CN116063748A - Hyaluronic acid gel and preparation method and application thereof - Google Patents

Hyaluronic acid gel and preparation method and application thereof Download PDF

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CN116063748A
CN116063748A CN202310212401.2A CN202310212401A CN116063748A CN 116063748 A CN116063748 A CN 116063748A CN 202310212401 A CN202310212401 A CN 202310212401A CN 116063748 A CN116063748 A CN 116063748A
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hyaluronic acid
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CN116063748B (en
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张田慧
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CHANGCHUN SINOBIOMATERIALS CO LTD
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Abstract

The invention belongs to the technical field of gel and preparation thereof, and relates to a hyaluronic acid gel, a preparation method and application thereof, wherein the hyaluronic acid gel comprises a cross-linked product of hyaluronic acid or salt thereof and a biodegradable cross-linking agent; the biodegradable crosslinking agent has the followingThe chemical formula is shown. The hyaluronic acid gel prepared by the invention combines the dual functions of hyaluronic acid and polylactide/polylactone, and has the advantages of low toxicity, less residue, low swelling degree, good enzyme resistance and good shaping property.
Figure ZY_1

Description

Hyaluronic acid gel and preparation method and application thereof
Technical Field
The invention belongs to the technical field of gel and preparation thereof, and relates to hyaluronic acid gel and a preparation method and application thereof.
Background
Hyaluronic Acid (HA) is a linear high molecular viscosity polysaccharide composed of D-glucuronic acid and N-acetylglucosamine linked repeatedly in disaccharide units, and is widely found in connective tissues such as joint synovial fluid, vitreous humor, skin, cell gap, cockscomb, etc. of humans and other vertebrates. HA is an endogenous substance in human body, HAs the characteristics of good biocompatibility, no toxicity, no immunogenicity, no irritation, higher safety, high viscoelasticity, non-newton rheological property, and the like, can be degraded and eliminated by injecting hyaluronidase, and is widely used as a soft tissue filler in beauty, for example, hyaluronic acid is injected into skin to increase the volume of soft tissues, so as to achieve the purposes of removing wrinkles or shaping, and the like.
However, since natural hyaluronic acid or its sodium salt has a small average molecular weight, and exists in a liquid form as it is, hyaluronic acid or its sodium salt is very easily decomposed under the action of hyaluronidase and free radicals in the body, and stability is poor, it is difficult to achieve a shaping effect and a filling effect lasts for a short time.
In order to overcome the defect, the cross-linking technology is applied to hyaluronic acid, namely, hyaluronic acid molecules are connected through a cross-linking agent to form hyaluronic acid gel with a relatively stable network structure, and the hyaluronic acid gel after cross-linking modification has the advantages of good viscoelastic property, water insolubility, high mechanical strength and long degradation time, can effectively prolong the filling maintenance time, and meanwhile, the hyaluronic acid gel with different cross-linking degrees has the characteristics of different viscosity, water retention, elasticity and the like, so that the requirements of different cosmetic filling can be better met.
The crosslinking agents used in the sodium hyaluronate gel products currently on the market are mainly: BDDE (butanediol diglycidyl ether) and DVS (divinyl sulfone). Sodium hyaluronate gels prepared by BDDE cross-linking are used, for example, in the Gaoder Meyer (Galderm) Rayleigh series, in the Allergan (Allergan) Qiao Yadeng (Juvederm) series, in the West French (Laboratoires Vivacy) Styleage series, in the Taiwan Kogyo-Haidemi (Hya-Dermis) series; whereas the U.S. genease company (Genzyme) Hylatform series, the German Aldrime company (Adoderm) Varioderm series, the Thai-OGmbH (Teoxane) teosyl series all use DVS as a cross-linker to prepare sodium hyaluronate gels.
Sodium hyaluronate gels prepared from BDDE crosslinker and DVS crosslinker have distinct physical properties: the DVS crosslinked product has hard texture and high crosslinking activity; BDDE crosslinked products are soft in texture and have a large expansion ratio. However, it is notable that both BDDE and DVS crosslinking agents have toxic or potential carcinogenic potential, and the crosslinked space network structure has a wrapping effect on the unreacted crosslinking agent, so that the difficulty in removing the crosslinking agent is high, and the sodium hyaluronate gel prepared by using BDDE crosslinking agent or DVS crosslinking agent has potential safety hazard.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the hyaluronic acid gel, wherein a brand-new biodegradable cross-linking agent is adopted, and the hyaluronic acid gel prepared by cross-linking the cross-linking agent and hyaluronic acid has the advantages of low toxicity, less residues, good enzyme resistance, high viscosity, good shaping property, difficult displacement, long in-vivo retention time and the like. Specifically, the crosslinking agent is a low-toxicity biodegradable epoxy crosslinking agent by modifying the tail end of polylactone/polylactide into epoxy groups to form polylactone epoxy derivatives/polylactide epoxy derivatives.
In order to avoid potential safety hazards caused by toxicity of the crosslinking agent, polymer materials with high safety such as polylactic acid or polycaprolactone are generally adopted at present to be directly used as a filler, or are compounded with hyaluronic acid and sodium carboxymethyl cellulose (CMC) carrier gel to be used as the filler.
Polylactic acid (PLA), also called polylactide, is a new generation of completely degradable polymer material which is rapidly developed in the 90 th century, and is polymerized by taking lactic acid as a monomer, and can be slowly degraded into lactic acid in human body through non-enzymatic hydrolysis, and finally degraded into CO 2 And H 2 O, no toxic or harmful effect. Lactic acid molecules include L-lactic acid (L-lactic acid) and D-lactic acid (D-lactic acid), and corresponding polylactic acids include poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA) and poly-D-lactic acid (PDLLA). PLL (phase locked loop)A is unique as a cosmetic injection material. This is because L-lactic acid, which is a monomer formed by hydrolysis of PLLA in the human body, is a substance that is originally present in the human body, and L-lactic acid can be completely metabolized into carbon dioxide or water in cells by lactic acid dehydrogenase (which is involved in L-lactic acid metabolism alone). Since there is no enzyme in the human body that metabolizes D-lactic acid, D-lactic acid degraded by PDLA can accumulate to cause acidosis, producing neurotoxic effects. The decomposition product of PLLA is L-lactic acid, which can effectively excite the activity of fibroblast in skin and activate the regeneration of self collagen. The PLLA material medical products currently approved are the AiWeilan child's needles under the Saint Boma flag and the soft-white angel under the Aimeike flag.
Polycaprolactone (PCL) is a common type of polylactone, when the polycaprolactone is used as a medical cosmetic filling material, the polycaprolactone is generally required to be prepared into microspheres with the size of a micron, the microspheres are further injected into deep tissues of skin through a syringe, then the PCL microspheres excite M2 type polarization of macrophages in the skin immune system through foreign body reaction of the PCL microspheres and 6-hydroxycaproic acid released in the degradation process of the PCL microspheres, and the macrophages in the M2 type polarization can highly express related cytokines such as TGF-beta (transforming growth factor) and the like to further induce fibroblasts to accelerate secretion of collagen, so that the tissue filling effect of injection sites is finally achieved. However, since the degradation rate of PCL is slow at the initial stage of implantation into skin tissue, the improvement effect of skin in the initial stage of treatment is not obvious for patients, and most of skin improvement of treated patients gradually starts to appear after 6 to 8 weeks after treatment. The microspheres prepared by compounding the collagen and the PCL in the current market can improve the treatment effect of the PCL microspheres in the initial stage of tissue filling, but the collagen used in the current medical market is mostly of animal origin, so that the microspheres have higher sensitization and immunogenicity risks. Meanwhile, because collagen is only dissolved in an aqueous phase solvent and is completely insoluble in an oil phase solvent, the microsphere needs to be prepared by a double-emulsion method, the preparation process is too complex, the cost is high, the large-scale production is not facilitated, at least more than two emulsifying agents are usually required to be introduced in the preparation process, the product purification difficulty is high, the biological safety is poor, and the application of the microsphere in the field of beauty and medical treatment is limited.
The invention prepares the polylactone epoxy derivative/polylactide epoxy derivative by connecting epoxy groups at the tail ends of the polylactide/polylactone through the design of a synthesis method. The polylactone epoxy derivative/polylactide epoxy derivative can be used as a cross-linking agent to prepare hyaluronic acid gel through the reaction of epoxy groups and groups in hyaluronic acid.
Specifically, the invention provides the following scheme:
a hyaluronic acid gel comprising a cross-link of hyaluronic acid or a salt thereof and a biodegradable cross-linking agent;
wherein the biodegradable crosslinking agent has a chemical formula represented by formula (a):
Figure SMS_1
formula (A)/(L)>
In the formula (A), R 1 Selected from C which is unsubstituted or optionally substituted by one, two or more Ra 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl;
each Ra, which are identical or different, are independently selected from OH, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 An aryl group;
r is a linking group;
m is an integer greater than or equal to 2;
x is an integer of 1 to 10;
n 1 to the degree of polymerization, n 1 Is an integer of 2 to 1000;
R 2 and R is 3 Identical or different, independently of one another, from H, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl.
According to the invention, R is selected from any one of the following structures: c (C) 1-16 Alkylene, C 2-16 Alkenylene, C 2-16 Alkynylene, C 3-8 Cycloalkylene, C 6-10 Arylene, 5-10 membered heteroarylene, or a structure represented by formula (B):
Figure SMS_2
(B)
In the formula (B), n 2 Is an integer of 0 or more;
R 4 and R is 5 Identical or different, independently of one another, from H, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl.
According to the present invention, the biodegradable crosslinking agent has a chemical formula represented by formula (I):
Figure SMS_3
formula (I)
In the formula (I), R 1 、m、x、n 1 、n 2 、R 2 、R 3 、R 4 And R is 5 Having the definition as described above.
According to the invention, m is an integer of 2-8, n 2 Is an integer of 0 to 12.
According to the invention, said R 1 Selected from C substituted with one, two or more Ra 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl.
According to the invention, said R 2 And R is 3 Identical or different, independently of one another, from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl.
According to the invention, said R 4 And R is 5 Identical or different, independently of one another, from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl.
According to the invention, said R 3 、R 4 And R is 5 Selected from H, x is 1, and the crosslinking agent has a chemical formula shown in formula (i):
Figure SMS_4
(i)
Wherein the R is 1 、R 2 、m、n 1 And n 2 Having the definition as described above.
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, the crosslinker having the formula (ii):
Figure SMS_5
(ii)
Wherein the R is 1 、m、n 1 、n 2 X has the definition as described above.
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, the crosslinker having the formula (iii):
Figure SMS_6
(iii)
Wherein the R is 1 、m、n 1 、n 2 X has the definition as described above.
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 2 0, the cross-linking agent has the formula [ ]iv) formula:
Figure SMS_7
(iv)
Wherein the R is 1 、m、n 1 Having the definition as described above.
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 1 Is 2, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (iv-1):
Figure SMS_8
(iv-1)
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 1 Is 4, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (iv-2):
Figure SMS_9
(iv-2)
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 2 0, the crosslinking agent having a chemical formula represented by formula (v):
Figure SMS_10
(v)
Wherein the R is 1 、m、n 1 Having the definition as described above.
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 1 Is 3, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (v-1):
Figure SMS_11
(v-1)
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 1 Is 6, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (v-2):
Figure SMS_12
(v-2)
According to the present invention, the hyaluronate is selected from one or more of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and tetrabutylammonium hyaluronate.
According to the invention, the hyaluronic acid or salt thereof has a molecular weight of 0.1-500 kilodaltons.
The invention also provides a preparation method of the hyaluronic acid gel, which comprises the following steps:
and (3) carrying out a crosslinking reaction on the biodegradable crosslinking agent and hyaluronic acid or salt thereof to obtain hyaluronic acid gel.
According to the present invention, the biodegradable crosslinking agent is prepared by a method comprising the steps of:
s1, reacting a compound shown in a formula (II) with a compound shown in a formula (III) in a solvent to obtain a polymer shown in a formula (IV);
Figure SMS_13
formula (II)
Figure SMS_14
Formula (III)
Figure SMS_15
(IV)
R 6 Selected from hydroxyl or halogen;
s2, reacting the polymer shown in the formula (IV) with an oxidant to obtain the biodegradable cross-linking agent shown in the formula (I).
The invention also provides the application of the hyaluronic acid gel in preparing products for industrial, pharmaceutical, medical cosmetology and cosmetic applications.
The invention has the beneficial effects that:
(1) According to the invention, the biodegradable cross-linking agent polylactide epoxy derivative/polylactone epoxy derivative is directly cross-linked with hyaluronic acid to form hyaluronic acid gel, so that the steps are simple, the operation difficulty is low, the reaction condition is mild, the reaction time is short, and the prepared hyaluronic acid gel combines the dual functions of hyaluronic acid and polylactide/polylactone, and has the advantages of low toxicity, less residue, low swelling degree, good enzyme resistance and good shaping property;
(2) The end of the biodegradable cross-linking agent epoxy derivative/polylactone epoxy derivative is epoxy group, alkane chain exists in the molecule, the flexibility is good, the cross-linking reaction is easy to occur with polysaccharide to form gel, the number of the epoxy groups in the molecule can be regulated and controlled because the number of the repeating units in the epoxy derivative is easy to regulate, and the gel performance prepared by the epoxy groups serving as the cross-linking agent and the polysaccharide is also easy to regulate and control; meanwhile, the biodegradable cross-linking agent polylactide epoxy derivative/polylactone epoxy derivative degraded product is nontoxic to cells, does not have adverse effect on tissues, and is safer to use.
Drawings
FIG. 1 shows nuclear magnetic hydrogen spectra of PLA500 and PLA500-2epo prepared in example 1 of the present invention;
FIG. 2 shows nuclear magnetic resonance hydrogen spectra of PCL500 and PCL500-2epo prepared in example 2 of the present invention;
FIG. 3 is a graph showing rheological properties of the hyaluronic acid gel A1 prepared in example 5 of the present invention;
FIG. 4 is a graph showing rheological properties of the hyaluronic acid gel A2 prepared in example 6 of the present invention;
FIG. 5 is a graph showing rheological properties of the hyaluronic acid gel A3 prepared in example 7 of the present invention;
FIG. 6 is a graph showing rheological properties of the hyaluronic acid gel A4 prepared in example 8 of the present invention;
FIG. 7 is a graph showing rheological properties of the hyaluronic acid gel B1 prepared in example 9 of the present invention;
FIG. 8 is a graph showing rheological properties of the hyaluronic acid gel B2 prepared in example 10 of the present invention;
FIG. 9 is a graph showing rheological properties of the hyaluronic acid gel B3 prepared in example 11 of the present invention;
FIG. 10 is a graph showing rheological properties of the hyaluronic acid gel B4 prepared in example 12 of the present invention;
FIG. 11 is a graph showing rheological properties of the hyaluronic acid gel C1 prepared in example 13 of the present invention.
Detailed Description
[ biodegradable Cross-linking agent ]
The invention firstly provides a biodegradable cross-linking agent, which has a chemical formula shown in a formula (A):
Figure SMS_16
(A)
In the formula (A), R 1 Selected from C which is unsubstituted or optionally substituted by one, two or more Ra 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl;
each Ra, which are identical or different, are independently selected from OH, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 An aryl group;
m is an integer greater than or equal to 2;
x is an integer of 1 to 10;
n 1 is the degree of polymerization;
r is a linking group;
R 2 And R is 3 Identical or different, independently of one another, from H, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl;
according to an embodiment of the present invention, R is selected from any one of the following structures: c (C) 1-16 Alkylene, C 2-16 Alkenylene, C 2-16 Alkynylene, C 3-8 Cycloalkylene, C 6-10 Arylene, 5-10 membered heteroarylene, or a structure represented by formula (B):
Figure SMS_17
(B)
In the formula (B), n 2 Is an integer of 0 or more;
R 4 and R is 5 Identical or different, independently of one another, from H, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl.
According to an embodiment of the present invention, the crosslinking agent has a chemical formula represented by formula (I):
Figure SMS_18
formula (I)
In the formula (I), R 1 ，m，x，n 1 ，n 2 ，R 2 ，R 3 ，R 4 And R is 5 Having the definition as described above.
The crosslinking agent of the invention is epoxy-terminated and has a degree of polymerization n 1 (c=o) - (C (R) 2 )(R 3 )) x -an O segment, which makes the cross-linking agent flexible and susceptible to cross-linking reaction with hyaluronic acid or a salt thereof; in addition, the polymerization degree is n 1 (c=o) - (C (R) 2 )(R 3 )) x The O chain segment is a biodegradable synthetic polymer chain segment, so that the cross-linking agent is biodegradable, and degradation products thereof are nontoxic to cells and do not have adverse effects on tissues; furthermore, the value of m is adjustable so that the number of epoxide groups in the molecule can be adjusted The gel performance of the hyaluronic acid prepared by using the hyaluronic acid as a cross-linking agent and the hyaluronic acid or the salt thereof is easy to regulate and control.
According to an embodiment of the present invention, m is an integer of 2 to 8, for example, 2, 3, 4, 5, 6, 7 or 8.
According to an embodiment of the invention, the n 1 Is an integer of 1 to 200, preferably the n 1 Is an integer of 2 to 20, further preferably, the n 1 Is an integer of 5 to 15, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
According to an embodiment of the invention, n 2 An integer of 0 to 12, e.g. n 2 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
According to an embodiment of the invention, the R 1 Selected from C substituted with one, two or more Ra 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl, ra having the definition as described above.
As an example, the R 1 -(O) m -selected from the group consisting of dehydrided pentaerythritol, ethylene glycol, 2-propanediol, 1, 4-butanediol, 1, 6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, trimethylolpropane or glycerol.
According to an embodiment of the invention, the R 2 And R is 3 Identical or different, independently of one another, from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl; preferably, said R 2 And R is 3 The same or different, independently of one another, from H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or phenyl; illustratively, R is 2 And R is 3 Identical and selected from H, or R 2 And R is 3 Different, one is selected from H, one is selected fromFrom methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or phenyl.
According to an embodiment of the invention, the R 4 And R is 5 Identical or different, independently of one another, from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl, preferably, the R 4 And R is 5 The same or different, independently of one another, are selected from H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or phenyl.
According to the invention, said R 3 、R 4 And R is 5 Selected from H, x is 1, and the crosslinking agent has a chemical formula shown in formula (i):
Figure SMS_19
(i)
Wherein the R is 1 、R 2 、m、n 1 And n 2 Having the definition as described above.
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, the crosslinker having the formula (ii):
Figure SMS_20
(ii)
Wherein the R is 1 、m、n 1 、n 2 X has the definition as described above.
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, the crosslinker having the formula (iii):
Figure SMS_21
(iii)
Wherein the R is 1 、m、n 1 、n 2 X has the definition as described above.
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 2 0, the crosslinking agent having a chemical formula represented by formula (iv):
Figure SMS_22
(iv)
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 1 Is 2, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (iv-1):
Figure SMS_23
(iv-1)
According to an embodiment of the invention, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 1 Is 4, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (iv-2):
Figure SMS_24
(iv-2)
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 2 0, the crosslinking agent having a chemical formula represented by formula (v):
Figure SMS_25
(v)
Wherein the R is 1 、m、n 1 Having the definition as described above.
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is1，n 1 Is 3, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (v-1):
Figure SMS_26
(v-1)
According to an embodiment of the invention, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 1 Is 6, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (v-2):
Figure SMS_27
formula (v-2).
[ preparation method of biodegradable Cross-linking agent ]
The invention further provides a preparation method of the biodegradable cross-linking agent, which comprises the following steps:
s1, reacting a compound shown in a formula (II) with a compound shown in a formula (III) in a solvent to obtain a polymer shown in a formula (IV);
Figure SMS_28
formula (II)
Figure SMS_29
Formula (III)
Figure SMS_30
(IV)
R 6 Selected from hydroxyl or halogen;
s2, reacting the polymer shown in the formula (IV) with an oxidant to obtain the biodegradable cross-linking agent shown in the formula (I).
Specifically, the reaction process of the present invention is as follows:
Figure SMS_31
Figure SMS_32
wherein R is 1 、R 2 、R 3 、R 4 、R 5 、R 6 、m、n 1 、n 2 And x is as defined above.
According to an embodiment of the present invention, the molecular weight of the compound represented by formula (II) is 300 to 10000, preferably the molecular weight of the compound represented by formula (II) is 500 to 2000, for example 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000.
According to an embodiment of the present invention, in step S1, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) is 1 (m-8 m), for example, 1:2, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60.
According to an embodiment of the present invention, the solvent in step S1 is selected from an organic solvent, preferably the organic solvent is selected from at least one of aromatic hydrocarbons, aliphatic hydrocarbons, alicyclic hydrocarbons, halogenated hydrocarbons, ethers, esters, ketones, acetonitrile, pyridine, dimethyl sulfoxide, N-dimethylformamide.
Preferably, the aromatic hydrocarbon is selected from one or more of benzene, toluene, xylene and ethylbenzene.
Preferably, the aliphatic hydrocarbon is selected from one or more of pentane, hexane, heptane and octane.
Preferably, the alicyclic hydrocarbon is selected from one or more of cyclohexane, cyclohexanone and toluene cyclohexanone.
Preferably, the halogenated hydrocarbon is selected from one or more of chlorobenzene, dichlorobenzene, dichloromethane, trichloromethane, carbon tetrachloride and dichloroethane.
Preferably, the ether is selected from one or more of diethyl ether, anisole, tetrahydrofuran, 2-methyltetrahydrofuran, 1, 3-dioxolane and 1, 4-dioxane.
Preferably, the ester is selected from one or more of methyl acetate, ethyl acetate and propyl acetate.
Preferably, the ketone is selected from one or more of acetone, methyl butanone and methyl isobutyl ketone.
According to an embodiment of the present invention, the reaction temperature in step S1 is 0 to 100 ℃. Exemplary values are any value in the range of any value or any two point values of 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 55 ℃, for example, 5 to 60 ℃, or 15 to 40 ℃.
According to an embodiment of the invention, the reaction time of step S1 is 6 h to 5 d, preferably 1 d to 3 d, for example 12 h, 2 d, 3 d, 4 d, 5 d.
According to an embodiment of the present invention, in step S1, the reaction to obtain the polymer represented by formula (IV) further comprises the following steps: and washing and drying the reactant, and removing the organic solvent to obtain the polymer.
According to an embodiment of the invention, in step S1, the washing comprises washing several times with a washing solvent.
According to an embodiment of the present invention, in step S1, the washing solvent is selected from saturated saline, naHCO 3 Aqueous solution, NH 4 One or more of the aqueous Cl solutions.
According to an embodiment of the invention, in step S1, the drying comprises adding a drying agent, preferably anhydrous MgSO, to the washed solution for drying 4 At least one of calcium chloride, calcium sulfate, anhydrous aluminum oxide, and silica gel, such as anhydrous MgSO 4 。
According to an embodiment of the present invention, in step S1, removing the organic solvent comprises subjecting the dried solution to distillation, for example, spin-steaming to remove the organic solvent.
According to an embodiment of the present invention, in step S2, the oxidizing agent is selected from one or more of hydrogen peroxide, peracetic acid, sodium hypochlorite, benzoyl peroxide, m-chloroperoxybenzoic acid, sodium percarbonate, sodium perborate, potassium perborate.
According to an embodiment of the present invention, in step S2, the reaction of the polymer represented by formula (IV) with the oxidizing agent is performed in an organic solvent or water.
According to an embodiment of the present invention, in step S2, the organic solvent is selected from at least one of aromatic hydrocarbons, aliphatic hydrocarbons, alicyclic hydrocarbons, halogenated hydrocarbons, alcohols, ethers, esters, ketones, glycol derivatives, acetonitrile, pyridine, dimethyl sulfoxide, N-dimethylformamide.
Preferably, the aromatic hydrocarbon is selected from one or more of benzene, toluene, xylene and ethylbenzene.
Preferably, the aliphatic hydrocarbon is selected from one or more of pentane, hexane, heptane and octane.
Preferably, the alicyclic hydrocarbon is selected from one or more of cyclohexane, cyclohexanone and toluene cyclohexanone.
Preferably, the halogenated hydrocarbon is selected from one or more of chlorobenzene, dichlorobenzene, dichloromethane, trichloromethane, carbon tetrachloride and dichloroethane.
Preferably, the alcohol is selected from one or more of methanol, ethanol, isopropanol, tert-butanol.
Preferably, the ether is selected from one or more of diethyl ether, anisole, tetrahydrofuran, 2-methyltetrahydrofuran, 1, 3-dioxolane and 1, 4-dioxane.
Preferably, the ester is selected from one or more of methyl acetate, ethyl acetate and propyl acetate.
Preferably, the ketone is selected from one or more of acetone, methyl butanone and methyl isobutyl ketone.
Preferably, the glycol derivative is selected from one or more of ethylene glycol monomethyl ether, ethylene glycol monoethyl ether and ethylene glycol monobutyl ether.
According to an embodiment of the present invention, in step S2, the molar ratio of the polymer represented by formula (IV) and the oxidizing agent is 1 (m-8 m), for example, 1:2, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60.
According to the embodiment of the invention, the reaction temperature of the step S2 is 0-65 ℃. Exemplary values are any value in the range of any value or any two point values of 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 55 ℃, for example, 5 to 60 ℃, or 15 to 40 ℃.
According to an embodiment of the invention, the reaction time of step S2 is 6 h to 10 d, preferably 1 d to 4 d, such as 1 d, 2 d, 3 d, 4 d.
According to an embodiment of the present invention, in step S2, the step of reacting the polymer represented by formula (IV) with an oxidizing agent further comprises the steps of: a quencher is added to the reaction solution to quench the unreacted oxidant, resulting in a crude solution.
According to an embodiment of the present invention, in step S2, the step of adding a quencher to the reaction solution further comprises the steps of: the reaction solution was cooled to room temperature.
According to an embodiment of the present invention, in step S2, the step of adding a quencher to the reaction solution further comprises the steps of: separating the crude solution, removing the water phase, and separating the organic phase to obtain the cross-linking.
According to an embodiment of the invention, in step S2, the separation of the organic phase comprises the steps of: the organic phase was passed through a silica gel column.
[ use of biodegradable Cross-linking agent ]
The invention also provides the use of the biodegradable cross-linking agent for cross-linking hyaluronic acid or a salt thereof.
[ hyaluronic acid gel ]
The invention also provides a hyaluronic acid gel, which comprises a cross-linked product of hyaluronic acid or a salt thereof and the biodegradable cross-linking agent.
According to an embodiment of the invention, the hyaluronate is selected from one or more of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate and tetrabutylammonium hyaluronate, for example sodium hyaluronate.
According to an embodiment of the invention, the hyaluronic acid or salt thereof has a molecular weight of 0.1-500 ten thousand daltons, preferably the hyaluronic acid or salt thereof has a molecular weight of 0.1-300 ten thousand daltons, e.g. the hyaluronic acid or salt thereof has a molecular weight of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 1 ten thousand, 10 ten thousand, 50 ten thousand, 100 ten thousand, 150 ten thousand, 200 ten thousand, 250 ten thousand, 300 ten thousand, 350 ten thousand, 400 ten thousand, 450 ten thousand or 500 ten thousand daltons.
[ method for producing hyaluronic acid gel ]
The invention also provides a preparation method of the hyaluronic acid gel, which comprises the following steps:
and (3) carrying out a crosslinking reaction on the biodegradable crosslinking agent and hyaluronic acid or salt thereof to obtain hyaluronic acid gel.
According to an embodiment of the present invention, the crosslinking is performed under alkaline conditions, which means that the pH is greater than 7, preferably the crosslinking is performed under conditions of pH greater than 8, further preferably the crosslinking is performed under conditions of pH greater than 9.
According to an embodiment of the present invention, the temperature of the crosslinking reaction is 0-80 ℃, preferably the temperature of the crosslinking reaction is 15-50 ℃, for example 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃.
According to an embodiment of the invention, the time of the crosslinking reaction is 1 h-7 d, preferably 1 h-24 h, for example 2 h, 5 h, 6 h, 7 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h.
According to an embodiment of the present invention, the crosslinking reaction is performed in a mixed solvent of an aqueous solution and an organic solvent selected from among water-miscible organic solvents, which may be selected from one or more of dimethyl sulfoxide (DMSO), 1' 4-Dioxane (DO), tetrahydrofuran (THF), N-Dimethylformamide (DMF), acetone, etc.
According to an embodiment of the present invention, the molar ratio of the repeating unit of hyaluronic acid or a salt thereof to the biodegradable crosslinking agent is (0.001-1000): 1, preferably the molar ratio of the repeating unit of hyaluronic acid or a salt thereof to the biodegradable crosslinking agent is (0.01-100): 1, further preferably the molar ratio of the repeating unit of hyaluronic acid or a salt thereof to the biodegradable crosslinking agent is (0.1-30): 1, for example 0.005:1, 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 600:1, 700:1, 900:1.
As an example, the molar ratio of the repeating unit of hyaluronic acid or a salt thereof to the biodegradable cross-linking agent is (1-30): 1, preferably (5-20): 1, for example, 1:1, 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1.
According to an embodiment of the invention, the molecular weight of the hyaluronic acid or salt thereof is 0.1-500 ten thousand daltons, preferably the molecular weight of the hyaluronic acid or salt thereof is 0.1-300 ten thousand daltons, e.g. the molecular weight of the hyaluronic acid or salt thereof is 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 1 ten thousand, 10 ten thousand, 50 ten thousand, 100 ten thousand, 150 ten thousand, 200 ten thousand, 250 ten thousand, 300 ten thousand, 350 ten thousand, 400 ten thousand, 450 ten thousand or 500 ten thousand daltons.
[ use of hyaluronic acid gel ]
The invention also provides an application of the hyaluronic acid gel in preparing products for industrial, pharmaceutical, medical cosmetology and cosmetic applications. In particular, it can be used for preparing soft tissue wound repair dressing, scaffold material for bone repair, viscoelastic for supporting in ophthalmic surgery, scaffold material for 3D bioprinting, etc.
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 to which the claimed subject matter belongs. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter of the present application. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the terms "include," as well as other forms, such as "comprising," "including," and "containing," are not limiting.
The term "C1-8 alkyl" is understood to mean preferably a linear or branched saturated monovalent hydrocarbon radical having 1,2, 3, 4, 5, 6, 7 or 8 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, or isomers thereof. In particular, the group has 1,2, 3 or 4 carbon atoms ("C1-4 alkyl"), such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly the group has 1,2 or 3 carbon atoms ("C1-3 alkyl"), such as methyl, ethyl, n-propyl or isopropyl.
The term "C 2-8 Alkenyl "refers to a straight or branched chain monovalent unsaturated aliphatic hydrocarbon group containing one, two or more double bonds. It will be appreciated that where the alkenyl group comprises more than one double bond, the double bonds may be separated from each other or conjugated. Non-limiting examples of alkenyl groups include vinyl, allyl, (E) -2-methylvinyl, (Z) -2-methylvinyl, (E) -but-2-enyl, (Z) -but-2-enyl, (E) -but-1-enyl, (Z) -but-1-enyl, pent-4-enyl, (E) -pent-3-enyl, (Z) -pent-3-enyl, (E) -pent-2-enyl, (Z) -pent-1-enyl, hex-5-enyl, (E) -hex-4-enyl, (E) -hex-3-enyl, (Z) -hex-3-enyl, (E) -hex-2-enyl, (Z) -hex-2-enyl, (E) -hex-1-enyl, (Z) -hex-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E) -1-methylprop-1-enyl, (Z) -1-methylpropan-1-enyl, 3-methyl1-methylbut-2-enyl, (Z) -1-methylbut-2-enyl, (E) -3-methylbut-1-enyl (Z) -3-methylbut-1-enyl, (E) -2-methylbut-1-enyl, (Z) -2-methylbut-1-enyl, (E) -1-methylbut-1-enyl, (Z) -1-methylbut-1-enyl, 1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl.
The term "C 2-8 Alkynyl "refers to a straight or branched monovalent unsaturated aliphatic hydrocarbon group containing one, two or more triple bonds. Non-limiting examples of alkynyl groups include ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl, hex-5-ynyl, 1-methylpropan-2-ynyl, 2-methylbutan-3-ynyl, 1-methylbutan-2-ynyl, 3-methylbutan-1-ynyl, pent-4-ynyl 1-ethylprop-2-ynyl, 3-methylpent-4-ynyl, 2-methylpent-4-ynyl, 1-methylpent-4-ynyl, 2-methylpent-3-ynyl, 1-methylpent-3-ynyl, 4-methylpent-2-ynyl, 1-methylpent-2-ynyl, 4-methylpent-1-ynyl, 3-methylpent-1-ynyl, 2-ethylbut-3-ynyl, 1-ethylbut-2-ynyl, 1-propylprop-2-ynyl, 1-isopropylprop-2-ynyl, 2, 2-dimethylbut-3-ynyl, 1-dimethylbut-2-ynyl or 3, 3-dimethylbut-1-ynyl. In particular, the alkynyl group is ethynyl, prop-1-ynyl or prop-2-ynyl.
The term "C 3-8 Cycloalkyl "refers to a saturated or partially unsaturated, monocyclic or polycyclic, cyclic hydrocarbon group, and a carbocycle may contain 3 to 8 carbon atoms. Carbocycles may be monocyclic or polycyclic. Carbocycles where there are multiple rings, these rings may form spiro, fused and bridged ring structures. For example, non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like; non-limiting examples of polycyclic carbocycles include decalinyl or isobornyl.
The term "C 6-10 Aryl "refers to a 6 to 10 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated electron system, such as phenyl and naphthyl.
The term "5-10 membered heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 10 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered (e.g., 5, 6, 7, 8, 9 or 10 membered), more preferably 5 or 6 membered. Non-limiting examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl, and the like, and their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazole, indazolyl, indolyl, isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, and the like; or an axcinyl group, an indolizinyl group, a purinyl group, etc., and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl and/or phenoxazinyl, and the like.
The compounds of the general formula of the present invention, as well as the methods for their preparation and use, will be described in further detail below in conjunction with the specific examples. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Example 1
Synthesis of an epoxy derivative of a PLA fragment having a double-ended hydroxyl molecular weight of 500 (PLA 500-2 epo)
The reaction formula for preparing PLA500-2epo is as follows:
Figure SMS_33
。
PLA with 10.0. 10.0 g hydroxyl groups at both ends and 500 molecular weight was placed in a 250 mL round bottom flask and 100. 100 mL anhydrous dichloromethane and 5.0 g K were added 2 CO 3 Stirring for 30 min, adding 3 mL acryloyl chloride, and stirring at normal temperature and in a dark place for reaction 24 h. After completion of the reaction, the reaction mixture was washed 3 times with saturated brine, followed by drying over anhydrous MgSO 4 Drying, collecting filtrate, and removing dichloromethane by rotary evaporation to obtain PLA with double bonds at two ends.
PLA with double bonds at 5.0. 5.0 g ends was placed in a 100 mL round bottom flask, 50 mL anhydrous methylene chloride and 3.54 g m-chloroperoxybenzoic acid were added, and the reflux reaction was 48 h at 40 ℃. After the completion of the reaction, the reaction solution was cooled to room temperature, and 10% of saturated aqueous sodium sulfite solution mL was added thereto, followed by stirring for 30 minutes to quench the residual m-chloroperoxybenzoic acid. Separating, removing the water phase, and passing the organic phase through a silica gel column to obtain PLA500-2epo.
Referring to FIG. 1, nuclear magnetic resonance spectra of PLA and PLA500-2epo prepared by this example show that after oxidation by m-chloroperoxybenzoic acid, a peak of epoxy group appears at 2.9-3.0 ppm, indicating successful synthesis of PLA500-2epo.
Example 2
Synthesis of epoxy derivatives of PCL fragment with 500 double-end hydroxyl molecular weight (PCL 500-2 epo)
The reaction formula for preparing PCL500-2epo is as follows:
Figure SMS_34
。
10.0. 10.0 g of a hydroxy terminated PCL having a molecular weight of 500 was placed in a 250 mL round bottom flask and 100 mL of anhydrous dichloromethane and 5.0 g of K were added 2 CO 3 Stirring for 30 min, adding 3 mL acryloyl chloride, and stirring at normal temperature and in a dark place for reaction 24 h. After completion of the reaction, the reaction mixture was washed 3 times with saturated brine, followed by drying over anhydrous MgSO 4 Drying, collecting filtrate, and removing dichloromethane by rotary evaporation to obtain PLA with double bonds at two ends.
5.0. 5.0 g double bond PCL was placed in a 100 mL round bottom flask, 50 mL anhydrous dichloromethane was added, and 3.54 g m-chloroperoxybenzoic acid was added. Reflux reaction 48 h at 40 ℃. After the completion of the reaction, the reaction solution was cooled to room temperature, and 10% of saturated aqueous sodium sulfite solution mL was added thereto, followed by stirring for 30 minutes to quench the residual m-chloroperoxybenzoic acid. Separating liquid and removing water phase. The organic phase was passed through a silica gel column to give PCL500-2epo.
Referring to FIG. 2, nuclear magnetic resonance spectra of PCL as a reaction raw material and PCL500-2epo prepared in this example show that an epoxy group peak appears at 2.9-3.0 ppm after oxidation by m-chloroperoxybenzoic acid, indicating that PCL500-2epo was successfully synthesized.
Example 3
Synthesis of an epoxy derivative of a PLA fragment having a molecular weight of 1000 at both terminal hydroxyl groups (PLA 1000-2 epo)
The reaction formula for preparing the PLA1000-2epo is as follows:
Figure SMS_35
。
PLA with a molecular weight of 1000 at the double end of 10.0. 10.0 g was placed in a 250 mL round bottom flask and 100. 100 mL anhydrous dichloromethane and 5.0 g K were added 2 CO 3 Stirring for 30 min, adding 1.5. 1.5 mL acryloyl chloride, and stirring at normal temperature and in a dark place for reaction 24 h. After completion of the reaction, the reaction mixture was washed 3 times with saturated brine, followed by drying over anhydrous MgSO 4 Drying, collecting filtrate, and removing dichloromethane by rotary evaporation to obtain PLA with double bonds at two ends.
PLA, 5.0. 5.0 g double-ended, was placed in a100 mL round bottom flask, 50 mL anhydrous methylene chloride was added, and 1.94. 1.94 g m-chloroperoxybenzoic acid was added. Reflux reaction 48 h at 40 ℃. After the completion of the reaction, the reaction solution was cooled to room temperature, and 10% of saturated aqueous sodium sulfite solution mL was added thereto, followed by stirring for 30 minutes to quench the residual m-chloroperoxybenzoic acid. Separating liquid and removing water phase. The organic phase was passed through a silica gel column to give PLA1000-2epo.
Example 4
Synthesis of epoxy derivatives of PCL fragment with a molecular weight of 1000 in terms of double-ended hydroxyl groups (PCL 1000-2 epo)
The reaction formula for preparing PCL1000-2epo is as follows:
Figure SMS_36
。
10.0. 10.0 g PCL having a double-ended hydroxyl molecular weight of 1000 was placed in a 250 mL round bottom flask and 100 mL anhydrous dichloromethane and 5.0 g K were added 2 CO 3 Stirring for 30 min, adding 1.5. 1.5 mL acryloyl chloride, and stirring at normal temperature and in a dark place for reaction 24 h. After completion of the reaction, the reaction mixture was washed 3 times with saturated brine, followed by drying over anhydrous MgSO 4 Drying, collecting filtrate, and removing dichloromethane by rotary evaporation to obtain PCL with double bonds at both ends.
5.0. 5.0 g double bond PCL was placed in a 100 mL round bottom flask, 50 mL anhydrous dichloromethane was added, and 1.94 g m-chloroperoxybenzoic acid was added. Reflux reaction 48 h at 40 ℃. After the completion of the reaction, the reaction solution was cooled to room temperature, and 10% of saturated aqueous sodium sulfite solution mL was added thereto, followed by stirring for 30 minutes to quench the residual m-chloroperoxybenzoic acid. Separating liquid and removing water phase. The organic phase was passed through a silica gel column to give PCL1000-2epo.
Examples 5 to 13
Examples 5 to 12 are methods for preparing crosslinked sodium hyaluronate gel by performing a crosslinking reaction with sodium hyaluronate using the biodegradable crosslinking agent prepared in examples 1 to 4; in contrast, example 13 is a method of preparing a crosslinked sodium hyaluronate gel by a crosslinking reaction with sodium hyaluronate using a commercially available crosslinking agent BDDE. The amounts of crosslinker and sodium hyaluronate fed are shown in table 1.
TABLE 1
Gel code Crosslinking agent and mass Mole number of crosslinker Sodium hyaluronate mass
Example 5 A1 PLA500-2epo (20 mg) 33.33 µmoL 100 mg
Example 6 A2 PLA500-2epo (25 mg) 41.67 µmoL 100 mg
Example 7 A3 PLA500-2epo (30 mg) 50.00 µmoL 100 mg
Example 8 A4 PLA1000-2epo (25 mg) 22.73 µmoL 100 mg
Example 9 B1 PCL500-2epo (20 mg) 33.33 µmoL 100 mg
Example 10 B2 PCL500-2epo (25 mg) 41.67 µmoL 100 mg
Example 11 B3 PCL500-2epo (30 mg) 50.00 µmoL 100 mg
Example 12 B4 PCL1000-2epo (25 mg) 22.73 µmoL 100 mg
Example 13 C1 BDDE (6.74 mg) 33.33 µmoL 100 mg
The crosslinking steps of examples 5-13 were: according to Table 1, a crosslinking agent and sodium hyaluronate powder (100. 100 mg) having a molecular weight of 250 ten thousand were dissolved in 1 mL of a 1% aqueous NaOH solution and DMSO mixed solution (volume ratio: 4:1), and stirred well enough and placed in a 50℃water bath to crosslink 6 h to obtain a bulk gel. The block gel was divided into small uniform blocks, and 3 volumes of PBS buffer was added to swell, and the buffer was changed every 4. 4 h until the pH of the gel was about 7.0, with an osmotic pressure of about 300 mOsmol/L. Homogenizing the fully swelled gel into granular gel with a homogenizer for 10 min at 8000 rpm and at 10 m/s. And filling the homogenized gel into a prefilled syringe, and sterilizing for 15 minutes at 121 ℃ to obtain the crosslinked hyaluronic acid gel, wherein the corresponding gel codes are A1, A2, A3, A4, B1, B2, B3, B4 and C1 respectively.
Example 14
Rheological Performance test
The rheological properties of the gels were all tested using an Anton Poar cube MCR 301 rheometer, and the gels prepared in examples 5-13 were placed on a test plate in oscillation mode with a gap between the rotor and the plate set to 0.3 mm, rotor diameter 25 mm, temperature: 25. DEG C, stress: 1%, frequency range: 0.1 to 10 g Hz. The elastic modulus (G ') and the viscous modulus (G ") at 1.0086 Hz are recorded, and from this the ratio of viscous to elastic (tanδ=g"/G') is calculated. FIGS. 3-11 are graphs of the rheological properties of the hyaluronic acid gels prepared in examples 5-13, in sequence, and Table 2 records the results of the G ', G ' ' and Tan delta values of the hyaluronic acid gels prepared in examples 5-13.
TABLE 2
Gel sample G’ G” Tan δ
A1 164.7 42.5 0.259
A2 205.2 21.4 0.104
A3 257.1 46.4 0.179
A4 239.3 22.8 0.095
B1 46.4 17.9 0.389
B2 82.4 27.4 0.332
B3 101.2 30.3 0.300
B4 89.8 27.7 0.308
C1 177 4.26 0.024
Referring to Table 2, it can be seen that Tan delta <1 for all gels prepared in examples 5-13, indicates that all gels tended to be elastic gels. Comparing the G 'values of all gels, A1> B1, A2> B2, A3> B3, A4> B4, shows that under the same conditions, the sodium hyaluronate gel prepared by the PLA epoxy derivative crosslinking agent has better elasticity than the sodium hyaluronate gel prepared by the PCL epoxy derivative crosslinking agent, and the highest G' value can reach 257.1. Comparing the G' values of all the gels, wherein A3 is greater than A2 is greater than A1, B3 is greater than B2 is greater than B1, and the elasticity of the sodium hyaluronate gel prepared by the PLA epoxy derivative crosslinking agent/PCL epoxy derivative crosslinking agent is greater along with the increase of the adding ratio of the crosslinking agent to the sodium hyaluronate within a certain range; meanwhile, the G' value of the sodium hyaluronate gel prepared by the PLA epoxy derivative crosslinking agent is at least equal to that of the gel prepared by the BDDE crosslinking agent and is basically larger than that of the gel prepared by the BDDE crosslinking agent; and the G' value of the sodium hyaluronate gel prepared by the PLA epoxy derivative crosslinking agent/PCL epoxy derivative crosslinking agent is larger than that of the gel prepared by the BDDE crosslinking agent.
Example 15
In vitro enzymolysis stability experiment
Precisely weighing the gel prepared in examples 5-12, respectively adding 2 mL of PBS (pH 7.0) and 3 mL hyaluronidase liquid (300U/mL), uniformly mixing, placing in a constant-temperature water bath at 37 ℃, uniformly mixing 50 mu L of the mixed liquid with 3 mL of PBS (pH 7.0) every 10 min, measuring the absorbance value at 232 and nm by an ultraviolet spectrophotometer, stopping measuring after the absorbance value is unchanged, and recording the time as the enzymolysis time of the gel, wherein the enzymolysis time of the gel is recorded in Table 4.
TABLE 4 Table 4
Gel sample A1 A2 A3 A4 B1 B2 B3 B4 C1
Enzymolysis time (min) 130 160 180 160 80 110 110 90 70
Referring to Table 4, the enzymolysis time A1> B1, A2> B2, A3> B3 and A4> B4 of the crosslinked sodium hyaluronate of different gels show that under the same conditions, the crosslinked sodium hyaluronate gel prepared by the PLA epoxy derivative crosslinking agent has better enzymolysis resistance and longer filling time in vivo. The enzymolysis time A1> B1> C1 shows that under the same condition, the enzyme resistance of the sodium hyaluronate gel prepared by the PLA epoxy derivative crosslinking agent/PCL epoxy derivative crosslinking agent is better than that of the sodium hyaluronate gel crosslinked by BDDE.
Example 16
Measurement of swelling degree
The specific detection method of the swelling degree is from crosslinked sodium hyaluronate for plastic surgery of a standard YY/T0962-2014. About 0.1 g of the cross-linked sodium hyaluronate gel prepared in examples 5-13, respectively, was placed on two petri dishes. Two dishes were placed in a dry box and weighed after constant weight at 80℃and designated m1. Water was added dropwise until the mixture swelled, and after removing excess water, the mixture was weighed and designated as m2. Calculating the swelling degree according to a swelling degree formula: q= (m 2-m 1)/m 1. The swelling degree of the crosslinked sodium hyaluronate gel is recorded in table 5.
TABLE 5
Gel sample A1 A2 A3 A4 B1 B2 B3 B4 C1
Sodium hyaluronate content (mg/mL) 21.2 20.15 22.07 21.54 20.54 21.85 21.38 20.77 21.32
Swelling degree 4.31 3.57 2.16 3.33 5.47 4.64 3.46 4.42 7.86
The swelling degree is a quantitative index for evaluating the gel structure, and is generally high in crosslinking degree and low in swelling degree of gel with compact structure, and the result shows that the swelling degree of the sodium hyaluronate gel prepared by the PLA epoxy derivative crosslinking agent/PCL epoxy derivative crosslinking agent is lower than that of the sodium hyaluronate gel crosslinked by BDDE.
The foregoing description of the specific embodiments of the present invention has been presented by way of example. However, the scope of the present invention is not limited to the above exemplary embodiments. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention should be included in the scope of protection of the claims of the present invention.

Claims (10)

1. A hyaluronic acid gel, characterized in that the hyaluronic acid gel comprises a cross-linked product of hyaluronic acid or a salt thereof and a biodegradable cross-linking agent;
wherein the biodegradable crosslinking agent has a chemical formula represented by formula (a):
Figure QLYQS_1
(A)
In the formula (A), R 1 Selected from C which is unsubstituted or optionally substituted by one, two or more Ra 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl;
each Ra, which are identical or different, are independently selected from OH, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 An aryl group;
m is an integer greater than or equal to 2;
x is an integer of 1 to 10;
n 1 is the degree of polymerization;
r is a linking group;
R 2 and R is 3 Identical or different, independently of one another, from H, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl.
2. The hyaluronic acid gel according to claim 1, characterized in that the biodegradable cross-linking agent has the formula (I):
Figure QLYQS_2
formula (I)
In the formula (I), n 2 Is an integer of 0 or more;
R 4 and R is 5 Identical or different, independently of one another, from H, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, halo C 1-8 Alkyl, C 3-8 Cycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl.
3. The hyaluronic acid gel according to claim 1 or 2, characterized in that m is an integer from 2 to 8, n 2 Is an integer of 0 to 12.
4. The hyaluronic acid gel according to claim 1 or 2, characterized in that R 1 Selected from C substituted with one, two or more Ra 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl;
and/or, the R 2 And R is 3 Identical or different, independently of one another, from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl.
5. The hyaluronic acid gel according to claim 2, characterized in that said R 4 And R is 5 Identical or different, independently of one another, from H, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 Cycloalkynyl radicals, C 6-14 Aryl, 5-14 membered heteroaryl.
6. The hyaluronic acid gel according to claim 2, characterized in that said R 3 、R 4 And R is 5 Selected from H, x is 1, and the crosslinking agent has a chemical formula shown in formula (i):
Figure QLYQS_3
(i)
Wherein the R is 1 、R 2 、m、n 1 And n 2 Having the definition as described above;
alternatively, the R 2 、R 3 、R 4 And R is 5 Selected from H, the crosslinker having the formula (ii):
Figure QLYQS_4
(ii)
Wherein the R is 1 、m、n 1 、n 2 X has the definition as described above;
alternatively, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, the crosslinker having the formula (iii):
Figure QLYQS_5
(iii)
Wherein the R is 1 、m、n 1 、n 2 X has the definition as described above;
alternatively, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 2 0, the crosslinking agent having a chemical formula represented by formula (iv):
Figure QLYQS_6
(iv)
Wherein the R is 1 、m、n 1 Having the definition as described above;
alternatively, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 1 Is 2, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (iv-1):
Figure QLYQS_7
(iv-1)
Alternatively, the R 2 、R 3 、R 4 And R is 5 Selected from H, x is 5, n 1 Is 4, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (iv-2):
Figure QLYQS_8
(iv-2)
Alternatively, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 2 0, the crosslinking agent having a chemical formula represented by formula (v):
Figure QLYQS_9
(v)
Wherein the R is 1 、m、n 1 Having the definition as described above;
alternatively, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 1 Is 3, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (v-1):
Figure QLYQS_10
(v-1)
Alternatively, the R 2 And R is 3 Respectively selected from H, CH 3 ，R 4 And R is 5 Selected from H, x is 1, n 1 Is 6, n 2 And 0, m is 2, and the crosslinking agent has a chemical formula shown in formula (v-2):
Figure QLYQS_11
formula (v-2).
7. The hyaluronic acid gel according to claim 1 or 2, characterized in that the hyaluronate is selected from one or more of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate and tetrabutylammonium hyaluronate;
And/or, the hyaluronic acid or salt thereof has a molecular weight of 0.1 ten thousand-500 ten thousand daltons.
8. The method for preparing a hyaluronic acid gel according to any of claims 1-7, characterized in that the method comprises the steps of:
and (3) carrying out a crosslinking reaction on the biodegradable crosslinking agent and hyaluronic acid or salt thereof to obtain hyaluronic acid gel.
9. The method of claim 8, wherein the biodegradable cross-linking agent is prepared by a method comprising the steps of:
s1, reacting a compound shown in a formula (II) with a compound shown in a formula (III) in a solvent to obtain a polymer shown in a formula (IV);
Figure QLYQS_12
formula (II)
Figure QLYQS_13
Formula (III)
Figure QLYQS_14
Formula (IV)>
R 6 Selected from hydroxyl or halogen;
s2, reacting the polymer shown in the formula (IV) with an oxidant to obtain the biodegradable cross-linking agent shown in the formula (I).
10. Use of the hyaluronic acid gel of any of claims 1-7 for the preparation of products for industrial, pharmaceutical, medical cosmetology and cosmetic use.
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FR3032617A1 (en) * 2015-02-16 2016-08-19 Benedicte Vincente Gavard Molliard Tauzin PROCESS FOR THE PREPARATION OF AN INJECTABLE HYDROGEL; HYDROGEL OBTAINED; USE OF THE HYDROGEL OBTAINED
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