CN110327310A - A kind of multicore is total to shell composite drug carried microsphere and its preparation method and application - Google Patents

Abstract

The invention discloses a multi-core co-shell composite drug-loaded microsphere and its preparation method and application. First, FNS/PHBV microspheres are prepared by an emulsified solvent evaporation method, and then coated on PVA/PHBV microspheres by a reverse suspension cross-linking method. In the CS hydrophilic polymer matrix, the hydrophobic drug is skillfully coated in the hydrophilic polymer with high efficiency, which improves the loading rate of the hydrophobic drug. The particle size of the prepared FNS/PHBV@PVA/CS drug-loaded microspheres meets the requirements of prostate targeted embolization therapy. It can be seen from the drug release curve that there is no burst release and sustained release in the whole drug release process. In vitro cell experiments and hemolysis experiments confirmed that it has good cytocompatibility and hemocompatibility. Experiments on rabbit ear embolism preliminarily show that it has good embolization effect and good tissue compatibility. Moreover, the preparation process is simple, the particle size is controllable, and it is suitable for batch industrial production. The invention effectively combines the physical embolism performance of the composite drug-loaded microspheres and the drug slow-release performance, and has potential application prospects in the embolization treatment of BPH.

Description

A multi-core co-shell composite drug-loaded microsphere and its preparation method and application

technical field

The invention belongs to the technical field of prostate interventional medicine, and in particular relates to a multi-core co-shell composite drug-loaded microsphere and its preparation method and application.

Background technique

Benign prostatic hyperplasia (BPH) is one of the common urinary system diseases in middle-aged and elderly men, mainly manifested as lower urinary tract symptoms such as frequent urination, dysuria, and urinary hesitancy. For mild patients, drug treatment is generally used to relieve symptoms, but it cannot be completely eradicated. The method of surgical treatment brings greater trauma to the patient and is prone to complications. Clinically, transurethral prostatectomy is considered to be the "gold standard" in the treatment of BPH, but the disadvantage of this method is that the prostate gland cannot be completely separated and removed from the surrounding capsule, and the remaining prostate gland will lead to postoperative recurrence , increasing the patient's physical and mental burden and economic pressure. In recent years, with the development of medical technology, Prostatic Artery Embolization (PAE) has gradually entered into clinical practice. It has many advantages such as minimally invasiveness, few complications and high curative effect. The choice of embolization material is the decisive factor in determining the success of embolization. Therefore, domestic and foreign scholars have done extensive and in-depth research on the synthesis, improvement and curative effect of embolic materials. At present, most of the embolic materials used clinically have a single treatment method, and can only perform one-time physical embolization, which cannot improve the efficiency of embolization. Drug-loaded embolic materials are the trend of tumor chemoembolization. For example, DC microspheres and HepaSphere microspheres can effectively combine physical embolization with drug therapy to increase the efficacy. However, there has not yet been an embolic material that combines physical embolization and drug therapy for embolization of BPH, and there are basically no reports related to it. Therefore, it is of great clinical significance to research and develop drug-loaded embolic materials for embolization therapy of BPH.

Clinically, Finasteride (Finasteride, FNS) has been used in the treatment of BPH. It is a 5α-reductase inhibitor that acts on 5α-reductase to prevent the conversion of testosterone into more active dihydrotestosterone. However, simple drug treatment has relatively large side effects, such as orthostatic hypotension, dizziness, decreased libido, and sexual dysfunction. If FNS can be effectively combined with embolic materials, it can reasonably and effectively control the release of drugs to the prostate lesion tissue while performing physical embolization therapy, and play a synergistic role in the treatment of BPH.

Polyvinyl alcohol (PVA) is a medical excipient that has been certified by the US FDA for clinical use. It has the characteristics of non-toxic, harmless, good biocompatibility and can be used as a drug carrier. However, it is a hydrophilic polymer, and FNS is highly hydrophobic. Direct drug loading will inevitably lead to a decrease in the drug loading rate, and there will inevitably be drug burst release.

Contents of the invention

In view of this, the present invention provides a multi-core co-shell composite drug-loaded microsphere and its preparation method and application, which have long-acting drug sustained release effect. In the present invention, FNS is loaded in poly(3-hydroxybutyrate-3-hydroxyvalerate) (Poly(3-hydroxybutyrate-3-hydroxyvalerate), PHBV), prepared into drug-loaded microspheres, and coated by emulsion polymerization The method is to coat it in PVA and chitosan (Chitosan, CS) composite matrix to form a composite drug-loaded microsphere with a multi-core co-shell structure, so as to improve the drug loading rate and prolong the sustained release time of the drug to meet the requirements of clinical application.

The drug-loaded microspheres have good sphericity, uniform particle size, and a multi-core co-shell structure, which can effectively avoid drug burst release. At the same time, the CS in the composite microspheres will produce a porous structure on the surface of the microspheres during the slow degradation process, which increases the chance of the drug-loaded PHBV microspheres inside the composite microspheres in contact with the external liquid environment, thereby facilitating the slow release of the drug. , prolong the drug action time. The use of the composite drug-loaded microspheres can avoid the loss of simple drugs in the process of blood vessel delivery. At the same time, the drug-loaded microspheres can be embolized to the prostate lesion by interventional means and release the drug continuously and slowly, which improves the utilization rate of the drug. Play the dual role of drug therapy and prostate blood vessel blocking embolism.

The first aspect of the present invention provides multi-core co-shell composite drug-loaded microspheres, the multi-core co-shell composite drug-loaded microspheres are PVA/CS composite polymer microspheres, and the inside of the PVA/CS composite polymer microspheres is coated with PHBV microspheres, the PHBV microspheres are loaded with FNS.

The second aspect of the present invention provides a method for preparing the above-mentioned multi-core co-shell composite drug-loaded microspheres, the steps comprising:

S1. Heat and dissolve the oil-soluble PHBV in an organic solvent, and then add FNS sonication to dissolve it in the organic solvent to obtain a drug-loaded PHBV solution;

S2. Add the drug-loaded PHBV solution obtained in step S1 into the polyvinyl alcohol solution, and stir to form an O/W emulsion;

S3. Add the O/W emulsion obtained in step S2 dropwise into the polyvinyl alcohol solution, stir until the organic solvent volatilizes to obtain the drug-loaded microspheres, and freeze-dry them after washing;

S4. Add the FNS/PHBV microspheres obtained in step S3 to the PVA/CS mixed solution containing Tween80, stir and sonicate to form a S/W mixed phase;

S5. Add the S/W mixed phase obtained in step S4 into the pre-emulsified oil phase, stir to form a S/W/O emulsion, add glutaraldehyde solution saturated with ether, add hydrochloric acid solution after stirring, and heat up to 40-65°C Carry out chemical cross-linking for 3-8 hours;

S6. The microspheres are collected by centrifugation and washed, and the free glutaraldehyde in the microspheres is removed with glycine or L-lysine solution, washed, freeze-dried, and sieved to obtain multi-core co-shell composite drug-loaded microspheres.

The third aspect of the present invention provides the application of the above multi-core co-shell composite drug-loaded microspheres in the treatment of prostate embolism.

The beneficial effects of the present invention are as follows:

1. The preparation method of the present invention is simple, and the prepared composite drug-loaded microspheres are smooth and round with good dispersibility, and the particle size can be regulated according to actual application requirements. Its physical and chemical properties also meet the needs of BPH embolization therapy.

2. With PVA/CS as the skeleton carrier, the drug-loaded PHBV microspheres with smaller particle size are coated, which reduces the burst release effect during the drug release process, and the drug release is stable and long-lasting, thereby increasing the local effective drug concentration.

3. The present invention uses the multi-core co-shell coating strategy to efficiently coat hydrophobic drugs in the hydrophilic polymer skeleton, which improves the loading rate of hydrophobic drugs and provides new ideas for the development of other new drug carriers.

4. The composite drug-loaded microspheres prepared by the present invention have good biocompatibility, are suitable for mass production, and have broad development and application prospects.

Description of drawings

Fig. 1 is FNS/PHBV microsphere FESEM figure (a), high magnification FESEM figure (b) and size distribution histogram (c) of the FNS/PHBV microsphere prepared in embodiment 1;

Figure 2(a) is the FESEM image and high magnification FESEM image (b) of the PVA/CS microspheres prepared in Comparative Example 1, FNS/PHBV@PVA/CS microspheres (c) and high magnification FESEM image (d) ;

Fig. 3 (a) is the cross-sectional FESEM figure and the high magnification FESEM figure (b) of the PVA/CS microsphere prepared in Comparative Example 1, and the FNS/PHBV@PVA/CS microsphere cross-sectional FESEM figure (c) prepared in Example 2 And high magnification FESEM image (d);

Fig. 4 is the FTIR spectrum of FNS, the PHBV prepared in Example 1, the FNS/PHBV microspheres, the PVA/CS microspheres prepared in Comparative Example 1 and the FNS/PHBV@PVA/CS microspheres prepared in Example 2;

Fig. 5 is the XRD spectrum of FNS, the PHBV prepared in Example 1, the FNS/PHBV microspheres, the PVA/CS microspheres prepared in Comparative Example 1 and the FNS/PHBV@PVA/CS microspheres prepared in Example 2;

Figure 6 is the drug release curve (a) of the FNS/PHBV@PVA/CS microspheres prepared in Example 2, the microsphere surface FESEM figure (b) and high magnification FESEM figure (c) after drug release 51 days, drug release 51 days Cross-sectional FESEM image (d) and high magnification FESEM image (e) of Tin Hau microspheres;

Fig. 7 is the result of the in vitro cytotoxicity test (MTT method) of the PVA/CS microspheres prepared in Comparative Example 1 and the FNS/PHBV@PVA/CS microspheres prepared in Example 2;

Fig. 8 is the dead and alive cell staining diagram of the PVA/CS microspheres prepared in Comparative Example 1 and the FNS/PHBV@PVA/CS microspheres prepared in Example 2;

Figure 9 is the FESEM image (a) and high magnification FESEM image (b) of the cell adhesion morphology on the surface of the microsphere after the co-culture of the PVA/CS microspheres prepared in Comparative Example 1 and HVSMCs for 1 day, and the FNS/CS microspheres prepared in Example 2. FESEM image (c) and high magnification FESEM image (d) of cell adhesion morphology on the surface of PHBV@PVA/CS microspheres co-cultured with HVSMCs for 1 day;

Figure 10 is the FESEM figure (a) and high magnification FESEM figure (b) of the cell adhesion morphology on the surface of the microsphere after the PVA/CS microspheres prepared in Comparative Example 1 and HVSMCs were co-cultured for 3 days, and the FNS/CS microspheres prepared in Example 2 FESEM image (c) and high magnification FESEM image (d) of cell adhesion morphology on the surface of PHBV@PVA/CS microspheres co-cultured with HVSMCs for 3 days;

Fig. 11 is the test result of the hemolysis rate of the PVA/CS microspheres prepared in Comparative Example 1 and the FNS/PHBV@PVA/CS microspheres prepared in Example 2;

Figure 12 is the in vitro fluidity test results of the FNS/PHBV@PVA/CS microspheres prepared in Example 2. (a) The microspheres are suspended in the syringe, (b) The microspheres pass through the catheter smoothly.

Fig. 13 is that the FNS/PHBV@PVA/CS microspheres prepared in Example 2 performed rabbit ear central artery plugging on New Zealand white rabbits for 0 days (a), 3 days (b), 7 days (c), and 15 days (d) and digital photos of the morphological changes of rabbit ears after 21 days (e);

Figure 14 is a HE staining photomicrograph of the central artery embolization of New Zealand white rabbits for 7 days with the PVA/CS microspheres prepared in Example 1 and the FNS/PHBV@PVA/CS microspheres prepared in Example 2;

Figure 15 is a HE staining photomicrograph of the central artery embolization of New Zealand white rabbits for 21 days with the PVA/CS microspheres prepared in Example 1 and the FNS/PHBV@PVA/CS microspheres prepared in Example 2;

Figure 16 shows New Zealand white rabbits using glycerol ((a)~(c)), PVA/CS microspheres prepared in Comparative Example 1 ((d)~(f)) and FNS/PHBV@PVA prepared in Example 2 Field emission transmission electron microscopy (FETEM) images of the ultrastructural changes of rabbit ears after 21 days of rabbit ear central artery embolization with /CS microspheres.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully and in detail below in conjunction with examples, but the protection scope of the present invention is not limited to the following specific examples.

Unless otherwise defined, all technical terms used hereinafter have the same meanings as commonly understood by those skilled in the art. The terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the protection scope of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

The first aspect of the present invention provides multi-core co-shell composite drug-loaded microspheres, the multi-core co-shell composite drug-loaded microspheres are PVA-CS composite polymer microspheres, and the PVA-CS composite polymer microspheres are coated with PHBV microspheres, the PHBV microspheres are loaded with FNS.

Preferably, the particle size of the multi-core co-shell composite drug-loaded microspheres is 100-300 μm.

The second aspect of the present invention provides a method for preparing the above-mentioned multi-core co-shell composite drug-loaded microspheres, the steps comprising:

S1. Heat and dissolve the oil-soluble PHBV in an organic solvent, and then add FNS sonication to dissolve it in the organic solvent to obtain a drug-loaded PHBV solution;

S2. Add the drug-loaded PHBV solution obtained in step S1 into the polyvinyl alcohol solution, and stir to form an O/W emulsion;

S3. Add the O/W emulsion obtained in step S2 dropwise into the polyvinyl alcohol solution, stir until the organic solvent volatilizes to obtain the drug-loaded microspheres, and freeze-dry them after washing;

S4. Add the FNS/PHBV microspheres obtained in step S3 to the PVA/CS mixed solution containing Tween80, stir and sonicate to form a S/W mixed phase;

S5. Add the S/W mixed phase obtained in step S4 into the pre-emulsified oil phase, stir to form a S/W/O emulsion, add glutaraldehyde solution saturated with ether, add hydrochloric acid solution after stirring, and heat up to 40-65°C Carry out chemical cross-linking for 3-8 hours;

S6. The microspheres are collected by centrifugation and washed, and the free glutaraldehyde in the microspheres is removed with glycine or L-lysine solution, washed, freeze-dried, and sieved to obtain multi-core co-shell composite drug-loaded microspheres.

Preferably, in step S1, the organic solvent is a mixture of dichloromethane and ethyl acetate, and the volume ratio of the dichloromethane and ethyl acetate is (1-4):1; the PHBV in the drug-loaded PHBV solution The concentration is 20-60mg/mL; the mass ratio of FNS to PHBV is 1:(1-5); the heating temperature is 30-60°C.

Preferably, in step S1, FNS is dissolved by ultrasonication for 5-10 min.

Preferably, in step S2, the volume ratio of the drug-loaded PHBV solution to the polyvinyl alcohol solution is 1: (3-8).

More preferably, in steps S2 and S3, the polyvinyl alcohol solution is a 1wt% polyvinyl alcohol 1788 solution.

More preferably, in step S2, the agitation is mechanically agitated by a high-speed homogenizer for 1-3 minutes, and the rotational speed of the mechanical agitation is 5000-20000 rpm/min.

Preferably, in step S3, the volume ratio of the O/W emulsion to the polyvinyl alcohol solution is 1:(20-80).

More preferably, in step S3, the stirring is magnetic stirring at a room temperature of 15-35° C., the magnetic stirring speed is 400-800 rpm/min, and the stirring time is 3-8 hours.

Preferably, in step S4, the concentration of PVA in the PVA/CS mixed solution is 30-80 mg/mL, and the mass percentage of CS in the PVA/CS mixed solution is 3%-10%.

Preferably, in step S4, the content of Tween80 in the PVA/CS solution is 0.2-1.5 wt%.

Preferably, in step S4, first magnetically stir for 10-30 min, and then sonicate for 30-60 min to form the S/W mixed phase.

Preferably, the mass percentage of the FNS/PHBV microspheres in the multi-core co-shell composite drug-loaded microspheres is 10-30%.

Preferably, in step S5, the oil phase is one or more of n-heptane, cyclohexane or petroleum ether.

Preferably, in step S5, after the S/W mixed phase is added to the pre-emulsified oil phase (O), it is stirred at 300-600 rpm/min for 30-60 min to form a S/W/O emulsion.

Preferably, in step S6, sieve with 60 and 150 mesh standard sieves to obtain microspheres with a certain particle size.

The third aspect of the present invention provides the application of the above multi-core co-shell composite drug-loaded microspheres in the treatment of prostate embolism.

The multi-core co-shell composite drug-loaded microspheres and their preparation methods and applications will be further described in detail in combination with specific examples.

Example 1

Weigh 160mg PHBV (PHV is 12mol%) in the mixed organic solvent that is equipped with 4mL dichloromethane/ethyl acetate (dichloromethane/ethyl acetate=70:30, v/v), the water bath is heated to 40 ℃ fully Dissolve, add 80mg FNS immediately after cooling, and dissolve the drug by ultrasonication for 8min. Add the above FNS/PHBV solution into a glass sample bottle containing 16mL 1wt% PVA1788, use a high-speed homogenizer to homogeneously stir at 8000rpm for 2min to form an O/W emulsion, and then add it to a three-necked flask containing 160mLPVA1788 after standing for 5min , stirred mechanically at 480 rpm for 5 h at room temperature, collected the drug-loaded microspheres by centrifugation, washed three times with deionized water, and stored after freeze-drying. The morphology and structure of the drug-loaded microspheres were observed and analyzed by field emission scanning electron microscopy (FESEM). It can be seen from Figure 1 that the particle size of the drug-loaded microspheres is uniform and the dispersion is good, with an average particle size of 7.0 μm (Figure 1(c)). Moreover, it can be seen from its microstructure (accompanying drawing 1 (b)) that the microsphere structure is loose and porous, which may be due to the fact that when the concentration of PHBV is low, the volatilization speed of the organic solvent is faster than the solidification speed of the microspheres to form a porous structure.

As a comparison, PHBV was dissolved in an organic solvent without adding FNS, and other PHBV microspheres were prepared by the above-mentioned preparation method.

Example 2

Weigh 0.8g of PVA124 and add 5.85mL of deionized water in a water bath to heat to dissolve, meanwhile weigh 0.04210g of CS (molecular weight is 110kDa) and dissolve in 5mL of 1wt% acetic acid solution. The two were mixed under magnetic stirring, and then a certain volume of Tween80 was added into the PVA/CS mixed solution to make the final concentration 0.5 wt%, and the magnetic stirring was continued for 20 min. Disperse the FNS/PHBV microspheres prepared in Example 1 in 3.5mL of 1wt% polyvinyl alcohol 1788 solution, then add the PVA/CS mixed solution dropwise under magnetic stirring, and then perform ultrasonication for 30min after magnetic stirring for 20min to form a uniform S/W mixed phase. The S/W mixed phase was slowly added dropwise to 72 mL of n-heptane with a mass fraction of 2.43% Span80 (pre-emulsified for 30 min), magnetically stirred at 350 rpm at room temperature for 30 min to form a S/W/O emulsion, and 25 wt. % glutaraldehyde solution (ether: 4.15 mL, glutaraldehyde: 2.6 mL), after stirring for 5 min, 1.8 mL of 1M hydrochloric acid solution was added, and the temperature was raised to 65°C for chemical crosslinking for 5 h. The microspheres were collected by centrifugation, washed three times with petroleum ether, absolute ethanol and deionized water, and then the free glutaraldehyde was removed with 4wt% glycine solution, washed with deionized water and freeze-dried to obtain the composite drug-loaded microspheres , respectively sieved with 60 and 150 mesh standard sieves to obtain microspheres with a certain particle size. FESEM results showed that the PVA/CS microspheres prepared in Comparative Example 1 had uniform particle size and regular shape, and there was no adhesion between the microspheres (accompanying drawing 2 (a)). And from its enlarged image (accompanying drawing 2 (b)), it can be seen that the surface of the microsphere is smooth and wrinkle-free. Attached Figures 2(c) and (d) are FESEM images of the surface of FNS/PHBV@PVA/CS microspheres. It can be seen from the figure that the microspheres are still spherical and round, with uniform particle size distribution and no adhesion between microspheres. . From the enlarged image (attached figure 2(d)), it can be clearly seen that the surface of the microspheres has become rough, and porous structures of different sizes are distributed. It can also be seen from some holes that the FNS/PHBV microspheres are coated on In the composite drug-loaded microspheres, the existence of these porous structures is conducive to the slow release of drugs from the FNS/PHBV microspheres inside the composite drug-loaded microspheres in contact with the external liquid environment. In addition, the cross-sectional morphology of the microspheres prepared in Comparative Example 1 and the present embodiment has also been characterized by FESEM. Accompanying drawing 3 (a), (b) is the entire cross-section and enlarged view of the PVA/CS microspheres. From the figure It can be seen that the interior of the microspheres is a solid structure, and the cross-sectional distribution has different degrees of pore structure, and the pore size is small. This is mainly due to the formation of bubbles formed by mechanical stirring during the preparation of the microspheres after the solidification of the microspheres. From the cross-sections of FNS/PHBV@PVA/CS microspheres shown in Figure 3(c) and (d), it can be seen that there are many pore structures distributed inside the microspheres, and the pore size is relatively large, and the FNS/PHBV microspheres mainly distributed in these pore structures. Figure 4 shows the FTIR spectra of Example 1, Comparative Example 1 and the PHBV microspheres, FNS/PHBV microspheres, PVA/CS microspheres, FNS/PHBV@PVA/CS microspheres and FNS of this embodiment. It can be seen from the spectrum that both FNS/PHBV microspheres and FNS/PHBV@PVA/CS microspheres have two characteristic absorption peaks of amino groups at 1675cm ^-1 and 1604cm ^-1 of FNS, while FNS/PHBV@PVA The -C=O absorption peak in the unique ester group of PHBV at 1733cm ^-1 appeared in /CS microspheres, which indicated that FNS/PHBV was successfully coated in PVA/CS. Accompanying drawing 5 shows the XRD pattern of above-mentioned medicine and microsphere. It can be seen from the results that FNS is a crystalline drug, and the characteristic peaks of FNS drugs clearly appeared in the FNS/PHBV microspheres, indicating that FNS exists in a crystalline form in FNS/PHBV. From the XRD pattern of FNS/PHBV@PVA/CS microspheres, it can also be seen that the characteristic peak of PHBV appeared at 2θ=13.4°, and the characteristic peak of FNS appeared at 16.8°, indicating that the FNS/PHBV microspheres were coated on In PVA/CS microsphere matrix.

Example 3

Weigh 30mg of FNS/PHBV@PVA/CS microspheres and disperse in 2mL of PBS solution containing 0.1wt% Tween80, transfer to a dialysis bag with a molecular weight cut-off of 3.5kDa, and then fill it into 15mL of PBS solution containing 0.1wt% Tween80 , placed on a constant temperature shaker at 37°C, oscillating at 150 rpm, and pipetting 2mL of PBS release solution into a centrifuge tube at regular intervals. The drug content in PBS was detected by high-performance liquid chromatography (HPLC) at 210nm, and the average value was obtained by repeating the operation 3 times, and the cumulative release curve was drawn. After each sampling, the same volume of fresh PBS solution was added to maintain a constant release volume.

Figure 6(a) shows the drug release curve of the composite drug-loaded microspheres. It can be seen from the figure that the entire drug release stage is relatively stable and has a good sustained release effect. The main reason is that PVA/CS as a coating layer greatly weakens the contact between the external PBS liquid and the FNS/PHBV microspheres inside the composite drug-loaded microspheres. At the same time, as time goes by, CS will slowly degrade. Holes are formed on the surface (Fig. 6(b), (c)), which facilitates the entry of PBS into the microspheres, and the FNS/PHBV microspheres will be slowly released as the PHBV degrades. From the FESEM images inside the composite drug-loaded microspheres (accompany (d), (e)), it can be seen that the FNS/PHBV microspheres in the composite drug-loaded microspheres were obviously eroded after 51 days of release, because PHBV is a biological Degradable materials, as the PHBV degrades, the drug will continue to be released from the pores of the composite microspheres.

Example 4

Prepare 10 mg/mL extracts of PVA/CS microspheres and FNS/PHBV/PVA/CS microspheres with DMEM medium containing 10% fetal bovine serum. Human vascular smooth muscle cells (HVSMCs) were used to test the cytotoxicity of the extract (CCK-8 method). The positive control group was 0.64wt% phenol solution, and the negative control group was DMEM medium with 10% fetal bovine serum. After culturing for 1, 2 and 3 days respectively, the absorbance was measured at 450 nm with a microplate reader, and the relative survival rate of the cells in the microsphere extract was calculated. After 1 and 3 days of culture, the effect of the microspheres on the viability of the cells was tested by direct contact culture with dead/live cell staining. Further, FESEM was used to observe the adhesion morphology of cells on the surface of microspheres after co-culture of HVSMCs and microspheres. The cytotoxicity of microspheres to HVSMCs detected by CCK-8 method is shown in Figure 7. The results showed that after 1, 2 and 3 days of culture, the relative survival rates of HVSMCs in the extracts of PVA/CS microspheres and FNS/PHBV@PVA/CS microspheres were above 94%, while the relative survival rates of cells in the positive control group It is below 10%, and it decreases obviously with the extension of time. It shows that PVA/CS microspheres and FNS/PHBV@PVA/CS microspheres have no cytotoxicity to cells. The cells were stained and observed by the dead/live cell staining method after 1 and 3 days of co-culture of the microspheres and the cells. Green represents live cells, and red represents dead cells (figure (8)). After 1 day of culture, there was no significant difference in the number of cells between the two microspheres and the negative control group, and there were basically no dead cells, while the number of cells in the positive control group was small and stained red, and the cell death rate was high. On the third day of cell culture, the number of cells in the two groups of microspheres and the negative control group increased significantly and were stained green, and there was no significant difference in the number of cells, while the cells in the positive control group were stained red, with more cell death, and The results were consistent with the results of CCK-8. The growth morphology of the cells on the two microspheres was further observed by FESEM, and the results are shown in Figure 9 and Figure 10. After 1 day of culture, a small amount of HVSMCs showed good adhesion and spreading on the two microspheres. (accompanying drawing (9)). After culturing for 3 days, the number of cells on the surface of the microspheres increased considerably and basically covered the entire microspheres, forming a single cell layer on the surface of the microspheres (figure (10)). The test results of these cell experiments showed that both groups of microspheres had good cytocompatibility.

Example 5

Hemolysis test is the most commonly used method to verify the hemocompatibility of materials, which mainly investigates the hemolysis effect of materials on red blood cells. Prepare diluted rabbit blood by diluting fresh anticoagulated rabbit blood and normal saline at a volume ratio of 4:5, and incubate 10 mg/mL of the two microspheres with 0.1 mL of diluted rabbit blood in an incubator at 37°C for 30 minutes Finally, after centrifuging at 1000rpm for 5min, take the supernatant and measure the absorbance at a wavelength of 545nm with a UV-visible spectrophotometer. Wherein, the negative control group is set as normal saline, and the positive control group is sterilized deionized water. The average value of each group of samples is repeated 5 times, and the hemolysis rate is calculated according to the following formula:

Hemolysis rate (%)=(X ₁ -X ₃ )/(X ₂ -X ₃ )×100%

In the _formula , X1 is the absorbance value _of the microsphere sample, X2 is the absorbance value of the positive control, and _X3 is the absorbance value of the negative control. The test results are shown in Figure 11. The average hemolysis rates of PVA/CS microspheres and FNS/PHBV@PVA/CS microspheres are 0.7417% and 1.6857%, respectively, far below the standard stipulated in ASTM F 756-08 (2000) , There is no hemolysis when the hemolysis rate is 0-2%. And from the digital photos of hemolysis, it can be seen that the positive control group is bright red after hemolysis, while the negative control and the two microsphere samples are almost colorless, indicating that the two microspheres have good blood compatibility.

Example 6

In vitro catheter fluidity testing of embolic materials is an important detection method to predict whether embolic materials will block catheters during in vivo embolization. Take an appropriate amount of FNS/PHBV@PVA/CS microspheres prepared in Example 1 and mix them evenly with saline, draw the microsphere suspension with a syringe and connect it to the 5Fr catheter, push the syringe, and observe the suspension state of the microspheres in the syringe and microsphere suspension through the catheter. As a result, as shown in accompanying drawing 12, the suspension of the embolic material in the syringe is good, and there is no phenomenon of microspheres clogging the catheter during the process of pushing the syringe (accompanying drawing 12 (a)), and it can be seen from the plate that the microspheres pass through the catheter in a state of The dispersion state (Fig. 12(b)) shows that the embolic microspheres have good in vitro circulation and are suitable for subsequent arterial embolization in vivo.

Example 7

New Zealand white rabbits were selected as animal embolization models, and the central arteries of bilateral rabbit ears were embolized to evaluate the embolization effect of embolization materials. All animal experiments were carried out in accordance with the "Regulations on the Administration of Experimental Animals of Hubei Province" and were reviewed and approved by the Ethics Committee of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. After New Zealand white rabbits were anesthetized, the rabbit hair was removed from the ears, and after being disinfected with iodine, the skin near the heart of the central artery of the rabbit ear was removed, the connective tissue around the central artery of the rabbit ear was removed, and the central artery was separated from the nerves and veins to expose After the central artery, use a vascular spacer to pass through the artery. After the blood vessel is congested, use a 22G arterial puncture needle to puncture the artery, and inject FNS/PHBV@PVA/CS microsphere-glycerol suspension from the proximal end. When the rabbit ear After the distal end of the central artery is completely occluded, the needle is pulled out, and the puncture site is pressed with a cotton ball for 15-20 minutes. During embolization at different time periods (0 days, 3 days, 7 days, 15 days and 21 days), the embolism status and infarction of rabbit ear vessels were observed. In the embolization experiment, the blank control group was glycerol, and the other experimental group was PVA/CS microspheres. as comparison. At a certain time (7 days, 21 days), the animals were sacrificed, the rabbit ears were cut off, and the rabbit ear infarction tissues containing the central artery were excised, soaked in 4% paraformaldehyde for 24 hours, dehydrated in conventional gradients, embedded in paraffin, and extracted from rabbit ears respectively. Transverse and longitudinal sections of the central artery were cut into 5 μm thin sections, stained with hematoxylin-eosin (HE), and observed histologically under a light microscope. Accompanying drawing 13 (a), (b), (c), (d) and (e) are respectively 0 day, 3 days, 7 days, 15 days and 21 days of rabbit ear morphological change results of rabbit ear embolism. After the embolization, it can be seen from Figure 13 (a) that there is an obvious ischemic area after the embolization of the microspheres in the central artery of the rabbit right ear, but there is no ischemic area after the embolization of glycerol in the central artery of the rabbit's left ear. This is because the glycerol With the blood flow into the blood circulation in the body. After 3 days of embolization, as shown in Figure 13(b), the central artery of the rabbit ear was blocked and blood flow could not enter the arterial branch, resulting in tissue edema. After 7 days of embolization, the tissue around the embolized vessel became darker, and there was a tendency of tissue necrosis (Fig. 13(c)). After 15 days of embolization, the tissue at the distal end of the rabbit ear became black, dry and thin, and the necrotic tissue tended to expand to the surrounding (Fig. 13(d)). After 21 days of embolization, a large area of necrosis appeared in the rabbit ears, and some tissues appeared large cracks due to drying up, which tended to fall off. The results show that the microspheres have successfully embolized the rabbit ears and the effect is very obvious. The results of HE staining are shown in Figure 14. The blood vessels (horizontal and vertical) of the blank control group were full of thrombi, because the blood coagulated in the central artery of the rabbit ear to form a thrombus after the New Zealand white rabbit was sacrificed. The layered structure of blood vessels around the thrombus was obvious, and there was no inflammatory reaction around the blood vessels. After PVA/CS microspheres embolize the central artery of the rabbit ear, it can be seen from the slices (transverse and longitudinal) that multiple microspheres are filled in the vascular tissue, and there is a certain extrusion with the vessel wall. The surrounding microspheres are filled with thrombus. The tissue had inflammatory infiltration, and the perivascular tissue became darker red after dense eosin staining, indicating that the perivascular tissue had degenerated necrosis after embolization. After embolization of vessels with FNS/PHBV@PVA/CS microspheres, the results of HE staining (transverse and longitudinal) were similar to those of PVA/CS microspheres, with less perivascular inflammatory response. After 21 days of embolization, HE staining analysis was carried out on the tissue sections after embolization of rabbit ears. It can be seen from Figure 15 that the blood vessels in the blank group were still full of thrombus, and a large number of microspheres were embolized in the blood vessels in the microsphere experimental group. Complete, and the area of dense eosin staining around the blood vessels expanded, indicating that the tissue necrosis was more serious. At the same time, it was found that the endothelial cells in the embolized blood vessels were hyperplastic, the blood vessel walls were thickened, and there were a small amount of blank microspheres in the blood vessels. caused by shedding.

Example 8

In order to observe the ultrastructural changes of rabbit ear tissues after embolization, the rabbit ear tissues embolized with glycerol, PVA/CS microspheres and FNS/PHBV@PVA/CS microspheres for 21 days in Example 7 were collected, and small pieces of rabbit ears were taken. Tissues were cut into small pieces of 1mm×1mm×1mm, fixed with 2.5wt% glutaraldehyde, washed with PBS several times, fixed with 1% osmium tetroxide, dehydrated with ethanol gradient, 100% ethanol/acetone (1:1, v/v) dehydration, soak in acetone/resin (1:1, v/v) for 1 hour, soak in pure resin, embed in neutral resin and slice, stain with toluidine blue, and trim to a certain thickness. The slices were picked up on a copper grid, stained with uranyl acetate and lead citrate, and observed under a field emission transmission electron microscope (FETEM). As can be seen from accompanying drawing 16, blank group (accompanying drawing 16 (a), (b), (c)) complete cell membrane can be seen in the fibroblasts around the central artery of the rabbit ear, and there are a large number of mitochondria, For organelles such as ribosomes, the nucleus is slightly indented, heterochromatin is gathered in the nuclear membrane, and euchromatin is distributed in a network. After embolization with PVA/CS microspheres and FNS/PHBV@PVA/CS microspheres, as shown in Figure 16(d)~(i), the nuclear membrane is ruptured and incomplete, mitochondria are swollen, and a large number of vacuoles, ribosomes, etc. The organelles dissolve and disappear, and the chromatin in the nucleus can be seen to shrink and aggregate into blocks. It shows that after embolization, the ultrastructure of rabbit ear tissue changes due to ischemia and hypoxia.

Comparative example 1

Weigh 0.8g of PVA124 and add 5.85mL of deionized water in a water bath to heat to dissolve, meanwhile weigh 0.04210g of CS (molecular weight is 110kDa) and dissolve in 5mL of 1wt% acetic acid solution. The two were mixed under magnetic stirring, and then a certain volume of Tween80 was added into the PVA/CS mixed solution to make the final concentration 0.5 wt%, and the magnetic stirring was continued for 20 min. The PVA/CS solution was slowly added dropwise to 72 mL of n-heptane with a mass fraction of 2.43% Span80 (pre-emulsified for 30 min), magnetically stirred at 350 rpm at room temperature for 30 min to form a W/O emulsion, and 25 wt% pentadiene saturated with ether was added Aldehyde solution (ether: 4.15mL, glutaraldehyde: 2.6mL), after stirring for 5min, 1.8mL of 1M hydrochloric acid solution was added, and the temperature was raised to 65°C for chemical crosslinking for 5h. The microspheres were collected by centrifugation, washed three times with petroleum ether, absolute ethanol and deionized water, and then the free glutaraldehyde was removed with 4wt% glycine solution, washed with deionized water and freeze-dried to obtain the composite drug-loaded microspheres , respectively sieved with 60 and 150 mesh standard sieves to obtain microspheres with a certain particle size.

While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies thereof, the present invention also intends to include these modifications and variations.

Claims (10)

1. A multi-core co-shell composite drug-loaded microsphere, characterized in that: the multi-core co-shell composite drug-loaded microsphere is a PVA/CS composite polymer microsphere, and the inside of the PVA/CS composite polymer microsphere is coated There are PHBV microspheres loaded with FNS. 2. The multi-core co-shell composite drug-loaded microsphere according to claim 1, characterized in that: the particle size of the multi-core co-shell composite drug-loaded microsphere is 100-300 μm. 3. The preparation method of multi-core co-shell composite drug-loaded microspheres according to claim 1 or 2, characterized in that: the steps include: S1. Heat and dissolve the oil-soluble PHBV in an organic solvent, and then add FNS sonication to dissolve it in the organic solvent to obtain a drug-loaded PHBV solution; S2. Add the drug-loaded PHBV solution obtained in step S1 into the polyvinyl alcohol solution, and stir to form an O/W emulsion; S3. Add the O/W emulsion obtained in step S2 dropwise into the polyvinyl alcohol solution, stir until the organic solvent volatilizes to obtain the drug-loaded microspheres, and freeze-dry them after washing; S4. Add the FNS/PHBV microspheres obtained in step S3 to the PVA/CS mixed solution containing Tween80, stir and sonicate to form a S/W mixed phase; S5. Add the S/W mixed phase obtained in step S4 into the pre-emulsified oil phase, stir to form a S/W/O emulsion, add glutaraldehyde solution saturated with ether, add hydrochloric acid solution after stirring, and heat up to 40-65°C Carry out chemical cross-linking for 3-8 hours; S6. The microspheres are collected by centrifugation and washed, and the free glutaraldehyde in the microspheres is removed with glycine or L-lysine solution, washed, freeze-dried, and sieved to obtain multi-core co-shell composite drug-loaded microspheres. 4. the preparation method of multi-core co-shell composite drug-loaded microspheres as claimed in claim 3, is characterized in that: in step S1, described organic solvent is the mixture of dichloromethane and ethyl acetate, described dichloromethane and The volume ratio of ethyl acetate is (1-4): 1; the concentration of PHBV in the drug-loaded PHBV solution is 20-60 mg/mL; the mass ratio of the FNS to PHBV is 1: (1-5); the The heating temperature is 30-60°C; 5. The preparation method of multi-core co-shell composite drug-loaded microspheres as claimed in claim 3, characterized in that: in step S2, the volume ratio of the drug-loaded PHBV solution to the polyvinyl alcohol solution is 1:(3～8 ). 6. The preparation method of multi-core co-shell composite drug-loaded microspheres as claimed in claim 3, characterized in that: the volume ratio of O/W emulsion and polyvinyl alcohol solution described in step S3 is 1: (20-80) . 7. The preparation method of multi-core co-shell composite drug-loaded microspheres as claimed in claim 3, characterized in that: in step S4, the concentration of PVA in the PVA/CS mixed solution is 30-80 mg/mL, and CS accounts for PVA The mass percentage of the /CS mixed solution is 3%-10%. 8. The method for preparing multi-core co-shell composite drug-loaded microspheres according to claim 3, characterized in that: the mass percentage of the FNS/PHBV microspheres in the multi-core co-shell composite drug-loaded microspheres is 10-30%. 9. The preparation method of multi-core co-shell composite drug-loaded microspheres as claimed in claim 3, characterized in that: in step S5, the oil phase is one or more of n-heptane, cyclohexane or petroleum ether. kind. 10. The application of the multi-core co-shell composite drug-loaded microsphere according to claim 1 or 2 in the treatment of prostate embolism.