CN111701081A

CN111701081A - Drug coating liquid for drug-coated balloon and drug-coated balloon

Info

Publication number: CN111701081A
Application number: CN202010581872.7A
Authority: CN
Inventors: 王玉记; 张启迪; 王彦明; 彭紫东; 阿依江; 冯琦琦
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2020-09-25
Anticipated expiration: 2040-06-23
Also published as: CN111701081B

Abstract

The invention provides a drug coating liquid for drug coating saccule, the drug coating saccule belongs to the technical field of pharmacy, the drug coating liquid of the invention takes pectin as excipient of the drug coating saccule, which is mixed with active drug (paclitaxel or docetaxel) to help the adhesion of the drug coating liquid and a blank saccule, when the drug coating saccule reaches the injury, the pectin helps the active drug to be quickly released from the saccule coating surface, and the effective drug concentration is achieved by deposition on the surface of the blood vessel wall, arginine-glycine-aspartic acid (RGD) peptide is a structure recognition motif of cell surface integrin, including α v β 3 and α 5 β 1, which is related to the process of anchoring extracellular matrix₂(CH₂)₆CH₃Can be used as target in blood vesselIntegrins on the skin cells enhance the absorption of the active agent while reducing the loss of the active agent during transport.

Description

Drug coating liquid for drug-coated balloon and drug-coated balloon

Technical Field

The invention relates to the technical field of pharmacy, in particular to a drug coating liquid for a drug coating balloon and the drug coating balloon.

Background

With the increasing use of Percutaneous Transluminal Coronary Angioplasty (PTCA), many associated devices and techniques have been improved, and in recent years drug-coated balloons (DCB) have attracted attention as compared to stent implantation and second generation drug-eluting stents. DCB is an angioplasty balloon directly coated with an antiproliferative active drug and an excipient (drug matrix or carrier) that serves to facilitate the transfer of the drug to the lesion. The medicine coating saccule is conveyed to the narrow part of the blood vessel through the catheter, and after being inflated, the saccule expands, and the medicine coated on the surface of the saccule is released rapidly in a certain period of time. Paclitaxel cell culture tests show that the drug blocks the effect of tubulin after being combined with the beta subunit of the tubulin, thereby blocking mitosis of cells, most of the surface coatings of the drug-coated saccule of peripheral arteries clinically used at present are paclitaxel (the structure is shown as formula I), and thereby the hyperplasia of intima of blood vessels is inhibited. While the effectiveness of DCB is not only determined by the active drug, other factors that are critical to the effectiveness and safety of the balloon are the remaining excipients in the coating in addition to the active drug, i.e., the formulation of the coating technique, which also indicates that the matrix is important in drug-coated balloons. Many excipients are also present, such as triethyl citrate butyryl, urea, shellac, iopromide, polysorbate/sorbitol, etc., each with a different function. And researches find that the matrix coating consisting of the mixture of lipophilic paclitaxel and hydrophilic spacers (excipients) is most effective, not only can improve the bioavailability of paclitaxel, but also can increase the contact area of the drug and the blood vessel wall and weaken the attraction between drug molecules so that the blood vessel wall can rapidly take in the drug.

The excipient adopted by the drug-coated balloon of the currently marketed product Dior II is shellac, and the structure is shown as formula II. The resin part constituting the shellac skeleton is a complex mixture of various sesquiterpene acids and hydroxy fatty acids, which can be separated into about 30% soft resin (monoester) and 70% hard resin (polyester consisting of several resin acid components). The shellac can be used as medicinal material and coating material to regulate disintegration property of tablet, and can protect inner layer medicine from moisture and reduce degradation. However, the most fatal problem exists in that shellac, as an excipient, is slightly disadvantageous in the critical step of rapid release. In the brief time of sacculus and vascular wall contact, the shellac can not release the taxol medicine of apparent percentage to pathological change vascular wall, but withdraws the sacculus after, wraps up taxol along with blood flows, reaches the rest positions of health and just can release the medicine completely, not only causes the reduction of effective part medicine absorption, can cause certain medicine whole body reaction moreover, leads to the fact certain influence to intact vascular wall. Therefore, the accurate and rapid release of the medicine at the key part is ensured, and the medicine loss is reduced, which is also a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a drug coating solution for a drug-coated balloon and the drug-coated balloon. The drug coating liquid for the drug-coated balloon rapidly releases the active drug into the external phase medium within 1min, and reduces the loss of the active drug in the transportation process.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a drug coating liquid for drug coating saccule, which comprises pectin and RGDS-OCH₂(CH₂)₆CH₃And an active agent; what is needed isThe active drug is paclitaxel or docetaxel.

Preferably, the pectin, RGDS-OCH are₂(CH₂)₆CH₃The mass ratio of the active medicine to the active medicine is (5.7-15): (57-150): 1500.

preferably, the pectin is added in the form of pectin dispersion liquid, and the mass concentration of the pectin dispersion liquid is 0.01-1.0 mg/mL; the active drug is added in the form of an active drug absolute ethyl alcohol solution, and the concentration of the active drug absolute ethyl alcohol solution is 5-50 mg/mL.

Preferably, the pectin dispersion is prepared by a method comprising the following steps:

adding pectin while adding water under the conditions of oil bath at 40 ℃ and stirring, finally adding glycerol, and adjusting the pH value to 7-7.5 by using alkali to obtain the pectin dispersion liquid.

The invention also provides a drug coating balloon which comprises a blank balloon and a drug coating coated on the blank balloon, wherein the drug coating is formed by coating the drug coating liquid in the technical scheme.

Preferably, the thickness of the drug coating is 1-2 mm.

Preferably, the blank balloon is made of polyethylene plastic or latex nylon.

Preferably, the diameter of the blank balloon is 3-6 mm.

The invention provides a drug coating liquid for drug coating saccule, which comprises pectin and RGDS-OCH₂(CH₂)₆CH₃The coating solution of the present invention uses pectin as an excipient for a drug-coated balloon, and helps the drug-coated balloon adhere to a blank balloon after mixing with an active drug, when the drug-coated balloon reaches a lesion, the pectin helps the active drug to be rapidly released from the balloon-coated surface and to be deposited on the surface of a blood vessel wall to reach an effective drug concentration, arginine-glycine-aspartic acid (RGD) peptide is a structural recognition motif for cell surface integrins, including α v β 3 and α 5 β 1, associated with the process of anchoring extracellular matrixThe RGDS-OCH obtained by connecting hydrophobic fatty alcohol chain and oligopeptide with hydrophilic property and containing RGD sequence according to phospholipid amphiphilic structure₂(CH₂)₆CH₃The fusion protein can be used as integrin on targeted vascular endothelial cells, enhances the absorption of active drugs and simultaneously reduces the loss of the active drugs in the delivery process.

Drawings

FIG. 1 shows pectin-paclitaxel paste (a) and pectin-RGDS-OCH₂(CH₂)₆CH₃-a slide map of paclitaxel paste (b);

FIG. 2 is a morphology chart of shellac-paclitaxel coating results;

FIG. 3 is a scanning electron microscope image of pectin lyophilized sample powder;

FIG. 4 is a scanning electron micrograph of paclitaxel powder;

FIG. 5 shows taxol-RGDS-OCH₂(CH₂)₆CH₃Scanning an electron microscope image;

FIG. 6 shows pectin-paclitaxel at 10^-3Scanning electron microscope images under mol/L concentration;

FIG. 7 shows pectin-paclitaxel at 10^-5Scanning electron microscope images under mol/L concentration;

FIG. 8 shows pectin-paclitaxel at 10^-6Scanning electron microscope images under mol/L concentration;

FIG. 9 shows pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel at 10^-3Scanning electron microscope images under mol/L concentration;

FIG. 10 shows pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel at 10^-5Scanning electron microscope images under mol/L concentration;

FIG. 11 shows pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel at 10^-6Scanning electron microscope images under mol/L concentration;

FIG. 12 shows shellac-paclitaxel at 10^-3Scanning electron microscope images under mol/L concentration;

FIG. 13 shows shellac-paclitaxel at 10^-4Scanning electron microscope images under mol/L concentration;

FIG. 14 shellac-paclitaxel in10^-5Scanning electron microscope images under mol/L concentration;

FIG. 15 shows shellac-paclitaxel at 10^-6Scanning electron microscope images under mol/L concentration;

FIG. 16 is a graph of the UV absorption spectrum of paclitaxel solution (20. mu.g/mL);

FIG. 17 is a high performance liquid chromatogram of a paclitaxel stock solution (60. mu.g/mL);

FIG. 18 is a standard curve of a standard solution of paclitaxel;

FIG. 19 is a shellac-paclitaxel release profile;

FIG. 20 is a release profile of pectin-paclitaxel;

FIG. 21 shows pectin-RGDS-OCH₂(CH₂)₆CH₃-a release profile of paclitaxel;

FIG. 22 is a pectin-paclitaxel rapid release profile;

FIG. 23 shows pectin-RGDS-OCH₂(CH₂)₆CH₃-a rapid release profile of paclitaxel;

FIG. 24 shows pre-sonication pectin-paclitaxel (a) and pectin-RGDS-OCH₂(CH₂)₆CH₃-picture of paclitaxel (b);

FIG. 25 shows pectin-RGDS-OCH₂(CH₂)₆CH₃-a rapid release profile of paclitaxel 2;

FIG. 26 shows pectin-RGDS-OCH₂(CH₂)₆CH₃-a rapid release profile of docetaxel.

Detailed Description

The invention provides a drug coating liquid for drug coating saccule, which comprises pectin and RGDS-OCH₂(CH₂)₆CH₃And an active agent; the active drug is paclitaxel or docetaxel.

In the invention, the RGDS-OCH₂(CH₂)₆CH₃Preferably prepared by a process comprising the steps of:

①Boc-Ser-OCH₂(CH₂)₆CH₃the preparation of (1):

dissolving Boc-Ser-OH 5mmol in 20mL of anhydrous THF, adding N-hydroxybenzotriazole (HOBt)5mmol under ice bath condition, dissolving completely, slowly adding Dicyclohexylcarbodiimide (DCC)6mmol, and stirring for 30min to obtain reaction solution A. Under ice-bath condition, 11mmol octanol (C)₈H₁₇OH) 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. Stirred in ice bath for 1h and then at room temperature for 48h, TLC (chloroform: methanol ═ 5:1) showed disappearance of Boc-Ser-OH; dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 50mL ethyl acetate and the resulting solution was sequentially saturated NaHCO₃Aqueous solution, saturated aqueous NaCl solution, 5% KHSO₄Aqueous solution, saturated aqueous NaCl solution, saturated aqueous NaHCO solution₃Washing with water solution and saturated NaCl water solution for three times, and washing ethyl acetate layer with anhydrous Na₂SO₄Drying, filtering to remove desiccant, and concentrating the filtrate under reduced pressure to dryness to obtain Boc-Ser-OCH₂(CH₂)₆CH₃。

②HCl·H-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

Boc-Ser-OCH₂(CH₂)₆CH₃Dissolving 1mmol with small amount of dry ethyl acetate, adding 4MHCl/EtOAc solution under stirring in ice bath, TLC (petroleum ether: acetone ═ 7:1) shows disappearance of raw material spot, pumping the solution with water pump, adding anhydrous ether, pumping the reaction solution with water pump again, repeating for three times to obtain HCl. H-Ser-OCH₂(CH₂)₆CH₃。

③Boc-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

Boc-Asp (OBzl) -OH 1.1mmol is dissolved in 10mL of anhydrous THF, N-hydroxybenzotriazole (HOBt)1mmol is added under the ice bath condition and is completely dissolved, Dicyclohexylcarbodiimide (DCC)1.1mmol is slowly added, and the mixture is stirred for 30min to obtain reaction liquid A. Under the ice bath condition, HCl, H-Ser-OCH₂(CH₂)₆CH₃Suspending 1mmol in 10mL of anhydrous THF, adding 0.5mL of N-methylmorpholine (NMM), adjusting pH to 8-9, adding to the reaction solution A, and dropwise adding N-methylmorpholine (NMM) to reactThe pH value of the solution is 8-9. Stirring was carried out in an ice bath for 1h and at room temperature for 12h, and TLC (petroleum ether: acetone ═ 4:1) showed a new spot. Dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 30mL ethyl acetate and the resulting solution was sequentially saturated NaHCO₃Aqueous solution, saturated aqueous NaCl solution, 5% KHSO₄Aqueous solution, saturated aqueous NaCl solution, saturated aqueous NaHCO solution₃Washing with water solution and saturated NaCl water solution for three times, and washing ethyl acetate layer with anhydrous Na₂SO₄Drying, filtering to remove drying agent, and concentrating the filtrate under reduced pressure to dryness to obtain Boc-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃。

④HCl·H-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

by using and preparing HCl, H-Ser-OCH₂(CH₂)₆CH₃The same method, using Boc-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃1mmol of HCl, H-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃。

⑤Boc-Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

by using a method similar to that for preparing Boc-Asp (Bzl) -Ser-OCH₂(CH₂)₆CH₃The same method is carried out by reacting Boc-Gly-OH1.2mmol with HCl. H-Asp (Bzl) -Ser-OCH₂(CH₂)₆CH₃1mmol of Boc-Gly-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃。

⑥HCl·H-Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

by using and preparing HCl, H-Ser-OCH₂(CH₂)₆CH₃The same method, using Boc-Gly-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃1mmol of HCl, H-Gly-Asp (Bzl) -Ser-OCH₂(CH₂)₆CH₃。

⑦Boc-Arg(NO₂)Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

by using a method similar to that for preparing Boc-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃In the same way, with Boc-Arg (NO)₂) OH1.2mmol and HCl. H-Gly-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃1mmol to yield Boc-Arg (NO)₂)-Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃。

⑧Boc-Arg-Gly-Asp-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

Boc-Arg (NO)₂)-Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃Dissolving 1mmol with methanol, adding appropriate amount of Pd/C, dropwise adding small amount of anhydrous formic acid, keeping the reaction system sealed, pumping out air with tee joint, introducing hydrogen gas in gas bag, pumping out air with tee joint, repeatedly replacing air in the reaction system, staying the tee joint in hydrogen gas state, keeping hydrogen environment at room temperature, stirring until the raw material point disappears, and monitoring by TLC. After the reaction is finished, the Pd/C is removed by reduced pressure filtration, the filtrate is dried by spinning, and the Boc-Arg-Gly-Asp-Ser-OCH is obtained by repeated scrubbing with diethyl ether₂(CH₂)₆CH₃。

⑨HCl·H-Arg-Gly-Asp-Ser-OCH₂(CH₂)₆CH₃(RGDS-OCH₂(CH₂)₆CH₃) The preparation of (1):

by using and preparing HCl, H-Ser-OCH₂(CH₂)₆CH₃The same method is carried out by Boc-Arg-Gly-Asp-Ser-OCH₂(CH₂)₆CH₃1mmol of the obtained product is HCl, H-Arg-Gly-Asp-Ser-OCH₂(CH₂)₆CH₃I.e. RGDS-OCH₂(CH₂)₆CH₃。

In the invention, the pectin and the RGDS-OCH are adopted₂(CH₂)₆CH₃The mass ratio of the active medicine to the active medicine is preferably (5.7-15): (57-150): 1500, more preferably 5.7: 57: 1500.

in the present invention, the pectin is preferably added in the form of a pectin dispersion, and the concentration of the pectin dispersion is preferably 0.01 to 1.0mg/mL, and more preferably 0.063 mg/mL. In the present invention, the pectin dispersion having a concentration of 0.063mg/mL is preferably prepared by a process comprising the steps of:

adding pectin while adding water under the conditions of oil bath at 40 ℃ and stirring, finally adding glycerol, and adjusting the pH value to 7-7.5 by using alkali to obtain the pectin dispersion liquid; the dosage ratio of pectin, water and glycerol is preferably 1.9 mg: 30mL of: 72 mu L of the solution; the alkali is preferably sodium hydroxide, and the concentration of the sodium hydroxide is preferably 2 mol/L.

In the invention, the active drug is preferably added in the form of an active drug absolute ethanol solution, and the concentration of the active drug absolute ethanol solution is preferably 5-50 mg/mL, and more preferably 15 mg/mL.

In the present invention, the drug coating solution for drug-coated balloons is preferably prepared by a method comprising the following steps:

preparing pectin dispersion liquid according to the technical scheme;

RGDS-OCH is added into the pectin dispersion liquid₂(CH₂)₆CH₃Uniformly mixed to form a light brown uniform liquid, namely pectin-RGDS-OCH₂(CH₂)₆CH₃A colloidal dispersion system;

in an EP tube, pectin-RGDS-OCH is added firstly₂(CH₂)₆CH₃Dispersing in colloid, adding anhydrous alcohol solution of taxol, layering, and manually stirring to obtain white paste, i.e. medicinal coating liquid for medicinal coating balloon.

Arginine-glycine-aspartic acid (RGD) peptide is a structural recognition motif for cell surface integrins, including α v β 3 and α 5 β 1, and for anchoring extracellular matrixThe processes are associated. The invention grafts hydrophobic aliphatic chain-OCH on RGD₂(CH₂)₆CH₃Connecting to obtain RGDS-OCH₂(CH₂)₆CH₃Has certain amphiphilicity, so that the hydrophilic pectin and the hydrophobic paclitaxel are uniformly mixed. Furthermore, due to the ability of peptide chain containing RGD sequence to be positioned on vascular endothelial cells, it is reasonable to believe that when pectin-RGDS-OCH₂(CH₂)₆CH₃RGDS-OCH release of paclitaxel from the balloon surface₂(CH₂)₆CH₃Can guide the paclitaxel to be delivered to the surface of endothelial cells, increase the positioning function and reduce the adverse reaction caused by the drug loss flowing to other parts along with the blood flow in the blood vessel.

In the invention, the thickness of the drug coating is preferably 1-2 mm.

In the invention, the blank balloon is preferably made of polyethylene plastic or latex nylon; the diameter of the blank balloon is preferably 3-6 mm, and more preferably 4 mm.

In the invention, the preparation method of the drug-coated balloon is preferably as follows: and coating the drug coating solution on the blank balloon, and evaporating the solvent to obtain the drug coating balloon.

The parameters of the coating are not particularly limited in the present invention as long as the thickness of the drug coating can meet the requirements.

The temperature and time for evaporating the solvent are not particularly limited in the present invention, as long as the solvent in the drug coating solution can be evaporated and removed.

The drug coating solution for drug-coated balloons and the drug-coated balloon provided by the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of paclitaxel stock solution:

weighing 6mg of paclitaxel by using a 1.5mL EP tube, adding 400 mu L of absolute ethyl alcohol for dissolving, performing vortex ultrasonic treatment until the paclitaxel is completely dissolved and clarified to obtain a paclitaxel stock solution with the concentration of 15mg/mL, sealing by using a para film, and storing in a refrigerator at 4 ℃.

Preparing a pectin colloid dispersion system:

weighing 1.9mg of powdered pectin by using a balance for later use; taking a 50mL eggplant bottle, adding powdered pectin while adding water under manual stirring in an oil bath at 40 ℃, wherein the total volume of the added water is 30mL, finally adding 72 mu L of glycerol, adjusting the pH value to 7-7.5 by using 2mol/LNaOH, and uniformly stirring to obtain light brown uniform liquid.

pectin-RGDS-OCH₂(CH₂)₆CH₃Preparation of colloidal dispersion system:

adding 19mg of RGDS-OCH into a pectin colloid dispersion system₂(CH₂)₆CH₃Mixing to obtain light brown uniform liquid.

pectin-RGDS-OCH₂(CH₂)₆CH₃Preparation of paclitaxel drug coating solution:

in a 1.5mL EP tube, 90 μ L of pectin-RGDS-OCH was added₂(CH₂)₆CH₃Colloid dispersed system, adding 100 μ L (15mg/mL) of prepared paclitaxel stock solution, layering, and manually stirring to obtain white paste.

Wherein, RGDS-OCH₂(CH₂)₆CH₃The preparation method comprises the following steps:

①Boc-Ser-OCH₂(CH₂)₆CH₃the preparation of (1):

dissolving Boc-Ser-OH 5mmol in 20ml of anhydrous THF, adding N-hydroxybenzotriazole (HOBt)5mmol under ice bath condition for complete dissolution, slowly adding Dicyclohexylcarbodiimide (DCC)6mmol, and stirring for 30min to obtain reaction liquid A. Under ice-bath condition, 11mmol octanol (C)₈H₁₇OH) is dissolved in 20mL of anhydrous THF and added into the reaction solution A, 1mL of N-methylmorpholine (NMM) is added, and the pH is adjusted to 8-9. Stirring in ice bath for 1h, and thenStirred at room temperature for 48h, TLC (chloroform: methanol ═ 5:1) showed disappearance of Boc-Ser-OH; dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 50mL ethyl acetate and the resulting solution was sequentially saturated NaHCO₃Aqueous solution, saturated aqueous NaCl solution, 5% KHSO₄Aqueous solution, saturated aqueous NaCl solution, saturated aqueous NaHCO solution₃Washing with water solution and saturated NaCl water solution for three times, and washing ethyl acetate layer with anhydrous Na₂SO₄Drying, filtering to remove desiccant, and concentrating the filtrate under reduced pressure to dryness to obtain Boc-Ser-OCH₂(CH₂)₆CH₃。

②HCl·H-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

③Boc-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

Boc-Asp (OBzl) -OH 1.1mmol is dissolved in 10mL of anhydrous THF, N-hydroxybenzotriazole (HOBt)1mmol is added under the ice bath condition and is completely dissolved, Dicyclohexylcarbodiimide (DCC)1.1mmol is slowly added, and the mixture is stirred for 30min to obtain reaction liquid A. Under the ice bath condition, HCl, H-Ser-OCH₂(CH₂)₆CH₃Suspending 1mmol of the suspension in 10mL of anhydrous THF, adding 0.5mL of N-methylmorpholine (NMM), adjusting the pH value to 8-9, adding the mixture into the reaction solution A, and dropwise adding N-methylmorpholine (NMM) to adjust the pH value of the reaction solution to 8-9. Stirring was carried out in an ice bath for 1h and at room temperature for 12h, and TLC (petroleum ether: acetone ═ 4:1) showed a new spot. Dicyclohexylurea (DCU) was filtered off and THF was distilled off under reduced pressure. The residue was dissolved in 30mL ethyl acetate and the resulting solution was sequentially saturated NaHCO₃Aqueous solution, saturated aqueous NaCl solution, 5% KHSO₄Aqueous solution, saturated aqueous NaCl solution, saturated aqueous NaHCO solution₃Aqueous solution, saturated aqueous NaCl solutionWashing three times, using anhydrous Na for an ethyl acetate layer₂SO₄Drying, filtering to remove drying agent, and concentrating the filtrate under reduced pressure to dryness to obtain Boc-Asp (OBzl) -Ser-OCH₂(CH₂)₆CH₃。

④HCl·H-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

⑤Boc-Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

⑥HCl·H-Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

⑦Boc-Arg(NO₂)Gly-Asp(OBzl)-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

⑧Boc-Arg-Gly-Asp-Ser-OCH₂(CH₂)₆CH₃The preparation of (1):

Comparative example 1

Preparation of shellac alkali salt:

weighing 1.9mg of powdered pale yellow shellac by balance, adding shellac into 50mL eggplant bottle in 40 deg.C oil bath while adding water under manual stirring, wherein the total volume of water is 10mL, adding 72 μ L of glycerol, adding saturated NaHCO₃Adjusting the pH value of the solution to 7, and stirring the solution uniformly to form a suspension.

Preparation of shellac-paclitaxel:

in a 1.5mL EP tube, 90. mu.L of shellac base salt suspension was added, 100. mu.L (15mg/mL) of the paclitaxel stock solution prepared in example 1 was added, and the mixture was vortexed to form a uniformly dispersed milky viscous liquid, which was stored in a 4 ℃ refrigerator.

Comparative example 2

Preparation of pectin-paclitaxel:

in a 1.5mL EP tube, 90. mu.L of the pectin colloid dispersion prepared in example 1 was added, and then 100. mu.L (15mg/mL) of the paclitaxel stock solution prepared in example 1 was added, and the mixture was layered and manually stirred to form a white paste.

Performance testing

First, coating observation

The pectin-RGDS-OCH prepared in example 1 was added₂(CH₂)₆CH₃The paclitaxel paste and the pectin-paclitaxel paste prepared in comparative example 2 were coated on glass slides, respectively, put into an incubator at 37 ℃ for 4 days, the solvent was evaporated, and the morphology on the glass slides was observed. FIG. 1 shows pectin-paclitaxel paste (a) and pectin-RGDS-OCH₂(CH₂)₆CH₃-slide morphology of paclitaxel paste (b). As can be seen from fig. 1: pectin-paclitaxel and pectin-RGDS-OCH₂(CH₂)₆CH₃The paclitaxel paste coating was uniform to the same extent.

Preparation of drug coating

Selecting three holes from six-hole plate, marking the name of each hole, namely pectin and pectin-RGDS-OCH₂(CH₂)₆CH₃And shellac. Under the names pectin, pectin-RGDS-OCH₂(CH₂)₆CH₃Uniformly coating pectin-paclitaxel paste, pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel paste, weight loss assay, giving: the mass on coating was 0.08203g and 0.07531g, respectively. In the pores of shellac, milky viscous liquid (shellac-taxol) having a certain fluidity was poured, and the mass of coating was 0.05592g as measured by a weight loss method.

The six well plate was placed in a 37 ℃ oven for 4 days until the solvent had evaporated and only a white deposit remained on the plate. Pasty pectin-paclitaxel and pectin-RGDS-OCH₂(CH₂)₆CH₃-taxus chinensisThe morphology of the alcohol white substance is the same as the coating observation result of fig. 1, while the morphology of the shellac-paclitaxel coating result is shown in fig. 2, wherein (a) in fig. 2 is taken out after being placed in a refrigerator and then mixed uniformly, and (b) is directly mixed; as can be seen from fig. 2: after the mixture is placed in a refrigerator, the mixture is uniform, the particle size of the particles is reduced, and the particles are finer, so that insoluble particles are almost not visible to naked eyes. At the same time, pectin-RGDS-OCH compares shellac-paclitaxel with pectin-paclitaxel paste₂(CH₂)₆CH₃The paclitaxel paste coating is more uniform.

Observation by scanning electron microscope

1. Preparation of scanning electron microscope sample

Pectin freeze-dried powder group: taking out 1mL of the pectin colloid dispersion system prepared in example 1, performing ultrasonic homogenization, freeze-drying to obtain powder, spraying the powder visible to the eyes on the surface of a silicon wafer, and directly observing.

A paclitaxel group: the visible paclitaxel powder is directly scattered on the surface of the silicon wafer and directly observed.

paclitaxel-RGDS-OCH₂(CH₂)₆CH₃Group (2): draw 100. mu.L of paclitaxel stock solution (15mg/mL) and mix with 0.1mg of RGDS-OCH₂(CH₂)₆CH₃After ultrasonic mixing is carried out uniformly, 10 mu L of the solution is absorbed by a pipette and dropped on a silicon wafer, the silicon wafer is placed on a watch glass and wrapped by a preservative film, and the glass wafer is placed in an incubator at 37 ℃ for 4 days and dried.

Pectin-paclitaxel base the calculated drug content of the pectin-paclitaxel paste (prepared according to the method of comparative example 2) is about 1-9 × 10^-3mol/L, so the paste is diluted by 100 times and 1000 times to obtain the medicine content of about 1-9 × 10^-5mol/L and 1-9 × 10^-6The samples of mol/L are all uniform milky white liquid, and the content of the drug is about 1 to 9 × 10^-3Coating pectin-paclitaxel paste with steel shovel to obtain visible amount of meat eye on silicon wafer, and diluting to obtain a medicinal composition with content of about 1-9 × 10^-5mol/L and 1-9 × 10^-6The uniform milky white pectin-paclitaxel liquid can be sucked by a gun head, so that 10 mu L of the uniform milky white pectin-paclitaxel liquid can be respectively sucked by a pipette gun and dripped on a silicon chip because of the surfaceThe surface tension is large, and the silicon wafer is in a milky white ball shape on the surface of the silicon wafer. Putting a piece of filter paper on the watch glass, dividing the watch glass into areas, marking the information of samples on the silicon chips, then sequentially putting 3 silicon chips, wrapping the watch glass with a preservative film, tying holes, and putting the watch glass into an incubator at 37 ℃ for 4 days to dry.

pectin-RGDS-OCH₂(CH₂)₆CH₃-a paclitaxel group: calculating to obtain pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel paste (pectin-RGDS-OCH prepared according to the method of example 1)₂(CH₂)₆CH₃Paclitaxel paste) is about 1-9 × 10^-3mol/L, so the paste is diluted by 100 times and 1000 times to obtain the medicine content of about 1-9 × 10^-5mol/L and 1-9 × 10^-6The samples of mol/L are all uniform milky white liquid, and the content of the drug is about 1 to 9 × 10^-3mol/L pectin-RGDS-OCH₂(CH₂)₆CH₃Coating paclitaxel paste on silicon wafer with visible amount of flesh and eye obtained by steel shovel, wherein the diluted medicine content is about 1-9 × 10^-5mol/L and 1-9 × 10^-6mol/L pectin-RGDS-OCH₂(CH₂)₆CH₃The paclitaxel uniform milky white liquid can be sucked by a gun head, so that 10 mu L of paclitaxel uniform milky white liquid is respectively sucked by a pipette gun and dripped on a silicon wafer, and milky white spheres are formed on the surface of the silicon wafer due to high surface tension. Putting a piece of filter paper on the watch glass, dividing the watch glass into areas, marking the information of samples on the silicon chips, then sequentially putting 3 silicon chips, wrapping the watch glass with a preservative film, tying holes, and putting the watch glass into an incubator at 37 ℃ for 4 days to dry.

Shellac-paclitaxel the calculated drug content of the shellac-paclitaxel cream viscous liquid (shellac-paclitaxel cream viscous liquid prepared according to the method of comparative example 1) was about 1-9 × 10^-3mol/L, so the milky viscous liquid is respectively diluted by 10 times, 100 times and 1000 times to obtain the medicine content of about 1-9 × 10^-4mol/L、1-9×10^-5mol/L、1-9×10^-6The samples of mol/L are all uniform milky white liquid, and the content of the drug is about 1 to 9 × 10^-3Coating mol/L shellac-paclitaxel milky viscous liquid on silicon wafer with visible amount of flesh eye by using steel shovel, and diluting to obtain medicine content of about1-9×10^-4mol/L、1-9×10^-5mol/L and 1-9 × 10^-6The even milk white shellac-taxol liquid in mol/L may be sucked with the gun head, so that 10 microliter liquid is sucked with the pipette and dropped onto the silicon chip in the form of milk white ball on the surface of the silicon chip owing to the great surface tension. Putting a piece of filter paper on the watch glass, dividing the watch glass into areas, marking the information of samples on the silicon chips, then sequentially putting 4 silicon chips, wrapping the watch glass with a preservative film, tying holes, and putting the watch glass into an incubator at 37 ℃ for 4 days to dry.

2. Observation of scanning electron microscope sample

Loading: and taking out the sample table on which the silicon wafer is placed from the acetone cylinder, wiping the sample table, then pasting the double-sided black glue, sequentially placing the silicon wafers, and marking numbers at the blank positions beside the silicon wafers for distinguishing.

Spraying gold: and putting the sample table into a metal spraying instrument, starting the metal spraying instrument, setting the metal spraying time of 50s, adjusting the display to the display pressure pa value, and starting vacuumizing. When the pressure is reduced to 20pa, the gas control is pressed, and when the pressure is reduced to below 10pa, the start is pressed, and the panel starts to display the gold spraying time. And (5) finishing spraying the gold for 50s, shutting down the machine, and taking out the sample table.

And (4) observation: and (5) after the sample table is assembled, putting the sample table into a sample groove of a scanning electron microscope. Clicking an H/L button, switching a high-low power lens, finding a sample visual field needing to be observed under the low power lens, and then switching to a high power lens to observe the form of the drug matrix in the sample. Continuously adjusting the brightness contrast by clicking ABCC (when the display of the le panel is lower than 7, clicking set to adjust the brightness of the visual field, observing the panel to always display 5kV, when a certain place is irradiated for a long time, a black shadow appears in the visual field, the resolution of the probe can be adjusted, the upper or mix is changed into lower), clicking the position of the Align stable probe, clicking slow3 to preview the image in the visual field, and clicking 1280 to photograph the current visual field. The photographed picture appears at the lower left corner, the required picture is stored in time, and the preview picture at the lower left corner is deleted.

Fig. 3 is a scanning electron micrograph of the pectin lyophilized sample powder, and it can be seen from fig. 3 that: the pectin freeze-dried sample powder is not uniformly dispersed, and the pectin with better dispersion should present spheres with the diameter of about 50nm, but the spheres are gathered together at some places; the pectin structure in the form of a rod can also be seen in the field.

FIG. 4 is a scanning electron micrograph of paclitaxel powder, which is shown in FIG. 4: the paclitaxel is in a spherical structure, and the diameter of the paclitaxel is 0.8-3 mu m; but some balls have gaps on the surface and are adhered to each other; still others exist in the form of disks and adhere to each other to form irregular shapes.

FIG. 5 shows taxol-RGDS-OCH₂(CH₂)₆CH₃Scanning electron micrographs, as can be seen in FIG. 5: paclitaxel and RGDS-OCH₂(CH₂)₆CH₃Are all in a spherical structure; uniformly distributed spherical objects exist in a visual field, and the diameters of the spherical objects are mainly different from 300nm to 700 nm; occasionally, small balls that come together are visible; the whole visual field forms fog, which shows that the two substances have certain adhesiveness after being formed and can be coated uniformly.

FIG. 6 shows pectin-paclitaxel at 10^-3Scanning electron micrograph under mol/L concentration, can be seen from FIG. 6: at a concentration of 10^- ³The dispersing degree of the mol/L pectin-paclitaxel is not uniform enough, the particle sizes are not uniform, when the magnification is small, a lot of lumpy or crystalline substances can be seen in the visual field, and rod-shaped structures with different sizes can be found after the magnification; however, the length of the dispersed rod-like objects is mainly between 0.7 and 7 μm, and the selection field varies to some extent.

FIG. 7 shows pectin-paclitaxel at 10^-5Scanning electron micrograph under mol/L concentration, can be seen from figure 7: at a concentration of 10^- ⁵The pectin-paclitaxel of mol/L can be seen in the visual field with little caking substance, which indicates that the aggregation is reduced, the visual field is mostly crystalline, and the pectin-paclitaxel is rod-shaped compound after being magnified, and the length is mainly between 0.7 and 3 mu m. Indicating that the thinner the concentration, the better the dispersion, the less aggregation, more rods were observed, and the rods decreased in length. Some spherical paclitaxel was seen between the rods, mainly between 0.7-1 μm in diameter.

FIG. 8 shows pectin-paclitaxel at 10^-6Scanning electron micrograph under mol/L concentration, can be seen from FIG. 8: at a concentration of 10^- ⁶mol/L pectin-violetThe dispersion degree of the fir alcohol is more uniform, the fir alcohol is mostly in a scattered rod shape, the length of the rod shape is mainly between 0.7 and 2 mu m, and the width of the rod shape is much smaller than that of the rod shape with high concentration, and is about 100 to 200 nm. The spheres are seen between the rod-like interlaces, the diameter is mainly between 300-900 nm, and the interlaces are uniform at high concentration. The whole visual field forms fog, which shows that the two substances have certain adhesiveness after being formed and can be coated uniformly.

FIG. 9 shows pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel at 10^-3Scanning electron micrograph under mol/L concentration, can be seen from FIG. 9: at a concentration of 10^-3mol/L pectin-RGDS-OCH₂(CH₂)₆CH₃The paclitaxel is not dispersed uniformly enough, when the magnification is smaller, more dispersed images can be seen in the visual field as crystalline, rod-shaped structures with different sizes can be found after the magnification, the length of the dispersed rods is mainly between 0.7 and 7 mu m, and certain changes exist due to different selected visual fields.

FIG. 10 shows pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel at 10^-5Scanning electron microscope images under mol/L concentration; as can be seen from fig. 10: at a concentration of 10^-5mol/L pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel shows less clumpy in the field of view at a smaller magnification, indicating less aggregation, and is mostly crystalline in the field of view, and is a rod-like compound after magnification, mainly between 0.7 and 3 μm in length. Indicating that the thinner the concentration, the better the dispersion, the less aggregation, more rods were observed, and the rods decreased in length. Some spheres were observed between the rods and consisted of paclitaxel and RGDS-OCH₂(CH₂)₆CH₃The diameter of the formed mixture is mainly 400-900 nm, the balls are uniformly mixed and then staggered with the rod-shaped pectin, and the gaps are in a fog shape, so that the three substances have certain adhesion after being mixed and can be uniformly coated.

FIG. 11 shows pectin-RGDS-OCH₂(CH₂)₆CH₃-violetThe taxol is in 10^-6Scanning electron microscope images under mol/L concentration; as can be seen from fig. 11: at a concentration of 10^-6mol/L pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel is mostly dispersed in the visual field as a fine rod or filamentous compound, the length of the fine rod is mainly between 0.7 and 2 μm, and the width of the rod is much smaller than that of the prior high concentration, about 100 nm. Furthermore, more balls and irregularities were observed between the intersections of the thin rods, which were made of paclitaxel and RGDS-OCH₂(CH₂)₆CH₃The diameter of the mixture is mainly 300-700 nm, and the cross-over ratio is uniform at high concentration. The balls are uniformly mixed and then staggered with the rod-shaped pectin, and the gaps are in a fog shape, which indicates that the three substances have certain adhesiveness after being mixed and can be uniformly coated.

FIG. 12 shows shellac-paclitaxel at 10^-3Scanning electron micrograph under mol/L concentration, can be seen from figure 12: at a concentration of 10^- ³The shellac in mol/L shellac-paclitaxel has a net structure, and when the concentration is higher, the aggregation is observed in the visual field when the magnification is lower, and after the magnification, the net interlacing can be seen, and the gaps are few.

FIG. 13 shows shellac-paclitaxel at 10^-4Scanning electron microscope images under mol/L concentration; as can be seen from fig. 13: the concentration is slightly reduced, less aggregation is observed when the magnification is low, reticular interlacing can be seen after the magnification, more gaps can be seen, and spherical paclitaxel can be seen in some gaps, and the diameter is about 500 nm.

FIG. 14 shellac-paclitaxel at 10^-5Scanning electron microscope images under mol/L concentration; as can be seen from fig. 14: the concentration is lower, less aggregation is observed at lower magnification, and many dispersed networks are seen after magnification, spaced more apart from each other.

FIG. 15 shows shellac-paclitaxel at 10^-6Scanning electron microscope images under mol/L concentration; as can be seen from fig. 15: the visual field is mostly divided into scattered filaments, some fusiform objects are occasionally seen, white particles are on a large area, paclitaxel may be contained, and some spherical particles are containedThe diameter of the paclitaxel is mainly 300-700 nm.

In vitro release curve determination of four and three drug coatings

1. Establishment of paclitaxel content determination method

Herein, the drug concentration of paclitaxel is determined using High Performance Liquid Chromatography (HPLC).

1.1 preparation of solutions of the Standard Curve

After 1.2mg of paclitaxel bulk drug was weighed using an analytical balance, 20mL of chromatographic acetonitrile solution was dissolved to make the paclitaxel solution concentration at the end 60 μ g/mL, as stock solution. Sequentially diluting paclitaxel stock solution (60 μ g/mL) to obtain paclitaxel standard solutions with concentration gradients of 0.5, 5, 10, 20, 30, and 60 μ g/mL.

1.2 examination of chromatographic conditions for paclitaxel detection

Taking a proper amount of 20 mu g/mL paclitaxel solution, taking chromatographic acetonitrile as a blank control, scanning an ultraviolet spectrophotometer within the wavelength range of 200-300 nm, and selecting the measurement wavelength according to the absorption spectrum result.

FIG. 16 is a graph showing the UV absorption spectrum of a paclitaxel solution (20. mu.g/mL). Based on the uv absorption spectrum results shown in the reference and fig. 16, the paclitaxel hplc conditions were determined as follows: detection wavelength: 228 nm; a chromatographic column: shimadzu ODSC-18 column (100 mm. times.4.6 mm, 5 μm); mobile phase: acetonitrile-water (60: 40, v: v); flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction amount: 10 μ L.

The paclitaxel stock solution (60 mug/mL) is used as a pre-experiment to record the peak time and the peak area, and the observation time length is determined. According to the results, the time of appearance of paclitaxel was determined so that the peak area of paclitaxel could be determined to calculate the content after mixing pectin and shellac.

The paclitaxel standard solutions with concentration gradients of 0.5, 5, 10, 20, 30, and 60 μ g/mL were extracted with a disposable syringe, and the sample was measured by the above-mentioned chromatographic method after passing through a 0.45 μm filter, and the results are shown in FIG. 17, in which FIG. 17 is a high performance liquid chromatogram of a paclitaxel stock solution (60 μ g/mL). The horizontal axis is paclitaxel concentration, the vertical axis is paclitaxel chromatogram peak area, a linear regression curve is established, and a standard curve is drawn as shown in fig. 18. FIG. 18 is a standard curve of a standard solution of paclitaxel, as can be seen from FIG. 18: the linear relation of the standard curve of the paclitaxel standard solution is good.

2. Establishment of release curve measuring method of drug coating

The in vitro release of the drug is carried out at a constant temperature of 37 ℃, and the release medium is deionized water. Coating the medicinal coating solution (shellac-paclitaxel, pectin-RGDS-OCH) prepared in the preparation process of medicinal coating₂(CH₂)₆CH₃-paclitaxel) into a six-well plate, adding 4mL of deionized water into each well, shaking at constant temperature of 37 ℃, and taking out all release media at regular time of 0h, 2h, 6h, 12h, 24h, 36h and 48h at 300rpm, respectively, and simultaneously supplementing deionized water with the same volume to calculate the concentration of the drug in water.

3. Determination of in vitro Release profiles of three drug coatings

4mL of the total release medium taken out each time was dispensed into 5mL of EP tubes, each 2mL, labeled with date and time and name, the puncture holes were sealed with para film, and the tubes were stored in a refrigerator at-20 ℃. After complete freezing, all samples from the EP tubes were lyophilized. Dissolving lyophilized powder in EP tube with 1mL of chromatographic acetonitrile, sucking 1mL of solution with disposable syringe, filtering with 0.45 μm filter membrane, and measuring by the above-mentioned chromatographic method. And recording the peak area of the chromatogram, substituting the peak area into a standard curve to calculate the content and drawing a cumulative release curve graph.

FIG. 19 is a shellac-paclitaxel release profile, as can be seen from FIG. 19: at 300rpm, almost no paclitaxel was released within 48h from the shellac-paclitaxel drug coating.

Fig. 20 is a release profile of pectin-paclitaxel, as can be seen from fig. 20: at 300rpm, paclitaxel in the pectin-paclitaxel drug coating was released up to 90% within 2h and up to 99% within 6 h. The release was fastest within 2h and stabilized after 6 h.

FIG. 21 shows pectin-RGDS-OCH₂(CH₂)₆CH₃-release profile of paclitaxel, as can be seen from fig. 21: at the rotating speed of 300rpm, pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel drug coating releases paclitaxel up to 90% within 2h and up to 99% within 6 h. The release was fastest within 2h and stabilized after 6 h. The release profile is similar to that of pectin-paclitaxel, indicating RGDS-OCH₂(CH₂)₆CH₃The addition of (a) does not alter the ability of the pectin to release paclitaxel.

To determine the drug loss that may occur during the course of the experiment and for experimental stringency, the amount of paclitaxel remaining in the drug coating after the end of the release time was determined, ensuring that the release profile was logical. And (3) putting the six-hole plate with all the external phase medium taken out into an incubator at 37 ℃ for drying, then adding acetonitrile, stirring and oscillating until all the paclitaxel in the coating enters the acetonitrile, then transferring into a 5mL EP tube, and rinsing with the acetonitrile until the transfer is complete, wherein the total volume of the acetonitrile used is 4 mL. 1mL of the solution was aspirated through a 0.45 μm filter using a disposable syringe and the sample was measured by the chromatography method described above. The chromatographic peak areas were recorded and substituted into the standard curve to calculate the content, the results are shown in table 1. As can be seen from table 1: the surface residue of paclitaxel in shellac-paclitaxel drug coating is 630.4 + -2.1 μ g, almost no release, and pectin-paclitaxel and pectin-RGDS-OCH₂(CH₂)₆CH₃The surface residual quantity of paclitaxel is 15.8 +/-2.4 mug and 18.6 +/-0.3 mug respectively, the release reaches 99 percent, and the result is consistent with the release curve.

Table 1: determination of residual amount on three surfaces

4. Optimized drug coating in vitro release profile determination

Drug-coated balloons (DCB) were first developed for coronary heart disease treatment, where the sites of application were coronary arteries, including primary coronary lesions, small coronary vessels, and branch lesions. The role of DCB in the treatment of arterial occlusive disease of the lower extremities (PAD) has recently been studied, the selected sites being mainly the femoropopliteal artery and the subclavian artery. The linear velocity of blood flow is inversely proportional to the total cross-sectional area of the vessel. The blood flow of different blood vessels is different, namely 18-22 cm/s of aorta and 7-8 cm/s of vena cava. Arterial blood flow rate also varies greatly with diastole and systole, and blood flow is proportional to pressure. Under a normal state, the blood flow of coronary artery is 300-400 mL/min, and under a motion state, the blood flow is increased by several times. And a plurality of cells exist in blood flow to scour the blood vessel wall, which is also the reason for a plurality of losses in the process of delivering the drug coating balloon. Therefore, when the drug coating is used for measuring the release curve, the rotating speed needs to be further increased to wash the drug coating coated on the surface of the six-hole plate, so that the release is accelerated.

Coating with medicinal coating (pectin-paclitaxel, pectin-RGDS-OCH)₂(CH₂)₆CH₃-paclitaxel) with 4mL deionized water per well, oscillating at constant temperature of 37 ℃ at 1200rpm, taking out all release media at regular time of 0min, 10min, 30min and 60min respectively, and simultaneously supplementing deionized water with the same volume to calculate the concentration of the drug in water.

4mL of the total release medium taken out each time was dispensed into 5mL of EP tubes, each 2mL, labeled with date and time and name, the puncture holes were sealed with para film, and the tubes were stored in a refrigerator at-20 ℃. After complete freezing, all samples from the EP tubes were lyophilized. 2mL of chromatographic acetonitrile was added to dissolve the sample, and 1mL of the sample was taken out of the solution through a 0.45 μm filter by a disposable syringe and subjected to sample injection measurement by the above-mentioned chromatographic method. And recording the peak area of the chromatogram, substituting the peak area into a standard curve to calculate the content and drawing a cumulative release curve graph.

Fig. 22 is a pectin-paclitaxel rapid release profile, as can be seen in fig. 22: at 1200rpm, paclitaxel in the pectin-paclitaxel drug coating was released up to 99% in 10min, after which it was almost not released.

FIG. 23 shows pectin-RGDS-OCH₂(CH₂)₆CH₃Rapid release profile of paclitaxel, as can be seen in fig. 23: at the rotating speed of 1200rpm, pectin-RGDS-OCH₂(CH₂)₆CH₃In the coating of paclitaxel drugsThe paclitaxel was released up to 99% in 10min, and then almost no longer released. The release curve is similar to the rapid release curve of pectin-paclitaxel, which indicates that the peptide chain is RGDS-OCH₂(CH₂)₆CH₃The addition of (a) does not alter the ability of the pectin to release paclitaxel.

Fifth, optimize experiment coating sacculus

By changing the proportion of ultrapure water for dissolving pectin and the using amount of absolute ethyl alcohol for dissolving paclitaxel, the two solutions are mixed to form a non-paste. The main method comprises the following steps: keeping the original dosage, sealing the paste after mixing uniformly, and storing in a refrigerator to obtain uniform emulsion. Then, extra ultrapure water is added for dissolution, and uniform vortex mixing is carried out, so that white emulsion capable of being subjected to liquid transfer treatment can be obtained. The optimized coating formula enables the coating process to be simpler, and then the coating is coated on a blank balloon to observe indexes such as the adhesion of the coating.

1. Pectin-paclitaxel coating process optimization

The white paste (pectin-paclitaxel in comparative example 2) was stored in a refrigerator at-4 ℃ for 2 days to obtain a white emulsion, and 400. mu.L of the white emulsion was taken out by a pipette gun and added with ultrapure water of the same volume to obtain a white liquid which was diluted and mixed uniformly and was very easy to be sucked by a pipette.

2. pectin-RGDS-OCH₂(CH₂)₆CH₃Optimization of paclitaxel coating Process

Taking improved pectin-paclitaxel white liquid 400 μ L (pectin-paclitaxel prepared in part 1 of the fifth part), adding 3mg RGDS-OCH₂(CH₂)₆CH₃A white turbidity was found to occur and the solution started to become colorless. Then, 400. mu.L of ultrapure water and 400. mu.L of absolute ethanol were added thereto, and sufficient sonication revealed that the white turbidity was gradually reduced and the solution began to turn white again.

FIG. 24 shows pre-sonication pectin-paclitaxel (a) and pectin-RGDS-OCH₂(CH₂)₆CH₃-picture of paclitaxel (b); as can be seen from fig. 24: the initial mixing of the RGDS carbon chain into pectin-paclitaxel may occurA white turbidity and the solution started to become colorless; the right picture is pectin-RGDS-OCH after ultrasonic treatment₂(CH₂)₆CH₃Paclitaxel became a homogeneous white emulsion, similar in morphology to pectin-paclitaxel homogeneous white emulsion.

Sixth, acute toxicity test

Since pectin eventually needs to enter the blood vessel, biosafety is a concern and acute toxicity testing is performed in mice. The active ingredient of the prior Dior II DCB paclitaxel is 3 mug/mm²A balloon with an effective length of 60mm and a diameter of 7mm, and a total coated area of 1320mm²Therefore, the amount of paclitaxel applied was about 4.0 mg. The amount of paclitaxel administered to the mice was calculated based on the reduced coefficients for adults and mice administered. Pectin according to the optimal proportion: paclitaxel was administered in pectin amounts.

1. Acute toxicity test of pectin colloid dispersion system

1.1 sample preparation

Pectin colloid dispersion system: dissolving 50mg of pectin powder in 1mL of ultrapure water by using a balance, and uniformly mixing by using ultrasonic waves to obtain light brown uniform liquid with a Tyndall effect.

1.2 animal experiments

10 healthy male ICR mice were weighed, numbered 1-10. The mouse status was observed by tail vein injection of 0.5 mL. Within 3 days, the mice work and rest normally, and the eating and activity states are normal. No abnormality occurred within 10 days.

Acute toxicity test of 2-pectin-paclitaxel

2.1 sample preparation

Pectin colloid dispersion system: weighing 50mg of pectin powder by using a balance, dissolving the pectin powder by using 4.5mL of ultrapure water, and uniformly mixing by using ultrasonic waves to obtain light brown uniform liquid with a Tyndall effect.

Paclitaxel solution: weighing 1.25mg pectin powder by using a balance, dissolving the pectin powder by using 0.5mL absolute ethyl alcohol, and uniformly mixing by using ultrasonic waves to obtain uniform and transparent liquid.

Adding 0.5mL of paclitaxel solution into a pectin colloid dispersion system, and uniformly mixing by ultrasonic to obtain a light brown uniform liquid as a dosing stock solution.

2.2 animal experiments

Example 2

Preparation of paclitaxel stock solution:

Preparing a pectin colloid dispersion system:

pectin-RGDS-OCH₂(CH₂)₆CH₃Preparation of colloidal dispersion system:

in a 1.5mL EP tube, 180. mu.L of pectin-RGDS-OCH was added₂(CH₂)₆CH₃Dispersing in colloid system, adding 100 μ L (15mg/mL) of paclitaxel stock solution, layering, and manually stirring to obtain white paste named pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel drug coating solution 2.

The RGDS-OCH₂(CH₂)₆CH₃The preparation method of (2) is the same as in example 1.

Adopt and carry outThe obtained pectin-RGDS-OCH was tested in the same manner as described above₂(CH₂)₆CH₃The fast release profile of paclitaxel drug coating solution 2, the results are shown in fig. 25. As can be seen from fig. 25: at the rotating speed of 1200rpm, pectin-RGDS-OCH₂(CH₂)₆CH₃Paclitaxel in the paclitaxel drug coating is released up to 99.5% in 10min, after which it is almost not released.

Example 3

Preparation of docetaxel stock solution:

weighing 6mg docetaxel with 1.5mL EP tube, adding 400 μ L anhydrous ethanol for dissolving, performing vortex ultrasonic treatment until the docetaxel is completely dissolved and clarified to obtain docetaxel stock solution with concentration of 15mg/mL, sealing with para film, and storing in a refrigerator at 4 deg.C.

Preparing a pectin colloid dispersion system:

pectin-RGDS-OCH₂(CH₂)₆CH₃Preparation of colloidal dispersion system:

pectin-RGDS-OCH₂(CH₂)₆CH₃-preparation of docetaxel drug coating solution:

in a 1.5mL EP tube, 90 μ L of pectin-RGDS-OCH was added₂(CH₂)₆CH₃Colloid dispersed system, adding 100 μ L (15mg/mL) of prepared docetaxel stock solution, layering, and manually stirring to obtain white paste.

pectin-RGDS-OCH testing of the Release Curve given in example 1₂(CH₂)₆CH₃The rapid release profile of docetaxel drug coating solution, the result is shown in fig. 26, and it can be seen from fig. 26 that: at the rotating speed of 1200rpm, pectin-RGDS-OCH₂(CH₂)₆CH₃Docetaxel drug coating released up to 99.5% in 10min, after which it was almost not released.

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

1. The drug coating solution for drug-coated balloons is characterized by comprising pectin and RGDS-OCH₂(CH₂)₆CH₃And an active agent; the active drug is paclitaxel or docetaxel.

2. The drug coating solution for drug-coated balloons of claim 1, characterized in that the pectin, RGDS-OCH₂(CH₂)₆CH₃The mass ratio of the active medicine to the active medicine is (5.7-15): (57-150): 1500.

3. the drug coating solution for drug-coated balloons according to claim 1 or 2, characterized in that the pectin is added in the form of a pectin dispersion solution having a mass concentration of 0.01 to 1.0 mg/mL; the active drug is added in the form of an active drug absolute ethyl alcohol solution, and the concentration of the active drug absolute ethyl alcohol solution is 5-50 mg/mL.

4. The drug coating solution for the drug layer balloon according to claim 3, wherein the pectin dispersion solution is prepared by a method comprising the following steps:

5. A drug-coated balloon, which is characterized by comprising a blank balloon and a drug coating coated on the blank balloon, wherein the drug coating is coated by the drug coating liquid according to any one of claims 1 to 4.

6. The drug-coated balloon of claim 5, wherein the drug coating has a thickness of 1-2 mm.

7. The drug-coated balloon of claim 5, wherein the blank balloon is made of polyethylene plastic or latex nylon.

8. The drug-coated balloon of claim 7, wherein the blank balloon has a diameter of 3-6 mm.