Micro-robot cluster capable of simultaneously delivering multiple drugs in targeted manner and preparation method of micro-robot cluster

Abstract

The invention provides a micro-robot cluster capable of simultaneously delivering multiple drugs in a targeted manner and a preparation method thereof. The micro-robot cluster comprises a plurality of micro-robots, and each micro-robot sequentially comprises a magnetic nanoparticle, a hydrogel layer and a pH response material shell from inside to outside; the hydrogel layer comprises dopamine (PDA) grafted 3-(methacryloyloxy) propyl trimethoxy silane located on the inner side and methacrylic acid wrapping the outer layer of the hydrogel layer; the hydrogel layer is used for packaging a plurality of medicines. According to the technical scheme, the micro-robot cluster can be driven by an external magnetic field to be conveyed to a specific position in a targeted mode in a complex simulated blood environment, combined delivery and controllable release of various drugs are achieved, and the micro-robot cluster has the advantages of being high in targeting performance, high in drug carrying capacity, intelligent in release and the like and can be applied to the field of precise drug therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted drug delivery, and in particular to a micro-robot cluster capable of simultaneously targetedly delivering multiple drugs and a preparation method thereof. Background Art

[0002] With the rapid development of precision medicine and targeted therapy technologies, drug delivery systems are constantly improving in the direction of high efficiency, precision and intelligence. In traditional drug delivery methods, drugs usually enter the body through oral or intravenous injection, but this non-specific delivery method often leads to uneven distribution of drugs in the body, making it difficult to concentrate on the lesion area. In addition, due to the lack of targeting of drugs, healthy tissues may also be damaged, resulting in increased toxic side effects and reduced therapeutic effects. Especially in the treatment of complex diseases such as cancer, inflammation or neurological diseases, the efficacy of a single drug is often limited, and multiple drugs are usually required to be used in combination to achieve synergistic treatment. Therefore, the development of a system that can achieve targeted delivery of multiple drugs has become one of the problems that need to be solved in the current field of drug delivery technology.

[0003] In recent years, microrobot technology has attracted widespread attention as an emerging intelligent drug carrier. Microrobots usually have the characteristics of small size, good biocompatibility and external controllable drive, and can achieve directional movement and precise drug delivery in complex body fluid environments. Driven by external physical fields (such as magnetic fields, light fields or ultrasound), microrobots can overcome biological barriers and complete cross-tissue drug transport, thereby increasing the cumulative concentration of drugs in the lesion site and significantly reducing the toxic side effects of healthy tissues. However, most microrobots currently still have problems such as limited drug loading, inaccurate release, and difficulty in achieving multi-drug synergistic delivery, which restricts their practical application in the treatment of complex diseases. Therefore, how to prepare a microrobot system with high drug loading, targeted transportation, multi-drug synergy and precise release functions has important research significance and application value for achieving efficient drug delivery and improving disease treatment effects. Summary of the invention

[0004] In response to the above technical problems, the present invention discloses a micro-robot cluster that can simultaneously deliver multiple drugs in a targeted manner and a preparation method thereof. Magnetic nanoparticles are used as the core of the micro-robot to give it magnetic responsiveness and driving ability; a hydrogel layer is introduced on the outside as a drug storage reservoir to encapsulate multiple drugs; the outermost layer is coated with a pH-responsive material to achieve intelligent and controllable release at the lesion site; this multi-level core-shell structure not only improves the drug loading capacity and targeted delivery efficiency, but also can utilize the lesion microenvironment to trigger release to achieve multi-drug synergistic treatment.

[0005] To this end, the technical solution adopted by the present invention is:

[0006] A microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner comprises, from the inside to the outside, magnetic nanoparticles, a hydrogel layer, and a pH-responsive material shell; the hydrogel layer comprises dopamine (PDA) grafted 3-(methacryloyloxy)propyltrimethoxysilane located on the inside and methacrylic acid wrapped on the outside; the hydrogel layer is used to encapsulate multiple drugs.

[0007] Using this technical solution, the microrobot is centered on magnetic nanoparticles, endowed with magnetic responsiveness and controllable driving functions, and can achieve precise movement and targeted positioning under the action of an external magnetic field. The hydrogel layer on the outside of the magnetic nanoparticles is modified as a drug-carrying layer. This drug-carrying layer can encapsulate various drugs, has high drug loading capacity and good biocompatibility, and can effectively encapsulate a variety of drugs to meet the needs of combined treatment. The pH-responsive material coated on the outermost layer serves as an intelligent release module, which can trigger the controlled release of drugs according to the lesion microenvironment (such as the acidic environment of tumor tissue), further improving the accuracy and efficiency of treatment. Driven by an external magnetic field, the microrobot cluster can be targeted and transported to a specific location in a complex simulated blood environment, and realize multi-drug joint delivery and synergistic release, significantly improving the therapeutic effect and reducing the toxic side effects of healthy tissues. The system has the characteristics of strong targeting, high drug loading capacity, controllable movement and intelligent release.

[0008] As a further improvement of the present invention, the magnetic nanoparticles are paramagnetic magnetite nanoparticles, which can be prepared by a solvothermal method.

[0009] As a further improvement of the present invention, the pH responsive shell is calcium phosphate.

[0010] As a further improvement of the present invention, the multiple drugs include adriamycin, glucose oxidase and MnCl 2 . Using this technical solution, by loading multiple drugs, the microrobot can achieve synergistic therapeutic effects in combined therapy under targeted delivery.

[0011] The present invention also discloses a method for preparing the microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner, comprising the following steps:

[0012] Step S1, preparation of Fe 3 O 4 Magnetic nanoparticles;

[0013] Step S2, by in situ polymerization on Fe 3 O 4 The surface of magnetic nanoparticles is coated with PDA layer to obtain Fe 3 O 4 @PDA nanoparticles, and then the obtained Fe 3 O 4After washing, the PDA nanoparticles were dispersed in a mixed solution of ethanol, deionized water and ammonia, and MPS was added to react to obtain Fe 3 O 4 @PDA-MPS nanoparticles. Then the hydrogel layer was anchored on the surface of the nanoparticles by precipitation polymerization, specifically: Fe 3 O 4 @PDA-MPS nanoparticles were dispersed in acetonitrile, stirred continuously, ultrasonicated, and MAAc, crosslinker and initiator were added under argon protection, and heated under reflux to obtain Fe 3 O 4 @PDA@PMAAc nanoparticles; after this step, the product can be washed and stored in deionized water for later use;

[0014] Step S3, by simulating the biomineralization process, 3 O 4 @PDA@PMAAc nanoparticles are deposited with a CaP shell layer on their surface. Specifically, the Fe 3 O 4 @PDA@PMAAc nanoparticles added with CaCl 2 The solution was stirred and then Na 2 HPO 4 Solution, continue stirring, obtain Fe 3 O 4 @PDA@PMAAc@CaP nanoparticles are microrobot clusters that can simultaneously deliver multiple drugs in a targeted manner.

[0015] As a further improvement of the present invention, step S1 comprises: adding sodium acetate and ethylene glycol to ferric chloride hexahydrate and stirring evenly; then adding polyethylene glycol thereto and performing ultrasonic treatment; finally placing the obtained solution into a reactor and reacting at 100-300° C. for 8-16 hours; after the reaction is completed, cooling to room temperature, washing, and obtaining Fe 3 O 4 Magnetic nanoparticles. Further, the mass ratio of ferric chloride hexahydrate to sodium acetate is 1:2-3. Further, the cleaning is performed by washing with deionized water for 2-5 times.

[0016] As a further improvement of the present invention, in step S2, the Fe 3 O 4 @PDA nanoparticles were prepared by the following steps: Fe 3 O 4 The magnetic nanoparticles were dispersed in an alkaline buffer solution, stirred and ultrasonicated, and then dopamine hydrochloride was added and reacted in an ice-water bath to obtain Fe 3 O 4 @PDA nanoparticles. Further, the Fe3 O 4 The mass ratio of magnetic nanoparticles to dopamine hydrochloride is 1:0.2-0.6.

[0017] As a further improvement of the present invention, in step S2, per mg of Fe 3 O 4 @PDA-MPS nanoparticles, the amount of MAAc used is 0.05-0.2 mL.

[0018] Furthermore, the cross-linking agent is MBA; the Fe 3 O 4 The mass ratio of PDA-MPS nanoparticles to MBA is 1:0.005-0.01.

[0019] The initiator is AIBN, the Fe 3 O 4 The mass ratio of PDA-MPS nanoparticles to AIBN is 1:0.001-0.015.

[0020] As a further improvement of the present invention, in step S3, per mg of Fe 3 O 4 @PDA@PMAAc nanoparticles, the CaCl 2 The dosage is 0.002-0.006mmol; per mg of Fe 3 O 4 @PDA@PMAAc nanoparticles, the Na 2 HPO 4 The dosage is 0.004-0.01mmol.

[0021] As a further improvement of the present invention, step S3 comprises: adding CaCl 2 Add various drugs to the solution, stir and dissolve to obtain CaCl 2 Mix the solutions; then 3 O 4 @PDA@PMAAc nanoparticles added with CaCl 2 The mixed solution was stirred and then Na 2 HPO 4 Solution, continue stirring, wash to obtain Fe 3 O 4 @PDA@PMAAc@CaP nanoparticles, i.e., microrobot clusters that can simultaneously deliver multiple drugs in a targeted manner; the multiple drugs include doxorubicin, glucose oxidase, and MnCl 2 .

[0022] As a further improvement of the present invention, each mg of Fe 3 O 4@PDA@PMAAc nanoparticles The dosage of doxorubicin is 0.1-0.2 mg, per mg of Fe 3 O 4 @PDA@PMAAc nanoparticles The amount of glucose oxidase used is 0.1-0.2 mg, per mg of Fe 3 O 4 @PDA@PMAAc nanoparticles MnCl 2 The dosage is 1-2mmol.

[0023] The present invention also discloses the use of the micro-robot cluster capable of simultaneously delivering multiple drugs in a targeted manner as described above in the preparation of targeted delivery drugs.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] First, using the technical solution of the present invention, the robot cluster uses magnetic nanoparticles as the core, which can be combined with external magnetic field drive to achieve efficient and accurate targeted delivery; at the same time, the hydrogel layer and pH-responsive material on the outside enable the microrobot to have a high drug loading capacity and intelligent and controllable drug release function, which significantly improves the accuracy and therapeutic effect of drug delivery. Load multiple drugs into the microrobot, adopt a multi-drug synergistic strategy, and achieve precise release through a controlled release mechanism, thereby enhancing the combined efficacy and improving the treatment efficiency;

[0026] Second, the microrobot of the present invention can move stably in a complex simulated blood environment, realize the combined delivery and coordinated release of multiple drugs, overcome the limitations of traditional drug delivery systems, and has the advantages of strong targeting, low toxic side effects and multi-drug synergistic treatment, providing a new, efficient, intelligent and controllable approach for precision drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of targeted therapy of a microrobot that can be used for simultaneous targeted delivery of multiple drugs according to the present invention.

[0028] Figure 2 This is the infrared spectrum of the nanoparticles in Example 1 of the present invention. The curves in the figure are as follows from bottom to top: Fe 3 O 4 , Fe 3 O 4 @PDA-MPS, Fe 3 O 4 @PDA@PMAAc,Fe 3 O 4 @PDA@PMAAc@CaP.

[0029] Figure 3 The Fe obtained in Example 1 of the present invention3 O 4 @SEM image of PDA-MPS nanoparticles.

[0030] Figure 4 It is a schematic diagram of the release of drugs from the shell under different pH conditions in Example 1 of the present invention.

[0031] Figure 5 It is a comparison of the DOX release effect of NV-DOX with or without CaP shell under pH control in Example 1 of the present invention.

[0032] Figure 6 1 is the release curve of NV-DOX in Example 1 of the present invention under different pH conditions.

[0033] Figure 7 This is the diameter change result of the PMAAc hydrogel under different pH conditions in Example 1 of the present invention.

[0034] Figure 8 This is a schematic diagram of the multi-drug combination therapy of Example 1 of the present invention.

[0035] Fig. 9 The glucose solution under different initial pH conditions in Example 1 of the present invention is X pH changes during 12 h of incubation as well as glucose solution alone and NV-GO X pH changes during 12 h of incubation.

[0036] Fig.10 It is the fluorescence intensity after the TPA solution of Example 1 of the present invention was incubated with different treatment groups for 6 hours.

[0037] Fig.11 It is the degradation efficiency of methylene blue after different treatments in Example 1 of the present invention.

[0038] Fig.12 Schematic diagram of the biochip of Example 1 of the present invention.

[0039] Fig.13 It is the passing rate of the distributed rolling and synchronous population in Example 1 of the present invention after passing through the complex channel between the starting position and the target position in the biochip.

[0040] Fig.14 These are the live / dead staining results of HepG2 cancer cells incubated for 24 hours after different treatments according to Example 1 of the present invention. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention are described in further detail below.

[0042] A microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner comprises a plurality of microrobots, wherein the microrobots are composed of magnetic nanoparticles, a hydrogel layer, and a pH-responsive material shell from the inside to the outside; the hydrogel layer comprises dopamine (PDA) grafted 3-(methacryloyloxy)propyltrimethoxysilane on the inside and methacrylic acid wrapped on the outside; the hydrogel layer is used to encapsulate multiple drugs. The magnetic nanoparticles are paramagnetic Fe 3 O 4 The magnetite nanoparticles can be prepared by a solvothermal method. The pH-responsive shell is calcium phosphate.

[0043] The preparation method of the micro robot comprises the following steps:

[0044] Step S1, adding sodium acetate and ethylene glycol to ferric chloride hexahydrate and stirring evenly; then adding polyethylene glycol thereto, and ultrasonically treating for half an hour; finally putting the obtained solution into a reactor, and reacting at 100-300° C. for 8-16 hours; after the reaction is completed, cooling to room temperature, and washing with deionized water for 2-5 times, thereby obtaining the final ferrosoferric oxide nanoparticles; wherein the mass ratio of ferric chloride hexahydrate to sodium acetate is 1:2-3.

[0045] Step S2, through an in-situ polymerization process, firstly in Fe 3 O 4 The surface is coated with a PDA layer and Fe 3 O 4 The nanoparticles were dispersed in a buffer solution adjusted to an alkaline pH, and after mechanical stirring and ultrasonic treatment, dopamine hydrochloride was added and the reaction was continued in an ice-water bath to obtain Fe 3 O 4 @PDA nanoparticles; Subsequently, the nanoparticles were washed with deionized water, dispersed in a mixed solution of ethanol, deionized water and ammonia, 2-5 mL of 3-(methacryloyloxy)propyltrimethoxysilane (MPS) was added and reacted for a period of time to obtain Fe 3 O 4 @PDA-MPS nanoparticles; then, the hydrogel layer was anchored on the surface of the nanoparticles by precipitation polymerization, and Fe 3 O 4 PDA-MPS nanoparticles were dispersed in acetonitrile, stirred continuously, and ultrasonicated. Methacrylic acid (MAAc), N,N'-methylenebisacrylamide crosslinker MBA, and initiator azobisisobutyronitrile AIBN were added under argon protection. The mixture was heated under reflux for a period of time to obtain Fe 3 O 4 The @PDA@PMAAc nanoparticles were finally washed and stored in deionized water for future use.

[0046] Among them, through Fe3 O 4 MPS was introduced onto the surface of the nanoparticles, successfully fixing the carbon-carbon double bonds on the surface of the particles. These double bonds provide cross-linking sites for the subsequent hydrogel layer. This surface functionalization method not only enhances the binding force between the nanoparticles and the hydrogel, but also improves the stability of the entire nanostructure; the hydrogel layer formed by precipitation polymerization using MAAc can effectively load drugs. The hydrogel itself improves the dispersion stability of the microrobots by surface modification onto the nanoparticles, providing the necessary conditions for the subsequent cluster movement. Secondly, the hydrophilic network structure of the hydrogel layer can effectively load a variety of drugs, accelerate drug release through swelling behavior in an acidic environment, and reduce drug leakage in normal tissues.

[0047] In step S2, if the amount of PDA is insufficient, the surface coverage will be uneven, affecting the subsequent modification effect of MPS; if the amount is too much, a too thick polymer film may be formed, reducing the magnetic response and dispersibility of the nanoparticles. 3 O 4 The mass ratio of PDA to dopamine hydrochloride is 1:0.2-0.6. A suitable amount of PDA is used to make the surface of the nanoparticles evenly covered without forming an excessively thick polymer film.

[0048] Per mg Fe 3 O 4 @PDA-MPS nanoparticles The dosage of MAAc is 0.05-0.2 mL, and the Fe 3 O 4 The mass ratio of PDA-MPS nanoparticles to MBA is 1:0.005-0.01. 3 O 4 The mass ratio of PDA-MPS nanoparticles to AIBN is 1:0.001-0.015.

[0049] Step S3, by simulating the biomineralization process, 3 O 4 @PDA@PMAAc nanoparticles are deposited with a CaP shell layer on their surface; the nanoparticles are then added with CaCl 2 After mechanical stirring, Na 2 HPO 4 The solution was stirred for a while to obtain Fe 3 O 4 @PDA@PMAAc@CaP nanoparticles; finally, washed with deionized water and stored in deionized water for later use; wherein, per mg Fe 3 O 4 @PDA@PMAAc nanoparticles CaCl 2The dosage is 0.002-0.006mmol, per mg of Fe 3 O 4 @PDA@PMAAc nanoparticles 2 HPO 4 The dosage is 0.004-0.01mmol.

[0050] In addition, the drug loading process of the microrobot is performed in step S3, including:

[0051] To CaCl 2 Add various drugs to the solution and stir to dissolve; similarly, add Fe 3 O 4 @PDA@PMAAc nanoparticles were added to CaCl 2 After mechanical stirring, Na 2 HPO 4 The solution is stirred for a period of time to obtain a drug-loaded microrobot; finally, the product is washed with deionized water and PBS for 3-5 times and stored in PBS for later use. The drug may be doxorubicin (DOX), glucose oxidase (GO X ) and MnCl 2 ; Among them, each mg of Fe 3 O 4 The dosage of DOX in the @PDA@PMAAc nanoparticles is 0.1-0.2 mg, and the microrobot loaded with DOX drug is defined as NV-DOX. 3 O 4 @PDA@PMAAc nanoparticles GO X The dosage is 0.1-0.2 mg, loaded with GO X The drug-carrying microrobot is defined as NV-GO X , per mg Fe 3 O 4 @PDA@PMAAc nanoparticles MnCl 2 The dosage is 1-2mmol, and MnCl is loaded 2 The drug-containing microrobots are defined as NV-Mn.

[0052] Driven by an external magnetic field, the microrobots can quickly self-organize and achieve controllable motion. Under a rotating magnetic field, the microrobots can form a stable cluster motion. 3 O 4 The surface of the nanoparticles is modified with a hydrogel layer, which can load different drugs and has a pH-responsive shell for controlled release. 3 O 4Nanoparticle surface modification (such as biocompatibility and dispersibility provided by the PDA layer, and functionalized sites introduced by the MPS and hydrogel layers) provides the necessary conditions for smooth and coordinated movement. After the microrobots loaded with different drugs are mixed to form clusters, the final drug delivery system is obtained, and the simultaneous release of three drugs will have a synergistic therapeutic effect, greatly improving the therapeutic effect.

[0053] The microrobot cluster loaded with three drugs in a certain proportion is added to a complex channel filled with plasma simulation fluid; the complex channel is placed in a three-dimensional Helmholtz electromagnetic coil, and the microrobot cluster moves to the target position with cancer cells under the control of a rotating magnetic field; the survival status of cancer cells is evaluated by incubation for 24 hours after drug administration. Figure 1 shown.

[0054] The following describes it in conjunction with specific embodiments.

[0055] Example 1

[0056] A microrobot cluster that can be used for simultaneous targeted delivery of multiple drugs includes a plurality of microrobots. The preparation steps of the microrobots include:

[0057] (1) Preparation of Fe by solvothermal method 3 O 4 Nanoparticles.

[0058] The specific steps include: 1.35g FeCl 3 6H 2 O and 3.6g NaAc were dissolved in 40mL ethylene glycol (EG) in turn and mixed under magnetic stirring for 30 minutes. Then 1g polyethylene glycol (PEG) was added and ultrasonic treatment was performed for 30 minutes to obtain a reddish brown turbid solution. The mixed solution was transferred to a 50mL autoclave, heated at 200℃ for 10 hours and then naturally cooled to room temperature. The product was washed with deionized water (DI water) 3 times, magnetically separated and stored in water for later use.

[0059] (2) In Fe 3 O 4 The surface is coated with a dopamine (PDA) layer by in-situ polymerization. The specific steps include:

[0060] Dissolve 0.12 g Tris in 100 mL DI water and add 1 m HCl dropwise to adjust the pH to 8.5. Mix the resulting buffer with 50 mg Fe 3 O 4The nanoparticles were poured into a 250 mL round-bottom flask, mechanically stirred and ultrasonically treated for 30 minutes. Then 0.02 g of dopamine hydrochloride was added, mechanically stirred and ultrasonically treated in an ice-water bath for 5 hours to obtain Fe 3 O 4 @PDA nanoparticles. After washing with DI water three times, Fe 3 O 4 @PDA nanoparticles were dispersed in a mixture of 160 mL ethanol, 40 mL DI water and 2 mL ammonia. After mechanical stirring for 15 minutes, 3 mL MPS was added and the Fe 3 O 4 @PDA-MPS nanoparticles. Finally, they were washed three times with ethanol and acetonitrile respectively, and stored in acetonitrile after magnetic separation for future use.

[0061] (3) Anchoring the hydrogel layer on the surface of the nanoparticles by precipitation polymerization. The specific steps include:

[0062] 15 mg Fe 3 O 4 The PDA-MPS nanoparticles were added to 100 mL of acetonitrile, mechanically stirred and ultrasonically treated for 30 minutes, while purging with argon to remove oxygen. Then 1.5 mL of methacrylic acid (MAAc), 0.1 g of N,N′-methylenebisacrylamide (MBA), and 0.015 g of azobisisobutyronitrile (AIBN) were added in sequence and quickly heated to boiling. After reflux reaction for 90 minutes, Fe 3 O 4 @PDA@PMAAc nanoparticles were washed with ethanol and DI water three times and then stored in DI water for future use.

[0063] (4) Depositing a CaP shell on the surface of the nanoparticles by simulating a biomineralization process. The specific steps include:

[0064] 15 mg Fe 3 O 4 @PDA@PMAAc nanoparticles were added with 2 mL 30 mmol CaCl 2 The solution was stirred mechanically overnight. Then, 4 mL of 20 mmol Na 2 HPO 4 The solution was further mechanically stirred for 2 hours to prepare Fe 3 O 4 @PDA@PMAAc@CaP nanoparticles. Finally, they were washed with DI water for 3 times and then magnetically separated and stored in DI water for future use.

[0065] During the mineralization process of step (4), various drugs can be loaded, such as DOX, GOX or MnCl 2 The specific operation is: DOX, GO X 、MnCl 2 Dissolve in 2 mL of 30 mM CaCl 2 The concentrations of Fe in the solution were 1 mg / mL, 1 mg / mL and 10 mM, respectively. Subsequently, 15 mg Fe 3 O 4 @PDA@PMAAc nanoparticles were mechanically stirred overnight. Then, 4 mL of 20 mM Na 2 HPO 4 The solution was further mechanically stirred for 2 hours to prepare NV-DOX and NV-GO. X Finally, the nanoparticles were washed with DI water and PBS for three times and then stored in PBS for future use.

[0066] In the above experiment, Fe 3 O 4 Nanoparticles, Fe 3 O 4 @PDA-MPS nanoparticles, Fe 3 O 4 @PDA@PMAAc nanoparticles, Fe 3 O 4 The infrared spectrum of @PDA@PMAAc@CaP nanoparticles is shown in Figure 2 As shown, SEM Figure 3 As shown. Figure 2 As shown, 588cm -1 The characteristic peak at 1650cm is attributed to the stretching vibration of Fe-O, while -1 and 3450cm -1 The absorption peaks at about 1700 cm-1 are attributed to the vibration modes of adsorbed water molecules and hydroxyl groups, respectively. The appearance of these characteristic peaks indicates that the synthesis of iron oxide nanoparticles has been successful. -1 and 2990cm -1 The multiple absorption peaks of 526 cm-1 correspond to the stretching vibrations of C=O and CH groups in polymethacrylic acid (PMAAc), which confirms the successful encapsulation of the nanoparticles by the hydrogel layer. -1 and about 1100cm -1New absorption bands appeared at , corresponding to the bending vibration and asymmetric stretching vibration of PO, respectively. This result shows that the phosphate composite material has been successfully formed. It can be seen that after the above steps, PDA, MPS, and PMAAc were successfully modified. The more MPS is modified, the more double bonds on the surface there are, and more molecules participate in the reaction during free radical polymerization, and the binding force is stronger.

[0067] The DOX release experiment was carried out on the prepared NV-DOX. In this experiment, 10 mg of NV-DOX was dispersed in 10 mL of PBS with pH values ​​of 7.2 and 5.5 and shaken in the dark at 37°C. At the set time point, the nanoparticles in the mixture were concentrated with a permanent magnet, 1 mL of the supernatant was taken out, and after measuring the DOX concentration with a UV-visible spectrophotometer, 1 mL of fresh PBS with the same pH was added to the corresponding mixture. At the same time, a comparative test of the DOX release of NV-DOX without CaP shell at different pH values ​​was carried out. The schematic diagram of the drug release from the shell under different pH conditions is shown in the figure. Figure 4 As shown in Figure 2, under pH control, the DOX release curves of NV-DOX with or without CaP shell were Figure 5 As shown. It can be seen that the CaP shell remains stable in a neutral environment, but will disintegrate in a weakly acidic environment, thereby achieving on-demand drug release and avoiding premature leakage. The schematic diagram is shown in Figure 3 As shown. Under pH 7.2, due to the lack of CaP shell barrier, the non-mineralized nanocarriers experienced significant drug leakage within 12 hours, with a release of approximately 38.7%. In contrast, the drug release of NV-DOX accounted for only about 10.5%, which was mainly attributed to the physical adsorption of DOX on the surface of the CaP shell, indicating that the CaP shell can effectively prevent premature drug leakage under physiological conditions. When the environmental pH value was adjusted to 5.5, the amount of DOX released from the nanocarrier increased significantly, with a release efficiency of approximately 77.9%, which can be intuitively judged by the change in the color of the medium. The experimental results show that the CaP shell has a significant barrier effect in controlling drug release, especially under physiological conditions, while effective drug release can be achieved in an acidic environment.

[0068] In order to further study the pH responsiveness of NV-DOX (without CaP shell), this experiment tested the release of NV-DOX under different pH conditions and the change in the diameter of PMAAc hydrogel under different pH conditions. Figure 6 As shown in Figure 2, the diameter of PMAAc hydrogels under different pH conditions changes as shown in Figure 7As shown. It can be seen that NV-DOX exhibits significant pH-responsive release behavior. Especially in the absence of a CaP shell, the nanoparticles still exhibit a certain degree of pH-dependent release characteristics, which is mainly attributed to the abundant pendant carboxyl structures in the hydrogel matrix. As the environmental pH changes, the PMAAc hydrogel undergoes significant volume changes, swelling under alkaline conditions and shrinking in an acidic environment. In addition, dynamic light scattering test results further confirmed this, showing that Fe 3 O 4 The hydrodynamic diameter of the @PDA@PMAAc nanoparticles decreases with decreasing pH. This property enables the hydrogel layer to act as a smart reservoir in the building block, triggering the controlled release of the drug under the acidic conditions of the tumor microenvironment, thereby enhancing its therapeutic effect.

[0069] For NV-GO X The release experiments of GO and NV-Mn nanoparticles were also carried out. When the pH value of the environment dropped to about 4-6, X and Mn 2+ It will be released after the CaP shell collapses. Under the catalysis of GO, glucose is consumed to produce acidic glucono-1,5-lactone and H 2 O 2 , thereby inhibiting the growth of cancer cells by starving them of nutrients. In addition, the generated acidic products further destroy the CaP shell and accelerate drug release. 2 O 2 In Mn 2+ Under the action of Mn, it is converted into active hydroxyl radicals (·OH) through Fenton-like reactions, exerting strong toxicity on tumors. In this way, combined therapy is expected to achieve the goal of promoting the development of Mn through glucose depletion starvation therapy and Mn 2+ The chemokinetic therapy mediated by Fenton-like reaction achieves synergistic effects, such as Figure 8 shown.

[0070] on GO X In the release experiment, 3 mg NV-GO X Dispersed in 30 mL of glucose solution (1 mg / mL) with initial pH values ​​of 7.2 and 5.5 and shaken at 37°C. At set time points, the nanoparticles in the mixture were concentrated with a permanent magnet, and the pH value of the supernatant was measured with a pH meter, and the nanoparticles were re-dispersed by shaking. At the same time, the initial pH value was 5.5 and only contained an equal amount of NV-GO. X or GO X The pH changes of glucose solution in the solution with NV-GO under different initial pH conditions were compared. X pH changes during 12 h of incubation as well as glucose solution alone and NV-GO XThe pH changes during the 12-hour incubation were as follows: Fig. 9 shown.

[0071] Validation of GO by evaluating changes in environmental pH X At 37℃, NV-GO X After adding glucose solution, the initial pH value of the mixture with an initial pH of 5.5 dropped to about 2.8 after 12 hours; while the initial pH value of the mixture with an initial pH of 7.2, pure glucose solution or pure NV-GO X The pH value of the solution did not change significantly. The above experiments prove that GO X Successful loading and pH-responsive release were achieved.

[0072] In Mn 2+ In the release experiment, the free radical probe TPA was first dissolved in 2 mm NaOH solution at a concentration of 0.5 mm, and then 25 mm NaHCO 3 Then 0.5 mg NV-Mn and 10 mm H 2 O 2 5 mL of the above solution was added and the pH value was adjusted to 7.2 and 5.5. After the mixture was shaken at 37 °C for 6 hours, the nanoparticles were concentrated with a permanent magnet and the fluorescence intensity of the supernatant was measured with a fluorescence spectrometer (λex = 312 nm, λem = 422 nm). 2 O 2 The fluorescence intensity changes in the mixed solution with a pH of 5.5 were compared and tested. Fig.10 shown.

[0073] Verify Mn 2+ Mediated Fenton-like reaction. Different from DOX and GO X , Mn 2+ Delivered in the form of manganese phosphate rather than ions. In an acidic environment, as the CaP shell disintegrates, the manganese phosphate dissolves and releases Mn 2+ HCO is widely present in the cellular environment. 3 - With the help of 2+ With H 2 O 2 Hydroxyl radicals (·OH) are generated through a Fenton-like reaction. The generation of ·OH was further confirmed by detecting hydroxynaphthalene (TPA) using a radical probe. TPA itself has no fluorescent signal, but it reacts with ·OH to generate fluorescent 2-hydroxy-p-hydroxynaphthoic acid.

[0074] In this example, the fluorescence intensity results after incubation of TPA solution with different treatment groups for 6 hours are as follows: Fig.10As shown. It can be seen that mixing TPA and H 2 O 2 A weaker fluorescence signal was detected when H 2 O 2 A small amount of ·OH is generated during the natural decomposition process; when NV-Mn is further added at pH 5.5, the fluorescence signal is significantly enhanced, confirming that Mn 2+ The mediated Fenton-like reaction accelerates the generation of ·OH. 2+ The release of α-glutamic acid showed significant pH dependence, which was consistent with the results of other drug release experiments.

[0075] (3) The microrobots loaded with three drugs are assembled into clusters through magnetic field manipulation, and are driven by the magnetic field to achieve targeted transportation, and then they accurately reach the designated location to complete the drug release task.

[0076] Example 2

[0077] Based on Example 1, Example 2 verifies the combined therapeutic effect of two drug-carrying microrobots forming a cluster. The specific experimental steps are as follows:

[0078] The macromolecular proteins (glucose oxidase, GO X ) and ions (Mn 2+ ) as the loaded drug, the steps of Example 1 are used to functionalize the microrobots, and it is expected that they will achieve synergistic effects through domino reactions. When the environmental pH value is reduced to about 4-6, GO X and Mn 2+ It will be released as the CaP shell dissolves. Under the catalytic action of GOX, glucose is consumed to generate acidic glucono-1,5-lactone and H 2 O 2 , which can inhibit cancer cell growth through "starvation therapy". In addition, the generated acidic products can accelerate the destruction of the CaP shell, thereby accelerating drug release. 2 O 2 In Mn 2+ Under the catalytic action of , it is converted into active hydroxyl radicals (·OH) through a Fenton-like reaction, exerting a strong toxic effect on the tumor, thereby achieving the synergistic treatment of "starvation therapy" and "chemodynamic therapy" through a domino reaction.

[0079] Methylene blue (MB) was used as an indicator to verify its degradation behavior by reacting with OH. 3- A certain concentration of MB was dissolved in the solution, and then 100 μg / mL of NV-GO was added. X or NV-Mn. The degradation efficiency of methylene blue after different treatments is shown in Fig.11 shown.

[0080] The results showed that the addition of NV-GO alone X After NV-GO or NV-Mn, the degradation degree of MB was low within 6 hours, which was mainly due to the physical adsorption of MB by nanoparticles. X The slight degradation increase caused by X The catalytic reaction produces H 2 O 2 However, when GO was added at a total concentration of 100 μg / mL (mass ratio 1:1) at pH 5.5, X and Mn 2+ The degradation efficiency of MB was significantly improved when the loaded microrobots were added. The absorbance of MB decreased sharply within the first 3 hours, and after 6 hours, almost all of the MB (>95%) was completely degraded. In contrast, no significant degradation effect was observed when the two nanocarriers were immersed in a pH 7.2 solution. This indicates that the generation of active hydroxyl radicals (·OH) only exhibits a strong and rapid reaction under acidic conditions. The above results verify that the domino reaction enhances the generation of active hydroxyl radicals, and further demonstrate the efficient synergistic effect of different drug-loaded microrobots.

[0081] Example 3

[0082] Based on Example 1, Example 3 verifies that a variety of drug-carrying robots are mixed to form a cluster and transported to a designated site to kill cancer cells. The specific experimental steps are as follows:

[0083] First, make a complex channel model (biochip) to simulate the vascular environment. The schematic diagram of the biochip is as follows Fig.12 As shown. In the biochip, four groups of experiments were designed to evaluate the performance of the microrobot cluster drug delivery system. In the "synchronous group" group, 10μg NV-DOX and 5μg NV-GO were added to the starting position on the left side of the chip. X The same number and type of nanocarriers were added to the “distributed rolling” group, but the control strategy was a weakly interacting rolling motion. In the “passive diffusion” group, the same number and type of nanocarriers were added to the target area seeded with HepG2 cancer cells. X and Mn 2+ ) of 100 μL solution, the drug dose was similar to that of NV-DOX and NV-GO XThe amount of drugs in the NV-Mn was the same as that in the control group. No substance was added to the control group. After all groups were treated, the biochip was placed in an incubator for 24 hours, and a live / dead cell staining experiment was performed on HepG2 cancer cells. Calcein-AM and propidium iodide (PI) were used to label live cells and dead cells with green and red fluorescence, respectively. The results of the passing rate of the distributed rolling and synchronized populations after passing through the complex channel between the starting position and the target position in the biochip are shown in Figure 2. Fig.13 shown.

[0084] The experimental results show that the weak clustering behavior of rolling motion causes the drug-carrying microrobots to navigate independently and it is difficult to form a stable mode. Most of the drug-carrying microrobots are scattered in complex environments, and only about 5% can reach the target location. Under the synchronous group control strategy, more than 90% of the drug-carrying microrobots can be accurately delivered to the target area, showing significant environmental adaptability and efficient targeting.

[0085] Further analysis of the survival status of cancer cells was performed. The live / dead staining results of HepG2 cancer cells after different treatments and 24 hours of incubation were as follows: Fig.14 As shown in the figure, it can be seen that passive diffusion of drugs has no significant effect on the growth and proliferation of HepG2 cancer cells, and drugs delivered by rolling motion can only kill a small number of cancer cells; while the functionalized drug-carrying units delivered by synchronous group control trigger domino reactions in the target area to achieve combined treatment, almost completely destroying cancer cells, showing excellent therapeutic effects and targeted delivery capabilities.

[0086] Example 4

[0087] Based on Example 1, the difference of this example is that in step (2), the amount of dopamine hydrochloride used is 0.01 g; in step (3), the amount of methacrylic acid (MAAc) used is 0.75 mL.

[0088] Fe 3 O 4 The infrared spectrum of @PDA@PMAAc@CaP nanoparticles is basically the same as that of Example 1, indicating that PDA, MPS, and PMAAc are successfully modified. Drug release experiments show that when the environmental pH value drops to about 4-6, DOX and GO X and Mn 2+ It will be released after the CaP shell disintegrates.

[0089] The microrobots loaded with three drugs are assembled into clusters through magnetic field manipulation, and are driven by the magnetic field to achieve targeted transportation, and complete the drug release task after accurately reaching the designated site. Example 5

[0090] Based on Example 1, the difference of this example is that in step (2), the amount of dopamine hydrochloride used is 0.03 g, and in step (3), the amount of methacrylic acid (MAAc) used is 3 mL.

[0091] Fe 3 O 4 The infrared spectrum of @PDA@PMAAc@CaP nanoparticles is basically the same as that of Example 1, indicating that PDA, MPS, and PMAAc are successfully modified. Drug release experiments show that when the environmental pH value drops to about 4-6, DOX and GO X and Mn 2+ It will be released after the CaP shell disintegrates.

[0092] Microrobots loaded with three drugs are assembled into clusters through magnetic field manipulation, and targeted transportation is achieved under the drive of the magnetic field. They complete the drug release task after accurately reaching the designated location.

[0093] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner, characterized in that: It includes several micro-robots, which are composed of magnetic nanoparticles, a hydrogel layer, and a pH-responsive material shell from the inside to the outside; the hydrogel layer includes dopamine-grafted 3-(methacryloyloxy)propyltrimethoxysilane located on the inside and methacrylic acid wrapped on the outside; the hydrogel layer is used to encapsulate drugs.

2. The microrobot cluster capable of simultaneously delivering multiple drugs according to claim 1, characterized in that: The magnetic nanoparticles are paramagnetic magnetite nanoparticles; and the pH-responsive shell is calcium phosphate.

3. The microrobot cluster capable of simultaneously delivering multiple drugs according to claim 2, characterized in that: The multiple drugs include doxorubicin, glucose oxidase and MnCl2.

4. The method for preparing a microrobot cluster capable of simultaneously delivering multiple drugs as claimed in claim 2 or 3, characterized in that: The steps include: Step S1, preparing Fe3O4 magnetic nanoparticles; Step S2, coating the surface of Fe3O4 magnetic nanoparticles with a PDA layer by in-situ polymerization to obtain Fe3O4@PDA nanoparticles, then washing the obtained Fe3O4@PDA nanoparticles and dispersing them in a mixed solution of ethanol, deionized water and ammonia water, adding MPS to react, and obtaining Fe3O4@PDA-MPS nanoparticles; then dispersing the Fe3O4@PDA-MPS nanoparticles in acetonitrile, continuously stirring and ultrasonically treating, and adding MAAc, a crosslinking agent and an initiator under argon protection, and heating and refluxing to obtain Fe3O4@PDA@PMAAc nanoparticles; Step S3, adding the Fe3O4@PDA@PMAAc nanoparticles to a CaCl2 solution and stirring, then adding a Na2HPO4 solution and continuing to stir to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles, which is a microrobot cluster that can simultaneously deliver multiple drugs in a targeted manner.

5. The method for preparing a microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner according to claim 4, characterized in that: Step S1 comprises: adding sodium acetate and ethylene glycol to ferric chloride hexahydrate and stirring evenly; then adding polyethylene glycol thereto and performing ultrasonic treatment; finally putting the obtained solution into a reactor and reacting at 100-300°C for 8-16 hours; after the reaction is completed, cooling to room temperature, washing, and obtaining Fe3O4 magnetic nanoparticles; the mass ratio of the ferric chloride hexahydrate to the sodium acetate is 1:2-3.

6. The method for preparing a microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner according to claim 5, characterized in that: In step S2, the Fe3O4@PDA nanoparticles are prepared by the following steps: Fe3O4 magnetic nanoparticles are dispersed in an alkaline buffer solution, and after stirring and ultrasonic treatment, dopamine hydrochloride is added and the reaction is continued in an ice water bath to obtain Fe3O4@PDA nanoparticles; the mass ratio of the Fe3O4 magnetic nanoparticles to dopamine hydrochloride is 1:0.2-0.

6.

7. The method for preparing a microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner according to claim 6, characterized in that: In step S2, the amount of MAAc used is 0.05-0.2 mL per mg of Fe3O4@PDA-MPS nanoparticles; the mass ratio of the Fe3O4@PDA-MPS nanoparticles to MBA is 1:0.005-0.01; the initiator is AIBN, and the mass ratio of the Fe3O4@PDA-MPS nanoparticles to AIBN is 1:0.001-0.015; In step S3, the amount of CaCl2 used is 0.002-0.006 mmol per milligram of Fe3O4@PDA@PMAAc nanoparticles; the amount of Na2HPO4 used is 0.004-0.01 mmol per milligram of Fe3O4@PDA@PMAAc nanoparticles.

8. The method for preparing a microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner according to claim 4, characterized in that: Step S3 includes: adding multiple drugs to the CaCl2 solution, stirring and dissolving to obtain a CaCl2 mixed solution; then adding the Fe3O4@PDA@PMAAc nanoparticles to the CaCl2 mixed solution, stirring, then adding Na2HPO4 solution, continuing to stir, and washing to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles, which can be a micro-robot cluster that can simultaneously target and deliver multiple drugs; the multiple drugs include doxorubicin, glucose oxidase and MnCl2.

9. The method for preparing a microrobot cluster capable of simultaneously delivering multiple drugs in a targeted manner according to claim 8, characterized in that: The amount of doxorubicin used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg, the amount of glucose oxidase used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg, and the amount of MnCl2 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 1-2 mmol.

10. Use of the microrobot cluster capable of simultaneously delivering multiple drugs as claimed in any one of claims 1 to 3 in the preparation of targeted delivery drugs.

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