Disclosure of Invention
The invention aims to provide a material which has good cell adhesion and bone repair functions, is simple to prepare, is low in price, and has good in vivo stability and even can be degraded and regulated.
The invention provides an application of a polypeptide polymer, a mimic or a pharmaceutically acceptable salt thereof, which is used for modifying the surface of or doping in a bone implant material; or for preparing bone repair materials.
In another preferred embodiment, the polypeptide polymer or polypeptide mimetic or pharmaceutically acceptable salt thereof is used as an active molecule associated with bone repair, is doped inside or modified on the surface of a bone implant material, and is used for promoting the osseointegration effect of the bone implant material.
In another preferred embodiment, the polypeptide polymer or polypeptide mimetic or pharmaceutically acceptable salt thereof is used to prepare a scaffold material for bone repair.
The polypeptide polymer or the polypeptide mimic or the pharmaceutically acceptable salt thereof can promote the functions of cell adhesion, proliferation, migration, differentiation and the like of cells related to bone repair in the application process, thereby promoting the osteogenesis repair effect and the osseointegration effect. Wherein the cells related to bone repair are one or more than two of osteoblasts, preosteoblasts, stem cells and macrophages.
In another preferred embodiment, the bone implant material is a non-degradable permanent implant material or a degradable implant material.
In another preferred embodiment, the non-degradable permanent implant material is selected from the group consisting of: porous titanium, titanium alloy, titanium nickel alloy, tantalum metal, magnesium alloy, stainless steel, cobalt chromium alloy, polymethyl methacrylate, polyurethane and polyether ether ketone.
In another preferred embodiment, the degradable implant material is selected from the group consisting of: poly (lactic-co-glycolic acid), polylactide, polyglycolide, polycaprolactone, alumina, zirconia, hydroxyapatite, tricalcium phosphate, bioglass, collagen, hyaluronic acid, chitosan, alginate, silk fibroin, fibrin.
In another preferred embodiment, the bone implant material is an inert implant material.
In another preferred embodiment, the bone repair material is in the form of one or a combination of two or more of hydrogel, electrospun film, 3D printed scaffold, porous scaffold.
In another preferred embodiment, the polypeptide polymer or mimetic thereof is a homopolymer, a copolymer or a copolymer selected from one or more of the following: alpha-amino acids, beta-amino acids, gamma-amino acids, oxazolines.
In another preferred embodiment, the polypeptide polymer or mimetic thereof is an oxazoline polymer, an alpha-amino acid polymer, an alpha/beta-amino acid polymer, a gamma-amino acid polymer, or a beta-amino acid polymer.
In another preferred embodiment, the polypeptide polymer or its mimic is a homopolymer, a binary copolymer or a multipolymer consisting of the following structures Aa, aa ', bb', cc ', cc ", dd', ee ', ff' and Gg, the total number of repeating units being a positive integer from 5 to 5000:
wherein r is independently at each occurrence 0, 1, 2, 3, 4, or 5; r' is independently at each occurrence 1, 2 or 3; r "is independently at each occurrence 0, 1, 2 or 3;
r in the formula1 、R2 、R3 、R4 、R5 And R is6 Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylhydroxy, C1-C6 alkoxy, C1-C6 alkylsulfonyl, C1-C6 alkylguanidino, C1-C6 alkyl ester, thio C1-C6 alkyl ester, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, C1-C6 alkyl-C6-C12 aryl, amino and
P1 to protect groups, the groups are independently selected at each occurrence from the following: t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethoxycarbonyl (Fmoc), phthaloyl (Pht), acetyl (Ac), trifluoroacetyl (Tfa), benzyl (Bn), triphenylmethyl (Tr);
P2 Independently at each occurrence selected from the following groups: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
x is independently selected at each occurrence from the following groups: no, hydrogen, amino, guanidino, hydroxy, carboxyl, amido, mercapto, methylthio, alkenyl, alkynyl, ester, aryl or 5-12 membered heterocyclic group;
l is independently selected at each occurrence from: -CHR'1 -、-CO-、-COO-、-S(＝O)2 -; q is an integer from 0 to 6;
R'1 independently at each occurrence selected from the group consisting of substituted or unsubstituted: hydrogen, amino, C1-C15 alkyl, C1-C15 alkylamino, C1-C-C15 alkylhydroxy, C1-C15 alkylaldehyde, C1-C15 alkyl ester, thio C1-C15 alkyl ester, C6-C15 aryl, C2-C15 alkenyl, C2-C15 alkynyl, -Rc-COO-Rc ', -Rc-CO-Rc', -Rc-O-Rc '-, -Rc-S-Rc', 5-15 membered heteroaryl, 5-12 membered heterocyclyl;
Ra and R isb Each occurrence is independently selected from the group consisting of substituted or unsubstituted: absence, hydrogen, C1-C15 alkyl, C1-C15 alkylamino, C1-C15 alkylhydroxy, C1-C15 alkylaldehyde, C1-C15 alkylsulfonyl, C2-C15 alkenyl, C2-C15 alkynyl, -Rc-COO-Rc ', -Rc-CO-Rc', -Rc-O-Rc '-, -Rc-S-Rc', C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 5-12 membered heterocyclyl, C6-C12 aryl, 5-12 membered heteroaryl;
Rc at each occurrence is independently selected from the group consisting of substituted or unsubstituted: C1-C15 alkylene, C2-C15 alkenylene, C2-C15 alkynylene, C3-C12 cycloalkylene, C4-C12 cycloalkenylene, 3-12 heterocyclylene, C6-C12 arylene, 5-12 heteroarylene;
rc' is independently selected from the following groups, substituted or unsubstituted, respectively, at each occurrence: C1-C15 alkyl, C1-C15 alkylamino, C2-C15 alkenyl, C2-C15 alkynyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 3-C12 heterocyclyl, C6-C12 aryl, 5-C12 heteroaryl,
each of the above substituents independently refers to a substitution with one or more substituents selected from the group consisting of: halogen, hydroxy, amino, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C8 cycloalkyl.
In another preferred embodiment, the total number of repeating units is from 5 to 100, preferably from 5 to 50.
In another preferred embodiment, R1 、R2 、R3 、R4 、R5 And R is6 Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkylhydroxy, C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylguanidino, C1-C4 alkyl ester, thio C1-C4 alkyl ester, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, phenyl, naphthyl, 5-6 membered heteroaryl, 5-6 membered heterocyclyl, C1-C4 alkyl-C6 aryl Amino and
in another preferred embodiment, X is each independently selected at the occurrence from the following groups: none, hydrogen, amino, guanidino, hydroxy, carboxyl, amido, mercapto, methylthio, alkenyl, alkynyl, ester (-COO-), phenyl or 5-6 membered heterocyclyl.
In another preferred embodiment, each occurrence of L is independently selected from: -CH2 -、-CO-、-COO-。
In another preferred embodiment, q is 0, 1, 2, 3, 4 or 5. When q is 0, (L) q is absent.
In another preferred embodiment, ra and Rb are each independently selected at the occurrence from the group consisting of substituted or unsubstituted: absence, hydrogen, C1-C6 alkyl, C1-C6 alkylamino, C1-C6 alkylhydroxy, C1-C6 alkylaldehyde, C1-C6 alkylsulfonyl, C2-C6 alkenyl, C2-C6 alkynyl, -Rc-COO-Rc ', -Rc-CO-Rc', -Rc-O-Rc '-, -Rc-S-Rc', C3-C6 cycloalkyl, C4-C6 cycloalkenyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl.
In another preferred embodiment, the pharmaceutically acceptable salt is the hydrochloride, bromate, trifluoroacetate, phosphate, lithium, sodium, or potassium salt of the polypeptide polymer or mimetic thereof.
In another preferred embodiment, the structures Aa, aa ', bb', cc ', cc ", dd', ee ', ff' and Gg show that the amino acid is in the L configuration, D configuration, or a mixture of both the D and L configurations.
In another preferred embodiment, the copolymer is a random copolymer, an alternating copolymer or a block copolymer. Preferably, it is a random copolymer.
The polypeptide polymer or polypeptide mimic also comprises a polypeptide polymer or polypeptide mimic derivative, wherein the derivative refers to that amino groups of a polymer side chain are changed into other functional groups such as guanidino groups and the like, or amino groups contained in the side chain are connected with other molecules such as drug molecules, fluorescent small molecules, protecting groups and the like through chemical reaction; the ends of the polymer are chemically modified, such as by attachment of fluorescent molecules, or drug molecules.
In another preferred embodiment, the polypeptide polymer or mimetic thereof is selected from the group consisting of:
in the method, in the process of the invention,
n is a positive integer of 5 to 5000;
a is a positive integer from 0 to 100;
x is more than or equal to 0% and less than or equal to 100%, y is more than or equal to 0% and less than or equal to 100%, and x+y=100%;
Rz selected from: halogen, carboxyl, active ester group, acyl chloride, alkylene oxide, mercapto, C2-C15 alkene group, C2-C15 alkynyl, azide, maleimide, o-dithiopyridyl (OPSS), cyclodextrin, adamantane;
Rs each at the occurrence is independently hydrogen orRt Each occurrence is independently selected from the following groups: C1-C15 alkyl, C1-C15 alkylamino, C2-C15 alkenyl, C2-C15 alkynyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 3-C12 heterocyclyl, C6-C12 aryl, 5-C12 heteroaryl;
Rw isThe wavy line indicates the junction; a' is a positive integer from 0 to 12; r in the formula11 、R12 、R13 And R is14 Each independently at each occurrence is selected from the group consisting of: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylhydroxy, C1-C6 alkoxy, C1-C6 alkylsulfonyl, C1-C6 alkylguanidino, C1-C6 alkyl ester, thio C1-C6 alkyl ester, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclyl, C1-C6 alkyl-C6-C12 aryl, amino and;
P1 to protect groups, the groups are independently selected at each occurrence from the following:t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethoxycarbonyl (Fmoc), phthaloyl (Pht), acetyl (Ac), trifluoroacetyl (Tfa), benzyl (Bn), triphenylmethyl (Tr);
P2 independently at each occurrence selected from the following groups: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted 5-12 membered heterocyclyl;
each of the above substituents independently refers to a substitution with one or more substituents selected from the group consisting of: halogen, hydroxy, amino, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C8 cycloalkyl.
In another preferred embodiment, the amino acid in the above structural unit is in the L-configuration, the D-configuration, or a mixture of the D-configuration and the L-configuration.
In another preferred embodiment, the structural anion is selected from the group consisting of: cl- 、Br- 、CF3 COO- 、H2 PO4- 、HPO42- 、PO43- 。
In the present invention, x and y are the ratio of the respective components, and x and y are calculated by dividing the number of repeating units of the corresponding component by the total number of repeating units of the polypeptide polymer or polypeptide mimetic.
In another preferred embodiment, the polypeptide polymer or mimetic thereof is selected from the group consisting of:
in the method, in the process of the invention,
n is a positive integer of 5 to 5000;
a is a positive integer from 0 to 100;
x is more than or equal to 0% and less than or equal to 100%, y is more than or equal to 0% and less than or equal to 100%, and x+y=100%;
Rz selected from: halogen, carboxyl, reactive ester groups, acid chloride, alkylene oxide, mercapto (-SH), C2-C15 alkene groups, C2-C15 alkynyl, azide, maleimide, o-dithiopyridinyl (OPSS), cyclic pastesAny one of essence and adamantane.
In another preferred embodiment, the amino acid in the above structural unit is in the L-configuration, the D-configuration, or a mixture of the D-configuration and the L-configuration.
In another preferred embodiment, the structural anion is selected from the group consisting of: cl- 、Br- 、CF3 COO- 、H2 PO4- 、HPO42- 、PO43- 。
In another preferred embodiment, the arrangement of the two repeating units in the above polymer structure is random, alternating or block, preferably random.
In another preferred embodiment, n is 5 to 100, preferably 5 to 50.
In another preferred embodiment, x: y is 0.01:0.99 to 0.99:0.01, preferably 0.05:0.95 to 0.95:0.05, even 1:9 to 9:1.
The main advantages of the invention include:
(1) The invention provides a material or a material composition which is easy to synthesize on a large scale, has low price, high in vivo stability or adjustable stability and supports the cell adhesion, bone repair and bone integration related to bone repair;
(2) The material or the material composition (polypeptide polymer and polypeptide mimic) supporting the active functions such as cell adhesion and the like provided by the invention can obtain the functions such as cell adhesion and the like of cells related to bone repair without specific amino acid sequences, and can be compared with the cell adhesion gold standard RGD polypeptide.
(3) The material or the material composition (polypeptide polymer and polypeptide mimic) for supporting cell adhesion provided by the invention can obviously improve the functions of cell adhesion and the like of inert materials, promote the compatibility of the materials and bone tissues, realize better osseointegration and bone repair effects, and has great clinical application advantages and values;
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions (e.g.those described in Sambrook et al, molecular cloning: A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989)) or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Preparation of alpha-amino acid polymers
Example 1: random open condition copolymerization of DL-norleucine-N-carboxyanhydride and N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxyanhydride initiated by lithium hexamethyldisilazide (LiHMDS)
Lithium hexamethyldisilazide (33.4 mg,0.2 mmol) was accurately weighed and prepared as a 0.1M solution in tetrahydrofuran (2 mL) for further use. DL-norleucine-N-carboxyanhydride (7.9 mg,0.05 mmol) and N-. Epsilon. -t-butoxycarbonyl-DL-lysine-N-carboxyanhydride (20.4 mg,0.075 mmol) (monomer ratio 6:4) were accurately weighed and dissolved in tetrahydrofuran (1 mL) in a reaction flask equipped with a stirrer. To a stirred reaction flask, 0.25mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M was added. The mixture was stirred at room temperature in a glove box for 5 minutes. After the polymerization reaction was completed, the reaction mixture was blocked overnight with triphenylmercaptoethylamine. Cold petroleum ether (40 mL) was poured into the reaction mixture, the white floc which separated out was collected by centrifugation, dried in air flow, redissolved in tetrahydrofuran (1.5 mL), and a large amount of cold petroleum ether was added for precipitation. This dissolution-precipitation process was repeated three times in total to obtain a purified copolymer. The polymer which was drained was added with 2mL of trifluoroacetic acid and 5% (v/v) triethylsilane, after shaking gently at room temperature overnight, excess trifluoroacetic acid was blown off, the resulting viscous liquid was dissolved in 0.5mL of methanol, 45mL of frozen diethyl ether was added to precipitate a white precipitate, and the dissolution-precipitation process was repeated three times, thereby obtaining a random polymer with deprotected side chain amino groups. The deprotected polymer was again dissolved in 5mL of ultrapure water and filtered and lyophilized for subsequent bioactivity testing. (yield 80%, GPC indicated that chain length was 32 mer).
Preparation of beta-amino acid polymers
Example 2: preparation of random beta-amino acid copolymer by co-initiating beta-lactam monomer NM and beta-lactam monomer CP by 2- (tritylthio) acetic acid-N-succinimidyl ester and hexamethyldisilazide lithium salt
R is
In a nitrogen-protected glove box, NM and CP were weighed, dried THF (tetrahydrofuran) was used as a solvent, and a magnet was added to the reaction flask, and 1.2mL NM (0.2M) and 0.8mL CP (0.2M) were stirred. Then, p-2- (tritylthio) acetic acid-N-succinimidyl ester (0.2M) and hexamethyldisilazide lithium salt (0.5M) were respectively prepared as co-initiators, and 100uL each was rapidly introduced into a reaction flask. The reaction was carried out at room temperature for 4 hours, and the reaction flask was taken out of the glove box and quenched with 1 drop of methanol. Petroleum ether (45 mL) was added to the reaction solution, and after precipitation of a white flocculent precipitate, the precipitate was collected by centrifugation, dissolved in THF (1 mL), and precipitated with petroleum ether. After repeating this three times, a polymer with protection was obtained. Then adding trifluoroacetic acid (2 mL) into the polymer with protection, shaking for 2 hours to remove the protective groups, blowing off most of the trifluoroacetic acid, adding ice methyl tertiary butyl ether (50 mL), precipitating white precipitate, centrifuging, collecting, dissolving with methanol (1 mL), precipitating with ice ethyl ether (50 mL), repeating the steps three times, pumping out the residual solvent by an oil pump, dissolving the sample with ultrapure water (5 mL) respectively, and finally freeze-drying to obtain the deprotected random beta-amino acid copolymer (yield 80%, and chain length 20mer is characterized by GPC).
Example 3: preparation of random beta-amino acid copolymer by co-initiating beta-lactam monomer MM and beta-lactam monomer CH by 2- (tritylthio) acetic acid-N-succinimidyl ester and hexamethyldisilazide lithium salt
R isThe experimental procedure is the same as in example 2, except that 1.2mL NM (0.2M) and 0.8mL CP (0.2M) are kept at concentrations that do not shift to 1.2mL MM and 0.8mL CH. The random β -amino acid copolymer (75% yield) was finally obtained, with a GPC characterized as chain length of 21 mer).
Example 4: preparation of random beta-amino acid copolymer by co-initiating beta-lactam monomer DM and beta-lactam monomer CH by 2- (tritylthio) acetic acid-N-succinimidyl ester and hexamethyldisilazide lithium salt
R is
The experimental procedure is the same as in example 2, except that 1.2mL NM (0.2M) and 0.8mL CP (0.2M) are kept at concentrations that do not shift to 1.2mL MM and 0.8mL CO. The random β -amino acid copolymer (yield 85%, GPC characterized by chain length 19 mer) was finally obtained.
Example 5: preparation of random beta-amino acid copolymer by co-initiating beta-lactam monomer MM and beta-lactam monomer CH by 2- (tritylthio) acetic acid-N-succinimidyl ester and hexamethyldisilazide lithium salt
R is
The experimental procedure was the same as in example 2, except that NM (0.2M) and 0.8mL CP (0.2M) were kept at concentrations that did not shift to 1.2mL DM and 0.8mL CH. The random β -amino acid copolymer (yield 85% and GPC characterization chain length 22 mer) was finally obtained.
Example 6: preparation of random beta-amino acid copolymer by CO-initiating beta-lactam monomer MM and beta-lactam monomer CO by 2- (tritylthio) acetic acid-N-succinimidyl ester and hexamethyldisilazide lithium salt
R is
The experimental procedure is the same as in example 5, except that 1.2mL NM (0.2M) and 0.8mL CP (0.2M) are kept at concentrations that do not shift to 1mL DM and 1mL CO. The random β -amino acid copolymer (yield 79% and GPC characterization chain length 21 mer) was finally obtained.
Example 7: preparation of random beta-amino acid copolymer by co-initiating N-benzyl chloroformate-beta-lactam-DL-lysine and beta-lactam monomer CH by 2- (tritylthio) acetic acid-N-succinimidyl ester and hexamethyldisilazide lithium salt
The experimental procedure was the same as in example 5, except that 1.2mL NM (0.2M) and 0.8mL CP (0.2M) were kept at concentrations that were not changed to 1.4mL benzyl N-chloroformate-beta-lactam-L-lysine and 0.6mL CH. The random β -amino acid copolymer (yield 82%, GPC characterized by chain length 18 mer) was finally obtained.
Preparation of alpha/beta-amino acid polymers
Example 8: preparation of random alpha/beta-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyl cyclic anhydride and DL-beta-glycine N-carboxyl thiocarbonyl cyclic anhydride with 2-trityl mercaptoethylamine
In a glove box protected by nitrogen, N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyl cyclic anhydride and DL-beta-glycine N-carboxyl thiocarbonyl cyclic anhydride are weighed, and dry N, N-dimethylformamide is taken as a solvent. The magneton was added to the reaction flask, and then 1.2mL of N-. Epsilon. -t-butoxycarbonyl-L-lysine-N-carboxycyclic anhydride (0.2M) and 0.8mL of DL-. Beta. -glycine N-carboxythiocarbonylcyclic anhydride (0.2M) were added and stirred. The initiator 2-trityl mercaptoethylamine was weighed and prepared into a solution (0.2M), 100. Mu.L of the solution was rapidly added into a reaction flask, the reaction was stirred at room temperature in a glove box and reacted for 3 days, the reaction solution was taken out of the glove box, cold petroleum ether (45 mL) was added, after precipitation of white flocculent precipitate, the solution was collected by centrifugation, dissolved in tetrahydrofuran (1 mL) and precipitated with cold petroleum ether, and the polymer with protected side chains was obtained after repeating the above steps three times. Adding trifluoroacetic acid (2 mL) into the polymer, shaking for 2 hours to remove protective groups, blowing off most of the trifluoroacetic acid, adding ice methyl tertiary butyl ether (50 mL), precipitating white precipitate, centrifuging, collecting, dissolving with methanol (1 mL), precipitating with ice diethyl ether (50 mL), repeating the steps three times, pumping out the residual solvent with an oil pump, dissolving the sample with ultrapure water (5 mL), and finally freeze-drying to obtain the deprotected random alpha/beta-amino acid copolymer (yield 72.5%, chain length 19mer is characterized by GPC)
Example 9: preparation of random alpha/beta-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-L-lysine-N-carboxyl cyclic anhydride and DL-beta-phenylalanine N-carboxyl thiocarbonyl cyclic anhydride with 2-trityl mercaptoethylamine
The experimental procedure was as in example 8, except that the concentration of DL-beta-glycine N-carboxythiocarbonyl cyclic anhydride (0.2M) was kept unchanged and was changed to DL-beta-phenylalanine N-carboxythiocarbonyl cyclic anhydride. The final random alpha/beta-amino acid copolymer (75% yield), GPC characterized as chain length 22mer, was obtained
Preparation of oxazoline polymers
Example 10: preparation of oxazoline copolymer by initiating N-epsilon-t-butoxycarbonyl-2- (aminopropyl) oxazoline and 2- (cyclohexyl) oxazoline with 3-triphenylmethyl propyl bromide
N-epsilon-t-butoxycarbonyl-2- (aminopropyl) oxazoline and 2- (cyclohexyl) oxazoline were weighed in a nitrogen-protected glove box with dried N, N-dimethylacetamide as solvent. The reaction flask was charged with the magneton, and then 0.6mL (1M) of N-. Epsilon. -t-butoxycarbonyl-2- (aminomethyl) oxazoline and 1.4mL (1M) 2- (cyclohexyl) oxazoline were weighed and added, respectively, to stir. The initiator 3-triphenylmethyl propyl bromide (1M) was then formulated. Finally, taking out the glove box after sealing the reaction bottle, stirring and reacting for 18 hours at 140 ℃, and obtaining relative molecular weight and PDI (PDI) by using GPC (gel permeation chromatography) standard with a small amount of reaction liquid; then cooling to room temperature, adding cold petroleum ether (45 mL), centrifuging and collecting after white flocculent precipitate is separated out, dissolving with tetrahydrofuran (2 mL), precipitating with cold petroleum ether, and repeating the steps three times to obtain polymer with side chain protection. Then adding trifluoroacetic acid (2 mL) and 40uL of triethylsilane into the polymer, shaking for 6 hours to remove protective groups, blowing off most of the trifluoroacetic acid, adding ice methyl tertiary butyl ether (45 mL), precipitating white precipitate, centrifuging, collecting, dissolving with methanol (1 mL), precipitating with ice ethyl ether (45 mL), repeating the steps three times, pumping out the residual solvent with an oil pump, dissolving a sample with ultrapure water (5 mL), and finally freeze-drying to obtain the deprotected oxazoline copolymer (the yield is more than 80%, and the chain length is 30+/-2 mer by GPC).
Example 11: preparation of oxazoline copolymer by initiating N-epsilon-t-butoxycarbonyl-2- (aminopropyl) oxazoline and 2- (isobutyl) oxazoline with 3-triphenylmethyl propyl bromide
The experimental procedure was the same as in example 10, except that 1.2mL of 2- (cyclohexyl) oxazoline was changed to 2- (isobutyl) oxazoline, while varying the amounts of initiator and monomer in terms of molar ratio, such that the chain length of the polymerization physicist was 20mer. The oxazoline copolymer is finally obtained (yield greater than 80%, GPC characterization chain length 20+ -2 mer)
Example 12: adhesion test of alpha-polypeptide Polymer to osteoblasts
In an alpha-MEM medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin, 2mM L-glutamine in 5% CO2 And culturing MC-3T3-E1 osteoblasts at 37 ℃. Observing when the cell proliferation coverage reaches 80-90% of the whole area of the culture dish, separating cells from the culture dish by trypsin, centrifuging to remove supernatant, and re-suspending in culture medium to obtain final cell concentration of 105 cells/mL. 10. Mu.L of the cell suspension was added to each individual well of the alpha-polypeptide polymer modified anti-cell adhesion layer slide prepared in example 1 (a layer of alpha-polypeptide polymer was coated on the slide with the anti-cell adhesion layer), wherein the surface of the non-modified alpha-polypeptide polymer was used as a control. The slides were placed in a petri dish and incubated at 37 ℃ for 2 hours to achieve initial attachment of the cells, then fresh medium was added to the petri dish to submerge the entire slide and the slide was incubated at 37 ℃ for 1 day. After removal of the excess medium, the cells were incubated with a dead-stained solution of cells containing 2. Mu.M calcein AM and 4. Mu.M ethidium bromide dimer-1 in the dark for 15 minutes. Cell fluorescence maps of different locations of each well were observed and photographed under a microscope and the images were analyzed using ImageJ software.
The observation result shows that the surface cells without modifying the alpha-polypeptide polymer have almost no adhesion, the surface modified by the alpha-polypeptide polymer and MC-3T3-E1 osteoblast have good adhesion and spreading morphology.
Example 13: adhesion test of beta-polypeptide Polymer, alpha/beta-amino acid copolymer to osteoblasts
The procedure was as in example 12, except that the β -polypeptide polymer prepared in examples 2-7 and the α/β -amino acid copolymer prepared in examples 8-9 were used to modify the surface of a slide having an anti-cell adhesion layer, and the α -polypeptide polymer was replaced with the β -polypeptide polymer and the α/β -amino acid copolymer.
The observation result shows that the surface cells of the unmodified polymer are hardly adhered, the surface modified by the beta-polypeptide polymer and the alpha/beta-amino acid copolymer, and the MC-3T3-E1 osteoblast has good adhesion and spreading morphology.
Example 14: adhesion test of polypeptide Polymer mimics to osteoblasts
The procedure was as in example 12 except that the polypeptide polymer mimetic prepared in example 10 was used to modify the surface of the slide with the anti-cell adhesion layer and the alpha-polypeptide polymer was replaced with the polypeptide polymer mimetic.
The observation result shows that the surface cells of the non-modified polypeptide polymer mimics hardly adhere, the surface modified by the polypeptide polymer mimics has good adhesion and spreading morphology, and the MC-3T3-E1 osteoblasts have good adhesion and spreading morphology.
Example 15: adhesion test of alpha-polypeptide Polymer to Stem cells
The procedure was as in example 12, except that MC-3T3-E1 osteoblasts were used in place of bone marrow mesenchymal stem cells.
The observation result shows that the surface cells without modifying the alpha-polypeptide polymer have almost no adhesion, the surface modified by the alpha-polypeptide polymer has good adhesion and spreading morphology.
Example 16: adhesion test of beta-polypeptide Polymer, alpha/beta-amino acid copolymer to Stem cells
The procedure was as in example 13, except that MC-3T3-E1 osteoblasts were used in place of bone marrow mesenchymal stem cells.
The observation result shows that the surface cells of the unmodified polymer are hardly adhered, the surface modified by the beta-polypeptide polymer and the alpha/beta-amino acid copolymer, and the bone marrow mesenchymal stem cells have good adhesion and spreading morphology.
Example 17: adhesion test of polypeptide Polymer mimics to Stem cells
The procedure was as in example 14, except that MC-3T3-E1 osteoblasts were used in place of bone marrow mesenchymal stem cells.
The observation result shows that the surface cells of the non-modified polypeptide polymer mimics hardly adhere, the surface modified by the polypeptide polymer mimics has good adhesion and spreading morphology.
Example 18: proliferation assay of osteoblasts with alpha-polypeptide polymers
In an alpha-MEM medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin, 2mM L-glutamine in 5% CO2 And culturing MC-3T3-E1 osteoblasts at 37 ℃. Observing when the cell proliferation coverage reaches 80-90% of the whole area of the culture dish, separating cells from the culture dish by trypsin, centrifuging to remove supernatant, and re-suspending in culture medium to obtain final cell concentration of 104 cells/mL. 100 μl of the cell suspension was added to each individual well of the α -polypeptide polymer, amino, RGD polypeptide (gold standard for cell adhesion peptides) modified slide prepared in example 1. The slides were placed in a petri dish and incubated at 37 ℃ for 2 hours to achieve initial attachment of the cells, then fresh medium was added to the petri dish to submerge the entire slide and the slide was incubated at 37 ℃ for 1, 3, 5 days. After removal of the medium, 50. Mu.l of Alamar-Blue detection solution was added to each well and incubated for 3 hours at 37℃in the absence of light. The liquid in each well was then transferred to a black, opaque 384 well plate and absorbance was measured for each well using a microplate reader at an excitation wavelength of 560nm and an emission wavelength of 590 nm.
The observation results show that on the third and fifth days, on the surface modified by the alpha-polypeptide polymer, the proliferation effect of MC-3T3-E1 osteoblasts is equivalent to that of RGD polypeptide modified by the cell adhesion gold standard, and the proliferation effect is obviously higher than that of amino surfaces.
Example 19: proliferation assay of osteoblasts with beta-polypeptide polymers, alpha/beta-amino acid copolymers
The procedure is as in example 18, except that the α -polypeptide polymer is replaced with the β -polypeptide polymer prepared in examples 2-7, and the α/β -amino acid copolymer prepared in examples 8-9.
The observation results show that on the third day and the fifth day, the proliferation effect of MC-3T3-E1 osteoblasts on the surface modified by the beta-polypeptide polymer and the alpha/beta-amino acid copolymer is equivalent to that of the surface modified by RGD polypeptide of the cell adhesion gold standard, and the proliferation effect is obviously higher than that of the amino surface.
Example 20: proliferation assay of osteoblasts with polypeptide polymer mimics
The procedure was as in example 18, except that the alpha-polypeptide polymer was replaced with the polypeptide polymer mimetic prepared in example 10.
The observation results show that on the third day and the fifth day, on the surface modified by the polypeptide polymer mimics, the proliferation effect of MC-3T3-E1 osteoblasts is equivalent to that of RGD polypeptide modified by the cell adhesion gold standard, and the proliferation effect is obviously higher than that of amino surfaces.
Example 21: migration test of alpha-polypeptide Polymer on osteoblasts
In an alpha-MEM medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin, 2mM L-glutamine in 5% CO2 And culturing MC-3T3-E1 osteoblasts at 37 ℃. When the cell proliferation coverage reached 80-90% of the whole dish area, cells were isolated from the dish using trypsin, the supernatant was removed by centrifugation, and then resuspended in medium to a final cell concentration of 104 cells/mL. 100. Mu.L of the cell suspension was added to each individual well of the alpha-polypeptide polymer prepared in example 1, KRR polypeptide (osteoblast selectively adhering polypeptide), RGD polypeptide (cell adhering gold standard) modified slide. The slides were placed in a petri dish and incubated at 37 ℃ for 2 hours to achieve initial cell attachment, then fresh medium was added to the petri dish to submerge the entire slideSlides were incubated at 37℃for 1 day. A standard 100. Mu.L disposable pipette tip was used to draw a straight line in the well of each cultured cell, remove the MC-3T3-E1 osteoblasts attached in the straight line, and incubate the slide at 37℃for 0h, 12h, 24h. After removal of the excess medium, the cells were incubated with a dead-stained solution of cells containing 2. Mu.M calcein AM and 4. Mu.M ethidium bromide dimer-1 in the dark for 15 minutes. Cell fluorescence maps of different locations of each well were observed and photographed under a microscope and the images were analyzed using ImageJ software.
The observation result shows that the migration speed of MC-3T3-E1 osteoblasts on the surface of the alpha-polypeptide polymer is equivalent to that of RGD modified surface and is faster than that of KRRR modified surface; 24h results showed that MC-3T3-E1 osteoblasts had been completely covered with the artificially set scratches and the KRRR surface was not completely covered. The alpha-polypeptide polymer and RGD modified surface is favorable for migration of MC-3T3-E1 osteoblasts and is superior to an osteogenic selective polypeptide KRRR.
Example 22: migration test of beta-polypeptide Polymer, alpha/beta-amino acid copolymer on osteoblasts
The procedure is as in example 21 except that the α -polypeptide polymer is replaced with the β -polypeptide polymer prepared in examples 2-7, and the α/β -amino acid copolymer prepared in examples 8-9.
The observation result shows that the migration speed of MC-3T3-E1 osteoblasts on the surfaces of the beta-polypeptide polymer and the alpha/beta-amino acid copolymer is equal to that of RGD modified surfaces, and the migration speed is faster than that of the surfaces modified by KSR; 24h results showed that MC-3T3-E1 osteoblasts had been completely covered with the artificially set scratches and the KRRR surface was not completely covered. The surface modified by the beta-polypeptide polymer, the alpha/beta-amino acid copolymer and the RGD is favorable for the migration of MC-3T3-E1 osteoblasts and is superior to an osteogenic selective polypeptide KRRR.
Example 23: migration test of polypeptide polymer mimics to osteoblasts
The procedure was as in example 18, except that the alpha-polypeptide polymer was replaced with the polypeptide polymer mimetic prepared in example 10. The observation result shows that the migration speed of MC-3T3-E1 osteoblasts on the surface of the polypeptide polymer mimic is equal to that of RGD modified surface and is faster than that of KRRR modified surface; 24h results showed that MC-3T3-E1 osteoblasts had been completely covered with the artificially set scratches and the KRRR surface was not completely covered. In conclusion, the polypeptide polymer mimics and RGD modified surfaces are favorable for migration of MC-3T3-E1 osteoblasts and are superior to the osteogenic selective polypeptide KRR.
Example 24: in vivo osteogenic Activity test of beta-polypeptide Polymer modified PEG hydrogels
The experiment was evaluated using an in situ skull defect model. The experimental animals were eight week female SD rats with a mass of about 180g-200g, and the rats were anesthetized by intraperitoneal injection with pentobarbital sodium at a concentration of 40 mg/kg. Rats were fixed to the console and the heads were shaved and sterilized with iodophor. A wound of about 5-8mm was cut with a knife in the center of the head, and a circular defect of 5mm in diameter was created in the skull by an electric drill with a drill of 5mm in diameter. The defect is a full-thickness defect but does not damage the dura mater. In the drilling process, the drill bit is required to be flushed with sterile normal saline to achieve the effect of cooling, so that thermal damage to bone tissues caused by overheating is avoided. Finally, 36 critical-sized skull defects were created in 18 rats and randomly implanted with the following 6 sets of hydrogel materials: (1) hydrogel material containing 5wt% peg (n=6); (2) The β -polypeptide polymer modified 5% peg hydrogel material prepared in example 6 (n=6); (3) hydrogel material containing 10wt% peg (n=6); (4) 10wt% peg hydrogel material modified by β -polypeptide polymer prepared in example 6 (n=6); (5) hydrogel material containing 20wt% peg (n=6); (6) The β -polypeptide polymer-modified 20wt% peg hydrogel material prepared in example 6 (n=6). The incision is closed with an absorbable suture after implantation of the material. After surgery, animals were allowed free access to murine food and water. Eight weeks after implantation, rats were sacrificed by intraperitoneal injection of excess sodium pentobarbital. Samples were collected and tissue was fixed with 4% paraformaldehyde solution. Bone repair of each set of implant materials was quantitatively analyzed using Micro-CT.
For the hydrogel material, the PEG hydrogel is formed by mixing four-arm PEG with acrylate as the end group and four-arm PEG with mercapto as the end group into glue. The hydrogel modified by the beta-polypeptide polymer is prepared by reacting four-arm PEG with end groups of acrylic ester with the beta-polypeptide polymer with end groups of sulfhydryl groups at room temperature for 1 hour, grafting the polymer to the tail end of part of PEG, and then mixing the polymer with the four-arm PEG with end groups of sulfhydryl groups to form the gel.
The results show that: for three hydrogel materials with different PEG contents, the volume of the new bone of the experimental group modified by the beta-polypeptide polymer is larger than that of the corresponding PEG hydrogel experimental group without the modification by the beta-polypeptide polymer.
Example 25: in vivo bone integration Activity test of beta-polypeptide Polymer modified titanium alloy implants
The experiment was evaluated using a femoral defect model. The experimental animals were eight week female SD rats with a mass of about 180g-200g, and the rats were anesthetized by intraperitoneal injection with pentobarbital sodium at a concentration of 40 mg/kg. Rats were fixed to the console and the legs were sterilized with iodophor after shaving. The joint part of the rear leg of the rat is cut by a surgical knife for 5-8mm of wound, then muscle tissue is cut by a knife at the cancellous bone part at the distal end of the femur, and the muscle at the cancellous bone part is pulled out by a stripper to expose the bone, and a small hole is drilled in advance on the leg bone by an electric drill with a drill with the diameter of 0.8 mm. Finally, 72 small hole defects were created in 36 rats and the following titanium alloy bone nails were randomly implanted: (1) unmodified titanium alloy bone nails (n=24); (2) Cell adhesion polypeptide RGD modified titanium alloy bone nail (n=24); (3) The beta-polypeptide polymer modified titanium alloy bone nail prepared in example 6 (n=24). After the bone screw is implanted, the wound is closed with absorbable suture. After surgery, animals were allowed free access to murine food and water. Rats were sacrificed by intraperitoneal injection of excess sodium pentobarbital at two time points of four weeks and eight weeks, respectively. Half of the collected samples of each group are fixed with 4% paraformaldehyde solution, the other half is not fixed, and the bone nails are directly pulled out of the bone tissues by a universal testing machine to test the biomechanical properties of the bone nails. Fixed bone tissue samples were quantitatively analyzed for bone repair of each set of implant materials using Micro-CT.
For the modified titanium alloy bone nail, firstly cleaning the nail in a UV/O3 cleaning machine, then modifying the polydopamine coating, and then immersing the nail in RGD polypeptide or beta-polypeptide polymer solution for reaction overnight to obtain the RGD polypeptide or beta-polypeptide polymer modified titanium alloy bone nail.
The results show that: from the quantitative results of Micro-CT, the histological staining results and biomechanical results show that compared with the experimental groups of titanium alloy bone nails modified by the beta-polypeptide polymer and the titanium alloy bone nails modified by RGD polypeptide, the experimental groups of titanium alloy bone nails modified by the beta-polypeptide polymer remarkably improve the osseointegration effect of the implant.
Example 26: in vivo bone integration Activity test of beta-polypeptide Polymer modified stainless Steel implants
The experimental method was the same as in example 5 except that the titanium alloy bone nail was replaced with a stainless steel bone nail. For the stainless steel bone nail with RGD polypeptide or beta-polypeptide polymer to be modified, firstly cleaning the nail in a UV/O3 cleaning machine, then modifying the polydopamine coating, and then immersing the nail in RGD polypeptide or polymer solution for reaction overnight to obtain the stainless steel bone nail with RGD polypeptide or polymer modification.
The results show that: the quantitative result of Micro-CT shows that compared with the experimental groups of titanium alloy bone nails without modification and RGD polypeptide modification, the experimental groups of titanium alloy bone nails with modification by the beta-polypeptide polymer significantly improve the osseointegration effect of the implant.
Example 27: testing of the repair Activity of fractured bones of beta-polypeptide Polymer-modified tobermorite implants
The experiment was evaluated using a fractured bone defect model. The experimental animals were eight week female SD rats with a mass of about 180g-200g, and the rats were anesthetized by intraperitoneal injection with pentobarbital sodium at a concentration of 40 mg/kg. Rats were fixed to the console and the legs were sterilized with iodophor after shaving. The joint part of the rear leg of the rat is cut with a surgical knife for 5-8mm of wound, then muscle tissue is cut at the middle part of the thigh by the knife, and the muscle at the part is pulled out by a stripper to expose the bone. The femur was then fixed up and down with a metal plate and 1cm of bone in the middle of the femur was removed using a jig saw. Finally, 24 large bone fracture defects were created in 12 rats and randomly implanted with the following materials: (1) non-modified tobermorite (n=12); (2) Beta-polypeptide polymer modified tobermorite prepared in example 6 (n=12). After implantation of the material, the wound is closed with absorbable sutures. After surgery, animals were allowed free access to murine food and water. Rats were sacrificed by intraperitoneal injection of excess sodium pentobarbital at two time points of four weeks and eight weeks, respectively. Samples were collected and tissue was fixed with 4% paraformaldehyde solution. Bone repair of each set of implant materials was quantitatively analyzed using Micro-CT.
For the modified tobermorite scaffold, a layer of polydopamine coating is modified on the scaffold, and then the modified tobermorite scaffold is immersed in a beta-polypeptide polymer solution for reaction overnight to obtain the beta-polypeptide polymer modified tobermorite scaffold.
The results show that: compared with the non-modified white tobermorite scaffold, the effect of repairing broken bones of the experimental group modified by the beta-polypeptide polymer is remarkably improved.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.