separation to improve the purity.
The invention also provides a pharmaceutical composition, which comprises the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, as well as pharmaceutically acceptable carriers and/or auxiliary materials.
Pharmaceutically acceptable carriers include, but are not limited to, sterile liquids, such as water, or animal, plant or artificially synthetic oils or mixtures thereof. Pharmaceutical excipients include but are not limited to starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, wetting agent, emulsifier or pH buffer, mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc.
The invention further provides a preparation containing the above pharmaceutical composition. The preparation can be a liquid preparation, a solid agent or a semi-solid agent. The liquid agent includes but is not limited to solutions and injections. The solid agent includes but is not limited to tablets and capsules. The semi-solid agent includes but is not limited to ointments and gels.
The invention further provides application of the application of the dual-effect anti caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt in preparation of anti-caries drugs.
The invention has the following beneficial effects:
The "dual-effect" anti-caries functional polypeptide with functions of resisting to cariogenic bacteria and promoting remineralization of the invention has the following functions: 0 an antibacterial effect, which can kill common pathogenic bacteria in oral cavity at a lower concentration; @ a reliable remineralization promoting effect, which can promote remineralization of early artificial caries; @ almost no toxicity to human oral epithelial cells in vitro; and @ a small molecular weight and a stable structure. The "dual-effect" anti-caries functional polypeptide has great potential in the field of caries prevention and treatment.
Figure 1 is an absorbance diagram showing effects of a polypeptide in on formation of a Streptococcus mutans biofilm in Embodiment 4;
Figure 2 is an absorbance diagram showing effects on a metabolic capacity of the formed Streptococcus mutans biofilm in Embodiment 5;
Figure 3 is a diagram showing detection results of surface microhardness of an enamel sample after a pH cycle in Embodiment 6;
Figure 4 is a cross-sectional microradiogram of an enamel sample after a pH cycle in Embodiment 6;
Figure 5 is a diagram showing results of each group of mineral losses before and after caries treatment shown by cross-sectional microradiography of an enamel sample after a pH cycle in Embodiment 6;
Figure 6 is a diagram showing results of caries depth of each group before and after cycle treatment shown by cross-sectional microradiography of an enamel sample after a pH cycle in Embodiment 6;
Figure 7 is a diagram showing results of mineral content at different caries depths before and after cycle treatment shown by cross-sectional microradiography of an enamel sample after a pH cycle in Embodiment 6;
Figure 8 is a diagram show polypeptide retention rates for a study on stability of the polypeptide in saliva in Embodiment 7; and
Figure 9 is an absorbance diagram of an in vitro cytotoxicity study of the polypeptide of Embodiment 8.
A dual-effect anti-caries polypeptide with functions of resisting to cariogenic bacteria and promoting remineralization, a polypeptide derivative or a polypeptide medicinal salt, wherein the polypeptide comprise an amino acid sequence a and an amino acid sequence b, the amino acid sequence a is Xn, X is S, T, Y, K, R, N or Q, and n is an integer selected from 5-8 for the number of amino acid repetitions; and the amino acid sequence b is an antibacterial peptide with an alpha double helix structure, positive charges, 8-12 amino acids, and alternate occurrence of hydrophilic and hydrophobic amino acids in the sequence.
In some preferred embodiments as follows, the amino acid sequence a: Xn, where X is S or T and n is 5 or 6.
In some preferred embodiments as follows, the amino acid sequence a is SSSSSS, SSSSS or TTTTTT, and more preferably the amino acid sequence a is SSSSSS.
In some preferred embodiments as follows, the amino acid sequence b is GLLWHLLHHLLH, LLRRLLRRLLRR or LLKKLLLKKLLKK, or a polypeptide having 80% or more identity to them.
According to the anti-caries polypeptide, the polypeptide derivative or the polypeptide medicinal salt, wherein the polypeptide comprises one of the following amino acid sequences, preferably SEQ ID NO. 1:
SEQIDNO.1: SSSSSSGLLWHLLHHLLH;
SEQ ID NO.2: SSSSSSLLRRLLRRLLRR;
SEQ ID NO.3: SSSSSSLLKKLLKKLLKK;
SEQ ID NO.4: SSSSSGLLWHLLHHLLH;
SEQ ID NO.5: SSSSSLLRRLLRRLLRR;
SEQ ID NO.6: SSSSSLLKKLLKKLLKK;
SEQ ID NO.7: TTTTTTGLLWHLLHHLLH;
SEQ ID NO.8: TTTTTTLLRRLLRRLLRR;
SEQ ID NO.9: TTTTTTLLKKLLKKLLKKo
According to the invention, polypeptide derivative is a modifier of the C-terminal amidation of the polypeptide, such as the C-terminal amidate of SEQ ID NO. 1-9, and the C-terminal amidation is more beneficial to the stability of the polypeptide.
According to the invention, the amino acid sequence b of the polypeptide has a small molecular weight and an alpha spiral structure, and can act on the cell wall of bacteria to cause perforation of the cell wall and death of bacteria, thereby playing an antibacterial role.
The inventors intend to connect a mineralized polypeptide to the antibacterial peptide based on the principle of protein splicing to obtain an anti-caries polypeptide with both functions of resisting to cariogenic bacteria and promoting remineralization. According to the research, the C-terminal of the antibacterial functional polypeptide plays an important role in exerting its antibacterial activity, so the mineralized fragment is connected to the N-terminal of the polypeptide. When the two sequences are changed, the antibacterial effect is significantly reduced or disappeared.
Further, the inventors of the present invention have found through research and summary that the groups -COOH, -CONH2, -OH, and -NH2 of the mineralized fragment can adsorb calcium and phosphorus ions, thus promoting the mineralization of tooth hard tissues. From high to low levels, the order of adsorption ability of the above groups is as follows: -COOH >-CONH2 ~-OH >-NH2. However, the positively and negatively charged groups -COOH of antibacterial functional polypeptides will affect their antibacterial function, so groups other than -COOH are preferred. Furthermore, the amino acids containing the -CONH2 group include asparagine and glutamine, the amino acids containing the -OH group include serine, threonine and tyrosine, and the amino acids containing the -NH2 group include lysine and arginine. As found in research and comparison of the above amino acids, serine has stronger calcium and phosphorus adsorption capacity and small side chain groups, which is a better choice. It is also found in the study that the mineralization ability of mineralized fragments increases with the increase of the number of amino acids, but the increase of the number of amino acids will affect the antibacterial effect of antibacterial functional polypeptides. Finally, the research by the inventors shows that when the number of amino acids of mineralized fragments is 5-8, especially 5 or 6, higher mineralization ability and antibacterial ability can be obtained.
Therefore, in some preferred embodiments of the present invention, GLLWHLLHHLLH is selected as the anti-cariogenic bacteria fragment. On this basis, the N-terminal mineralization promoting fragment SSSSSS is added, and the C-terminal of the polypeptide is amidated to construct the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, thereby generating better caries prevention and treatment effect.
The anti-caries polypeptide or the derivative of the present invention can be further prepared into medicinal salts according to demands such as facilitating the preparation of preparations. These methods are commonly used in the field of pharmaceutical manufacturing.
The anti-caries polypeptide or the derivative of the present invention can be prepared by using the method of synthesizing polypeptide known in the prior art, and the present invention also provides a specific synthesis method in the embodiment.
The contents of the present invention will be further explained and explained by specific examples below for definition of the technical scheme and beneficial effects of the present invention. However, those skilled in the art should understand that these examples are only some preferred options of the present invention and are not limiting the scope of the above-mentioned subject matter of the present invention. Any modification made without departing from the spirit and principles of the present invention, as well as equivalent substitutions or improvements made in accordance with the general technical knowledge and customary means in the art, shall be included in the scope of protection of the present invention.
EMBODIMENT 1:
The amino acid sequence of the anti-caries polypeptide of the present invention is as follows: SEQ ID NO. 1-9.
The polypeptide is prepared with the following methods:
1. Taking Fmoc-His (Trt)-Wang Resin as the resin (carrier);
2. Fully swelling the resin with DCM;
3. Removing a Fmoc-protective group with an appropriate concentration of DBLK (hexahydropyridine + DMF);
4. Carrying out washing for several times with DMF to remove DBLK;
5. Weighing suitable condensing agents and activators (HBTU, NMM) and the second Fmoc-protected amino acid (Fomc-His (trt)-OH) at the C terminal for condensation;
6. Using a ninhydrin detection method to ensure complete connection;
7. Carrying out cleaning with DMF for several times to remove various residual residues and an activator condensing agent;
8. Carrying out condensation according to the amino acid sequence of the polypeptide, wherein the method can be refers to in Step 3-7;
9. After connection of all amino acids, removing the last Fmoc-protecting group by the method of Step 3 and 4;
10. Carrying out cracking with a TFA cutting solution to remove resin and amino acid protective groups, and obtaining a crude product;
11. Using spectrometry to confirm that the product is correct (a molecular weight conforms to theoretical value); and
12. Treating the crude product with purification and separation to improve the purity.
EMBODIMENT 2
Preparation of A C-Terminal amidated derivative:
The preparation is carried out with reference to the method described in Embodiment 1, except that Rink-Amide-Am Resin is selected as the resin (carrier).
EMBODIMENT 3 Measurement of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the "dual-effect" polypeptide
MIC (Minimal inhibitory concentration) refers to the minimum concentration at which antibacterial agents can inhibit the growth of bacteria in the culture medium in vitro. Minimal bactericidal concentration (MBC) refers to the minimum concentration of antibacterial drugs that can kill living bacteria in the culture medium in vitro. MIC and MBC are indicators of antibacterial activity of drugs, showing the ability of drugs to inhibit and kill pathogenic microorganisms. Streptococcus mutans UA159, provided by the National Key Laboratory of Oral Disease Research, was used as the main oral cariogenic bacteria in the experiment. The tested polypeptide is a C-terminal amidate of SEQ ID NO. 1 (hereinafter referred to as SH18).
The experimental steps for MIC and MBC measurement are as follows:
1. Selecting and culturing single colonies overnight in a 37 °C constant temperature anaerobic culture tank in a 10mL BHI liquid medium(80% N2, 10% H2, 10% C02).
2. Sucking 100 pL of bacterial liquid into a 10 mL BHI liquid medium and placing it in a constant temperature anaerobic culture tank (80% N2, 10% H2, 10% C02) for culture of 6 hours, and diluting the bacterial liquid to 2x106 CFU/mL for later use.
3. Adding the polypeptide into a U-shaped 96-well plate by a 2-fold dilution method, with 20 pL per well.
4. Adding 80 pL of the BHI medium and 100 pL of a spare bacterial liquid to each well to make the final concentration of polypeptide reach 512 IM~1 M.
5, 96-well plate were incubated in 37 °C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% C02) for 24h.
6. MIC is the lowest polypeptide concentration clarified in the well plate.
7. Sucking100 L of the bacterial liquid in a clear hole and coating it on a BHI plate, and carrying out overnight culture in a 37 °C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% C02) for 24h.
8. MBC is the lowest polypeptide concentration for colony-free growth on the plate.
9. Repeating the experiment at least 3 times.
Results: SH18 has an antibacterial effect, with MIC of 64 pM and MBC of 128 tM.
EMBODIMENT 4 Effects of "dual-effect" polypeptide on formation of a Streptococcus mutans biofilm
Minimal biofilm inhibitory concentration (MBIC) is an indicator denoting the ability of a medicine to resist biofilm formation. MBIC5o refers to the lowest concentration at which antibacterial drugs can inhibit the formation of more than 50% biofilm in the culture medium in vitro tests. Streptococcus mutans UA159 was used in the experiment. The test polypeptide was SH18. Aseptic deionized water was used as negative control.
The experimental steps are as follows:
1. Selecting and culturing single colonies overnight in a 37 °C constant temperature anaerobic culture tank in a 1OmL BHI liquid medium(80% N2, 10% H2, 10% C02).
2. Sucking100pL of the bacterial liquid into the 10mL BHI liquid medium and placing it in constant temperature anaerobic culture tank (80% N2, 10% H2, 10% C02) for culture of 6 hours, and diluting the bacterial liquid to 2x 106 CFU/mL for later use.
3. Adding the polypeptide into a 96-well plate by a 2-fold dilution method, with 20 ptL per well.
4. Adding a 80 tL BHIS (BHI plus 2% sucrose) medium and a 100 pL standby bacteria solution to each well, so that the final concentration of polypeptide is 512 pM-1 ptM, and using aseptic deionized water as negative control.
5. Putting the 96-well plate in 37 °C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% C02) for culture of 24h.
6. Sucking the culture solution away and detecting the amount of biofilm formed: washing it twice with PBS, fixing with methanol for 15min, dyeing with 0.1% crystal violet for 5min, decolorizing with 95% ethanol for 30min, and measuring the absorbance at 595nm.
7. MBICso is the lowest polypeptide concentration with absorbance reduced by more than 50% compared with negative control group.
8. Repeating the experiment at least 3 times.
As shown in figure 1, the abscissa indicates the concentration of polypeptide, the ordinate indicates the absorbance, and the absorbance reflects the amount of biofilm. The higher the absorbance is, the more the amount of biofilm will be. As shown in figure 1, the amount of biofilm formed when SH18 concentration is 16-512 tM is significantly lower than that of negative control group (polypeptide concentration of 0 pM), and the difference is statistically significant (P < 0.05). The dotted line in figure 1 indicates the amount of biofilm formed is 50% of that of the negative control group. Obviously, the MBIC5oof SH18 is 16 pM.
EXAMPLE 5 Effects of "dual-effect" polypeptide on metabolic capacity of formed Streptococcus mutans biofilm
SMIC (Sessile minimal inhibitory concentration) is an indicator that reflects the effect of drugs on the metabolic capacity of formed biofims. Among them, SMICo refers to the lowest concentration at which antibacterial drugs can reduce the metabolic capacity of biofilm by more than 50% in vitro tests. Streptococcus mutans UA159 was used in the experiment. The test polypeptide was SH18. Aseptic deionized water was used as negative control.
The experimental steps are as follows:
1. Selecting and culturing single colonies overnight in a 37 °C constant temperature anaerobic culture tank in a lOmL BHI liquid medium(80% N2, 10% H2, 10% C02).
2. SuckinglOpL of the bacterial liquid into the 10 mL BHI liquid medium and placing it in constant temperature anaerobic culture tank (80% N2, 10% H2, 10% C02) for culture of 6 hours, and diluting the bacterial liquid to 1x106 CFU/mL adding it into a 96 well plate, and carrying out constant temperature anaerobic culture at 37 °C for 24 hours.
3. Discarding a supernatant, washing the biofilm with PBS for 3 times, and adding the polypeptide into 96-well plate by a 2-fold dilution method, with 20 pL per well.
4. Adding a 180 tL BHIS medium, so that the final concentration of polypeptide is 512 pM-1 pM, and using aseptic deionized water as negative control.
5. Putting the 96-well plate in 37 °C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% C02) for culture of 24h.
6. Sucking the culture solution and detecting the metabolic capacity of the biofilm: MTT incubation for 2 hours, DMSO decolorization for 30min, and measurement of the absorbance at 540nm.
7. MBIC50 is the lowest polypeptide concentration that inhibits the metabolic ability of biofilm by more than 50%.
8. Repeating the experiment at least 3 times.
As shown in figure 2, the abscissa indicates the concentration of polypeptide, the ordinate indicates the absorbance, and the absorbance reflects the amount of biofilm. The higher the absorbance is, the more the amount of biofilm will be. As shown in figure 2, the amount of biofilm formed when SH18 concentration is 128-512 M is significantly lower than that of negative control group (polypeptide concentration of 0jM), and the difference is statistically significant (P < 0.05). The dotted line in figure 2 indicates the amount of biofilm formed is 50% of that of the negative control group. Obviously, the MBIC5o of SH18 is 128 tM.
EXAMPLE 6 Effects of "dual-effect" polypeptide on remineralization of demineralized enamel
In this example, the remineralization effect of "dual-effect" polypeptide on early artificial enamel caries was observed through classical pH cycle in vitro.
The experimental steps are as follows:
1. Preparation of enamel samples: Fresh extracted bovine incisors were selected and enamel samples of bovine teeth were prepared. Under flowing water, aluminum oxide paste was used to remove enamel surface staining, tartar and irregular surface. After ultrasonic washing in deionized water for 20 minutes, it was stored in PBS containing 0.05% thymol and placed in refrigerator at 4 °C for later use. The crown root was separated, and ultrasonic cleaning was conducted on the crown tooth tissue for 20 minutes. It was naturally dried. The crown tissue with smooth surface, no fluorosis, no pigment and no crack was selected for the next operation. The crown part was cut into enamel blocks with a size of about 5x5x2 mm by a hard tissue high-speed cutting machine, and the lip enamel was grinding flattened and polished by a polishing machine and sequential use of 800#-1200#-2400# silicon carbide water frosted paper under running water, for removal of about 100 tm of surface enamel and elimination of surface organic pollutants and irregular enamel patterns. After 20 minutes of ultrasonic washing, the teeth were naturally dried. Epoxy resin was used to embed the teeth. A 4 mmx4 mm window area was retained in the center of the lip surface of the enamel block by using a sealing film. The part outside the window area was covered with acid-fast nail polish, which was evenly coated twice. Through the surface microhardness baseline (i. e. SHMo), enamel blocks with hardness values ranging from 340 to 380KHN were screened out for the next experiment.
2. Preparation of artificial early enamel caries: Bovine enamel samples were demineralized in a specific volume of demineralized solution (demineralized solution: 2.2 mM Ca (N03)2, 2.2 mM KH2PO4, 50 mM acetic acid, 5.0 mM NaN3, 0.5 ppm NaF, pH 4.5) according to the ratio of enamel fenestration area surface area to solution 2mm 2 lml. The early demineralized enamel caries were formed in the fenestration area of bovine enamel samples after stirring with magnetic agitator (100 rpm) and demineralization at 37 °C for 72 hours.
3. Microhardness measurement of early enamel caries: The surface microhardness value of enamel samples forming early caries was measured again and recorded as SMHi, and 30 enamel blocks with surface microhardness value ranging from 140 to 220 KHN were screened out to enter the next remineralization cycle experiment. One side of the fenestration area of each sample was covered with a 4x2 mm sealing film and sealed with acid-fast nail polish, which was taken as a morphological control for early enamel caries before the remineralization cycle.
4. Remineralization cycle experiment: 30 enamel samples with early caries were randomly divided into 3 groups with 10 samples in each group, which were divided into experimental group: SH18 polypeptide group; negative control group: DDW group. Cycling under in vitro pH cycling conditions was carried out for 12 days, wherein in vitro pH cycling per day included 2 hours of demineralization (demineralization solution: 2.2 mM Ca (N03)2, 2.2 mM KH2PO4, 50 mM acetic acid, 1.0 mM NaN3, pH 4.5) for 4 times of 5 minutes, and the rest of the time was spent on soaking in remineralization solution (remineralization solution: 1.5 mM CaCl2, 0.9 mM KH2PO4,130 mM KCl, 1.0 mM NaN3, mM HEPES, pH 7.0) for about 22 hours/day. It was stirred in a 37 °C closed thermostat with a magnetic stirrer at 100 rpm.
5. Results Detection Index
5.1 Surface microhardness
The parameters of the surface microhardness tester are set as before, and the surface microhardness of the fenestration area of enamel samples after pH cycle in vitro is measured again. Each enamel sample is measured at five points, and the average value is the surface microhardness value of the sample after pH cycle treatment, which is recorded as SMH2. After comparison of the enamel samples of normal bovine enamel, early enamel caries after demineralization and pH cycling in vitro, the final recovery percentage of surface microhardness (SMHR%) of each sample can be calculated: SMHR%=(SMH2-SMH1)/(SMHi-SMHo) x 100%.
5.2 Transection microradiography
Samples are recycled and taken out, rinsed with deionized water, treated with ultrasound oscillation for 20 minutes, and then dried naturally. The enamel samples were sliced perpendicularly to the fenestration area using a hard tissue cutting machine. Each section consists of two parts: the early artificial caries part before and after in vitro pH cycle and the remineralization cycle treatment part. The slices are about 250 pm thick, and then the slices are polished into slices about 100 pm thick using imported sanding paper under polishing machine flowing water. Finally, the slices washed with deionized water were exposed to CuK X-ray, 20kV, 20mA for 25s. After imaging, the images were analyzed with Transversal Microadiography Software 2006 (Inspektor Research Systems BV, Netherlands), so that the changes of caries depth and mineral contents of the samples were obtained.
Results: figure 3 display the results of surface microhardness test after circulation. The recovery percentage of surface microhardness value in the experimental group is significantly higher than that in the negative control group (P < 0.05). The results of cross-sectional microradiography are shown in figure 4-7. (1) Before and after the in vitro pH cycle, the surface layer of enamel caries in negative control group, i. e. DDW group, did not change significantly, nor did the depth of caries change significantly, while the surface layer of enamel caries in experimental group SH18 increased significantly, and the depth of caries decreased. Figure 4 displays the specific detection results; (2) Before and after in vitro pH cycle, the mineral loss of enamel samples in DDW group did not change significantly, while the mineral loss of enamel samples in polypeptide SH18 group decreased significantly, and there was a statistical difference compared to that before in vitro pH cycle treatment (P < 0.05), as shown in figure 5; (3) Before and after in vitro pH cycle, the caries depth of enamel samples in DDW group did not change significantly, while the caries depth of enamel samples in SH18 group significantly decreased, and there was statistical difference compared to that before the in vitro pH cycle treatment (P < 0.05), as shown in figure 6; (4) The mineral content of enamel samples in different caries depth after pH cycling in vitro showed that there was no significant difference between SH18 group and DDW group at 30 pm of caries surface layer, but the mineral content of enamel samples in SH18 group was significantly higher than that in DDW group at 30-100 tm of caries, and the difference was statistically significant (P < 0.05). In a word, the results of in vitro pH cycle confirm that the dual effect polypeptide SH18 has the function of promoting remineralization of demineralized enamel.
EXAMPLE 7 Study on stability of "dual-effect" polypeptide in saliva
At present, many studies on protein and polypeptide drugs show that, the structural stability of protein polypeptide drugs is of great significance to function exertion and long-term clinical research and application of them. Moreover, since the functional polypeptide designed and synthesized by the invention focuses on the research field of oral caries, it is particularly necessary to detect the structural stability of the saliva protein bionic anti-caries functional polypeptide designed and synthesized by the invention. For this reason, we measured the stability of the polypeptide by observing the retention rate of the polypeptide SH18 in saliva through reverse-phase high performance liquid chromatography.
The experimental steps are as follows:
1. Upon examination and approval by the Medical Ethics Society of Sichuan University, collecting saliva of healthy patients before meals in the morning and centrifuged at 12000 rpm and 4 °C for 20min.
2. Using a 0.45 pm filter element to filter bacteria and carrying out standing at 37 °C for 1h.
3. Adding the 512 pM polypeptide into saliva for incubation at 37 °C; and detecting the polypeptide content by reversed-phase high performance liquid chromatography at min, 15min and 30min.
Results are shown in figure 8, wherein the retention rate of polypeptide incubated in saliva for half an hour was still above 94%.
EXAMPLE 8 Cytotoxicity study of "dual-effect" polypeptide
The effect of polypeptide on the activity of human oral keratinocytes (HOK) was observed, so that whether the polypeptide had cytotoxicity was observed. The activity of HOK was determined by Cell Counting Kit-8 (CCK-8, China), and the test polypeptide was SH18.
The specific steps are as follows:
1. Inoculating HOKs in a 96-well plate with 2x103 cells per well, wherein the culture coverage area was about 50%; and using a DMEM medium with 20% fetal bovine serum (FBS) for culture.
2. Adding a culture solution containing a polypeptide with a concentration of MBC, 2MBC or 4MBC to cells, and carrying out culture of the polypeptide treated and untreated (negative control) cells in a C02 incubator (5% C02, 37C constant temperature) for 0.5, 1, 2, 4, 6 and 24 h, wherein aseptic deionized water was negative control.
3. Operating cells at each time point according to an Am-blue kit instruction manual.
4. Reading the value at 450nm using an enzyme labeling instrument.
The experimental results are shown in figure 9. The obtained data is the absorbance of the liquid in each well. The higher the absorbance is, the better the cell activity will be. When the polypeptide treatment concentration was MBC, 2MBC and 4MBC and the treatment time is 24 h, the polypeptide treatment had little effect on cell proliferation. After calculation by variance analysis, all the P values are higher than 0.05, which has no statistical significance, indicating that polypeptide treatment has no obvious effect on cell vitality.
In conclusion, the "dual-effect" anti-carious functional polypeptide with functions of resisting to bacteria and promoting remineralization of the invention have remarkable antibacterial effects in resisting to cariogenic bacteria, can inhibit the growth of Streptococcus mutans, inhibit the formation of Streptococcus mutans biofilm, and reduce the metabolic capacity of the formed Streptococcus mutans biofilm; it can promote remineralization of demineralized enamel and reduce caries depth and mineral loss of demineralized enamel; and in addition, the polypeptide has high stability and no obvious cytotoxicity. The polypeptide is of a high research value in the field of caries prevention and treatment.
The above content only involves the preferred embodiments of the present invention and is only illustrative and not limiting to the present invention; it should be understood by those skilled in that art, a lot of changes, modification, and even equivalents may be made thereto within the spirit and scope as defined by the claims of the present invention, but will fall within the protection scope of the present invention.
Sequence listing
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<212> PRT
< 213 > Artificial Sequence
<400> 3
Ser Ser Ser Ser Ser Ser Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu
1 5 10 15
Lys Lys
<210> 4
<211> 17
<212> PRT
< 213 > Artificial Sequence
<400> 4
Ser Ser Ser Ser Ser Gly Leu Leu Trp His Leu Leu His His Leu Leu
1 5 10 15
His
<210> 5
<211> 17
<212> PRT
< 213 > Artificial Sequence
<400> 5
Ser Ser Ser Ser Ser Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg
1 5 10 15
Arg
<210> 6
<211> 17
<212> PRT
< 213 > Artificial Sequence
<400> 6
Ser Ser Ser Ser Ser Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys
1 5 10 15
Lys
<210> 7
<211> 18
<212> PRT
< 213 > Artificial Sequence
<400> 7
Thr Thr Thr Thr Thr Thr Gly Leu Leu Trp His Leu Leu His His Leu
1 5 10 15
Leu His
<210> 8
<211> 18
<212> PRT
< 213 > Artificial Sequence
<400> 8
Thr Thr Thr Thr Thr Thr Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu
1 5 10 15
Arg Arg
<210> 9
<211> 18
<212> PRT
< 213 > Artificial Sequence
<400> 9
Thr Thr Thr Thr Thr Thr Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu
1 5 10 15
Lys Lys
Claims (10)
1. A dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, a polypeptide derivative or a polypeptide medicinal salt, wherein the polypeptide comprise an amino acid sequence a: Xn, X is S, T, Y, K, R, N or Q, and n is an integer selected from 5-8 for the number of amino acid repetitions; and
an amino acid sequence b which is an antibacterial peptide with an alpha double helix structure, positive charges, 8-12 amino acids, and alternate occurrence of hydrophilic and hydrophobic amino acids in the sequence.
2. The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt according to claim 1, wherein the amino acid sequence a is Xn, X is S or T, and n is 5 or 6.
3. The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt according to claim 1, wherein the amino acid sequence a is SSSSSS, SSSSS or TTTTTT.
4. The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt according to claim 1, wherein the amino acid sequence b is GLLWHLLHHLLH, LLRRLLRRLLRR or LLKKLLLKKLLKK, or a polypeptide having 80% or more identity to them.
5. The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt according to claim 1, wherein the polypeptide comprises one of the following amino acid sequences, preferably SEQ ID NO. 1:
SEQIDNO.1: SSSSSSGLLWHLLHHLLH;
SEQ ID NO.2: SSSSSSLLRRLLRRLLRR;
SEQ ID NO.3: SSSSSSLLKKLLKKLLKK;
SEQ ID NO.4: SSSSSGLLWHLLHHLLH;
SEQ ID NO.5: SSSSSLLRRLLRRLLRR;
SEQ ID NO.6: SSSSSLLKKLLKKLLKK;
SEQ ID NO.7: TTTTTTGLLWHLLHHLLH;
SEQ ID NO.8: TTTTTTLLRRLLRRLLRR;
SEQ ID NO.9: TTTTTTLLKKLLKKLLKKo
6. The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt according to claim 1, wherein the polypeptide derivative is a modifier of the C-terminal amidation of the polypeptide.
7. The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt according to claim 6, wherein the polypeptide derivative is a C terminal amidate of SEQ ID NO.1-9, preferably a C terminal amidate of SEQ ID NO.1: SSSSSSGLLWHLLHHLLH-NH2.
8. The dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt according to claim 1, wherein the salt includes, but is not limited to, hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate, organic amine salt, and the like.
9. A pharmaceutical composition, wherein the composition according to any more of claims 1-8 comprises the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt, as well as pharmaceutically acceptable carriers and/or auxiliary materials.
10. Application of the dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, the polypeptide derivative or the polypeptide medicinal salt in preparation of anti-caries drugs according to any one of claims 1-8.
-1/5-
Figure 1
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Figure 2
Figure 3
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Figure 4
Figure 5
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Figure 6
Figure 7
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Figure 8
Figure 9
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020102985A AU2020102985A4 (en) | 2020-10-23 | 2020-10-23 | Dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, polypeptide derivative and salt thereof, and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020102985A AU2020102985A4 (en) | 2020-10-23 | 2020-10-23 | Dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, polypeptide derivative and salt thereof, and application |
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| Publication Number | Publication Date |
|---|---|
| AU2020102985A4 true AU2020102985A4 (en) | 2020-12-24 |
Family
ID=73838805
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020102985A Active AU2020102985A4 (en) | 2020-10-23 | 2020-10-23 | Dual-effect anti-caries polypeptide with functions of resisting to bacteria and promoting remineralization, polypeptide derivative and salt thereof, and application |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2020102985A4 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716812A (en) * | 2021-01-11 | 2021-04-30 | 天津医科大学口腔医院 | Biofilm-resistant remineralizing material for root canal irrigation and blocking treatment of dental caries and preparation method thereof |
| CN114699325A (en) * | 2022-02-21 | 2022-07-05 | 南昌大学附属口腔医院(江西省口腔医院) | Novel enamel bonding method for preventing orthodontic tooth enamel damage |
-
2020
- 2020-10-23 AU AU2020102985A patent/AU2020102985A4/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716812A (en) * | 2021-01-11 | 2021-04-30 | 天津医科大学口腔医院 | Biofilm-resistant remineralizing material for root canal irrigation and blocking treatment of dental caries and preparation method thereof |
| CN114699325A (en) * | 2022-02-21 | 2022-07-05 | 南昌大学附属口腔医院(江西省口腔医院) | Novel enamel bonding method for preventing orthodontic tooth enamel damage |
| CN114699325B (en) * | 2022-02-21 | 2023-12-29 | 南昌大学附属口腔医院(江西省口腔医院) | Novel enamel bonding method for preventing orthodontic enamel damage |
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