CN114224846B

CN114224846B - Drug-loaded fiber microsphere and preparation method and application thereof

Info

Publication number: CN114224846B
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: 2023-03-14
Anticipated expiration: 2041-11-18
Also published as: CN114224846A

Abstract

The invention relates to a drug-loaded fiber microsphere and a preparation method and application thereof ³⁺ Modifying HA on the surface of the PLGA short fiber to obtain functional short fiber PGH SFs; PGH SFs and DOX are blended in an aqueous solution containing 0.1wt% of gelatin, and the obtained dispersion is sequentially subjected to electrospray and glutaraldehyde crosslinking to obtain the drug-loaded fiber microsphere DOX @ PGH with the MR imaging and targeting specificity functions. 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 invention relates to a drug-loaded fiber microsphere, which is prepared from raw materials containing functionalized short fibers PGH SFs and drugs by electrospray and crosslinking; wherein the surface of the functional short fiber PGH SFs is modified with Gd ³⁺ 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 chelating Gd through PEI-DTPA on the surface of the PLGA short fibers ³⁺ And modifying HA, further uniformly mixing with DOX, and then sequentially performing electrospray and glutaraldehyde crosslinking to obtain the modified HA.

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 0.1wt% 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 =80 kDa-810 kDa) in a solvent to obtain a PLGA spinning solution, and performing electrostatic spinning to obtain the PLGA nanofiber membrane; 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 =85 to 124 kDa) is 0.2 to 0.5wt%; the mass-volume ratio of the PLGA fiber membrane to the PVA aqueous solution is 90-120mg.

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, re-dispersing, centrifuging, and repeating the operation for 3 times.

The mass ratio of the PLGA short fiber to the EDC in the step (2) is 8-10; the molar ratio of EDC to NHS is 1.0-1.2; a molar ratio of PEI (Mw =20 kDa-25 kDa) to DTPA of 1 to 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 70-80mg:6-8mL; the gadolinium salt is Gd (NO) ₃ ) ₃ ·6H ₂ O; the Gd ³⁺ 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; the mass ratio of PG SFs to HA is 1.5-2.0; 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 uses Scanning Electron Microscope (SEM) and nuclear magnetic resonance hydrogen spectrum ( ¹ The 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 563.8nm; the average diameter of the PLGA short fiber obtained after the homogenization treatment is 1.7 μm, 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 had a porous structure, and the short fibers were connected to each other by crosslinking, thereby ensuring the stability of the microsphere structure.

(2) ¹ H NMR characterization:

¹ the 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 DTPA ₂ And 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 DOX @ PGH which is not crosslinked releases almost all the DOX in a short time, the DOX accumulated release amount of the DOX @ PGH which is crosslinked for 1h at 24h is 76%, the long-term slow release of the DOX is not achieved, and the microspheres which are crosslinked for 6h may cause the DOX release amount to be very small due to excessive crosslinking. The accumulative release amount of the drug crosslinked for 3h, namely DOX @ PGH, in 120h is 52.2%, so that the long-term stable sustained release of the anticancer drug DOX is achieved, and therefore, the crosslinking for 3h is selected for the experiments and the characterization of the microspheres involved in the experiment.

(6) Fluorescence microscopy test:

a fluorogram is shown in FIG. 7a, DOX @ PGH has stable red fluorescence, indicating 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 material ₁ The MR imaging effect is gradually enhanced; as shown in FIG. 8b, the relaxation rate r of DOX @ PGH was found by linear fitting of the Gd concentration of the material to the reciprocal of the relaxation time ₁ ＝17.7mM ^-1 s ^-1 。

(9) In vitro cytotoxicity and anticancer activity assay:

the cytotoxicity determination result is shown in fig. 9a, the cell viability of the PGH with different concentrations after being co-cultured with 4T1 cells for 24h exceeds 90%, which indicates that the fibrous material has good cell compatibility. The results of the anticancer activity assay 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 had the strongest killing power on 4T1 cells, and DOX @ PGH + HA group was the weakest. When the concentration of 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 the 4T1 cells was quantitatively analyzed by ICP-OES, as shown in fig. 10, at the same Gd concentration, the Gd content of the 4T1 cells in the PGH group was significantly increased compared to the PGH + HA group, and with the increase of the Gd concentration, the Gd content of the 4T1 cells in the PGH group was gradually increased, and the trend was greater than that of the PGH + HA group, which indicates that CD44 receptors on the surface of the HA-pretreated 4T1 cells were occupied by too much HA, and the microspheres could not accurately identify the cells, resulting in a decrease in the binding efficiency of the 4T1 cells. This result indicates that HA modification confers significant targeting specificity to the fibrous microspheres.

(10) Scratch test results:

the image of the scratch experiment and the cell quantitative migration area result are shown in FIGS. 11a-b, the migration rate of the control group after 24h culture reaches 57.4%, which indicates that the 4T1 cell HAs higher transfer capacity, which is obviously higher than PGH, DOX @ PGH + HA groups, and indicates that the prepared multifunctional microspheres have a certain inhibition effect on the transfer of the 4T1 cell. 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, of the original. 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:

as shown in FIG. 12, it is evident that the MR signal intensity at the tumor site of the mice is significantly increased and tends to be weakened with the lapse of time after the injection of DOX @ PGH and DOX @ PGH + HA. For DOX @ PGH and DOX @ PGH + HA, the MR signal intensity was still much stronger on day 5 than initially. By quantifying the signal-to-noise ratio at the tumor site, DOX @ PGH was found to consistently exhibit a stronger MR signal intensity than DOX @ PGH + HA, suggesting that DOX @ PGH HAs a longer residence time at the tumor site due to mediation by HA.

(13) Evaluation of tumor treatment effect:

statistics of relative tumor volume and body weight for five groups of nude mice are shown in figure 13, mice in the DOX group drop their body weight dramatically at the beginning of treatment, causing strong side effects. Furthermore, tumor volume in all cases showed an upward trend, especially in the control group, which was 12.2-fold 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 obtains the best antitumor 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, thereby ensuring that DOX directly acts on the tumor cells once released, enhancing the accumulation and bioavailability of DOX at tumor sites, and further greatly reducing the toxic and side effects of DOX on normal tissues.

(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 other experimental groups, and in particular, 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 is characterized in that the fiber microsphere surface is modified with HA, so that the fiber microsphere surface is endowed with targeting capability, cancer cells can be firmly anchored by combining the advantages of the size and the structure of the fiber microsphere surface, and the metastasis of the tumor cells is inhibited (firstly, in an in vitro cell experiment, a microscopic picture of a cell scratch experiment and a 3D cell sphere culture experiment and a corresponding quantitative statistical result prove that the fiber microsphere modified by HA can inhibit the migration of the cells, then, on an in vivo animal experiment level, the comparison of two groups of experiments shows that the MR signal intensity of a tumor part is longer to be monitored under the condition that the HA blocking is not carried out on the tumor cells, namely, the stagnation time of the fiber microsphere 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 result of the number of the lung tissue tumor knots is combined to show that the fiber microsphere material can effectively inhibit the metastasis of the tumor cells).

(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 at different magnifications of DOX @ PGH in example 1 of the present invention;

FIG. 4 shows the preparation of PEI-DTPA in example 1 of the present invention ¹ H 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 DOX @ PGH cumulative release profile in PBS solution (pH = 7.4) for DOX @ PGH at different 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 shows T of DOX @ PGH at different concentrations in example 1 of the present invention ₁ Weighted MR image (a) and Gd concentration and T ₁ A linear fit of the inverse relaxation times (b);

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

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

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 migration rates 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 + HA ₁ Weighted MR imaging (a) and MR signal-to-noise ratio (b) of the corresponding tumor region;

FIG. 13 is the curves of 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 810 kDa) was purchased from the firm Dai handle bio materials Ltd. PEI (Mw =20 kDa-25 kDa) and PVA (Mw = 85-124 kDa) were purchased from Sigma-Aldrich trade 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 ·6H ₂ O, DMF, THF, gelatin, glutaraldehyde (25% aqueous solution) were purchased from the national pharmaceutical group chemicals ltd (shanghai, china). HA (Mw = 5830) was purchased from bio-technology limited, source east of township (zhenjiang, china). A regenerated cellulose dialysis membrane with a molecular weight cutoff of 3000 was purchased from Fisher (Pittsburgh, pa.). DMEM medium, fetal calf serum, penicillin-streptomycin and trypsin 0.25% solution were purchased from Hangzhou Jinuo biomedical technology, 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) Soaking the obtained PLGA nano fiber membrane in ultrapure water for 5-10 min, then tearing off the membrane from the aluminum foil paper, and cutting the membrane into pieces with the area of about 0.25cm ² Then transferred to an aqueous solution containing 0.2wt% PVA, followed by homogenization treatment with a homogenizer at 16000rpm for 30 min; 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 the rotating speed of 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 3h. 50mg of PEI was dissolved in 5mL of ultrapure waterAdding 3mL of 40mg DTPA-containing aqueous solution into water, magnetically stirring, reacting for 1h to obtain PEI-DTPA solution, slowly adding into PLGA short fiber dispersion solution, reacting for 48h, and adding 1mL of 100mg Gd (NO) into the solution ₃ ) ₃ ·6H ₂ And 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 (60 mg) were then redispersed in 6mL of water, to which the HA solution was added dropwise and magnetic stirring was continued at room temperature for 48h. Then, the mixed solution was centrifuged, washed with water, and lyophilized to obtain PGH SFs.

(5) PGH SFs and DOX are taken to be mixed in an aqueous solution containing 0.1wt% 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 to obtain 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 563.8nm; 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 had a porous structure, and the short fibers were connected to each other by crosslinking, thereby ensuring the stability of the microsphere structure.

Example 3

To determine the successful preparation of PEI-DTPA and the amount of DTPA attached to PEI, example 1, step (C:)2) The PEI-DTPA solution obtained in (1) was lyophilized by dialysis, and 5mg of the solution was weighed out and dissolved in 600. Mu. L D ₂ In O, carrying out ¹ And H NMR characterization. As a result, 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 DTPA ₂ And 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 heating 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, 2mg of each drug-loaded fibrous microsphere cross-linked for different times was added to a dialysis bag with a molecular weight cut-off of 3000, and then the dialysis bag was tightly sealed and immersed in 9mL 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 long-term stable sustained 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 DOX @ PGH with different concentrations by using nuclear magnetic resonance imager ₁ Relaxation time sum T ₁ Weighted imaging effects, as shown in FIG. 8a, with increasing material concentration, T ₁ The MR imaging effect is gradually enhanced; as shown in FIG. 8b, the relaxation rate r of DOX @ PGH was found by linear fitting of Gd concentration and reciprocal of relaxation time ₁ ＝17.7mM ^-1 s ^-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 plate ⁴ Cell/well 4T1 cell suspension, incubated at 37 ℃ and 5% CO ₂ Overnight in the incubator, after the cells were fully adherent, the original medium was aspirated away, 100. Mu.L of medium containing PGH (0.25, 0.5, 1, 2, 4, 8 mg/mL) at various concentrations was added, the medium was returned to the incubator and incubated for 24h, then the medium was aspirated away, washed with PBS 3 times, 100. Mu.L of serum-free medium containing 10% of CCK-8 was added to each well, and after incubation for 3h, the absorbance of the well plate at 450nm was measured 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 after 24h of coculture with 4T1 cells was over 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 after co-incubation of free DOX, DOX @ PGH + HA and DOX @ PGH with 4T1 cells was compared at the same DOX concentration. First, 4T1 cells were treated with 10 ⁴ The density of each well is planted in a 96-well culture plate, the culture is adhered to the wall overnight, the original culture medium is discarded, and 100 mu L of the culture medium containing the same DOX concentration is added into each wellMedium of pure DOX or DOX @ PGH (DOX concentration gradient 2.5, 5, 10, 20, 40, 80. Mu.g/mL). For the DOX @ PGH + HA group, HA (2 mM) was incubated with the cells for 2h before replacing the medium with the medium containing DOX @ PGH. Culturing the cells and the material in the incubator for 24 hours, removing the medium by aspiration, washing with PBS 3 times, adding 100. Mu.L of a serum-free medium containing 10% of CCK-8 per well, incubating for 3 hours, and measuring the absorbance of the well plate at 450nm using an microplate reader to calculate the cell viability. For each fiber material concentration, 5 parallel holes were tested. The results of the anticancer activity assay are shown in fig. 9b, and 4T1 cell viability was decreased in each group with increasing DOX concentration. Among them, free DOX had the strongest killing power on 4T1 cells, and DOX @ PGH + HA group was the weakest. When the concentration of 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 10 ⁵ One/well was seeded in 12-well plates and cultured overnight to allow cells to adhere. Then, the medium of each well was replaced with fresh medium containing PGH at different Gd concentrations (3, 6, 12. Mu.g/mL) and cultured with the cells for a further 4h. For the PGH + HA group, HA (2 mM) was incubated with the cells for 2h to ensure that the CD44 receptor on the 4T1 cell surface was 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 24h. Finally, the sample was diluted with PBS and Gd concentration was measured with ICP-OES. As shown in fig. 10, under the material treatment with the same Gd concentration, the Gd content of the 4T1 cells of the PGH group was significantly increased compared to the PGH + HA group, and with the increase of the Gd concentration, the Gd content of the 4T1 cells of the PGH group was gradually increased, and the trend was greater than that of the PGH + HA group, which indicates that CD44 receptors on the surface of HA-pretreated 4T1 cells were already occupied by too much HA, and the fiber microspheres could not accurately recognize the cells, resulting in a decrease in the binding efficiency to the 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, namely PGH, DOX @ PGH + HA and DOX @ PGH sets. The same strategy, for the DOX @ PGH + HA group, requires that cells be previously co-conferred with HA (2 mM) for 2h, ensuring that the cell surface CD44 receptor HAs been occupied by excess HA, and then allowing the cells to incubate with DOX @ PGH. First, 4T1 cells in good state were cultured at 2X 10 ⁵ Density of individual/well in 12-well plate, put at 5% CO ₂ After incubation for 24h at 37 ℃, the plates were scored with a 100 μ L pipette tip for one cell scratch on each plate, then washed with PBS to remove floating cells, and finally 1mL PBS was added to each plate and the scratched wounds were observed under a microscope and photographed (noted as 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 12h and 24h thereafter. The scratch area of the cells in each photograph was calculated by ImageJ software. As shown in FIGS. 11a-b, the control group had a migration rate of 57.4% after 24h of culture, indicating that the 4T1 cells had a higher metastatic potential, which was significantly higher than the PGH, DOX @ PGH + HA groups, indicating that the prepared multifunctional microspheres had a certain inhibitory effect on the metastasis 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. Then the cooled and solidified agar block is gently peeled off from the mold, transferred to a 12-well plate, added with DMEM medium and letThe agar blocks were soaked in the medium for 30min. After soaking, the medium was aspirated, and 190. Mu.L of the medium containing 8X 10 was added to each well on the agar block ⁵ Complete medium of 4T1 cells. After 15min, 2.5mL of complete medium was added to each well and placed at 5% CO ₂ And 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 (2 mg/mL) was added as an experimental group. Similarly, cells of the DOX @ PGH + HA group were previously co-administered with HA (2 mM) for 2h, followed by co-incubation with DOX @ PGH. At different time points (24 h, 72h and 120 h), 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, of the original. 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 μ L physiological saline (Gd concentration is 5 mM) containing DOX @ PGH or DOX @ PGH + HA into the tumor of the 4T1 tumor-bearing nude mouse, and carrying out MR imaging (30 min, 1h, 1 day, 3 days, 5 days) on the 4T1 tumor-bearing nude mouse before and after DOX @ PGH or DOX @ PGH + HA injection at different time points by scanning with a nuclear magnetic resonance imager. For the DOX @ PGH + HA group, HA (24mg, 100. Mu.L physiological saline) was injected intratumorally 2h before the injection of DOX @ PGH. As shown in FIG. 12, it was revealed that the mouse injected with DOX @ PGH and DOX @ PGH + HA showed a significant increase in the MR signal intensity at the tumor site and tended to decrease with the passage of time. 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, DOX @ PGH was found to consistently exhibit a 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

And (3) preparing the prepared DOX @ PGH into a solution with the DOX concentration of 1mg/mL by using a sterile PBS buffer solution, wherein 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 before DOX @ PGH injection. Changes in body weight and tumor volume of the mice were recorded over 14 days of treatment. As a result, as shown in fig. 13, the body weight of mice in the DOX group decreased sharply at the start of the treatment, causing strong side effects. Furthermore, tumor volume in all cases showed an upward trend, especially in the control group, which was 12.2-fold 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 antitumor effect, relative to tumor volume is lower than the DOX @ PGH + HA group, because DOX @ PGH is firmly anchored on tumor cells through the binding of HA and CD44 receptor, ensures that DOX acts directly on tumor cells once released, and enhances the accumulation and bioavailability of DOX at tumor sites.

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 other experimental groups, especially in the DOX @ PGH group, which 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 nano fiber membrane in ultrapure water for 5-10 min, then tearing off the membrane from the aluminum foil paper, and cutting the membrane into pieces with the area of about 0.25cm ² Then transferring the square fragments into a PVA-containing solution, wherein the concentration of PVA is 0.2wt%, and then carrying out homogenization treatment for 30min by using a homogenizer at the rotating speed of 16000rpm; after homogenizing, carrying out centrifugal treatment on the homogenized solution, centrifuging at 1000rpm for 1min, and discarding precipitates which are mostly fiber blocks which are not homogenized and completely crushed; 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 ·6H ₂ And 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 HA was dissolved in 4mL water and EDC (26mg, 1mL water) and NHS (15mg, 1mL water) were added with magnetic stirring to activate the carboxyl groups of surface HA. The prepared PG SFs (60 mg) 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 48h. Then, the mixed solution was centrifuged, washed with water, and lyophilized to obtain PGH SFs.

(5) Dispersing PGH SFs in an aqueous solution containing 0.1wt% 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 are porous and have a size comparable to that of the drug-loaded microspheres.

Claims

1. A preparation method of a 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; wherein the concentration of PVA in the aqueous solution containing PVA is 0.2 to 0.5wt%; the mass-volume ratio of the PLGA fiber membrane to the PVA aqueous solution is 90-120mg; the homogenizing treatment process comprises the following steps: the rotation speed of the homogenizer is 16000 to 16800rpm/min, and the homogenization treatment is 30 to 40min; the centrifugation parameters were: centrifuging at 1000 to 1200rpm/min for 1 to 3min, 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;

(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; the mass ratio of the PLGA short fiber to the EDC is 8 to 10; the molar ratio of EDC to NHS is 1.0-1.2; the molar ratio of PEI to DTPA is 1;

(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; the mass volume ratio of the PLGA short fiber to the PEI-DTPA solution is 70-80mg:6-8mL; the gadolinium salt is Gd (NO) ₃ ) ₃ ·6H ₂ O; the Gd ³⁺ The molar ratio of the compound to DTPA is 1 to 2;

(4) Dissolving hyaluronic acid 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; the molar ratio of the HA to the EDC to the NHS is 1; the mass ratio of PG SFs to HA is 1.5 to 2.0;

(5) Dispersing PGH SFs in an aqueous solution containing 0.1wt% gelatin, adding the medicine, uniformly mixing, electrically spraying, freeze-drying and crosslinking to obtain medicine-carrying fiber microspheres; the mass ratio of the PGH SFs to the medicine is 3 to 5.

2. The preparation method according to claim 1, wherein the activation time in the step (2) is 2 to 4 hours; the stirring reaction time is 1 to 3 hours, and the temperature is room temperature.

3. The preparation method according to claim 1, wherein the stirring reaction time in the step (3) is 1 to 3 days, and then the gadolinium salt aqueous solution is added, and the stirring reaction is continued for 12 to 18 hours.

4. The method according to claim 1, wherein the reaction time in the step (4) is 1 to 3 days with stirring and the temperature is room temperature.

5. The method according to claim 1, wherein the drug in the step (5) is DOX; the technological parameters of the electric spraying are as follows: 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 ℃, and the humidity is 30%; the crosslinking is glutaraldehyde crosslinking.

6. The drug-loaded fiber microsphere prepared by the method of claim 1, wherein the fiber microsphere is prepared from raw materials containing functionalized short fibers PGH SFs and drugs by electrospray and crosslinking; wherein the surface of the functional short fiber PGH SFs is modified with Gd ³⁺ And HA; the drug is DOX.

7. The use of the drug-loaded fiber microsphere of claim 6 in the preparation of an antitumor drug.