A folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel, preparation method and application thereof in preparing in vitro controlled-release drugs

Abstract

The present invention discloses a folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel, a preparation method and its application in preparing in vitro controlled-release drugs. The core-shell microgel is used for targeted delivery of the anti-tumor drug DOX, and comprises a PDA core layer, a P(NIPAM‑AAm‑GMA) shell layer and a targeting molecule FA grafted on the surface of the shell layer. The P(NIPAM‑AAm‑GMA) shell layer has a hydrophilic polymer network and a large number of pores, which can not only enhance the physiological stability of PDA, but also give it the characteristics of temperature-sensitive drug release, thereby optimizing the drug controlled-release performance of PDA; FA is grafted on the P(NIPAM‑AAm‑GMA) shell layer, so that it can recognize tumor cells with overexpression of folate receptors and improve its anti-tumor effect.

Description

Technical Field

[0001] The invention relates to a folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel, a preparation method and application thereof in preparing an in vitro controlled-release drug, and belongs to the field of drug delivery. Background Art

[0002] Cancer is one of the leading causes of death in today's society, and its incidence and mortality are closely related to human living standards. Egen et al. reported in 2017 that the incidence and mortality of cancer in poor urban residents are much higher than those in the rich, which is probably because the disease is diagnosed in the final stage of cancer and the access to timely and effective treatment is limited. Although there are many cancer treatment methods, local surgery / radiation therapy and systemic chemotherapy, patients still suffer from side effects such as intestinal problems, nervous system damage or fatigue. The development of nanomedicine is very effective for cancer treatment and has opened a new stage for targeted systemic therapy.

[0003] The enhanced permeability and retention effect (EPR effect) is an effective way for nano drug delivery bodies to bring drug molecules to tumors. Tumor tissues need blood to provide nutrients and blood for their excessive cell growth, so new blood vessels will form as the tumor grows, but these blood vessels are often poorly formed, resulting in "leakage". The infiltrated vascular system and poor lymphatic drainage at the tumor allow relatively large nanoparticles to enter cancer cells but cannot be effectively taken away, thereby increasing accumulation at the tumor. This effect allows a large number of nanomaterials to be used as drug carriers.

[0004] Nanoparticles with good stability and water solubility can improve the pharmacokinetics of drugs, reduce the toxic side effects of drugs, and have a good protective effect on active drug molecules, thus playing an important role in drug delivery systems. Among them, polydopamine (PDA) is a main pigment of true melanin with good biocompatibility, which is formed by the oxidative polymerization of dopamine (DA) under alkaline conditions. The surface of PDA contains a large number of functional groups such as amino, hydroxyl and carbonyl groups, which can provide a variety of active sites for binding with other functional biomolecules or anti-tumor drugs. Zhang et al. designed a copper sulfide@polydopamine-folic acid / doxorubicin nanocomposite for chemotherapy and photothermal therapy of cancer cells. Doxorubicin (DOX) is loaded on the surface of the material through electrostatic forces and π-π stacking interactions, so the nanocomposite can release drug molecules in response to changes in pH [Non-patent literature 1]. In addition, PDA is an outstanding photosensitizer that can absorb near infrared (IR) light and convert it into heat. For example, Li et al. designed a multifunctional nanocomposite of AuNSs@PDA-Ce6 conjugated with dihydrochlorin and coated with PDA for photoacoustic imaging, photothermal therapy and photodynamic therapy. Under the irradiation of 808nm laser, 4T1 tumor cells can be completely eliminated and lung metastasis can be effectively inhibited [Non-patent Document 2]. Although PDA has excellent performance, it still has the disadvantage of chemical instability in biomedical applications. In order to improve its physiological stability, it is often modified on its surface with substances such as polyethylene glycol that can keep it stable in the body [Non-patent Document 3].

[0005] For drug carriers, researchers tend to develop multiple stimulus responsiveness to meet the requirements of the complex physiological environment in the human body. Stimuli-responsive polymers can improve the bioavailability of drugs, and the outer layer coated with a stimulus-responsive polymer shell can not only solve the physiological stability of PDA, but also give it a more intelligent drug release mechanism [Non-patent literature 4 and 5].

[0006] Non-patent literature 1: Li J, Huang S, Shi W, etal.Pt nanoparticledecorated carbonnanotubes nanocomposite based sensing platform for the monitoring of cell-secreted dopamine[J]. Sensors and Actuators B: Chemical, 2021, 330(1):129311-129344;

[0007] Non-patent Document 2: Grze′skowiak B F, Maziukiewicz D, A, et al. Polyamidoamine Dendrimers Decorated Multifunctional Polydopamine Nanoparticles for Targeted Chemo-and Photothermal Therapy of Liver Cancer Model[J]. International Journal of Molecular Sciences, 2021, 22(2): 738-754;

[0008] Non-patent Document 3: Li Z W, Yang F, Wu D, et al. Ce6-Conjugated and polydopamine-coated gold nanostars with enhanced photoacoustic imaging and photothermal / photodynamic therapy to inhibit lung metastasis of breast cancer[J]. Nanoscale, 2020, 12(43): 22173-22184;

[0009] Non-patent Document 4: Zhang S, Liu X, Sun Q, et al. CuS@PDA-FA nanocomposites: a dual-stimuli-responsive DOX delivery vehicle with ultrahigh loading level for synergistic photothermal-chemotherapies on breast cancer[J]. Journal of Materials Chemistry B, 2020, 8(7): 1396-1407;

[0010] Non-patent literature 5: Liu L, Wang S, Qi P, et al. Dopamine-modified poly (ε-caprolactone) micelles for pH controlled delivery of bortezomib [J]. International Journal of Pharmaceutics, 2020, 590: 119885-119895. Summary of the invention

[0011] Based on the current mainstream pH-responsive PDA drug delivery system, the present invention designs a temperature, pH, and NIR triple-responsive core-shell microgel based on PDA in combination with a thermosensitive polymer for targeted delivery of the anti-tumor drug DOX. The present invention provides a core-shell microgel with PDA as the core and P(NIPAM-AAm-GMA) as the shell, and grafted with a targeting molecule FA for multimodal chemotherapy and photothermal therapy; the material is loaded with DOX, and the MTT experiment and cell uptake experiment using Hep G2 liver cancer cells as the model cell line are carried out, confirming the biocompatibility of the material and the targeting effect of FA.

[0012] The technical solution adopted by the present invention is:

[0013] A folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel, comprising polydopamine;

[0014] Polydopamine is coated with a polymer obtained by copolymerization of a silane coupling agent containing a C=C double bond, N-isopropylacrylamide, acrylamide and glycidyl methacrylate, and the polymer is grafted onto the surface of polydopamine through the -Si-O bond of the silane coupling agent;

[0015] and folic acid linked to glycidyl methacrylate via an amide bond.

[0016] The preparation method of the above-mentioned folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel comprises the following steps:

[0017] S1: Synthesis of PDA microspheres: dopamine hydrochloride is oxidized and self-polymerized under alkaline conditions, and the PDA microspheres are purified after the reaction is completed;

[0018] S2: Synthesis and modification of PDA-SiO2 microspheres: PDA microspheres are hydrolyzed and condensed with TEOS through a solution-gel reaction, and after the reaction is completed, the PDA microspheres are centrifuged, washed, and vacuum dried to obtain PDA-SiO2 microspheres; then the PDA-SiO2 microspheres are dispersed in toluene and a silane coupling agent is added dropwise, and after the reaction is completed, the modified PDA-SiO2 microspheres, i.e., PDA-SiO2-ene microspheres, are obtained by washing and vacuum drying;

[0019] S3: Synthesis of PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel: PDA-SiO2-ene, NIPAM, AAm, GMA, MBA and SDS were dispersed in deionized water, heated under nitrogen environment, and then APS and SPS were added dropwise to the system. After the reaction was completed, the PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel was obtained by dialysis and removal of impurities.

[0020] S4: Synthesis of FA-grafted PDA@P(NIPAM-AAm-GMA) core-shell microgels:

[0021] After the PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel was amino-functionalized in one solution, FA was activated in another solution, and then the activated FA was grafted onto the amino-functionalized PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel through amidation reaction to prepare FA-PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel;

[0022] S5: Synthesis of FA-PDA@P(NIPAM-AAm-GMA) core-shell microgels:

[0023] FA-PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel was dispersed in deionized water and swelled, and then HF aqueous solution was added and stirred. After the reaction was completed, it was dialyzed to remove impurities and freeze-dried to obtain FA-PDA@P(NIPAM-AAm-GMA) core-shell microgel.

[0024] Preferably,

[0025] In step S1, the specific method for synthesizing PDA microspheres is:

[0026] Pour 40 mL of anhydrous ethanol, 3 mL of ammonia water and 90 mL of ultrapure water into a beaker and mix well. Quickly add 10 mL to a concentration of 50 mg mL -1 The dopamine hydrochloride aqueous solution was magnetically stirred at room temperature for 24 hours. During the reaction, the color of the solution changed from colorless to brown and finally stabilized to black. After the reaction, the PDA microspheres were purified by centrifugation and acetone precipitation, respectively, and the process was repeated 6 times to obtain PDA microspheres, which were freeze-dried. The time for each centrifugation was 5 minutes and the centrifugal speed was 12000 rpm. Each time the acetone was purified, the amount of acetone was twice the volume of the PDA solution.

[0027] Preferably,

[0028] In step S2, the specific method for synthesizing PDA-SiO2 microspheres is:

[0029] The PDA microspheres were ultrasonically dispersed in 80 mL of anhydrous ethanol, and after being evenly dispersed, 0.5 mL of TEOS was slowly added dropwise and magnetically stirred for 6 hours. After the reaction was completed, the PDA-SiO2 microspheres were obtained by centrifugation, washing, and vacuum drying, wherein the centrifugation time was 5 minutes and the centrifugal speed was 9000 rpm;

[0030] The specific method of PDA-SiO2 microsphere modification is:

[0031] 0.13 g of PDA-SiO2 microspheres were ultrasonically dispersed in 25 mL of toluene, 3 mL of KH570 was slowly added, and then ultrasonicated at 45°C for 30 minutes, and then transferred to an oil bath and magnetically stirred at 45°C for 4 hours; after the reaction, the mixture was centrifuged, and washed with toluene and anhydrous ethanol in turn to remove excess KH570, and then the precipitate was vacuum dried at 45°C overnight to obtain PDA-SiO2-ene, wherein the centrifugal treatment time was 5 minutes and the centrifugal speed was 9000 rpm.

[0032] Preferably,

[0033] In step S3, the specific method for synthesizing PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel is:

[0034] The ground PDA-SiO2-ene, NIPAM, AAm, GMA, MBA and 1 mg SDS were ultrasonically dispersed in deionized water. After uniform dispersion, they were injected into a three-necked flask in a nitrogen atmosphere. The temperature was raised to 60 °C. After the temperature stabilized, 1 mL was continuously added dropwise at a concentration of 3 mg mL -1 of APS and 1 mL, the concentration is 2.5 mg mL -1 The SPS was reacted for 6 hours, and then the heat source was removed. After cooling to room temperature, the mixture was encapsulated in a dialysis bag, and the water was changed every 6 hours for 3 consecutive days. The residue in the dialysis bag was removed to obtain PDA-SiO2@P(NIPAM-AAm-GMA) microgel.

[0035] Preferably,

[0036] In step S4, the specific method of amino functionalization treatment of PDA-SiO2@P(NIPAM-AAm-GMA) is as follows:

[0037] 30 mg of ground PDA-SiO2@P(NIPAM-AAm-GMA) microgel powder was evenly dispersed by ultrasonic in 20 mL of PBS buffer with a pH of 6.0, 1 mL of EA was added, and the mixture was magnetically stirred at room temperature for 24 h to obtain amino-functionalized microgels.

[0038] The specific method of FA activation is:

[0039] 5 mg of FA was ultrasonically dispersed in PBS buffer with a pH of 6.0, and then EDC and NHS were added sequentially for activation for 15 min;

[0040] The specific method of grafting is:

[0041] The pH of the activated solution was adjusted to 7.2-7.4 with a 0.1 M NaOH solution, and then quickly added to the amino-functionalized solution, and the reaction was continued for 24 hours under light-proof conditions. After the reaction, the solution was encapsulated in a dialysis bag and soaked for 2 days, and the water was changed every 6 hours. The residue in the dialysis bag was freeze-dried to obtain FA-PDA@P (NIPAM-AAm-GMA) core-shell microgel.

[0042] Preferably,

[0043] In step S5, the specific method for synthesizing FA-PDA@P(NIPAM-AAm-GMA) core-shell microgel is:

[0044] 50 mg of ground FA-PDA-SiO2@P (NIPAM-AAm-GMA) microgel powder was ultrasonically dispersed uniformly in 20 mL of deionized water. After swelling overnight, 1 mL of 5 wt.% HF aqueous solution was added in a fume hood. After open magnetic stirring at room temperature for 24 hours, the powder was dialyzed to remove impurities and freeze-dried to obtain FA-PDA@P (NIPAM-AAm-GMA) core-shell microgel.

[0045] Application of the above folic acid grafted polydopamine@thermosensitive polymer core-shell microgel in the preparation of in vitro drug controlled release drugs.

[0046] Preferably, the folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel is loaded with the anti-tumor drug doxorubicin DOX.

[0047] Preferably,

[0048] The specific method of loading the anti-tumor drug doxorubicin DOX on the folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel is as follows:

[0049] 15 mg FA-PDA@P(NIPAM-AAm-GMA) core-shell microgels were ultrasonically dispersed in 20 mL of PBS buffer with a pH of 8.0, and then 5 mL of DOX solution with a concentration of 1 mg mL-1 was added. The mixture was stirred for 24 h in the dark and then dialyzed to remove free drug molecules.

[0050] The beneficial effects of the present invention are:

[0051] 1. PDA-SiO2 microspheres introduce C=C double bond functional groups through hydroxyl hydrolysis condensation, so that the core surface can be coated with a uniform P(NIPAM-AAm-GMA) shell layer, which makes up for the defect that the PDA surface lacks sufficient active functional groups to initiate polymerization reaction;

[0052] 2. The P(NIPAM-AAm-GMA) shell has a hydrophilic polymer network and a large number of pores, which can not only enhance the physiological stability of PDA, but also give it the characteristics of temperature-sensitive drug release, thus optimizing the drug controlled release performance of PDA;

[0053] 3. FA was grafted onto the shell of P(NIPAM-AAm-GMA), enabling it to recognize tumor cells that overexpress folate receptors and improve its anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the synthesis of FA-PDA@P(NIPAM-AAm-GMA) core-shell microgel;

[0055] Figure 2 Infrared spectra of PDA (a), PDA-SiO2 (b), PDA-SiO2-ene (c), PDA-SiO2@P(NIPAM-AAm-GMA (d), PDA@P(NIPAM-AAM-GMA) (e);

[0056] Figure 3 UV spectra of FA (a), FA-PDA@P(NIPAM-AAm-GMA) (b) and PDA (c);

[0057] Figure 4 Thermogravimetric curves of PDA (a), PDA-SiO2 (b), PDA-SiO2-ene (c), FA-PDA-SiO2@P(NIPAM-AAm-GMA) (d) and FA-PDA@P(NIPAM-AAm-GMA) (e) microgels;

[0058] Figure 5 Scanning electron micrographs of PDA (a), PDA-SiO2 (b), PDA-SiO2@P (NIPAM-AAm-GMA) core-shell microgel (c) and PDA@P (NIPAM-AAm-GMA) core-shell microgel (d); (e) EDS image and element distribution map of PDA@P (NIPAM-AAm-GMA) microgel;

[0059] Figure 6Transmission electron microscopy images of PDA (a), PDA-SiO2 (b), PDA-SiO2@P(NIPAM-AAm-GMA) microgel (c, d) and PDA@P(NIPAM-AAm-GMA) microgel (e);

[0060] Figure 7 (a) Transmittance of microgel solution at 500nm wavelength changes with temperature; (b) Transmittance of PNAG-3 at 25℃ and 39℃; (c) Particle size of microgel changes with temperature; (d) Physical appearance of PNAG-3 at room temperature (left) and 39℃ (right);

[0061] Figure 8 (a) 808nm laser (5.12W, cm -2 ) under irradiation, the temperature curves of PDA (a), PDA-SiO2 (b), FA-PDA-SiO2@p(NIPAM-AAm-GMA) microgel (c), FA-PDA-SiO2@p(NIPAM-AAm-GMA) microgel (d) and the temperature curve of PBS buffer (e), (all samples were 1 mg mL -1 , 2mL);

[0062] (b) FA-P(NIPAM-AAm-GMA) microgel (0.5 mg mL -1 , 2mL) was exposed to 808nm laser and switched on and off for 5 cycles;

[0063] Fig. 9 (a) Potential diagram of PDA; (b) pH-dependent DOX release of PDA; (c) DOX release rate of FA-PNAG-3 under different conditions; (d) Cumulative DOX release rate of FA-PNAG-3 dispersed in PBS (pH 5.5 or 7.4) under laser on / off; standard error is used as error bar (n=3);

[0064] Fig.10 (a) Biocompatibility of FA-PNAG-3. (b) Survival rate of Hep G2 cells after treatment with DOX or DOX-FA-PNAG-3.

[0065] Fig.11 Fluorescence microscopy images of Hep G2 liver cancer cells incubated with DOX-FA-PNAG-3 and DOX-PNAG-3. DETAILED DESCRIPTION

[0066] The present invention will be specifically described below in conjunction with the accompanying drawings and embodiments.

[0067] Example

[0068] This embodiment is a method for preparing folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel, and the specific process is as follows:

[0069] 1. Synthesis and purification of PDA microspheres

[0070] Pour 40 mL of anhydrous ethanol, 3 mL of ammonia water and 90 mL of ultrapure water into a beaker and mix well. Quickly add 10 mL to a concentration of 50 mg mL -1 The dopamine hydrochloride aqueous solution was magnetically stirred at room temperature for 24 hours. During the reaction, the color of the solution changed from colorless to brown and finally stabilized to black. After the reaction, the PDA microspheres were purified by centrifugation and acetone precipitation, respectively, and the process was repeated 6 times to obtain PDA microspheres, which were freeze-dried. The time for each centrifugation was 5 minutes and the centrifugal speed was 12000 rpm. Each time the acetone was purified, the amount of acetone was twice the volume of the PDA solution.

[0071] 2. PDA surface silica shell modification and KH570 modification

[0072] The PDA microspheres were ultrasonically dispersed in 80 mL of anhydrous ethanol, and after uniform dispersion, 0.5 mL of TEOS was slowly added dropwise and magnetically stirred for 6 hours. After the reaction was completed, the PDA-SiO2 microspheres were obtained by centrifugation, washing, and vacuum drying, wherein the centrifugal treatment time was 5 minutes and the centrifugal speed was 9000 rpm.

[0073] 0.13 g of PDA-SiO2 microspheres were ultrasonically dispersed in 25 mL of toluene, 3 mL of KH570 was slowly added, and then ultrasonicated at 45°C for 30 minutes, and then transferred to an oil bath and magnetically stirred at 45°C for 4 hours; after the reaction, the mixture was centrifuged, and washed with toluene and anhydrous ethanol in turn to remove excess KH570, and then the precipitate was vacuum dried at 45°C overnight to obtain PDA-SiO2-ene, wherein the centrifugal treatment time was 5 minutes and the centrifugal speed was 9000 rpm.

[0074] 3. Synthesis of PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel

[0075] Core-shell microgels with PDA-SiO2-ene as the core were synthesized by emulsion polymerization. Specifically, PDA-SiO2-ene, NIPAM, AAm, GMA, MBA and 1 mg SDS were ultrasonically dispersed in deionized water and injected into a three-necked flask (equipped with a condenser to maintain a constant temperature) in a nitrogen environment after being evenly dispersed. The temperature was raised to 60°C, and after the temperature stabilized, 1 mL APS (3 mg mL -1 ) and 1 mL SPS (2.5 mg mL -1After the reaction continued for 6 hours, the heat source was removed, and after cooling to room temperature, the mixture was packaged into a dialysis bag (molecular weight cutoff, MWCO = 8000-14000), and the water was changed every 6 hours for 3 consecutive days to remove free monomers, initiators and other impurities, and PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel was obtained.

[0076] In order to control the low critical solution temperature (LCST) of the core-shell microgel, the amount of AAm was adjusted while keeping the amounts of other reagents constant, and the above experiment was repeated. The specific feed ratio is shown in Table 1.

[0077] Table 1: Feed ratios for synthesis of core-shell microgels

[0078]

[0079] 4. FA-grafted PDA@P(NIPAM-AAm-GMA) core-shell microgel

[0080] The trace amount of GMA in the PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel is only used to graft the targeting molecule FA. 30 mg of PDA-SiO2@P(NIPAM-AAm-GMA) was ultrasonically dispersed in 20 mL of PBS buffer (pH = 6.0), 1 mL of EA (excess) was added, and the mixture was magnetically stirred at room temperature for 24 hours to obtain amino-functionalized core-shell microgels.

[0081] In the experiment, the carboxyl group of FA was activated by EDC / NHS, and then grafted on the surface of the core-shell microgel through an amidation reaction. Specifically, 5 mg of FA was ultrasonically dispersed in PBS buffer (pH = 6.0), and then EDC and NHS were added in sequence for activation for 15 minutes. After the pH was adjusted to between 7.2 and 7.4 with a 0.1M sodium hydroxide solution, it was quickly added to the above microgel solution, and the reaction lasted for 24 hours under light-proof conditions. After the reaction was completed, the FA-PDA-SiO2@P(NIPAM-AAm-GMA) microgel solution was encapsulated in a dialysis bag with MWCO = 3500 and soaked for 2 days, the water was changed every 6 hours, and finally freeze-dried for use.

[0082] The SiO2 intermediate layer is for the convenience of coating the thermosensitive polymer network. In order not to affect the role of PDA in subsequent drug loading and release experiments, HF is used to eliminate it. Specifically, 50 mg of FA-PDA-SiO2@P(NIPAM-AAm-GMA) microgel freeze-dried powder was ultrasonically dispersed in 20 mL of deionized water. After swelling overnight, 1 mL of diluted HF aqueous solution (5 wt.%) was added in a fume hood. After open magnetic stirring at room temperature for 24 hours, dialysis was performed to remove impurities, and FA-PDA@P(NIPAM-AAm-GMA) core-shell microgel was obtained by freeze drying.

[0083] Photothermal performance testing

[0084] In order to study the photothermal conversion performance of the material, 1 mg mL -1 PDA, PDA-SiO2, FA-PDA-SiO2@P(NIPAM-AAm-GMA) microgel, and FA-PDA@P(NIPAM-AAm-GMA) microgel dispersions were prepared, and 2 mL was taken and transferred to a quartz cuvette. The light source (808 nm, 5.12 W cm-2) was separated from the cuvette by 2 cm, and the temperature rise of each material dispersion over time was recorded within 3 minutes. After 3 minutes, the laser was turned off and the temperature drop of the material was recorded. For comparison, PBS buffer was used as a blank control group and the same operation was performed.

[0085] In order to study the thermal stability of FA-PDA@P(NIPAM-AAm-GMA) core-shell microgels, 2 mL of microgel solution (0.5 mg mL -1 ) After ultrasonic dispersion is uniform, move to a cuvette, record the temperature after thirty seconds and the first minute under laser irradiation, then turn off the heat source and record the temperature after the second minute and the third minute. Repeat the operation 5 times.

[0086] Drug loading and controlled release

[0087] 15 mg of FA-grafted PNAG-3 (named as FA-PNAG-3) was ultrasonically dispersed in 20 mL of PBS buffer (pH 8.0), and then 5 mL of DOX solution (1 mg mL -1 ), the mixture was stirred in the dark for 24 hours and then dialyzed to remove free drug molecules. The dialysate was 100 mL PBS buffer (pH 8.0), and the absorbance at 480 nm was measured by ultraviolet spectroscopy. The unloaded DOX content was obtained by the DOX standard working curve, and the drug loading rate and encapsulation rate of the material were calculated accordingly. The formula is as follows:

[0088]

[0089] Wherein, m0 and m1 are the initial mass of DOX used and the mass of DOX removed by dialysis, respectively, and w0 is the mass of the carrier.

[0090] As a comparison, 15 mg of PDA was ultrasonically dispersed in a PBS buffer solution at pH 8.0, and 5 mL of DOX aqueous solution (1 mg mL -1 ), stirred for 24 hours in dark conditions. After the reaction, the free drug molecules were removed by centrifugation washing with PBS buffer, and the drug loading rate and encapsulation rate of PDA were calculated by the same operation as above.

[0091] The controlled release experiments of DOX were carried out under different temperature and pH conditions: 25°C, pH 5.5 and pH 7.4; 39°C, pH 5.5 and pH 7.4. Specifically, 6 mL of DOX-loaded core-shell microgels (0.5 mg mL -1 ) were encapsulated in a dialysis bag and immersed in 30 mL PBS buffer at 25°C or 39°C. At the specified time interval, 3 mL of dialysate was taken out, the drug release amount was calculated by the standard curve, and 3 mL of fresh PBS buffer was added. For comparison, PDA with the same drug content was subjected to the same operation at 25°C. In addition, in order to explore the effect of 808 nm laser radiation on drug release, drug release experiments were carried out at 25°C, pH 5.5 or 7.4, respectively. The laser was irradiated for 5 minutes at 60 minutes, 180 minutes, 420 minutes, 660 minutes and 780 minutes, 3 mL of dialysate was taken to detect the DOX concentration, and 3 mL of fresh PBS buffer was added.

[0092] In vitro cell experiments

[0093] The cells selected for the experiment were Hep G2 liver cancer cells with high expression of folate receptors purchased from the American Type Culture Collection (ATCC). The cell culture, cytotoxicity study and uptake study were described as follows.

[0094] Cell culture

[0095] Hep G2 hepatoma cells were purchased from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum in a humidified incubator (37°C and 5% CO2).

[0096] Cytotoxicity studies

[0097] Hep G2 liver cancer cells were used as a model cell line for MTT assay to investigate the cytotoxicity of free DOX and DOX-loaded FA-PNAG-3. 5The cells were inoculated at a density of 100 μg / mL in a 96-well plate and incubated overnight at 37°C with 5% CO2. Different concentrations of free DOX and DOX-loaded FA-PNAG were added to the cells (the drug loading rate was calculated so that the concentration of doxorubicin in the above two groups of materials was the same: 0.5, 1, 2, 4, 8 μg mL -1 ) for 24 hours. MTT (20 μL, 5 mg mL -1 ) was added to each well and cultured for 4 hours before being discarded. 100 μL DMSO was added to dissolve the generated MTT-formazan crystals, and the absorbance of the resulting solution at 570 nm was recorded using a microplate reader. To reduce experimental errors, the experiment was repeated three times. In addition, to confirm the biocompatibility of FA-PNAG-3, concentrations of 15, 30, 60, 120, and 240 μg mL -1 The above experiment was performed with FA-PNAG-3. The cell survival rate was calculated by the following formula (untreated Hep G2 liver cancer cells in cell culture medium were used as the control group).

[0098]

[0099] Cellular uptake studies

[0100] Hep G2 liver cancer cells were seeded in 6-well plates (3×10 5 ) for 16 h, and treated the cells with DOX-loaded FA-PNAG-3 or PNAG-3 (2 μg mL-1). Hep G2 liver cancer cells were fixed with 4% paraformaldehyde for 15 min, rinsed with PBS buffer, and 1 mL of 2 μg mL-1 phalloidin-FITC was added. After DPAI staining (1 mg mL -1 , 10 min) and washed again with PBS buffer, and the cell images were observed under a fluorescence microscope.

[0101] Results and Discussion

[0102] Infrared spectroscopy

[0103] The synthesis process of core-shell microgels is as follows Figure 1 As shown, dopamine hydrochloride is oxidized and self-polymerized into PDA microspheres under alkaline conditions, and then the abundant hydroxyl functional groups on its surface are used to hydrolyze and condense tetraethyl orthosilicate through a solution-gel reaction to form a SiO2 shell. The PDA surface lacks sufficient active functional groups to initiate polymerization reactions, making it difficult to coat a uniform polymer shell. The SiO2 intermediate layer can make up for this defect by introducing C=C double bond functional groups through hydroxyl hydrolysis and condensation. After coating with the thermosensitive polymer shell layer P (NIPAM-AAm-GMA), FA is grafted to eliminate the silica shell to obtain the final product. The entire synthesis process can be verified by infrared spectroscopy ( Figure 1 ). Figure 2 The infrared spectrum of PDA shows 3400cm -1 The NH / OH stretching vibration peak is at 1620 cm -1 and 1508cm -1 The peaks at 1099cm correspond to the stretching vibration and NH shear vibration peaks of the aromatic ring. -1 The characteristic peak at 2940cm corresponds to the asymmetric Si-O-Si stretching vibration peak of PDA-SiO2 microspheres. Since the surface of PDA-SiO2 contains abundant hydroxyl functional groups, KH570 can be used to introduce polymerizable carbon-carbon double bonds on the surface of PDA-SiO2. In the infrared spectrum of organically modified PDA-SiO2-ene, 2940cm -1 and 2881cm -1 The absorption peak at 1329 cm is attributed to CH stretching vibration. -1 The peak at 2983 cm is the -C-CO-C skeleton vibration peak derived from the methacryloyl group. In addition, the weakening of the Si-O-Si absorption peak also confirms the hydrolysis and condensation reaction between KH570 and the silica shell. -1 and 2975cm -1 represents the stretching vibration of the CH bond, with an absorption peak at 1537 cm -1 (stretching vibration peak of NH bond), 1650cm -1 (stretching vibration peak of C=O bond) and 1099cm -1 (Stretching vibration peak of Si-O-Si bond), confirming that the P(NIPAM-GMA-AAm) thermosensitive layer is polymerized on the PDA-SiO2-ene surface. In addition, the Si-O-Si stretching vibration peak disappears in the P(NIPAM-AAM-GMA) spectrum, indicating that the SiO2 shell is eliminated by HF.

[0104] UV spectroscopy analysis

[0105] like Figure 3 As shown, FA shows a characteristic absorption peak at 285 nm, PDA shows a broad plasma absorption peak in the near-infrared range, and FA-P (NIPAM-AAm-GMA) core-shell microgel integrates the characteristics of both, confirming that FA is grafted on the surface of the amino-modified P (NIPAM-AAm-GMA) microgel.

[0106] Thermogravimetric analysis

[0107] like Figure 4As shown, the weight loss of PDA is about 47%, which is mainly caused by the high residual rate during the pyrolysis process. The mass loss of PDA-SiO2 is reduced to 31% because the silica shell has a high heat resistance. This change can also confirm that the surface of PDA is coated with a silica shell, which hinders its thermal decomposition. The mass loss of PDA-SiO2-ene after organic modification of the silica shell surface reaches 45%, indicating that the KH570 grafting rate is about 14%. For the core-shell microgel FA-PDA-SiO2@P(NIPAM-AAm-GMA), its thermogravimetric loss is mainly divided into three stages. Before 250°C, it is the desorption and dehydration process of water, and then to 450°C is mainly the decomposition of the polymer chain. The subsequent trace loss almost consistent with PDA-SiO2-ene is the dehydroxylation of nanoparticles. The final mass residue is 12%, which confirms the presence of a large amount of polymer on the silica shell surface. For FA-PDA@P(NIPAM-AAm-GMA), the mass residue is 3.88%, which not only confirms the existence of surface polymer, but also indicates that the SiO2 shell has been eliminated by HF.

[0108] Morphology analysis

[0109] Scanning electron microscopy and transmission electron microscopy images can be used to analyze the morphological characteristics of the prepared materials and can also further confirm the synthesis status of the materials. Figure 5 Middle a area and Figure 6 The a region in the middle is the scanning electron microscope and transmission electron microscope images of PDA, which intuitively shows that the spherical PDA is monodisperse with a particle size of about 156nm. When coated with a SiO2 shell, its surface becomes rough and the size is about 166nm ( Figure 5 Middle B area, Figure 6 As the middle layer of SiO2, the PDA surface can be evenly coated with a polymer layer, such as Figure 5 As shown in the c and d regions, PDA-SiO2@P(NIPAM-AAm-GMA) and PDA@P(NIPAM-AAm-GMA) are both uniformly spherical, and the coating of the polymer layer makes the surface of the material smooth. Transmission electron microscopy can further verify the existence of the polymer layer, such as Figure 6 As shown in the c and d regions, the core-shell microgel shows an obvious core-shell structure, and the thickness of the gel layer is about 35nm. When HF is used to eliminate the SiO2 shell layer, the structure of the material does not change significantly, as shown in Figure 6 EDS spectrum can be used to analyze the element composition and content of the sample, such as Figure 5 As shown in the middle e area, the C, N, and O elements of PDA@P(NIPAM-AAm-GMA) account for 41.03, 23.4, and 35.57 wt %, respectively, and the presence of Si element could not be detected, confirming that the silicon shell has been eliminated by HF.

[0110] Temperature Sensitive Performance Analysis

[0111] Temperature sensitivity is the key to controlled drug release of core-shell microgels. The LCST of core-shell microgels can be detected by turbidity detection and dynamic light scattering. Figure 7 As shown in the middle a area, the LCST of PNAG-1 is about 31.5℃, the LCST of PNAG-2 is about 41℃, the LCST of PNAG-3 is about 38.5℃, the LCST of PNAG-4 is about 36℃, and the LCST of PNAG-5 is about 34℃. Figure 7 As shown in the c area in the figure, dynamic light scattering can also draw approximate conclusions based on the trend of sample particle size changes with temperature, confirming that the LCST of the sample is related to the content of the hydrophilic comonomer AAm. Due to the interaction between the core-shell microgel solution and water molecules, the polymer shell is fully stretched, so the measured particle size is larger than the TEM image under dry conditions. PNIPAM relies on amide groups to form hydrogen bonds with water molecules, and its LCST is around 32°C. The addition of AAm can provide more amide groups. The higher the content, the greater the hydrogen bonding effect with water molecules. Therefore, the LCST increases with the increase of AAm content. In order to verify that the material can continuously perform swelling / contraction behavior, we continuously subjected the dialyzed PNAG-3 to a heating (above its LCST)-cooling (room temperature) cycle, as shown in Figure 7 As shown in area b, the sample has good thermal stability. Figure 7 The middle d area is the actual picture of PNAG-3 after dialysis at room temperature and 39°C. The thermosensitive properties of the material can also be seen with the naked eye. In general, the LCST of PDA@P(NIPAM-AAm-GMA) can be adjusted by AAm, and the material has good thermal stability and can be used as a drug delivery carrier to stably control the release of drugs.

[0112] Photothermal performance research

[0113] In order to study the photothermal conversion performance of the material, a power of 5.12 W cm -2 The 808 nm near-infrared laser irradiation concentration was 1 mg mL -1 The PDA, PDA-SiO2, FA-PDA-SiO2@p(NIPAM-AAm-GMA) microgel and FA-PDA-SiO2@p(NIPAM-AAm-GMA) microgel dispersions were recorded to record the temperature changes over time when the materials were exposed to laser (laser on) and when they were naturally cooled (laser off), such as Figure 8As shown in area a, the temperature of the PBS buffer in the blank control group rose by about 11°C after 3 minutes of laser irradiation, while that of PDA rose by 60°C, showing strong photothermal conversion performance. The thermal conductivity of the SiO2 shell is low, and evenly coating the PDA surface will affect the thermal conductivity of the material, such as Figure 8 As shown in curve b in region a, the temperature of PDA-SiO2 rises to 72°C (temperature difference 51°C). When the thermosensitive polymer shell is coated, the thermal conductivity is further reduced, as shown in Figure 8 As shown in curve c in region a, the temperature of FA-PDA-SiO2@p(NIPAM-AAm-GMA) microgel increased from 20°C to 57°C within 3 minutes under laser irradiation, and dropped back to 23°C after 12 minutes. When the silica shell was removed, the temperature of FA-P(NIPAM-AAm-GMA) microgel increased by 44°C within 3 minutes, proving that it is an outstanding photothermal material.

[0114] In addition, we also studied the FA-P(NIPAM-AAm-GMA) microgels (0.5 mg mL -1 )'s light-to-heat conversion stability, such as Figure 8 As shown in the middle b area, the time-temperature curve of five consecutive laser on-off operations did not show any attenuation phenomenon. The material has good thermal stability and can be used in photothermal therapy.

[0115] Drug loading and controlled release studies

[0116] The surface of PDA contains abundant amino, phenyl and hydroxyl functional groups, which provide a large number of active sites for bonding target molecules. PDA was dispersed in PBS buffer with different pH values ​​and its Zeta potential was tested. Fig. 9 As shown in the a region, the isoelectric point of PDA is about 2.9. When the solution pH is higher than 2.9, PDA is negatively charged due to the deprotonation of the phenolic functional groups on its surface. According to literature reports, the pKa value of DOX is about 8.3, and it is positively charged due to the amine groups in the PBS buffer at pH = 8.5. In the PBS buffer at pH 8.5, PDA is loaded with DOX through electrostatic interactions, π-π stacking and hydrophobic interactions, with a drug loading rate of 25.8% and an encapsulation efficiency of 77.5%. The cross-linked polymer shell can also provide a large number of pores to store drug molecules. FA-PNAG-3 was selected for the same drug loading process, and its final drug loading rate and encapsulation efficiency were 31.6% and 94.9%, respectively.

[0117] The DOX-loaded PDA was dispersed in PBS buffer (pH = 5.5 or 7.4) to measure its release properties. Fig. 9As shown in the b area, under the conditions of pH 5.5 and 25°C, the cumulative drug release rate within 30 hours is 36%, while under the conditions of pH 7.4 and 25°C, it is only 22%, which confirms the good pH-sensitive drug controlled release performance of PDA. The polymer shell layer P (NIPAM-AAm-GMA) can not only improve the stability of PDA, but also give it temperature-sensitive drug controlled release performance. Fig. 9 As shown in the middle c area, the cumulative drug release rate of FA-PNAG-3 at pH 5.5 and 25°C is only 24.1%, while when the temperature is 39°C, its cumulative drug release rate increases to 75.6%. The temperature-sensitive polymer shell is similar to a switch. When the temperature is lower than its LCST, the polymer layer fully swells, hindering the transfer of drug molecules. The drug release of FA-PNAG-3 depends only on the concentration diffusion between the PDA itself and the periphery of the polymer layer. When the temperature rises above its LCST, the polymer layer shrinks, which not only shortens the distance for PDA to release drugs, but also allows the drugs loaded in the gel layer itself to be released in large quantities, greatly improving the drug release efficiency. The cumulative drug release rates at pH 7.4, 25°C and 39°C are 13.6% and 51.1%, respectively. The difference from the above comparison is mainly due to the pH sensitivity of PDA. In general, FA-PNAG-3 has good temperature and pH-sensitive drug controlled release properties.

[0118] Based on the above research on the photothermal properties of the material, we further explored the effect of 808nm near-infrared laser radiation on drug release, such as Fig. 9 As shown in the middle d area, whether under pH 5.5 or 7.4 conditions, 808nm laser radiation can promote the rapid release of DOX in a short time. For example, under pH 5.5 and 25°C, the release rate of DOX was only 6.8% after 60 minutes, while after 5 minutes of laser radiation, the DOX release rate increased to 20.8%. This confirms that the material has drug sustained-release properties and can promote drug release in a short time through external 808nm laser radiation to achieve on-demand drug delivery, making it an outstanding drug release carrier.

[0119] In vitro toxicity studies

[0120] MTT analysis selected Hep G2 liver cancer cells with high expression of folate receptor as the research object. Fig.10 As shown in region a, FA-PNAG-3 has good biocompatibility even at a concentration of 240 μg mL -1 , the cell survival rate was still above 90%. The high cell survival rate within the experimental concentration range confirmed that the cytotoxicity of the carrier itself was negligible, and the cell survival rate of subsequent toxicity experiments was only related to the antitumor drug DOX. Fig.10As shown in area b, DOX has high cytotoxicity to Hep G2 cells, and the cytotoxicity of FA-PNAG-3 loaded with DOX (calculated based on the drug loading rate to make it the same as the concentration of free DOX) is significantly higher than that of free DOX, confirming the active targeting performance of FA. FA-PNAG-3 can transport DOX into tumor cells, resulting in a higher DOX concentration in the targeted area, resulting in an anti-tumor efficiency that is significantly higher than that of free DOX.

[0121] Cellular uptake studies

[0122] Because DOX itself has red fluorescence, the tumor selectivity of DOX-loaded FA-PNAG-3 was directly observed by laser confocal microscopy. Fig.11 Fluorescence microscopy images of DOX-FA-PNAG-3 and DOX-PNAG-3 were shown. Hep G2 cells were cultured for 3 h and 6 h, respectively, with a DOX concentration of 2 μg mL -1 . For DOX-PNAG-3, its red fluorescence intensity is weak, and even after 6 hours of incubation, it is still lower than that of DOX-FA-PNAG-3 after 3 hours, which is mainly attributed to the lack of active targeting effect of the material. The red fluorescence intensity of DOX-FA-PNAG-3 is higher. Only a small part of it was taken up by Hep G2 cells after 3 hours of incubation. As the incubation time increases, the red fluorescence signal is further enhanced because the carrier can gradually pass through the cell membrane and release DOX into the cytoplasm, which not only confirms the targeting effect of FA, but also reflects the cumulative release of the encapsulated drug DOX.

[0123] in conclusion

[0124] In this work, we designed a temperature, pH, and NIR triple-responsive drug carrier FA-PDA@P(NIPAM-AAm-GMA) for combined targeted chemotherapy and hyperthermia. PDA microspheres have pH-responsive drug release characteristics and photothermal conversion properties. They are simple to prepare and can be purified in large quantities by acetone precipitation. Dynamic light scattering and turbidity point detection confirmed that the LCST of the shell P(NIPAM-AAm-GMA) is at 38.5℃. Compared with the drug loading rate of 25.8% and the encapsulation efficiency of 77.5% of the PDA carrier, the addition of the thermosensitive polymer shell increased the drug loading rate to 31.6% and the encapsulation efficiency to 94.9%. The cumulative drug release rate of FA-PDA@P(NIPAM-AAm-GMA) at pH 5.5 and 25℃ was only 24.1%, while it was as high as 75.9% at pH 5.5 and 39℃. Under external 808nm laser irradiation, the core-shell microgel can increase 44℃ within 3 minutes, which not only meets the conditions for thermal ablation of cancer cells, but also promotes the release of DOX in a short time. In addition, MTT analysis confirmed the biocompatibility of the carrier, and the uptake experiment confirmed the targeting of the carrier. In general, the triple responsiveness

[0125] FA-PDA@P(NIPAM-AAm-GMA) has broad application prospects in the field of drug delivery.

[0126] The above is only a preferred implementation of the patent of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the patent of the present invention. These improvements and modifications should also be regarded as the scope of protection of the patent of the present invention.

Claims

1. A method for preparing folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel, characterized in that: The steps include: S1: oxidizing and self-polymerizing dopamine hydrochloride under alkaline conditions, and purifying to obtain PDA microspheres after the reaction is completed; S2: PDA microspheres are hydrolyzed and condensed with TEOS through a solution-gel reaction, and after the reaction is completed, the microspheres are centrifuged, washed, and vacuum dried to obtain PDA-SiO2 microspheres; then the PDA-SiO2 microspheres are dispersed in toluene and a silane coupling agent is added dropwise, and after the reaction is completed, the microspheres are washed and vacuum dried to obtain modified PDA-SiO2 microspheres, i.e., PDA-SiO2-ene microspheres; S3: PDA-SiO2-ene, NIPAM, AAm, GMA, MBA and SDS were dispersed in deionized water, heated under nitrogen environment, and then APS and SPS were added dropwise to the system. After the reaction was completed, the system was dialyzed to remove impurities and obtain PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel; S4: PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel is amino-functionalized in one solution, FA is activated in another solution, and then the activated FA is grafted onto the amino-functionalized PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel through an amidation reaction to prepare FA-PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel; S5: Disperse FA-PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel in deionized water to swell, then add HF aqueous solution and stir. After the reaction is completed, dialyze to remove impurities and freeze-dry to obtain FA-PDA@P(NIPAM-AAm-GMA) core-shell microgel.

2. The method for preparing a folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 1, characterized in that: In step S1, the specific method for synthesizing PDA microspheres is: The anhydrous ethanol, ammonia water and ultrapure water were mixed evenly, and then the dopamine hydrochloride aqueous solution was added and stirred for reaction. After the reaction was completed, the PDA microspheres were purified and freeze-dried to obtain the PDA microspheres.

3. The method for preparing a folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 1, characterized in that: In step S2, The specific method for synthesizing PDA-SiO2 microspheres is: PDA microspheres are ultrasonically dispersed in anhydrous ethanol, and after being evenly dispersed, TEOS is added dropwise and magnetically stirred. After the reaction is completed, PDA-SiO2 microspheres are obtained by centrifugation, washing, and vacuum drying. The specific method of PDA-SiO2 microsphere modification is: PDA-SiO2 microspheres were ultrasonically dispersed in toluene, KH570 was added dropwise, and then heated and ultrasonically treated. The mixture was then transferred to an oil bath and magnetically stirred until the reaction was completed. The mixture was centrifuged and washed to remove excess KH570, and then the precipitate was vacuum dried to obtain PDA-SiO2-ene. During the modification process of PDA-SiO2 microspheres, the temperature was controlled at 40-50°C.

4. The method for preparing a folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 1, characterized in that: In step S3, the specific method for synthesizing PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel is: The ground PDA-SiO2-ene, NIPAM, AAm, GMA, MBA and SDS were ultrasonically dispersed in deionized water, heated to 50-70 °C under nitrogen environment, and then APS and SPS were added dropwise. After the reaction, it was cooled to room temperature and dialyzed to remove impurities to obtain PDA-SiO2@P(NIPAM-AAm-GMA) microgel.

5. The method for preparing a folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 1, characterized in that: In step S4, The specific method of amino functionalization treatment of PDA-SiO2@P(NIPAM-AAm-GMA) is as follows: The ground PDA-SiO2@P(NIPAM-AAm-GMA) microgel powder was evenly dispersed by ultrasonic in PBS buffer, EA was added, and the mixture was magnetically stirred at room temperature to obtain amino-functionalized microgel. The specific method of FA activation is: FA was ultrasonically dispersed in PBS buffer, and then EDC and NHS were added sequentially for activation for 10-20 min; The specific method of grafting is: The pH of the activated solution was adjusted to 7.2-7.4 with NaOH solution, and then quickly added to the amino functionalized solution for reaction; after the reaction, the solution was dialyzed to remove impurities, and FA-PDA-SiO2@P(NIPAM-AAm-GMA) core-shell microgel was obtained after freeze-drying.

6. The method for preparing a folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 1, characterized in that: In step S5, the specific method for synthesizing FA-PDA@P(NIPAM-AAm-GMA) core-shell microgel is: The ground FA-PDA-SiO2@P(NIPAM-AAm-GMA) microgel powder was ultrasonically dispersed in deionized water, HF aqueous solution was added after swelling, magnetic stirring was performed for reaction, and then dialyzed to remove impurities, and freeze-dried to obtain FA-PDA@P(NIPAM-AAm-GMA) core-shell microgel.

7. A folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel, characterized in that: The method is prepared by any one of claims 1 to 6.

8. Use of the folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 7 in the preparation of in vitro controlled-release drugs.

9. Use of the folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 8 in the preparation of in vitro controlled-release drugs, characterized in that: The folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel is loaded with the anti-tumor drug doxorubicin DOX.

10. Use of the folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel according to claim 9 in the preparation of in vitro controlled-release drugs, characterized in that: The specific method of loading the anti-tumor drug doxorubicin DOX on the folic acid-grafted polydopamine@thermosensitive polymer core-shell microgel is: FA-PDA@P(NIPAM-AAm-GMA) core-shell microgels were ultrasonically dispersed in PBS buffer, and then DOX solution was added and stirred for reaction. After the reaction, the free drug molecules were removed by dialysis.

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