CN111671971B

CN111671971B - Polypeptide single-layer film with 6% primary amino group exposure and preparation method and application thereof

Info

Publication number: CN111671971B
Application number: CN202010753429.3A
Authority: CN
Inventors: 李天铎; 许静; 马慧君; 班青; 张震
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2022-01-25
Anticipated expiration: 2040-07-30
Also published as: CN111671971A

Abstract

The invention provides a polypeptide monolayer film with a primary amino group exposure of 6% and a preparation method and application thereof. The polypeptide is composed of polypeptide molecules with a molecular weight of (1.48±0.2)×10 ⁵ g/mol, the thickness of the monolayer film is 14.6±0.1nm, and the exposure of primary amino groups on the surface of the film is 6±0.3%. The Zeta potential of the film was -4.48±0.1 mV; the contact angle of the film was 45±1°. The polypeptide monolayer film of the invention has good stability, the exposure of primary amino groups on the film surface is precisely controlled at 6.0±0.3%, can be applied to the surface coating material of oral implants, can carry antibiotics and antibacterial drugs and is stable Release, the local drug-loaded coating of the polypeptide monolayer film has the advantages of less systemic side effects and strong antibacterial properties. It is evenly and tightly combined with the pure titanium material, and is not easy to separate and fall off.

Description

Polypeptide single-layer film with 6% primary amino group exposure and preparation method and application thereof

Technical Field

The invention belongs to the field of natural polymers, relates to a polypeptide single-layer film, and a preparation method and application thereof, and particularly relates to a polypeptide single-layer film with 6% of primary amino group exposure, and a preparation method and application thereof.

Background

Collagen polypeptide is a water-soluble protein obtained by chemical thermal degradation of collagen. It is one of the most commonly used biopolymers due to its excellent biocompatibility, plasticity, viscosity, abundance and low cost. The collagen polypeptide is used as a biodegradable and renewable resource and is widely applied to preparation of medical materials, bionic materials, packaging and coating materials. The biological immobilized coating is often applied to the field of biological bionic scaffolds, solves the carrying problem of biomolecules such as enzymes, lactose, polydopamine and the like, drug molecules, synthetic macromolecules or organic small molecules, and has the advantages of easy and accurate control of carrying capacity and the like if the collagen polypeptide is prepared into a polypeptide single-layer film.

However, the thickness of the bio-immobilization coating in the prior art is thick and difficult to control, and the layer thickness of the bio-immobilization coating is generally larger than 100 nm. The collagen polypeptide molecules contain a plurality of polar groups such as amino groups, carboxyl groups, hydroxyl groups and the like, so that the collagen polypeptide molecules generate stronger intermolecular hydrogen bonds to form a net structure, and then form a brittle film after dehydration; in addition, the groups form hydrogen bonds with water molecules, so that the polypeptide film is easy to absorb water. These properties result in the collagen polypeptide material becoming brittle and readily soluble in water, limiting its use in some applications.

The secondary structure of natural biological macromolecules can influence the exposure of functional groups on polypeptide molecules, so that the physical and chemical properties of the surface of the membrane, such as chemistry, hydrophilicity and electrical properties, can be influenced, and the biological immobilized coating molecular layer can be applied to the fields of preparation of cardiovascular and cerebrovascular stents and the like by changing the chemical properties, the hydrophilicity and the electrical properties of the surface of the molecular layer membrane.

Limited by the complexity of natural biological macromolecular structure, the research on the surface properties of single-layer films of polypeptide molecules is rarely reported, so the application of the polypeptide molecules is limited. The research on the surface properties of the single-layer film of the polypeptide molecules is enhanced, so that the next modification of the polypeptide molecules is facilitated, and the defects of the polypeptide molecules can be further overcome.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a polypeptide single-layer film with 4% of primary amino group exposure, and a preparation method and application thereof. The invention improves the properties of surface charge, hydrophilicity and hydrophobicity and the like of the film by changing the exposure of primary amino group on the surface of the polypeptide single-layer film, takes the polypeptide single-layer film as the medicine-carrying coating of the oral implant, and can further provide a foundation for realizing controllable grafting of various biological agents.

In the present invention, the exposure of the primary amino group means: primary amino group molar amount per collagen polypeptide (g).

In order to achieve the purpose, the invention adopts the following technical scheme:

with 6% primary amino group exposureA polypeptide monolayer wherein the polypeptide has a molecular weight of (1.48. + -. 0.2). times.10⁵The single-layer film is 14.6 +/-0.1 nm in thickness, the primary amino group exposure on the film surface is 6 +/-0.3%, and the Zeta potential of the polypeptide single-layer film is-4.48 +/-0.1 mV; the contact angle of the film was 45 ± 1 °.

Preferably, the polypeptide is a collagen polypeptide.

Preferably, the composition of the amino acids of the polypeptide is glycine (Gly): 7.30 +/-0.5%; valine (Vla): 17.48 plus or minus 0.5 percent; isoleucine (Ile): 36.97 +/-0.5%; leucine (Leu): 13.85 plus or minus 0.5 percent; tyrosine (Tyr): 2.68 plus or minus 0.5 percent; phenylalanine (Phe): 1.5 plus or minus 0.5 percent; lysine (Lys): 4.41 plus or minus 0.5 percent; histidine (His): 0.45 plus or minus 0.5 percent; arginine (Arg): 3.45 plus or minus 0.5 percent; proline (Pro): 5.96 plus or minus 0.5 percent; cysteine (Cys): 5.95 +/-0.5 percent.

Preferably, the secondary structure content of the monofilm polypeptide is: the alpha-helix is 9.93 plus or minus 0.16 percent; the beta-sheet is 35.45 plus or minus 0.18 percent; beta-turn is 19.55 plus or minus 0.13%; random coil is 35.34 + -0.24%. The invention adopts a Circular Dichrograph (CD) to characterize the secondary structure of the single-layer membrane polypeptide.

Preferably, the primary amino group exposure of the film surface is 5.78%.

Preferably, the monolayer of the polypeptide consists of closely packed nanoparticles, and the average particle size of the spherical nanoparticles is 40 +/-2 nm.

The invention also provides a composite membrane containing the polypeptide monolayer membrane: the film comprises a polyethyleneimine film and a polypeptide single-layer film, wherein polyethyleneimine and polypeptide molecules are combined through ionic bonds, the thickness of the polyethyleneimine film is 0.25-0.38 nm, and the thickness of the polypeptide single-layer film is 14.6 +/-0.1 nm.

The invention also provides a preparation method of the polypeptide monolayer film, which is characterized by comprising the following steps:

(1) preparing a polypeptide solution at a certain temperature, adding a surfactant Sodium Undecyl Sulfate (SUS) to obtain a polypeptide-SUS mixed solution, and keeping the temperature for later use, wherein the concentration of SUS in the mixed solution is 6.52 mmol/L;

(2) polishing the surface of the titanium sheet, immersing the titanium sheet into a mixed acid solution for treatment, flushing the titanium sheet to be neutral, drying the titanium sheet after drying the titanium sheet by using nitrogen;

(3) immersing the dried titanium sheet into a Polyethyleneimine (PEI) aqueous solution for treatment, washing with water, drying by blowing with nitrogen, and drying to obtain a positive ionization titanium sheet deposited with PEI;

(4) and (2) immersing the positively ionized titanium sheet into the polypeptide-SUS mixed solution obtained in the step (1), depositing for 8-12 min, then pulling the titanium sheet in deionized water for 20-25 times, and blow-drying by using high-purity nitrogen gas to obtain the polypeptide single-layer film.

Preferably, the temperature in step (1) and the deposition process temperature in step (4) are both 50 ℃.

Preferably, in step (1), the concentration of the collagen polypeptide solution is 4% wt.

Preferably, in step (1), the preparation method of the collagen polypeptide solution comprises: mixing collagen polypeptide and deionized water, swelling at room temperature for 0.5 hr, heating to 50 deg.C, stirring for 2 hr until collagen polypeptide is completely dissolved; the pH was then adjusted to 10.00. + -. 0.02.

Preferably, in the step (2), after the titanium sheet is polished by using metallographic abrasive paper, the titanium sheet is ultrasonically cleaned by deionized water, absolute ethyl alcohol and acetone for 15min respectively in sequence, then dried by using high-purity nitrogen and dried in an oven at 60 ℃ for 12 h. Further preferably, the grinding and polishing method comprises the following steps: and (3) sequentially grinding and polishing by using metallographic abrasive paper according to the sequence of 800, 1500, 3000, 5000 and 7000 meshes.

Preferably, in the step (2), the mixed acid solution is 30% H by mass with the volume ratio of 1:1₂O₂And 98% H₂SO₄The treatment time of the mixed solution of (1) was 1 hour.

Preferably, in the step (3), the treatment time of the titanium sheet in the PEI aqueous solution is 20-40 minutes.

According to the invention, the collagen polypeptide with a regular structure is obtained by a commercial polypeptide product through a dialysis method.

The existing oral implant (dental implant) is generally made of pure titanium material. The antibacterial modification is carried out on the surface of the titanium implant, and the loaded local drug-loaded coating has the advantages of small toxic and side effects on the whole body, strong antibacterial property and the like.

The invention also provides application of the polypeptide single-layer film as a local drug-loaded coating in a dental implant made of pure titanium material.

The polypeptide single-layer film can be loaded with antibiotics such as gentamicin and metronidazole and broad-spectrum antibacterial agents such as chlorhexidine and chloroxylenol, can stably release the drugs, and has the drug loading rate of about 5-6 mu g/mm. The polypeptide single-layer film constructs a local drug-loaded antibacterial coating on the surface of the oral implant, and the implant is endowed with antibacterial performance through surface modification, so that postoperative infection is prevented or early intervention treatment is carried out on peri-implantitis.

The polypeptide single-layer film local drug-loaded coating has the advantages of small toxic and side effects on the whole body, strong antibacterial property and the like, is uniformly and tightly combined with a pure titanium material, and is not easy to separate and fall off.

The invention has the beneficial effects that:

the polypeptide single-layer film has good stability, the exposure of primary amino group on the surface of the film is accurately controlled to be 6.0 +/-0.3%, and the polypeptide single-layer film can be applied to a coating material on the surface of an oral implant, can carry antibiotics and antibacterial drugs and can be stably released. Has certain hydrophilic property, can improve the attachment and proliferation of cells at the planting position, and promotes the healing of the injury position.

The polypeptide single-layer film can carry antibiotics such as gentamicin and metronidazole and broad-spectrum antibacterial agents such as chlorhexidine and chloroxylenol, and can stably release the medicines. The polypeptide single-layer film constructs a local drug-loaded antibacterial coating on the surface of the oral implant, and the implant is endowed with antibacterial performance through surface modification, so that postoperative infection is prevented or early intervention treatment is carried out on peri-implantitis.

Drawings

FIG. 1 is a graph of the effect of polypeptide concentration on ovality;

FIG. 2 is an AFM image of a collagen polypeptide molecular layer film obtained with 4% concentration of collagen polypeptide;

FIG. 3 is the fluorescence intensity for different numbers of pulls;

FIG. 4 is an AFM image of a monolayer film G-SUS of the polypeptide;

FIG. 5 is a high resolution N1s XPS spectrum (a, G-SUS, b, G-SDS) of a single layer film of a polypeptide_6％C, 4% polypeptide film);

FIG. 6 is the product Tetraphenylethylene (TPE) -Isothiocyanate (ITC) TPE-CH₃(a),TPE-N₃(b) TPE-ITC (c)¹H NMR spectrum;

FIG. 7 shows the result of CCK-8 detection of polypeptide monolayer G-SUS;

FIG. 8 shows MTT detection results of a polypeptide single-layer film G-SUS;

FIG. 9 is a bar graph of cell survival (a, control, b, G-SUS) and the fraction of cells surviving each group after cell cloning experiments with different samples;

FIG. 10 is fluorescence microscope images (a, b) of a G-SUS polypeptide single-layer membrane before and after immersion in physiological saline for 7 days; fluorescence microscope image (c) of the sample after 15 days in the incubator.

The specific implementation mode is as follows:

the collagen polypeptide used in the examples of the present invention is a commercially available polypeptide product (A.R.) having a molecular weight of about 5.00X 10⁴～1.80×10⁵g/mol, polypeptide (1.48 + -0.2). times.10) with molecular weight obtained by dialysis⁵g/mol. Other reagents not specifically mentioned are common commercial products.

Collagen polypeptide is amphoteric polyelectrolyte, and can be agglomerated into spherical particles at isoelectric point. By utilizing the aggregation behavior of the collagen polypeptide, the collagen polypeptide with small molecular weight passes through the semipermeable membrane by adjusting factors such as temperature, concentration, pH, ionic strength and the like, thereby achieving the aim of separating from the collagen polypeptide with larger molecular weight. The research results of gel electrophoresis and laser particle size analyzer show that the dialysis bag with 5 ten thousand specifications has collagen polypeptide dialysis concentration of 2%, dialysis temperature of 45 deg.C, and NaCl concentration of 0.9 mol.L^-1Can prepare collagen polypeptide with narrow molecular weight distribution.

Collagen polypeptide CP, CA, M before and after dialysis_WComparison with Isoelectric Point (IP) is shown in Table 1, and the amino acid type ratio before and after dialysis is shown in Table 2. GPC test nodeThe results show the weight average molecular weight M of the dialyzed collagen polypeptide_w＝1.48×10⁵g·mol^-1，M_w/M_n1.43. The Content of Protein (CP) in the collagen polypeptide is 83.38% and the content of amino acid (CA) is 4.95 × 10 measured by Kjeldahl method^-4mol·g^-1The primary amino group quantifier shows that the dialyzed collagen polypeptide molecule contains 4.95 multiplied by 10 according to the measurement result at 50 DEG C^-4g·mol^-1The molecular structure of the collagen polypeptide is not obviously changed before and after dialysis. Preparing collagen polypeptide into 5% water solution with conductivity of 5.98 μ S cm^-1The self conductivity of the deionized water is 2.06 mu S cm^-1The above results indicate that the collagen polypeptide having a small molecular weight and the inorganic salts mixed in the collagen polypeptide are dialyzed out.

Table 1.

Table 2.

Example 1

A preparation method of a polypeptide monolayer film comprises the following steps:

(1) preparing 50mL of collagen polypeptide solution with the concentration of 4% wt: accurately weighing collagen polypeptide in 100mL of the solution in a three-neck flask, accurately weighing deionized water, pouring the deionized water into the three-neck flask, swelling the solution at room temperature for 0.5h, putting the three-neck flask into a water bath at 50 +/-1 ℃, heating and stirring the solution for 2h to completely dissolve the collagen polypeptide, then adjusting the pH of the solution to 10.00 +/-0.02 by using 2mol/L of sodium hydroxide, and stabilizing the solution in the water bath for 0.5 h.

(2) Adding a surfactant SUS into the collagen polypeptide solution to obtain a collagen polypeptide-SUS mixed solution, wherein the concentration of SUS in the mixed solution is 6.52mmol/L (at 50 ℃, the mass fraction of SUS in the mixed solution is 6%); and stabilizing in a water bath for 6h for later use.

(3) Cutting size of 1cm × 1cAnd (3) sequentially grinding and polishing a rectangular titanium sheet with the m multiplied by 1mm by using metallographic abrasive paper according to the sequence of 800, 1500, 3000, 5000 and 7000 meshes, sequentially ultrasonically cleaning the titanium sheet by using deionized water, absolute ethyl alcohol and acetone for 15min respectively, blow-drying by using high-purity nitrogen, and drying in an oven at 60 ℃ for 12h for later use. Preparation of 30% H₂O₂And 98% H₂SO₄Cooling the mixed acid solution with the volume ratio of 1:1 to room temperature, treating the treated titanium sheet for 1 hour by using the mixed acid, then washing the titanium sheet to be neutral by using tap water, washing the titanium sheet for 5 times by using deionized water, finally drying the titanium sheet for 12 hours in a 60 ℃ oven after drying the titanium sheet by using high-purity nitrogen for later use.

(4) Preparing 1mg/mL PEI polyethyleneimine aqueous solution, treating the acid-etched titanium sheet with the PEI solution for 0.5h at room temperature, cleaning the titanium sheet with deionized water for 5 times to remove the electric charge which is not firmly bonded, and finally drying the titanium sheet with high-purity nitrogen in an oven at 60 ℃ for 12h for later use. Putting the positively ionized titanium sheet into a deposition box, respectively pouring the prepared polypeptide solutions of different systems into the deposition box, depositing for 10min at 50 ℃, then pulling the titanium sheet in deionized water for 20 times, drying the titanium sheet by using high-purity nitrogen, and storing the titanium sheet in nitrogen.

The obtained polypeptide monolayer was labeled as G-SUS.

Comparative example 1

Preparing a collagen polypeptide solution with the concentration of 1-5 wt%, calculating the mass of the required collagen polypeptide and the volume of deionized water, accurately weighing the collagen polypeptide in a 50mL three-neck flask, accurately weighing the deionized water, pouring the deionized water into the three-neck flask, swelling for 0.5h at room temperature, heating and stirring the three-neck flask in a water bath at 50 ℃ for 2h to completely dissolve the collagen polypeptide, and then adjusting the pH of the solution to 10.00 +/-0.02 by using 1mol/L sodium hydroxide for later use.

The collagen polypeptide solutions of different concentrations are subjected to circular dichroism spectroscopy (CD) characterization, usually with a molar extinction coefficient difference Δ ε (M)^-1·cm^-1) And molar ellipticity θ to measure the magnitude of circular dichroism. The CD test was performed on a Chirascan system (photophysics, UK) using a nitrogen purge at a flow rate of 35 mL/min. The protein concentration in all solutions was diluted to 0.16mg/mL and the mixed samples equilibrated at 50 ℃ 1h, while measuring at 50 ℃ 200. mu.L of the solution in a 1mm sample cell, the measurement temperature was maintained at 50 ℃. Recording the spectrum in the range of 190-260 nm, the resolution is 0.2nm, and scanning is carried out for 6 times. Data processing: the spectra of the buffer solution were subtracted to correct for baseline, the CD spectra were normalized in molar ovality, and the secondary structure content was calculated using peak regression calculation and continue fitting program. The results of the effect of polypeptide concentration on its secondary structure are shown in FIG. 1 and Table 3.

TABLE 3

As shown in Table 3 and FIG. 1, the structures of α -helix, Antiparallell β -sheet and parallell β -sheet show a tendency of increasing first and then decreasing as the mass concentration of the polypeptide increases from 1% to 5%, and the maximum is reached at a concentration of 4%; the beta-turn, random coil structure shows a tendency of decreasing first and then increasing, reaching a minimum at a concentration of 4%. The results indicate that at 4% the secondary structure of the polypeptide molecule is greatly changed. This concentration is well at the interface between the contact concentration of the polypeptide molecule and the entanglement concentration. Therefore, in the present invention, when preparing a collagen polypeptide monolayer film, the mass concentration of the polypeptide is preferably 4%.

Comparative example 2

Compared with the embodiment 1, the difference of the preparation method of the polypeptide single-layer film is that no surfactant is added in the preparation process of the single-layer film, only the polypeptide is deposited on the positively ionized titanium sheet, and the other conditions are the same as the embodiment 1.

The polypeptide solution with the concentration of 4% is deposited on a titanium metal sheet treated by PEI, the deposition temperature is 50 ℃, the deposition time is 10min, the lifting frequency is 20 times, and the polypeptide molecules are loosely arranged, which is shown in figure 2 in detail. The resulting polypeptide monolayer was labeled G.

Comparative example 3

(2) Adding a surfactant SDS into the collagen polypeptide solution to obtain a collagen polypeptide-SDS mixed solution, wherein the concentration of SDS in the mixed solution is 8.32 (6% wt) mmol/L; and stabilizing in a water bath for 6h for later use.

(3) Cutting a rectangular titanium sheet with the size of 1cm multiplied by 1mm, using metallographic abrasive paper to sequentially polish and polish the titanium sheet according to the order of 800, 1500, 3000, 5000 and 7000 meshes, sequentially using deionized water, absolute ethyl alcohol and acetone to ultrasonically clean the titanium sheet for 15min respectively, then using high-purity nitrogen to blow dry the titanium sheet, and drying the titanium sheet in an oven at the temperature of 60 ℃ for 12h for later use. Preparation of 30% H₂O₂And 98% H₂SO₄Cooling the mixed acid solution with the volume ratio of 1:1 to room temperature, treating the treated titanium sheet for 1 hour by using the mixed acid, then flushing the titanium sheet to be neutral by using tap water, cleaning the titanium sheet for 5 times by using deionized water, finally drying the titanium sheet in a 60 ℃ oven for 12 hours for later use after drying the titanium sheet by using high-purity nitrogen

The obtained single-layer membrane of the polypeptide is marked as G-SDS_6％。

1. Polypeptide monolayer thickness determination

After collagen polypeptide is deposited by the PEI-treated titanium sheet, the-COO in the polypeptide molecule^-with-NH in PEI₃ ⁺Strong ionic bonds can be formed. To verify that the collagen polypeptide molecule is adsorbed to the collagen polypeptide via ionic bond rather than physical bondAnd (3) bottom binding, and measuring the fluorescence intensity of the polypeptide monolayer film at different pulling times in the deposition process. As the number of times of pulling increases (5 to 20 times), the polypeptide physically adsorbed to the substrate is washed away and the polypeptide bound by ionic bonds is firmly immobilized on the substrate. As can be seen from FIG. 3, after 15 times of pulling, the fluorescence intensity was no longer decreased, indicating that the collagen polypeptide physically adsorbed to the substrate was removed.

The film surface morphology was studied using a Multimode8 type AFM (Bruker, Germany). And (3) placing the polypeptide monolayer film sample on a workbench, and testing the appearance of the sample in a Peak Force mode. Measurement of film thickness: when the polypeptide single-layer film is prepared by using a deposition method, a tin foil is used for wrapping half of the titanium sheet, so that the titanium sheet is not polluted by the solution. In the test, the boundary of the titanium sheet was found by an optical auxiliary system provided in an atomic force microscope, and then the test range was set to 20 μm so as to span the substrate and the sample region, and an AFM tip was used to scan along the boundary, and 3 different regions were scanned from the height corresponding to the film substrate up to the bottom of the boundary to obtain an average film thickness. The scanning speed is 0.977Hz, the scanning ranges are 20 μm, 10 μm, 5 μm and 1 μm, and the data processing software is NanoScope Analysis carried by AFM.

Single-layer film of the polypeptide obtained in comparative example 3 (G-SDS)_6％) Has an average thickness of about 14.2nm, and the collagen polypeptide monolayer is composed of closely packed nanoparticles having an average particle diameter of about 60 nm. The thickness of the G-SUS polypeptide monolayer film obtained in the example of the invention is 14.6 +/-0.1 nm, the average particle size of the spherical nanoparticles is about 40nm, and AFM images of the G-SUS polypeptide monolayer film are shown in Table 4.

2. Determination of primary amino group exposure on surface of polypeptide single-layer membrane

The samples obtained in example 1 and comparative examples 2 and 3 were subjected to XPS characterization, and the N element thereof was subjected to peak separation treatment. High resolution spectra of N1s core region (from 396 to 402eV) and primary amino group exposure as shown in fig. 5. Primary amino group exposure: G-SUS, 5.78%; G-SDS 6%, 13.13%; g, 2.89%. The binding energy of the primary amine was 400.05eV, the amide bond 398.89eV, and the secondary amine was 398.26 eV. XPS data allows semi-quantitative analysis of functional groups by detecting changes in binding energy and local chemical state. The results of using CasaXPS to peak N1s high resolution spectra and calculating primary amino group content XPS and Raman show that amino group exposure in a single film of collagen polypeptide is not only related to increased β -sheet and random coil structures, but also to non-covalent interactions of collagen polypeptide and surfactant.

3. Determination of film surface wettability and Charge Properties

The water Contact Angle (CA) of the sample was measured at room temperature using a DSA-100 type optical contact angle measuring instrument (Kruss Co., Germany) for the film sample. 2mL of deionized water was dropped onto the sample using an automatic dispensing controller and CA was automatically determined using the Laplace-Young fitting algorithm. The average CA value was obtained by measuring the sample at five different positions, and an image was taken with a digital camera (sony corporation, japan). The Zeta potential of the membrane surface was determined using a surfass electrokinetic solid surface analyzer.

1mM Na was used₂SO₄The Zeta potential of the membrane surface was measured using the solution as an electrolyte. Zeta potential of 4 wt.% polypeptide monolayer film: -15.6 mV; G-SDS_6％Zeta potential of polypeptide monolayer film: 4.907 mV; the potential of the single-layer film of G-SUS polypeptide was-4.48 mV. The result shows that 4 wt.% of polypeptide monolayer membrane has lower potential, poor hydrophilicity and easy adsorption of protein and the like, and can not be used for medicine loading in organisms; G-SDS_6％The nano-particles have high potential and strong hydrophilicity, are not easy to adsorb protein, can promote cell activity, and can be used for in vivo drug loading such as cardiovascular and cerebrovascular stent coatings; the G-SUS polypeptide single-layer membrane potential is between the two, can not be used for intravascular drug loading, but can be used as an oral antibacterial drug carrier and applied to an oral implant drug loading coating.

The wettability of the surface of the polypeptide monolayer film can be directly reflected by the contact angle value of water. The pure Ti sheet shows hydrophobicity, the contact angle is 101.4 +/-0.2 degrees, and the contact angle displayed on the surface of the 4 wt.% collagen polypeptide single-layer film is 56.1 +/-1.2 degrees. The contact angle of the surface of the single-layer film of the G-SUS collagen polypeptide is about 45 degrees. And G-SDS_6％The contact angle of the polypeptide single-layer film is 10 degrees. The G-SUS polypeptide single-layer film under the exposure has weaker hydrophilicity, and can ensure the stable release of antibacterial drugs.

4. Calculation of content of secondary structure of polypeptide in polypeptide monolayer

The content of secondary structures in the SUS polypeptide single-layer film, alpha-helix, was characterized by using a Circular Dichrograph (CD) to be 9.93 +/-0.16%; the beta-sheet is 35.45 plus or minus 0.18 percent; beta-turn is 19.55 plus or minus 0.13%; random coil is 35.34 + -0.24%. The increase of the beta-sheet structure can help to reduce the thickness of the collagen polypeptide layer, is beneficial to the extension of the polypeptide molecular chain, and promotes the exposure of primary amino groups. The amount of primary amino group exposed influences the zeta potential, contact angle and other properties.

5. Characterization of primary amino distribution points on membrane surface

And (3) probe synthesis: synthesis of Tetraphenylethylene (TPE) -Isothiocyanate (ITC) as a primary amino group-responsive fluorescent probe molecule, the primary amino group distribution on the surface of the polypeptide monolayer film is visually characterized. In particular to an adduct of 1- [4- (methyl isothiocyanate) phenyl ] -1,2, 2-triphenylethylene (TPE-ITC), Tetraphenylethylene (TPE) and Isothiocyanate (ITC).

The synthesis steps are as shown in the formula (1), and are divided into 5 steps: (ii) in a 250mL two-necked round-bottom flask, in N₂Next, 5.05g (30mmol) of diphenylmethane was dissolved in 100mL of distilled tetrahydrofuran. After the mixture was cooled to 0 deg.C, 15mL (2.5M in hexane, 37.5mmol) of n-butyllithium were slowly added via syringe. The mixture was stirred at 0 ℃ for 1 hour. 4.91g (25mmol) of 4-methylbenzophenone were then added to the reaction mixture. The mixture was warmed to room temperature and stirred for 6 hours. Compound 3 was synthesized.

② the reaction mixture was quenched with a saturated ammonium chloride solution and then extracted with carbon dichloride. The organic layer was collected and concentrated. The crude product and 0.20g of p-toluenesulfonic acid were dissolved in 100mL of toluene. The mixture was heated to reflux for 4 hours. After cooling to room temperature, the reaction mixture was extracted with carbon dichloride. The organic layer was collected and concentrated. The crude product was purified by silica gel chromatography using hexane as eluent to give 4 as a white solid.

③ in a 250mL round bottom flask, a solution of 5.20g (15.0mmol) of 4, 2.94g (16.0mmol) of N-bromosuccinimide, 0.036g of benzoyl peroxide in 80mL of carbon tetrachloride was refluxed for 12 hours. After completion of the reaction, the mixture was extracted with dichloromethane and water. The organic layers were combined and dried over anhydrous magnesium sulfate. The crude product was purified by silica gel chromatography using hexane as eluent to give 5 as a white solid.

Tetra (R) in a 250mL two-necked round-bottom flask, in N₂Next, 1.70g (4mmol) of 5 and 0.39g (6mmol) of sodium azide were dissolved in dimethyl sulfoxide. The mixture was stirred at room temperature overnight (25 ℃, 48 h). Then a large amount (100mL) of water was added and the solution was extracted three times with ether. The organic layers were combined and dried over anhydrous magnesium sulfate. The crude product was purified by silica gel chromatography using hexane/chloroform (v/v ═ 3:1) as eluent to give 6 as a white solid.

Fifthly, the azido-functionalized tetraphenylethylene (6; 0.330g, 0.852mmol) and triphenylphosphine (0.112g, 0.426mmol) were added to a two-necked flask, which was evacuated under vacuum and flushed with dry nitrogen three times. Carbon disulfide (0.55g, 7.242mmol) and distilled dichloromethane (50mL) were added to the flask and stirred. The resulting reaction mixture was refluxed overnight, and then the solvent was removed under reduced pressure. The crude product was precipitated with cold ether (250mL), filtered and washed three times. And finally, drying the product in vacuum to obtain TPE-ITC which is a white solid.

The synthesized product (tetraphenylethylene (TPE) -Isothiocyanate (ITC)) was first subjected to nuclear magnetic hydrogen spectroscopy characterization. Of the product¹H NMR was obtained by AVANCE II 400 NMR spectrometer (Bruker, Germany) by placing 0.5cm of sample to be tested into a nuclear magnetic tube, adding 0.6mL of deuterated chloroform to dissolve it completely, measuring by manual shimming at room temperature with Tetramethylsilane (TMS) as internal standard, and scanning 64 times¹The H NMR spectrum was processed using MestReNova software, and the results are shown in fig. 6.

Characterization of the product by NMR spectroscopy (FIG. 6a)¹H NMR(CDCl₃400MHz), δ (TMS, ppm): 7.15-6.98(m, 15H), 6.89(s, 4H), 2.24(s, 3H); (FIG. 6b)¹H NMR(CDCl₃，400MHz)，δ(TMS，ppm)：7.12-6.90(m，19H)，4.24(s，2H) (ii) a (FIG. 6c)¹H NMR(400MHz，CDCl₃) δ (ppm): 6.90-7.15(m, 19H), 4.61(s, 2H). For example, due to the resonance of the methylene unit between the TPE and ITC units, the product is¹The H NMR (fig. 6c) spectrum shows a peak at δ 4.16.

Synthesis of TPE-ITC molecular probes for primary amino imaging and functionalization is demonstrated, where the reactive ITC groups are sensitive to primary amino groups. Therefore, TPE-ITC is a typical fluorescent molecule with aggregation-induced emission (AIE) properties. The AIE properties of TPE-ITCs allow TPE-polypeptide bioconjugates to produce intense fluorescence by attaching a large number of AIE tags to the collagen polypeptide chains. The fluorescence output of the bioconjugates can be greatly increased (up to 2 orders of magnitude) by simply increasing their Degree of Labeling (DL). The AIE probe strategy is an efficient method for real-time observation of primary amino groups. Its advantages are simple operation, low cost and high efficiency. Furthermore, further tuning of the structure of the AIE fluorophore will still help in the development of specific probes for surface functional group detection.

The primary amino group on the surface of the collagen polypeptide membrane is marked by the synthesized TPE-ITC, and the marking process is shown as the formula (2).

The marking method comprises the following specific steps: preparing TPE-ITC/DMSO solution with concentration of 0.8mg/mL, sucking 0.5mL of the solution by using a 1mL syringe, and dripping 9 drops of the solution to 5mL of Na₂CO₃/NaHCO₃And (4) in the buffer solution, carrying out ultrasonic treatment on the mixed solution for 10min, and uniformly dispersing. And (2) placing the polypeptide single-layer film into a deposition box, slowly pouring the ultrasonic mixed solution into the deposition box, reacting for 2 hours at 50 ℃, pulling in DMSO for 10 times to remove the unlabeled TPE-ITC after the reaction is finished, and finally drying by using high-purity nitrogen and storing in nitrogen.

Laser scanning confocal microscope (CLSM) images of the samples were obtained from a TCS SP8 STED 3X confocal laser scanning microscope (laika, germany) equipped with an argon ion laser and two photomultiplier tubes. A resonant scanner is used with an ultra-sensitive HyDTM probe. Exciting the sample by using 405nm laser, and detecting fluorescence at 430-493 nm. The results show that CLSM results are consistent with those of XPS analysis.

6. Membrane biocompatibility testing

The membrane samples were assayed for cellular compatibility using cholecystokinin octapeptide (CCK-8) and tetramethylazodicarbonyl blue (MTT). The test material was prepared in the same size as the wells in a 12-well cell culture plate. Pure Ti sheet and G-SUS single layer film samples were placed in the wells, using three parallel wells per sample. Human umbilical vein endothelial cells (HUVECs, 5X 10)⁵cells/mL) were seeded in each well at 37 ℃ with 5% CO₂And 10% Fetal Bovine Serum (FBS) in RPMI 1640 medium for 24 hours. Subsequently, the cells were washed twice with the serum-free essential Medium Eagle (MEM), and 15. mu.L of CCK-8 solution was added to each well containing 100. mu.L of serum-free MEM. At 37 deg.C, 5% CO₂After 1h of incubation, 100. mu.L of the mixture was transferred to another 12-well plate because the residual G-SUS monolayer affects the absorbance value at 450 nm. The absorbance of the mixed solution was measured at 450nm using an iMark microplate reader with 655nm as reference, and wells containing cells and medium only were used as controls. The cell viability calculation formula is as follows:

Viability_CCK-8＝(Sample abs_450-655nm/Positive control abs_450-655nm)×100

HUVECs cell viability was determined by MTT assay in addition to CCK-8 assay. The cell viability was calculated by the following formula. Non-single membrane cells were used as controls.

Viability_MTT＝(Sample abs_570-655nm/control abs_570-655nm)×100

The results of CCK-8 analysis showed that the presence of G-SUS as a modified surface had no effect on cell viability and growth compared to the control group (FIG. 7). MTT assay results also showed that G-SUS single-layer membrane was almost non-toxic to HUVEC (fig. 8).

Cell cloning experiments: MCF-7 cells were cultured in 60mm dishes at 37 ℃ with 5% CO₂And DMEM for 24 hours, then the cells were subjected to 2 different treatments: blank control group and G-SUS sheetA membrane. After 8h, cells were washed 3 times with PBS buffer (10mM, pH 7.4). Subsequently, the cells were incubated at 37 ℃ in fresh cell culture medium at 5% CO₂DMEM for another 10 days, then fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. More than 50 colonies per cell were counted. The mean survival score was obtained from three parallel experiments.

Survival score ═ (number of colony forming cells)/(number of cell inoculation × inoculation efficiency)

During the culture process, G-SUS showed the highest cell attachment and proliferation ability due to the exposure of amino group, which is advantageous for the viability of cells. After two different treatments of the cells (control, G-SUS repeated twice), colonies of cells were counted after 8 hours (fig. 9). The number of colonies in the control group, G-SUS group, was only slightly different, which indicates that trace amounts of surfactant in the single-layer membrane of the pro-polypeptide had no effect on cell viability. The polypeptide monolayer film obtained by the invention has excellent cell compatibility on the surface.

7. Membrane stability test

The stabilization of the collagen polypeptide monolayer was carried out on a DMI3000B inverted fluorescence microscope (come, germany) equipped with a leia DFC 450C type CCD. After the polypeptide single-layer membrane G-SUS is placed in normal saline at room temperature and soaked for 7 days, a sample is dried by using high-purity nitrogen for later use. The G-SUS is continuously placed in a biochemical incubator at 40 ℃ for soaking for 15 days, and then is dried by high-purity nitrogen for later use. Before observation, the fluorescent module is opened, and the machine is preheated for 15min before use. The method comprises the following steps of cleaning a glass slide, taking a sample to be detected on the cleaned glass slide, placing the sample to be detected on an objective table for fixation, roughly adjusting the height of the objective table, finely adjusting focusing, finding the clearest sample details in a bright field, observing the fluorescent point distribution condition by using a fluorescence module, observing the fluorescent point distribution condition by using 50X, amplifying the multiple in sequence, observing the fluorescent point distribution, comparing the fluorescent point distribution condition before and after soaking the collagen polypeptide single-layer film, and analyzing the stability of the collagen polypeptide single-layer film intuitively. The results are shown in fig. 10, where the distribution of green fluorescence was not reduced after one week of soaking; the samples were placed in an incubator at 40 ℃ for 15 days, and the distribution of fluorescence spots did not change significantly. Combining the above results, it can be concluded that a relatively stable G-SUS monolayer was formed on the Ti surface due to electrostatic and other non-covalent interactions between PEI and collagen polypeptides.

Claims

1. the preparation method of the polypeptide monolayer film that a primary amino group exposure amount is 6%, is characterized in that, comprises the following steps:

(1) At a certain temperature, prepare a collagen polypeptide solution with a concentration of 4% wt, and then add the surfactant sodium undecyl sulfate to obtain a polypeptide-sodium undecyl sulfate mixed solution. The concentration of sodium monoalkyl sulfate is 6.52mmol/L;

(2) Grind and polish the surface of the titanium sheet, immerse it in a mixed acid solution, rinse it to neutrality, dry it with nitrogen, and then dry it;

(3) immersing the dried titanium sheet in an aqueous solution of polyethyleneimine for treatment, rinsing with water, drying with nitrogen, and then drying to obtain a positively ionized titanium sheet deposited with polyethyleneimine;

(4) Immerse the positively ionized titanium sheet in the polypeptide-sodium undecyl sulfate mixed solution obtained in step (1), deposit it for 8-12 minutes, and then pull it in deionized water for 20-25 times, using high-purity After drying with nitrogen, the polypeptide monolayer film was obtained;

The polypeptide is composed of polypeptide molecules with a molecular weight of (1.48±0.2)×10 ⁵ g/mol, the thickness of the monolayer film is 14.6±0.1nm, and the exposure of primary amino groups on the surface of the film is 6±0.3%. The Zeta potential of the film was -4.48±0.1mV; the contact angle of the film was 45±1°;

The secondary structure content of the polypeptide in the polypeptide monolayer film is: α-helix is 9.93 ± 0.16%; β- sheet is 35.45 ± 0.18%; β- turn is 19.55 ± 0.13%; random coil is 35.34 ± 0.24%.

2 . The preparation method according to claim 1 , wherein the temperature in step (1) and the deposition process temperature in step (4) are both 50° C. 3 .

3 . The preparation method according to claim 1 , wherein in step (1), the preparation method of the collagen polypeptide solution is as follows: mixing collagen polypeptide and deionized water, swelling at room temperature for 0.5 hours, and heating to 50° C. 4 . , and stirred for 2 hours, the collagen polypeptide was completely dissolved; then the pH was adjusted to 10.00 ± 0.02.

4. The preparation method according to any one of claims 1 to 3, wherein in step (2), after the titanium sheet is ground and polished with metallographic sandpaper, deionized water, absolute ethanol and acetone are used for ultrasonic cleaning in sequence. The titanium sheets were dried for 15 min each, then blown dry with high-purity nitrogen, and then dried in an oven at 60 °C for 12 h.

5 . The preparation method according to claim 4 , wherein in step (2), the grinding and polishing method is as follows: using metallographic sandpaper in the order of 800, 1500, 3000, 5000, and 7000 meshes in sequence. 6 .

6. The preparation method according to claim 4, wherein in step (2), the mixed acid solution is a mixture of 30% H ₂ O ₂ and 98% H ₂ SO ₄ with a mass fraction of 1:1 in volume ratio solution, the treatment time is 1 hour.

7 . The preparation method according to claim 1 , wherein in step (3), the treatment time of the titanium sheet in the polyethyleneimine aqueous solution is 20 to 40 minutes. 8 .

8. The preparation method according to claim 1, wherein the amino acid composition of the polypeptide is glycine (Gly): 7.30±0.5%; valine (Vla): 17.48±0.5%; isoleucine Acid (Ile): 36.97±0.5%; Leucine (Leu): 13.85±0.5%; Tyrosine (Tyr): 2.68±0.5%; Phenylalanine (Phe): 1.5±0.5%; Lysine (Lys): 4.41±0.5%; Histidine (His): 0.45±0.5%; Arginine (Arg): 3.45±0.5%; Proline (Pro): 5.96±0.5%; Cysteine ( Cys): 5.95±0.5%.

9. preparation method according to claim 1, is characterized in that, the primary amino group exposure of film surface is 5.78%; The polypeptide monolayer film is made up of close-packed nanoparticles, and the average particle size of spherical nanoparticles is 40%. ±2 nm.

10. A composite film containing a polypeptide monolayer film is characterized in that, comprising a polyethylenimine film and the polypeptide monolayer film prepared by the method of any one of claims 1 to 9, the polyethylenimine and the polypeptide molecule are mixed. The thickness of the polyethyleneimine film is 0.25~0.38nm, and the thickness of the polypeptide monolayer is 14.6±0.1nm.

11. The application of the polypeptide monolayer film prepared by the method of any one of claims 1 to 9 or the composite film of claim 10 as a local drug-loaded coating in a dental implant of pure titanium material.