CN116271186B

CN116271186B - Drug-loaded embolism microsphere and preparation method thereof

Info

Publication number: CN116271186B
Application number: CN202310117812.3A
Authority: CN
Inventors: 张展鹏
Original assignee: Shanghai Ruining Biotechnology Co ltd
Current assignee: Shanghai Ruining Biotechnology Co ltd
Priority date: 2023-02-15
Filing date: 2023-02-15
Publication date: 2024-08-09
Anticipated expiration: 2043-02-15
Also published as: CN116271186A

Abstract

The invention belongs to the technical field of medical appliances, in particular to a drug-loaded embolic microsphere and a preparation method thereof, the gelatin and polyethylene glycol derivative are used as water phase materials to prepare microemulsion with oil phase containing emulsifier, and then the microemulsion is crosslinked by aldehyde crosslinking agent and photo-crosslinked to form the double-network hydrogel microsphere. The degradable medicine-carrying embolism microsphere can rapidly and automatically adsorb medicines under the electrostatic action, the medicine carrying performance can be adjusted through the component proportion, and the high medicine carrying capacity and the slow release of the medicines can be realized. The drug-loaded embolism microsphere provided by the invention has good biocompatibility, can be completely degraded, absorbed and metabolized by a human body, and the degradation period can be adjusted through the component proportion, so that the vascular access can be ensured within a controllable period of time after the microsphere is used for embolism.

Description

Drug-loaded embolism microsphere and preparation method thereof

Technical Field

The invention belongs to the technical field of medical instruments, and further belongs to the technical field of medical biopolymer materials, in particular to a drug-loadable embolism microsphere and a preparation method thereof.

Background

Interventional therapy is an emerging treatment method between surgical treatment and medical treatment, which is characterized in that under the condition of not exposing focus, a micro channel with the diameter of a few millimeters is made on blood vessels and skin, or the focus is treated by the guidance of image equipment (such as angiography machine, fluoroscopy machine, CT, MR, B ultrasonic and the like) through the original pipeline of a human body, and the minimally invasive treatment method has the advantages of small trauma, simplicity, safety, effectiveness, fewer complications and obvious reduction of hospitalization time.

The number of liver cancer attacks in China exceeds 40 ten thousand per year, 53% of the world accounts for, and more than 70% of patients are in middle and late stages when diagnosed, interventional therapy becomes the most important treatment means which can not cut liver cancer by operation, and ductal arterial chemoembolization (TACE) is the most frequently and most mature interventional technology used at present, one of the characteristics of the treatment is that the concentration of perfusion medicines is high, for example, the hepatic arterial perfusion of liver cancer is 100-400 times higher than that of medicines administered by vein, and high-concentration chemotherapy can play a role in killing a large amount of tumor cells and also can alleviate systemic adverse reactions, so the treatment becomes one of the important methods of anticancer treatment; secondly, the vascular embolism effect, after the tumor blood vessel is blocked, the tumor tissue is denatured and necrotized due to ischemia. In the interventional therapy of treating tumor cells, embolic materials play an important role.

The first generation solid embolism products are gelatin sponge and polyvinyl alcohol (PVA) particles with irregular shapes, but the solid embolism products have the defects of easy aggregation, difficult injection, incomplete embolism of blood vessels and the like in clinical use due to the irregular shapes; the second generation is a spherical blank microsphere with regular shape, such as a polyvinyl alcohol microsphere, a sodium alginate microsphere and the like, is easy to push for clinical use, and has better embolism effect than the first generation; the third generation of embolism material is medicine-carrying embolism microsphere, which has the characteristics of regular shape and uniform particle size of the second generation of product, and has good elasticity, and can be injected through a thinner catheter. In addition, aiming at the fact that most first-line anti-cancer drugs are drugs with positive charges, the existing drug-loaded microspheres can modify the chemical structure of the microspheres by adding anionic functional groups, so that the purposes of loading the anti-cancer drugs through charge and releasing the drugs after embolism are achieved. The most dominant drug eluting microspheres CALLISPHERES, HEPASPHERE and DC head in the current market have a highest drug loading of 25-40 mg of epirubicin per 1ml microsphere. However, none of these microspheres is degradable, and its clinical disadvantages are very evident: 1) The process of microsphere embolism easily causes the situation of mistaking embolism, namely, the embolism microsphere flows back to other blood vessels, and causes permanent injury to normal organ tissues; 2) After the permanent embolism of the target blood vessel is realized, necrosis of the blood vessel is easy to cause, and the generation of new collateral blood capillary is easy to cause, so that the subsequent interventional treatment can not be carried out through the blood vessel again; 3) Non-degradable materials can produce long-term foreign body reactions.

In the existing patent inventions, preparation of degradable embolic microspheres is also proposed, and the degradable embolic microspheres mainly comprise gelatin microspheres synthesized by natural polymers such as the published patent CN115245591A and hyaluronic acid microspheres such as the published patent CN115414522A, however, due to poor mechanical properties of natural polymer materials, deformation and rupture easily occur in the use process, and the degradation rate of natural polymers is difficult to control.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the degradable hydrogel embolism microsphere with high drug-loading efficiency and ideal drug slow-release performance, and the microsphere has excellent mechanical property and good biocompatibility. The specific technical scheme of the invention is as follows:

the invention provides a drug-carrying embolism microsphere, which is formed by crosslinking an aldehyde crosslinking agent and a photoinitiator after microemulsion is prepared by taking gelatin, polyethylene glycol derivatives and a drug-carrying functional monomer as water phase materials and an oil phase containing an emulsifier; the gelatin is A-type or B-type gelatin with jelly strength ranging from 100 g to 300g Bloom; the polyethylene glycol derivative is poly (ethylene glycol) diacrylate PEGDA or poly (ethylene glycol) methacrylate; the molecular weight of the polyethylene glycol derivative is between 200 and 1000000 Da; the drug-carrying embolic microspheres are spherical and have a particle size ranging from 100 to 1000 μm, preferably, the drug-carrying embolic microspheres can be screened and stored separately in different particle size ranges of, for example, 100 to 300 μm,300 to 500 μm or 500 to 700 μm, and further preferably, have a diameter ranging from 100 to 300 μm.

Preferably, the mass ratio of the gelatin to the polyethylene glycol derivative to the drug-loadable functional monomer is 1: (0.1-10): (0.1-1).

Preferably, the aldehyde cross-linking agent is selected from one or more of formaldehyde, glutaraldehyde and aldehyde dextran sulfate; wherein, the aldehyde cross-linking agent is preferably a mixture of glutaraldehyde and aldehyde dextran sulfate as a mixed cross-linking agent; compared with an aldehyde cross-linking agent of Shan Wu dialdehyde components, the slow release effect of the drug-loaded embolic microsphere can be further improved by adding the aldehyde dextran sulfate as the aldehyde cross-linking agent after mixing; further preferably, in the mixed cross-linking agent, the mass ratio of glutaraldehyde to the aldehyde dextran sulfate is 5:2.

Preferably, the drug-loadable functional monomer is 2-acrylamide-2-methylpropanesulfonic Acid (AMPS) or methacrylic sulfonic acid, more preferably AMPS.

Preferably, the photoinitiator is photoinitiator 2959.

Preferably, the emulsifier concentration in the oil phase is from 0.1% to 5%, more preferably from 0.5% to 2%.

Preferably, the volume ratio of the aqueous phase to the oil phase is 1:10 to 1:20, more preferably 1:10 to 1:12.

Preferably, the emulsifier is selected from one or more of span and tween; the oil phase is selected from one or more of soybean oil, sesame oil, liquid paraffin, silicone oil, or water-immiscible organic solvent.

The drug-loaded embolic microsphere disclosed by the invention can be prepared by the following steps:

Step S1, gelatin, polyethylene glycol derivatives and functional monomers capable of carrying medicines are dissolved in water and placed in a water bath with the temperature of 50-90 ℃ to be stirred for 30 minutes to prepare aqueous phase solution;

Step S2, adding the aqueous phase solution prepared in the step S1 into the oil phase containing the emulsifying agent at 50-70 ℃, stirring, then reducing the temperature to 2-8 ℃ and continuously stirring;

Step S3, adding an aldehyde cross-linking agent and a photoinitiator into the microsphere obtained in the step S2, applying ultraviolet irradiation, and continuously reacting for a period of time at 2-8 ℃;

and S4, washing the microspheres obtained in the step S3 by using a washing liquid, and removing an oil phase to obtain the gelatin-polyethylene glycol composite microspheres.

The drug-loaded embolic microsphere prepared by the method can be further stored in a freeze-drying way or in physiological saline or buffer solution.

The invention also discloses application of the drug-loaded embolic microsphere in preparation of an anti-tumor preparation, and specifically, the drug-loaded embolic microsphere is mixed with an anti-tumor drug solution with positive charges, and drugs are adsorbed through ionization. Preferably, the positively charged drug is selected from one or more of the group consisting of ib Li Tikang, epirubicin, daunorubicin.

The beneficial effects are that:

Firstly, in order to realize the degradability of the drug-loaded embolism microsphere, the application adopts the natural polymer material gelatin as a main degradable preparation raw material, but at the same time, as the prior art mentioned in the background art, because the mechanical property of the natural polymer gelatin is poor, deformation and fracture easily occur when the natural polymer gelatin is extruded, in order to overcome the limitation of the natural polymer gelatin, the application adopts the polyethylene glycol derivative composite gelatin to prepare the microsphere so as to improve the compression modulus of the microsphere; it should be noted that, although the compression modulus of the gelatin microsphere with a single component can be improved by increasing the concentration of the gelatin in a certain concentration range, the concentration of the gelatin microsphere generally cannot exceed 40%, and the too high concentration of the gelatin can lead to the problems that the viscosity of an aqueous solution is too high, so that the emulsification process is easy to agglomerate, the particle size of the microsphere is difficult to control, and the particle size is too large, and on the other hand, if the emulsification process is easy to cause air to wrap, a hollow porous structure is formed in the prepared microsphere, so that the compression modulus of the microsphere is reduced; therefore, the application adopts polyethylene glycol derivative composite gelatin to prepare the microsphere so as to improve the mechanical property of the microsphere; therefore, the microsphere disclosed by the application has controllable mechanical properties and can meet the requirements of catheter injection of various specifications. The microsphere provided by the application has excellent mechanical properties, and the elastic modulus is 50-1000 kPa.

Secondly, the microsphere has a negatively charged structure from gelatin and a drug-carrying monomer, when aldehyde dextran sulfate is used as a cross-linking agent, the cross-linking agent also carries a negatively charged sulfate group, has extremely strong affinity to drugs with higher positive charge groups, such as daunorubicin, and has high drug-carrying capacity; the adopted drug-loaded monomer has negative-electricity group sulfonate, so that the adsorption quantity can be better regulated and controlled, and the negative charge density of the final microsphere is controlled by regulating the feeding ratio of the drug-loaded monomer, so that the drug adsorption level same as that of the commercial microsphere is maintained;

Again, the composite drug-loaded embolic microsphere prepared by the invention is used for slow release of drugs, and has the advantages compared with the traditional drug-loaded embolic microsphere DC/LC bead in the prior art that: the drug-loaded embolic microsphere provided by the invention has different negative groups and has different adsorption capacities on drugs, and after being implanted into a human body, the drug-loaded embolic microsphere can realize slow and stable release of the drugs, avoid abrupt release or too slow release of the drugs, improve the therapeutic effect of the drugs and reduce systemic toxicity caused by the drugs. Specifically, the charged groups of the drug-loaded embolic microspheres provided by the invention have diversity of types and distribution; because carboxyl groups of amino acids of gelatin, sulfonate groups of glucan and sulfonate groups of drug-carrying monomers have different electrostatic acting forces, chemical environments are different, and steric hindrance is diversified, the drugs have different adsorption constants and desorption constants, and finally, the adsorption and release behaviors of the drugs are different. The traditional drug-loaded embolic microsphere DC/LC bead adsorbs the drug through sulfonate with high charge density and adsorption strength, so that most of the drug cannot be released; the invention can release the absorbed medicine to a greater extent.

In addition, the composite drug-loaded embolism microsphere prepared by the invention has good biocompatibility and can be degraded in vivo, and is completely degraded, absorbed and metabolized by human body; the degradation speed can be realized by the proportion of different gelatin-polyethylene glycol, and the degradation time can be controlled within the time range of 1-90 days; thereby ensuring that the vascular recanalization can be carried out within a controllable period of time after the microsphere embolism, reducing the risk caused by the error embolism, reducing the generation of collateral new capillary vessels and the reservation of main blood vessels, and facilitating the subsequent treatment again.

Finally, the preparation process of the microsphere is simple, and compared with the high-temperature reaction and the use of toxic reagents and solvents related to the traditional preparation process of the PVA microsphere, the preparation process is safer and easier to control and has lower cost.

Drawings

FIG. 1 is a partial microsphere light spectrum prepared in experiment set 6 of example 1;

Fig. 2 is a graph showing drug release profiles of hydrogel microspheres prepared from all experimental groups in example 4.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are illustrative only and are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

The percentages referred to in the following examples are mass percentages; the gelatin starting materials used were 250g bloom, type b, produced from Sigma); PEGDA referred to in the examples below are all poly (ethylene glycol) diacrylates of number average molecular weight mn=700, produced from Sigma;

Example 1 preparation of gelatin-polyethylene glycol double network hydrogel microspheres

1. Preparing a solution

Aqueous phase: respectively preparing 10ml of PBS composite solution according to water phase raw materials in different experimental groups by referring to Table 1, wherein the specific dissolution mode is that the PBS composite solution is heated and dissolved in a water bath at 70 ℃, and the dissolution is promoted by stirring; operating in a dark place;

An oil phase: liquid paraffin, 0.5% span80, 10ml, specifically melting mode is that preheating in 70 ℃ water bath, operating in a dark place.

2. The preparation method of the microsphere comprises the following steps:

adding the oil phase into a beaker, and stirring in a water bath at 70 ℃; adding the water phase, and stirring for 30 minutes to realize emulsification; after emulsification, transferring to ice bath, cooling to 2-8deg.C, then slowly adding cross-linking agent, and irradiating with ultraviolet rays, stirring for 1hr for cross-linking. Washing with ice water containing surfactant, solid-liquid separating, sieving to collect microspheres with different particle diameters, and packaging.

3. And (3) testing the mechanical properties of the microspheres:

Microspheres with the diameter ranging from 700 um to 1000um are selected to be piled up in a mould, and are placed in a texture analyzer to measure the compression modulus of the microspheres, so that the mechanical property of the microspheres is represented.

TABLE 1

Analysis of results:

The difference between the experimental group 1 and the experimental group 2 is only the concentration of gelatin, and the compression modulus values of the experimental group 1 and the experimental group 2 are compared, so that the compression modulus of the pure gelatin microsphere is improved along with the rising of the concentration of gelatin; comparing experiment group 4 with experiment group 2, it can be known that replacing part of gelatin in experiment group 2 with PEG with equal mass concentration can further improve compression modulus, and the effect is more obvious than increasing gelatin with corresponding concentration; the PEG-gelatin composite double-crosslinked hydrogel structure can obviously improve the compression modulus of the microsphere, thereby breaking the limitation of a single gelatin network on the crosslinking density and the compression modulus;

The difference between the experimental group 2 and the experimental group 3 is only that the cross-linking agent is different, and the comparison of the compression modulus values of the two shows that under the same conditions of the raw materials and the particle size range, the mechanical property of the hydrogel microsphere crosslinked by glutaraldehyde is slightly better than that of the hydrogel microsphere crosslinked by aldehyde dextran sulfate.

And observing the microsphere state under a light microscope: the hydrogel microspheres with regular round spheres can be obtained from each experimental group. An experimental group 6 microsphere light map is shown in FIG. 1. The microsphere surface was observed to be smooth and uniform in particle size.

Example 2 gelatin-polyethylene glycol double network hydrogel microspheres carrying Li Tikang

1. Preparing a solution

Aqueous phase: respectively preparing 10ml of PBS composite solution according to water phase raw materials in different experimental groups in a specific dissolution mode by heating and dissolving in a water bath at 70 ℃ and promoting dissolution by stirring according to the reference table 2; operating in a dark place;

2. The preparation method of the microsphere comprises the following steps:

3. Microsphere medicine carrying step:

Selecting 1ml of microsphere sample with diameter ranging from 100-300um, adding into 5ml of solution Li Tikang mg/ml of solution, performing drug adsorption, measuring the drug concentration of supernatant at 30min, 1hr, 2hr, and 3hr, and when the drug concentration does not change, the corresponding drug adsorption amount is the maximum drug loading amount.

TABLE 2

Analysis of results:

Compared with the gelatin single hydrogel network of the experimental group 7, the introduction of polyethylene glycol and AMPS hydrogel network in the experimental group 8 can increase the adsorption sites of the medicine, so that the medicine carrying capacity of the double-network hydrogel microsphere can be improved. The difference between the experimental group 8 and the experimental group 9 is only that the aldehyde dextran sulfate is used as the cross-linking agent, and the comparison of the drug loading data of the two groups shows that the influence of the aldehyde dextran sulfate as the cross-linking agent on the drug loading is not obvious for the Li Tikang, the difference between the experimental group 8 and the experimental group 9 is only that the configuration concentration of the gelatin, and the comparison of the drug loading data of the two groups shows that the influence of the proportion of the gelatin on the drug loading is small.

Example 3 epirubicin-loaded gelatin-polyethylene glycol double network hydrogel microspheres

1. Preparing a solution

Aqueous phase: referring to table 3, the aqueous phase raw materials in different experimental groups were respectively prepared into 10ml of PBS composite solution, specifically, the dissolution was carried out by heating in a water bath at 70 ℃ and accelerating the dissolution by stirring; operating in a dark place;

2. The preparation method of the microsphere comprises the following steps:

3. Microsphere medicine carrying step:

selecting 1ml of microsphere sample with diameter of 100-300um, adding into 10ml of epirubicin solution with diameter of 10mg/ml, performing drug adsorption, measuring the drug concentration of supernatant at 30min, 1hr, 2hr, and 3hr, and making the corresponding drug adsorption amount be maximum drug loading amount when the drug concentration does not change.

TABLE 3 Table 3

Analysis of results:

By comparing experimental group 11 with experimental group 12, or comparing experimental groups 13 and 14, it is known that the double-network hydrogel can increase the drug adsorption amount compared to the single-network gelatin hydrogel. The single network polyethylene glycol hydrogels of experimental group 15 and experimental group 16 also had lower drug adsorption than the dual network system. Comparing experiment group 12 with experiment group 14 or comparing experiment group 11 with experiment group 12, it is known that the use of the aldehyde-modified dextran sulfate as a cross-linking agent can effectively increase the drug adsorption amount.

Example 4 daunorubicin-loaded gelatin-polyethylene glycol double network hydrogel microspheres

1. Preparing a solution

Aqueous phase: respectively preparing 10ml of PBS composite solution according to water phase raw materials in different experimental groups in a specific dissolution mode by heating and dissolving in a water bath at 70 ℃ and promoting dissolution by stirring according to the reference table 4; operating in a dark place;

2. The preparation method of the microsphere comprises the following steps:

3. Microsphere medicine carrying step:

Selecting 1ml of microsphere sample with size of 100-300um, adding into 5ml of daunorubicin solution with concentration of 20mg/ml for drug adsorption, measuring the drug concentration of supernatant at 30min, 1hr, 2hr, and 3hr, and obtaining maximum drug loading when the drug concentration does not change.

4. Microsphere drug release testing step:

After the absorption reaches the plateau phase, all the supernatant is sucked away, 5ml of physiological saline is added, and the mixture is placed in a water area shaking table at 37 ℃ for shaking, so that a drug release experiment is carried out. At 30min,1hr,2hr,3hr,1d,2d,3d,4d,7d,8d,9d,10d,11d time points, centrifuging, removing all supernatant with a pipette, supplementing 5ml physiological saline, shaking again, and standing on a shaker. The concentration of daunorubicin in the supernatant taken at each time point was measured by ultraviolet rays, and thus a drug release profile was calculated as shown in fig. 2.

TABLE 4 Table 4

Analysis of results

From the results of the drug adsorption experiments, the dual-network hydrogel microspheres can adsorb a large amount of daunorubicin drugs, and the drug adsorption amount is less sensitive to the composition ratio of the dual-network hydrogel.

From the drug release profile, it can be seen that the double hydrogel network of experiment 17, experiment 18, experiment 19 can achieve a slower and sustained release of the drug, and the therapeutic effect can be maintained for a longer period of time; whereas the single hydrogel network system of experimental group 20, 22 exhibited a faster drug release. In addition, the aldehyde dextran sulfate as a cross-linking agent has very important influence on the slow release of the medicine: the release of the drug was very rapid as in experimental group 21 without the use of the aldehyde dextran sulfate.

Example 5 in vitro degradation of gelatin-polyethylene glycol double network hydrogel microspheres

The operation steps are as follows: the aqueous gel microspheres prepared in experimental groups 17-22 of example 4 were placed in centrifuge tubes, 10mL of PBS buffer (pH 7.4) was added, and then placed in 60℃water with shaking. Taking out and centrifuging at a fixed time point, removing supernatant, weighing the mass of the residual microspheres, comparing the mass with the initial mass, calculating the degradation rate, adding 10mLPBS buffer solution (pH 7.4) again, and placing in a water bath with 60 ℃ for continuous oscillation.

TABLE 5

Experimental group	21-Day degradation rate%
		17	31％
18	26％
		19	37％
20	61％
		21	76％
22	56％

Claims

1. The drug-carrying embolism microsphere is characterized in that the drug-carrying embolism microsphere is prepared by taking gelatin, polyethylene glycol derivatives and drug-carrying functional monomers as water phase materials to prepare microemulsion with oil phase containing emulsifying agent, and then crosslinking the microemulsion by aldehyde crosslinking agent and photoinitiator; the gelatin is selected from A type gelatin or B type gelatin with jelly strength in the range of 100-300 g Bloom; the polyethylene glycol derivative is poly (ethylene glycol) diacrylate PEGDA or poly (ethylene glycol) methacrylate; the molecular weight of the polyethylene glycol derivative is 200-1000000 Da; the drug-loaded embolism microsphere is spherical, and the particle size range is 100-1000 mu m;

the aldehyde cross-linking agent is a mixture of glutaraldehyde and aldehyde dextran sulfate, and is used as a mixed cross-linking agent;

The medicine-carrying functional monomer is 2-acrylamide-2-methylpropanesulfonic acid AMPS or methacrylic sulfonic acid.

2. The drug-loadable embolic microsphere according to claim 1, wherein the drug-loadable embolic microsphere has a particle size ranging from 100-300 μιη.

3. The drug-loadable embolic microsphere according to claim 1, wherein the mass ratio of gelatin, polyethylene glycol derivative and drug-loadable functional monomer is 1: (0.1 to 10): (0.1-1).

4. The drug-loadable embolic microsphere according to claim 1, wherein the mass ratio of glutaraldehyde to the aldehyde-linked dextran sulfate in the mixed cross-linking agent is 5:2.

5. The drug-loaded embolic microsphere according to claim 1, wherein the emulsifier is selected from one or more of span, tween; the oil phase is selected from one or more of soybean oil, sesame oil, liquid paraffin, silicone oil, or water-immiscible organic solvent.

6. A method of preparing a drug-loadable embolic microsphere as in claim 1, wherein the preparing step comprises:

Step S2, adding the aqueous phase solution prepared in the step S1 into the oil phase containing the emulsifier at 50-70 ℃, stirring, then reducing the temperature to 2-8 ℃ and continuously stirring;

7. Use of the drug-loadable embolic microsphere according to any one of claims 1-5 for the preparation of embolic formulations, wherein the drug-loadable embolic microsphere is mixed with a positively charged drug solution and loaded with a drug by ionization for the preparation of embolic formulations.