CN115429928A

CN115429928A - Drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere and drug-loaded embolism microsphere

Info

Publication number: CN115429928A
Application number: CN202210952059.5A
Authority: CN
Inventors: 纪建松; 殳高峰; 陈敏江; 方世记; 赵中伟; 陈为谦; 陈晓晓; 郭小菊
Original assignee: Lishui Central Hospital
Current assignee: Lishui Central Hospital
Priority date: 2022-08-09
Filing date: 2022-08-09
Publication date: 2022-12-06

Abstract

The invention discloses a drug-loaded monodisperse calcium carbonate-gelatin composite embolization microsphere and a drug-loaded embolization microsphere. The drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere with uniform particle size and controllable size is prepared based on the microfluidic technology, so that the embolism effect on tumor blood supply arteries with different sizes can be remarkably improved, and ectopic embolism can be effectively avoided; compared with the common gelatin microspheres, the calcium carbonate-gelatin composite embolism microspheres have higher drug loading capacity, can prolong the release time of the drug, and can further play a longer-acting anti-liver cancer effect; compared with the traditional medicine-carrying embolism microsphere, the calcium carbonate-gelatin composite embolism microsphere after medicine carrying can effectively neutralize lactic acid accumulated at the tumor part after embolism, can realize effective synergy of tumor microenvironment regulation, embolism treatment and local chemotherapy, and further remarkably reduces the recurrence and metastasis rate of tumors.

Description

Drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere and drug-loaded embolism microsphere

Technical Field

The invention relates to the field of biomedical materials, in particular to a monodisperse calcium carbonate-gelatin composite embolism microsphere capable of carrying medicine and a medicine-carrying embolism microsphere.

Background

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality, has the characteristics of hidden morbidity, high malignancy degree, rapid progress and the like, and more than 70 percent of patients are in the middle and late stage at the time of clinical diagnosis, so that the best time for radical surgical resection is lost. Transcatheter hepatic artery chemoembolization (TACE) has gradually become the first treatment scheme for unresectable liver cancer in clinic, and the treatment aims are achieved by mainly blocking blood supply arteries of tumors and combining local chemotherapy to enable the tumors to be necrotized and atrophied in a short time.

In the liver cancer embolism treatment, the embolization agent can block blood supply of tumor tissues and inhibit lactic acid from being discharged from tumor parts, and the lactic acid highly expressed in a tumor microenvironment can enable tumor cells to enter a dormant state when the tumor cells lack nutrition supply, so that the tumor cells are protected from being influenced by glucose deficiency, the sensitivity of the tumor cells to chemotherapeutic drugs can be obviously reduced, and the recurrence rate and the metastasis rate of liver cancer are further improved. Research shows that the treatment scheme of injecting sodium bicarbonate solution to the tumor part to neutralize lactic acid before embolizing blood supply artery can obviously improve the embolization treatment effect of liver cancer. However, sodium bicarbonate is a water-soluble small molecule, and is easily lost from the tumor site, making it difficult to maintain a slightly alkaline environment in the tumor site for a long period of time.

The drug-loaded embolism microsphere is an embolism agent which is commonly used in clinic at present, and compared with the traditional iodized oil/chemotherapeutic drug embolism agent, the drug-loaded embolism microsphere can maintain the concentration of chemotherapeutic drugs at tumor parts for a longer time, improve the curative effect of chemotherapy and reduce the toxic and side effects of the drugs on the whole body. However, the existing embolization microspheres have the problems of non-uniform particle size and uncontrollable size, which seriously affect the embolization treatment effect of liver cancer, and are mainly clinically expressed as follows: the microspheres with overlarge sizes are difficult to effectively embolize the liver cancer blood supply artery endings, so that the embolization effect is not thorough; microspheres that are too small in size may enter the vein through the venous anastomosis branch, leading to embolization of the lung and other tissues, resulting in ischemic necrosis of non-target organs, with serious complications.

Gelatin is a natural polymer material, has the advantages of good biocompatibility, low price, biodegradability and the like, and is widely applied to embolism treatment. In addition, the preparation method of the gelatin microsphere is simple, the gelatin microsphere prepared by crosslinking is smooth in appearance and spherical, can be better adapted to blood vessels, has good elasticity after water absorption, and can achieve better embolism effect. However, the traditional gelatin microspheres have limited loading capacity for chemotherapeutic drugs, and effective regulation of tumor acid microenvironment is difficult to realize.

Patent CN202111363386.9 discloses a gelatin composite embolism microsphere containing nano calcium carbonate, a preparation method thereof and a drug-loaded embolism microsphere, wherein the gelatin and calcium carbonate are adopted to successfully prepare the drug-loaded embolism microsphere, and the drug-loaded embolism microsphere has the advantages of simple preparation method, low cost, good reproducibility and the like, but has the following defects: the prepared embolism microsphere has insufficient particle size uniformity and difficult particle size control.

There is therefore a need for improvements in the prior art to provide a more reliable solution.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problem to be solved by the invention is to provide a drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere with a tumor acid microenvironment regulation function and a drug-loaded embolism microsphere based on the same.

In order to solve the technical problems, the invention adopts the technical scheme that: a monodisperse calcium carbonate-gelatin composite embolism microsphere capable of carrying medicine comprises calcium carbonate nanoparticles and gelatin, the microsphere has a tumor acidic microenvironment regulation function, and is prepared by a microfluidic-based method, and the specific preparation method comprises the following steps:

s1, building a microchannel reactor, wherein the microchannel reactor comprises a droplet generation tube, a mobile phase input tube and a disperse phase input tube which are communicated with the input end of the droplet production tube, and a sample receiving tube which is communicated with the output end of the droplet production tube;

s2, preparing a gelatin water solution containing calcium carbonate nanoparticles as a dispersion phase;

s3, preparing a mobile phase;

s4, respectively injecting the prepared dispersed phase and the prepared mobile phase into a dispersed phase input pipe and a mobile phase input pipe, so that the dispersed phase is sheared by the mobile phase in a liquid drop generating pipe, and the W/O type calcium carbonate-gelatin liquid drops are continuously generated;

and S5, preparing the calcium carbonate-gelatin composite embolization microsphere by using the collected calcium carbonate-gelatin liquid drops.

Preferably, the calcium carbonate-gelatin composite embolization microspheres comprise the following components in percentage by mass: 3-30% of calcium carbonate nanoparticles and 70-90% of gelatin;

the calcium carbonate-gelatin composite embolism microsphere has the average grain size of 50-300 mu m and the coefficient of variation of less than 5%.

Preferably, the step S1 specifically includes:

s1-1, providing a glass sample application capillary tube and two transparent silica gel hoses, respectively inserting two ends of the glass sample application capillary tube into the two transparent silica gel hoses, wherein the glass sample application capillary tube is used as a liquid drop generating tube, and the transparent silica gel hoses connected with the front end and the rear end of the glass sample application capillary tube are respectively used as a mobile phase input tube and a sample receiving tube; further preferably, the length of the glass spotting capillary is about 7cm, the length of the transparent silica gel hose is about 20cm, and the depth of the glass spotting capillary inserted into the transparent silica gel hose is about 10mm;

s1-2, enabling an injection needle to penetrate through a transparent silica gel hose and be inserted into a glass sample application capillary, and enabling the injection needle to serve as a disperse phase input tube; it is further preferred that the needle is inserted into the glass spotting capillary to a depth of about 10mm;

s1-3, placing the injection needle, the glass sample application capillary and the parts, connected with the glass sample application capillary, of the two transparent silica gel hoses on a glass slide, fixing the joints through AB glue sealing, and building to obtain the micro-channel reactor.

Preferably, the step S2 specifically includes:

s2-1, preparing calcium carbonate nano suspension:

s2-1-1, dissolving polyacrylic acid and calcium chloride in deionized water, and stirring at normal temperature to obtain a mixed solution;

s2-1-2, dissolving sodium carbonate in deionized water, adding the obtained sodium carbonate solution into the mixed solution obtained in the step S2-1-1, and stirring at normal temperature;

s2-1-3, after the reaction is finished, centrifuging, collecting the precipitate, washing the precipitate with deionized water, and then dispersing the precipitate in the deionized water again to obtain calcium carbonate nano suspension;

s2-2, adding gelatin into the calcium carbonate nano suspension prepared in the step S2-1-3, heating and stirring to completely dissolve the gelatin, and preparing a gelatin water solution containing calcium carbonate nano particles, namely a dispersion phase.

Preferably, the step S3 specifically includes: adding an emulsifier into liquid paraffin, and uniformly stirring at room temperature to obtain a mobile phase.

Preferably, the step S4 specifically includes:

s4-1, respectively filling the dispersed phase prepared in the step S2 and the mobile phase prepared in the step S3 into syringes, connecting the syringes filled with the dispersed phase with a dispersed phase input pipe, heating the syringes filled with the mobile phase and then connecting the syringes filled with the mobile phase input pipe, controlling the dispersed phase to be injected into a droplet generation pipe at a flow rate of 5-50 mu L/min and the mobile phase at a flow rate of 100-700 mu L/min, and enabling the dispersed phase to be sheared by the mobile phase in the droplet generation pipe to continuously generate W/O type calcium carbonate-gelatin droplets.

Preferably, the step S5 specifically includes:

s5-1, collecting the calcium carbonate-gelatin droplets discharged from the sample receiving pipe through a glass bottle containing a mobile phase, wherein the calcium carbonate-gelatin droplets are prepared in the step S4, and stirring the calcium carbonate-gelatin droplets under the ice-bath condition in the collecting process;

s5-2, adding a cross-linking agent into the mixture obtained in the step S5-1, and continuously stirring for reaction to obtain a calcium carbonate-gelatin composite embolism microsphere crude product;

s5-3, centrifuging the crude product of the calcium carbonate-gelatin composite embolism microsphere obtained in the step S5-2, taking a precipitate, and sequentially washing the precipitate by using isopropanol with volume fractions of 50%, 70% and 100% respectively to remove residual mobile phase and cross-linking agent;

and S5-4, naturally drying the product obtained in the step S5-3 to obtain the calcium carbonate-gelatin composite embolism microsphere.

Preferably, the inner diameter of the glass sample application capillary is 0.4-1.1mm, and the injection needle is any one of 34G, 32G, 30G and 27G.

Preferably, the emulsifier is one or more of Span 20, span 40, span60 and Span 80, and the cross-linking agent is one or more of formaldehyde solution with mass fraction of 6%, glutaraldehyde solution with mass fraction of 25% or genipin solution with mass fraction of 5%.

Preferably, the polyacrylic acid in step S2-1 has an average molecular weight of 2kDa,3kDa,5kDa or 10kDa.

Preferably, in the step S2-2, the concentration of the calcium carbonate nanoparticles is 5-30mg/mL, and the concentration of the gelatin is 95-200mg/mL.

Preferably, the crosslinking time in S5-2 is 2-24h.

The invention also provides a drug-loaded embolization microsphere, which comprises the drug-loaded monodisperse calcium carbonate-gelatin composite embolization microsphere and a drug loaded on the microsphere, wherein the drug comprises one or more of adriamycin, epirubicin, cisplatin and gemcitabine.

The invention has the beneficial effects that:

1. the drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere with uniform particle size and controllable size is prepared based on the microfluidic technology, so that the embolism effect on tumor blood supply arteries with different sizes can be remarkably improved, and ectopic embolism can be effectively avoided;

2. in the invention, calcium carbonate nanoparticles with negative charges are introduced into the calcium carbonate-gelatin composite embolism microspheres prepared from the gelatin microspheres, compared with the common gelatin microspheres, the calcium carbonate-gelatin composite embolism microspheres have higher drug loading rate, can prolong the release time of the drug, and can further play a longer-acting anti-liver cancer effect;

3. compared with the traditional medicine-carrying embolism microsphere, the calcium carbonate-gelatin composite embolism microsphere after medicine carrying can effectively neutralize lactic acid accumulated at the tumor part after embolism, can realize effective cooperation of tumor microenvironment regulation, embolism treatment and local chemotherapy, and further remarkably reduces the recurrence and metastasis rate of tumor.

Drawings

FIG. 1: the structure of the microchannel reactor is shown schematically.

FIG. 2 is a schematic diagram: influence of mobile phase flow rate on preparation of monodisperse calcium carbonate-gelatin composite embolic microspheres. (A) Optical microscope photos of the monodisperse calcium carbonate-gelatin composite embolism microsphere prepared under different mobile phase flow rates; (B) The monodisperse calcium carbonate-gelatin composite embolism microsphere prepared under different mobile phase flow rates has the average particle size and the Coefficient of Variation (CV) value.

FIG. 3: the effect of the dispersed phase flow rate on the preparation of monodisperse calcium carbonate-gelatin composite embolic microspheres. (A) Optical microscope photographs of monodisperse calcium carbonate-gelatin composite embolic microspheres prepared at different dispersion phase flow rates; (B) The monodisperse calcium carbonate-gelatin composite embolism microsphere prepared under different dispersion phase flow rates has the average particle size and the Coefficient of Variation (CV) value.

FIG. 4: the in vivo embolization effect of the monodisperse calcium carbonate-gelatin composite embolization microsphere. (A) DSA picture of monodisperse calcium carbonate-gelatin composite embolism microsphere before embolism rabbit left kidney; (B) DSA picture of monodisperse calcium carbonate-gelatin composite embolization microsphere after embolizing left kidney of rabbit; (C) CT angiogram of rabbit kidney at day 14 post-embolism; (E) photographic images of the left and right kidneys of rabbits at day 14 post-embolism; (E) H & E staining pattern of rabbit left kidney at day 14 post-embolism; (F) H & E staining pattern of rabbit right kidney at day 14 post-embolism.

FIG. 5 shows the measurement results of the efficacy of monodisperse calcium carbonate-gelatin composite embolization microspheres and monodisperse ordinary gelatin embolization microspheres on chemotherapeutic drug doxorubicin.

FIG. 6: the monodisperse calcium carbonate-gelatin composite embolism microsphere can adjust the micro-acid environment of the tumor and improve the measurement result of the chemotherapy sensitivity of the tumor cells.

Description of reference numerals:

1-mobile phase input pipe; 2-dispersed phase input tube; 3-a droplet generating tube; 4-sample receiving tube.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The test methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified. The following examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

A drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere comprises the following components by mass percent: 3-30% of calcium carbonate nanoparticles and 70-90% of gelatin, wherein the microspheres are prepared by a microfluidic-based method, and the specific preparation method comprises the following steps:

s1, building a microchannel reactor, wherein the microchannel reactor comprises a droplet generation tube 3, a mobile phase input tube 1 and a disperse phase input tube 2 which are communicated with the input end of the droplet production tube 3, and a sample receiving tube 4 which is communicated with the output end of the droplet production tube 3; the method specifically comprises the following steps:

s1-1, providing a glass sample application capillary tube with the length of about 7cm and the inner diameter of 0.5mm and two transparent silica gel hoses with the length of about 20cm, respectively inserting two ends of the glass sample application capillary tube into the two transparent silica gel hoses, wherein the insertion depth is about 10mm, the glass sample application capillary tube is used as a liquid drop generating tube, and the transparent silica gel hoses connected with the front end and the rear end of the glass sample application capillary tube are respectively used as a mobile phase input tube and a sample receiving tube;

s1-2, a 32G injection needle penetrates through a transparent silica gel hose and is inserted into a glass sample application capillary, the depth of the injection needle inserted into the glass sample application capillary is about 10mm (namely, the distance between the tail end of the injection needle and the left port of the glass sample application capillary is about 10 mm), and the injection needle is used as a disperse phase input tube;

s1-3, placing the injection needle, the glass sample application capillary and the parts, connected with the glass sample application capillary, of the two transparent silica gel hoses on a glass slide, fixing the joints by using AB glue in a sealing manner, and constructing to obtain the microchannel reactor, wherein the structure of the microchannel reactor is shown in figure 1.

s2-1, preparing calcium carbonate nano suspension:

s2-1-1, dissolving polyacrylic acid with the molecular weight of 2kDa and (0.5M) and calcium chloride (0.5M) in 50mL of deionized water, and stirring for 1h at normal temperature to obtain a mixed solution;

s2-1-2, dissolving sodium carbonate (0.5M) in 50mL of deionized water, then adding the obtained sodium carbonate solution into the mixed solution obtained in the step S2-1-1, and stirring for 1h at normal temperature;

s2-1-3, after the reaction is finished, centrifuging at a high speed of 12000rpm for 10min, collecting precipitates, washing the precipitates for 3 times by using deionized water, and then dispersing the precipitates in the deionized water again to obtain calcium carbonate nano suspension with the concentration of 5 mg/mL;

s2-2, adding 300mg of gelatin into 3mL of the calcium carbonate nano suspension prepared in the step S2-1-3, heating to 55 ℃, stirring to completely dissolve the gelatin, and preparing a gelatin water solution containing calcium carbonate nanoparticles, namely a dispersed phase.

S3, preparing a mobile phase:

50mL of liquid paraffin is taken, 5g of emulsifier Span 80 is added into the liquid paraffin, and the mixture is stirred uniformly at room temperature to dissolve the emulsifier, so that a mobile phase is obtained.

S4, preparing calcium carbonate-gelatin liquid drops;

s4-1, respectively filling the dispersed phase prepared in the step S2 and the mobile phase prepared in the step S3 into syringes, connecting the syringes filled with the dispersed phase with a dispersed phase input pipe, heating the syringes filled with the mobile phase to 55 ℃, then connecting the syringes filled with the mobile phase with a mobile phase input pipe, controlling the flow rate of the dispersed phase to be 5-50 mu L/min and the flow rate of the mobile phase to be 100-700 mu L/min, and injecting the dispersed phase into a droplet generation pipe so that the dispersed phase is sheared by the mobile phase in the droplet generation pipe to continuously generate W/O type calcium carbonate-gelatin droplets.

In the step, four groups of experiments are performed, wherein the flow of the dispersed phase in each group of experiments is controlled to be 10 mu L/min, and the flow of the mobile phase in the first group of experiments to the fourth group of experiments is controlled to be 100, 200, 300 and 400 mu L/min respectively.

And S5, preparing the calcium carbonate-gelatin composite embolism microsphere by using the collected calcium carbonate-gelatin liquid drops.

S5-1, collecting the calcium carbonate-gelatin droplets discharged from the sample receiving pipe through a glass bottle containing 20mL of mobile phase and prepared in the step S4, continuously collecting for 30min, putting the glass bottle under an ice bath condition in the collection process, and stirring the calcium carbonate-gelatin droplets at a low speed to condense the calcium carbonate-gelatin droplets into gel and prevent the gel microspheres from being adhered to each other;

s5-2, adding 0.5mL of 25% glutaraldehyde into the mixture obtained in the step S5-1 to react and crosslink the gelatin, and stirring for 6 hours to obtain a crude product of the calcium carbonate-gelatin composite embolic microsphere;

s5-3, centrifuging the crude product of the calcium carbonate-gelatin composite embolism microsphere obtained in the step S5-2 at 1000rpm for 10min, taking the precipitate, and sequentially washing the precipitate by using isopropanol with volume fractions of 50%, 70% and 100% respectively to remove residual mobile phase and cross-linking agent;

s5-4, naturally drying the product obtained in the step S5-3 to obtain the calcium carbonate-gelatin composite embolism microsphere.

The morphology of the first to fourth groups of calcium carbonate-gelatin composite embolization microspheres prepared in this example was characterized by an optical microscope. The results show that the calcium carbonate-gelatin composite embolism microsphere prepared by the micro-fluidic technology has complete shape and uniform particle size, and the average particle size has the tendency of decreasing with the increase of the flow velocity of the mobile phase (figure 2A).

The first to fourth groups of calcium carbonate-gelatin composite embolization microspheres prepared in this example were subjected to particle size statistics using image J software, and the Coefficient of Variation (CV) of the microspheres was further calculated, wherein the CV value was calculated by the following formula: CV = (standard deviation/average) × 100%. The results show that when the flow rate of the mobile phase was increased from 100, 200, 300 to 400. Mu.L/min, the average particle size of the calcium carbonate-gelatin composite embolization microspheres was reduced from 133.7. + -. 6.5, 116.7. + -. 4.6, 95.9. + -. 3.5 to 76.3. + -. 3.7. Mu.m, but the CV values were not more than 5%, further illustrating that the microspheres prepared in this example were uniform in size and good in monodispersity (FIG. 2B).

Example 2

A drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere, the preparation method comprises the following steps:

s1, building a micro-channel reactor, and specifically comprising the following steps:

s2-1, preparing calcium carbonate nano suspension:

S3, preparing a mobile phase:

50mL of liquid paraffin was taken, 5g of Span 80 emulsifier was added thereto, and the mixture was stirred at room temperature to dissolve the emulsifier, thereby obtaining a mobile phase.

S4, preparing calcium carbonate-gelatin liquid drops;

s4-1, respectively filling the dispersed phase prepared in the step S2 and the mobile phase prepared in the step S3 into an injector, connecting the injector filled with the dispersed phase with a dispersed phase input pipe, heating the injector filled with the mobile phase to 55 ℃, then connecting the injector with the mobile phase input pipe, controlling the dispersed phase to be injected into a droplet generation pipe at a flow rate of 5-50 mu L/min and the mobile phase to be injected into the droplet generation pipe at a flow rate of 100-700 mu L/min, and enabling the dispersed phase to be sheared by the mobile phase in the droplet generation pipe to continuously generate W/O type calcium carbonate-gelatin droplets.

In the step, four groups of experiments are carried out, wherein the flow rate of the dispersed phase in each group of experiments is controlled to be 30 mu L/min, and the flow rates of the mobile phase in the first group of experiments to the fourth group of experiments are respectively controlled to be 5, 15, 20 and 30 mu L/min.

s5-2, adding 0.5mL of 25% glutaraldehyde into the mixture obtained in the step S5-1 to react and crosslink gelatin, and stirring to react for 6 hours to obtain a crude product of the calcium carbonate-gelatin composite embolism microsphere;

s5-3, centrifuging the crude product of the calcium carbonate-gelatin composite embolism microsphere obtained in the step S5-2 at 1000rpm for 10min, taking a precipitate, and sequentially washing the precipitate by using isopropanol with volume fractions of 50%, 70% and 100% respectively to remove residual mobile phase and cross-linking agent;

The morphology of the first to fourth groups of calcium carbonate-gelatin composite embolization microspheres prepared in this example was characterized using an optical microscope. The results show that the calcium carbonate-gelatin composite embolization microspheres prepared by the microfluidic technology have complete shapes and uniform particle sizes, and the average particle size tends to increase with the increase of the flow velocity of the disperse phase (fig. 3A).

The first to fourth groups of calcium carbonate-gelatin composite embolization microspheres prepared in this example were subjected to particle size statistics using image J software, and the Coefficient of Variation (CV) of the microspheres was further calculated. The results show that when the flow rate of the dispersed phase was increased from 5, 15, 20 to 30. Mu.L/min, the average particle size of the calcium carbonate-gelatin composite embolization microspheres was increased from 88.2. + -. 3.7, 105.7. + -. 3.2, 112.7. + -. 4.4 to 132.3. + -. 5.0. Mu.m, but the CV values were not more than 5%, further illustrating that the microspheres prepared in this example were uniform in size and good in monodispersity (FIG. 3B).

EXAMPLE 3 embolization Effect test of monodisperse calcium carbonate-gelatin composite embolizing microspheres

Monodisperse calcium carbonate-gelatin composite embolization microspheres were prepared according to the method of the third set of experiments of example 1 (10. Mu.L/min for the dispersed phase and 300. Mu.L/min for the mobile phase), and the embolization effect of the microspheres in vivo was examined using New Zealand white rabbit kidney embolization model. New Zealand big ear white rabbits are taken and fasted for 12 hours before operation. Firstly, the rabbit is anesthetized by injecting a sodium pentobarbital solution into the ear margin vein, the hair of the inguinal at the right side of the rabbit is cleaned and disinfected, then the skin of the inguinal is cut according to the principle of aseptic operation, and a small opening is cut at the right femoral artery. Then, with the aid of a Digital Subtraction Angiography (DSA), a microcatheter was passed through the cut-out small opening into the entrance of the renal artery, and the renal artery before microsphere embolization was angiographically performed by injecting an iohexol solution. Then, injecting the monodisperse calcium carbonate-gelatin composite embolism microsphere dispersed in the physiological saline into the left kidney through a microcatheter, and simultaneously injecting iohexol solution for embolism angiography, wherein no obvious blood vessel is seen at the end point of embolism angiography. As can be seen from FIGS. 4A and 4B, the left renal blood vessels can be clearly seen by injecting iohexol before embolization, but the left renal blood vessels disappear by injecting iohexol again after microsphere embolization, indicating that the renal artery of the left kidney is completely embolized by the monodisperse calcium carbonate-gelatin composite embolization microspheres. On 14 days after embolization, the long-term embolization effect of the calcium carbonate-gelatin composite embolization microsphere was observed by using CT angiography technique. As shown in FIG. 4C, the right kidney without embolism can be clearly seen, while the left kidney with embolism can not disappear, which indicates that the monodisperse calcium carbonate-gelatin composite embolization microsphere has good long-term embolization effect on renal artery. Meanwhile, the kidney was removed 14 days after embolization and was photographed, and the left kidney after embolization was found to be white, indicating that the left kidney had suffered severe ischemic infarction (as shown in fig. 4D). Further, the collected kidney tissues were subjected to H & E staining, and the results (fig. 4E and 4F) showed that the left kidney after embolization of the composite monodisperse calcium carbonate-gelatin composite embolization microsphere had an obvious coagulative necrosis manifestation, while the right kidney without embolization had no obvious change, further indicating that the monodisperse calcium carbonate-gelatin composite embolization microsphere had a good embolization effect.

Example 4

A drug-loaded embolization microsphere, which comprises the drug-loaded monodisperse calcium carbonate-gelatin composite embolization microsphere of example 1 or example 2 and a drug loaded on the microsphere, wherein the drug comprises one or more of adriamycin, epirubicin, cisplatin and gemcitabine.

Example 5 drug loading potency test of monodisperse calcium carbonate-gelatin composite embolic microspheres for doxorubicin

Monodisperse calcium carbonate-gelatin composite embolic microspheres were prepared according to the method of the third set of experimental groups of example 1 (flow rate of dispersed phase was 10 μ L/min, flow rate of mobile phase was 300 μ L/min).

The monodisperse common gelatin microsphere prepared by the same method (without calcium carbonate nanoparticles) is used as a contrast, and the preparation method of the monodisperse common gelatin microsphere comprises the following steps:

s1, building a micro-channel reactor, and is the same as the embodiment 1.

S2, preparing the monodisperse common gelatin composite embolism microsphere, which specifically comprises the following steps:

s2-1, adding 300mg of gelatin into 3mL of deionized water, heating to 55 ℃, stirring to completely dissolve the gelatin, and preparing a gelatin aqueous solution, namely a dispersion phase;

s2-2, measuring 50mL of liquid paraffin, adding 5g of Span 80, and stirring at room temperature to dissolve the liquid paraffin to form a mobile phase;

s2-3, respectively filling the dispersed phase prepared in the step S2-1 and the mobile phase prepared in the step S2-2 into syringes, connecting the syringes filled with the dispersed phase with a dispersed phase input pipe, heating the syringes filled with the mobile phase to 55 ℃, then connecting the syringes filled with the mobile phase input pipe, and controlling the flow rates of the dispersed phase and the mobile phase to be 10 mu L/min and 300 mu L/min respectively, so that the dispersed phase is sheared by the mobile phase in a microchannel reactor to continuously generate W/O type gelatin droplets with uniform size;

s2-4: collecting calcium carbonate-gelatin droplets discharged from the sample receiving pipe through a glass bottle containing 20mL of mobile phase and prepared in the step S2-3, continuously collecting for 30min, putting the glass bottle under an ice bath condition in the collection process, and stirring the calcium carbonate-gelatin droplets at a low speed to condense the calcium carbonate-gelatin droplets into gel and prevent the gel microspheres from being adhered to each other;

s2-5: adding 0.5mL of 25% glutaraldehyde into the mixture system obtained in the step S2-4 to react and crosslink the gelatin, and stirring for 6 hours to obtain a crude product of the common gelatin embolism microsphere;

s026: centrifuging the crude product of the gelatin embolism microsphere obtained in the step S2-5 at 1000rpm for 10min, taking precipitate, and sequentially washing the obtained precipitate with isopropanol with volume fractions of 50%, 70% and 100% for 3 times to remove residual mobile phase and crosslinking agent

S027: and (5) naturally drying the product obtained in the step S026 to obtain the monodisperse common gelatin embolism microsphere.

The monodisperse ordinary gelatin embolization microspheres prepared according to the method of this example were determined to have an average particle size of 131.7. + -. 4.1. Mu.m, and a CV value of 3.1%.

Then, the drug-loading efficiency of the monodisperse calcium carbonate-gelatin composite embolization microspheres and the monodisperse common gelatin microspheres to the adriamycin is detected by adopting an ultraviolet spectrophotometry.

The method comprises the following specific steps: precisely weighing 20mg of microsphere sample, placing the microsphere sample in a 1.5mL centrifuge tube, and respectively adding 0.4mL of adriamycin aqueous solution with the concentration of 2.5,5,7.5 and 10mg/mL into each tube; then, placing the centrifuge tube in an ultrasonic cleaner, performing water bath ultrasonic treatment for 10min to fully disperse the microspheres in adriamycin aqueous solution, centrifuging at low speed of 1500rpm for 10min, collecting supernatant, washing the microspheres for 3 times by using PBS (phosphate buffer solution) with pH7.4 to remove the unencapsulated adriamycin, centrifuging, combining the supernatants, measuring the absorbance of the supernatant at 480nm by adopting an ultraviolet spectrophotometry, substituting the absorbance value into a standard curve equation to calculate the content of the adriamycin in the supernatant, and further calculating the drug loading rate of the microspheres to the adriamycin: drug loading = (input amount of adriamycin before drug loading-content of adriamycin in supernatant)/weight of drug loaded microsphere × 100%.

It was determined that the drug loading of the monodisperse calcium carbonate-gelatin composite embolization microspheres to doxorubicin was 4.46 + -0.17%, 8.39 + -0.15%, 12.18 + -0.11% and 15.8 + -0.19%, respectively, significantly higher than that of the monodisperse ordinary gelatin microspheres, such as 1.71 + -0.23%, 3.16 + -0.40%, 4.49 + -0.36 and 6.06 + -0.57%, when the doxorubicin solution concentration was 2.5,5,7.5 and 10mg/mL (FIG. 5). The result also proves that the problem of too low drug loading of the existing gelatin embolism microsphere can be effectively solved by introducing the calcium carbonate nano particles into a monodisperse gelatin microsphere system.

Example 6 measurement of monodisperse calcium carbonate-gelatin composite embolization microspheres for regulating tumor microacid environment and improving in vitro anti-tumor efficacy

Monodisperse calcium carbonate-gelatin composite embolic microspheres were prepared according to the method of the third set of experimental groups in example 1, and whether the monodisperse calcium carbonate-gelatin composite embolic microspheres can reverse chemotherapy resistance caused by lactic acid was examined using the MTT method. The method comprises the following specific steps: precisely weighing 200mg of microsphere sample, placing the microsphere sample in 4mL of DMEM medium containing 20mM lactic acid, standing for 24h at 37 ℃, centrifuging to obtain supernatant, and filtering and sterilizing the obtained supernatant for later use; regularly digesting LM3 hepatoma cell at 1 × 10 ⁴ The density of each well is inoculated in a 96-well cell plate, and each well volume is about 0.2mL; after the cells are completely attached to the wall after 24 hours of incubation, replacing the culture medium in the cell plate with a fresh culture medium, a fresh culture medium containing 20mM lactic acid or a culture medium treated by the microspheres in the same volume; after the cells were assigned for 1h, 5 μ L of free doxorubicin solutions at different concentrations were added to the above 3 groups of experimental cells (final drug concentrations of 0.5,1,2,4,8 and 16 μ g/mL, respectively), each group being provided with 3 parallel wells; after 24h incubation, 200 mu L (5 mg/mL) of MTT solution is added into each well, the culture is continued for 4h, the supernatant is discarded, 200 mu L of DMSO is added into each well, the absorbance A of each well at 570nm is measured by a microplate reader, and the cell survival rate is calculated.

As shown in fig. 6, it can be seen that the presence of lactic acid can significantly reduce the killing ability of doxorubicin to tumor cells, indicating that the acid microenvironment of breast tumors can lead to resistance to chemotherapy. When the culture medium containing lactic acid is treated by monodisperse calcium carbonate-gelatin composite embolism microsphere, the killing effect of adriamycin on tumor cells is obviously improved, and the death rate of the cells is equivalent to that of a normal culture medium group. The result also shows that the monodisperse calcium carbonate-gelatin composite embolism microsphere can effectively remove lactic acid, thereby reversing chemotherapy resistance in an acidic microenvironment for diagnosis and treatment, and further providing possibility for realizing more efficient liver cancer TACE treatment.

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.

Claims

1. A drug-loaded monodisperse calcium carbonate-gelatin composite embolism microsphere comprises calcium carbonate nanoparticles and gelatin, and is characterized in that the microsphere is prepared by a microfluidic-based method, and the specific preparation method comprises the following steps:

s3, preparing a mobile phase;

2. The drug-loadable monodisperse calcium carbonate-gelatin composite embolic microsphere of claim 1, wherein the calcium carbonate-gelatin composite embolic microsphere comprises the following components by mass percent: 3-30% of calcium carbonate nanoparticles and 70-90% of gelatin;

3. The drug-loaded monodisperse calcium carbonate-gelatin composite embolic microsphere of claim 2, wherein the step S1 specifically comprises:

s1-1, providing a glass sample application capillary tube and two transparent silica gel hoses, respectively inserting two ends of the glass sample application capillary tube into the two transparent silica gel hoses, wherein the glass sample application capillary tube is used as a liquid drop generating tube, and the transparent silica gel hoses connected with the front end and the rear end of the glass sample application capillary tube are respectively used as a mobile phase input tube and a sample receiving tube;

s1-2, enabling an injection needle to penetrate through a transparent silica gel hose and be inserted into a glass sample application capillary, and enabling the injection needle to serve as a disperse phase input tube;

s1-3, placing the injection needle, the glass sample application capillary and the parts, connected with the glass sample application capillary, of the two transparent silica gel hoses on a glass slide, fixing the joints by using AB glue in a sealing manner, and building to obtain the microchannel reactor.

4. The drug-loadable monodisperse calcium carbonate-gelatin composite embolic microsphere of claim 3, wherein the step S2 specifically comprises:

s2-1, preparing calcium carbonate nano suspension:

5. The drug-loadable monodisperse calcium carbonate-gelatin composite embolic microsphere of claim 4, wherein the step S3 specifically comprises: taking liquid paraffin, adding an emulsifier, and uniformly stirring at room temperature to obtain a mobile phase.

6. The drug-loadable monodisperse calcium carbonate-gelatin composite embolic microsphere of claim 5, wherein the step S4 specifically comprises:

s4-1, respectively filling the dispersed phase prepared in the step S2 and the mobile phase prepared in the step S3 into an injector, connecting the injector filled with the dispersed phase with a dispersed phase input pipe, heating the injector filled with the mobile phase, connecting the injector with the mobile phase input pipe, controlling the dispersed phase to be injected into a droplet generation pipe at a flow rate of 5-50 mu L/min and the mobile phase to be injected into the droplet generation pipe at a flow rate of 100-700 mu L/min, and enabling the dispersed phase to be sheared by the mobile phase in the droplet generation pipe to continuously generate W/O type calcium carbonate-gelatin droplets.

7. The drug-loadable monodisperse calcium carbonate-gelatin composite embolic microsphere of claim 6, wherein the step S5 specifically comprises:

s5-1, collecting the calcium carbonate-gelatin droplets which are discharged from the sample receiving pipe and prepared in the step S4 through a glass bottle containing a mobile phase, placing the glass bottle under an ice bath condition in the collecting process, and stirring the calcium carbonate-gelatin droplets;

s5-3, centrifuging the crude product of the calcium carbonate-gelatin composite embolism microsphere obtained in the step S5-2, taking a precipitate, and sequentially washing the precipitate by using isopropanol with volume fractions of 50%, 70% and 100%;

8. The drug-loaded monodisperse calcium carbonate-gelatin composite embolization microsphere of claim 3, wherein the inner diameter of the glass sample application capillary is 0.4-1.1mm, and the injection needle is any one of 34G, 32G, 30G and 27G.

9. The drug-loadable monodisperse calcium carbonate-gelatin composite embolic microsphere of claim 7, wherein the emulsifier is a mixture of one or more of Span 20, span 40, span60 and Span 80, and the cross-linking agent is a mixture of one or more of a 6% mass fraction formaldehyde solution, a 25% mass fraction glutaraldehyde solution, or a 5% mass fraction genipin solution.

10. A drug-loaded embolization microsphere comprising the drug-loadable monodisperse calcium carbonate-gelatin composite embolization microsphere as claimed in any one of claims 1 to 9, and a drug loaded on the microsphere, wherein the drug comprises one or more of doxorubicin, epirubicin, cisplatin and gemcitabine.