CN115012221A

CN115012221A - Nanofiber material for controlling stem cell differentiation and preparation method and application thereof

Info

Publication number: CN115012221A
Application number: CN202110246141.1A
Authority: CN
Inventors: 喻长远; 刘爽; 柳朝永; 游长江; 覃小艳
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2022-09-06
Anticipated expiration: 2041-03-05
Also published as: CN115012221B

Abstract

The invention provides a nanofiber material for controlling stem cell differentiation and a preparation method and application thereof. The modified nanofiber material can immobilize the controlled-release nanocapsules on the premise of not changing the polymer property of the nanofiber body, and can induce stem cell differentiation. Specifically, according to the requirement of inducing stem cell differentiation, the tail end of the hydrophilic side chain is coupled with a controllable release nanocapsule wrapping the cell signal factor, so that the specific immobilized cell signal factor is ensured. The modified nanofiber immobilized with the cell signal factors is used as a scaffold to prepare a three-dimensional culture matrix, the pH value (7.0-8.5) of the microenvironment of the matrix is adjusted, the cell signal factors (such as BMP-2) are controllably released, and stem cell differentiation is induced.

Description

Nanofiber material for controlling stem cell differentiation and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nanofiber materials, and particularly relates to a nanofiber material for controlling stem cell differentiation, 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 including signal proteins such as transforming growth factors and the like according to a certain proportion; (3) the culture substrate needs to have a hardness similar to that of the tissue.

At present, a full-artificial stem cell culture medium with determined components gradually replaces biological products, 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 can obtain a base material with excellent biocompatibility through surface modification on the premise of not changing the mechanical properties of the material, and the base material is used for various functional tissue engineering scaffolds. In addition to promoting the adhesion growth of stem cells on biomedical materials, artificial materials for regenerative medicine also need to control the spatial distribution of stem cell differentiation for transformation into organoids. Therefore, there is a need in the art for an interfacial treatment material that controls the spatial distribution of stem cell differentiation. Stem cell differentiation can be determined by detecting differentiation markers. Staining for alkaline phosphatase (ALP) is a reliable detection method for osteogenic differentiation.

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. The surfaces of the materials are provided with micro/nano structures, such as electrospinning nano fibers and irregular porous materials which are commonly used for supporting stem cell culture matrixes. The substrate with the micron/nanometer structure is subjected to biocompatibility treatment, and the solution dip dyeing covering has a good effect. For hydrophobic substrate materials, biocompatibility modification is usually performed by covering the surface with artificial macromolecules and natural biological macromolecules such as bovine serum albumin. Biofunctionalization of hydrophobic substrates using amphiphilic block polymers is also a common method. However, the conventional polypropylene oxide is weak in hydrophobicity and cannot satisfy the case where a strong hydrophobic interaction is required. Furthermore, the type of the block moiety is fixed, and it is difficult to flexibly modify the block moiety depending on the properties of the substrate. Any adjustment of the copolymerization ratio in the block copolymer requires the re-synthesis of the main chain.

In the application of biomedical materials, the controllable release of active effective components influencing the stem cell differentiation in time and space is of great significance. The spatial and temporal distribution of signal factors in the extracellular matrix is controlled, which is beneficial to understanding the specific mechanism of the signal factors for regulating and controlling the stem cell differentiation and the synergistic mechanism among the signal factors, thereby controlling the fate of the stem cells on the biomedical materials and promoting the stem cells to be differentiated into target tissues. Bone Morphogenetic Proteins (BMPs) are a group of highly conserved functional proteins with similar structures, belonging to the TGF-beta family. The BMP is used as a transforming growth factor, is not limited to osteogenic directed differentiation of stem cells, but also is closely related to development of central nervous tissue and differentiation of other stem cells, including embryonic stem cells, germ line stem cells, hematopoietic stem cells and intestinal stem cells.

Naturally occurring cell signaling factors (cytokines) that control stem cell differentiation and maintenance of sternness are often shown to have significant domain limiting effects, thereby ensuring that sternness maintenance and differentiation of stem cells occurs within a controlled temporal and spatial range. These cell signaling factors are prone to non-specific adsorption, aggregation and loss of activity. The cell signal factor, such as BMP2, encapsulated by the polymer nano capsule can protect the cell signal factor from being contacted with each other and gathered. The encapsulation of the polymer nanocapsule can also avoid the nonspecific adsorption loss of cell signal factors in the stem cell culture matrix, and greatly reduce the required dosage. However, since the cell signal factors encapsulated by the nanocapsules can freely diffuse in the culture system, the domain-limiting effect necessary for the differentiation control of the stem cells is lost. In regenerative medicine application, in order to make the artificial matrix completely reproduce the confinement effect of the cell signal factors, the cell signal factors wrapped by the nano capsules need to be distributed in the artificial matrix in a controllable manner.

Disclosure of Invention

The invention aims to provide a modified nanofiber material capable of realizing the spatial controllable distribution of cell signaling factors, a preparation method thereof and application thereof in controlling the fate of stem cells. The modified nanofiber material is obtained by modifying the surface of a nanofiber with an amphiphilic polymer (through hydrophobic interaction) and then immobilizing cell signaling factors wrapped by nanocapsules. The amphiphilic polymer is characterized in that a hydrophilic component and a hydrophobic component are introduced, wherein the hydrophobic component is used for forming firm surface modification with the hydrophobic surface of the nanofiber material through hydrophobic effect, and the hydrophilic component provides biocompatibility and carries a functional group capable of specifically immobilizing a cell signal factor wrapped by a nanocapsule. The amphiphilic polymer is used for carrying out surface modification treatment on the hydrophobic nanofiber material, so that the hydrophobic nanofiber material has good biocompatibility. The nanocapsule wrapped with the cell signal factor is immobilized on the nanofiber material after the surface modification treatment, and the differentiation direction of the stem cells can be adjusted by using the nanocapsule as a biological functional scaffold of a cell culture matrix. Under specific environment, for example, external conditions such as pH value, salinity, temperature, oxidation-reduction condition, etc., cell signal factors influencing the fate of stem cells are separated from the surface of the modified nano fiber material scaffold, thereby controlling the differentiation of the stem cells.

In the present invention, the term "stem cell fate" refers to differentiation and proliferation of stem cells.

In the present invention, the term "immobilization" refers to immobilization of nanocapsules on the nanofiber surface, and includes immobilization on the nanofiber surface by "nonspecific adsorption" or "specific coupling" (also referred to as "specific immobilization (covalent bonding)" and "nonspecific immobilization (nonspecific adsorption))", which can keep immobilized biological substances from inactivation.

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

a modified nanofiber material comprises a hydrophobic nanofiber material, nanocapsules wrapped with cell signaling factors and an amphiphilic polymer, wherein the amphiphilic polymer comprises a hydrophobic group and a hydrophilic group, the amphiphilic polymer is bonded to the surface of the hydrophobic nanofiber material through at least part of the hydrophobic group on the amphiphilic polymer by means of hydrophobic interaction, and at least part of the hydrophilic group of the amphiphilic polymer is connected with the nanocapsules wrapped with the cell signaling factors through covalent bonds.

According to the invention, the hydrophobic nanofiber material is selected from at least one of Polycaprolactone (PCL), polylactic acid (PLA), Polyester (PET), polylactic acid-polyester copolymer and polylactic acid-polycaprolactone copolymer.

According to the invention, the diameter of the hydrophobic nanofiber material is 200-800 nm.

According to the present invention, the hydrophobic nanofiber material may be obtained commercially or may be prepared by methods known in the art, such as by electrospinning.

According to the present invention, the cell signaling factor is selected from at least one of protein cell signaling factors, illustratively, bone morphogenetic protein BMP-2, Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF), and the like.

According to the invention, the nanocapsule encapsulating the cell signaling factor is a polymer, preferably a cross-linked polymer, such as a degradable cross-linked polymer, and the monomer is at least one of acrylamide, N- (3-aminopropyl) methacrylamide and N, N-methylene bisacrylamide. The cross-linking agent is, for example, a polylactide-b-polyethylene glycol-b-polylactide-diacrylate triblock copolymer, glycerol dimethacrylate.

According to the invention, the particle size of the nanocapsules is 17-25 nm.

According to the present invention, in the amphiphilic polymer, a hydrophobic group and a hydrophilic group are introduced into a side chain of the polymer by a grafting method. The main chain of the polymer contains at least one heteroatom such as oxygen, nitrogen, sulfur, silicon and the like besides carbon atoms. The main chain of the polymer is 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 polysilicon rubber-polyamide main chain, a polyethyleneimine main chain, a polyamino acid main chain and the like.

According to the invention, the polyamino acid is poly-L-lysine, artificially synthesized poly-D-lysine and the like.

According to the present invention, the amount of the hydrophilic group-containing side chains in the amphiphilic polymer is 2 to 98 mol%, preferably 5 to 90 mol%, more preferably 10 to 80 mol%, based on the total amount of the side chains.

According to the invention, the amphiphilic polymer has a molar percentage of side chains containing hydrophobic groups of 2% to 98%, preferably 5% to 90%, more preferably 10% to 80%, of the total number of side chains.

According to the present invention, the total amount of hydrophilic groups and hydrophobic groups in the amphiphilic polymer is 5 to 100 mol%, preferably 20 to 100 mol%, more preferably 40 to 100 mol%, based on the total amount of all side chains.

According to the invention, the hydrophobic group of the amphiphilic polymer is C _4-25 A hydrocarbyl group. The hydrophilic group of the amphiphilic polymer is polyethylene glycol.

According to the present invention, the polyethylene glycol is a chain polyethylene glycol, 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.

The invention also provides a preparation method of the modified nanofiber material, which comprises the following steps:

1) preparation of amphiphilic polymers in which the hydrophilic group is bound to a reactive group Z which can undergo a click chemistry reaction ¹ ；

2) Putting the hydrophobic nanofiber material into a solution of an amphiphilic polymer to obtain the nanofiber material modified by the amphiphilic polymer;

3) preparing nanocapsules wrapped with cell signaling factors, and introducing reactive groups Z which generate click chemical reaction on the surfaces of the nanocapsules ² ；

4) Mixing the nanofiber material modified by the amphiphilic polymer in the step 2) with the nanocapsule obtained in the step 3), wherein the reactive group Z in the amphiphilic polymer is ¹ With reactive groups Z on the nanocapsules ² And carrying out click chemical reaction to obtain the modified nanofiber material.

In one embodiment, in step 4), the amphiphilic polymer modified nanofiber material in step 2) is mixed with protein, so that the protein is adsorbed on the nanofiber material, and the nonspecific adsorption is reduced. The protein is, for example, bovine serum albumin BSA. And mixing with the nano-capsule obtained in the step 3).

According to the invention, in the step 1), the preparation method of the amphiphilic polymer comprises the following steps:

having a reactive group Y in a side chain ¹ And X ¹ With the compound R-X ² One end of which has a reactive group Y ² The other end has a reactive group Z which can generate click chemistry reaction ¹ Reacting the polyethylene glycol; wherein, Y ¹ And Y ² Can react to make the linking of the reactive group Z which can generate click chemistry reaction ¹ The polyethylene glycol of (a) is attached to a side chain of the polymer; x ¹ And X ² A reaction occurs to attach the R group to the side chain of the polymer.

According to the invention, the method can carry out one-pot reaction on the raw materials or carry out the reaction in two steps.

According to the invention, the reactive group X ¹ 、X ² 、Y ¹ 、Y ² For example, selected from the group consisting of hydroxyl, amino, carboxyl, aldehyde, keto, ester, thiol, maleimide, α -halocarbonyl. Wherein the reactive group X ¹ And X ² 、Y ¹ And Y ² Mutually reactive groups, a reaction can occur.

For example, amino groups react with carboxyl groups to give amide linking groups, or amino groups react with aldehyde or ketone groups to give schiff base linking groups, or hydroxyl groups react with carboxyl groups to give ester linking groups, or hydroxyl groups react with hydroxyl groups to give ether linking groups by dehydration, or maleimide groups react with thiol groups, or thiol groups react with α -halocarbonyl groups by substitution, or amino groups react with ester groups to give amide linking groups.

As an example, the one end has a reactive group Y ² The other end has a reactive group Z which can generate click chemistry reaction ¹ The 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 Y ² A reactive group Z capable of undergoing a click chemistry reaction ¹ Can be directly connected, i.e., as a capping group, or can be connected through any spacer group, depending on the reactive group Y employed and conventional methods in the art ² A reactive group Z capable of undergoing a click chemistry reaction ¹ Introduced into both ends of the polyethylene glycol. In the presence of polyethylene glycol and a reactive group Y ² Reactive group Z capable of undergoing click chemistry reaction ¹ The spacer group therebetween may be any as long as it does not interfere with the preparation of the amphiphilic polymer according to the invention, and is, for example, C _1-12 Alkyl, ester, amide, ketone, and the like.

According to the invention, said reactive group Y ¹ And X ¹ The 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 Y ¹ And X ¹ Preferably, the end group of the side chain comprises a reactive group Y ¹ And X ¹ . Said group Y having a reactive group ¹ And X ¹ The polymers of (a) are, for example: having reactive groups Y ¹ And X ¹ Polyether, polyester, polyamide, polyurethane, polysulfide rubber, polysilicone rubber-polyamide, polyethyleneimine, polyamino acid, etc.;

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

According to the invention, said reactive group Y ¹ And X ¹ Of the polymer side chain has a reactive group X ¹ 、Y ¹ The same or different. When the reactive group X ¹ 、Y ¹ In 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-X ² And (4) reacting. When the reactive group X is ¹ 、Y ¹ At different times, one of the reactive groups may be reacted with polyethylene glycol having a reactive group and the other reactive group with the compound R-X ² And (4) reacting.

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, said X ¹ And X ² When a reaction is carried out, or the Y ¹ And Y ² The reaction may be carried out, for example, under the acceleration of a coupling agent. For example, the condensation of an amino group with a carboxyl group in the presence of a coupling agent to give an amide linkage, or the condensation of a hydroxyl group with a carboxyl group in the presence of a coupling agent to give an ester linkageA 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.

According to the present invention, in the step 2), the amphiphilic polymer obtained in the step 1) is dissolved in a solvent, such as a phosphate buffer containing 20% ethanol, to obtain a solution of the amphiphilic polymer.

According to the invention, in the step 2), the hydrophobic nanofiber material is placed into a solution of the amphiphilic polymer to be soaked for a certain time, for example, 1-24 hours, so as to obtain the nanofiber material modified by the amphiphilic polymer; the concentration of the amphiphilic polymer solution is, for example, 1 to 10 mg/ml.

According to the invention, in the step 3), the nanocapsule coated with the cell signaling factor is prepared by a method commonly used in the field.

According to the invention, in the step 3), a reactive group Z which can generate click chemistry reaction is introduced on the surface of the nano capsule ² The method of (b) is also carried out by methods commonly used in the art. For example, the following substances are selected to react with the nanocapsules: one end of the substance is provided with a click chemistry reaction group Z ² And one end bearing a group that can react with a free group (e.g., an amino group) on the outer shell of the nanocapsule.

According to the invention, the reactive group Z ¹ 、Z ² Comprises the following steps: azido, alkynyl, tetrazine and double bonds, wherein the azido and the alkynyl are subjected to click reaction, and the tetrazine and the double bonds are subjected to click reaction;

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, the alkyne group is generally at the end group), or cyclotension catalyzed azide-alkyne cycloaddition (SPAAC reaction, the alkyne group is in the middle of the tension ring). The alkynyl group may be located in a ring, and may be, for example, an azabicyclooctanyl group.

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.

The linking group resulting from the click reaction of azido and alkynyl is a triazolyl group, e.g.

Wherein R is ⁵ Selected from H, C _1-6 Alkyl, dotted line represents C _3-10 A carbocyclic ring; the carbocyclic ring may or may not be present.

The linking group resulting from the click reaction of tetrazines with double bonds being a diazacyclo, e.g.

Wherein the dotted line represents C _3-10 A carbocyclic ring, which may or may not be present.

The invention also provides application of the modified nanofiber material in controlling stem cell fate.

The invention has the beneficial effects that:

the invention provides a modified nanofiber material capable of realizing space controllable distribution of cell signaling factors, a preparation method thereof and application of the modified nanofiber material in controlling stem cell fate. The modified nanofiber material can immobilize the controlled-release nanocapsules on the premise of not changing the polymer property of the nanofiber body, and can induce stem cell differentiation. Specifically, according to the requirement of inducing stem cell differentiation, the tail end of the hydrophilic side chain is coupled with a controllable release nanocapsule wrapping the cell signal factor, so that the specific immobilized cell signal factor is ensured. The modified nanofiber immobilized with the cell signal factors is used as a scaffold to prepare a three-dimensional culture matrix, the pH value (7.0-8.5) of the microenvironment of the matrix is adjusted, the cell signal factors (such as BMP-2) are controllably released, and stem cell differentiation is induced.

The nanofiber modified by the amphiphilic polymer can greatly reduce nonspecific adsorption, but can allow cells to be adhered and proliferated.

Drawings

FIG. 1 is OA-PLL-PEG-N prepared in example 1 ₃ (I) Is/are as follows ¹ H NMR spectrum.

FIG. 2 is OA-PLL-PEG-N prepared in example 2 ₃ (II) of ¹ H NMR spectrum.

FIG. 3 is OA-PLL-PEG-N prepared in example 3 ₃ (III) of ¹ H NMR spectrum.

FIG. 4 is OA-PLL-PEG-N prepared in example 4 ₃ (IV) of ¹ H NMR spectrum.

Figure 5 is the water contact angle test results for biofunctionalized PCL.

FIG. 6 shows the hydrodynamic size distribution of dynamic light scattering measurement n (BMP-2).

FIG. 7 shows PCL, PCL-OPP-DBCO-n ( ^FITC BSA) and PCL-OPP-DBCO-n (BMP-2) scaffolds.

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.

Example 1

Synthesis of oleic acid-poly-L-lysine-polyethylene glycol-azide amphiphilic Polymer (I) (oleic acid 13.5%, polyethylene glycol azide 41.7%)

Oleic acid (OA, available from Sigma-aldrich) and carboxy-polyethylene glycol-azide (HOOC-PEG-N) were activated with 8 times the molar amount of poly-L-lysine (PLL, available from Shanghai leaves) repeat unit carbodiimide hydrochloride (EDC, available from Sigma-aldrich) ₃ Commercially available from Shanghai 3A Chemicals Co.) one-step Synthesis of OA-PLL-PEG-N ₃ (I) .1. the The synthesis steps are as follows:

1) 90 mg/18. mu. mol of carboxy-polyethylene glycol-azide (HOOC-PEG-N) was weighed ₃ A molecular weight of 5000Da),

2) sucking 6.4. mu.l/18. mu. mol oleic acid (OA molecular weight 282.5g/mol, density 0.813 g/ml); (ii) a

3) 54.7 mg/288. mu. mol carbodiimide hydrochloride (EDC molecular weight 191.70) was weighed;

4) weighing poly-L-lysine hydrobromide (PLLHBr, molecular weight 30-70KDa) 7.5mg/0.15 μmol;

adding the substances in 1), 2) and 3) into a mixed solution of 1ml of dimethylformamide and 1ml of HEPES buffer solution (pH7.0), mixing, stirring with magnetons for 1h to activate carboxyl, adding the substance in 4), and reacting for 24 h;

5) and (3) purification: the dialysis membrane is selected from 14KDa cut-off molecular weight, and the dialysate is dialyzed for 48 hours at normal temperature by using 30% ethanol water solution;

6) freeze-drying: the purified sample was dried using a low temperature freeze dryer and subjected to nmr spectroscopy for qualitative and quantitative analysis (solvent is deuterated methanol, MeOD).

The freeze-dried powder synthesized by the method is oleic acid-poly-L-lysine-polyethylene glycol-azide amphiphilic polymer (I), and the hydrogen nuclear magnetic resonance spectrum shows that the proportion of oleic acid is 13.5 percent and the proportion of PEG is 41.7 percent, and the freeze-dried powder is named as OA-PLL-PEG-N ₃ (I) In that respect As shown in fig. 1.

Example 2

Synthesis of oleic acid-poly (L-lysine) -polyethylene glycol-azide amphiphilic Polymer (II) (oleic acid 12.5%, polyethylene glycol azide 33.4%)

Similar to example 1, except that a 10-fold molar amount of poly-L-lysine (PLL, available from Shanghai leaves) repeating unit of carbodiimide hydrochloride (EDC, available from Sigma-aldrich) was used, that is, 64mg of EDC was weighed, activated oleic acid (OA, available from Sigma-aldrich) and carboxy-polyethylene glycol-azide (HOOC-PEG-N) were added ₃ Commercially available from Shanghai 3A Chemicals) to synthesize OA-PLL-PEG-N in one step ₃ (II)。

The freeze-dried powder obtained by synthesis is oleic acid-poly-L-lysine-polyethylene glycol-azide amphiphilic polymer (II), and nuclear magnetic resonance hydrogen spectrum shows that the proportion of oleic acid is 12.5 percent, the proportion of PEG is 33.4 percent, and the freeze-dried powder is named asOA-PLL-PEG-N ₃ (II). As shown in fig. 2.

Example 3

Synthesis of oleic acid-poly (L-lysine) -polyethylene glycol-azide amphiphilic polymer (III) (oleic acid 24%, polyethylene glycol azide 31.4%)

Similar to example 1, except that EDC/NHS-sulfo (N-hydroxysuccinimide sulfonate, available from Shanghai carbofuran) was used in combination in a molar ratio of 2: 1; carbodiimide hydrochloride 2 times the molar amount of poly-L-lysine repeating units, i.e. 12.8mg EDC and 8.46mg NHS-sulfo were weighed out. Activating carboxyl of oleic acid and carboxyl-polyethylene glycol-azide to synthesize OA-PLL-PEG-N in one step ₃ (III)。

The synthesized freeze-dried powder is oleic acid-poly-L-lysine-polyethylene glycol-azide amphiphilic polymer (III), and the nuclear magnetic resonance hydrogen spectrum shows that the proportion of oleic acid is 24 percent and the proportion of PEG is 31.4 percent, and the freeze-dried powder is named as OA-PLL-PEG-N ₃ (III). As shown in fig. 3.

Example 4

Synthesis of oleic acid-poly (L-lysine) -polyethylene glycol-azide amphiphilic polymer (IV) (oleic acid 36%, polyethylene glycol azide 20.2%)

Similar to example 3, except DMF and NaHCO were used ₃ The reaction was carried out in a 3:1 by volume solution of carbodiimide hydrochloride 2 times the molar amount of poly-L-lysine repeating units, i.e. 12.8mg EDC and 8.46mg NHS-sulfo were weighed out. Activating carboxyl of oleic acid and carboxyl-polyethylene glycol-azide to synthesize OA-PLL-PEG-N in one step ₃ (IV)。

The synthesized freeze-dried powder is oleic acid-poly-L-lysine-polyethylene glycol-azide amphiphilic polymer (IV), and nuclear magnetic resonance hydrogen spectrum shows that the proportion of oleic acid is 36 percent, the proportion of PEG is 20.2 percent, and the freeze-dried powder is named as OA-PLL-PEG-N ₃ (IV). As shown in fig. 4.

Example 5

Functionalization of surface of nanofiber by oleic acid-poly-L-lysine-polyethylene glycol-azide amphiphilic polymer

Selection of OA-PLL-PEG-N prepared in example 4 ₃ (IV) (OPP for short) as amphiphilic polymer to functionalize Polycaprolactone (PCL) nanofiber (PCL-OPP for short))。

The PCL nanofibers were purchased from Qingdao Nuokang environmental protection science and technology, and were dried in a vacuum oven at room temperature for 7 days before use to remove the non-volatile solvents and impurities. Weighing 2mg of OPP, and dissolving in 1ml of 1 XPBS solution containing 20% ethanol, wherein the final concentration is 2 mg/ml; washing the PCL nano-fiber for 5 times by using 1 XPBS solution to remove impurity influence, then soaking the PCL nano-fiber in 1ml of solution containing OPP, incubating for 3h at room temperature, taking out the PCL nano-fiber, repeatedly washing for 5 times by using the 1 XPBS solution, and finally drying for 48h in vacuum oven at room temperature in vacuum to obtain the dried PCL-OPP.

The surface hydrophilicity and hydrophobicity of the PCL-OPP are tested by static water contact angle, as shown in figure 5, the obtained water contact angle of the PCL-OPP is 31.45 degrees, which is obviously reduced compared with 122.65 degrees of the PCL, and the hydrophilicity is obviously improved. Surface element composition analysis of PCL and PCL-OPP was performed by X-ray photoelectron spectroscopy. PCL has two peaks, including C1s (285eV) and O1s (531.5eV) as the main elements constituting PCL. After the OPP functionalization, an N1s peak (399.5eV) appears in the spectrum of the PCL-OPP nanofiber, which is a nitrogen element component introduced by the OPP functionalization. As a control, no nitrogen element was detected on unfunctionalized PCL (see data in table 1). Analysis of the quantitative atomic chemistry of PCL-OPP, while elemental nitrogen (0.91%) was detected on OPP-functionalized PCL-OPP scaffolds, confirmed the successful modification of OPP on PCL. These results indicate that OPP successfully modifies PCL.

TABLE 1 results of quantitative analysis of atomic chemical composition of PCL-OPP

Example 6 adsorption analysis of oleic acid-poly-L-lysine-polyethylene glycol-azide amphiphilic Polymer functionalized nanofibers for proteins

Bovine serum albumin BSA (BSA) labeled with FITC (fluorescein isothiocyanate) for PCL and PCL-OPP ^FITC BSA) as an initial criterion for bioaffinity and cellular compatibility.

Cutting the PCL nano-fiber into a square with the side length of 1cm according to the following stepsThe protocol of example 5 produces functionalized PCL nanofibers; 0.3mg/ml of the preparation ^FITC BSA solution, two pieces of cleaned PCL nano-fiber and one piece of PCL-OPP are soaked in the above solution ^FITC BSA solution was incubated at 37 ℃ for 2h, followed by fluorescence detection. And finally, performing optical density quantitative analysis on the obtained fluorescence image by adopting Gel-Pro Analyzer image analysis software. Control (Control) was PBS soaked blank PCL. The result shows that the protein is adsorbed by the PCL scaffold in the initial 2h in a higher amount, and strong green fluorescence is shown. The PCL bracket is functionally modified by OPP, so that green fluorescence is weakened. This indicates that even though the functionalization of PCL by OPP changes its surface to hydrophilic, it does not enhance its adsorption ability to proteins but reduces non-specific adsorption of proteins. Showing that the polyethylene glycol molecule covering exists on the surface of the protein, and the protein adsorption is hindered by steric hindrance and entropy increase. On the other hand, the appearance of weak green fluorescence indicates that the OPP surface modification does not completely block the nonspecific adsorption of PCL-OPP to protein, so that the adhesion and proliferation of cells on the scaffold can be supported.

Quantitative analysis showed that the protein adsorption by PCL-OPP scaffolds was reduced by 60% relative to unfunctionalized PCL scaffolds (./p < 0.05). The adsorption and reduction of protein show that the PCL-OPP scaffold not only maintains good biological affinity and cell compatibility, but also reduces non-specific adsorption, and can be used for cell culture.

EXAMPLE 7 preparation of nanocapsules encapsulating cell signaling factor

(1) preparation of pH-responsive degradable nanocapsule-BMP-2-coated nanocapsule n (BMP-2)

Mixing initiators of Ammonium Persulfate (APS) and Tetramethylethylenediamine (TEMED), monomer acrylamide (AAm) and N- (3-aminopropyl) methacrylamide (APm), a cross-linking agent of polylactide-b-polyethylene glycol-b-polylactide-diacrylate triblock copolymer (AI102) and Glycerol Dimethacrylate (GDMA) with BMP-2, wherein the molar ratio of BMP-2, AAm, APm, AI102 and GDMA is 1:4600:400:400: 100. The components with the molar ratio are mixed and then undergo in-situ free radical polymerization on the surface of BMP-2 to form a pH-responsive degradable nanocapsule, namely a nanocapsule n (BMP-2) wrapping the BMP-2. Dynamic Laser Scattering (DLS) data showed that the diameter of the BMP-2 encapsulating nanocapsule n (BMP-2) was 19.3nm, consistent with the expected 20nm range (shown in FIG. 6).

(2) Preparation of DBCO modified BMP-2 coated nanocapsule

The method for carrying out Dibenzocyclooctyne (DBCO) grafting modification on a nano-capsule shell layer of a nano-capsule n (BMP-2) coated with BMP-2 comprises the following steps: 5mg of DBCO-PEG-NHS (purchased from Sienna Rexi Biotechnology Co., Ltd., model R-2016-2k) was dissolved in 100. mu.l of DMSO to a final concentration of 25. mu.M; mixing and vortexing at the DBCO-PEG-NHS (BMP-2) molar ratio of 25:1, adjusting pH to 8.5-9.0, and incubating at room temperature for 2 h. Before use, the micro ultrafiltration tube (MWCO ═ 100kDa) is balanced once by water and 1 XPBS respectively, then the prepared n (BMP-2) is placed in the micro ultrafiltration tube, 200 mul of 1 XPBS is supplemented for centrifugal filtration, the temperature is 4 ℃, 5500rpm is 10min is carried out for the first time, 200 mul of 1 XPBS is supplemented for centrifugal filtration, the temperature is 4 ℃, 5500rpm is carried out, 6min is carried out for the second time, then protein solution in the ultrafiltration tube is collected, and the concentration is determined by a BCA protein analysis kit. The results of the spectrophotometer confirmed the successful grafting of DBCO and quantified a grafting molar ratio of DBCO to n (BMP-2) of 10: 1. The prepared DBCO modified BMP-2-coated nanocapsule is named as DBCO-n (BMP-2) and is used for subsequent specific immobilization on the surface of PCL-OPP.

(3) Non-degradable nanocapsules-encapsulation ^FITC Preparation of nanocapsules of BSA

The method for preparing the nano capsule n (BMP-2) coated with the BMP-2 in the step (1) is adopted to prepare the coating ^FITC The BSA nanocapsule is characterized in that a crosslinking agent is N, N-methylene Bisacrylamide (BIS) to replace a crosslinking agent AI102 and a crosslinking agent GDMA, FITC marked bovine serum albumin BSA is used to replace BMP-2, and the non-degradable nanocapsule coated with the FITC marked bovine serum albumin BSA is prepared and named as N (N: (B) (B)) ^FITC BSA) _BIS 。

(4) DBCO-modified wraps ^FITC Preparation of nanocapsules of BSA

Wrapping the package by adopting the method of the step (2) ^FITC Carrying out DBCO grafting modification on the BSA nanocapsule to obtain a DBCO modified bagWrapping paper ^FITC Nanocapsules of BSA, named DBCO-n (R) ((R)) ^FITC BSA) _BIS For subsequent specific immobilization on PCL and PCL-OPP surfaces.

Example 8 specific immobilization of DBCO-modified nanocapsules on nanofibers

With DBCO-n: ( ^FITC BSA) _BIS Specific immobilization of DBCO-modified nanocapsules on nanofibers will be described by loading BSA (bovine serum albumin) -blocked PCL and PCL-OPP as examples. Firstly, 3 wt% BSA solution (not labeled by FITC and 30mg/ml) is prepared, two PCL and one PCL-OPP are respectively put into the solution for soaking, and incubation is carried out for 2h at 37 ℃, thus obtaining BSA blocked PCL and PCL-OPP, both labeled as BSA Block. Correspondingly, unblocked PCL and PCL-OPP are labeled "No BSA Block". 20 μ l of DBCO-n (C:) ^FITC BSA) _BIS (0.5mg/ml) of 1 XPBS solution was coated on the BSA blocked PCL and PCL-OPP samples, sealed, placed in a refrigerator at 4 ℃ and incubated for 12h in the absence of light, followed by fluorescence detection and optical density quantification. The result shows that after 3% BSA blocking, the unfunctionalized PCL only has weak fluorescence, which indicates that the BSA blocking greatly reduces the DBCO-n (DBCO-n) on the PCL nanofiber surface ^FITC BSA) _BIS Non-specific adsorption of (2). On the BSA-blocked PCL-OPP scaffold, DBCO-n (A), (B) and (C) were observed ^FITC BSA) _BIS Strong fluorescent signal. This indicates that DBCO-n (C-CO-n) is lower than that of PCL in the case where PCL-OPP shows a weaker nonspecific protein-adsorbing ability than PCL ^FITC BSA) _BIS Is specifically coupled to the scaffold surface of PCL-OPP by click chemistry reaction. Quantitative analysis shows that after blank control experiment results are deducted, the PCL-OPP bracket is used for DBCO-n: (A), (B), (C), (D) and D) ^FITC BSA) _BIS The immobilization amount of the polymer is 2.6 times (p) of that of the PCL bracket<0.05). Comparison of results on BSA blocked PCL scaffold, DBCO-n (C: (B)) ^FITC BSA) _BIS The nonspecific adsorption of the protein is reduced by about 65%, which shows that the BSA blocking can effectively reduce the nonspecific adsorption of the protein, thereby obviously showing the specificity of the nanocapsule immobilization. The DBCO-n (supported) prepared above ^FITC BSA) _BIS PCL-OPP (PCL-OPP-DBCO-n) (for short) ^FITC BSA) _BIS 。

Example 9 specific immobilization of DBCO-modified nanocapsules on nanofibers control experiment one

With DBCO-n: ( ^FITC BSA) _BIS PCL without BSA blocking and PCL-OPP were loaded as control experiments to demonstrate the specific immobilization of DBCO-modified nanocapsules on nanofibers. Similar to example 8, except that 20. mu.l of 0.5mg/ml n (g)/(b) ^FITC BSA) _BIS With DBCO-n: ( ^FITC BSA) _BIS Uniformly covering PCL-OPP without BSA blocking, sealing, placing in a refrigerator at 4 ℃, keeping out of the sun, incubating for 12h, and then carrying out fluorescence detection and optical density quantitative analysis. The results show that, in the absence of BSA blocking, the PCL-OPP scaffold is paired with n: ( ^FITC BSA) _BIS The non-specific adsorption of (a) results in stronger fluorescence but contains DBCO-n (b:) ^FITC BSA) _BIS The PCL-OPP scaffold of (A) showed 1.5 times stronger fluorescence intensity. This phenomenon demonstrates that DBCO-n (C) is present in the presence of non-specific adsorption ^FITC BSA) _BIS Can also be specifically coupled to the surface of the PCL-OPP scaffold by click reaction, so that the PCL-OPP scaffold is coupled to DBCO-n ((II)) ^FITC BSA) _BIS The immobilized amount of (c) is higher than that of the control group<0.001)。

In the following examples, to obtain specific immobilization of the nanocapsules, the nanofibrous scaffold used was PCL-OPP, n (without DBCO modification) was used ^FITC BSA) _BIS DBCO-modified DBCO-n (C) was used as an experimental control group ^FITC BSA) _BIS As experimental group.

Example 10 specific immobilization of DBCO-modified nanocapsules on nanofibers control experiment II

By n: ( ^FITC BSA) _BIS With DBCO-n: ( ^FITC BSA) _BIS The specific immobilization of DBCO-modified nanocapsules on nanofibers was demonstrated using BSA-blocked PCL-OPP as a control experiment. Similar to example 9, 20. mu.l of 0.5mg/ml of n ( ^FITC BSA) _BIS And DBCO-n ( ^FITC BSA) _BIS Uniformly covering the BSA blocked PCL-OPP, sealing, placing in a refrigerator at 4 ℃, incubating for 12h in the dark, and then performing fluorescence detection and optical density quantitative analysis. The results show that the control PCL-OPP scaffold pair was blocked with 3% BSAn( ^FITC BSA) _BIS The non-specific adsorption of (A) is reduced to be basically similar to the background fluorescence, which indicates that no obvious adsorption exists. In the substantial absence of non-specific adsorption, the PCL-OPP scaffold exhibited a two-fold increase in fluorescence signal (. p)<0.05). This phenomenon is identified as DBCO-n: ( ^FITC BSA) _BIS Indeed, it was specifically coupled to the PCL-OPP scaffold surface by click reaction. Although the PCL-OPP scaffold prepared by the current method can not completely block nonspecific adsorption, the non-specific adsorption can be greatly reduced.

Example 11 controlled differentiation of Stem cells on a nanofiber scaffold

DBCO-n (BMP-2) -immobilized PCL-OPP scaffolds (with DBCO-n (BMP-2)) prepared according to the method of example 10 in place of DBCO-n (BMP-2)) in example 10 ^FITC BSA) _BIS ) The scaffolds were cut into circles of 1.5cm diameter and placed in 24-well plates. PCL-OPP-DBCO-n (prepared by the same method) ^FITC BSA) as a negative control group, and PCL as a blank control group; cell count at 5X 10 ⁴ Rat bone marrow mesenchymal stem cells (BMSCs) are implanted into each hole, an osteogenesis induction culture medium (a conventional culture medium contains 100nmol/L of dexamethasone, 50 mu g/ml of ascorbic acid and 10mmol/L of beta-sodium glycerophosphate) is replaced after the cells are cultured for 24 hours for adherence, and the solution is replaced once every three days to induce the rat bone marrow mesenchymal stem cells to differentiate into osteoblasts. On day 14, the cell samples in the well plates were stained using an alkaline phosphatase (ALP) staining kit, which stained ALP as a blue-violet substance as a marker of osteogenic differentiation, and the results are shown in fig. 7. After 14 days of induction, no obvious bluish purple substances are generated in the PCL and PCL-OPP-DBCO-n (BSA) control group. A clear bluish purple substance was observed in the PCL-OPP-DBCO-n (BMP-2) test group. The results prove that the prepared PCL-OPP-DBCO-n (BMP-2) composite scaffold can induce stem cells to carry out osteogenic differentiation.

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 without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A modified nanofiber material, comprising a hydrophobic nanofiber material, nanocapsules wrapped with cell signaling factors and an amphiphilic polymer, wherein the amphiphilic polymer comprises a hydrophobic group and a hydrophilic group, the amphiphilic polymer is bonded to the surface of the hydrophobic nanofiber material through at least part of the hydrophobic group on the amphiphilic polymer by means of hydrophobic interaction, and at least part of the hydrophilic group of the amphiphilic polymer is connected with the nanocapsules wrapped with the cell signaling factors through covalent bonds.

2. The modified nanofibrous material of claim 1, wherein the hydrophobic nanofibrous material is selected from at least one of Polycaprolactone (PCL), polylactic acid (PLA), Polyester (PET), polylactic acid-polyester copolymer, polylactic acid-polycaprolactone copolymer;

the cell signaling factor is selected from at least one of protein cell signaling factors, exemplarily, the cell signaling factor is selected from at least one of bone morphogenetic protein BMP-2, Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF) and Nerve Growth Factor (NGF);

the nanocapsule encapsulating the cell signaling factor is a polymer, such as a degradable cross-linked polymer, and the monomer is at least one of acrylamide, N- (3-aminopropyl) methacrylamide and N, N-methylene bisacrylamide; the cross-linking agent is, for example, a polylactide-b-polyethylene glycol-b-polylactide-diacrylate triblock copolymer, glycerol dimethacrylate.

3. The modified nanofiber material of claim 1 or 2, wherein in the amphiphilic polymer, a hydrophobic group and a hydrophilic group are introduced into a side chain of the polymer by a grafting method; the main chain of the polymer is 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 and a polyamino acid main chain.

4. The modified nanofiber material of claim 1, wherein in the amphiphilic polymer, the amount of the side chains containing hydrophilic groups accounts for 2-98% of the total amount of all the side chains in a molar percentage; the mole percentage of the side chain containing the hydrophobic group in the total amount of all the side chains is 2-98%; the total amount of the hydrophilic group and the hydrophobic group accounts for 5 to 100 percent of the total amount of all the side chains in a molar percentage.

5. The modified nanofiber material of claim 1, wherein the hydrophobic group of the amphiphilic polymer is C _4-25 A hydrocarbyl group; the hydrophilic group of the amphiphilic polymer is polyethylene glycol.

6. A method of preparing a modified nanofibrous material of any of claims 1 to 5, the method comprising the steps of:

4) Mixing the nanofiber material modified by the amphiphilic polymer in the step 2) with the nanocapsule obtained in the step 3), wherein the reactive group Z in the amphiphilic polymer ¹ With reactive groups Z on the nanocapsules ² And carrying out click chemical reaction to obtain the modified nanofiber material.

7. The preparation method according to claim 6, wherein, in the step 4), the amphiphilic polymer modified nanofiber material in the step 2) is mixed with the protein, and then mixed with the nanocapsule obtained in the step 3).

8. The production method according to claim 6 or 7, wherein in the step 1), the production method of the amphiphilic polymer comprises the steps of:

having a reactive group Y in a side chain ¹ And X ¹ With the compound R-X ² One end of which has a reactive group Y ² The other end has a reactive group Z which can generate click chemistry reaction ¹ Reacting the polyethylene glycol; wherein Y is ¹ And Y ² Capable of reacting so as to link a reactive group Z capable of reacting in a click chemistry ¹ The polyethylene glycol of (a) is attached to a side chain of the polymer; x ¹ And X ² Reacting to attach the R group to a side chain of the polymer; the reactive group X ¹ 、X ² 、Y ¹ 、Y ² Selected from the group consisting of hydroxyl, amino, carboxyl, aldehyde, keto, ester, thiol, maleimide, alpha-halocarbonyl; wherein the reactive group X ¹ And X ² 、Y ¹ And Y ² Mutually reactive groups, a reaction can occur.

9. The method of claim 6, wherein the reactive group Z ¹ 、Z ² Comprises the following steps: azido, alkynyl, tetrazine and double bonds, wherein the azido and the alkynyl have click reaction, and the tetrazine and the double bonds have click reaction.

10. Use of the modified nanofibrous material of any of claims 1 to 5 to control stem cell fate.