阅读说明：本技术 一种用于促进干细胞界面粘附生长的双亲性聚合物及其制备方法和用途 (Amphiphilic polymer for promoting stem cell interface adhesion growth and preparation method and application thereof ) 是由 喻长远 李泽浩 游长江 许立达 李政泰 李丽丹 于 2019-09-04 设计创作，主要内容包括：本发明提供一种用于促进干细胞界面粘附生长的双亲性聚合物及其制备方法和用途,所述双亲性聚合物通过引入亲水性成分和疏水性成分,其中疏水性成分用于与材料的疏水表面通过疏水作用形成稳固的表面覆盖,亲水性成分提供生物相容性并带有促进干细胞粘附生长的多肽。所述双亲性聚合物用于对疏水性基材进行表面改性处理,使其具有良好的生物相容性且能促进干细胞的长时间附着生长并维持其干性。(The invention provides an amphiphilic polymer for promoting stem cell interface adhesion growth and a preparation method and application thereof. The amphiphilic polymer is used for carrying out surface modification treatment on a hydrophobic substrate, so that the amphiphilic polymer has good biocompatibility and can promote the long-term adhesion growth of stem cells and maintain the dryness of the stem cells.)

1. An amphiphilic polymer, wherein the polymer is prepared from the following raw materials:

having a reactive group Y in a side chain¹And X¹Of (2) a polymer of (2), a compound R-X²One end of which has a reactive group Y²Polyethylene glycol with a polypeptide at the other end, optionally a reactive group Y at one end²The other end has a reaction-inert group R²Polyethylene glycol of (2); wherein, Y¹And Y²Capable of reacting to polyethylene glycol having attached thereto a polypeptide and optionally a reactive inert group R²The polyethylene glycol of (a) is attached to a side chain of the polymer; x¹And X²Reacting to attach the R group to a side chain of a polymer, said polypeptide comprising a polypeptide sequence that promotes cell adhesion growth.

2. A method for preparing an amphiphilic polymer, wherein the method comprises the steps of:

having a reactive group Y in a side chain¹And X¹With a compound R-X²One end of which has a reactive group Y²Polyethylene glycol with a polypeptide at the other end, optionally a reactive group Y at one end²The other end has a reaction-inert group R²Reacting the polyethylene glycol; wherein, Y¹And Y²Capable of reacting to polyethylene glycol having attached thereto a polypeptide and optionally a reactive inert group R²The polyethylene glycol of (a) is attached to a side chain of the polymer; x¹And X²Reacting to attach the R group to a side chain of a polymer, said polypeptide comprising a polypeptide sequence that promotes cell adhesion growth.

3. The amphiphilic polymer according to claim 1 or the production method according to claim 2, wherein the amphiphilic polymer can be produced by a one-pot method comprising:

having a reactive group Y in a side chain¹And X¹With the compound R-X²One end of which has a reactive group Y²Polyethylene glycol having a polypeptide at the other end, and optionally a reactive group Y at one end²The other end has a reaction-inert group R²The amphiphilic polymer is prepared by reacting the polyethylene glycol.

Preferably, the amphiphilic polymer may be prepared by a two-step process comprising:

having a reactive group Y in a side chain¹And X¹With the compound R-X²Reacting the above product with a reactive group Y at one end²The other end is provided withPolyethylene glycol of a polypeptide and optionally a reactive group Y at one end²The other end has a reaction-inert group R²The polyethylene glycol is reacted to prepare the amphiphilic polymer; alternatively, the first and second electrodes may be,

having a reactive group Y in a side chain¹And X¹With a reactive group Y at one end²Polyethylene glycol having a polypeptide at the other end, and optionally a reactive group Y at one end²The other end has a reaction-inert group R²Is reacted with a compound R-X²And carrying out reaction to prepare the amphiphilic polymer.

4. The amphiphilic polymer according to claim 1 or 3 or the production method according to claim 2 or 3, wherein the one terminal has a reactive group Y²The polyethylene glycol with the polypeptide at the other end can be prepared by the following method:

one end has a reactive group Y²The other end having a reactive group Z¹With a reactive group Z at one end²Wherein Z is¹、Z²A reaction occurs to link the polyethylene glycol to the polypeptide.

Preferably, the polypeptide sequence for promoting cell adhesion growth is at least one of RGD, IKVAV, RYVVLPR, RNIAEIIKDI, CCRRIKVAVWLC, CSRARKQAASKAVSAVSADR, and artificially synthesized positive charge-rich sequence KR, RKR, KKRK and the like.

Preferably, the reactive group X¹、X²、Y¹、Y²、Z¹、Z²For example, at least one selected from the group consisting of a hydroxyl group, an amino group, a carboxyl group, an aldehyde group, a ketone group, an ester group, a thiol group, maleimide, an α -halocarbonyl group, an alkynyl group, an alkenyl group, an azido group, a tetrazinyl group, and the like. Wherein the reactive group X¹And X²、Y¹And Y²、Z¹And Z²Mutually reactive groups, which may be reacted, but Y²、Z²No reaction takes place.

Preferably, the polyethylene glycol is a chain polyethylene glycol, and preferably, the number of repeating units is an integer between 1 and 600, preferably an integer between 2 and 300, and more preferably an integer between 4 and 200.

Preferably, the reaction-inert group R²Is C_1-6An alkoxy group.

Preferably, the hydrophobic group R is C_4-25Alkyl radical, C_4-25Alkenyl radical, C_4-25Alkynyl, C_6-36An aryl group;

preferably, said reactive group Y is¹And X¹The polymer of (a) has a main chain comprising carbon atoms and at least one heteroatom such as oxygen, nitrogen, sulfur, silicon, etc.; the side chain comprising a reactive group Y¹And X¹. Said group Y having a reactive group¹And X¹The polymers of (a) are, for example: having reactive groups Y¹And X¹Polyether, polyester, polyamide, polyurethane, polysulfide rubber, polysilicone rubber-polyamide, polyethyleneimine, polyamino acid, etc.;

preferably, the polyamino acid is poly-L-lysine, and artificially synthesized poly-D-lysine, and the like.

Preferably, said reactive group Y is¹And X¹The number of the main chain repeating units of the polymer of (1) is an integer of 2 to 2000, preferably 2 to 1000, more preferably 2 to 500.

Preferably, the side chains of the amphiphilic polymer include four types of side chains:

1) containing unreacted reactive groups Y¹And X¹The side chain of (a) is the side chain 1,

2) the side chain containing the group R, namely the side chain 2,

3) contains a side chain of polyethylene glycol with one end being polypeptide, a side chain 3,

4) containing a reactive inert group R at one end²The side chain of the polyethylene glycol of (2) is the side chain 4.

Preferably, in the amphiphilic polymer, the mole percentage of the side chain 3 to the total amount of the side chains 3 and 4 is 0.1% to 100%, preferably 1% to 100%, more preferably 10% to 100%.

Preferably, in the amphiphilic polymer, the molar percentage of the total amount of side chains 3 and 4 is 2% to 98%, preferably 5% to 90%, more preferably 10% to 80%, of the total amount of all side chains.

Preferably, in the amphiphilic polymer, the mole percentage of the side chains 2 to the total amount of all side chains is 2% to 98%, preferably 5% to 90%, more preferably 10% to 80%.

Preferably, in the amphiphilic polymer, the total amount of side chains 2, 3 and 4 is 5% to 100%, preferably 20% to 100%, more preferably 40% to 100%, by mole based on the total amount of all side chains.

5. An amphiphilic polymer, wherein the structure of the polymer is shown as formula I:

the polymer comprises a main chain and side chains, wherein the main chain comprises carbon atoms and at least one heteroatom, and at least part of the side chains respectively comprise an A group, a B group and an optional D group;

the A group is-X-R, wherein X is a linking group and R is a hydrophobic group;

the B group is-Y-L¹-PEG-L²-Z-R¹The D group is-Y-L¹-PEG-L²-R²Wherein Y is a linking group, Z is a linking group, PEG is a polyethylene glycol chain segment, R is a polyethylene glycol chain segment¹Being a polypeptide containing a polypeptide sequence which promotes cell adhesion growth, L¹、L²Is a direct bond or a spacer group, R²Are reactive inert groups.

6. The amphiphilic polymer of claim 5 wherein X, Y, Z is the following group:

-CO-NH-、-O-、-CO-O-、-S-S-、-R³R⁴C＝N-、any one of a connecting group obtained by click reaction of azido and alkynyl and a connecting group obtained by click reaction of tetrazine and double bonds, wherein the sequence of the groups can be reversed; wherein R is³、R⁴Are independently selected from H, C_1-6An alkyl group;

preferably, the number of repeating units of the polyethylene glycol is an integer between 1 and 600, preferably an integer between 2 and 300, more preferably an integer between 4 and 200;

preferably, the polypeptide sequence that promotes cell adhesion growth is, for example: RGD, IKVAV, or RYVVLPR, RNIAEIIKDI, CCRRIKVAVWLC, CSRARKQAASKAVSADDR, and artificially synthesized positive charge-rich sequences KR, RKR, KKRK, etc.;

preferably, L¹、L²Is a direct bond or an optional reactive group Y²、X¹A spacer group of a polyethylene glycol segment is introduced.

Preferably, said R is²Can be C_1-6Alkoxy radicals, such as methoxy, ethoxy.

Preferably, R is C_4-25Alkyl radical, C_4-25Alkenyl radical, C_4-25Alkynyl, C_6-36An aryl group;

preferably, the main chain of the polymer contains at least one hetero atom of oxygen, nitrogen, sulfur, silicon and the like in addition to carbon atoms. The main chain structure includes, for example: at least one of a polyether main chain, a polyester main chain, a polyamide main chain, a polyurethane main chain, a polysulfide rubber main chain, a polysilicone rubber-polyamide main chain, a polyethyleneimine main chain, a polyamino acid main chain and the like;

preferably, the polyamino acid is poly-L-lysine, and artificially synthesized poly-D-lysine, and the like.

Preferably, the mole percentage of side chains containing a B group to the total amount of side chains containing a B group and a D group in the polymer is 0.1% to 100%, preferably 1% to 100%, more preferably 10% to 100%.

Preferably, the polymer has from 2% to 98%, preferably from 5% to 90%, more preferably from 10% to 80% of the total number of side chains comprising the group B and optionally the group D.

Preferably, the polymer has a percentage of the number of side chains containing the A group to the total number of side chains in the range of 2% to 98%, preferably 5% to 90%, more preferably 10% to 80%.

Preferably, the percentage of the total number of side chains in the polymer that are present as a sum of the number of side chains containing a groups and the number of side chains containing B groups and optionally D groups is between 5% and 100%, preferably between 20% and 100%, more preferably between 40% and 100%.

7. Use of the amphiphilic polymer according to any one of claims 1, 3-6 for the modification treatment of surface hydrophobic substrates or in the field of stem cell differentiation and tissue engineering.

8. A method of modifying a surface hydrophobic substrate, the method comprising the steps of:

contacting the surface hydrophobic substrate to be treated with the amphiphilic polymer of any one of claims 1, 3-6 to effect a modification treatment of the surface hydrophobic substrate.

Preferably, the contacting may be, for example, contacting the surface hydrophobic substrate to be treated with a dispersion of the amphiphilic polymer described above. The dispersion may be an aqueous solution, an organic solution or a mixed solution of an organic solvent and water of the modified polymer.

Preferably, the concentration of the amphiphilic polymer in the aqueous solution, the organic solution or the mixed solution of the organic solvent and water of the amphiphilic polymer is less than 1000 g/L.

9. The article of claim 8 having a surface hydrophobic substrate modified.

10. The article of claim 9, wherein the surface hydrophobic substrate comprises a biodegradable material and a non-degradable material.

Preferably, the biodegradable material includes polylactic acid, polyester, polycaprolactone, and polylactic acid-polyester copolymer, polylactic acid-polycaprolactone copolymer, and the like.

Preferably, the non-biodegradable materials include olefinic and diene polymers, polystyrene, polyvinyl halides, polyvinylidene fluoride, and the like, as well as polyethers, polyesters, polyamides, polyurethanes, polysulfide rubber, polysilicone rubber, and silane-based polymers, such as polydimethylsiloxane.

Preferably, the surface hydrophobic substrate may be obtained by surface modification of a hydrophilic substrate.

Technical Field

The invention belongs to the field of polymers, and particularly relates to an amphiphilic polymer for promoting stem cell interface adhesion growth, and a preparation method and application thereof.

Background

Regenerative medicine requires reliable mass culture of stem cells and artificial tissues for repair and replacement of tissues and organs. Biomedical materials that promote the adherent growth of cells and stem cells have been widely used in stem cell culture. The stem cell culture environment must meet the harsh requirements of the stem cell culture environment on various aspects such as nutrient components, growth factors, osmotic pressure and the like, and is specifically embodied in that: (1) the culture substrate must be a hydrosol system; (2) the matrix needs to contain a plurality of signal channel regulating factors according to a certain proportion, including Transforming Growth Factor (TGF), Noggin, Wnt and other signal proteins; (3) the culture substrate needs to have an elastic modulus (hardness) similar to that of the tissue.

At present, biological products are gradually replaced by a fully artificial stem cell culture medium with determined components, and typical artificial hydrosols include polyacrylamide-polyethylene glycol derivatives, self-assembly polypeptides and the like. Biomedical materials for regenerative medicine are required to maintain the activity of biomolecules and to have low cytotoxicity, that is, to have good biocompatibility. The interface modification performance obtains a base material with excellent biocompatibility through surface modification on the premise of not changing the mechanical property of a material body, and the base material is used for various functional tissue engineering scaffolds. For artificial materials used in regenerative medicine, it is vital to be able to promote adherent growth of stem cells on biomedical materials, and that such adherent growth does not lead to uncontrolled stem cell differentiation. Therefore, there is a need in the art for an interfacial treatment material that can maintain the dryness of stem cells for a long period of time. Maintenance of sternness can be known by examining the expression of specific stem cell markers, or factors such as Oct-4, SOX-2, SSEA-4 and NANOG.

To obtain a stable biocompatible protective layer, hydrophilic functional groups can be covalently attached to the substrate surface by chemical means. The coupling reaction of carboxylic acids with amine and alcohol groups is commonly used to covalently attach polyethylene glycol diamines or mucopolysaccharide sulfonates to the substrate surface. For hydrophobic substrate materials, biocompatible modification is usually performed by covering the surface with artificial macromolecules and natural biological macromolecules such as albumin.F-127 is a typical synthetic polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock polymer material in which the polypropylene oxide (PPO) is more hydrophobic than the polyethylene oxide (PEO) to cover hydrophobic surfaces. However, the use of amphiphilic block polymers for biofunctionalizing hydrophobic substrates has its limitations. For example, polypropylene oxide is less hydrophobic and does not satisfy the situation where strong hydrophobic interactions are required; the type of the block part is fixed, and flexible change is difficult to carry out according to the property of the base material; any adjustment of the copolymerization ratio in the block copolymer requires the re-synthesis of the main chain.

A large number of biomedical materials have surface hydrophobicity, including biodegradable materials (such as common polylactic acid, polycaprolactone, and polylactic-polycaprolactone) and non-degradable materials (such as polyvinylidene fluoride) and the like, which can be large planar, or two-dimensional micro/nano structured patterns on solid phase surfaces, or three-dimensional micro/nano structures such as microbeads, nanoparticles, nanofibers, nano two-dimensional materials, irregular porous materials, and the like. The substrate with the micro-nano structure is subjected to biocompatibility treatment, and has a good effect of being covered by solution dip dyeing.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide an amphiphilic polymer for promoting the adhesion growth of stem cell interfaces, and a preparation method and application thereof, wherein the amphiphilic polymer is prepared by introducing a hydrophilic component and a hydrophobic component, the hydrophobic component is used for forming firm surface coverage with the hydrophobic surface of a material through hydrophobic interaction, and the hydrophilic component provides biocompatibility and carries a polypeptide for promoting the adhesion growth of stem cells. The amphiphilic polymer is used for carrying out surface modification treatment on a hydrophobic substrate, so that the amphiphilic polymer has good biocompatibility and can promote the long-term adhesion growth of stem cells and maintain the dryness of the stem cells.

The purpose of the invention is realized by the following technical scheme:

an amphiphilic polymer prepared from the following starting materials: having a reactive group Y in a side chain¹And X¹Of (2) a polymer of (2), a compound R-X²One end of which has a reactive group Y²Polyethylene glycol with a polypeptide at the other end, optionally a reactive group Y at one end²The other end has a reaction-inert group R²Polyethylene glycol of (2); wherein, Y¹And Y²Capable of reacting to polyethylene glycol having attached thereto a polypeptide and optionally a reactive inert group R²The polyethylene glycol of (a) is attached to a side chain of the polymer; x¹And X²A reaction occurs to attach the R group to the side chain of the polymer.

According to the invention, the polypeptide comprises a polypeptide sequence promoting cell adhesion growth, such as at least one of RGD, IKVAV, RYVVLPR, RNIAEIIKDI, CCRRIKVAVWLC, CSRARKQAASKAVSADR, and artificially synthesized positive charge-rich sequences KR, RKR, KKRK, etc.

According to the invention, the amphiphilic polymer may be prepared by a one-pot process comprising:

having a reactive group Y in a side chain¹And X¹With the compound R-X²One end of which has a reactive group Y²Polyethylene glycol having a polypeptide at the other end, and optionally a reactive group Y at one end²The other end has a reaction-inert group R²The amphiphilic polymer is prepared by reacting the polyethylene glycol.

According to the invention, the amphiphilic polymer can be prepared by a two-step process comprising:

having a reactive group Y in a side chain¹And X¹With the compound R-X²Reacting the above product with a reactive group Y at one end²Polyethylene glycol having a polypeptide at the other end, and optionally a reactive group Y at one end²The other end has a reaction-inert group R²The polyethylene glycol is reacted to prepare the amphiphilic polymer; alternatively, the first and second electrodes may be,

having a reactive group Y in a side chain¹And X¹With a reactive group Y at one end²Polyethylene glycol having a polypeptide at the other end, and optionally a reactive group Y at one end²The other end has a reaction-inert group R²Is reacted with a compound R-X²And carrying out reaction to prepare the amphiphilic polymer.

According to the invention, said one end has a reactive group Y²The polyethylene glycol with the polypeptide at the other end can be prepared by the following method:

one end has a reactive group Y²The other end having a reactive group Z¹With a reactive group Z at one end²Wherein Z is¹、Z²A reaction occurs to link the polyethylene glycol to the polypeptide.

According to the invention, the reactive group X¹、X²、Y¹、Y²、Z¹、Z²For example, selected from the group consisting of hydroxyl, amino, carboxyl, aldehyde, keto, ester, thiol, maleimide, α -halocarbonyl, alkynyl, alkenyl, azido, tetrazinyl. Wherein the reactive group X¹And X²、Y¹And Y²、Z¹And Z²Mutually reactive groups, which may be reacted, but Y²、Z²No reaction takes place.

For example, an amino group and a carboxyl group are subjected to a condensation reaction to obtain an amide linking group, or an amino group and an aldehyde group or a ketone group are subjected to a reaction to obtain a schiff base linking group, or a hydroxyl group and a carboxyl group are subjected to a condensation reaction to obtain an ester linking group, or a hydroxyl group and a hydroxyl group are dehydrated to obtain an ether linking group, or maleimide and a mercapto group are subjected to an addition reaction, or a substitution reaction of a mercapto group and an α -halocarbonyl group, or an amino group and an ester group are subjected to a reaction to obtain an amide linking group, or an alkynyl group and an azide group are subjected to a click reaction to obtain a linking group.

Wherein the click reaction of an alkynyl group with an azido group is a reaction known in the art, for example: azide-alkyne cycloaddition catalyzed by metal ions (e.g., cu (i)) (Sharpless reaction, with the alkynyl group typically at the end group), or cyclotension catalyzed azide-alkyne cycloaddition (SPAAC reaction, with the alkynyl group in the middle of the strained ring).

Where the click reaction of an alkenyl group with a tetrazine group is a reaction known in the art, for example the cycloaddition reaction of a cyclic olefin with a tetrazine group.

Illustratively, when X¹When it is amino, X²Is at least one of carboxyl, aldehyde group, ketone group and ester group; when X is present¹When it is hydroxy, X²At least one of carboxyl and hydroxyl; when X is present¹When it is mercapto, X²Is at least one of maleimide and alpha-halogenated carbonyl; when X is present¹When it is alkynyl, X²Is azido; when X is present¹When it is alkenyl, X²Is tetrazinyl. The opposite is also true, for example, when X²When it is amino, X¹Is at least one of carboxyl, aldehyde group, ketone group and ester group.

Illustratively, when Y is¹When it is amino, Y²Is at least one of carboxyl, aldehyde group, ketone group and ester group; when Y is¹When it is hydroxy, Y²At least one of carboxyl and hydroxyl; when Y is¹When it is mercapto, Y²Is at least one of maleimide and alpha-halogenated carbonyl; when Y is¹When it is alkynyl, Y²Is azido; when Y is¹When it is alkenyl, Y²Is tetrazinyl. The reverse is also true, for example, when Y²When it is amino, Y¹Is at least one of carboxyl, aldehyde group, ketone group and ester group.

Is exemplified byWhen Z is¹When it is amino, Z²Is at least one of carboxyl, aldehyde group, ketone group and ester group; when Z is¹When it is hydroxy, Z²At least one of carboxyl and hydroxyl; when Z is¹When it is mercapto, Z²Is at least one of maleimide and alpha-halogenated carbonyl; when Z is¹When it is alkynyl, Z²Is azido; when Z is¹When it is alkenyl, Z²Is tetrazinyl. The opposite is also true, for example, when Z²When it is amino, Z¹Is at least one of carboxyl, aldehyde group, ketone group and ester group.

In one embodiment, the polyethylene glycol is a chain polyethylene glycol, and preferably, the number of repeating units is an integer between 1 and 600, preferably an integer between 2 and 300, and more preferably an integer between 4 and 200.

As an example, the one end has a reactive group Y²The other end having a reactive group Z¹The polyethylene glycol(s) of (a) can be commercially available or can be prepared by a method conventional in the art. In the presence of polyethylene glycol and a reactive group Y²、Z¹Can be directly connected, i.e. as a capping group, or can be connected through any spacer group, depending on the reactive group Y which is to be treated by methods conventional in the art²、Z¹Introduced into both ends of the polyethylene glycol. In the presence of polyethylene glycol and a reactive group Y²、Z¹The spacer group therebetween may be any as long as it does not interfere with the preparation of the amphiphilic polymer of the present invention, and is, for example, C_1-12Alkyl, ester, amide, ketone, and the like.

As an example, the one end has a reactive group Y²The other end has a reaction-inert group R²The polyethylene glycol(s) of (a) can be commercially available or can be prepared by a method conventional in the art. In the presence of polyethylene glycol and a reactive group Y²A reaction-inert group R²Can be directly connected, i.e., as a capping group, or can be connected through any spacer group, depending on the reactive group Y being treated by methods conventional in the art²A reaction-inert group R²Introduction ofTo both ends of the polyethylene glycol. In the presence of polyethylene glycol and a reactive group Y²A reaction-inert group R²The spacer group therebetween may be arbitrary as long as it does not interfere with the preparation of the amphiphilic polymer according to the present invention.

In one embodiment, the reaction inert group R²Can be C_1-6Alkoxy radicals, such as methoxy, ethoxy.

In one embodiment, the hydrophobic group R is C_4-25Alkyl radical, C_4-25Alkenyl radical, C_4-25Alkynyl, C_6-36An aryl group;

in one embodiment, the reactive group Y is¹And X¹The polymer of (a) has a main chain comprising carbon atoms and at least one heteroatom such as oxygen, nitrogen, sulfur, silicon, etc.; the side chain comprising a reactive group Y¹And X¹Preferably, the end group of the side chain comprises a reactive group Y¹And X¹. Said group Y having a reactive group¹And X¹The polymers of (a) are, for example: having reactive groups Y¹And X¹Polyether, polyester, polyamide, polyurethane, polysulfide rubber, polysilicone rubber-polyamide, polyethyleneimine, polyamino acid, etc.;

in one embodiment, the polyamino acid is poly-L-lysine, and artificially synthesized poly-D-lysine, and the like.

According to the invention, said reactive group Y¹And X¹The number of the main chain repeating units of the polymer of (1) is an integer of 2 to 2000, preferably 2 to 1000, more preferably 2 to 500.

According to the invention, said reactive group Y¹And X¹Of the polymer side chain has a reactive group X¹、Y¹The same or different. When the reactive group X is¹、Y¹In the same way, the above reactive group moiety is reacted with polyethylene glycol having a reactive group, and the moiety is reacted with the compound R-X²And (4) reacting. When the reactive group X is¹、Y¹When different, one of the reactive groups can be reacted withPolyethylene glycol reaction of a reactive group with another reactive group with a compound R-X²And (4) reacting.

In one embodiment, the backbone of the amphiphilic polymer is the reactant (having the reactive group Y)¹And X¹The side chains of the amphiphilic polymer include the following four types of side chains:

1) containing unreacted reactive groups Y¹And X¹The side chain of (a) is the side chain 1,

2) the side chain containing the group R, namely the side chain 2,

3) contains a side chain of polyethylene glycol with one end being polypeptide (-polyethylene glycol-polypeptide), a side chain 3,

4) containing a reactive inert group R at one end²Polyethylene glycol (-polyethylene glycol-R)²) The side chain of (3) is the side chain 4.

In one embodiment, the amphiphilic polymer has a mole percentage of side chains 3 to the total of side chains 3 and side chains 4 of 0.1% to 100%, preferably 1% to 100%, more preferably 10% to 100%.

In one embodiment, the amphiphilic polymer has a molar percentage of the total amount of side chains 3 and 4 to the total amount of all side chains of 2% to 98%, preferably 5% to 90%, more preferably 10% to 80%.

In one embodiment, the amphiphilic polymer has 2% to 98%, preferably 5% to 90%, more preferably 10% to 80% of side chains 2 by mole based on the total amount of side chains.

In one embodiment, the amphiphilic polymer has a molar percentage of the total amount of side chains 2, 3 and 4 to the total amount of all side chains of 5% to 100%, preferably 20% to 100%, more preferably 40% to 100%.

According to the present invention, in the above steps, the reaction is a conventional reaction step in the art, and the reaction temperature is 10 to 40 ℃ for example.

According to the invention, X¹And X²When a reaction is carried out, or the Y¹And Y²When a reaction is carried out, or the Z¹And Z²The reaction may be carried out, for example, under the acceleration of a coupling agent. For example, an amino group may be condensed with a carboxyl group in the presence of a coupling agent to provide an amide linking group, or a hydroxyl group may be condensed with a carboxyl group in the presence of a coupling agent to provide an ester linking group. The coupling agent is for example a carbodiimide derivative selected from 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, or N, N-dicyclohexylcarbodiimide, added in a molar ratio to the reactants of from 1 to 1000, preferably from 1 to 500, more preferably from 1 to 100.

The invention also provides a preparation method of the amphiphilic polymer, wherein the method comprises the following steps:

having a reactive group Y in a side chain¹And X¹With a compound R-X²One end of which has a reactive group Y²Polyethylene glycol with a polypeptide at the other end, optionally a reactive group Y at one end²The other end has a reaction-inert group R²Reacting the polyethylene glycol; wherein, Y¹And Y²Capable of reacting to polyethylene glycol having attached thereto a polypeptide and optionally a reactive inert group R²The polyethylene glycol of (a) is attached to a side chain of the polymer; x¹And X²A reaction occurs to attach the R group to the side chain of the polymer.

According to the method, the raw materials can be subjected to one-pot reaction or two-step reaction.

The invention also provides an amphiphilic polymer, wherein the structure of the polymer is shown as the formula I:

the polymer comprises a main chain and side chains, wherein the main chain comprises carbon atoms and at least one heteroatom, and at least part of the side chains respectively comprise an A group, a B group and an optional D group;

the A group is-X-R, wherein X is a linking group and R is a hydrophobic group;

the B group is-Y-L¹-PEG-L²-Z-R¹The D group is-Y-L¹-PEG-L²-R²Wherein Y is a linking group, Z is a linking group, PEG is polyethylene glycol, R is¹Being a polypeptide containing a polypeptide sequence which promotes cell adhesion growth, L¹、L²Is a direct bond or a spacer group, R²Are reactive inert groups.

In one embodiment, the group in X or Y is as follows:

-CO-NH-、-O-、-CO-O-、-S-S-、-R³R⁴C＝N-、any one of a connecting group obtained by click reaction of azido and alkynyl and a connecting group obtained by click reaction of tetrazine and double bonds, wherein the sequence of the groups can be reversed, for example, -CO-NH-represents-CO-NH-or-NH-CO-; wherein R is³、R⁴Same or different, independently from each other selected from H, C_1-6An alkyl group;

wherein the connecting group obtained by click reaction of azido and alkynyl is triazolyl, e.g. triazolylWherein R is⁵Selected from H, C_1-6Alkyl, dotted line represents C_3-10A carbocyclic ring; the carbocyclic ring may or may not be present.

Wherein the linking group resulting from the click reaction of the tetrazine with the double bond is a diazacyclo group, e.g.Wherein the dotted line represents C_3-10A carbocyclic ring, which may or may not be present.

In one embodiment, the X is formed by reacting a reactive group X on a polymer side chain¹With compounds R-X²Reactive group X in (1)²The reaction is carried out. The reactive group X¹、X²For example selected from hydroxy, amino, carboxyAt least one of a group, an aldehyde group, a ketone group, an ester group, a thiol group, a maleimide group, an α -halocarbonyl group, an alkynyl group, an alkenyl group, an azido group, and a tetrazinyl group. For example, reacting an amino group on a polymer side chain with R-COOH gives-NH-CO-.

In one embodiment, the Y is formed by reacting a reactive group Y on a polymer side chain¹With a reactive group Y at one end²With a polyethylene glycol of (a), the reactive group Y¹、Y²For example, at least one selected from the group consisting of a hydroxyl group, an amino group, a carboxyl group, an aldehyde group, a ketone group, an ester group, a thiol group, maleimide, an α -halocarbonyl group, an alkynyl group, an alkenyl group, an azido group, and a tetrazinyl group. For example, reacting an amino group on a polymer side chain with polyethylene glycol having a succinimide ester group at one end to give-NH-CO-.

The PEG is a polyethylene glycol segment, and the number of repeating units of the PEG is an integer between 1 and 600, preferably an integer between 2 and 300, and more preferably an integer between 4 and 200.

In one embodiment, the polypeptide sequence that promotes cell adhesion growth may be or include, for example, the following: RGD, IKVAV, or RYVVLPR, RNIAEIIKDI, CCRRIKVAVWLC, CSRARKQAASKAVSADDR, and artificially synthesized positive charge-rich sequences KR, RKR, KKRK, etc.;

in one embodiment, Z is the following group:

-CO-NH-、-O-、-CO-O-、-S-S-、-R³R⁴C＝N-、any one of a connecting group obtained by click reaction of azido and alkynyl and a connecting group obtained by click reaction of tetrazine and double bonds, wherein the sequence of the groups can be reversed, for example, -CO-NH-represents-CO-NH-or-NH-CO-; wherein R is³、R⁴Same or different, independently from each other selected from H, C_1-6An alkyl group;

in one embodiment, the linking group Z may be a reactive group Z attached through one end of a polyethylene glycol¹E.g. selected from hydroxy, amino, carboxy, aldehyde groupsAt least one of a ketone group, an ester group, a thiol group, a maleimide group, an α -halocarbonyl group, an alkynyl group, an alkenyl group, an azido group, and a tetrazinyl group, and a reactive group Z on a modified or unmodified amino acid at one end of the polypeptide²(e.g., hydroxyl, amino, carboxyl, aldehyde, keto, ester, thiol, maleimide, α -halocarbonyl, alkynyl, alkenyl, azido, tetrazinyl). For example, polyethylene glycol with maleimide at one end and polypeptide with modified or unmodified cysteine at one end are subjected to addition reaction to obtain the following connecting groups:

in one embodiment, L¹、L²Is a direct bond or an optional reactive group Y²、X¹A spacer group of a polyethylene glycol segment is introduced.

In one embodiment, said R is²Can be C_1-6Alkoxy radicals, such as methoxy, ethoxy.

In one embodiment, R is C_4-25Alkyl radical, C_4-25Alkenyl radical, C_4-25Alkynyl, C_6-36An aryl group;

in one embodiment, the polymer contains at least one heteroatom selected from oxygen, nitrogen, sulfur, silicon, and the like in addition to carbon atoms in the backbone. The main chain structure includes, for example: at least one of a polyether main chain, a polyester main chain, a polyamide main chain, a polyurethane main chain, a polysulfide rubber main chain, a polysilicone rubber-polyamide main chain, a polyethyleneimine main chain, a polyamino acid main chain and the like;

in one embodiment, the polyamino acid is poly-L-lysine, and artificially synthesized poly-D-lysine, and the like.

In one embodiment, the mole percentage of side chains containing a B group to the total amount of side chains containing a B group and a D group in the polymer is 0.1% to 100%, preferably 1% to 100%, more preferably 10% to 100%.

The number of side chains containing the B group and optionally the D group is from 2% to 98%, preferably from 5% to 90%, more preferably from 10% to 80% of the total number of side chains.

The percentage of the number of side chains containing the A group to the total number of side chains is 2% to 98%, preferably 5% to 90%, more preferably 10% to 80%.

The percentage of the sum of the number of side chains containing a group and the number of side chains containing B groups and optionally D groups to the total number of side chains is 5% to 100%, preferably 20% to 100%, more preferably 40% to 100%.

The invention also provides the application of the amphiphilic polymer, which is used for modifying the surface hydrophobic substrate or used in the stem cell differentiation and tissue engineering fields.

The invention also provides a method for modifying a surface hydrophobic substrate, comprising the following steps:

and (3) contacting the surface hydrophobic substrate to be treated with the amphiphilic polymer to realize the modification treatment of the surface hydrophobic substrate.

According to the invention, the contacting can be, for example, a contacting of the surface hydrophobic substrate to be treated with a dispersion of the amphiphilic polymer described above. The dispersion may be an aqueous solution, an organic solution or a mixed solution of an organic solvent and water of the modified polymer.

According to the invention, the concentration of the amphiphilic polymer in the aqueous solution, the organic solution or the mixed solution of the organic solvent and water of the amphiphilic polymer is less than 1000g/L, preferably less than 200g/L, more preferably less than 10 g/L. The volume ratio of the organic solvent to water in the mixed solution is not particularly limited, and is, for example, 1 to 99:99-1, for example, 10 to 90: 90-10.

According to the invention, the aqueous solution comprises pure water or a buffered saline solution, such as phosphate buffered saline, HEPES buffered solution, citric acid-trisodium citrate buffer. The pH of the buffered salt solution is 2-12, preferably 7.4.

According to the invention, the organic solution is an alcohol, a ketone, an ether, an ester, a sulfone, an alkyl-substituted amide, a halogenated organic solvent, an aromatic organic solvent, and their alkyl derivatives, alkenyl derivatives, alkynyl derivatives, aryl and heteroaryl derivatives.

The invention also provides a product obtained by modifying the surface hydrophobic substrate.

According to the present invention, the surface hydrophobic substrate refers to a substrate having a water contact angle of the substrate surface of more than 60 °, such as more than 90 °.

According to the invention, the surface hydrophobic substrate comprises a biodegradable material and a non-degradable material.

Wherein the biodegradable material comprises polylactic acid, polyester, polycaprolactone, polylactic acid-polyester copolymer, polylactic acid-polycaprolactone copolymer and the like.

The non-biodegradable material includes alkene and diene polymers, polystyrene, polyvinyl halide, polyvinylidene fluoride, etc., and polyether, polyester, polyamide, polyurethane, polysulfide rubber, polysilicon rubber, silane-based polymer, etc., such as polydimethylsiloxane.

According to the present invention, the surface hydrophobic substrate can be obtained by surface modification of a hydrophilic substrate, for example, forming a hydrophobic self-assembled monomolecular film on a metal such as gold, silver, stainless steel, etc. using a compound having a mercapto group; the compound with sulfhydryl group is C_4-25Alkyl mercaptan, C_4-25Alkenyl mercaptan, C_4-25Alkynyl thiols and mercapto-substituted aryl groups.

According to the present invention, the surface hydrophobic substrate may also be obtained by surface-modifying a hydrophilic inorganic material and an organic material with silane, the inorganic material including: metal oxides such as titanium oxide, zinc oxide, aluminum oxide alloy, tin oxide, and the like; inorganic oxides such as quartz, glass, ITO glass, etc.; silicates, aluminosilicates such as mica, silicon carbide, and the like; the organic material includes surface-oxidation-treated polyether, polysulfone, polyimide, polystyrene, polyvinylidene fluoride, silane-based polymer, and the like, such as polydimethylsiloxane; the surface oxidation treatment includes plasma treatment and corona discharge treatment.

According to the invention, the silanes include methylsilane, dimethylsilane, diethylsilane; also chlorosilanes, such as dimethylchlorosilane, dimethyldichlorosilane; also siloxanes such as dimethyldimethoxysilane, dimethyldiethoxysilane, diethyldimethoxysilane; the silane may optionally contain an aromatic group.

According to the present invention, the hydrophobic substrate is a plane, and may also be a two-dimensional micro/nano-structured pattern, or a three-dimensional micro/nano-structure, such as one of micro-beads, nano-particles, nano-fibers, nano-two-dimensional materials, irregular porous materials, or the like, or a combination thereof.

[ terms and explanations ]

The alkyl group in the present invention represents a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms, for example, methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, etc.

The alkenyl group in the present invention represents a linear, branched or cyclic alkenyl group having 2 to 12 carbon atoms, for example, ethylene, propylene, isopropylene, butene, etc. Preferably, the number of double bonds is an integer from 1 to 6.

The alkynyl group in the present invention represents a linear, branched or cyclic alkynyl group having 2 to 12 carbon atoms, for example, acetylene, propyne, butyne and the like. Preferably, the number of acetylenic bonds is an integer from 1 to 6.

The aryl group of the present invention refers to a monocyclic, or polycyclic, fused aromatic group having 6 to 36 carbon atoms, and representative aryl groups include: phenyl, naphthyl, pyrenyl, and the like.

The terms "carbocycle", "carbocyclyl" or "carbocyclyl" refer to a monovalent non-aromatic, saturated or partially unsaturated ring having from 3 to 12 carbon atoms in a single ring or from 7 to 12 carbon atoms in a bicyclic ring. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.

The amino group in the invention represents the group-NH₂、-NHR⁷or-NR⁷ ₂Wherein R is⁷Independently selected from H, alkyl, aryl, heteroaryl, heterocyclic radical.

The ether group of the invention represents a group-OR⁸Wherein R is⁸Independently selected from C_1-6Alkyl, - (CH)₂-CH₂O)n-CH₂-CH₃(n is greater than 2); examples of the ether group include methyl ether, ethyl ether, propyl ether, isopropyl ether, butyl ether, isobutyl ether, tert-butyl ether, polyoxyethylene ether group having an ethylene oxide number of 9 to 12, and the like.

The term "reactive inert group" refers to a group which is not susceptible to chemical reaction with other groups, e.g. C_1-6Alkoxy radicals, such as methoxy, ethoxy.

The term "reactive group" may also be referred to as a "reactive group," which refers to a functional group that can form a chemical bond with another "reactive group. Suitable chemical bonds are well known in the art and may be, for example: hydroxyl, amino, carboxyl, aldehyde, ketone, ester, sulfhydryl, maleimide, alpha-halocarbonyl, alkynyl, alkenyl, azido and tetrazine.

The term "linking group" refers to a group that links any two groups together, which is a group formed by the reaction of two "reactive groups".

The term "spacer group" refers to a group that may be formed when a reactive group or a reactive inert group is introduced at the end of a polyethylene glycol chain by conventional reaction. The groups depend on the preparation method used for introducing the groups.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, selenocysteine, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. One amino acid that may be particularly used is citrulline, which is a derivative of arginine and is involved in the formation of urine in the liver. Amino acid mimetics refer to chemical compounds that differ in structure from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid. The term "unnatural amino acid" is intended to mean the "D" stereochemical form of the above-mentioned 20 naturally occurring amino acids. It is further understood that the term unnatural amino acid includes homologs of natural amino acids or D-isomers thereof, as well as synthetically modified forms of natural amino acids. Synthetically modified forms include, but are not limited to, amino acids with side chains shortened or lengthened by up to 2 carbon atoms, amino acids comprising optionally substituted aryl groups and amino acids comprising halogenated groups, preferably halogenated alkyl and aryl groups and also N-substituted amino acids, such as N-methyl-alanine. The amino acid or peptide may be linked to the linker/spacer or cell-binding agent via the terminal amine or terminal carboxylic acid of the amino acid or peptide. Amino acids can also be linked to a linker/spacer or cell-binding agent through a side chain reactive group such as, but not limited to, the thiol group of cysteine, the epsilon amine of lysine, or the side chain hydroxyl group of serine or threonine. In addition, the synthetically modified amino acid may also have a reactive group introduced from the α carbon, for example, any of an azide group, an alkyne group, a carbonyl group, an aldehyde group, an alkene group, and a tetrazine group.

Amino acids and peptides may be protected by protecting groups. Protecting groups are atoms or chemical moieties that protect the N-terminus of an amino acid or peptide from undesired reactions and may be used during synthesis. The protecting group should remain attached to the N-terminus throughout the synthesis and can be removed by chemical or other conditions that selectively effect its removal after synthesis of the drug conjugate is complete.

Suitable protecting groups for N-terminal protection are well known in the field of peptide chemistry. Exemplary protecting groups include, but are not limited to, methyl ester, t-butyl ester, methyl 9-fluorenylcarbamate (Fmoc), and benzyloxycarbonyl (Cbz).

The invention has the beneficial effects that:

the amphiphilic polymer is used for carrying out one-step surface treatment on a hydrophobic material, avoids the complexity and low efficiency of a multi-step method in the prior art, can carry out high-efficiency interface modification on hydrophobic surfaces with various structures, ensures the adhesion growth and maintenance of the dryness of stem cells, and is used for tissue engineering of stem cell culture, differentiation and organ reconstruction. The amphiphilic polymer has the following advantages: (1) strong hydrophobicity can be obtained by using strong hydrophobic side chain modification; (2) the kind of the hydrophobic side chain can be flexibly selected; (3) the grafting modification does not need to synthesize a main chain again, and only modifies the side chain.

Drawings

FIG. 1 is an electron micrograph (a) and diameter distribution (b) of nanofibers used for the cell adhesion functionalization of the present invention.

Fig. 2 is a result of detecting a stem cell dry marker of a human mesenchymal stem cell after the human mesenchymal stem cell grows on a nanofiber membrane of the present invention.

Detailed Description

The preparation method of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

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

The HEPES buffer used in the following examples was a buffer solution having a concentration of 100mM and pH 7.5.

Optionally indicating the presence or absence of the stated feature, and also indicating that the stated feature must be present, although the particular choice may be arbitrary.

EXAMPLE 1 Synthesis of amphiphilic graft Polymer OA25-PLL-PEG3k-RGD50

7.5mg of poly-L-lysine hydrobromide (PLL, average molecular weight 2.2kDa, from Shanghai leaves) was weighed into a2 ml centrifuge tube and dissolved in 100. mu.l of HEPES buffer to obtain a solution (I).

54mg of maleimide-polyethylene glycol-succinimidyl ester (NHS-PEG3k-MAL, average molecular weight 3000Da, available from Sigma-aldrich Co.) (polyethylene glycol with terminal group modification of maleimide and succinimidyl ester, respectively) was weighed and dissolved in 100. mu. l N, N-dimethylformamide (analytically pure, available from Beijing chemical industries) to give a solution (II).

Then 12.6mg of hexapeptide containing N-terminal acetyl-modified cysteine and RGD sequence with the amino acid sequence CGRGDS (Ac-CGRGDS, molecular weight 636, available from Nanjing King Shirui Biotech Co., Ltd.) was weighed and dissolved in 100. mu.l of HEPES buffer solution to obtain solution (III).

Mu.l of oleic acid (OA, MW 282.5 from Sigma-aldrich) was measured in a glass vial and placed in 100. mu. l N, N-dimethylformamide to give a solution (IV).

Solution (two) and solution (three) were mixed at room temperature for 15 minutes, after which solution (one) was added to the above mixed solution, uniformly shaken using a shaker at room temperature, reacted for 4 hours, followed by addition of solution (four), and 28mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, molecular weight 191.7, available from Sigma-aldrich) was immediately added to the resulting mixed solution. Shaking overnight for 12 hours, 100. mu.l of 10mM cysteine HEPES buffer was added for 30 minutes to terminate the reaction. The resulting reaction solution was transferred to a semipermeable membrane having a cut-off molecular weight of 14kDa, and dialyzed against 2 liters of deionized water for 48 hours. The obtained solution is frozen and then placed in a freeze dryer to prepare 41mg of freeze-dried powder. Sealing and storing at-20 deg.C.

The freeze-dried powder is a functionalized amphiphilic polymer grafted and modified by 25% of oleic acid and 50% of polyethylene glycol 3000-GRGDS, is named OA25-PLL-PEG3k-RGD50, and has a specific structural formula shown in the specification.

Wherein the number of unmodified side chains is represented by p, the number of side chains connected with polyethylene glycol and polypeptide is represented by q, the number of side chains connected with hydrophobic groups is represented by r, and the polypeptide with cell adhesion function is connected with the polyethylene glycol through addition reaction of maleimide at one end of the polyethylene glycol with the average molecular weight of 3000 and sulfydryl in cysteine.

EXAMPLE 2 Synthesis of amphiphilic graft Polymer OA25-PLL-PEG2k-RGD30

Similar to example 1, except that 20mg of polyethylene glycol (NHS-PEG2k-MAL, average molecular weight 2000Da, available from Sigma-aldrich Co.) with terminal group modification of maleimide and succinimide ester, respectively, were weighed out and dissolved in 100. mu. l N, N-dimethylformamide to give a solution (two).

7.6mg of hexapeptide having an amino acid sequence of CGRGDS (Ac-CGRGDS, molecular weight 636, available from Nanjing King-Sirui Biotech Co., Ltd.) linked to N-terminal acetyl-modified cysteine was weighed and dissolved in 100. mu.l of HEPES buffer to obtain a solution (III).

The obtained lyophilized powder is a functionalized amphiphilic polymer grafted and modified by 25% oleic acid and 30% polyethylene glycol 2000-GRGDS, and is called OA25-PLL-PEG2k-RGD 30.

Example 3 OA25-PLL-PEG2k-OMe30-PEG2k-RGD30 was synthesized.

Similar to example 2, except that 20mg of polyethylene glycol with terminal group modification of succinimidyl ester and methoxy group respectively (NHS-PEG2k-OMe, polyethylene glycol average molecular weight 2000Da, available from Nanocs, USA) and 20mg of polyethylene glycol with terminal group modification of maleimide and succinimidyl ester respectively (NHS-PEG2k-MAL, polyethylene glycol average molecular weight 2000Da, available from Sigma-aldrich) were weighed and dissolved in 100. mu. l N, N-dimethylformamide to obtain a solution (II).

The obtained lyophilized powder is a functionalized amphiphilic polymer grafted and modified by 25% oleic acid, 30% polyethylene glycol 2000-OMe and 30% polyethylene glycol 2000-GRGDS, and is called OA25-PLL-PEG2k-OMe30-PEG2k-RGD 30.

EXAMPLE 4 Synthesis of amphiphilic graft Polymer OA25-PLL-PEG3k-IKVAV30

Similar to example 1, except that 9.1mg of a heptapeptide having an N-acetyl-modified cysteine at one end thereof and having an amino acid sequence of CIKVAVS (Ac-CIKVAVS, molecular weight 761, available from Ostrey Biotech Co., Ltd., Nanjing) was weighed and dissolved in 100. mu.l of HEPES buffer to obtain a solution (III).

The obtained white lyophilized powder is a functionalized amphiphilic polymer grafted and modified by 25% oleic acid and 30% polyethylene glycol 3000-IKVAVS, and is called OA25-PLL-PEG3k-IKVAV 30.

EXAMPLE 5 Synthesis of amphiphilic graft Polymer OA50-PLL-PEG3k-IKVAV30

Similar to example 1, except that 9.1mg of a heptapeptide having an N-acetyl-modified cysteine at one end thereof and having an amino acid sequence of CIKVAVS (Ac-CIKVAVS, molecular weight 761, available from Ostrey Biotech Co., Ltd., Nanjing) was weighed and dissolved in 100. mu.l of HEPES buffer to obtain a solution (III);

and 6.4. mu.l of oleic acid (MW 282.5, from Sigma-aldrich) was measured and placed in 100. mu. l N, N-dimethylformamide to give a solution (IV).

The obtained white lyophilized powder is a functionalized amphiphilic polymer grafted and modified by 50% oleic acid and 30% polyethylene glycol 3000-IKVAVS, and is called OA50-PLL-PEG3k-IKVAV 30.

EXAMPLE 6 Synthesis of amphiphilic graft Polymer OA50-PLL-PEG3k-IKVAV50

Similar to example 5, except that 15.2mg of heptapeptide having an N-acetyl-modified cysteine at one end thereof and having an amino acid sequence of CIKVAVS (Ac-CIKVAVS, molecular weight 761, available from Ostrey Biotech Co., Ltd., Nanjing) was weighed and dissolved in 100. mu.l of HEPES buffer to obtain a solution (III).

The obtained white lyophilized powder is a functionalized amphiphilic polymer grafted and modified by 50% oleic acid and 50% polyethylene glycol 3000-IKVAVS, and is called OA50-PLL-PEG3k-IKVAV 50.

EXAMPLE 7 Synthesis of amphiphilic graft Polymer Py25-PLL-PEG3k-RGD30

7.5mg of poly-L-lysine hydrobromide (PLL, average molecular weight 22500Da, from Shanghai leaves) was weighed out using a2 ml centrifuge tube and dissolved in 100. mu.l of HEPES buffer to obtain solution one.

54mg of polyethylene glycol (NHS-PEG3k-MAL, average molecular weight 3000Da, available from Sigma-aldrich) with terminal group modified to maleimide and succinimidyl ester, respectively, were weighed and dissolved in 100. mu. l N, N-dimethylformamide to give a solution (two).

7.6mg of hexapeptide having N-acetyl-modified cysteine at one end and having an amino acid sequence of CGRGDS (Ac-CGRGDS, molecular weight 636, available from Nanjing King-Shirui Biotech Co., Ltd.) was weighed and dissolved in 100. mu.l of HEPES buffer solution to obtain a solution (III).

2.6mg of 1-pyrenebutanoic acid (molecular weight 288.5, from Sigma-aldrich) was weighed out in a glass vial and placed in 100. mu.l of dimethylformamide to give a solution (IV).

Solution (two) and solution (three) were mixed at room temperature for 15 minutes, after which solution (one) was added to the above mixed solution, uniformly shaken using a shaker at room temperature, reacted for 4 hours, followed by addition of solution (four), and 28mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, molecular weight 191.7, available from Sigma-aldrich) was immediately added to the resulting mixed solution. Shaking overnight for 12 hours, 100. mu.l of 10mM cysteine HEPES buffer was added for 30 minutes to terminate the reaction. The resulting reaction solution was transferred to a semipermeable membrane having a cut-off molecular weight of 14kDa, and dialyzed against 2 liters of deionized water for 48 hours. The obtained solution is frozen and then placed in a freeze dryer to prepare 29mg of freeze-dried powder. Sealing and storing at-20 deg.C.

The white powder is a functionalized amphiphilic polymer grafted and modified by 25% of pyrene and 30% of polyethylene glycol 3000-GRGDS, and is called Py25-PLL-PEG3k-RGD 30.

EXAMPLE 8 Synthesis of amphiphilic graft Polymer Py25-PLL-PEG3k-IKVAV30

Similar to example 7, except that 9.1mg of a heptapeptide having an N-acetyl-modified cysteine at one end thereof and having an amino acid sequence of CIKVAVS (Ac-CIKVAVS, molecular weight 761, available from Ostrey Biotech Co., Ltd., Nanjing) was weighed and dissolved in 100. mu.l of HEPES buffer to obtain a solution (III).

The obtained white freeze-dried powder is a functionalized amphiphilic polymer grafted and modified by 25% pyrene and 30% polyethylene glycol 3000-IKVAVS and is called Py25-PLL-PEG3k-IKVAV 30.

EXAMPLE 9 Synthesis of amphiphilic graft Polymer Py50-PLL-PEG3k-IKVAV50

Similar to example 8, except that 15.2mg of heptapeptide having an N-acetyl-modified cysteine at one end thereof and having an amino acid sequence of CIKVAVS (Ac-CIKVAVS, molecular weight 761, available from Ostrey Biotech Ltd., Nanjing) was weighed to prepare a solution (III);

5.2mg of 1-pyrenebutanoic acid (purchased from Sigma-aldrich) was weighed and placed in 100. mu.l of dimethylformamide to obtain a solution (IV).

The obtained white lyophilized powder is a functionalized amphiphilic polymer grafted and modified by 50% pyrene and 50% polyethylene glycol 3000-IKVAVS, and is called Py50-PLL-PEG3k-IKVAV 50.

Example 10 modification of L-polylactic acid nanofiber with amphiphilic functional Polymer Py25-PLL-PEG3k-IKVAV30

10g of L-polylactic acid (PLLA, weight average molecular weight 1X 10)⁵Denna gang biotechnology limited), was added to methylene chloride (analytically pure, beijing chemical plant), magnetically stirred for 8 hours, and after completely dissolved, N-dimethylformamide was added so that the volume ratio of methylene chloride to N, N-dimethylformamide was 6: 4. stirring was continued for 30 minutes, and the volume of the solution was controlled so that the final concentration of the polymer in the mixed solvent was 4% by weight by volume. The electrostatic spinning equipment (model TL-BM-300, Shenzhen Shangli Nali micro-nano science and technology Limited) is adopted for spinning. The solution was drawn into a syringe and the flow rate was controlled by a syringe pump. A stainless steel single nozzle with an inner diameter of 0.32mm is adopted, a disc-shaped wire collector with a diameter of 20cm is used, and an aluminum foil with a thickness of 0.40mm is paved on the wire collector. 0- +40kV high-voltage direct-current power supply. The spinning parameters are as follows: the flow rate is 0.5mL/h, the voltage is +8.5kV, the receiving distance is 15cm, and the rotating speed of the rotating disc is 60 rpm. The nozzle makes 10cm reciprocating motion 20 times with the center of the turntable as the starting point, and the linear velocity of the nozzle is 5 cm/min. The environmental temperature is controlled to be 25 ℃ and the relative humidity is controlled to be 30 percent. Taking the electrospun fiber and the aluminum foil out of the turntable, putting the electrospun fiber and the aluminum foil into a vacuum oven, drying the electrospun fiber and the aluminum foil at the temperature of 60 ℃ for 8 hours, and completely removing the solvent. And annealing to room temperature and taking out. The average diameter of the electrospun fiber was 410nm (a and b in FIG. 1), and the average thickness of the electrospun fiber membrane was 22 μm. The water contact angle of the electrospun fiber membrane measured on the aluminum foil substrate is 110 degrees, and the electrospun fiber membrane shows strong hydrophobicity. 1mg Py25-PLL-PEG3k-IKVAV30 was purified from 5mL of N, N-dimethylformamide: dissolving the mixed solvent of water (50: 50 volume ratio), immersing the PLLA electrospun fiber membrane and the aluminum foil into the solution, standing for 8 hours at room temperature, taking out, putting into a vacuum oven, drying for 20 hours at 60 ℃, and completely removing the solvent. And annealing to room temperature and taking out. The electrospun fiber membrane was measured to have a water contact angle of 58 ° which showed increased hydrophilicity compared to the untreated nanofiber membrane. The PLLA electrospun fiber membrane and the aluminum foil substrate thereof are cut into blocks of 12mm multiplied by 12mm by a slicer, and the nanofiber membrane is transferred to a glass substrate of 10mm multiplied by 10mm and completely covers the substrate. Storing in a sterilizing box at room temperature for later use.

Example 11 adherent growth of Stem cells in amphiphilic Polymer-modified Poly (L-lactic acid) nanofibers

Extracting mesenchymal stem cells from umbilical cord tissues of healthy people, and culturing and amplifying the obtained stem cells in vitro. The method comprises the following specific steps: collecting umbilical cords of healthy people (Beijing Anzhen hospital biological sample library, obtained according to informed terms); removing the blood vessels and the outer membrane in the umbilical cord; shearing umbilical cord tissue to about 2mm³Small pieces of size; planting the cut small pieces in a culture dish (diameter is 100mm) added with serum-free mesenchymal stem cell culture solution, and placing at 37 deg.C and saturated humidity with 5% CO₂Culturing in an incubator; half a half of the culture solution is changed every 4 days, the cells are about 80 percent confluent after being cultured for 15 days, and the changed waste culture solution is collected and stored in a refrigerator at the temperature of minus 80 ℃; when the cells were about 80% confluent, the cells were digested with 0.25% trypsin at 1X 10⁵The cell number/mL is inoculated in a culture dish for subculture, after 5 passages, the high-purity mesenchymal stem cells are obtained by separation and purification, and the purity of the mesenchymal stem cells is analyzed by a FACS Calibur flow cytometer (BD Biosciences). Subjecting the obtained human mesenchymal stem cells to extraction at a ratio of 1 × 10⁵Cell number/mL was seeded on Py25-PLL-PEG3000-IKVAV30 modified PLLA nanofiber substrate obtained in example 8, using feeder cells freeConditioned culture of the layers. Specifically, the medium used contained 20% of KSR (Thermo Fisher Co., Ltd.), 1 XNEAA (non-essential amino acid, Thermo Fisher Co., Ltd.), 2mM L-glutamic acid and 0.1mM D-MEMF12(CT-D6421) in dissolved 2-mercaptoethanol at 37 ℃ under 5% CO₂. The cells were passaged every 3 days using dissociation solution (containing 0.25% trypsin, 1mg/ml collagenase IV solution, CaCl dissolved in 1mM phosphate buffer solution)₂(ii) a All from Thermo Fisher) and dissociated into small cell masses (consisting of about 50-100 cells) with a pipette, and seeded onto another piece of the amphiphilic functional polymer Py25-PLL-PEG3000-IKVAV30 modified poly (l-lactic acid) nanofiber substrate. Repeating the above work to obtain 10 generations of human mesenchymal stem cells. And separating the stem cells of each generation from the nanofiber substrate by using a dissociation solution, dispersing the stem cells into a phosphate buffer solution, and analyzing the expression of the mesenchymal stem cell surface markers CD29, CD44, CD73, CD90 and CD105 of each generation on the cell surface. The results of the FACS Calibur flow cytometer analysis of passage 1, 3, 5, 7, 10 are shown in figure 2. The results show that the dryness of the human mesenchymal stem cells is well maintained on the Py25-PLL-PEG3000-IKVAV30 modified poly-L-lactic acid nanofiber substrate.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.