CN114224846A

CN114224846A - Drug-loaded fiber microsphere and preparation method and application thereof

Info

Publication number: CN114224846A
Application number: CN202111371571.2A
Authority: CN
Inventors: 沈明武; 张蓓蕾; 史向阳
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2021-11-18
Filing date: 2021-11-18
Publication date: 2022-03-25
Anticipated expiration: 2041-11-18
Also published as: CN114224846B

Abstract

The invention relates to a drug-loaded fiber microsphere and a preparation method and application thereof³⁺Modifying HA on the surface of the PLGA short fiber to obtain functional short fiber PGH SFs; PGH SFs and DOX are blended in aqueous solution containing 0.1 wt% of gelatin, and the obtained dispersion liquid is crosslinked by electrospray and glutaraldehyde in sequenceObtaining the drug-loaded fiber microsphere DOX @ PGH with the functions of MR imaging and targeting specificity. The preparation process is simple and easy to operate; the prepared multifunctional drug-loaded fiber microsphere can be used for anchoring of a primary tumor model, long-acting chemotherapy and magnetic resonance imaging diagnosis, and further achieves the effects of treating tumors and inhibiting tumor metastasis.

Description

Drug-loaded fiber microsphere and preparation method and application thereof

Technical Field

The invention belongs to the field of antitumor drugs, and particularly relates to a drug-loaded fiber microsphere as well as a preparation method and application thereof.

Background

Metastasis and invasion are the leading causes of death in cancer patients. More than 90% of patients are diagnosed, the metastatic invasive in-situ tumor cells are already shed and enter a blood circulation system to form circulating tumor cells, and the circulating tumor cells are planted to distant organs of a body along with blood circulation to establish metastatic lesions, so that the primary tumor is metastatic and spread in the human body. Therefore, developing a platform that can both treat tumors and fundamentally inhibit the occurrence of in situ tumor metastasis and invasion is a promising strategy for improving survival rates of patients diagnosed.

The electrostatic spinning short fiber is processed by the electrostatic spinning nanofiber membrane through a homogenization technology. Compared with the nanofiber membrane, the short fiber has received more attention due to small size and good dispersibility, and becomes a transition platform for converting a two-dimensional structure fiber membrane into a three-dimensional structure, which is a great progress in the field of tissue engineering and also provides more possibility for the electrostatic spinning technology in the aspect of tumor treatment.

The research on the preparation of the multifunctional drug-loaded fiber microspheres by combining electrostatic spinning, a homogenizing technology and an electrospray technology and the research on the application of the multifunctional drug-loaded fiber microspheres in tumor treatment and tumor metastasis inhibition are not reported yet.

Disclosure of Invention

The invention aims to solve the technical problem of providing a drug-loaded fiber microsphere, a preparation method and application thereof, and fills the technical blank of the drug-loaded fiber microsphere in the prior art.

The drug-loaded fiber microsphere is prepared by carrying out electrospray and crosslinking on raw materials containing functional short fibers PGH SFs and drugs; wherein the surface of the functional short fiber PGH SFs is modified with Gd³⁺And HA.

The drug is DOX.

The drug-loaded fiber microsphere is prepared by homogenizing a PLGA nano fiber membrane to obtain PLGA short fibers, and the surfaces of the PLGA short fibers are chelated with Gd through PEI-DTPA³⁺Modifying HA, further mixing with DOX, and sequentially introducingAnd performing electrospraying and glutaraldehyde crosslinking.

The preparation method of the drug-loaded fiber microsphere comprises the following steps:

(1) adding the polylactic acid-glycolic acid PLGA nano fiber membrane into an aqueous solution containing polyvinyl alcohol PVA for homogenization treatment, centrifuging, and discarding the precipitate; centrifuging the supernatant, collecting the precipitate, and freeze-drying to obtain PLGA short fibers;

(2) dispersing PLGA short fiber in water, and activating by (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS to obtain PLGA short fiber dispersion liquid; dissolving polyethyleneimine PEI in water, adding diethylenetriaminepentaacetic dianhydride DTPA, and stirring for reaction to obtain a PEI-DTPA solution;

(3) mixing the PEI-DTPA solution and the PLGA short fiber dispersion liquid, stirring for reaction, then adding a gadolinium salt aqueous solution, continuously stirring for reaction, centrifuging, washing with water, and freeze-drying to obtain PG SFs;

(4) dissolving hyaluronic acid HA in water, adding EDC and NHS for activation to obtain an activated carboxyl HA solution, then dropwise adding the activated carboxyl HA solution into PG SFs dispersion liquid, stirring for reaction, centrifuging, washing with water, and freeze-drying to obtain PGH SFs;

(5) dispersing PGH SFs in an aqueous solution containing 0.1 wt% of gelatin, adding the medicine, uniformly mixing, electrically spraying, freeze-drying and crosslinking to obtain the medicine-carrying fiber microspheres.

The preferred mode of the above preparation method is as follows:

the PLGA nanofiber membrane obtained in the step (1) is prepared by dissolving PLGA (Mw is 80 kDa-810 kDa) in a solvent to obtain a PLGA spinning solution, and performing electrostatic spinning to obtain the PLGA nanofiber membrane; wherein the electrostatic spinning process conditions are as follows: the spinning voltage is 20kV, the flow rate of the injection pump is 0.6mL/h, the receiving distance is 15cm, the ambient temperature is 25 ℃, and the humidity is 30%.

In the aqueous solution containing PVA in the step (1), the concentration of PVA (Mw is 85-124 kDa) is 0.2-0.5 wt%; the mass-volume ratio of the PLGA fiber membrane to the PVA water solution is 90-120mg:40-50 mL.

The homogenization treatment process in the step (1) comprises the following steps: the rotating speed of the homogenizer is 16000-16800 rpm/min, and the homogenizing treatment is 30-40 min; the centrifugation parameters were: centrifuging at 1000-1200 rpm/min for 1-3 min, and discarding the precipitate; the parameters of the supernatant fluid centrifugation are as follows: centrifuging at 5000-6000 rpm/min for 3-5 min, collecting precipitate, dispersing again, centrifuging, and repeating the operation for 3 times.

The mass ratio of the PLGA short fibers to the EDC in the step (2) is 8-10: 1; the molar ratio of EDC to NHS is 1: 1.0-1.2; the molar ratio of PEI (Mw is 20kDa to 25kDa) to DTPA is 1: 40-50; the activation time is 2-4 h; the stirring reaction time is 1-3 h, and the temperature is room temperature.

The mass-volume ratio of the PLGA short fiber to the PEI-DTPA solution in the step (3) is 70-80 mg: 6-8 mL; the gadolinium salt is Gd (NO)₃)₃·6H₂O; the Gd³⁺The molar ratio of the DTPA to the DTPA is 1-2: 1; the stirring reaction time is 1 to 3 days, then gadolinium salt aqueous solution is added, and the stirring reaction is continued for 12 to 18 hours.

The molar ratio of HA, EDC and NHS in the step (4) is 1: 18-20; the mass ratio of PG SFs to HA is 1.5-2.0: 1; the reaction is stirred for 1 to 3 days at room temperature.

The mass ratio of PGH SFs to the medicines in the step (5) is 3-5: 1; the drug is DOX; the technological parameters of the electric spraying are as follows: the voltage is 10kV, the flow rate of an injection pump is 2mL/h, the receiving distance is 10cm, an aluminum foil soaked in liquid nitrogen is used as a collecting device, the ambient temperature is 25 ℃, and the humidity is 30%; the crosslinking is glutaraldehyde crosslinking.

The invention provides application of the drug-loaded fiber microspheres in preparation of antitumor drugs.

Further, the anti-tumor means includes in situ treatment of tumors and inhibition of tumor metastasis.

The invention combines electrostatic spinning, homogenization treatment and electrospray technology to obtain drug-loaded fiber microspheres which can firmly anchor tumor cells under the guidance of a targeting mechanism and inhibit the metastasis of the tumor cells; the microsphere porous structure can efficiently load anticancer drugs, increase the accumulation of the drugs on tumor parts, achieve long-term stable slow release, and effectively inhibit the metastasis and invasion of tumors while efficiently treating in-situ tumors.

The invention makesBy Scanning Electron Microscope (SEM), NMR, Hydrogen Spectroscopy (C)¹The prepared drug-loaded fiber microspheres are characterized by methods such as H NMR (nuclear magnetic resonance), inductively coupled plasma spectroscopy (ICP-OES), thermogravimetric analysis (TGA) and the like, and then various performances and application potentials of the multifunctional drug-loaded fiber microspheres prepared in the invention in cancer treatment are evaluated by tests such as drug slow release experiments, magnetic resonance imaging performances, CCK-8 cell viability and the like and in-vivo tumor treatment, and the specific test results are as follows:

(1) and (4) SEM test:

the SEM photograph is shown in FIG. 2, which shows that the PLGA nano-fiber has smooth surface and uniform appearance, and the average diameter of the fiber is 563.8 nm; the average diameter of the PLGA short fiber obtained after homogenization treatment is 1.7 mu m, and the increase of the diameter of the short fiber is probably caused by the phenomenon that the fiber absorbs water and swells in the homogenization treatment process; as shown in FIG. 3, the obtained DOX @ PGH has a porous structure, and due to the crosslinking effect, the short fibers are mutually connected, so that the stability of the microsphere structure is ensured.

(2)¹H NMR characterization:

¹the results of H NMR analysis are shown in FIG. 4, in which 2.1ppm to 3.0ppm are characteristic peaks of hyperbranched PEI, and 3.3ppm and 3.2ppm are CH in DTPA₂And it was found by calculation that 27 DTPAs were modified per PEI.

(4) TGA test and ICP-OES test:

TGA results are shown in FIG. 5, where the weight loss of PLGA, PG, PGH SFs was 99.4%, 90.1% and 82.8% at 600 ℃, respectively, and thus the modified PEI-DTPA-Gd and HA content in PGH SFs was 9.3% and 7.3%, respectively. Wherein, the content of Gd in PGH SFs is 3.6 percent through ICP-OES determination.

(5) Testing the slow release performance of the medicine:

the test result of the drug sustained release experiment is shown in fig. 6, and the fiber microspheres with different crosslinking time have different release behaviors for DOX. The cumulative release of DOX at 120h for crosslinked microspheres of 0h, 1h, 3h and 6h was 98.7%, 91.0%, 52.2% and 15.2%. It is clear that the uncrosslinked DOX @ PGH released almost all DOX in a short time, and that the cumulative release of DOX at 24h for 1h of crosslinked DOX @ PGH was 76% and did not achieve a long-term slow release of DOX, whereas the crosslinked 6h microspheres may have very little release of DOX due to excessive crosslinking. The accumulative release amount of the cross-linked 3h DOX @ PGH medicament in 120h is 52.2 percent, so that the long-term stable slow release of the anticancer medicament DOX is achieved, and the cross-linked 3h is selected for the experiments and the characterization of the microspheres involved in the experiments.

(6) Fluorescence microscopy test:

the fluorescence photograph is shown in FIG. 7a, and DOX @ PGH has stable red fluorescence, which indicates the successful loading of DOX; the diameter of the fiber microspheres in the fluorescent photograph was counted using ImageJ software, and the average diameter of the fiber microspheres was 118.8 μm, as shown in fig. 7 b.

(7) Magnetic resonance imaging performance testing:

as shown in FIG. 8a, T increases with the concentration of the material₁The MR imaging effect is gradually enhanced; as shown in FIG. 8b, the relaxation rate r of DOX @ PGH is known by linear fitting of the Gd concentration of the material to the reciprocal of the relaxation time₁＝17.7mM^-1s^-1。

(9) In vitro cytotoxicity and anticancer activity assay:

the cytotoxicity determination results are shown in fig. 9a, and the cell viability of the PGH with different concentrations and 4T1 cells after 24h co-culture exceeds 90%, which indicates that the fibrous material has good cell compatibility. The anticancer activity measurement results are shown in fig. 9b, and it can be seen that 4T1 cell viability was decreased in each group with the increase of DOX concentration. Among them, free DOX was the most potent on 4T1 cells, and DOX @ PGH + HA group was the least potent. When the concentration of the DOX exceeds 20 mug/mL, the cell activity after the DOX @ PGH treatment is lower than 50%, which indicates that the prepared drug-loaded fiber microsphere DOX @ PGH has a good killing effect on cancer cells.

(10) Evaluation of target specificity:

the Gd content of 4T1 cells was quantitatively analyzed by ICP-OES, and as shown in fig. 10, the Gd content of 4T1 cells in the PGH group was significantly increased compared to the PGH + HA group at the same Gd concentration, and with the increase of Gd concentration, the Gd content of 4T1 cells in the PGH group was gradually increased, which was more trend than in the PGH + HA group, indicating that CD44 receptors on the surface of HA-pretreated 4T1 cells were already occupied by excessive HA, and microspheres failed to accurately identify cells, resulting in decreased binding efficiency to 4T1 cells. This result indicates that HA modification confers significant targeting specificity to the fibrous microspheres.

(10) Scratch test results:

the images of the scratch test and the results of the quantitative migration area of the cells are shown in fig. 11a-b, the migration rate of the control group after 24h culture reaches 57.4%, which indicates that the 4T1 cells have higher transfer capacity, which is obviously higher than that of the PGH, DOX @ PGH and DOX @ PGH + HA groups, and indicates that the prepared multifunctional microspheres have a certain inhibition effect on the transfer of 4T1 cells. In particular, DOX @ PGH HAs the strongest metastasis inhibition effect on 4T1 cells under the conditions of killing DOX load and targeted specific HA modification.

(11) Results of 3D cell pellet experiments:

the anti-tumor metastasis function and the anti-cancer effect of the fiber microsphere are further proved by a plate culture experiment of an in vitro three-dimensional multicellular tumor sphere. As shown in FIGS. 11c-d, at 120h, the control tumor spheroids proliferated rapidly during plating and grew to 2 times the original diameter. Tumor spheroids co-cultured with PGH had a slightly reduced diameter growth to 1.7 times the original diameter. The growth of tumor spheroids treated with DOX @ PGH + HA and DOX @ PGH was significantly inhibited and the diameters increased to 1.5 and 0.5 times, respectively. In particular, DOX @ PGH showed the strongest ability to inhibit tumor spheroid growth, significantly lower than the relative diameter of the control group.

(12) In vivo MR imaging test:

the results of in vivo MR imaging in animals are shown in FIG. 12, and it is clear that the MR signal intensity at the tumor site is significantly increased and tends to be decreased with the lapse of time in mice injected with DOX @ PGH and DOX @ PGH + HA. For DOX @ PGH and DOX @ PGH + HA, the MR signal intensity was still much stronger at day 5 than initially. By quantifying the signal-to-noise ratio at the tumor site, it was found that DOX @ PGH consistently showed a stronger MR signal intensity than DOX @ PGH + HA, indicating that DOX @ PGH HAs a longer residence time at the tumor site due to HA mediation.

(13) Evaluation of tumor treatment effect:

statistics of relative tumor volume and body weight of five groups of nude mice are shown in fig. 13, and mice in the DOX group had a sharp weight drop at the beginning of treatment, causing strong side effects. In addition, tumor volume in all cases showed an upward trend, especially in the control group, which was 12.2-fold higher than the initial tumor volume, but only increased 1.9-fold after treatment with DOX @ PGH. It is also clear that the tumor growth rate of the drug-loaded fiber microsphere group is lower than that of the drug-free fiber microsphere group. The PGH group showed a slightly lower tumor volume increase than the PBS group, probably due to the modification of HA, which allowed the microspheres to anchor directionally to the tumor site, preventing the random growth of tumor cells. DOX @ PGH HAs the best anti-tumor effect, and the relative tumor volume is lower than that of the DOX @ PGH + HA group, because DOX @ PGH is firmly anchored on tumor cells through the combination of HA and CD44 receptors, the DOX is ensured to directly act on the tumor cells once being released, the accumulation and bioavailability of the DOX at the tumor part are enhanced, and the toxic and side effects of the DOX on normal tissues are greatly reduced.

(14) Evaluation of inhibition of metastasis:

after 14 days of various treatments, lung tissue was removed from each group of 4T1 tumor-bearing nude mice and photographed. As shown in fig. 14, the highest number of tumor nodules were present in the lungs of the control group, indicating that the 4TI tumor is a malignancy with a high degree of metastatic character. In contrast, the number of tumor nodules in the lung was reduced in the other experimental groups, and in particular, the DOX @ PGH group showed the strongest inhibition of tumor metastasis with the least number of tumor nodules. It is noted that PGH, although less effective in inhibiting tumor growth, was successful in immobilizing tumor cells in situ to inhibit tumor distant metastasis, probably due to the inherent porous structure of the fibrous microspheres themselves and the CD 44-mediated targeting effect.

Advantageous effects

(1) The invention has simple preparation process, controllable size, low cost, degradability in vivo and good biological safety because the main material is PLGA fiber microspheres, and has industrial prospect.

(2) According to the invention, the nanofiber membrane is processed into the porous fiber microsphere material through the homogenization treatment and the electrospray technology, so that the conversion from a two-dimensional structure to a three-dimensional structure of the electrostatic spinning nanofiber membrane is successfully realized, and the injectability of the fiber material is realized.

(3) The fiber microsphere material modified on the surface of the fiber microsphere HAs targeting capacity, so that the fiber microsphere material can firmly anchor cancer cells and inhibit the metastasis of tumor cells by combining the advantages of the size and the structure of the fiber microsphere material (firstly, the microscopic photos of a cell scratch experiment and a 3D cell ball culture experiment and the corresponding quantitative statistical results prove that the fiber microsphere material modified by HA can inhibit the migration of the cells in an in vitro cell experiment, then, the comparison of two groups of experiments on an in vivo animal experiment level shows that the duration of the MR signal intensity of the tumor part is longer when monitoring the tumor part under the condition of not blocking the HA of the tumor cells, namely, the stagnation time of the fiber microsphere material at the tumor part is longer, so that the fiber microsphere material can be judged to be firmly anchored at the tumor part, in addition, the lung metastasis condition of a mouse is evaluated, and the comparison of the quantitative statistical results of the lung tissue photo and the tumor knot number of the lung tissue shows that the fiber microsphere material can effectively inhibit the tumor cells Transfer).

(4) The drug-loaded fiber microsphere prepared by the invention not only can effectively inhibit the growth of in-situ tumor, but also can effectively inhibit the metastasis of tumor (the conclusion that the fiber microsphere material can effectively inhibit the lung metastasis can be obtained through in vitro cell experiments and in vivo lung metastasis conditions, and in addition, the result of cell experiment cytotoxicity experiments and the comparison of a tumor volume statistical graph of a treated mouse show that the fiber microsphere loaded with the anti-cancer drug DOX has the smallest tumor volume which is obviously smaller than that of a control group, so the growth of in-situ tumor is effectively inhibited), and the drug-loaded fiber microsphere has wide application prospect in the field of cancer treatment.

Drawings

FIG. 1 is a schematic diagram of the preparation of the multifunctional drug-loaded fiber microsphere of the present invention;

FIG. 2 shows SEM images of a PLGA nanofiber membrane and a PLGA staple fiber in example 1, and c and d are diameter distribution diagrams of the PLGA nanofiber membrane and the PLGA staple fiber;

FIG. 3 a, b, c are SEM images of DOX @ PGH at different magnifications in example 1 of the invention;

FIG. 4 shows the preparation of PEI-DTPA in example 1 of the present invention¹H NMR spectrum;

FIG. 5 shows the results of thermogravimetric analysis of the multifunctional staple fibers (PLGA, PG and PGH SFs) in example 1 of the present invention;

FIG. 6 is a plot of the cumulative release of DOX drug from DOX @ PGH in PBS (pH 7.4) at various cross-linking times in example 1 of the present invention;

FIG. 7 is a photograph taken by a fluorescence microscope of DOX @ PGH in example 1 of the present invention (a) and a result of statistical distribution of diameters (b);

FIG. 8 is a graph showing T at various concentrations of DOX @ PGH in example 1 of the present invention₁Weighted MR image (a) and Gd concentration and T₁A linear fit of the inverse relaxation times (b);

FIG. 9 shows the results of PGH cell compatibility and anticancer activity tests using DOX @ PGH, a is the cell viability test result of 4T1 cells incubated with different concentrations of PGH for 24h, and b is the cell viability test result of 4T1 cells incubated with different concentrations of DOX, DOX @ PGH, or DOX @ PGH + HA for 24 h;

fig. 10 is a PGH target specificity validation result, which is a determination of Gd content of 4T1 cells after incubation with PGH and PGH + HA containing different Gd concentrations for 4 h;

fig. 11a is a microscopic photograph of 4T1 cells incubated with different fibrous microsphere materials for 12h and 24h, and b is a statistical result of the mobility of 4T1 cells for 12h and 24h in a cell scratch experiment; c is a microscope photo of different materials and the 3D cell balls after being subjected to planar culture for different time, and D is a cell ball spreading diameter change curve;

FIG. 12 is T of 4T1 tumor-bearing nude mice at different time points after intratumoral injection of DOX @ PGH and DOX @ PGH + HA₁Weighted MR imaging (a) and MR signal-to-noise ratio (b) of the corresponding tumor region;

FIG. 13 is the weight change (a) and the relative volume change (b) of the tumor of 4T1 tumor-bearing nude mice treated with different materials;

FIG. 14 is a photograph of lung tissue (a) and statistics of lung metastatic nodules (b) of 4T1 tumor-bearing nude mice after 14 days of treatment with different materials;

FIG. 15 is an SEM photograph of PGH in comparative example 1 of the present invention at various magnifications.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Unless otherwise specified, all chemical reagents were commercially available and used without further purification. PLGA (Mw 80kDa to 810kDa) is available from dendri, gordonia bio materials ltd. PEI (Mw ═ 20kDa to 25kDa) and PVA (Mw ═ 85 to 124kDa) were purchased from Sigma-Aldrich trade company ltd (shanghai, china). DOX was purchased from Beijing Huafeng pharmaceutical Co., Ltd (Beijing, China). DTPA was purchased from TCI development Inc. (Shanghai, China). EDC and NHS were purchased from carbofuran technologies ltd (shanghai, china). Gd (NO)₃)₃·6H₂O, DMF, THF, gelatin, glutaraldehyde (25% aqueous solution) were purchased from the national pharmaceutical group chemicals ltd (shanghai, china). HA (Mw 5830) was purchased from zhenjiang eastern biotechnology limited (zhenjiang, china). A regenerated cellulose dialysis membrane with a molecular weight cutoff of 3000 was purchased from Fisher (Pittsburgh, Pa.). DMEM medium, fetal bovine serum, penicillin-streptomycin, and trypsin 0.25% solution were purchased from hangzhou jinuo biomedical technologies, inc (hangzhou, china). Murine 4T1 breast cancer cells were provided by the institute of biochemistry and cell biology, national academy of sciences (Shanghai, China). The Cell Counting Kit-8(CCK-8) assay Kit was purchased from 7Sea Biotech Co., Ltd. (Shanghai, China).

Example 1

(1) Adding 0.5g of PLGA into 2mL of a mixed solution of N, N-dimethylformamide DMF and tetrahydrofuran THF, wherein the volume ratio of DMF to THF is 1:3, and magnetically stirring for 4-8 h to obtain a PLGA spinning solution; and then carrying out electrostatic spinning (the spinning voltage is 20kV, the flow rate of an injection pump is 0.6mL/h, the receiving distance is 15cm, the ambient temperature is 25 ℃, the humidity is 30%), and carrying out vacuum drying for 48h to obtain the PLGA nanofiber membrane.

(2) Will be provided withSoaking the obtained PLGA nano fiber membrane in ultrapure water for 5-10 min, then taking off the PLGA nano fiber membrane from the aluminum foil paper, and cutting the PLGA nano fiber membrane into pieces with the area of about 0.25cm²Then transferring the square fragments into an aqueous solution containing 0.2 wt% of PVA, and then carrying out homogenization treatment for 30min by using a homogenizer at the rotating speed of 16000 rpm; after the homogenization treatment is finished, centrifuging the homogenized solution at 1000rpm for 1min, wherein the precipitate is a fiber block which is not homogenized and completely crushed, and discarding the precipitate; centrifuging the upper layer solution at 6000rpm for 3min, and collecting precipitate, wherein the main target product of the precipitate is PLGA short fiber; and dispersing the obtained precipitate in ultrapure water, oscillating and dispersing by a vortex mixer, centrifuging, dispersing again, centrifuging, repeating the steps for 3 times, removing the PVA dispersing agent on the surface of the fiber, and freeze-drying to obtain the PLGA short fiber.

(3) 80mg of PLGA short fiber is dispersed in 8mL of ultrapure water, 1mL of aqueous solution containing 9.6mg of EDC is added, magnetic stirring reaction is carried out for 30min, 1mL of aqueous solution containing 6.8mg of NHS is added, and magnetic stirring reaction is carried out for 3 h. Dissolving 50mg PEI in 5mL of ultrapure water, adding 3mL of 40mg DTPA-containing aqueous solution, magnetically stirring, reacting for 1h to obtain PEI-DTPA solution, slowly adding the PEI-DTPA solution to the PLGA short fiber dispersion solution, reacting for 48h, and adding 1mL of 100mg Gd (NO) into the solution₃)₃·6H₂And reacting the mixture for 12 hours by using an O aqueous solution, and then centrifuging, washing and freeze-drying the reaction product to obtain the PG SFs.

(4) 40mg of HA was dissolved in 4mL of water and EDC (26mg, 1mL of water) and NHS (15mg, 1mL of water) were added with magnetic stirring to activate the carboxyl group of HA. The resulting PG SFs (60mg) were then redispersed in 6mL of water, to which the HA solution was added dropwise and magnetic stirring was continued at room temperature for 48 h. Then, the mixed solution was centrifuged, washed with water, and lyophilized to obtain PGH SFs.

(5) PGH SFs and DOX are mixed in an aqueous solution containing 0.1 wt% of gelatin, the concentrations of the PGH SFs and the DOX are respectively 15mg/mL and 4mg/mL, the mixture is vibrated and dispersed evenly by a vortex mixer, the mixture is sucked into an injector, electric injection is carried out (the voltage is 10kV, the flow rate of the injection pump is 2mL/h, the receiving distance is 10cm, an aluminum foil soaked in liquid nitrogen is used as a collecting device, the ambient temperature is 25 ℃, the humidity is 30 percent), freeze-drying is carried out, and glutaraldehyde crosslinking is carried out for 3h, so as to obtain the DOX @ PGH.

Example 2

And (3) observing the shapes and the sizes of the PLGA nano fiber membrane and the PLGA short fibers obtained in the step (1) in the example 1 and the functionalized drug-loaded fiber microspheres obtained in the step (5) in the example 1 by adopting SEM, carrying out gold spraying treatment on the fiber sample before shooting, wherein the gold spraying time is 60s, and carrying out statistical analysis on the diameter distribution of the nano fiber membrane and the short fibers by using ImageJ software. As shown in fig. 2, it can be seen that the PLGA nanofibers have smooth surfaces and uniform morphology, and the average diameter of the fibers is about 563.8 nm; the average diameter of the PLGA short fiber obtained after homogenization treatment is 1.7 mu m, and the increase of the diameter of the short fiber is caused by the phenomenon that the fiber absorbs water and swells in the homogenization treatment process. As shown in FIG. 3, the obtained DOX @ PGH has a porous structure, and due to the crosslinking effect, the short fibers are mutually connected, so that the stability of the microsphere structure is ensured.

Example 3

To confirm the successful preparation of PEI-DTPA and the amount of DTPA attached to PEI, 5mg of the PEI-DTPA solution obtained in step (2) of example 1 was lyophilized by dialysis and weighed out in 600. mu. L D₂In O, carrying out¹And H NMR characterization. As shown in FIG. 4, 2.1ppm to 3.0ppm are characteristic peaks of hyperbranched PEI, and 3.3ppm and 3.2ppm are CH in DTPA₂And it was found by calculation that 27 DTPAs were modified per PEI.

Example 4

5mg of PLGA, PG and PGH SFs were weighed respectively and subjected to TGA test under nitrogen condition at a temperature range of 50-600 ℃ and a temperature rise rate of 10 ℃/min, and the results are shown in FIG. 5. The modification amounts of PEI-DTPA-Gd and HA on the short fiber were 9.3% and 7.3%, respectively. 1mg of PGH SFs obtained in the step (4) of example 1 was weighed, added to 1mL of aqua regia, digested for 24 hours with the aqua regia, and then 3mL of ultrapure water was added, and the Gd concentration in the solution was measured by ICP, whereby the Gd content chelated by the PGH SFs was calculated to be 3.6%.

Example 5

To investigate the effect of cross-linking time on drug release performance, DOX @ PGH lyophilized in step (5) of example 1 was cross-linked with glutaraldehyde for 0h, 1h, 3h and 6h, respectively, and 2mg of each drug-loaded fiber microsphere cross-linked for different times was added to a dialysis bag having a molecular weight cut-off of 3000, and then the dialysis bag was tightly sealed and immersed in 9mL of PBS (pH 7.4), followed by being placed in a constant temperature shaker at 37 ℃ and 160 rpm. At the set time point, 1mL of external buffer was removed and 1mL of PBS was added. And testing the light absorption value of the extracted solution at 480nm by using an ultraviolet-visible spectrophotometer (UV-vis), and calculating the concentration and the accumulated release amount of the DOX according to a DOX concentration-light absorption value standard curve. The results are shown in fig. 6, the non-crosslinked drug loaded microspheres released DOX almost completely in a very short time; the release amount of DOX after crosslinking for 1h is up to 76% in 24h, and the stable effect of long-term slow release cannot be achieved; the fiber microspheres may be over-crosslinked after 6h of crosslinking, resulting in release of only 15.2% of DOX at 120 h; the fiber microspheres can realize the long-term stable slow release of DOX after crosslinking for 3h, and the cumulative release amount of the drug for 120h is 52.2%. Therefore, too long or too short a crosslinking time is not favorable for long-term stable release of DOX, and 3h is a relatively moderate crosslinking time. The characterization of the microspheres in this experiment was that the microspheres were selected to be crosslinked for 3 hours.

Example 6

The loading condition of DOX in example 1 is observed under a fluorescent microscope, and the size of the drug-loaded fiber microspheres is counted by using ImageJ software. As shown in fig. 7a, the red fluorescence photograph illustrates the successful loading of DOX; as shown in FIG. 7b, the average diameter of DOX @ PGH was 118.8. mu.m.

Example 7

Testing T of different concentrations of DOX @ PGH by using nuclear magnetic resonance imager₁Relaxation time sum T₁Weighted imaging effects, as shown in FIG. 8a, with increasing material concentration, T₁The MR imaging effect is gradually enhanced; as shown in FIG. 8b, the relaxation rate r of DOX @ PGH is found by linear fitting of Gd concentration to the reciprocal of the relaxation time₁＝17.7mM^-1s^-1。

Example 8

The viability of 4T1 cells after incubation with various concentrations of PGH was determined using CCK-8 to characterize the cellular compatibility of PGH. First, 100. mu.L of a 10-density suspension was added to each well of a 96-well plate⁴4T1 cell suspension/well, put at 37 ℃ and 5% CO₂In an incubatorAt night, after the cells are completely attached to the wall, the original culture medium is removed by suction, 100 mu L of culture medium containing PGH (0.25, 0.5, 1, 2, 4, 8mg/mL) with different concentrations is added, the culture medium is placed back into the incubator for continuous culture for 24 hours, the culture medium is removed by suction, PBS is washed for 3 times, 100 mu L of serum-free culture medium containing 10% CCK-8 is added into each well, and after continuous incubation for 3 hours, the absorbance value of the well plate at 450nm is tested by using a microplate reader. For each fiber material concentration, 5 parallel holes were tested. As shown in FIG. 9a, the cell viability of the PGH with different concentrations and 4T1 cells after 24h co-culture all exceeded 90%, indicating that the material has good cell compatibility.

Furthermore, to analyze the in vitro anti-cancer activity of DOX @ PGH, a similar protocol was used to determine the viability of 4T1 cells. Cell viability was compared after co-incubation of free DOX, DOX @ PGH + HA and DOX @ PGH with 4T1 cells at the same DOX concentration. First, 4T1 cells were cultured at 10⁴The cells were plated at a density of one well in 96-well plates and incubated overnight for adherence, the original medium was discarded, and 100. mu.L of medium containing either pure DOX or DOX @ PGH at the same DOX concentration (DOX concentration gradient of 2.5, 5, 10, 20, 40, 80. mu.g/mL) was added to each well. For the DOX @ PGH + HA group, HA (2mM) was incubated with the cells for 2h before replacing the medium with the medium containing DOX @ PGH. Continuously culturing the cells and the materials in an incubator for 24h, removing the culture medium by suction, washing with PBS for 3 times, adding 100 mu L of serum-free culture medium containing 10% CCK-8 into each hole, continuously incubating for 3h, testing the light absorption value of the hole plate at 450nm by using an enzyme-labeling instrument, and calculating the cell activity. For each fiber material concentration, 5 parallel holes were tested. The anticancer activity assay results are shown in fig. 9b, where 4T1 cell viability decreased with increasing DOX concentration for each group. Among them, free DOX was the most potent on 4T1 cells, and DOX @ PGH + HA group was the least potent. When the concentration of the DOX exceeds 20 mug/mL, the cell activity after the DOX @ PGH treatment is lower than 50%, which indicates that the prepared drug-loaded fiber microsphere DOX @ PGH has a good killing effect on cancer cells.

Example 9

4T1 cells were plated at a density of 2X 10⁵One/well was seeded in 12-well plates and cultured overnight to allow cells to adhere. Then, fresh PGH containing different Gd concentrations (3, 6, 12. mu.g/mL) was usedThe medium was replaced in each well and cultured with the cells for a further 4 h. For the PGH + HA group, HA (2mM) was incubated with cells for 2h to ensure that CD44 receptors on the surface of 4T1 cells were blocked, and then the medium was replaced with PGH-containing medium. After that, the medium was carefully removed, the cells were washed 3 times with PBS, treated with trypsin, suspended and counted. The remaining cells were collected by centrifugation and treated with 1mL of aqua regia solution for 24 h. Finally, the sample was diluted with PBS and the concentration of Gd was measured with ICP-OES. As shown in fig. 10, under the material treatment with the same Gd concentration, Gd content of 4T1 cells of the PGH group was significantly increased compared to the PGH + HA group, and with the increase of Gd concentration, Gd content of 4T1 cells of the PGH group was gradually increased, which was more trend than that of the PGH + HA group, indicating that CD44 receptor on the surface of HA-pretreated 4T1 cells had been occupied by too much HA, and that the fiber microspheres could not accurately recognize the cells, resulting in a decrease in binding efficiency to 4T1 cells. This result indicates that HA modification confers significant targeting specificity to the fibrous microspheres.

Example 10

The cell scratch test is to verify the inhibition effect of the multifunctional fiber microspheres and the cell metastasis under the chemotherapy effect of the further combination of the anticancer drugs. Here, 3 sets of experiments were established, PGH, DOX @ PGH + HA and DOX @ PGH sets, respectively. The same strategy, for the DOX @ PGH + HA group, required that cells were previously co-assigned with HA (2mM) for 2h to ensure that the cell surface CD44 receptor had been occupied by excess HA, and then the cells were incubated with DOX @ PGH. First, 4T1 cells in good state were cultured at 2X 10⁵The density of each hole is planted in a 12-hole plate and is put in 5 percent CO₂After incubation for 24h at 37 ℃, the plates were scratched with a 100 μ L pipette tip to remove floating cells, washed with PBS, and 1mL PBS was added to each plate, and the scratched wounds were observed under a microscope and photographed (0 h). After the observation was completed, the cells were cultured with complete medium containing PGH, DOX @ PGH + HA and DOX @ PGH, respectively, and the cells were observed for scratching and photographed at the next 12h and 24 h. The scratch area of the cells in each photograph was calculated by ImageJ software. As shown in FIGS. 11a-b, the control group reached 57.4% mobility after 24h incubation, indicating that 4T1 cellsThe transfer capacity is higher than that of PGH, DOX @ PGH and DOX @ PGH + HA groups, and the prepared multifunctional microspheres have certain inhibition effect on transfer of 4T1 cells. In particular, DOX @ PGH HAs the strongest metastasis inhibition effect on 4T1 cells under the conditions of killing DOX load and targeted specific HA modification.

Example 11

The in vitro culture of the 3D cell spheres by the template method is to further study the anti-metastasis and killing degree of the prepared multifunctional fiber microspheres on tumor cells. First, 0.25g of agarose was weighed into a glass vial and sterilized. Meanwhile, tools such as templates, tweezers and the like used in the experiment are soaked in alcohol and subjected to ultraviolet sterilization. After completion of sterilization, 12.5mL of physiological saline was added to the agarose, and the mixture was dissolved by heating. When the solution is clear and transparent, 500. mu.L of agarose solution is slowly dropped into the mold, and then naturally cooled until the solution is completely solidified. The cooled and solidified agar block was then gently peeled off the mold, transferred to a 12-well plate, and DMEM medium was added to allow the agar block to soak in the medium for 30 min. After soaking, the medium was aspirated, and 190. mu.L of the medium containing 8X 10 was added to each well on the agar block⁵Complete medium of 4T1 cells. After 15min, 2.5mL of complete medium was added to each well and placed in 5% CO₂And incubating in an incubator at 37 ℃.

After 3 days of culture, 3D multicellular spheroids were formed, pipetted gently, and suspended single scattered cells were filtered off. The formed multicellular spheroids were photographed using an inverted phase contrast microscope (as 0 h). Thereafter, 1mL of complete medium containing PGH and DOX @ PGH (2mg/mL) was added as an experimental group. Similarly, cells from the DOX @ PGH + HA group were previously assigned HA (2mM) for 2h before incubation with DOX @ PGH. At different time points (24h, 72h and 120h), the 12-well plate was removed, washed with PBS, replaced with fresh medium (1 mL per well), and photographed under a light microscope. The deployed diameter of the three-dimensional multicellular spheroids was measured using Image J software. As shown in FIGS. 11c-d, at 120h, the control tumor spheroids proliferated rapidly during plating and grew to 2 times the original diameter. Tumor spheroids co-cultured with PGH had a slightly reduced diameter growth to 1.7 times the original diameter. The growth of tumor spheroids treated with DOX @ PGH + HA and DOX @ PGH was significantly inhibited and the diameters increased to 1.5 and 0.5 times, respectively. In particular, DOX @ PGH showed the strongest ability to inhibit tumor spheroid growth, significantly lower than the relative diameter of the control group.

Example 12

Injecting 100 mu L of physiological saline (Gd concentration is 5mM) containing DOX @ PGH or DOX @ PGH + HA into 4T1 tumor-bearing nude mice, and carrying out MR imaging (30min, 1h, 1 day, 3 days and 5 days) on different time points on 4T1 tumor-bearing nude mice before and after DOX @ PGH or DOX @ PGH + HA injection through scanning of a nuclear magnetic resonance imager. For the DOX @ PGH + HA group, HA (24mg, 100. mu.L saline) was injected intratumorally 2h prior to DOX @ PGH injection. As shown in FIG. 12, it was evident that the MR signal intensity at the tumor site was significantly increased and tended to decrease with the passage of time in the mice injected with DOX @ PGH and DOX @ PGH + HA. For DOX @ PGH and DOX @ PGH + HA, the MR signal intensity was still much stronger at day 5 than initially. By quantifying the signal-to-noise ratio at the tumor site, it was found that DOX @ PGH consistently showed stronger MR signal intensity than DOX @ PGH + HA. This demonstrates the specificity of targeting by HA, which results in a longer residence time of the material at the tumor site, facilitating anchoring and enhanced treatment of the primary tumor.

Example 13

The prepared DOX @ PGH is prepared into a solution with the DOX concentration of 1mg/mL by using a sterile PBS buffer solution, and the weight of the microspheres without drug loading is equal to that of the microspheres with drug loading. The 4T1 tumor-bearing nude mice were divided into 5 groups, and 100. mu.L of the solution was injected into each tumor-bearing nude mouse by intratumoral injection: (1) PBS, (2) PGH, (3) free DOX, (4) DOX @ PGH + HA and (5) DOX @ PGH. For the DOX @ PGH + HA group, HA (24mg, 100. mu.L PBS) was injected intratumorally 2h prior to DOX @ PGH injection. Changes in body weight and tumor volume of the mice were recorded over 14 days of treatment. The results are shown in figure 13, where mice in the DOX group had a dramatic weight loss at the beginning of treatment, causing strong side effects. In addition, tumor volume in all cases showed an upward trend, especially in the control group, which was 12.2-fold higher than the initial tumor volume, but only increased 1.9-fold after treatment with DOX @ PGH. It is also clear that the tumor growth rate of the drug-loaded fiber microsphere group is lower than that of the drug-free fiber microsphere group. The tumor volume growth was slightly lower in the PGH group than in the PBS group, probably due to the modification of HA, allowing directional anchoring of the microspheres at the tumor site, preventing tumor cell growth. DOX @ PGH achieves the best anti-tumor effect, relative to tumor volume, lower than the DOX @ PGH + HA group, because DOX @ PGH is anchored tenaciously to tumor cells by binding of HA to CD44 receptors, ensuring that DOX acts directly on tumor cells once released, enhancing DOX accumulation and bioavailability at the tumor site.

Example 14

After 14 days of treatment, the mice were dissected, the lungs were photographed, and tumor metastasis in the lungs was observed and tumor nodule counts were counted. As shown in fig. 14, the highest number of tumor nodules were present in the lungs of the control group, indicating that the 4TI tumor is a malignancy with a high degree of metastatic character. In contrast, the number of tumor nodules in the lung was reduced in the other experimental groups, especially the DOX @ PGH group showed the strongest inhibition of tumor metastasis and the least number of tumor nodules in the lung tissue. It is noted that PGH, although less effective in inhibiting tumor growth, was successful in immobilizing tumor cells in situ to inhibit tumor distant metastasis, probably due to the inherent porous structure of the fibrous microspheres themselves and the CD 44-mediated targeting effect.

Comparative example 1

This comparative example provides a method of preparing an unloaded fiber microsphere (control):

(1) adding 0.5g of PLGA into 2mL of mixed solution of DMF and THF, wherein the volume ratio of DMF to THF is 1:3, and magnetically stirring for 4-8 h to obtain PLGA spinning solution; and then carrying out electrostatic spinning (the spinning voltage is 20kV, the flow rate of an injection pump is 0.6mL/h, the receiving distance is 15cm, the ambient temperature is 25 ℃, the humidity is 30%), and carrying out vacuum drying for 48h to obtain the PLGA nanofiber membrane. Soaking the obtained PLGA nanofiber membrane in ultrapure water for 5-10 min, then taking off the PLGA nanofiber membrane from aluminum foil paper, and cutting the PLGA nanofiber membrane into pieces with the area of about 0.25cm²Then transferring the square fragments into a PVA-containing solution, wherein the concentration of PVA is 0.2 wt%, and then carrying out homogenization treatment for 30min by using a homogenizer at the rotating speed of 16000 rpm; after the homogenization is finishedCentrifuging the homogenized solution at 1000rpm for 1min, and discarding the precipitate, wherein the precipitate is mostly a fiber block which is not homogenized and completely broken; centrifuging the upper layer solution for 3min at the rotating speed of 6000rpm, and collecting precipitates, wherein the main target product PLGA short fibers are precipitated; and dispersing the obtained precipitate in ultrapure water, oscillating and dispersing by a vortex mixer, centrifuging, dispersing again, centrifuging, repeating the steps for 3 times, removing the PVA dispersing agent on the surface of the fiber, and freeze-drying to obtain the PLGA short fiber.

(2) Dissolving 50mg of PEI in 5mL of ultrapure water, then adding 3mL of DTPA aqueous solution containing 40mg of PEI into the ultrapure water, magnetically stirring the solution, and reacting the solution for 1 hour to obtain a PEI-DTPA solution;

(3) dispersing 80mg PLGA short fiber in 8mL of ultrapure water, adding 1mL of aqueous solution containing 9.6mg EDC, reacting for 30min under magnetic stirring, adding 1mL of aqueous solution containing 6.8mg NHS, reacting for 3h under magnetic stirring, slowly adding PEI-DTPA solution obtained in the step (3), reacting for 48h, adding 1mL of PEI-DTPA solution containing 100mg Gd (NO) into the solution, reacting for 48h, and adding 1mL of PEI-DTPA solution containing 100mg Gd (NO)₃)₃·6H₂And reacting the mixture for 12 hours by using an O aqueous solution, and then centrifuging, washing and freeze-drying the reaction product to obtain the PG SFs.

(4) 40mg of HA was dissolved in 4mL of water and EDC (26mg, 1mL of water) and NHS (15mg, 1mL of water) were added with magnetic stirring to activate the carboxyl groups of surface HA. The prepared PG SFs (60mg) were then redispersed in 6mL of water. Thereafter, the above HA solution was added dropwise to the dispersion of PG SF and magnetic stirring was continued at room temperature for 48 h. Then, the mixed solution was centrifuged, washed with water, and lyophilized to obtain PGH SFs.

(5) Dispersing PGH SFs in an aqueous solution containing 0.1 wt% of gelatin to form a PGH SFs dispersion liquid with the concentration of 15mg/mL, sucking the PGH SFs dispersion liquid into an injector, performing electronic injection (the voltage is 10kV, the flow rate of the injection pump is 2mL/h, the receiving distance is 10cm, an aluminum foil immersed in liquid nitrogen is used as a collecting device, the ambient temperature is 25 ℃, the humidity is 30%), performing freeze-drying, and performing glutaraldehyde crosslinking for 3h to obtain PGH.

The morphology of the PGH obtained in step (5) of comparative example 1 was characterized by SEM, and the results are shown in fig. 15, where the fibrous microspheres obtained by electrospray technique were porous and their size was also comparable to that of the drug-loaded microspheres.

Claims

1. The drug-loaded fiber microsphere is characterized in that the fiber microsphere is obtained by carrying out electrospray and crosslinking on raw materials containing functionalized short fiber PGH SFs and drugs; wherein the surface of the functional short fiber PGH SFs is modified with Gd³⁺And HA.

2. The microsphere of claim 1, wherein the drug is DOX.

3. A preparation method of drug-loaded fiber microspheres comprises the following steps:

(1) adding the PLGA nano-fiber membrane into an aqueous solution containing PVA for homogenization treatment, centrifuging, and discarding the precipitate; centrifuging the supernatant, collecting the precipitate, and freeze-drying to obtain PLGA short fibers;

(2) dispersing PLGA short fiber in water, and activating by EDC and NHS to obtain PLGA short fiber dispersion liquid; dissolving PEI in water, adding DTPA, and stirring for reaction to obtain a PEI-DTPA solution;

(4) dissolving HA in water, adding EDC and NHS for activation to obtain an activated HA solution, then dropwise adding the activated HA solution into PG SFs dispersion liquid, stirring for reaction, centrifuging, washing with water, and freeze-drying to obtain PGH SFs;

4. The preparation method according to claim 3, wherein in the aqueous solution containing PVA in the step (1), the concentration of PVA is 0.2-0.5 wt%; the mass-volume ratio of the PLGA fiber membrane to the PVA water solution is 90-120mg:40-50 mL.

5. The preparation method according to claim 3, wherein the homogenization treatment in the step (1) comprises the following steps: the rotating speed of the homogenizer is 16000-16800 rpm/min, and the homogenizing treatment is 30-40 min; the centrifugation parameters in the step (1) are as follows: centrifuging at 1000-1200 rpm/min for 1-3 min, and discarding the precipitate; and then the centrifugal parameters of the supernatant fluid are as follows: centrifuging at 5000-6000 rpm/min for 3-5 min, collecting precipitate, dispersing again, centrifuging, and repeating the operation for 3 times.

6. The preparation method according to claim 3, wherein the mass ratio of the PLGA staple fiber to the EDC in the step (2) is 8-10: 1; the molar ratio of EDC to NHS is 1: 1.0-1.2; the molar ratio of the PEI to the DTPA is 1: 40-50; the activation time is 2-4 h; the stirring reaction time is 1-3 h, and the temperature is room temperature.

7. The preparation method according to claim 3, wherein the mass-to-volume ratio of the PLGA staple fiber to the PEI-DTPA solution in the step (3) is 70-80 mg: 6-8 mL; the gadolinium salt is Gd (NO)₃)₃·6H₂O; the Gd³⁺The molar ratio of the DTPA to the DTPA is 1-2: 1; the stirring reaction time is 1 to 3 days, then gadolinium salt aqueous solution is added, and the stirring reaction is continued for 12 to 18 hours.

8. The preparation method according to claim 3, wherein the molar ratio of HA, EDC and NHS in the step (4) is 1: 18-20; the mass ratio of PG SFs to HA is 1.5-2.0: 1; the reaction is stirred for 1 to 3 days at room temperature.

9. The preparation method according to claim 3, wherein the mass ratio of PGH SFs to the drug in the step (5) is 3-5: 1; the drug is DOX; the technological parameters of the electric spraying are as follows: the voltage is 10kV, the flow rate of an injection pump is 2mL/h, the receiving distance is 10cm, an aluminum foil soaked in liquid nitrogen is used as a collecting device, the ambient temperature is 25 ℃, and the humidity is 30%; the crosslinking is glutaraldehyde crosslinking.

10. The use of the drug-loaded fiber microsphere of claim 1 in the preparation of an antitumor drug.