CN113648427A - Hyaluronic acid-ES 2-AF peptide conjugate and preparation method and application thereof - Google Patents
Hyaluronic acid-ES 2-AF peptide conjugate and preparation method and application thereof Download PDFInfo
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Abstract
The application provides a hyaluronic acid-ES 2-AF peptide conjugate, a preparation method and application thereof, wherein the hyaluronic acid-ES 2-AF peptide conjugate comprises an ES2-AF peptide and Hyaluronic Acid (HA), and a connecting bridge formed by 4-Carboxybenzaldehyde (CBA) and adipic Acid Dihydrazide (ADH), wherein the connecting bridge connects the ES2-AF peptide and the hyaluronic acid. The hyaluronic acid-ES 2-AF peptide conjugate provided by the invention HAs good stability and bioactivity, and HAs higher stability and stronger bioactivity compared with ES2-AF peptide and HA-ES2-AF conjugate, thereby having good value of practical application.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to a hyaluronic acid-ES 2-AF peptide conjugate and a preparation method and application thereof.
Background
The information disclosed in this background of the invention is intended to enhance an understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known as prior art to a person skilled in the art.
Endostatin (ES, "IVRRADRAAVP") was originally isolated from the supernatant of a cultured murine vascular endothelial cell tumor cell line, a novel angiogenesis inhibitor containing 183 amino acids and having a molecular weight of 20kDa, has the ability to inhibit tumor angiogenesis, and exhibits potent anti-endothelial cell activity, including inhibition of endothelial cell proliferation, migration, adhesion, and the like. The selective antagonism of the hexapeptide Anti-Flt1 ("GNQWFI") introduced into VEGF receptor 1 may better target VEGF receptors in tumor vascular endothelial cells, inhibiting endothelial cell migration and lumen formation. The existing ES2-AF peptide synthesized by connecting and bridging the ES-2 and the Anti-Flt1 through five glycines through chemical solid phase can better inhibit the tumor angiogenesis through the multi-target point combined action. Although the ES2-AF peptide chain is prolonged, the application is still limited due to the defects of short half-life in vivo, poor stability and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a hyaluronic acid-ES 2-AF peptide conjugate and a preparation method and application thereof. The Hyaluronic acid-ES 2-AF peptide conjugate is successfully prepared by connecting an anti-vascular endothelial-derived ES2-AF peptide synthesized by a chemical solid phase to a hydrophilic Hyaluronic Acid (HA) molecular skeleton by using a derivative obtained by connecting Adipic Acid Dihydrazide (ADH) and 4-carboxybenzaldehyde (4-carboxybenzaza-ldehyde, CBA) as an acid-sensitive connecting bridge, wherein the connecting bridge is abbreviated as ADH-CBA, and the Hyaluronic acid-ES 2-AF peptide conjugate HAs good stability and bioactivity, so that the Hyaluronic acid-ES 2-AF peptide conjugate HAs good practical application value.
Specifically, the present invention provides the following technical features, and one or a combination of the following technical features constitutes the technical solution of the present invention.
In a first aspect of the invention, the invention provides a hyaluronic acid-ES 2-AF peptide conjugate comprising an ES2-AF peptide and Hyaluronic Acid (HA), and a connecting bridge formed by 4-Carboxybenzaldehyde (CBA) and adipic Acid Dihydrazide (ADH) (i.e., ADH-CBA), wherein the connecting bridge connects the ES2-AF peptide and hyaluronic acid.
In an embodiment of the present invention, in the connecting bridge, 4-carboxybenzaldehyde and adipic acid dihydrazide are connected by an amide bond.
In an embodiment of the invention, the linker bridge is linked to hyaluronic acid via an amide bond and the linker bridge is linked to the ES2-AF peptide via an imine or azomethine bond.
In an embodiment of the invention, the ES2-AF peptide has the sequence shown in SEQ ID NO.1 (IVRRADRAAVPGGGGGGNQWFI).
In some preferred embodiments of the invention, ES2-AF HAs a molecular weight of 2254.53Da and HA HAs a molecular weight of 57000 Da.
The hyaluronic acid-ES 2-AF peptide conjugate HAs a simple structure of HA-ADH-CBA-ES 2-AF, which is called HEA for short. In the embodiment of the invention, the hyaluronic acid-ES 2-AF peptide conjugate can be represented in a structure simplified form, and can also be represented by HEA.
In the embodiment of the invention, ADH-CBA is used as a connecting bridge to connect the ES2-AF peptide and hyaluronic acid, so that the ES2-AF peptide can be released from the HEA conjugate under the acidic condition in a tumor microenvironment to be in a free state and quickly reach endothelial cells around tumors to play an anti-angiogenesis effect.
In a second aspect of the present invention, the present invention provides a method for preparing the conjugate of hyaluronic acid-ES 2-AF peptide as described in the first aspect above, comprising: carrying out amidation reaction on adipic dihydrazide and hyaluronic acid to be connected to the hyaluronic acid to obtain an HA-ADH conjugate, carrying out amidation reaction on 4-carboxybenzaldehyde and free amino of adipic dihydrazide in the HA-ADH conjugate to obtain an HA-ADH-CBA conjugate, adding ES2-AF peptide, and carrying out Schiff base reaction on the amino of ES2-AF peptide and aldehyde group in 4-carboxybenzaldehyde in the HA-ADH-CBA conjugate to obtain the hyaluronic acid.
In an embodiment of the present invention, each reaction may be followed by a purification step. The skilled person can select a suitable purification method for the conjugate of the invention by methods known in the art. By way of example, in some embodiments of the present invention, the present invention provides a superior purification method, which includes dialysis and lyophilization. For example, in some embodiments, dialysis may be performed by a dialysis bag method, the reaction product is poured into a pre-treated dialysis bag, and after dialysis for one or more times with water or an ethanol/water mixture, impurities are removed, filtration with a filter membrane is performed, and then pre-freezing is performed, and then freeze-drying is performed in a freeze-dryer.
Wherein, after the HEA is obtained by purification, a spherical structure with good dispersion and uniform granularity can be formed in a dissolving medium by ultrasonic treatment.
Further, the method for preparing the conjugate of hyaluronic acid-ES 2-AF peptide comprises the following steps:
preparation of HA-ADH conjugate: dissolving HA in water, adding ADH and EDCI, completely dissolving, adjusting pH to acidity, adjusting pH to neutrality after reaction is finished, and purifying a reaction product to obtain an HA-ADH conjugate;
preparation of HA-ADH-CBA conjugate: dissolving CBA in dimethyl sulfoxide, adding EDCI and NHS as catalysts to activate carboxyl in CBA; dissolving HA-ADH in water, slowly adding the CBA solution for continuous reaction, and after the reaction is finished, purifying a reaction product to obtain an HA-ADH-CBA conjugate;
preparation of HEA conjugates: dissolving HA-ADH-CBA in a mixed solution of a buffer solution (pH is 7.4) and dimethyl sulfoxide, adding ES2-AF, reacting overnight at room temperature, and purifying a reaction product after the reaction is finished to obtain an HEA conjugate; through ultrasonic treatment, a spherical structure with good dispersion and uniform granularity can be formed in a dissolving medium.
In the process of preparing the HA-ADH conjugate, the pH is adjusted to be acidic, specifically, an acidic solution (such as HCl) is added to adjust the pH to be about 4.75; adjusting pH to neutral by adding alkaline solution (such as NaOH) to adjust pH to about 7.0; after the adipic acid dihydrazide solution is added, the reaction time is controlled to be about 6 hours; the reaction product purification step includes dialysis and lyophilization steps.
During the preparation of the HA-ADH-CBA conjugate, the activation time is controlled to be 2 h; slowly adding the 4-carboxybenzaldehyde solution for continuous reaction, and controlling the reaction time to be 12 h; the reaction product purification step comprises dialysis and freeze-drying.
In the process of preparing the HEA conjugate, the buffer solution is citric acid-disodium hydrogen phosphate buffer solution or disodium hydrogen phosphate-potassium dihydrogen phosphate, and the reaction time is controlled to be 12 hours after ES2-AF is added; the reaction product purification step comprises dialysis and drying; the ultrasonic power is 40W, and the time is 5 min.
In a third aspect of the invention, the invention provides a pharmaceutical composition comprising a conjugate of hyaluronic acid-ES 2-AF peptide as described in the first aspect above.
In a fourth aspect of the invention, the invention provides a pharmaceutical formulation comprising the conjugate of hyaluronic acid-ES 2-AF peptide as described in the first aspect above and at least one pharmaceutically acceptable adjuvant or pharmaceutical carrier.
In some embodiments of the present invention, the pharmaceutical composition or pharmaceutical preparation of the present invention may be administered parenterally or parenterally, including but not limited to injectable implants, emulsions, liposomes, microcapsules, microspheres, nanoparticles, and the like.
The common pharmaceutically acceptable excipients are excipients, such as solvents, binders, fillers, wetting agents, and the like, which can be optionally added when preparing a specific dosage form. If necessary, flavoring agent, colorant, stabilizer, lubricant, and other adjuvants can be added. One skilled in the art can formulate the HEA of the invention into a pharmaceutical composition or pharmaceutical formulation using techniques well known in the art. For example, the pharmaceutical preparation may be prepared according to the modern pharmaceutical preparation series compiled by Shenyang pharmaceutical university. And, in addition to those mentioned in the present invention, suitable pharmaceutical Excipients may also be of other types known in the art, for example as described in the Handbook of pharmaceutical Excipients (Handbook of pharmaceutical Excipients) by the authors Paul J Sheskey et al, which has been amended to the eighth edition previously, the first edition being disclosed in 1986 and the eighth edition in 2017.
The pharmaceutical carrier may be a pharmaceutically acceptable solvent, suspending agent or carrier for the delivery of the pharmaceutically active ingredient into an animal or human body. The carrier may be a liquid or solid and is selected according to the intended mode of administration. Proteins and liposomes are also drug carriers.
In a fifth aspect of the invention, the invention provides the use of the conjugate of hyaluronic acid-ES 2-AF peptide of the first aspect above or the pharmaceutical composition of the third aspect above or the pharmaceutical preparation of the fourth aspect above in the preparation of a medicament and/or an anti-tumor medicament for the treatment of a disease associated with angiogenesis.
In the present invention, the diseases associated with angiogenesis include, but are not limited to, diabetic retinopathy, age-related macular degeneration, arthritis, and the like; such tumors include, but are not limited to, melanoma, breast, lung, colon, ovarian, and renal tumors.
During tumorigenesis, large blood vessels are required to deliver oxygen and nutrients to maintain rapid growth of tumor cells. If the generation and development of the tumor are to be inhibited, the nutrition supply is cut off, namely the formation of new blood vessels is inhibited, and finally the purpose of starving the tumor is achieved. Diabetic Retinopathy (DR) is one of the common complications of diabetes. Studies have shown that retinal neovascularization is one of the major pathogenic causes of DR disease, and thus inhibition of neovascularization is effective in inhibiting DR.
In some embodiments of the invention, the agent for treating a disease associated with angiogenesis is an anti-angiogenesis agent, an agent that inhibits endothelial cell migration, and/or an agent that inhibits luminal formation of endothelial cells.
In a sixth aspect of the invention, the invention provides a method of treating a disease associated with angiogenesis, comprising administering to a subject an effective dose of a HEA conjugate of the invention or a pharmaceutical composition or pharmaceutical formulation comprising the conjugate. Wherein treating a disease associated with neovascularization comprises anti-neovascularization, an agent that inhibits endothelial cell migration, and/or inhibits endothelial cell lumen formation.
The term "subject" refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment. By therapeutically effective amount is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.
Compared with the prior art, the invention has the advantages that: compared with ES2-AF peptide, the hyaluronic acid-ES 2-AF peptide conjugate enhances the anti-angiogenesis and anti-tumor activities of the ES2-AF peptide, integrates the targeting property of hyaluronic acid molecules, and enables the obtained conjugate to have stronger biological activity, higher targeting property and stronger stability; compared with HA-CO-NH- (ES2-AF) synthesized by the inventor in the previous period, the HEA shows stronger biological activity and better application potential due to the change of the connecting bridge.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: nuclear magnetic resonance hydrogen spectrum of HEA conjugate in example 1;
FIG. 2: particle size morphology of HEA conjugate in example 1;
FIG. 3: the results of the inhibitory effect of ES2-AF, HEA conjugate and HA-ES2-AF conjugate on endothelial cell proliferation in Experimental example 1;
FIG. 4: the results of the inhibition of endothelial cell migration by ES2-AF, HEA conjugate and HA-ES2-AF conjugate in Experimental example 2;
FIG. 5: the results of the inhibition of endothelial cell lumen formation by ES2-AF, HEA conjugate and HA-ES2-AF conjugate in Experimental example 3;
FIG. 6: the results of the study of the targeting affinity of HEA conjugates to endothelial cells in experimental example 4.
FIG. 7: the results of evaluation of the environmental-responsive release behavior of the HEA conjugate in experimental example 5.
Detailed Description
The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
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. The reagents or starting materials used in the present application can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present application can be used in the conventional manner in the art or in the product specification. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred embodiments and materials described herein are intended to be exemplary only.
The sequence information of the ES2-AF peptide is shown in SEQ ID NO.1(IVRRADRAAVPGGGGGGNQWFI), the molecular weight is 2254.53Da, the ES2-AF peptide is disclosed in Chinese patent CN107261124A and can be obtained according to the method in the patent, and meanwhile, the HA-ES2-AF conjugate related to the following experimental example is prepared according to the method in CN107261124A example 2. The disclosure in chinese patent CN107261124A is hereby incorporated by reference into the present application.
Example 1: preparation of hyaluronic acid-ES 2-AF peptide conjugate (HA-ADH-CBA-ES 2-AF, HEA)
The preparation steps are as follows:
(1) dissolving a proper amount of HA in deionized water to obtain an HA solution, sequentially adding 5 times of equivalent of ADH and EDCI, stirring for dissolving, then adjusting the pH value of the reaction solution to 4.75, reacting at room temperature for 6 hours, adjusting the pH value to 7.00 after the reaction is finished, filling the reaction solution into a dialysis bag (with the molecular weight cutoff of 3500Da), dialyzing for two days with deionized water three times a day, removing unreacted substances, filtering with a 0.22 mu m filter membrane, pre-freezing, and freeze-drying in a freeze-dryer to obtain the HA-ADH conjugate.
(2) Dissolving an appropriate amount of HA-ADH in deionized water to obtain an HA-ADH solution, additionally weighing an appropriate amount of CBA, dissolving in dimethyl sulfoxide, and respectively adding EDCI and NHS as catalysts in a ratio of 5: 1, reacting for 2 hours, finally dropwise adding the mixture into completely dissolved HA-ADH, reacting for 12 hours at room temperature, then putting the mixture into a dialysis bag (molecular weight cut-off 500), dialyzing the mixture for three times by using an ethanol/water (V: V ═ 1:4) mixed solution, and then continuously dialyzing the mixture for three times by using deionized water to remove unreacted impurities. Filtering with 0.22 μm filter membrane, pre-freezing, and lyophilizing in a lyophilizer to obtain HA-ADH-CBA conjugate.
(3) An appropriate amount of HA-ADH-CBA conjugate was dissolved in a mixture of buffer (pH 7.4) and DMSO (V: 1), 1 equivalent of anti-neoangiogenic peptide ES2-AF was added, and the mixture was reacted at room temperature for 12 hours and dialyzed three times against pH 7.4 buffer to remove unreacted impurities. Filtering with 0.22 μm filter membrane, pre-freezing, and lyophilizing in a lyophilizer to obtain HEA conjugate. Through ultrasonic treatment, a spherical structure with good dispersion and uniform granularity can be formed in a dissolving medium.
By using1H NMR identifies structures of HA, HA-ADH, HA-ADH-CBA and HEA, results are shown in figure 1, and the HEA conjugate is successfully prepared. The spherical structure, as a result, is shown in FIG. 2.
Experimental example 1: comparison of the inhibitory Effect of ES2-AF peptide, HEA conjugate and HA-ES2-AF conjugate on endothelial cell proliferation
The experimental procedure was as follows:
(1) experimental drugs: ES2-AF peptide, the HEA conjugate prepared in example 1, and the HA-ES2-AF conjugate were found to have the same concentration of ES2-AF peptide in the three groups of drugs.
(2) The experimental method comprises the following steps: the CCK-8 method is adopted to explore the influence of the preparation and the HEA conjugate on the proliferation of endothelial cells. Taking EAhy926 endothelial cells in logarithmic growth phase, digesting, centrifuging, re-suspending, diluting to 5 × 104And (4) taking 100 mu L/well, inoculating the solution into a 96-well plate, and adding ES2-AF solutions with the final concentration of 5, 50, 250, 500 and 1000 mu g/mL of ES2-AF, HEA and HA-ES2-AF after overnight adherence. Wells containing DMEM medium alone were set as a blank control, and wells containing cells without drug-containing medium were set as a negative control. Placing in an incubator, continuing incubation for 24h, discarding the culture medium, adding 100 μ L of a minimal medium containing 20% CCK-8 into each well, continuing incubation for 1-2h until the color becomes orange, taking out, setting the wavelength to 450nm by using an enzyme-labeling instrument, and measuring the absorption value (OD) of each well. The cell inhibition rate was calculated using the following formula:
the cell inhibition ratio (%) (blank control group-experimental group)/blank control group × 100%,
the results of the experiments on the inhibition of endothelial cell proliferation are shown in FIG. 3. As can be seen from the figure, ES2-AF, HEA and HA-ES2-AF all have obvious inhibition effects on the proliferation of EAhy926 cells, and the inhibition rate is increased in a dose-dependent manner with the increase of the peptide concentration. It was also found that HEA was more strongly inhibited than ES2-AF and HA-ES2-AF with increasing concentration.
Experimental example 2: comparison of the inhibitory Effect of ES2-AF peptide, HEA conjugate and HA-ES2-AF conjugate on endothelial cell migration
The experimental procedure was as follows:
(1) experimental drugs: ES2-AF peptide, the HEA conjugate prepared in example 1, and the HA-ES2-AF conjugate were found to have the same concentration of ES2-AF peptide in the three groups of drugs.
(2) The experimental method comprises the following steps: the effect of the preparation on endothelial cell migration was explored using a cell scratch experiment. 200 mu L of yellow gun head needs to be put in a refrigerator for precooling at 4 ℃ in advance, EAhy926 cells in logarithmic growth phase are taken, digested, centrifuged and resuspended, and then diluted into 2 multiplied by 105And (2) inoculating 1 mL/well into a 6-well plate, after overnight adherence, lightly drawing three parallel straight lines on the cell surface by using a cold yellow gun head, washing redundant cells by using PBS, adding ES2-AF, HEA conjugate and HA-ES2-AF conjugate with the final concentration of 5, 50, 250, 500 and 1000 mu g/mL of ES2-AF to each well, setting the wells with cells and without a drug-containing culture medium as negative control groups, continuously incubating for 24h, respectively placing the negative control groups in an inverted fluorescence microscope for observation and photographing at 0h and 24h, and performing subsequent data processing by using software Image J.
The results of the experiments on the inhibition of endothelial cell migration are shown in FIG. 4. As can be seen from the figure, the ES2-AF, HEA and HA-ES2-AF conjugates all significantly reduced cell migration, and as the concentration increased, the HEA was more able to inhibit endothelial cell migration than the ES2-AF and HA-ES2-AF conjugates.
Experimental example 3: comparison of the inhibitory Effect of ES2-AF peptide, the HEA conjugate prepared in example 1, and the HA-ES2-AF conjugate on endothelial cell lumen formation
The experimental procedure was as follows:
(1) experimental drugs: ES2-AF peptide, the HEA conjugate prepared in example 1, and the HA-ES2-AF conjugate, the ES2-AF peptide concentrations were identical in both drugs.
(2) The experimental method comprises the following steps: the effect of the formulation on the anti-angiogenesis of EAhy926 cells was explored using a lumen formation experiment. The tip and 96-well plate need to be pre-cooled overnight at 4 ℃ in advance, and Matrigel is also pre-melted at 4 ℃. Taking 50 mu L/well matrixThe gel was spread evenly into a 96-well plate and gently tapped to remove air bubbles for subsequent observation. Incubating in cell incubator for 40min, collecting EAhy926 cells in logarithmic growth phase after matrix gel is completely solidified, digesting, centrifuging, re-suspending, and diluting to 4 × 105Inoculate at 100. mu.L/well per mL. ES2-AF peptide, HEA conjugate and HA-ES2-AF conjugate with ES2-AF final concentration of 5, 250 and 1000 mug/mL are added, a well with cells and without a drug-containing culture medium is only used as a negative control group, after the cells are cultured for 6 hours, the cells are placed under an inverted fluorescence microscope to observe the generation condition of a cellular vascular network and take pictures, data statistics is carried out by using software Image J, and then mapping analysis is carried out.
The results of the inhibition lumen formation experiments are shown in fig. 5. As can be seen, the ES2-AF, HEA and HA-ES2-AF conjugates all inhibited the tubule formation of endothelial cell EAhy926 and the inhibitory ability increased with increasing concentration. HEA was more potent at inhibiting endothelial cell luminal formation than ES2-AF and HA-ES2-AF conjugates
Experimental example 4: example 1 study of the affinity of the HEA conjugates prepared for targeting endothelial cells
The experimental procedure was as follows:
(1) experimental drugs: doxorubicin (Doxorubicin, DOX), DOX-labeled HEA (DOX-HEA) conjugate prepared in example 1, with consistent concentrations of DOX in both groups.
(2) The experimental method comprises the following steps: that is, 0.1mg of HEA was dissolved in a buffer solution of pH 7.4 to a concentration of 4mg/mL, and 1.5 equivalents of DOX & HCl was added to the above solution, reacted overnight at room temperature, and then placed in a dialysis bag (molecular weight cut-off 3500Da) and dialyzed against pH 7.4 buffer solution until colorless, followed by lyophilization, to obtain DOX fluorescence-labeled conjugate DOX-HEA.
Blocking ligand-receptor mediated endocytosis behavior by a receptor saturation method, and verifying whether the hyaluronic acid-ES 2-AF peptide conjugate has hyaluronic acid receptor targeting affinity by using the fluorescence intensity of each group, wherein the specific operation steps are as follows:
namely, EAhy926 cells in logarithmic growth phase are respectively taken, digested, centrifuged, resuspended and diluted to 10 multiplied by 104Inoculating 1mL of the mixture into a confocal dish, adhering the mixture to the wall overnight, washing the mixture with PBS for three times, and adding the mixture with the final concentration of 2 mu g/mLFree DOX or DOX-labeled HEA. And (2) taking the cells incubated for 2 hours in advance by using a high-concentration HA solution as a receptor blocking group, continuously incubating the cells for 1, 2 or 4 hours, discarding the culture medium, washing the cells for three times by using PBS (phosphate buffer solution), fixing the cells for 30min at room temperature by using 1mL of 4% paraformaldehyde, continuously washing the cells for three times by using PBS, adding a proper amount of DAPI (Dairy PI) for dyeing for 10min, discarding the dye, washing the cells by using PBS, adding 1mL of PBS for infiltrating the cells, and finally, observing the uptake condition of the cells under a confocal microscope and taking pictures.
The results of the targeting affinity experiments for endothelial cells are shown in FIG. 6. As can be seen from the figure, HA in each group with the increase of the uptake amount along with the prolongation of the time is incubated in advance, the receptor is saturated, the uptake amount is obviously reduced, the green fluorescence is obviously reduced, and the targeted affinity of the HEA conjugate to the endothelial cells is proved.
Experimental example 5: evaluation of the environmental-responsive Release behavior of the HEA conjugates prepared in example 1
The experimental procedure was as follows:
(1) experimental drugs: ES2-AF peptide, HEA conjugate prepared in example 1
(2) The experimental method comprises the following steps: to verify whether the synthesized HEA is responsive in acidic environment, the cumulative amount of ES2-AF released was measured using fluorescence spectrophotometry. In vitro simulated acid-sensitive experiments were performed by placing 1mL of HEA at 500. mu.g/mL ES2-AF concentration in 10mL of buffer containing different pH values (7.4, 6.8, 5.0). Each solution was incubated at 37 ℃ in a shaker and the release medium was sampled at different time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72h) and after each 0.5mL removal, an equal amount of fresh release medium was added. The ES2-AF content was determined and plotted as a time-release curve using fluorescence spectrophotometry using a multifunctional microplate reader.
The evaluation results of the environmentally responsive release behavior of the conjugate are shown in FIG. 7. As can be seen from the figure, almost no ES2-AF is released under the neutral environment with the pH value of 7.4, and the cumulative release rate is increased along with the increase of acidity, which indicates that the HEA conjugate has better acid-sensitive responsiveness, can be rapidly released in the tumor microenvironment and plays a better role.
Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university
<120> hyaluronic acid-ES 2-AF peptide conjugate and preparation method and application thereof
<130> 202124814
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> PRT
<213> Artificial
<400> 1
Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Gly Gly Gly Gly Gly
1 5 10 15
Gly Asn Gln Trp Phe Ile
20
Claims (10)
1. A hyaluronic acid-ES 2-AF peptide conjugate, comprising an ES2-AF peptide and hyaluronic acid, and a connecting bridge formed by 4-carboxybenzaldehyde and adipic acid dihydrazide, wherein the connecting bridge connects the ES2-AF peptide and hyaluronic acid.
2. The conjugate of hyaluronic acid-ES 2-AF peptide according to claim 1, wherein in the connecting bridge 4-carboxybenzaldehyde is amide-bonded to adipic acid dihydrazide.
3. The hyaluronic acid-ES 2-AF peptide conjugate according to claim 1 or 2, wherein the linker bridge is linked to hyaluronic acid via an amide bond, and the linker bridge is linked to the ES2-AF peptide via an imine or azomethine bond.
4. The hyaluronic acid-ES 2-AF peptide conjugate of any of claims 1 to 3, wherein the sequence of the ES2-AF peptide is shown in SEQ ID No. 1.
5. A method for preparing the hyaluronic acid-ES 2-AF peptide conjugate as claimed in any of claims 1 to 4, wherein adipic acid dihydrazide is amidated with HA to obtain HA-ADH conjugate, 4-carboxybenzaldehyde is amidated with the free amino group of ADH in HA-ADH conjugate to obtain HA-ADH-CBA conjugate, ES2-AF peptide is added to perform Schiff base reaction with the CBA end in HA-ADH-CBA conjugate to obtain the hyaluronic acid-ES 2-AF peptide conjugate.
6. The method of claim 5, wherein each reaction step is followed by a purification step.
7. The method of claim 6, wherein the purifying comprises performing dialysis and lyophilization.
8. A pharmaceutical composition comprising the hyaluronic acid-ES 2-AF peptide conjugate of any of claims 1-4.
9. A pharmaceutical formulation comprising the hyaluronic acid-ES 2-AF peptide conjugate of any of claims 1-4 and at least one pharmaceutically acceptable adjuvant or pharmaceutical carrier.
10. Use of the hyaluronic acid-ES 2-AF peptide conjugate of any of claims 1-4 or the pharmaceutical composition of claim 8 or the pharmaceutical formulation of claim 9 for the manufacture of a medicament and/or an anti-tumor medicament for the treatment of a disease associated with angiogenesis;
preferably, the diseases associated with angiogenesis include diabetic retinopathy, age-related macular degeneration and arthritis;
preferably, the medicament for treating the disease related to the angiogenesis is an anti-angiogenesis medicament, an endothelial cell migration inhibition medicament and/or an endothelial cell lumen formation inhibition medicament;
preferably, the tumor comprises melanoma, breast tumor, lung tumor, colon tumor, ovarian tumor and kidney tumor.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114209816A (en) * | 2021-12-22 | 2022-03-22 | 山东大学 | Glycerol glucoside-superoxide dismutase conjugate as well as preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1950395A (en) * | 2004-03-02 | 2007-04-18 | 美国陶氏益农公司 | Insecticidal toxin complex fusion proteins |
| KR20120005280A (en) * | 2010-07-08 | 2012-01-16 | 포항공과대학교 산학협력단 | Hyaluronic acid derivative, hyaluronic acid-peptide conjugate, and preparing the same |
| KR20120089506A (en) * | 2010-12-10 | 2012-08-13 | 포항공과대학교 산학협력단 | Hyaluronic acid-protein conjugate, and preparation method thereof |
| CN105153428A (en) * | 2015-07-02 | 2015-12-16 | 天津大学 | PH responsive polymeric micelle for mucus infiltration and preparation method thereof |
| CN107261124A (en) * | 2017-06-09 | 2017-10-20 | 山东大学 | A kind of preparation method and the application of angiogenesis inhibitor peptide and its hyaluronic acid decorated thing |
| CN111356764A (en) * | 2017-09-25 | 2020-06-30 | 卡斯西部储备大学 | Compositions and methods for lowering serum cholesterol and PCSK9 |
-
2021
- 2021-08-20 CN CN202110961783.XA patent/CN113648427B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1950395A (en) * | 2004-03-02 | 2007-04-18 | 美国陶氏益农公司 | Insecticidal toxin complex fusion proteins |
| KR20120005280A (en) * | 2010-07-08 | 2012-01-16 | 포항공과대학교 산학협력단 | Hyaluronic acid derivative, hyaluronic acid-peptide conjugate, and preparing the same |
| KR20120089506A (en) * | 2010-12-10 | 2012-08-13 | 포항공과대학교 산학협력단 | Hyaluronic acid-protein conjugate, and preparation method thereof |
| CN105153428A (en) * | 2015-07-02 | 2015-12-16 | 天津大学 | PH responsive polymeric micelle for mucus infiltration and preparation method thereof |
| CN107261124A (en) * | 2017-06-09 | 2017-10-20 | 山东大学 | A kind of preparation method and the application of angiogenesis inhibitor peptide and its hyaluronic acid decorated thing |
| CN111356764A (en) * | 2017-09-25 | 2020-06-30 | 卡斯西部储备大学 | Compositions and methods for lowering serum cholesterol and PCSK9 |
Non-Patent Citations (2)
| Title |
|---|
| 李祥子;胡平静;朱振铎;朱国兴;沈小平;汪茗;孙玉;冯德香;: "pH响应型纳米药物载体的释药机制及性能研究进展" * |
| 李祥子;胡平静;朱振铎;朱国兴;沈小平;汪茗;孙玉;冯德香;: "pH响应型纳米药物载体的释药机制及性能研究进展", 无机化学学报, no. 08 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114209816A (en) * | 2021-12-22 | 2022-03-22 | 山东大学 | Glycerol glucoside-superoxide dismutase conjugate as well as preparation method and application thereof |
| CN114209816B (en) * | 2021-12-22 | 2023-11-24 | 山东大学 | Glycerol glucoside-superoxide dismutase conjugate and preparation method and application thereof |
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