阅读说明：本技术 一类基于天然胆固醇无规共聚物、制备方法及其应用 (Random copolymer based on natural cholesterol, preparation method and application thereof ) 是由 盛瑞隆 史向阳 李茂全 王昭 于 2021-01-11 设计创作，主要内容包括：本发明涉及一类基于天然胆固醇无规共聚物、制备方法及其应用,该无规共聚物中疏水砌块来源于生物相容性的天然甾体化合物胆固醇,进而通过与聚甲基丙烯酸N,N-二甲氨基乙酯(PDMAEMA)进行阳离子开环聚合后加入功能化的胺获得。本发明的基于天然胆固醇无规共聚物其分子结构为两亲性,具有分子量和亲疏水比例可调控的优点,共聚物具有明显较高的各向同性和非液晶性,而且与已知的PDMAEMA相比,具有较好的疏水特性。且可以采用溶剂涂膜法制备层层组装Layer-by-Layer材料。本发明所提供的制备方法具有简便、高效、原料易得的优点,有望作为低成本、高生物相容性的生物修复材料在医学组织工程中得到实际应用。(The invention relates to a random copolymer based on natural cholesterol, a preparation method and application thereof, wherein a hydrophobic building block in the random copolymer is derived from biocompatible natural steroid compound cholesterol, and is obtained by adding functionalized amine after cationic ring-opening polymerization with poly N, N-dimethylaminoethyl methacrylate (PDMAEMA). The molecular structure of the random copolymer based on natural cholesterol is amphiphilic, the random copolymer based on natural cholesterol has the advantages of adjustable molecular weight and hydrophilic-hydrophobic ratio, the copolymer has obviously higher isotropy and non-liquid crystal property, and compared with the known PDMAEMA, the random copolymer based on natural cholesterol has better hydrophobic property. And a Layer-by-Layer assembled Layer-Layer material can be prepared by adopting a solvent coating method. The preparation method provided by the invention has the advantages of simplicity, convenience, high efficiency and easily obtained raw materials, and is expected to be practically applied to medical tissue engineering as a biological repair material with low cost and high biocompatibility.)

1. A random copolymer based on natural cholesterol, which is shown in a formula (I),

wherein m and n are natural numbers, m is 1-200, and n is 10-200.

2. A random copolymer based on natural cholesterol, which is shown in a formula (I),

wherein m and n are natural numbers, m is 10-80, and n is 10-80.

3. The method for preparing a natural cholesterol random copolymer according to any one of claims 1 to 2, comprising the steps of:

(1) adding dodecyl mercaptan DDMAT, azobisisobutyronitrile, cholesterol-6C-methacrylic acid monomer, DMAEMA monomer and toluene into a dry Schlenk reaction tube with a preset stirrer;

(2) performing liquid nitrogen cooling, vacuumizing, melting and introducing nitrogen for circulation for three times to remove residual oxygen in the reaction tube;

(3) immersing the reaction tube into a preheated oil bath for reaction, dripping the concentrated reaction liquid into a nonpolar organic solvent for precipitation, repeatedly dissolving the concentrated reaction liquid twice by the polar organic solvent and the nonpolar organic solvent, then precipitating and purifying the polymer, and finally drying in vacuum.

4. The preparation method according to claim 3, wherein the raw materials are used in the following amounts:

dodecyl mercaptan DDMAT: 109.4mg, 0.3 mmol;

azobisisobutyronitrile: 16.4mg, 0.1 mmol;

cholesterol-6C-methacrylic acid monomer: 3.33g, 6 mmol;

DMAEMA monomer: 4-67 mmol.

5. The preparation method according to claim 4, wherein the raw materials are used in the following amounts:

dodecyl mercaptan DDMAT: 109.4mg, 0.3 mmol;

azobisisobutyronitrile: 16.4mg, 0.1 mmol;

cholesterol-6C-methacrylic acid monomer: 3.33g, 6 mmol;

DMAEMA monomer: 6-26 mmol.

6. The preparation method according to claim 3, wherein the reaction temperature in the preparation process of the natural cholesterol random copolymer is 50-100 ℃ and the reaction time is 8-24 hours.

7. The preparation method of claim 6, wherein the reaction temperature in the preparation process of the natural cholesterol random copolymer is 60-80 ℃ and the reaction time is 10-15 h.

8. The preparation method according to claim 3, wherein the polar organic solvent is selected from any one or a combination of several of the following: tetrahydrofuran, 1, 4-dioxane, trichloromethane, methanol, ethanol, acetonitrile and acetone.

9. The preparation method according to claim 3, wherein the nonpolar organic solvent is selected from any one or a combination of several of the following: n-hexane, cyclohexane, petroleum ether, dipropyl ether and diisopropyl ether.

10. Use of a random copolymer of natural cholesterol as claimed in any one of claims 1 to 2 for the preparation of a pro-gene transfection/somatomedin release/antibacterial/antitumor interface biomaterial.

Technical Field

The invention relates to the technical field of interface biomaterials and biomedicines for novel natural cholesterol random copolymers, in particular to a natural cholesterol random copolymer based, a preparation method and application thereof.

Background

Research and development of biocompatible interfacial biomaterials has become one of the most popular research topics in the interdisciplinary fields of life science and material science. Under normal physiological conditions, cells/tissues have specific microenvironment to maintain their associated functions, and under pathological conditions, defects or lesions in the tissues can lead to destruction of the microenvironment and abnormal physiological signals. The interface biomaterial can simulate the cell/tissue microenvironment and reconstruct the components and the form of the microenvironment, and research the mechanical response and the signal transduction of the cells on the biomaterial interface can guide the realization of the repair of damaged tissues, so the research on the novel interface biomaterial has important significance. In order to improve the biocompatibility of the interface biomaterial, enrich the biological function of the material and realize the bionic effect, the preparation of the functionalized polymer interface biomaterial by adopting a biocompatible natural product as a modification means has become a research hotspot in the field. Through a modification approach of a biocompatible natural product, a series of high polymer/polymer interface biomaterials with wide application prospects such as promotion of gene transfection, growth promotion factor release, antibiosis, tumor resistance and the like can be prepared in a bionic manner. However, the currently used high molecular materials have poor designability and tailorability, single chemical property, wide molecular weight distribution and difficult performance regulation. These disadvantages limit the application prospects of the prepared materials in complex pathological microenvironment. The natural products are introduced into the high polymer materials for modification and preparation of functional synthetic high polymer materials, development of repair-treatment intelligent materials is gradually a hot point, and a series of bionic composite interface materials can be further prepared by means of 'drug-material combination' and used for repair medicine and regenerative medicine application. With the emergence of more high molecular polymer synthesis methods and the improvement of processing technologies, the problems can be solved in the future, and accurate micro-scale bionics can be realized.

As a green renewable natural product, cholesterol has the characteristics of natural source and hydrophobic structure, and can be synthesized into novel biocompatible functional polymers or surfactants by further modifying functional fragments, such as water-soluble fragments, and a series of previous research works show that the functional molecules/polymers have been primarily applied to the construction of functional drugs and gene carrier biomaterials (Biomacromolecules 2016,17, 98-110; International Journal of molecular science.2018,19,369; Chinese patent: CN 102161688; CN 101870719; CN 103541 214214). The interface biomaterial has the advantages of low toxicity, no immunogenicity, in-vivo degradability and absorbability, effective combination with biological tissues, long-term stable existence in blood circulation and reduction of nonspecific interaction with components such as proteins in blood. Introduction of an amino-containing functional group having good biocompatibility into a cholesterol derivative is an effective method for improving biocompatibility and enhancing biological tissue binding property.

Poly (N, N-dimethylaminoethyl methacrylate) (PDMAEMA) is a tertiary amino polymer with good biocompatibility, wherein the tertiary amine pKa is about 7.5, and also has a certain proton buffering capacity, and can carry out cell membrane adhesion through protonation effect. DMAEMA monomer can obtain polymers with controllable molecular weight and distribution through living radical polymerization, and can obtain various topological structures (blocks, stars, grafts and the like) through reasonable molecular design. The molecular weight, composite size, preparation conditions, pH, ionic strength, temperature, etc. of PDMAEMA all affect the interfacial efficiency of PDMAEMA. The toxicity and the interfacial hydrophilicity of the PDMAEMA modified by the hydrophilic block are simultaneously reduced, and the toxicity and the interfacial adhesion efficiency of the polymer are greatly improved compared with those of a pure DMAEMA homopolymer by introducing a hydrophobic segment in the polymer, such as Polycaprolactone (PCL). Thus, hydrophobically modified DMAEMA polymers have attracted extensive attention in the modification of monomers. However, to date, many functionalized DMAEMA polymers have molecular structures that are not conducive to their further manipulation, and their preparation and isolation methods are difficult to precisely control and model, thus limiting their large-scale synthesis and use. Therefore, the hydrophobic modified DMAEMA polymer and the derivative thereof of natural products (such as cholesterol and the like) which have the molecular structure controllability, lower cytotoxicity and good biocompatibility are further explored and developed, and the molecular function diversity of the hydrophobic modified DMAEMA polymer and the application of the hydrophobic modified DMAEMA polymer as biomedical materials can be enriched and expanded to a great extent.

On the other hand, in order to meet the actual requirements of the biological interface material, it is necessary to establish a high-efficiency, easy-to-operate, structurally-controllable preparation method of the natural cholesterol-derived cationic functional polymer, and conventionally synthesize and prepare the controllable polymer in large quantities, so as to further and deeply perform the performance research of the novel interface biological material. No relevant report is found about the natural cholesterol-based random copolymer, the preparation method and the application thereof.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a random copolymer based on natural cholesterol, a preparation method and application thereof.

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

in a first aspect, the present invention provides a random copolymer based on natural cholesterol, represented by the general chemical structure formula (I):

the random copolymer based on natural cholesterol has a repeating unit of m, and a molecular connection structural segment of n is shown in the formula, wherein m and n are selected from natural numbers of 1-200, preferably m is selected from natural numbers of 10-80, and n is selected from natural numbers of 10-200, preferably n is selected from natural numbers of 10-80.

In a second aspect, the present invention provides a process for preparing a natural cholesterol based random copolymer as described above, comprising the steps of:

specifically, the method comprises the following steps: dodecyl mercaptan DDMAT (109.4mg, 0.3mmol), azobisisobutyronitrile (16.4mg, 0.1mmol), cholesterol-6C-methacrylic acid monomer (3.33g, 6mmol), DMAEMA monomer (4-67 mmol) and freshly distilled toluene (20 mL) are added into a dry Schlenk reaction tube with a preset stirrer, and residual oxygen in the reaction tube is removed through three times of liquid nitrogen freezing-vacuumizing-melting nitrogen circulation. Immersing the reaction tube in an oil bath preheated to a certain reaction temperature for reaction for a certain time, concentrating the reaction liquid to be dropped into a nonpolar organic solvent for precipitation, purifying the polymer by a method of repeatedly dissolving and precipitating a polar solvent and a nonpolar solvent twice, and finally drying in vacuum to obtain a light yellow powdery product based on the natural cholesterol random copolymer.

Further, the charging proportion of dodecyl mercaptan DDMAT (109.4mg, 0.3mmol), azobisisobutyronitrile (16.4mg, 0.1mmol) and cholesterol-6C-methacrylic acid monomer (3.33g, 6mmol) in the above preparation method is fixed, while the charging proportion of DMAEMA monomer is (4-67 mmol), preferably 6-26 mmol.

Further, the reaction temperature described in the above production method is 50 to 100 ℃, particularly preferably 60 to 80 ℃.

Further, the reaction time described in the above production method is 8 to 24 hours, particularly preferably 10 to 15 hours.

Further, the polar organic solvent includes tetrahydrofuran, 1, 4-dioxane, chloroform, methanol, ethanol, acetonitrile, acetone, and a mixed solvent thereof.

Further, the nonpolar solvent includes n-hexane, cyclohexane, petroleum ether, dipropyl ether, diisopropyl ether and mixed solvents thereof.

In a third aspect, the invention provides an application of the natural cholesterol random copolymer in preparing a gene transfection-promoting interface biological material/growth factor release interface biological material/antibacterial interface biological material/anti-tumor interface biological material.

The invention has the advantages that:

1. the synthetic method of the interface biomaterial based on the natural cholesterol random copolymer is simple and efficient, the functional molecular main body can be derived from a large amount of existing cholesterol natural products, the rest molecular structure fragments can be derived from organic chemical raw materials which can be industrially prepared in a large amount, and the synthetic method is easy to popularize, low in cost, capable of being prepared in a large scale and good in industrial productivity.

2. The random copolymer interface biomaterial based on natural cholesterol provided by the invention has amphipathy and interface activity, the polymer has higher isotropy and non-liquid crystal property, can be used as a bioactive interface material, and has better hydrophobic property compared with the known PDMAEMA.

3. The interface biomaterial based on the natural cholesterol random copolymer provided by the invention has good biocompatibility, can be used for preparing Layer-by-Layer assembled Layer-by-Layer materials by adopting a solvent coating method, is expected to be practically applied in medical tissue engineering as a bioremediation material, and has good application prospect.

Drawings

FIG. 1 is the liquid crystal phase/non-liquid crystal phase morphology of a natural cholesterol random copolymer-based interface biomaterial P1-P4 at different temperatures in examples 1-4.

FIG. 2 is a Dataphysis OCA20(German) optical video imaging and contact angle data based on cross-sectional contact angles of natural cholesterol random copolymer interface biomaterials P1-P4 of one class of examples 1-4.

FIG. 3 is MTT cytotoxicity assessment data for a class of natural cholesterol random copolymer-based interfacial biomaterials as described in examples 1-4.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1 Synthesis of random copolymer of natural cholesterol P1(PDMAEMA18-PMCHOL16)

Dodecyl mercaptan DDMAT (109.4mg, 0.3mmol), azobisisobutyronitrile (16.4mg, 0.1mmol), cholesterol-6C-methacrylic acid monomer (3.33g, 6mmol), DMAEMA monomer (0.91g,6mmol) and freshly distilled toluene (20 mL) were added to a dry Schlenk reaction tube with a pre-stirrer and the residual oxygen in the reaction tube was removed by three cycles of liquid nitrogen freezing-evacuation-melting nitrogen gas. The reaction tube was immersed in an oil bath preheated to 80 ℃ for 12 hours, the reaction solution was concentrated and dropped into petroleum ether for precipitation, and the polymer was purified by two repeated dissolution-precipitation with methanol-cyclohexane, and finally dried under vacuum to obtain 2.26g of a pale yellow powdery product based on natural cholesterol random copolymer P1 in 82% yield.

Chemical Structure of Polymer P1

Example 2 Synthesis of random copolymer of Natural Cholesterol P2(PDMAEMA30-PMCHOL16)

Dodecyl mercaptan DDMAT (109.4mg, 0.3mmol), azobisisobutyronitrile (16.4mg, 0.1mmol), cholesterol-6C-methacrylic acid monomer (3.33g, 6mmol), DMAEMA monomer (1.81g,12mmol) and freshly distilled toluene (20 mL) were added to a dry Schlenk reaction tube with a pre-stirrer and the residual oxygen in the reaction tube was removed by three cycles of liquid nitrogen freezing-evacuation-melting nitrogen gas. The reaction tube was immersed in an oil bath preheated to 80 ℃ for 10 hours, the reaction solution was concentrated and dropped into cyclohexane for precipitation, and the polymer was purified by twice repeated dissolution-precipitation with acetone-n-hexane, and finally dried under vacuum to obtain 2.11g of a pale yellow powdery product based on natural cholesterol random copolymer P2 in a yield of 76%.

Chemical Structure of Polymer P2

Example 3 Synthesis of random copolymer of Natural Cholesterol P3(PDMAEMA43-PMCHOL16)

Dodecyl mercaptan DDMAT (109.4mg, 0.3mmol), azobisisobutyronitrile (16.4mg, 0.1mmol), cholesterol-6C-methacrylic acid monomer (3.33g, 6mmol), DMAEMA monomer (2.41g, 18mmol) and freshly distilled toluene (20 mL) were added to a dry Schlenk reaction tube with a pre-stirrer and the residual oxygen in the reaction tube was removed by three cycles of liquid nitrogen freezing-evacuation-melting nitrogen gas. The reaction tube is immersed in an oil bath preheated to 80 ℃ for reaction for 12 hours, the concentrated reaction liquid is dropped into n-hexane for precipitation, the polymer is purified by twice repeated dissolution-precipitation methods of acetonitrile-n-hexane, and finally vacuum drying is carried out to obtain 1.98g of light yellow powdery product based on natural cholesterol random copolymer P3 with the yield of 72 percent.

Chemical Structure of Polymer P3

Example 4 Synthesis of random copolymer of Natural Cholesterol P4(PDMAEMA72-PMCHOL16)

Dodecyl mercaptan DDMAT (109.4mg, 0.3mmol), azobisisobutyronitrile (16.4mg, 0.1mmol), cholesterol-6C-methacrylic acid monomer (3.33g, 6mmol), DMAEMA monomer (3.58g,24mmol and 20mL of freshly distilled toluene were added to a dry Schlenk reaction tube with a stirrer, and subjected to three cycles of liquid nitrogen freezing-evacuation-melting with nitrogen to remove residual oxygen in the reaction tube, the reaction tube was immersed in an oil bath preheated to 90 ℃ for 12 hours, the reaction solution was concentrated and dropped into n-hexane for precipitation, and the polymer was purified by twice repeated dissolution-precipitation with methanol-diisopropyl ether, and finally dried under vacuum to give 2.35g of a pale yellow powdery product based on natural cholesterol random copolymer P4 in 84% yield.

Chemical Structure of Polymer P4

Characterization data in Table 1, examples 1-4, based on natural cholesterol random copolymer (P1-P4)

Note: m_n,NMRRepresents a nuclear magnetic number average molecular weight; m_n,GPCRepresents the number average molecular weight of gel exclusion chromatography; PDI represents the polymer molecular weight distribution.

Example 5 solution Properties of Natural Cholesterol random copolymer interface-based biomaterials

The interfacial biomaterial based on natural cholesterol random copolymer synthesized in examples 1-4, P1-P4(5mg), was added to 2mL of purified water. Putting the nano-composite material into an ultrasonic cleaner with the power of 100W and setting the power for 20min in advance, ultrasonically dispersing, then fixing the volume to 10mL by pure water, incubating for 30min at room temperature to obtain micron-sized suspension of the natural cholesterol random copolymer interface biomaterial P1-P4, and then taking 2mL of the sample in a polypropylene ultrasonic centrifuge tube for photographing.

Example 6 liquid Crystal Performance testing of Natural Cholesterol random copolymer interface-based biomaterials

The liquid crystal phase/non-liquid crystal phase morphology of the natural cholesterol random copolymer interface-based biomaterial (P1-P4) at different temperatures was tested using a hot stage polarization microscope (POM) and observed by an Olympus BX51 polarization microscope equipped with a Linkam LTS350 hot stage. Firstly, slowly heating 0.5mg of natural cholesterol-based random copolymer interface biomaterial P1-P4 to 165-180 ℃, carrying out isothermal treatment for 10min to completely convert a liquid crystal phase into an isotropic phase, then reducing the temperature to 30-45 ℃ at a cooling speed of 1 ℃/min, carrying out isothermal treatment for 1-12 h, and then carrying out observation and shooting a liquid crystal phase texture picture.

As a result: as shown in FIG. 1, it can be seen from the image obtained by hot stage polarization microscope (POM) that the interfacial biomaterial P1-P4 based on the natural cholesterol random copolymer has non-liquid crystal property and isotropy, the preparation of the interfacial biomaterial is not facilitated by the anisotropy of the liquid crystal phase of the natural cholesterol, and the non-liquid crystal property and isotropy of P1-P4 indicate that the copolymer has good film-forming property.

Example 7 interfacial hydropathy Performance test based on Natural Cholesterol random copolymer interfacial biomaterials

An interface contact angle experiment based on natural cholesterol random copolymer interface biomaterials as described in examples 1-4 of the present invention: 2.0mg of natural cholesterol random copolymer P1-P4 is fully dissolved in 1mL of organic solvent dichloromethane, then the solution is dropped on the surface of a common medical glass slide to enable the solvent to naturally volatilize to prepare a natural cholesterol random copolymer film, deionized water is dropped on the film, then a Dataphysis OCA20(German) optical video contact angle instrument is adopted to measure the contact angle between water and the natural cholesterol random copolymer film, and the average value measured in 3 times is adopted as the final data in the experiment. And simultaneously, the appearance of water drops on the interface of the natural cholesterol random copolymer film is shot.

As a result: as shown in FIG. 2, the ductility of the water drop increases with the contact angle of P1 of 102.9 + -1.6 o, the contact angle of P2 of 98.5 + -3.1 o, the contact angle of P3 of 93.1 + -1.2 o, and the contact angle of P4 of 95.9 + -0.5 o. The result shows that the interface hydrophilic characteristic of the natural cholesterol random copolymer interface biomaterial can be effectively improved by increasing the proportion of PDMAEMA, so that hydrophilic and hydrophobic regulation and control of cell adhesion and growth performance on the interface biomaterial matrix in future are facilitated.

Example 8 cytotoxicity test based on Natural Cholesterol random copolymer interfacial biomaterials

MTT method cytotoxicity evaluation of a class of natural cholesterol random copolymer-based interfacial biomaterials described in examples 1-4 of the present invention: first, human hepatoma cell HepG2 was added at 5X 10 per well⁴The density of the cells is inoculated in a 96-well culture plate, after 12 hours of culture in an incubator at 37 ℃, different concentrations of the interfacial biomaterial based on the natural cholesterol random copolymer are added, and the culture is continued for 24 hours at 37 ℃. Then, 20. mu.L of MTT (5mg/mL) solution was added and the mixture was left at 37 ℃ for 4 hours. Using a Biotek ELX-800 microplate reader at 490nm wavelengthAnd (6) analyzing. The HepG2 cytotoxicity experimental data obtained in the patent are statistically analyzed by a t-test method, and the average value and the standard deviation are obtained by further processing the test results of each sample for 3 times. Relative survival (%) of HepG2 cells (sample OD 490-sample OD 630)/(control OD 490-control OD630) × 100%.

As a result: as shown in figure 3, the P1-P4 has low cytotoxicity in human hepatoma cell HepG2, the cytotoxicity can be regulated and controlled by regulating the proportion of PDMAEMA, and the random copolymer based on natural cholesterol has low cytotoxicity and good biocompatibility, is beneficial to hydrophilic and hydrophobic regulation and control of cell adhesion and growth performance on the interface biomaterial matrix in the future, and is expected to be applied to the aspect of medical tissue repair engineering.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.