CN115806736B - MMP enzyme-responsive injectable polyamino acid hydrogel and preparation method and application thereof - Google Patents
MMP enzyme-responsive injectable polyamino acid hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an injectable polyamino acid hydrogel with MMP enzyme response, a preparation method and application thereof, wherein the preparation raw material comprises polyamino acid with a structure shown in a formula I and polypeptides with azide modification at two ends. The polyamino acid hydrogel can be rapidly glued at a lower concentration, and the glue forming time and the mechanical strength are controllable; the gel forming time and mechanical strength of the hydrogel are changed by changing the concentration of the polyamino acid having the structure of formula I and/or the polypeptide with azide modification at both ends. The polyamino acid hydrogel can be degraded in response in the presence of MMP-2 and MMP-9, and is beneficial to the application of the polyamino acid hydrogel in biomedicine, such as drug slow release.
Description
Technical Field
The invention belongs to the technical field of macromolecules, and particularly relates to an injectable polyamino acid hydrogel with MMP enzyme response, and a preparation method and application thereof.
Background
Chemically crosslinked hydrogels are crosslinked by covalent bonds, the structure of which is typically a three-dimensional network structure, and which typically contain a significant amount of water within the hydrogel. According to different sources of the composition components, the chemical hydrogel can be divided into synthetic polymer hydrogel and natural polymer hydrogel, wherein the natural polymer hydrogel comprises protein-based hydrogel, polyester and polyamino acid hydrogel. In the natural polymer hydrogel, natural amino acid is used as a basic structural unit, such as an amide structure similar to protein, such as poly leucine, poly glutamic acid, poly aspartic acid, poly lysine and the like, and the natural polymer hydrogel is a biodegradable material with good performance, and degradation products are small amino acid molecules and can be degraded into water and carbon dioxide, so that the natural polymer hydrogel has the advantages of good biocompatibility, in vivo degradability, responsiveness, in vivo absorption and the like, and has wide application prospects in the aspects of drug slow release, 3D tissue engineering and the like.
Matrix Metalloproteinases (MMPs) are zinc-dependent endopeptidases that are present in the extracellular matrix and can degrade various protein components of the extracellular matrix, which are essential factors for cell proliferation. Meanwhile, the content of the matrix metalloproteinase in the tumor microenvironment is far higher than that in common tissues, and the matrix metalloproteinase can participate in the occurrence, development and metastasis of tumors. At present, a lot of researches on preparing a responsive material by utilizing the targeting of MMP are carried out, and the responsive material is applied to the aspects of 3D tissue culture, drug slow release and the like. However, the materials reported at present have certain limitations in application, such as incapability of timely cleaning, certain toxicity, and great limitation on application in cell culture, tissue engineering and drug slow release.
Disclosure of Invention
In view of the above, the invention aims to provide an injectable polyamino acid hydrogel with MMP enzyme response and a preparation method thereof, and the hydrogel has good MMP responsiveness, can be rapidly degraded in the presence of MMP-2 and MMP-9, and is beneficial to drug release.
The invention provides an injectable polyamino acid hydrogel responded by MMP enzyme, which is prepared from polyamino acid with a structure shown in formula I and polypeptides with azide modification at two ends;
wherein x is more than or equal to 10 and less than or equal to 400; y is more than or equal to 3 and less than or equal to 120; the ratio of (x+y) is more than or equal to 10 and less than or equal to 500.
In the present invention, x is a polymerization degree, x is 10.ltoreq.400, preferably 50.ltoreq.x.ltoreq.350; more preferably 100.ltoreq.x.ltoreq.300.
Y is the degree of polymerization, y is not less than 3 and not more than 120, preferably not less than 5 and not more than 50, and more preferably not less than 6 and not more than 20.
In the invention, the polyamino acid with the structure shown in the formula I is poly (L-glutamic acid) modified by aza-dibenzocyclooctyne.
In the present invention, the polypeptide sequence of the polypeptide with azide modification at both ends is selected from KEPVGLIGEK, KELPGVIGEK, PLGLAG, KCGPQGIWGQCK, GPQGIWGQ, GPQGIAGQ, APGL, GDQGIAGF, VPMSMRGG, VPMSMRG, VPMSMR, RPMSMR, IPVSLRSG, SGESPAYYTA, PAYYTA, GTAGLIGQ, VPLSLYSG, IPENFFGV, PPGAYHGA or GPPGVVGP.
In the present invention, the preparation raw material further includes a solvent selected from physiological saline, a buffer solution, a tissue culture solution or body fluid.
In the present invention, the polyamino acid having the structure of formula I is prepared as follows:
poly (L-glutamic acid) is mixed with an organic solvent, and primary amino modified azadibenzocyclooctyne is grafted on the carboxyl of a poly (L-glutamic acid) side chain under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) to obtain the polyamino acid with the structure shown in the formula I.
In the present invention, the edc·hcl and NHS are capable of activating the side chain carboxyl group of poly (L-glutamic acid). The molar ratio of EDC, HCl and NHS to carboxyl of poly (L-glutamic acid) is (0.07-0.4): 1.
in the invention, poly (L-glutamic acid) is prepared by taking N-hexylamine as an initiator, taking N, N-dimethylformamide as a solvent, and initiating the ring-opening polymerization of gamma-benzyl-L-glutamate-N-carboxyl cyclic anhydride monomer.
In the present invention, the molar ratio of the primary amino-modified azadibenzocyclooctyne to the carboxyl group of the poly (L-glutamic acid) is (0.05 to 0.3): 1.
the invention provides a preparation method of the injectable polyamino acid hydrogel, which comprises the following steps:
mixing a polyamino acid solution with a structure shown in a formula I with a polypeptide solution with azide modification at two ends to form a polyamino acid hydrogel.
In the invention, the fraction of the polyamino acid solution with the structure of formula I is 2-20%; the mass fraction of the polypeptide solution with two ends being azide modified is 2-20%. The molar ratio of side chain cyclooctyne in the polyamino acid with the structure shown in the formula I to azide in the polypeptide with azide modification at two ends is 1:1.
Copper-free click reaction is carried out between cyclooctyne in the polyamino acid with the structure shown in the formula I and azide in the polypeptide with azide modification at two ends, so as to form hydrogel.
The solvent in the polypeptide solution and the polyamino acid solution with the structure shown in the formula I is independently selected from physiological saline, buffer solution, tissue culture solution or body fluid.
In the present invention, the azide-modified polypeptide solution and the polyamino acid solution having the structure of formula I are preferably mixed in equimolar amounts.
In the present invention, the temperature at which the polyamino acid solution having the structure of formula I and the polypeptide solution having azide modification at both ends are mixed is 10 to 40℃and preferably 10 to 37 ℃.
In the invention, the gel forming time of the polyamino acid gel is reduced along with the increase of the mass fraction of the poly (L-glutamic acid) solution with the side group being modified by the azadibenzocyclooctyne and/or the polypeptide solution with the two ends being modified by the azide. As the polymerization degree of poly (L-glutamic acid) modified by aza-dibenzocyclooctyne as the side group increases, the gel forming time of the polyamino acid hydrogel decreases. The mechanical strength of the polyamino acid hydrogel increases as the mass fraction of the poly (L-glutamic acid) solution modified by the aza-dibenzocyclooctyne as the side group and/or the polypeptide solution modified by the azide as the two ends increases.
In the present invention, the injectable polyamino acid hydrogel is capable of rapid degradation in the presence of MMP-2, MMP-9.
The invention provides an application of the injectable polyamino acid hydrogel in drug slow release.
The invention provides a polyamino acid hydrogel, which is prepared from polyamino acid with a structure shown in a formula I and polypeptides with azide modification at two ends. The polyamino acid hydrogel can be rapidly glued at a lower concentration, and the glue forming time and the mechanical strength are controllable; the gel forming time and mechanical strength of the hydrogel are changed by changing the concentration of the polyamino acid having the structure of formula I and/or the polypeptide with azide modification at both ends. The polyamino acid hydrogel can be degraded in response in the presence of MMP-2 and MMP-9, and is beneficial to the application of the polyamino acid hydrogel in biomedicine, such as drug slow release.
Drawings
FIG. 1 is a flow chart of a 6% by mass polyamino acid hydrogel provided in example 7 of this invention;
FIG. 2 is a flow chart of 5% by mass of a polyamino acid gel as provided in example 8 of this invention;
FIG. 3 is a degradation chart of 4% by mass of polyamino acid gel in PBS as provided in example 9 of the invention;
FIG. 4 is a graph showing the degradation of 5% by mass of polyamino acid gel in a collagen type IV enzyme solution provided in comparative example 1 of the present invention;
FIG. 5 shows a 5% by mass polyamino acid gel (polypeptide N) as provided in example 10 of the invention 3 -KEPVGLIGEK-N 3 ) Degradation profile in collagen type IV enzyme solution;
FIG. 6 shows a 5% by mass polyamino acid gel (polypeptide N) as provided in example 11 of the invention 3 -KELPGVIGEK-N 3 ) Degradation profile in collagen type IV enzyme solution;
FIG. 7 is a graph showing the degradation of 5% by mass of a polyamino acid-PEG hydrogel provided in comparative example 2 in a collagen IV type enzyme solution;
FIG. 8 is a graph showing the cytotoxicity of poly (glutamic acid) modified with azadibenzocyclooctyne as a side group according to example 12 of the present invention;
FIG. 9 is a cryoelectron microscopic view of 5% by mass of polyamino acid hydrogel provided in example 14 of the present invention;
FIG. 10 is a graph showing the release profile of 5% by mass of a polyamino acid gel in a collagen type IV enzyme solution provided in example 15 of the present invention;
FIG. 11 is a graph showing the release profile of 5% by mass of a polyamino acid-PEG hydrogel provided in comparative example 3 in a collagen type IV enzyme solution.
Detailed Description
To further illustrate the present invention, the following describes in detail an MMP enzyme-responsive injectable polyamino acid hydrogel, and methods of preparation and use thereof, provided by the present invention, in conjunction with the examples, which are not to be construed as limiting the scope of protection of the present invention.
Example 1
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared with a PBS buffer at pH 7.4 to give a mass fraction of 6%. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, mixing, wherein the gel forming time of the mixture is measured by a test tube inversion method by a vortex instrument for 5s, the gel forming time is recorded, and the gel forming time of the polyamino acid injectable hydrogel with the mass fraction of 6% at 37 ℃ is 110+/-5 s.
Example 2
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, mixing, wherein the gel forming time of the mixture is measured by a test tube inversion method by a vortex instrument for 5 seconds, the gel forming time is recorded, and the gel forming time of the polyamino acid injectable hydrogel with the mass fraction of 5% at 37 ℃ is 210+/-5 seconds.
Example 3
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 4% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The pH of the polypeptide solution was adjusted to pH=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio1:1, mixing, wherein the gel forming time of the mixture is measured by a test tube inversion method by a vortex instrument for 5s, the gel forming time is recorded, and the gel forming time of the polyamino acid injectable hydrogel with the mass fraction of 4% at 37 ℃ is 820+/-5 s.
Example 4
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 3% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, mixing by a vortex instrument, wherein the gel forming time is measured by a test tube inversion method for 5s, the gel forming time is recorded, and the gel forming time of the polyamino acid injectable hydrogel with the mass fraction of 3% at 37 ℃ is 2215+/-5 s.
Example 5
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=200) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, mixing, wherein the gel forming time of the mixture is measured by a test tube inversion method by a vortex instrument for 5 seconds, the gel forming time is recorded, and the gel forming time of the polyamino acid injectable hydrogel with the mass fraction of 5% at 37 ℃ is 1200+/-5 seconds.
Example 6
Poly (L-glutamic acid) (degree of polymerization=100) modified with aza-dibenzocyclooctyne as side group and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, mixing by a vortex instrument for 5s, determining the gel forming time by adopting a test tube inversion method, recording the gel forming time, wherein the gel forming time of the polyamino acid injectable hydrogel with the mass fraction of 5% at 37 ℃ is 2340+/-5 s.
Example 7
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared with a PBS buffer at pH 7.4 to give a mass fraction of 6%. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, vortex machine mixing for 3s, and rapidly transferring to a rotary rheometer to measure the mechanical strength (i.e. viscoelasticity modulus), and the result is shown in fig. 1, wherein one curve is loss energy G ", one curve is storage modulus G ', and when G' is larger than G", the gel formation is started, and the gel formation point tested by the instrument and the stable mechanical strength after the gel formation can be seen in the figure, and particularly, the elastic modulus is stable at 1875Pa. As a result, the mass fraction was 6%, and the mechanical strength of the polyamino acid injectable hydrogel was 1875Pa.
Example 8
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, vortex for 3s, and rapidly transfer to an rotameter to determine the mechanical strength, the mass fraction is 5% and the mechanical strength of the polyamino acid injectable hydrogel is 991Pa, see FIG. 2.
Example 9
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 4% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1, vortex for 3s, and rapidly transfer to an rotameter to determine the mechanical strength, as shown in fig. 3, the mass fraction is 4% and the mechanical strength of the polyamino acid injectable hydrogel is 293Pa.
Comparative example 1
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. Benefit (benefit)The polypeptide solution was adjusted to ph=7.4 with NaOH solution, and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex instrument mixing for 5s, pipette 100 μl into a small dish with known mass, analytical balance weighing after 30min, adding 2mL PBS buffer solution, placing in a shaking table at 37deg.C, changing PBS at intervals, weighing, recording mass, and as a result, referring to FIG. 4, polyamino acid injectable hydrogel has not degraded for 16 days, showing that the hydrogel has no responsiveness to MMP.
Example 10
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex instrument mixing for 5s, transferring 100 mu L of the mixture to a small dish with known mass by using a liquid transferring gun, weighing by using an analytical balance after 30min, adding 2mL of collagen IV type enzyme solution, placing the mixture in a shaking table with constant temperature at 37 ℃, changing the collagen IV type enzyme solution at intervals, weighing and recording the mass, and as a result, referring to figure 5, the polyamino acid injectable hydrogel is completely degraded for 3 days, and has better MMP responsiveness.
Example 11
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KELPGVIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex instrument mixing for 5s, transferring 100 mu L of the mixture to a small dish with known mass by using a liquid transferring gun, weighing by using an analytical balance after 30min, adding 2mL of collagen IV type enzyme solution, placing the mixture in a shaking table with constant temperature at 37 ℃, changing the collagen IV type enzyme solution at intervals, weighing and recording the mass, and as a result, referring to figure 6, the polyamino acid injectable hydrogel is completely degraded for 4 days, and has better MMP responsiveness.
Comparative example 2
Poly (L-glutamic acid) (polymerization degree=300) with pendant azadibenzocyclooctyne modified and PEG with both ends azide modified were each formulated as a 5% mass fraction solution with PBS buffer at pH 7.4. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex instrument mixing for 5s, pipette 100 μl into small dish with known mass, analytical balance weighing after 30min, adding 2mL collagen IV enzyme solution, placing in shaking table at 37deg.C constant temperature, changing collagen IV enzyme solution at intervals, weighing and recording mass, and as a result, referring to fig. 7, polyamino acid injectable hydrogel has not been degraded for 16 days, which shows that the hydrogel has no responsiveness to MMP.
Example 12
Mouse fibroblasts 3T3 were grown in 96-well plates at 7000 cells per well, 200 μl of complete medium per well (90% DMEM medium+10% neonatal calf serum) and placed in an incubator for 24h incubation. After 24h, the plates were removed, 20 μl of PBS buffer at pH 7.4 and poly (L-glutamic acid) with pendant azadibenzocyclooctyne modified groups at different concentrations (polymerization = 300) were added to each plate and incubated in an incubator for 24h. After 24 hours, the culture plate is taken out, the culture medium is sucked away, PBS is used for washing 2-3 times, 10% of CCK-8 solution is added in a dark place, the culture plate is placed in an incubator for incubation for 1 hour, and the absorbance at 450nm is tested by an enzyme-labeled instrument. Cytotoxicity of materials at different concentrations (500 mg/mL, 250mg/mL, 125mg/mL, 62.5mg/mL, 31.25mg/mL, 15.625 mg/mL) on 3T3 cells see FIG. 8, and experimental results show that poly (L-glutamic acid) modified with azadibenzocyclooctyne as a side group is not cytotoxic to normal fibroblasts.
Example 13
Mouse fibroblasts 3T3 were grown in 96-well plates at 7000 cells per well, 200 μl of complete medium per well (90% DMEM medium+10% neonatal calf serum) and placed in an incubator for 24h incubation. After 24h, the plates were removed and 20. Mu.L of PBS buffer at pH 7.4 and different concentrations of polypeptide (N were added to each plate 3 -KEPVGLIGEK-N 3 ) The solution was placed in an incubator for incubation for 24h. Taking out the culture plate after 24 hours, sucking the culture medium, washing 2-3 times with PBS, adding 10% CCK-8 solution in dark placeIncubate for 1h in incubator and test absorbance at 450nm with a microplate reader. Experimental results indicate that the polypeptide (N) 3 -KEPVGLIGEK-N 3 ) There is no cytotoxicity to normal fibroblasts.
Example 14
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solution with the mass fraction of 5% is prepared by using PBS buffer solution with the pH of 7.4 respectively, the solution and the PBS buffer solution are mixed in an equimolar manner, the mixture is uniformly mixed by using a vortex meter and then is formed into gel at 37 ℃, and after the gel is stabilized for 1h, a frozen electron microscope image is shown in figure 9.
Example 15
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The antitumor drug oxaliplatin was mixed in poly (L-glutamic acid) (polymerization degree=300) using a pH meter polypeptide solution adjusted to ph=7.4 with NaOH solution, the final concentration of oxaliplatin was 1.0mg/mL and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex instrument mixing for 5s, pipetting 200 μl into a small dish of known mass with a pipette, adding 2mL of collagen IV type enzyme solution, placing in a shaking table at 37deg.C, changing collagen IV type enzyme solution at intervals, and collecting release liquid. The release results are shown in fig. 10, in which oxaliplatin is slowly released within five days, and the total release amount is 94%.
Example 16
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The antitumor drug oxaliplatin was mixed in poly (L-glutamic acid) (polymerization degree=300) using a pH meter polypeptide solution adjusted to ph=7.4 with NaOH solution, the final concentration of oxaliplatin was 1.0mg/mL and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex mixing for 5s, transferring 200 μl to a small dish with known mass with a pipette, adding 2mL PBS buffer solution, placing in a shaking table with constant temperature of 37deg.C, and changing PBS buffer at intervalsThe solution was washed and the release solution was collected. The release results showed that oxaliplatin release was incomplete and the total release amount was 32% for 10 days.
Comparative example 3
Poly (L-glutamic acid) (polymerization degree=300) with pendant azadibenzocyclooctyne modified and PEG with both ends azide modified were each formulated as a 5% mass fraction solution with PBS buffer at pH 7.4. The antitumor drug oxaliplatin was mixed in poly (L-glutamic acid) (polymerization degree=300) using a pH meter polypeptide solution adjusted to ph=7.4 with NaOH solution, the final concentration of oxaliplatin was 1.0mg/mL and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex instrument mixing for 5s, pipetting 200 μl into a small dish of known mass with a pipette, adding 2mL of collagen IV type enzyme solution, placing in a shaking table at 37deg.C, changing collagen IV type enzyme solution at intervals, and collecting release liquid. The release results showed that oxaliplatin release was incomplete and the total release amount was 42% for 10 days.
Comparative example 4
Poly (L-glutamic acid) (polymerization degree=300) with pendant azadibenzocyclooctyne modified and PEG with both ends azide modified were each formulated as a 5% mass fraction solution with PBS buffer at pH 7.4. The pH value of the polypeptide solution is regulated to be 7.4 by NaOH solution, the antitumor drug oxaliplatin is mixed in poly (L-glutamic acid) (polymerization degree=300), the final concentration of oxaliplatin is 1.0mg/mL, then the two polymer solutions are mixed according to the molar ratio of 1:1, a vortex machine is used for mixing for 5 seconds, 200 mu L of the polypeptide solution is removed from a small dish with known mass by a pipette, 2mL of PBS buffer solution is added, the PBS buffer solution is placed in a shaking table with constant temperature of 37 ℃, the PBS buffer solution is replaced at intervals, and the release solution is collected. The release results showed that oxaliplatin release was incomplete and the total release amount was 43% for 10 days.
Example 17
Poly (L-glutamic acid) modified with aza-dibenzocyclooctyne as side group (degree of polymerization=300) and polypeptide N 3 -KEPVGLIGEK-N 3 The solutions were prepared as 5% by mass solutions with PBS buffer solutions having a pH of 7.4, respectively. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, and Cy 3-coat anti-rate IgG (h+l) anti-body was mixed in poly (L-glutamic acid) (polymerization degree=300)Cy 3-coat anti-rate IgG (H+L) anti-body final concentration was 0.5mg/mL and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortex instrument mixing for 5s, transferring 200mL to a small dish with known mass by a liquid transferring gun, adding 2mL of collagen IV type enzyme solution, placing in a shaking table with constant temperature and shaking at 37 ℃, replacing the collagen IV type enzyme solution at intervals, and collecting release liquid. The release results show that Cy 3-coat anti-rate IgG (H+L) anti-ibody is completely released in the hydrogel for about three days.
Comparative example 5
Poly (L-glutamic acid) (polymerization degree=300) with pendant azadibenzocyclooctyne modified and PEG with both ends azide modified were each formulated as a 5% mass fraction solution with PBS buffer at pH 7.4. The polypeptide solution was adjusted to ph=7.4 with NaOH solution using a pH meter, cy 3-coat anti-ratigg (h+l) anti-ibody was mixed in poly (L-glutamic acid) (polymerization degree=300), cy 3-coat anti-ratigg (h+l) anti-ibody final concentration was 0.5mg/mL and then the two polymer solutions were mixed in a molar ratio of 1:1 mixing, vortexing for 5s, pipetting 200 μl into a cuvette of known mass with a pipette, adding 2mL of PBS buffer, placing into a shaking table at 37deg.C with constant temperature, changing PBS buffer at intervals, and collecting the release solution. The release result shows that Cy 3-coat anti-rate IgG (H+L) anti-ibody is not completely released in the hydrogel, and the total release amount is 53% in 10 days.
From the above examples, the present invention provides a polyamino acid hydrogel prepared from a polyamino acid having the structure of formula I and a polypeptide having azide modifications at both ends. The polyamino acid hydrogel can be rapidly glued at a lower concentration, and the glue forming time and the mechanical strength are controllable; the gel forming time and mechanical strength of the hydrogel are changed by changing the concentration of the polyamino acid having the structure of formula I and/or the polypeptide with azide modification at both ends. The polyamino acid hydrogel can be degraded in response in the presence of MMP-2 and MMP-9, and is beneficial to the application of the polyamino acid hydrogel in biomedicine, such as drug slow release. The experimental results show that: the gel forming time is 110+/-5 s to 2340+/-5 s; the mechanical strength is 290-1900 Pa; has no cytotoxicity to normal fibroblasts; oxaliplatin is slowly released within 5 days, and Cy 3-coat anti-rate IgG (H+L) anti-ibody is slowly released within 3 days.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. An injectable polyamino acid hydrogel with MMP enzyme response is characterized in that the preparation raw material comprises polyamino acid with a structure shown in a formula I and polypeptides with azide modification at two ends;
a formula I;
wherein x is more than or equal to 10 and less than or equal to 400; y is more than or equal to 3 and less than or equal to 120; the ratio of (x+y) is more than or equal to 10 and less than or equal to 500;
the polypeptide sequence of the polypeptide with the azide modified ends is selected from KEPVGLIGEK, KELPGVIGEK, PLGLAG, KCGPQGIWGQCK, GPQGIWGQ, GPQGIAGQ, APGL, GDQGIAGF, VPMSMRGG, VPMSMRG, VPMSMR, RPMSMR, IPVSLRSG, SGESPAYYTA, PAYYTA, GTAGLIGQ, VPLSLYSG, IPENFFGV, PPGAYHGA or GPPGVVGP.
2. The injectable polyamino acid hydrogel of claim 1 further comprising a solvent selected from the group consisting of physiological saline, a buffer solution, a tissue culture solution, and a body fluid.
3. The injectable polyaminohydrogel of claim 1 wherein the polyamino acid having the structure of formula i is prepared by the following method:
mixing poly (L-glutamic acid) with an organic solvent, and grafting primary amino modified azadibenzocyclooctyne on the carboxyl of a poly (L-glutamic acid) side chain under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain the polyamino acid with the structure shown in the formula I.
4. The injectable polyamino acid hydrogel of claim 3 wherein the molar ratio of primary amino-modified azadibenzocyclooctyne to carboxyl groups of poly (L-glutamic acid) is (0.05 to 0.3): 1.
5. the injectable polyamino acid hydrogel of claim 3 wherein the poly (L-glutamic acid) is prepared by ring opening polymerization of gamma-benzyl-L-glutamate-N-carboxycyclic anhydride monomer using N-hexylamine as an initiator and N, N-dimethylformamide as a solvent.
6. A method of preparing the injectable polyamino acid hydrogel of claim 1 comprising the steps of:
mixing a polyamino acid solution with a structure shown in a formula I with a polypeptide solution with azide modification at two ends to form polyamino acid hydrogel;
a formula I;
wherein x is more than or equal to 10 and less than or equal to 400; y is more than or equal to 3 and less than or equal to 120; the ratio of (x+y) is more than or equal to 10 and less than or equal to 500;
the polypeptide sequence of the polypeptide with the azide modified ends is selected from KEPVGLIGEK, KELPGVIGEK, PLGLAG, KCGPQGIWGQCK, GPQGIWGQ, GPQGIAGQ, APGL, GDQGIAGF, VPMSMRGG, VPMSMRG, VPMSMR, RPMSMR, IPVSLRSG, SGESPAYYTA, PAYYTA, GTAGLIGQ, VPLSLYSG, IPENFFGV, PPGAYHGA or GPPGVVGP.
7. The method according to claim 6, wherein the fraction of the polyamino acid solution having the structure of formula i is 2 to 20%;
the mass fraction of the polypeptide solution with two ends being azide modified is 2-20%.
8. The method according to claim 6, wherein the temperature of the mixing is 10-40 ℃.
9. Use of an injectable polyamino acid gel according to any one of claims 1 to 5 for the preparation of a drug-eluting material.
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