CN113350296A - PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan and preparation method and application thereof - Google Patents

PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan and preparation method and application thereof Download PDF

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CN113350296A
CN113350296A CN202110652104.0A CN202110652104A CN113350296A CN 113350296 A CN113350296 A CN 113350296A CN 202110652104 A CN202110652104 A CN 202110652104A CN 113350296 A CN113350296 A CN 113350296A
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chitosan
sequence peptide
pad4 inhibitor
crgd
crgdv
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CN113350296B (en
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王玉记
王彦明
贾翌江
阿依江
朱迪
卢玉
冯琦琦
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Capital Medical University
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Abstract

The invention provides a PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan, a preparation method and application thereof, belonging to the technical field of antitumor drugs. The CRGD sequence peptide modified chitosan is used as a drug carrier, wherein the chitosan has good biocompatibility and biodegradability, the RGD sequence peptide in the CRGD sequence peptide can be specifically combined on a corresponding receptor over-expressed by a target cell, and the targeting effect achieved by targeting drug delivery to a target region can be achieved by utilizing the specific combination of the receptor on the surface of a tumor cell and a ligand of a targeting preparation. According to the invention, the PAD4 inhibitor is loaded by using CRGD sequence peptide modified chitosan, so that the selectivity of the PAD4 inhibitor on target tumor cells can be enhanced, adverse drug reactions are reduced, and the PAD4 inhibitor has good antitumor activity.

Description

PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan and preparation method and application thereof
Technical Field
The invention relates to the technical field of antitumor drugs, and particularly relates to a PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan, and a preparation method and application thereof.
Background
Cancer is a leading disease of death in most countries, cancer cells grow uncontrollably, nutrients drawn from host cells can grow and multiply indefinitely, and the variety of cancer cells is very diverse, thus being very challenging to treat cancer.
The polypeptide arginine deiminase 4 (PAD 4) is one of the important targets of cancer. PAD4 is used as a adjuvant inhibitor of p53 and can inhibit the expression of cancer suppressor genes (such as p21/CDKN1A and GADD45) together with histone deacetylase HDAC 2. PAD4 has lower bioavailability.
To improve the bioavailability of a drug, it is common practice to deliver the drug using a drug carrier. To date, there are many more mature drug carriers, including water-soluble high molecular weight polymer carriers, polymer nanoparticles, polymer micelles, dendrimers, liposomes, viral particles, carbon nanotubes, and graphene oxide, but the active targeting of these carriers is often not high.
Disclosure of Invention
In view of the above, the invention aims to provide a PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan, and a preparation method and application thereof.
In order to achieve the purpose of the invention, the invention provides the following technical scheme:
the invention provides a PAD4 inhibitor loaded on CRGD sequence peptide modified chitosan, which comprises a CRGD sequence peptide modified chitosan carrier and a PAD4 inhibitor loaded on the surface of the CRGD sequence peptide modified chitosan carrier;
the cysteine in the CRGD sequence peptide is linked to the chitosan surface via acryloyl chloride.
Preferably, the CRGD sequence peptide includes one or more of CRGDV, CRGDs and CRGDF.
Preferably, the PAD4 inhibitor comprises one or more of PAD4 inhibitor, 4B, PAD4 inhibitor, 3B, PAD4 inhibitor, 3B-OH.
Preferably, the PAD4 inhibitor in the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan has a loading amount of 12-18 wt%.
The invention provides a preparation method of the PAD4 inhibitor loaded on the CRGD sequence peptide modified chitosan, which comprises the following steps:
(1) mixing chitosan, acryloyl chloride, a first acid-binding agent and a first organic solvent, and carrying out nucleophilic substitution reaction to obtain acryloyl chloride modified chitosan;
(2) mixing the chitosan modified by acryloyl chloride, the CRGD sequence peptide, a second acid-binding agent and a second organic solvent, and carrying out Michael addition reaction to obtain a CRGD sequence peptide modified chitosan carrier;
(3) and stirring and mixing the CRGD sequence peptide modified chitosan carrier, the PAD4 inhibitor and a third organic solvent to obtain the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan.
Preferably, the molar ratio of the chitosan to the acryloyl chloride is 1.5-3: 1, and the molar ratio of the chitosan to the CRGD sequence peptide is 1-2: 1.8.
Preferably, the molar ratio of the chitosan to the PAD4 inhibitor is 1: 1-5.
Preferably, the time of the nucleophilic substitution reaction is 12-24 h.
Preferably, the time of the Michael addition reaction is 12-24 h.
The invention provides application of the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan in preparation of antitumor drugs.
The invention provides a PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan, which takes the CRGD sequence peptide modified chitosan as a drug carrier, wherein the chitosan has good biocompatibility and biodegradability, the RGD sequence peptide in the CRGD sequence peptide can be specifically combined on a corresponding receptor overexpressed by a target cell, and the targeting effect achieved by targeting drug delivery to a target region can be achieved by utilizing the specific combination of the receptor on the surface of a tumor cell and a ligand of a targeting preparation. According to the invention, the PAD4 inhibitor is loaded by using CRGD sequence peptide modified chitosan, so that the selectivity of the PAD4 inhibitor on target tumor cells can be enhanced, adverse drug reactions are reduced, and the PAD4 inhibitor has good antitumor activity. The results of the examples show that the CRGD sequence peptide modified chitosan loaded PAD4 inhibitor provided by the invention has good anti-tumor effect through activity evaluation.
The invention provides a preparation method of the PAD4 inhibitor loaded by CRGD sequence peptide modified chitosan, which is simple to operate and easy to realize industrial batch production.
Drawings
FIG. 1 is a schematic structural diagram of CRGD sequence peptide modified chitosan loaded PAD4 inhibitor;
FIG. 2 is an infrared spectrum of chitosan;
FIG. 3 is an infrared spectrum of chitosan-CRGDV;
FIG. 4 is an infrared spectrum of chitosan-CRGDF;
FIG. 5 is an infrared spectrum of chitosan-CRGDS;
FIG. 6 is an infrared spectrum of FIG. 4B;
FIG. 7 is an infrared spectrum of CRGDV-chitosan-4B;
FIG. 8 is an infrared spectrum of FIG. 8B;
FIG. 9 is an infrared spectrum of CRGDV-chitosan-3B;
FIG. 10 is an infrared spectrum of 3B-OH;
FIG. 11 is an infrared spectrum of CRGDV-chitosan-3B-OH;
FIG. 12 is a drawing of chitosan1H NMR chart;
FIG. 13 is a drawing of chitosan-acryloyl chloride1H NMR chart;
FIG. 14 is a drawing of chitosan-CRGDV1A HNMR map;
FIG. 15 is of chitosan-CRGDF1H NMR chart;
FIG. 16 is a drawing of chitosan-CRGDS1H NMR chart;
FIG. 17 is a drawing of CRGDV-chitosan-4B1H NMR chart;
FIG. 18 is a drawing showing CRGDV-chitosan-3B1H NMR chart;
FIG. 19 is of CRGDV-chitosan-3B-OH1H NMR chart;
FIG. 20 is a transmission electron micrograph of chitosan;
FIG. 21 is a transmission electron micrograph of chitosan-CRGDV;
FIG. 22 is a transmission electron micrograph of chitosan-CRGDF;
FIG. 23 is a transmission electron micrograph of chitosan-CRGDS;
FIG. 24 shows chitosan-CRGDV: transmission electron micrograph of 1: 1;
FIG. 25 shows chitosan-CRGDV: transmission electron micrographs of 4B 1: 2;
FIG. 26 is a drawing of chitosan-CRGDV: transmission electron micrographs of 4B 1: 3;
FIG. 27 shows chitosan-CRGDV: transmission electron micrographs of 4B 1: 4;
FIG. 28 is chitosan-CRGDV: transmission electron micrographs of 4B 1: 5;
fig. 29 is chitosan: transmission electron micrographs of 4B 1: 3;
FIG. 30 shows chitosan-CRGDV: transmission electron micrograph of 1: 3;
FIG. 31 is chitosan-CRGDV: transmission electron micrograph of 3B-OH ═ 1: 3;
FIG. 32 is a standard graph of 4B (4-carboxyphenylboronic acid-Orn (Cl) -NBzl).
Detailed Description
The invention provides a PAD4 inhibitor loaded on CRGD sequence peptide modified chitosan, which comprises a CRGD sequence peptide modified chitosan carrier and a PAD4 inhibitor loaded on the surface of the CRGD sequence peptide modified chitosan carrier;
the cysteine in the CRGD sequence peptide is linked to the chitosan surface via acryloyl chloride.
In the present invention, the CRGD sequence peptide preferably includes one or more of CRGDV, CRGDs and CRGDF. In the present invention, the CRGDV is cysteine-arginine-glycine-aspartic acid-valine, the CRGDS is cysteine-arginine-glycine-aspartic acid-phenylalanine, and the CRGDF is cysteine-arginine-glycine-aspartic acid-serine.
In the present invention, the PAD4 inhibitor preferably comprises one or more of PAD4 inhibitor 4B, PAD4 inhibitor 3B, PAD4 inhibitor 3B-OH. In the present invention, the PAD4 inhibitor 4B is(s) - (4- ((1- (benzylamino) -5- (2-chloroacetamide) -1-oxopentan-2-yl) carbonyl) phenyl) boronic acid, and the PAD4 inhibitor 4B has the formula shown in formula 1:
Figure BDA0003111999830000031
in the present invention, the PAD4 inhibitor 3B is(s) - (3- ((1- (benzylamino) -5- (2-chloroacetamide) -1-oxopentan-2-yl) carbonyl) phenyl) boronic acid, and the PAD4 inhibitor 3B has the formula 2:
Figure BDA0003111999830000041
in the invention, the PAD4 inhibitor 3B-OH is(s) -4- ((2- (3-boron amide) -5- (2-chloroacetamide) pentanal) methyl) benzoic acid, and the PAD4 inhibitor 3B-OH is put into a structural formula shown in formula 3:
Figure BDA0003111999830000042
in the invention, the PAD4 inhibitor in the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan preferably has a loading amount of 12-18 wt%, and preferably 14.2 wt%.
The invention provides a preparation method of the PAD4 inhibitor loaded on the CRGD sequence peptide modified chitosan, which comprises the following steps:
(1) mixing chitosan, acryloyl chloride, a first acid-binding agent and a first organic solvent, and carrying out nucleophilic substitution reaction to obtain acryloyl chloride modified chitosan;
(2) mixing the chitosan modified by acryloyl chloride, the CRGD sequence peptide, a second acid-binding agent and a second organic solvent, and carrying out Michael addition reaction to obtain a CRGD sequence peptide modified chitosan carrier;
(3) and stirring and mixing the CRGD sequence peptide modified chitosan carrier, the PAD4 inhibitor and a third organic solvent to obtain the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan.
The method comprises the steps of mixing chitosan, acryloyl chloride, a first acid binding agent and a first organic solvent, and carrying out nucleophilic substitution reaction to obtain the acryloyl chloride modified chitosan. In the present invention, the first organic solvent is preferably N, N-dimethylformamide. In the present invention, the first acid scavenger is preferably triethylamine.
The invention has no special requirements on the type of the chitosan, and the chitosan which is generally sold in the market in the field can be used. In the invention, the molar ratio of the chitosan to the acryloyl chloride is preferably 1.5-3: 1, more preferably 2:1, and the molar ratio of the chitosan to the first acid-binding agent is preferably 1-2: 1.8. In the present invention, the acryloyl chloride is preferably added as a solution of acryloyl chloride in methylene chloride.
In the present invention, the mixing is preferably performed in the following manner: mixing chitosan, a first acid-binding agent and a first organic solvent, cooling to 0 ℃, and then dropwise adding a dichloromethane solution of acryloyl chloride.
In the invention, the nucleophilic substitution reaction is preferably carried out under the condition of stirring, the temperature of the nucleophilic substitution reaction is preferably room temperature, and the time is preferably 12-24 h, and more preferably 16-20 h. In the nucleophilic reaction process, the acryloyl chloride and amino or hydroxyl on the chitosan are subjected to nucleophilic substitution.
In the present invention, after the nucleophilic substitution reaction, the present invention preferably performs a post-treatment of the resulting nucleophilic substitution reaction liquid, the post-treatment preferably comprising the steps of:
and (3) sequentially dialyzing and freeze-drying the obtained nucleophilic substitution reaction liquid to obtain the acryloyl chloride modified chitosan solid. The dialysis is preferably carried out by using distilled water, and the cut-off molecular weight of the dialysis bag is 700-1000 Da during dialysis. In the present invention, the dialysis time is preferably 48 hours. The present invention does not require any particular means for lyophilization, and those well known to those skilled in the art can be used.
After the chitosan modified by the acryloyl chloride is obtained, the chitosan modified by the acryloyl chloride, the CRGD sequence peptide, a second acid-binding agent and a second organic solvent are mixed for Michael addition reaction to obtain the chitosan carrier modified by the CRGD sequence peptide.
In the present invention, the CRGD sequence peptide preferably includes one or more of CRGDV, CRGDs and CRGDF. In the present invention, the preparation method of CRGDV preferably comprises the following steps:
(1) soaking Fmoc-Val-Wang resin by using an organic solvent to obtain swelling Fmoc-Val-Wang resin;
(2) carrying out deprotection reaction on the swollen Fmoc-Val-Wang resin to obtain deprotected Fmoc-Val-Wang resin;
(3) mixing the deprotected Fmoc-Val-Wang resin, Fmoc-Asp (OtBu), a condensing agent and a coupling agent to perform solid-phase synthesis;
(4) repeating the steps (2) to (3) until the last amino acid is deprotected;
(5) and (4) mixing the product obtained in the step (4) with methanol, removing the methanol, and adding a cracking solution to perform cracking reaction to obtain the CRGDV.
The method uses an organic solvent to soak Fmoc-Val-Wang resin to obtain the swelling Fmoc-Val-Wang resin. In the present invention, the organic solvent is preferably anhydrous DMF; the swelling time is preferably 3-6 h.
The swelling Fmoc-Val-Wang resin is subjected to deprotection reaction to obtain the deprotected Fmoc-Val-Wang resin. In the present invention, it is preferable that the swollen Fmoc-Val-Wang resin is subjected to suction filtration under reduced pressure to remove the organic solvent before the deprotection reaction. In the invention, the deprotection reagent is preferably anhydrous DMF and piperidine, and the volume ratio of the anhydrous DMF to the piperidine is preferably 4: 1. In the invention, the deprotection reaction is preferably carried out in two times, firstly, a deprotection reagent is added once, after oscillation reaction is carried out for 3min, the deprotection reagent is removed by vacuum filtration, then, a deprotection reagent is added once, oscillation reaction is carried out for 8min, and the deprotection reagent is removed by vacuum filtration. After removing the deprotection reagent, the invention preferably washes the deprotected Fmoc-Val-Wang resin, and the washing detergent is preferably anhydrous DMF and CH in sequence2Cl2And anhydrous DMF, the number of washes per detergent is preferably 2.
After the washing, the present invention preferably uses the indetrione method for detecting-NH2It is dark purple.
The invention mixes the deprotected Fmoc-Val-Wang resin, Fmoc-Asp (OtBu), a condensing agent and a coupling agent to carry out solid phase synthesis. In the present invention, the condensing agent is preferably HBTU, the coupling agent is preferably anhydrous DMF and N-methylmorpholine, and the volume ratio of the anhydrous DMF to the N-methylmorpholine is preferably 95: 5. In the present invention, the time for the solid phase synthesis is preferably 45 min.
After the solid phase synthesis, the solid phase synthesis product is preferably washed, and the washing detergent is preferably anhydrous DMF and CH in sequence2Cl2And anhydrous DMF, the number of washes per detergent is preferably 2.
After the washing, the resin is preferably detected by an indetrione method, and is colorless and transparent.
The invention repeats steps (2) - (3) until the last amino acid is deprotected.
The product obtained in the step (4) is mixed with methanol, and after the methanol is removed, cracking liquid is added for cracking reaction to obtain CRGDV. In the present invention, the lysis solution is preferably trifluoroacetic acid: water: triisopropylsilane: 1, 2-ethanedithiol 92.5:2.5:2.5: 2.5. In the present invention, the cleavage reaction is preferably performed under ice bath conditions, and the time of the cleavage reaction is preferably 2.5.
In the present invention, after the cleavage reaction, it is preferable that the reaction solution obtained in the present invention is filtered, and trifluoroacetic acid in the filtrate is blown dry with nitrogen. And adding glacial ethyl ether into the filtrate for washing, washing for three times, centrifuging and removing supernatant to obtain CRGDV.
In the present invention, the CRGDF is prepared in a similar manner to the CRGDV except that Fmoc-Val-Wang resin of step (1) is replaced with Fmoc-Val-Wang resin.
In the present invention, the CRGDS is prepared in a similar manner to the CRGDV except that Fmoc-Val-Wang resin of step (1) is replaced with Fmoc-Ser-Wang resin.
The preparation method comprises the steps of mixing the chitosan modified by acryloyl chloride, CRGD sequence peptide, triethylamine and a second organic solvent, and carrying out Michael addition reaction to obtain the CRGD sequence peptide modified chitosan carrier. In the present invention, the second organic solvent is preferably dimethyl sulfoxide and/or dioxane, and when the second organic solvent is dimethyl sulfoxide and dioxane, the volume ratio of dimethyl sulfoxide and dioxane is preferably 1:1. In the present invention, the second acid scavenger is preferably triethylamine.
In the present invention, the molar ratio of the chitosan to the CRGD-sequence peptide is preferably 1:1.8, and the molar ratio of the chitosan to the second acid scavenger is preferably 1: 0.7. In the invention, the Michael addition reaction is preferably carried out under the condition of stirring, the temperature of the Michael addition reaction is preferably room temperature, and the time is preferably 12-24 h, and more preferably 16-20 h. In the invention, the double bond of acryloyl chloride connected to chitosan in nucleophilic substitution reaction and cysteine side chain sulfhydryl in CRGD peptide chain produce Michael addition reaction.
In the present invention, after the michael addition reaction, the present invention preferably subjects the obtained michael addition reaction liquid to a post-treatment, which preferably comprises the steps of:
and (3) dialyzing and freeze-drying the obtained Michael addition reaction liquid in sequence to obtain the CRGD sequence peptide modified chitosan carrier solid. The dialysis is preferably carried out by using distilled water, and the cut-off molecular weight of the dialysis bag is 700-1000 Da during dialysis. In the present invention, the dialysis time is preferably 48 hours. The present invention does not require any particular means for lyophilization, and those well known to those skilled in the art can be used.
After the CRGD sequence peptide modified chitosan carrier is obtained, the CRGD sequence peptide modified chitosan carrier, the PAD4 inhibitor and a third organic solvent are stirred and mixed to obtain the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan. In the present invention, the PAD4 inhibitor preferably comprises one or more of PAD4 inhibitor 4B, PAD4 inhibitor 3B, PAD4 inhibitor 3B-OH. The source of the PAD4 inhibitor, 4B, PAD4 inhibitor, 3B, PAD4 inhibitor, 3B-OH is not particularly required in the present invention, and any of the above materials conventionally commercially available in the art or prepared by itself may be used.
In the present invention, the third organic solvent is preferably dimethyl sulfoxide and/or dioxane, and when the second organic solvent is dimethyl sulfoxide and dioxane, the volume ratio of dimethyl sulfoxide and dioxane is preferably 1:1.
In the invention, the molar ratio of the chitosan to the PAD4 inhibitor is preferably 1: 1-5, more preferably 1: 2-4, and even more preferably 1: 3.
In the present invention, the temperature of the stirring and mixing is preferably room temperature, and the time is preferably 8 hours.
In the present invention, after the stirring and mixing, the present invention preferably performs a post-treatment of the obtained michael addition reaction liquid, and the post-treatment preferably comprises the steps of:
and (3) dialyzing and freeze-drying the obtained Michael addition reaction liquid in sequence to obtain the CRGD sequence peptide modified chitosan carrier solid. The dialysis is preferably carried out by using distilled water, and the cut-off molecular weight of the dialysis bag is 700-1000 Da during dialysis. In the present invention, the dialysis time is preferably 24 hours. The present invention does not require any particular means for lyophilization, and those well known to those skilled in the art can be used.
In the present invention, the structural scheme of the CRGD sequence peptide-modified chitosan-loaded PAD4 inhibitor is shown in fig. 1.
The invention provides application of the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan in preparation of antitumor drugs.
The PAD4 inhibitor loaded on CRGD sequence peptide modified chitosan provided by the present invention and the preparation method and application thereof are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Synthesis of (mono) CRGD sequence peptides
(1) Solid phase synthesis of CRGDV
1) Swelling: 300mg of Fmoc-Val-Wang resin was soaked in 10mL of anhydrous DMF for 3h in a solid phase synthesis tube to swell.
2) Deprotection: the anhydrous DMF was removed by suction filtration under reduced pressure, and 8 to 10mL of a deprotecting agent (anhydrous DMF: hexahydropyridine ═ 4:1) was added thereto, followed by shaking reaction for 3min, then the deprotecting agent was removed by suction filtration under reduced pressure, and 8 to 10mL of a deprotecting agent was added thereto, followed by shaking reaction for 8 min.
3) Washing: vacuum filtering to remove deprotection agent, sequentially adding anhydrous DMF and CH2Cl2And anhydrous DMF washes, twice for each solution.
4) Color development: indantrione method for detecting resin (detection-NH)2) It is dark purple.
5) Coupling: Fmoc-Asp (OtBu) and HBTU (condensation agent) were weighed, dissolved with a coupling agent (anhydrous DMF: N-methylmorpholine ═ 95:5), added to a solid phase synthesis tube, and reacted for 45min with shaking.
6) Washing: filtering under reduced pressure to remove coupling agent, sequentially adding anhydrous DMF and CH2Cl2And anhydrous DMF washes, twice for each solution.
7) Color development: the resin is detected by an indetrione method and is colorless and transparent.
Repeating the steps of 2-7 until the last amino acid is synthesized, and then carrying out deprotection.
8) Methanol shrinkage: methanol was added to the solid phase synthesis tube, the methanol was removed by suction filtration under reduced pressure, and this was repeated three times, and the resin was transferred to a 10mL reagent bottle.
9) Cracking: 3mL of a lysate (trifluoroacetic acid: water: triisopropylsilane: 1, 2-ethanedithiol: 92.5:2.5:2.5:2.5) was put into a reagent bottle, and the reaction was stirred in an ice-water bath for 2.5 hours. After the reaction is finished, filtering is carried out, and trifluoroacetic acid in the filtrate is dried by nitrogen. And adding glacial ethyl ether into the filtrate for washing, washing for three times, centrifuging and removing supernatant to obtain CRGDV.
(2) Solid phase synthesis of CRGDF
The same preparation method as CRGDV is adopted, and 300mg of Fmoc-Phe-Wang resin is used as a raw material to obtain CRGDF.
(3) Solid phase synthesis of CRGDS
The same preparation method as CRGDV is adopted, and 300mg Fmoc-Ser-Wang resin is used as a raw material to obtain CRGDS.
Synthesis of (di) PAD4 inhibitors
(1) Synthesis of 4B (4-carboxyphenylboronic acid-Orn (Cl) -NBzl)
1) Preparation of Boc-Orn (Cbz) -NBzl:
Boc-Orn (Cbz) -OH 10mmol is dissolved in 20mL anhydrous Tetrahydrofuran (THF), 12mmol of N-hydroxybenzotriazole (HOBt) is added under ice bath condition to be completely dissolved, 12mmol of Dicyclohexylcarbodiimide (DCC) is slowly added, and stirring is carried out for 30min, thus obtaining reaction liquid A. Under the ice bath condition, 12mmol of benzylamine is dissolved in 20mL of anhydrous THF and added into the reaction solution A, 1 mLN-methylmorpholine (NMM) is added, and the pH is adjusted to 8-9. The mixture was stirred in ice bath for 1h and further stirred at room temperature for 48h, and TLC (chloroform: methanol ═ 20:1) showed disappearance of Boc-Orn (Cbz) -OH. Dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 50ml of Ethyl Acetate (EA), and the resulting solution was successively saturated with NaHCO3Aqueous solution, saturated NaCl aqueous solution, 5% KHSO4Aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3Washing with water solution and saturated NaCl water solution for three times, and washing ethyl acetate layer with anhydrous Na2SO4Drying, filtering under reduced pressure to remove the drying agent, and concentrating the filtrate under reduced pressure to dryness to obtain the compound Boc-Orn (Cbz) -NBzl.
2) Preparation of HCl. H-Orn (Cbz) -NBzl:
Boc-Orn (Cbz) -NBzl (10mmol) was dissolved in a small amount of dry ethyl acetate and 4N HCl/EtOAc solution, TLC (EA: H) was added with stirring in an ice bath2O: HAc 4:1:2d) showed disappearance of the starting material spot, the solution was drained with a water pump, anhydrous ether was added, the reaction solution was again drained with a water pump, and the process was repeated three times to obtain HCl · H-orn (cbz) -NBzl.
3) Preparation of 4-carboxyphenylboronic acid-Orn (Cbz) -NBzl:
dissolving 10mmol of 4-carboxyphenylboronic acid in 20mL of anhydrous Tetrahydrofuran (THF), adding 12mmol of N-hydroxybenzotriazole (HOBt) under an ice bath condition, completely dissolving, slowly adding 12mmol of Dicyclohexylcarbodiimide (DCC), and stirring for 30min to obtain a reaction solution A. Under the ice-bath condition, 12mmol of HCl & H-Orn (Cbz) -NBzl is dissolved in 20mL of anhydrous THF and added into the reaction solution A, 1 mLN-methylmorpholine (NMM) is added, and the pH is adjusted to 8-9. Stirring was carried out in an ice bath for 1h and at room temperature for 48h, and TLC (chloroform: methanol ═ 20:1) showed disappearance of 4-carboxyphenylboronic acid. Dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 50ml of Ethyl Acetate (EA), and the resulting solution was successively treated with saturated aqueous NaHCO3 solution, saturated aqueous NaCl solution, and 5% KHSO4Aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3Washing the aqueous solution and saturated NaCl aqueous solution for three times, drying the ethyl acetate layer by using anhydrous Na2SO4, performing suction filtration under reduced pressure to remove the drying agent, and concentrating the filtrate under reduced pressure to be dry to obtain the compound 4-carboxyphenylboronic acid-Orn (Cbz) -NBzl.
4) Preparation of 4-carboxyphenylboronic acid-Orn-NBzl:
stirring and dissolving 4-carboxyphenylboronic acid-Orn (Cbz) -NBzl 10mmol by using a proper amount of methanol, adding a proper amount of Pd/C, keeping the reaction system sealed, pumping out air by using a tee joint, introducing hydrogen filled in an air bag, pumping out air by using the tee joint, repeatedly replacing the air in the reaction system in the way, finally keeping the tee joint in a hydrogen introducing state, keeping a hydrogen environment at room temperature, stirring until a raw material point disappears, and monitoring by TLC. After the reaction is finished, the Pd/C is removed by reduced pressure filtration, and the filtrate is concentrated to be dry by reduced pressure to obtain the compound 4-carboxyl phenylboronic acid-Orn-NBzl.
5) Preparation of 4B (4-carboxyphenylboronic acid-Orn (Cl) -NBzl):
reacting 4-carboxy1mmol of phenylboronic acid-Orn-NBzl is dissolved with a suitable amount of absolute methanol under stirring, 5mmol of ethyl 2-chloroacetimidoate hydrochloride are added in an ice-water bath, and the pH is adjusted to 10 with N, N-Diisopropylethylamine (DIPEA). Stirring at room temperature for 12H, TLC (EA: H)2O: HAc ═ 4:1:2d) showed disappearance of 4-carboxyphenylboronic acid-Orn-NBzl, which was concentrated to dryness under reduced pressure and purified by C18 column chromatography to give 4B (4-carboxyphenylboronic acid-Orn (cl) -NBzl).
(2) Synthesis of 3B (3-carboxyphenylboronic acid-Orn (Cl) -NBzl)
3B (3-Carboxyphenylboronic acid-Orn (Cl) -NBzl) was obtained from 3-carboxyphenylboronic acid by the same method as that for 4B (4-Carboxyphenylboronic acid-Orn (Cl) -NBzl).
(3) Synthesis of 3B-OH (3-carboxyphenylboronic acid-Orn (Cl) -NBzl)
1) Preparation of 3-carboxyphenylboronic acid-Orn (Boc) -OBzl:
dissolving 10mmol of 3-carboxyphenylboronic acid in 20mL of anhydrous Tetrahydrofuran (THF), adding 12mmol of N-hydroxybenzotriazole (HOBt) under an ice bath condition, completely dissolving, slowly adding 12mmol of Dicyclohexylcarbodiimide (DCC), and stirring for 30min to obtain a reaction solution A. Under the ice-bath condition, 12mmol of HCl & H-Orn (Boc) -OBzl is dissolved in 20mL of anhydrous THF and added into the reaction solution A, and 1 mLN-methylmorpholine (NMM) is added to adjust the pH value to 8-9. Stirring was carried out in an ice bath for 1h and at room temperature for 48h, and TLC (chloroform: methanol ═ 20:1) showed disappearance of 3-carboxyphenylboronic acid. Dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 50ml of Ethyl Acetate (EA), and the resulting solution was successively treated with saturated aqueous NaHCO3 solution, saturated aqueous NaCl solution, and 5% KHSO4Aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3Washing the aqueous solution and saturated NaCl aqueous solution for three times, drying the ethyl acetate layer by using anhydrous Na2SO4, performing suction filtration under reduced pressure to remove a drying agent, and concentrating the filtrate under reduced pressure to be dry to obtain the compound 3-carboxyl phenylboronic acid-Orn (Boc) -OBzl.
2) Preparation of 3-carboxyphenylboronic acid-Orn (Boc) -OH:
stirring and dissolving 10mmol of 3-carboxyphenylboronic acid-Orn (Boc) -OBzl with a proper amount of methanol, adding a proper amount of Pd/C, keeping the reaction system sealed, pumping out air by using a tee joint, introducing hydrogen filled in a gas bag, pumping out air by using the tee joint, repeatedly replacing the air in the reaction system in such a way, finally staying the tee joint in a hydrogen introducing state, keeping a hydrogen environment at room temperature, stirring until a raw material point disappears, and monitoring by TLC. After the reaction is finished, the Pd/C is removed by filtering under reduced pressure, and the filtrate is concentrated to be dry under reduced pressure to obtain the compound 3-carboxyl phenylboronic acid-Orn (Boc) -OH.
3) Preparation of methyl 3-carboxyphenylboronic acid-Orn (Boc) -4-aminobenzoate:
dissolving 3-carboxyphenylboronic acid-Orn (Boc) -OH 10mmol in 20mL of anhydrous Tetrahydrofuran (THF), adding N-hydroxybenzotriazole (HOBt)12mmol under ice bath condition, completely dissolving, slowly adding Dicyclohexylcarbodiimide (DCC)12mmol, and stirring for 30min to obtain reaction liquid A. Dissolving 12mmol of 4-methyl aminobenzoate in 20mL of anhydrous THF under an ice bath condition, adding the mixture into the reaction solution A, adding 1 mLN-methylmorpholine (NMM), and adjusting the pH value to 8-9. The mixture was stirred in ice bath for 1h and at room temperature for 48h, and TLC (chloroform: methanol ═ 20:1) showed disappearance of 3-carboxyphenylboronic acid-orn (boc) -OH. Dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 50ml of Ethyl Acetate (EA), and the resulting solution was successively saturated with NaHCO3Aqueous solution, saturated NaCl aqueous solution, 5% KHSO4Aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3Washing with water solution and saturated NaCl water solution for three times, and washing ethyl acetate layer with anhydrous Na2SO4Drying, filtering under reduced pressure to remove the drying agent, and concentrating the filtrate under reduced pressure to dryness to obtain the compound 3-carboxyphenylboronic acid-Orn (Boc) -4-methyl aminobenzoate.
4) Preparation of 3-carboxyphenylboronic acid-Orn (Boc) -4-aminobenzoic acid:
3-carboxyphenylboronic acid-Orn (Boc) -4-aminobenzoic acid methyl ester was dissolved with an appropriate amount of methanol with stirring, the pH was adjusted to 13 with 2N NaOH solution in an ice-water bath, and the reaction was carried out for 6 hours in an ice-water bath, and TLC (chloroform: methanol ═ 20:1) showed disappearance of 3-carboxyphenylboronic acid-Orn (Boc) -4-aminobenzoic acid methyl ester. With saturated KHSO4Adjusting pH of the reaction solution to neutral with water solution, concentrating under reduced pressure to dry, dissolving the residue with appropriate amount of saturated NaCl water solution, and dissolving with saturated KHSO4Adjusting pH of the aqueous solution to 2, extracting the water layer with ethyl acetate for 3 times, and extracting the ethyl acetate layer with anhydrous Na2SO4Drying, filtering under reduced pressure to remove desiccant, and concentrating the filtrate under reduced pressure to dryness to obtain compound 3-carboxyphenylboronic acid-Orn ((R))Boc) -4-aminobenzoic acid.
5) Preparation of 3-carboxyphenylboronic acid-Orn-4-aminobenzoic acid:
the 3-carboxyphenylboronic acid-Orn (Boc) -4-aminobenzoic acid was dissolved in a small amount of dry ethyl acetate, 4N HCl/EtOAc solution was added with stirring in an ice bath, TLC (EA: H2O: HAc ═ 4:1:2d) showed the disappearance of the starting material spot, the solution was drained with a water pump, anhydrous ether was added, the reaction was again drained with a water pump three times, and 3-carboxyphenylboronic acid-Orn-4-aminobenzoic acid was obtained.
6) Preparation of 3B-OH (3-carboxyphenylboronic acid-Orn (Cl) -4-aminobenzoic acid):
1mmol of 3-carboxyphenylboronic acid-Orn-4-aminobenzoic acid was dissolved with stirring in an appropriate amount of absolute methanol, 5mmol of ethyl 2-chloroacetimidoate hydrochloride were added in an ice-water bath, and the pH was adjusted to 10 with N, N-Diisopropylethylamine (DIPEA). After stirring at room temperature for 12 hours, TLC (EA: H2O: HAc. RTM. 4:1:2d) showed disappearance of 3-carboxyphenylboronic acid-Orn-4-aminobenzoic acid, concentration was carried out under reduced pressure until dryness, and C18 column chromatography was carried out to purify the resulting product to obtain 3B-OH (3-carboxyphenylboronic acid-Orn (Cl) -4-aminobenzoic acid).
Example 2 Synthesis of CRGDV-Chitosan-4B
Synthesis of (I) chitosan-acryloyl chloride
First, chitosan (0.6g, 0.20mmol) and triethylamine (Et3N, 50. mu.L, 0.36mmol) were dissolved in 20mL of N, N-Dimethylformamide (DMF). After cooling at 0 deg.C, acryloyl chloride (8 μ L, 0.10mmol) in 8mL of methylene chloride solution was added dropwise to the stirred solution. The reaction was carried out at room temperature for 24 hours and then dialyzed against distilled water (molecular weight cut-off of 700-1000 Da) for 48 hours. A pale yellow solid was obtained after lyophilization.
Synthesis of (di) chitosan-CRGDV
Chitosan-acryloyl chloride (0.3g, 0.10mmol) and CRGDV (98mg, 0.18mmol) were dissolved in 5mL of dimethyl sulfoxide and Et3N (10. mu.L, 0.07mmol) was added at room temperature. The reaction is stirred at room temperature for 12-24 hours and dialyzed against distilled water (MWCO: 700-1000 Da) for 48 hours. After lyophilization, a pale yellow solid was obtained.
(III) Synthesis of CRGDV-Chitosan-4B
PAD4 inhibitor 4B (5mg, 10. mu. mol) was dissolved in 5mL of anhydrous DMSO together with varying amounts of chitosan-CRGDV (32mg, 10. mu. mol; 16mg, 5. mu. mol; 10.56mg, 3.3. mu. mol; 8mg, 2.5. mu. mol; 6.4mg, 2. mu. mol). The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 4B: chitosan-CRGDV-chitosan-4B of 1:1, 2:1, 3:1, 4:1, 5: 1.
Example 3 Synthesis of CRGDV-Chitosan-3B
PAD4 inhibitor 3B (5mg, 10. mu. mol) and chitosan-CRGDV (10.56mg, 3.3. mu. mol) were dissolved in 5mL of anhydrous DMSO. The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 3B: chitosan-CRGDV-chitosan-3B at 3: 1.
Example 4 Synthesis of CRGDV-Chitosan-3B-OH
PAD4 inhibitor 3B-OH was dissolved in 5mL of anhydrous DMSO together with chitosan-CRGDV (10.56mg, 3.3. mu. mol). The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 3B-OH: chitosan-CRGDV-chitosan-3B-OH, 3: 1.
EXAMPLE 5 Synthesis of CRGDF-chitosan-4B
Synthesis of chitosan-CRGDF
Chitosan-acryloyl chloride (0.3g, 0.10mmol) and CRGDF (107mg, 0.18mmol) were dissolved in 5mL dimethyl sulfoxide (DMSO) and Et3N (10. mu.L, 0.07mmol) was added at room temperature. The reaction was stirred at room temperature for 24 hours and dialyzed against distilled water (MWCO: 1000Da) for 48 hours. After lyophilization, a pale yellow solid was obtained.
Synthesis of (II) CRGDF-chitosan-4B
PAD4 inhibitor 4B (5mg, 10. mu. mol) and chitosan-CRGDF (3.3. mu. mol) were dissolved in 5mL of anhydrous DMSO. The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 4B: chitosan-CRGDF ═ 3:1 CRGDF-chitosan-4B.
EXAMPLE 6 Synthesis of CRGDF-chitosan-3B
PAD4 inhibitor 3B (5mg, 10. mu. mol) and chitosan-CRGDF (3.3. mu. mol) were dissolved in 5mL of anhydrous DMSO. The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 3B: chitosan-CRGDF ═ 3:1 CRGDF-chitosan-3B.
EXAMPLE 7 Synthesis of CRGDF-chitosan-3B-OH
PAD4 inhibitor 3B-OH was dissolved in 5mL of anhydrous DMSO together with chitosan-CRGDF (3.3. mu. mol). The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 3B-OH: chitosan-CRGDF ═ 3:1 CRGDF-chitosan-3B-OH.
Example 8 Synthesis of CRGDS-Chitosan-4B
Synthesis of (I) Chitosan-CRGDS
Chitosan-acryloyl chloride (0.3g, 0.10mmol) and CRGDS (104mg, 0.18mmol) were dissolved in 5mL dimethyl sulfoxide (DMSO) and Et3N (10. mu.L, 0.07mmol) was added at room temperature. The reaction was stirred at room temperature for 24 hours and dialyzed against distilled water (MWCO: 1000Da) for 48 hours. After lyophilization, a pale yellow solid was obtained.
Synthesis of (di) CRGDS-chitosan-4B
PAD4 inhibitor 4B (5mg, 10. mu. mol) and chitosan-CRGDS (3.3. mu. mol) were dissolved in 5mL of anhydrous DMSO. The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 4B: chitosan-CRGDS-chitosan-4B at 3: 1.
Example 9 Synthesis of CRGDS-Chitosan-3B
PAD4 inhibitor 3B (5mg, 10. mu. mol) and chitosan-CRGDS (3.3. mu. mol) were dissolved in 5mL of anhydrous DMSO. The reaction was stirred at room temperature for 8h, and then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24 h. The resulting solution was then lyophilized to obtain 3B: chitosan-CRGDS-chitosan-3B at 3: 1.
Example 10 Synthesis of CRGDS-Chitosan-3B-OH
The PAD4 inhibitor 3B-OH (10 μmol with chitosan-CRGDF (3.3 μmol) was dissolved in 5mL anhydrous DMSO the reaction was stirred at room temperature for 8h, then the reaction mixture was dialyzed against distilled water (MWCO: 1000Da) for 24h, then the resulting solution was lyophilized to obtain CRGDS-chitosan-3B-OH of 3B-OH: chitosan-CRGDS ═ 3: 1.
Structural characterization
Zeta potential and particle diameter
The Zeta potential and the particle size of the above partial products and intermediate products were tested, and the results are shown in table 1.
TABLE 1 Zeta potential and particle size of some of the products, intermediates
Zeta(mV) Particle size (nm)
Chitosan 27.3 233.2±23.36
Chitosan-CRGDV 36.0 458.3±81.38
Chitosan-CRGDF 31.7 413.6±133.5
Chitosan-CRGDS 21.9 195.5±50.44
Chitosan 4B ═ 1:3 31.7 /
chitosan-CRGDV: 4B ═ 1:1 / /
chitosan-CRGDV: 4B ═ 1:2 6.82 209.1±26.33
chitosan-CRGDV: 4B ═ 1:3 10.4 221.1±36.82
chitosan-CRGDV: 4B ═ 1:4 27.8 /
chitosan-CRGDV: 4B ═ 1:5 12.2 262.3±37.43
chitosan-CRGDV: 3B ═ 1:3 12.7 202.3±58.8
chitosan-CRGDV: 3B-OH ═ 1:3 30.3 188.9±63.09
In Table 1,/means that no valid data could be detected.
(II) Infrared Spectrum
The infrared spectrogram of chitosan is shown in FIG. 2;
the infrared spectrum of chitosan-CRGDV is shown in FIG. 3;
the infrared spectrogram of chitosan-CRGDF is shown in FIG. 4;
the infrared spectrogram of chitosan-CRGDS is shown in FIG. 5;
the infrared spectrum of 4B is shown in FIG. 6;
the infrared spectrum of CRGDV-chitosan-4B is shown in FIG. 7;
the infrared spectrum of 3B is shown in FIG. 8;
the infrared spectrum of CRGDV-chitosan-3B is shown in FIG. 9;
the infrared spectrum of 3B-OH is shown in FIG. 10;
the infrared spectrum of CRGDV-chitosan-3B-OH is shown in FIG. 11.
As can be seen from the above IR spectrogram, the IR spectrogram of chitosan in FIG. 2 is 1670cm more than the IR spectrogram of chitosan-CRGD series in FIGS. 3, 4 and 5-1The absorption of (b), here characteristic of the carbonyl group on the amide bond, demonstrates that chitosan is grafted with CRGD sequence peptide. In FIGS. 7, 9 and 11, the IR absorption changes in 1020cm compared with the IR spectra of chitosan-CRGDV in FIG. 3-1The absorption in the vicinity was significantly enhanced, demonstrating the successful attachment of the chitosan-CRGD series to the PAD4 inhibitor.
(III) nuclear magnetic resonance hydrogen spectrum
Process for preparing chitosan1HNMR map (300MHz, D2O) is shown in FIG. 12;
process for preparing chitosan-acryloyl chloride1H NMR chart (300MHz, D)2O) is shown in FIG. 13;
of chitosan-CRGDV1HNMR picture (300MHz, D)2O) is shown in FIG. 14;
process for preparing chitosan-CRGDF1H NMR chart (300MHz, D)2O) is shown in FIG. 15;
process for preparing chitosan-CRGDS1H NMR chart (300MHz, D)2O) is shown in FIG. 16;
preparation of CRGDV-chitosan-4B1H NMR chart (300MHz, D)2O) is shown in FIG. 17;
preparation of CRGDV-chitosan-3B1H NMR chart (300MHz, D)2O) is shown in FIG. 18;
of CRGDV-chitosan-3B-OH1H NMR chart (300MHz, D)2O) is shown in fig. 19.
(IV) Transmission Electron microscopy
The transmission electron micrograph (concentration: 0.1mg/mL) of chitosan is shown in FIG. 20;
the transmission electron micrograph (concentration: 0.1mg/mL) of chitosan-CRGDV is shown in FIG. 21;
the transmission electron micrograph (concentration: 0.1mg/mL) of chitosan-CRGDF is shown in FIG. 22;
the transmission electron micrograph (concentration: 0.1mg/mL) of chitosan-CRGDS is shown in FIG. 23;
chitosan-CRGDV: a transmission electron micrograph (0.1mg/mL) of 1:1 of 4B is shown in fig. 24;
chitosan-CRGDV: a transmission electron micrograph (0.1mg/mL) of 1:2 of 4B is shown in fig. 25;
chitosan-CRGDV: a transmission electron micrograph (0.1mg/mL) of 4B ═ 1:3 is shown in fig. 26;
chitosan-CRGDV: a transmission electron micrograph (0.1mg/mL) of 1:4 (4B) is shown in fig. 27;
chitosan-CRGDV: a transmission electron micrograph (0.1mg/mL) of 1:5 (4B) is shown in fig. 28;
and (3) chitosan: a transmission electron micrograph (0.1mg/mL) of 1:3 of 4B is shown in fig. 29;
chitosan-CRGDV: a transmission electron micrograph (0.1mg/mL) of 1:3 (3B) is shown in fig. 30;
chitosan-CRGDV: a transmission electron micrograph (0.1mg/mL) of 3B — OH ═ 1:3 is shown in fig. 31.
As can be seen from fig. 20, chitosan does not form nanoparticles with a uniform distribution of structural sizes. FIGS. 21-23 show that nanoparticles with uniform structural size distribution can be formed after chitosan is grafted with CRGDV/F/S. FIGS. 24-28 show that chitosan-CRGDV: when 4B is 1:4, nanoparticles with compact structure and uniform size can be formed, and as can be seen from fig. 27, the nanoparticles have a more compact inner layer and a more loose outer layer, indicating that CRGDV and PAD4 inhibitor 4B encapsulates chitosan to form nanoparticles. Fig. 29 shows that chitosan: when 4B is 1:3, nanoparticles having a compact structure and a uniform size can be formed. Fig. 30, 31 show chitosan-CRGDV: 3B/3B-OH is 1:3, and can form nano particles with compact structure and uniform size.
Performance testing
CRGDV-Chitosan-4B (Chitosan-CRGDV: 4B ═ 1:3) drug loading test
After 600. mu.g of 4B (4-carboxyphenylboronic acid-Orn (Cl) -NBzl) was weighed using an analytical balance, 4mL of ultrapure water was dissolved to finally obtain a 4B (4-carboxyphenylboronic acid-Orn (Cl) -NBzl) concentration of 150. mu.g/mL as a stock solution. Sequentially diluting to obtain 4B (4-carboxyphenylboronic acid-Orn (Cl) -NBzl) standard solutions with concentration gradients of 9.38, 18.75, 37.5, 75 and 150 mu g/mL respectively, and then respectively measuring ultraviolet absorption spectrograms to obtain standard curves. As shown in fig. 32.
The standard curve linear fit equation for 4B (4-carboxyphenylboronic acid-orn (cl) -NBzl) is y ═ 0.026x +0.0694, and R2 ═ 0.9982. 640 μ g of CRGDV-chitosan-4B (chitosan-CRGDV: 4B ═ 1:3) was weighed, and 4mL of ultrapure water was taken to dissolve the CRGDV-chitosan-4B so that the CRGDV-chitosan-4B concentration was finally 150 μ g/mL, the absorbance was measured to be 0.259, and when the concentration of 4B was calculated to be 7.3 μ g/mL, the drug loading amount of 4B in CRGDV-chitosan-4B was 7.3/150 ═ 4.7%. When CRGDV-chitosan-4B (chitosan-CRGDV: 4B ═ 1:5)1370 μ g was weighed and dissolved in 4mL of ultrapure water to give a CRGDV-chitosan-4B concentration of 342.5 μ g/mL, the absorbance was measured as 1.334, and the concentration of 4B was calculated as 48.6 μ g/mL, the drug loading of 4B in CRGDV-chitosan-4B was 48.6/342.5 ═ 14.2%.
(II) in vitro tumor cell Activity
Evaluating the in vitro anti-cell proliferation activity of HCT-116 and S180 cells with different tested drugs, performing statistical analysis on the concentration of the tested compounds with inhibition rate, and calculating the IC of the positive drug and nano drug-carrying system by using a regression analysis-probability statistical method in SPSS50The results are shown in Table 2.
TABLE 2 IC of the in vitro anti-cell-proliferation Activity of the test drugs50Value of
Test drug HCT-116 S180
DOX 0.39±0.14 0.40±0.24
4B 41.3±7.2 36.8±3.8
3B 53.4±6.2 86.4±4.1
3B-OH 67.5±4.2 73.2±6.9
CRGDV-Chitosan ND ND
CRGDV-Chitosan-4B 4.7±1.4 6.2±1.8
CRGDV-Chitosan-3B 15.6±1.3 18.9±3.4
CRGDV-Chitosan-3B-OH 21.3±3.8 23.9±4.3
CRGDF-chitosan ND ND
CRGDF-chitosan-4B 6.8±2.1 9.4±2.5
CRGDF-chitosan-3B 20.2±3.1 28.7±4.2
CRGDF-chitosan-3B-OH 23.5±2.9 29.3±4.6
CRGDS-chitosan ND ND
CRGDS-Chitosan-4B 10.7±2.3 20.9±4.9
CRGDS-Chitosan-3B 21.6±1.9 29.4±5.3
CRGDS-Chitosan-3B-OH 26.4±1.5 34.1±4.5
Note: ND stands for not detectable, n is 9.
As can be seen from Table 2, the PAD4 inhibitor loaded on the CRGD sequence peptide modified chitosan provided by the invention has good in vivo biological activity.
(III) evaluation of antitumor Activity in vivo
Negative control group: normal Saline (NS), 10 per group, the route of administration was tail vein injection; positive control group: adriamycin (DOX), 10 in each group, with the dosage of 2.0 mu mol/kg, and the administration route is intraperitoneal injection; test group: PAD4 inhibitor 4B, CRGDV-chitosan, CRGDV-chitosan-4B, 5 groups, 10 each, 4B dose of 10.0 μmol/kg, CRGDV-chitosan-4B group 4B dose of 2.0 μmol/kg, administration route of tail vein injection.
Experimental procedure
The tumor source used by the transplanted mouse S180 sarcoma model is S180 mouse fibrosarcoma cells, which are purchased from animal experiment center of department of medicine of Beijing university and maintained by self passage. Inoculating S180 mouse fibrosarcoma cells into an abdominal cavity of an SPF male ICR mouse, carrying out self-passage, anesthetizing the mouse with S180 ascites tumor after passage for one week with a proper amount of ether, removing the neck of the mouse, killing the mouse, soaking and disinfecting the mouse in 75% alcohol for 1min, then splitting the abdominal cavity of the mouse, collecting ascites S180 tumor liquid, centrifuging the tumor liquid for 10min at 1000rpm, discarding supernatant, washing with normal saline to remove non-cell fragments, tissues and floating blood, fully mixing the above to obtain S180 cell suspension, diluting to a certain multiple, uniformly mixing with freshly prepared 0.2% trypan blue at a volume ratio of 9:1, and dyeing. Live cells will not be stained blue, while dead cells will be stained pale blue.
Counting by using an erythrocyte counting plate, and calculating the cell concentration and the cell survival rate according to the following formulas:
cell concentration (cell number/mL) 4 Large Square Living cell number/4X 104X dilution multiple fineThe cell survival rate was live cell number/(live cell number + dead cell number) × 100%
Diluting cell suspension with survival rate greater than 90% to 2 × 10 with physiological saline7And (2) each mL, wherein inoculation of the experiment should be completed as soon as possible, when inoculation is performed, the mouse is grabbed by the left hand to be fixed, the right hand-held syringe is punctured into the right armpit of the mouse to the depth of about 2mm below the skin, a small cavity is separated by using a needle in a blunt manner, 0.2mL of prepared cell suspension is injected, and the cell suspension is mixed uniformly before each injection. And finishing the establishment of the animal model. After the model is established, the mice are observed every day, and are grouped after most mice axilla can see soybean-sized solid tumors, the experiment is randomly grouped on the 5 th day after inoculation, so that the tumor sizes of the mice in each group are evenly distributed, administration is started on the same day, 1 time per day, and 7 times of administration is performed from the 11 th day. And taking out the mice 24h after the last administration, weighing each group of mice, removing eyeballs after ether anesthesia for blood sampling, then removing necks for sacrifice, fixing the growth parts of solid tumors at the armpits of the mice by using forceps at the left hand, cutting the skin by using scissors at the right hand, fully exposing tumor tissues, carrying out blunt separation along the skin and the upper limbs, taking out the tumor tissues, and weighing. Then, the heart, liver, spleen, kidney, brain and other organs of the mouse are dissected and taken out in sequence and weighed.
Results and analysis of the experiments
In situ tumor tissue mass is expressed as mean ± SD g, and statistical comparisons between groups were made by t-test. The inhibition rate was [ (average tumor weight of negative control group-average tumor weight of test compound)/(average tumor weight of negative control group)/] × 100%.
The results of evaluating the tumor proliferation inhibitory activity in test drug bodies in the transplanted mouse S180 sarcoma model are shown in Table 3.
TABLE 3 evaluation of tumor proliferation inhibitory Activity in test drug bodies
Test drug Tumor weight (g) Inhibition ratio (%)
NS 1.91±0.55
DOX 0.84±0.14a 56.0％
4B 1.34±0.22a 29.8％
3B 1.45±0.42a 24.1％
3B-OH 1.52±0.34a 20.4％
CRGDV-Chitosan 1.87±0.56 2.1％
CRGDV-Chitosan-4B 0.97±0.43a 49.2％
CRGDV-Chitosan-3B 1.31±0.33a 31.4％
CRGDV-Chitosan-3B-OH 1.43±0.41a 25.1％
CRGDF-chitosan 1.81±0.55 5.2％
CRGDF-chitosan-4B 1.09±0.44a 42.9％
CRGDF-chitosan-3B 1.37±0.34a 28.3％
CRGDF-chitosan-3B-OH 1.52±0.37a 20.4％
CRGDS-chitosan 1.77±0.39 7.3％
CRGDS-Chitosan-4B 1.27±0.35a 33.5％
CRGDS-Chitosan-3B 1.46±0.39a 23.6％
CRGDS-Chitosan-3B-OH 1.62±0.54 15.2％
Note: dox tail vein 0.2mg/kg a
4B,3B and 3B-OH tail vein 10.0 mu mol/kg
CRGDV/F/S-Chitosan-4B, CRGDV/F/S-Chitosan-3B and CRGDV/F/S-Chitosan-3B-OH, 2.0. mu. mol/kg
CRGDV/F/S-chitosan 0.2mg/kg
n-10, by t test, a: p <0.01 compared to NS;
as can be seen from Table 3, the PAD4 inhibitor loaded on the CRGD sequence peptide modified chitosan provided by the invention has good in vivo tumor proliferation inhibition activity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A PAD4 inhibitor loaded on CRGD sequence peptide modified chitosan, which comprises a CRGD sequence peptide modified chitosan carrier and a PAD4 inhibitor loaded on the surface of the CRGD sequence peptide modified chitosan carrier;
the cysteine in the CRGD sequence peptide is linked to the chitosan surface via acryloyl chloride.
2. The CRGD sequence peptide-modified chitosan-loaded PAD4 inhibitor of claim 1, wherein the CRGD sequence peptide comprises one or more of CRGDV, CRGDs and CRGDF.
3. The CRGD sequence peptide-modified chitosan-loaded PAD4 inhibitor according to claim 1, wherein the PAD4 inhibitor comprises one or more of PAD4 inhibitor 4B, PAD4 inhibitor 3B, PAD4 inhibitor 3B-OH.
4. The CRGD sequence peptide modified chitosan loaded PAD4 inhibitor according to claim 1 or 3, wherein the PAD4 inhibitor loading of the CRGD sequence peptide modified chitosan loaded PAD4 inhibitor is 12-18 wt%.
5. A method for preparing a CRGD sequence peptide modified chitosan loaded PAD4 inhibitor as claimed in any one of claims 1 to 4, comprising the steps of:
(1) mixing chitosan, acryloyl chloride, a first acid-binding agent and a first organic solvent, and carrying out nucleophilic substitution reaction to obtain acryloyl chloride modified chitosan;
(2) mixing the chitosan modified by acryloyl chloride, the CRGD sequence peptide, a second acid-binding agent and a second organic solvent, and carrying out Michael addition reaction to obtain a CRGD sequence peptide modified chitosan carrier;
(3) and stirring and mixing the CRGD sequence peptide modified chitosan carrier, the PAD4 inhibitor and a third organic solvent to obtain the PAD4 inhibitor loaded by the CRGD sequence peptide modified chitosan.
6. The method according to claim 5, wherein the molar ratio of chitosan to acryloyl chloride is 1.5-3: 1, and the molar ratio of chitosan to CRGD sequence peptide is 1-2: 1.8.
7. The method of claim 5, wherein the molar ratio of chitosan to PAD4 inhibitor is 1: 1-5.
8. The preparation method according to claim 5, wherein the nucleophilic substitution reaction time is 12-24 h.
9. The preparation method according to claim 5, wherein the Michael addition reaction time is 12-24 h.
10. Use of the CRGD sequence peptide-modified chitosan-loaded PAD4 inhibitor of any one of claims 1 to 4 or the CRGD sequence peptide-modified chitosan-loaded PAD4 inhibitor prepared by the preparation method of any one of claims 5 to 9 in preparation of an anti-tumor drug.
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CN101690823A (en) * 2009-11-03 2010-04-07 首都医科大学 Method for preparing RGDV-containing cytosine arabinoside conjugate pharmacosome and application as antitumor agent
CN105884862A (en) * 2014-12-16 2016-08-24 首都医科大学 RGD tetrapeptide modified beta-carboline acyl-tryptophan and preparation, nanostructure, activity and application thereof
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CN101690823A (en) * 2009-11-03 2010-04-07 首都医科大学 Method for preparing RGDV-containing cytosine arabinoside conjugate pharmacosome and application as antitumor agent
CN105884862A (en) * 2014-12-16 2016-08-24 首都医科大学 RGD tetrapeptide modified beta-carboline acyl-tryptophan and preparation, nanostructure, activity and application thereof
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