Micro-robot swarm for simultaneous targeted delivery of multiple drugs and method of making the same

Abstract

The application provides a micro-robot cluster capable of simultaneously targeting and delivering multiple drugs and a preparation method thereof, which comprises a plurality of micro-robots, the micro-robots sequentially comprise a magnetic nanoparticle, a hydrogel layer and a pH-responsive material shell from inside to outside; the hydrogel layer comprises dopamine (PDA) grafted 3-(methacryloyloxy)propyl trimethoxysilane on the inside and methacrylic acid wrapped on the outside; and the hydrogel layer is used for encapsulating multiple drugs. The micro-robot cluster of the technical scheme can be driven by an external magnetic field, transported to a specific position in a complex simulated blood environment, and realize combined delivery and controllable release of multiple drugs, has the advantages of strong targeting, high drug loading capacity and intelligent release, and can be applied to the field of precision drug therapy.

Description

Technical Field

[0001] This invention relates to the field of drug targeted delivery technology, and in particular to a microrobot cluster capable of simultaneously targeting and delivering multiple drugs and its preparation method. Background Technology

[0002] With the rapid development of precision medicine and targeted therapy technologies, drug delivery systems are continuously advancing towards higher efficiency, precision, and intelligence. In traditional drug delivery methods, drugs are typically administered orally or intravenously. However, this non-specific delivery often results in uneven drug distribution within the body, making it difficult to concentrate the drug on the lesion area. Furthermore, due to the lack of drug targeting, healthy tissue may be damaged, leading to increased toxicity and reduced therapeutic efficacy. Especially in treating complex diseases such as cancer, inflammation, or neurological disorders, the efficacy of a single drug is often limited, typically requiring the combined use of multiple drugs for synergistic treatment. Therefore, developing a system capable of multi-drug targeted delivery has become one of the most pressing challenges in the field of drug delivery technology.

[0003] In recent years, microrobot technology has attracted widespread attention as an emerging intelligent drug delivery system. Microrobots typically possess characteristics such as small size, good biocompatibility, and externally controllable actuation, enabling them to achieve directional movement and precise drug delivery in complex bodily 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 drug concentration at the lesion site and significantly reducing the toxic side effects in healthy tissues. However, most current microrobots still suffer from limited drug loading capacity, insufficient release precision, and difficulty in achieving multi-drug synergistic delivery, which restricts their practical application in the treatment of complex diseases. Therefore, developing a microrobot system with high drug loading capacity, targeted delivery, multi-drug synergy, and precise release capabilities is of significant research importance and application value for achieving efficient drug delivery and improving disease treatment outcomes. Summary of the Invention

[0004] To address the above technical problems, this invention discloses a microrobot cluster capable of simultaneously targeting and delivering multiple drugs and its preparation method. Magnetic nanoparticles are used as the core of the microrobot, endowing them with magnetic responsiveness and driving ability. A hydrogel layer is introduced on the outside as a drug 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 drug loading capacity and targeted delivery efficiency, but also utilizes the lesion microenvironment to trigger release, achieving multi-drug synergistic treatment.

[0005] The technical solution adopted by this invention is as follows:

[0006] A cluster of microrobots capable of simultaneously targeting and delivering multiple drugs comprises, from the inside out, magnetic nanoparticles, a hydrogel layer, and a pH-responsive material shell; the hydrogel layer includes dopamine (PDA) grafted with 3-(methacryloyloxy)propyltrimethoxysilane on the inner side and methacrylic acid encapsulated on the outer layer; the hydrogel layer is used to encapsulate multiple drugs.

[0007] This technology utilizes magnetic nanoparticles as the core of a microrobot, endowing them with magnetic responsiveness and controllable actuation capabilities, enabling precise movement and targeted positioning under the influence of an external magnetic field. A hydrogel layer is modified on the outer side of the magnetic nanoparticles, serving as a drug-carrying layer. This layer can encapsulate various drugs, possessing high drug-loading capacity and good biocompatibility, effectively encapsulating multiple drugs to meet the needs of combined therapy. The outermost layer, coated with a pH-responsive material, acts as a smart release module, triggering controlled drug release based on the lesion microenvironment (such as the acidic environment of tumor tissue), further improving the precision and efficiency of treatment. Driven by an external magnetic field, the microrobot cluster can target and transport drugs to specific locations in a complex simulated blood environment, achieving multi-drug combined delivery and synergistic release, significantly improving treatment efficacy and reducing toxic side effects on healthy tissues. This system features 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 doxorubicin, glucose oxidase, and MnCl2. Using this technical solution, by loading multiple drugs, the microrobot can achieve synergistic therapeutic effects in combination therapy with targeted delivery.

[0011] This invention also discloses a method for preparing a microrobot cluster capable of simultaneously targeting and delivering multiple drugs, as described above, comprising the following steps:

[0012] Step S1: Prepare Fe3O4 magnetic nanoparticles;

[0013] Step S2 involves coating a PDA layer onto the surface of Fe3O4 magnetic nanoparticles via in-situ polymerization to obtain Fe3O4@PDA nanoparticles. The Fe3O4@PDA nanoparticles are then washed and dispersed in a mixed solution of ethanol, deionized water, and ammonia. MPS is added to initiate the reaction, yielding Fe3O4@PDA-MPS nanoparticles. A hydrogel layer is then anchored onto the nanoparticle surface via precipitation polymerization. Specifically, Fe3O4@PDA-MPS nanoparticles are dispersed in acetonitrile, continuously stirred, and sonicated. MAAc, a crosslinking agent, and an initiator are added under argon protection, and the mixture is heated to reflux to obtain Fe3O4@PDA@PMAAc nanoparticles. After this step, the product can be washed and stored in deionized water for later use.

[0014] Step S3 involves depositing a CaP shell on the surface of Fe3O4@PDA@PMAAc nanoparticles by simulating a biomineralization process. Specifically, the Fe3O4@PDA@PMAAc nanoparticles are added to a CaCl2 solution and stirred, followed by the addition of a Na2HPO4 solution and continued stirring to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles, which constitute a cluster of microrobots capable of simultaneously targeting and delivering multiple drugs.

[0015] As a further improvement of the present invention, step S1 includes: adding sodium acetate and ethylene glycol to ferric chloride hexahydrate and stirring until homogeneous; then adding polyethylene glycol and performing ultrasonic treatment; finally, placing the resulting solution into a reaction vessel and reacting at 100-300℃ for 8-16 hours; after the reaction is completed, cooling to room temperature and washing to obtain Fe3O4 magnetic nanoparticles. Further, the mass ratio of ferric chloride hexahydrate to sodium acetate is 1:2-3. Further, the washing is performed using deionized water 2-5 times.

[0016] As a further improvement of the present invention, 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. Further, the mass ratio of the Fe3O4 magnetic nanoparticles to dopamine hydrochloride is 1:0.2-0.6.

[0017] As a further improvement of the present invention, in step S2, the amount of MAAc used is 0.05-0.2 mL per milligram of Fe3O4@PDA-MPS nanoparticles.

[0018] Furthermore, the crosslinking agent is MBA; the mass ratio of Fe3O4@PDA-MPS nanoparticles to MBA is 1:0.005-0.01.

[0019] The initiator is AIBN, and the mass ratio of Fe3O4@PDA-MPS nanoparticles to AIBN is 1:0.001-0.015.

[0020] As a further improvement of the present invention, in step S3, the amount of CaCl2 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.002-0.006 mmol; the amount of Na2HPO4 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.004-0.01 mmol.

[0021] As a further improvement of the present invention, step S3 includes: adding multiple drugs to a CaCl2 solution, stirring and dissolving to obtain a CaCl2 mixed solution; then adding the Fe3O4@PDA@PMAAc nanoparticles to the CaCl2 mixed solution, stirring, adding Na2HPO4 solution, continuing stirring, and washing to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles, which can simultaneously target and deliver multiple drugs to a cluster of microrobots; the multiple drugs include doxorubicin, glucose oxidase and MnCl2.

[0022] As a further improvement of the present invention, the amount of doxorubicin per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg, the amount of glucose oxidase per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg, and the amount of MnCl2 per milligram of Fe3O4@PDA@PMAAc nanoparticles is 1-2 mmol.

[0023] The present invention also discloses the use of the microrobot clusters described above, capable of simultaneously targeting and delivering multiple drugs, in the preparation of targeted drug delivery.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] First, employing the technical solution of this invention, the robot cluster, with magnetic nanoparticles as its core, can be combined with external magnetic field drive to achieve efficient and precise targeted delivery. Simultaneously, the outer hydrogel layer and pH-responsive material endow the microrobots with high drug loading capacity and intelligent, controllable drug release capabilities, significantly improving the accuracy of drug delivery and therapeutic efficacy. By loading multiple drugs into the microrobots and employing a multi-drug synergistic strategy, and achieving precise release through a controllable release mechanism, the combined therapeutic effect is enhanced, and treatment efficiency is improved.

[0026] Secondly, the microrobot of this invention can move stably in a complex simulated blood environment, realizing the combined delivery and synergistic release of multiple drugs, overcoming the limitations of traditional drug delivery systems. It has the advantages of strong targeting, low toxicity and side effects, and synergistic treatment of multiple drugs, providing a new, efficient, intelligent and controllable approach for precision drug therapy. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the targeted therapy using a microrobot that can be used to simultaneously deliver 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, from bottom to top, are: Fe3O4, Fe3O4@PDA-MPS, Fe3O4@PDA@PMAAc, Fe3O4@PDA@PMAAc@CaP.

[0029] Figure 3 This is a SEM image of the Fe3O4@PDA-MPS nanoparticles obtained in Example 1 of this invention.

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

[0031] Figure 5 This is a comparison of the DOX release effect of NV-DOX with and without a CaP shell under pH control, according to Example 1 of the present invention.

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

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

[0034] Figure 8 This is a schematic diagram of a combination of multiple drugs in Embodiment 1 of the present invention.

[0035] Figure 9 This refers to glucose solutions under different initial pH conditions in Example 1 of the present invention, in combination with NV-GO. X pH changes over 12 hours of incubation, and glucose solution and NV-GO alone X pH changes during 12 hours of incubation.

[0036] Figure 10 The fluorescence intensity is the result of incubating the TPA solution of Example 1 of this invention with different treatment groups for 6 hours.

[0037] Figure 11This refers to the degradation efficiency of methylene blue after different treatments in Example 1 of the present invention.

[0038] Figure 12 This is a schematic diagram of the biochip in Embodiment 1 of the present invention.

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

[0040] Figure 14 These are the live / dead staining results of HepG2 cancer cells incubated for 24 hours after different treatments in Example 1 of this invention. Detailed Implementation

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

[0042] A cluster of microrobots capable of simultaneously targeting and delivering multiple drugs comprises several microrobots, each consisting of, from the inside out, magnetic nanoparticles, a hydrogel layer, and a pH-responsive material shell. The hydrogel layer comprises dopamine (PDA) grafted with 3-(methacryloyloxy)propyltrimethoxysilane on the inner side and methacrylic acid encapsulated on the outer layer. The hydrogel layer encapsulates multiple drugs. The magnetic nanoparticles are paramagnetic Fe3O4 magnetite nanoparticles, which can be prepared using a solvothermal method. The pH-responsive shell is calcium phosphate.

[0043] The method for fabricating this microrobot includes the following steps:

[0044] Step S1: Add sodium acetate and ethylene glycol to ferric chloride hexahydrate and stir until homogeneous; then add polyethylene glycol and sonicate for half an hour; finally, place the resulting solution into a reaction vessel and react at 100-300℃ for 8-16 hours; after the reaction is completed, cool to room temperature and wash with deionized water 2-5 times to obtain the final iron oxide nanoparticles; wherein the mass ratio of ferric chloride hexahydrate to sodium acetate is 1:2-3.

[0045] Step S2: Using an in-situ polymerization process, a PDA layer is first coated onto the surface of Fe3O4. Fe3O4 nanoparticles are then dispersed in a buffer solution adjusted to an alkaline pH. After mechanical stirring and ultrasonic treatment, dopamine hydrochloride is added, and the reaction is continued in an ice-water bath to obtain Fe3O4@PDA nanoparticles. Subsequently, the nanoparticles are washed with deionized water and dispersed in a mixed solution of ethanol, deionized water, and ammonia. 2-5 mL of 3-(methacryloyloxy)propyltrimethoxysilane (MPS) is added, and the reaction is carried out for a period of time to obtain F… Fe3O4@PDA-MPS nanoparticles were then used. Next, a hydrogel layer was anchored on the surface of the nanoparticles via precipitation polymerization. The Fe3O4@PDA-MPS nanoparticles were dispersed in acetonitrile, continuously stirred, and sonicated. Under argon protection, methacrylic acid (MAAc), N,N'-methylenebisacrylamide crosslinking agent MBA, and initiator azobisisobutyronitrile (AIBN) were added. After heating the mixture under reflux for a period of time, Fe3O4@PDA@PMAAc nanoparticles were obtained. Finally, the nanoparticles were washed and stored in deionized water for later use.

[0046] In this study, carbon-carbon double bonds were successfully immobilized on the surface of Fe3O4 nanoparticles by introducing MPS. These double bonds provide cross-linking sites for the subsequent hydrogel layer. This surface functionalization method not only enhances the binding force between nanoparticles and 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, through surface modification onto the nanoparticles, improves the dispersion stability of the microrobots, providing the necessary conditions for subsequent swarming motion. Furthermore, the hydrophilic network structure of the hydrogel layer can effectively load various drugs and accelerate drug release through swelling behavior in acidic environments, while reducing drug leakage into normal tissues.

[0047] In step S2, insufficient PDA dosage will result in uneven surface coverage, affecting the subsequent MPS modification effect; excessive dosage may form an overly thick polymer film, reducing the magnetic response and dispersibility of the nanoparticles. In this invention, the mass ratio of Fe3O4 to dopamine hydrochloride is 1:0.2-0.6. By using an appropriate amount of PDA, the surface of the nanoparticles is uniformly covered without forming an overly thick polymer film.

[0048] The amount of MAAc used per milligram of Fe3O4@PDA-MPS nanoparticles is 0.05-0.2 mL, the mass ratio of Fe3O4@PDA-MPS nanoparticles to MBA is 1:0.005-0.01, and the mass ratio of Fe3O4@PDA-MPS nanoparticles to AIBN is 1:0.001-0.015.

[0049] Step S3: A CaP shell is deposited on the surface of Fe3O4@PDA@PMAAc nanoparticles by simulating a biomineralization process. The nanoparticles are added to a CaCl2 solution, and after mechanical stirring, a Na2HPO4 solution is slowly added at a constant rate. Stirring is continued for a period of time to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles. Finally, the nanoparticles are washed with deionized water and stored in deionized water for later use. The amount of CaCl2 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.002-0.006 mmol, and the amount of Na2HPO4 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.004-0.01 mmol.

[0050] In addition, the drug delivery process of the microrobot is carried out in step S3, including:

[0051] Multiple drugs were added to a CaCl2 solution and stirred until dissolved. Similarly, Fe3O4@PDA@PMAAc nanoparticles were added to the CaCl2 solution, and after mechanical stirring, Na2HPO4 solution was slowly added at a constant rate, with stirring continued for a period of time to obtain drug-loaded microrobots. Finally, the product was washed 3-5 times with deionized water and PBS and stored in PBS for later use. The drugs may be doxorubicin (DOX) or glucose oxidase (GO). X ) and MnCl2; wherein, the amount of DOX used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg, the microrobot loaded with DOX drug is defined as NV-DOX, and the amount of DOX used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg. X The dosage is 0.1-0.2 mg, loaded with GO. X The microrobots for drugs are defined as NV-GO X The amount of MnCl2 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 1-2 mmol, and the microrobot loaded with MnCl2 drug is defined as NV-Mn.

[0052] Driven by an external magnetic field, microrobots can rapidly self-organize and achieve controllable motion. A rotating magnetic field can induce stable cluster motion among the microrobots. By modifying the surface of Fe3O4 nanoparticles with a hydrogel layer, it is possible to achieve controlled release of different drugs from a pH-responsive shell. Surface modifications of the Fe3O4 nanoparticles (such as the biocompatibility and dispersibility provided by the PDA layer, and the functionalized sites introduced by the MPS and hydrogel layers) provide the necessary conditions for smooth and coordinated movement. Mixing microrobots loaded with different drugs to form a cluster yields the final drug delivery system, and the simultaneous release of the three drugs results in a synergistic therapeutic effect, significantly improving treatment efficacy.

[0053] The process involved adding a cluster of microrobots, pre-loaded with three drugs in specific proportions, into a complex channel filled with simulated plasma. This channel was then placed within a three-dimensional Helmholtz electromagnetic coil, and the cluster was moved to a target location containing cancer cells under the control of a rotating magnetic field. Cancer cell survival was assessed through a 24-hour incubation period following drug administration. A schematic diagram of the drug-loaded robot cluster movement and targeted therapy is shown below. Figure 1 As shown.

[0054] The following description uses specific examples to illustrate the point.

[0055] Example 1

[0056] A cluster of microrobots for simultaneous targeted delivery of multiple drugs, comprising several microrobots, wherein the fabrication steps of the microrobots include:

[0057] (1) Fe3O4 nanoparticles were prepared by solvothermal method.

[0058] The specific steps include: dissolving 1.35g FeCl3·6H2O and 3.6g NaAc sequentially in 40mL ethylene glycol (EG) and mixing for 30 minutes under magnetic stirring. Then, 1g polyethylene glycol (PEG) is added, and the mixture is sonicated for 30 minutes to obtain a reddish-brown turbid liquid. The mixture is transferred to a 50mL high-pressure reactor, heated to 200℃ for 10 hours, and then naturally cooled to room temperature. The product is washed three times with deionized water (DI water), magnetically separated, and stored in water for later use.

[0059] (2) A dopamine (PDA) layer was coated onto the surface of Fe3O4 via in-situ polymerization. Specific steps included:

[0060] 0.12 g Tris was dissolved in 100 mL of DI water, and the pH was adjusted to 8.5 by adding 1 mL of HCl dropwise. The resulting buffer solution was then added to a 250 mL round-bottom flask along with 50 mg of Fe3O4 nanoparticles. The mixture was mechanically stirred and sonicated for 30 minutes. Next, 0.02 g of dopamine hydrochloride was added, and the mixture was mechanically stirred and sonicated for 5 hours in an ice-water bath to obtain Fe3O4@PDA nanoparticles. Subsequently, after washing three times with DI water, the Fe3O4@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 of MPS was added, and the reaction was allowed to proceed for 24 hours to obtain Fe3O4@PDA-MPS nanoparticles. Finally, the nanoparticles were washed three times each with ethanol and acetonitrile, magnetically separated, and stored in acetonitrile for later use.

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

[0062] 15 mg of Fe3O4@PDA-MPS nanoparticles were added to 100 mL of acetonitrile, mechanically stirred, and sonicated for 30 minutes while continuously 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 sequentially, and the mixture was rapidly heated to boiling. After reflux for 90 minutes, Fe3O4@PDA@PMAAc nanoparticles were obtained. After washing with ethanol and DI water three times, the nanoparticles were stored in DI water for later use.

[0063] (4) A CaP shell is deposited on the surface of nanoparticles by simulating a biomineralization process. Specific steps include:

[0064] 15 mg of Fe3O4@PDA@PMAAc nanoparticles were added to 2 mL of 30 mmol CaCl2 solution and mechanically stirred overnight. Subsequently, 4 mL of 20 mmol Na2HPO4 solution was injected into the mixture at a rate of 1 mL / h using a syringe pump, and mechanical stirring was continued for 2 hours to prepare Fe3O4@PDA@PMAAc@CaP nanoparticles. Finally, after washing three times with DI water, magnetic separation was performed, and the nanoparticles were stored in DI water for later use.

[0065] In the mineralization process of step (4), various drugs can be loaded, such as DOX and GO. X Or MnCl2. The specific steps are: add DOX and GO... XMnCl2 and Fe3O4@PDA@PMAAc nanoparticles were dissolved in 2 mL of 30 mmol / L CaCl2 solution, with concentrations of 1 mg / mL, 1 mg / mL, and 10 mmol / L, respectively. Then, 15 mg of Fe3O4@PDA@PMAAc nanoparticles were added, and the mixture was mechanically stirred overnight. Next, 4 mL of 20 mmol / L Na2HPO4 solution was injected into the mixture using a syringe pump at a rate of 1 mL / h, and the mixture was further mechanically stirred for 2 hours to prepare NV-DOX and NV-GO. X And NV-Mn nanoparticles. Finally, after washing three times each with DI water and PBS, it was stored in PBS for later use.

[0066] The infrared spectra of Fe3O4 nanoparticles, Fe3O4@PDA-MPS nanoparticles, Fe3O4@PDA@PMAAc nanoparticles, and Fe3O4@PDA@PMAAc@CaP nanoparticles in the above experiments are shown below. Figure 2 As shown, SEM Figure 3 As shown. Figure 2 As shown, 588cm -1 The characteristic peak at 1650 cm⁻¹ is attributed to the stretching vibration of Fe-O, while the peak at 1650 cm⁻¹ is attributed to the stretching vibration of Fe-O. -1 and 3450cm -1 The absorption peaks at approximately 1700 cm⁻¹ are attributed to the vibrational modes of adsorbed water molecules and hydroxyl groups, respectively. The appearance of these characteristic peaks indicates the successful synthesis of iron oxide nanoparticles. Furthermore, the absorption peak at approximately 1700 cm⁻¹... -1 and 2990cm -1 Multiple absorption peaks correspond to the stretching vibrations of C=O and CH groups in polymethacrylic acid (PMAAc), confirming the successful encapsulation of nanoparticles by the hydrogel layer. After biomineralization treatment, the 526 cm⁻¹... -1 Approximately 1100cm -1 New absorption bands appeared, corresponding to the bending vibration and asymmetric tensile vibration of PO, respectively, indicating that the phosphate composite material has been successfully formed. It is evident that PDA, MPS, and PMAAc modification was successful after the above steps. The more MPS added, the more double bonds are present on the surface, resulting in more molecules participating in the free radical polymerization reaction and stronger binding forces.

[0067] DOX release experiments were conducted 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 set time points, the nanoparticles in the mixture were concentrated using a permanent magnet. 1 mL of the supernatant was collected, and the DOX concentration was measured using a UV-Vis spectrophotometer. Then, 1 mL of fresh PBS with the same pH was added to the corresponding mixture. Simultaneously, the DOX release of NV-DOX without a CaP shell at different pH values ​​was compared. Schematic diagrams of drug release from the shell under different pH conditions are shown below. Figure 4 As shown, under pH control, the DOX release curves of NV-DOX with and without a CaP shell are as follows. Figure 5 As shown in the diagram, the CaP shell remains stable in a neutral environment but disintegrates in a weakly acidic environment, thus enabling on-demand drug release and preventing premature leakage. Figure 3 As shown, under pH 7.2 conditions, the non-mineralized nanocarrier experienced significant drug leakage within 12 hours due to the lack of a CaP shell, with a release rate of approximately 38.7%. In contrast, the drug release rate of NV-DOX was only about 10.5%, mainly attributed to the physical adsorption of DOX on the CaP shell surface. This indicates that the CaP shell can effectively prevent premature drug leakage under physiological conditions. When the ambient pH was adjusted to 5.5, the amount of DOX released by the nanocarrier increased significantly, with a release efficiency of approximately 77.9%, which could be visually observed through changes in the color of the medium. The experimental results demonstrate that the CaP shell has a significant barrier effect in controlling drug release, especially under physiological conditions, while achieving effective drug release in acidic environments.

[0068] To further investigate the pH responsiveness of NV-DOX (without a CaP shell), this experiment tested the release of NV-DOX under different pH conditions and the changes in the diameter of the PMAAc hydrogel under different pH conditions. The release curves of NV-DOX under different pH conditions are shown below. Figure 6 As shown, the diameter changes of PMAAc hydrogels under different pH conditions are as follows: Figure 7As shown, 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, primarily attributed to the abundant pendant carboxyl structures in the hydrogel matrix. With changes in ambient pH, the PMAAc hydrogel undergoes significant volume changes, expanding under alkaline conditions and shrinking under acidic conditions. Furthermore, dynamic light scattering tests further confirm this, showing that the hydrodynamic diameter of the Fe3O4@PDA@PMAAc nanoparticles decreases with decreasing pH. This property allows the hydrogel layer to act as a smart reservoir within the building block, triggering controlled drug release under the acidic conditions of the tumor microenvironment, thereby enhancing its therapeutic effect.

[0069] For NV-GO X Release experiments were also conducted with NV-Mn nanoparticles. When the ambient pH value dropped to approximately 4-6, GO... X and Mn 2+ It will be released after the CaP shell disintegrates. Under the catalysis of GO, glucose is consumed, generating acidic gluconic acid-1,5-lactone and H2O2, thereby inhibiting the growth of cancer cells by depriving them of nutrients. Furthermore, the generated acidic products further damage the CaP shell, accelerating drug release. A large amount of H2O2 is released into the Mn... 2+ Under the influence of [unclear], it is converted into reactive hydroxyl radicals (·OH) via a Fenton-like reaction, exerting strong toxicity on tumors. In this way, combination therapy holds promise through glucose depletion starvation therapy and [unclear] 2+ Fenton-mediated chemokinetics achieve synergistic effects, such as... Figure 8 As shown.

[0070] on GO X In the release experiment, 3 mg of NV-GO was used. X The nanoparticles were 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 using a permanent magnet. After measuring the pH value of the supernatant using a pH meter, the nanoparticles were redispersed by shaking. Simultaneously, for a mixture with an initial pH of 5.5 and containing only an equal amount of NV-GO... X or GO X The pH changes in the solution were compared and tested. Glucose solutions under different initial pH conditions were compared with NV-GO. X pH changes over 12 hours of incubation, and glucose solution and NV-GO alone X pH changes during 12 hours of incubation Figure 9 As shown.

[0071] GO was validated by assessing changes in environmental pH. XThe encapsulation and release. At 37°C, NV-GO... X After the addition of glucose solution, the mixture with an initial pH of 5.5 decreased to approximately 2.8 after 12 hours; while the mixture with an initial pH of 7.2, pure glucose solution, or pure NV-GO... X The pH of the solution did not change significantly. The above experiment proves that GO X Successfully loaded and achieved pH-responsive release.

[0072] In Mn 2+ In the release experiment, the free radical probe TPA was first dissolved in 2 mm NaOH solution to a concentration of 0.5 mm, followed by the addition of 25 mm NaHCO3 solution. Then, 0.5 mg NV-Mn and 10 mm H2O2 were added to 5 mL of the above solution, and the pH was adjusted to 7.2 and 5.5, respectively. After shaking the mixture at 37 °C for 6 hours, the nanoparticles were concentrated using a permanent magnet, and the fluorescence intensity of the supernatant was measured using a fluorescence spectrometer (λex = 312 nm, λem = 422 nm). Simultaneously, the fluorescence intensity changes in the blank TPA solution and the mixed solution at pH 5.5 containing only equal amounts of NV-Mn or H2O2 were compared. The results are as follows: Figure 10 As shown.

[0073] Verify Mn 2+ Mediated Fenton-like reactions. Unlike DOX and GO. X Mn 2+ It is delivered in the form of manganese phosphate, rather than in an ionic state. In an acidic environment, as the CaP shell disintegrates, manganese phosphate dissolves, releasing Mn. 2+ HCO3 is widely present in the cellular environment. - With the assistance of Mn 2+ Hydroxyl radicals (·OH) are generated by reacting with H₂O₂ via a Fenton-like reaction. The generation of ·OH was further confirmed by detecting hydroxynaphthalene (TPA) using a radical probe. TPA itself does not show a fluorescent signal, but reacts with ·OH to generate fluorescent 2-hydroxy-p-hydroxynaphthalic acid.

[0074] In this embodiment, the fluorescence intensity results of TPA solution after incubation with different treatment groups for 6 hours are as follows: Figure 10 As shown, a weak fluorescence signal was detected when TPA and H2O2 were mixed alone, which is attributed to the generation of a small amount of ·OH during the natural decomposition of H2O2. When NV-Mn was further added at pH 5.5, the fluorescence signal significantly increased, confirming the presence of Mn. 2+ The Fenton-like mediated reaction accelerated the formation of ·OH. Furthermore, Mn... 2+ The release of the drug showed a significant pH dependence, consistent with the results of other drug release experiments.

[0075] (3) The microrobots loaded with three drugs are assembled into a cluster by magnetic field control and targeted transport under magnetic field drive, and the drugs are released after accurately reaching the designated location.

[0076] Example 2

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

[0078] Large molecular weight proteins (glucose oxidase, GO) were selected. X ) and ions (Mn 2+ As the loaded drug, the steps of Example 1 were used to functionalize the microrobots, with the expectation that they would achieve synergistic effects through a domino effect. When the ambient pH decreased to approximately 4-6, GO... X and Mn 2+ It is released as the CaP shell dissolves. Under the catalysis of GOX, glucose is consumed, generating acidic gluconic acid-1,5-lactone and H2O2, which can inhibit cancer cell growth through "starvation therapy." Furthermore, the generated acidic products can accelerate the destruction of the CaP shell, thereby speeding up drug release. Simultaneously, abundant H2O2 in Mn... 2+ Under the catalysis of [a substance], it is converted into active hydroxyl radicals (·OH) through a Fenton-like reaction, exerting a strong toxic effect on tumors, thereby achieving synergistic treatment of "starvation therapy" and "chemokinetic therapy" through a domino reaction.

[0079] Methylene blue (MB) was used as an indicator, and its degradation behavior was verified by its reaction with ·OH. First, a mixture containing glucose and HCO3- was prepared. 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 as follows: Figure 11 As shown.

[0080] The results show that adding NV-GO alone X After NV-Mn, the degradation rate of MB was low within 6 hours, mainly due to the physical adsorption of MB by nanoparticles. NV-GO, on the other hand... X The slight increase in degradation caused is attributed to GO X The catalytic reaction produces H2O2. However, when GO is added simultaneously at a total concentration of 100 μg / mL (mass ratio 1:1) under pH 5.5 conditions... X and Mn 2+When loaded with microrobots, the degradation efficiency of MB was significantly improved. Within the first 3 hours, the absorbance of MB decreased sharply, and after 6 hours, almost all MB (>95%) was completely degraded. Conversely, no significant degradation effect was observed when either nanocarrier was immersed in a pH 7.2 solution. This indicates that the generation of active hydroxyl radicals (·OH) exhibits a potent and rapid reaction only under acidic conditions. These results validate that the domino reaction enhances the generation of active hydroxyl radicals and further demonstrate the highly efficient synergistic effect of different drug-loaded microrobots.

[0081] Example 3

[0082] Building upon Example 1, Example 3 verified that a cluster of various drug-carrying robots, when mixed, could be targeted and delivered to specific sites to kill cancer cells. The specific experimental steps are as follows:

[0083] First, a complex channel model (biochip) is created to simulate the vascular environment. A schematic diagram of the biochip is shown below. Figure 12 As shown. Four sets of experiments were designed within the biochip to evaluate the performance of the microrobot swarm drug delivery system. In the "synchronous population" group, 10 μg of NV-DOX and 5 μg of NV-GO were added to the starting position on the left side of the chip. X 5 μg NV-Mn (dissolved in 100 μL PBS) was added, and a simultaneous population control strategy was used to deliver the nanocarriers to the target area seeded with HepG2 cancer cells. In the "distribution rolling" group, the same number and type of nanocarriers were added, but the control strategy was a weakly interacting rolling motion. In the "passive diffusion" group, nanocarriers containing pure drugs (DOX, GO) were added. X and Mn 2+ A 100 μL solution of NV-DOX and NV-GO was used, with the drug dosage being similar to that of NV-DOX and NV-GO. X The amount of drug contained in NV-Mn was equal to that in the "control" group. No substance was added in the "control" group. After treatment, the biochip was incubated 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 and dead cells with green and red fluorescence, respectively. The throughput results of the distributed rolling and synchronous populations after passing through the complex channels between the starting and target positions in the biochip are shown below. Figure 13 As shown.

[0084] Experimental results show that the weak swarming behavior of rolling motion leads to independent navigation of drug-carrying microrobots and difficulty in forming stable patterns. Most drug-carrying microrobots are scattered in complex environments, with only about 5% able to reach the target location. However, under the synchronous swarm control strategy, more than 90% of the drug-carrying microrobots can accurately deliver drugs to the target area, demonstrating significant environmental adaptability and efficient targeting.

[0085] Further analysis of cancer cell survival status revealed the live / dead staining results of HepG2 cancer cells after 24 hours of incubation following different treatments. Figure 14 As shown, passive diffusion drugs have 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 functionalized drug delivery units delivered by synchronous population control trigger a domino effect in the target area to achieve combined therapy, almost completely destroying cancer cells, demonstrating excellent therapeutic effect and targeted delivery capability.

[0086] Example 4

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

[0088] The infrared spectrum of the obtained Fe3O4@PDA@PMAAc@CaP nanoparticles was basically the same as that of Example 1, indicating successful modification with PDA, MPS, and PMAAc. Drug release experiments showed that when the ambient pH value dropped to approximately 4-6, DOX and GO... X and Mn 2+ It will be released after the CaP shell disintegrates.

[0089] Microrobots carrying three drugs were assembled into a cluster using magnetic field manipulation. Driven by the magnetic field, they achieved targeted delivery, precisely reaching the designated site to release the drugs. Example 5

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

[0091] The infrared spectrum of the obtained Fe3O4@PDA@PMAAc@CaP nanoparticles was basically the same as that of Example 1, indicating successful modification with PDA, MPS, and PMAAc. Drug release experiments showed that when the ambient pH value dropped to approximately 4-6, DOX and GO... X and Mn 2+ It will be released after the CaP shell disintegrates.

[0092] Microrobots carrying three drugs are assembled into a cluster by magnetic field manipulation, and then targeted transport is achieved under magnetic field drive, accurately reaching the designated site to complete the drug release task.

[0093] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A cluster of microrobots capable of simultaneously targeting and delivering multiple drugs, characterized in that: It comprises several microrobots, which, from the inside out, consist of magnetic nanoparticles, a hydrogel layer, and a pH-responsive material shell; the hydrogel layer includes dopamine-grafted 3-(methacryloyloxy)propyltrimethoxysilane on the inner side and methacrylic acid wrapped around it on the outer layer; the hydrogel layer is used to encapsulate drugs; The microrobot cluster capable of simultaneously targeting and delivering multiple drugs was prepared using the following steps: Step S1: Prepare Fe3O4 magnetic nanoparticles; Step S2: A PDA layer is coated onto the surface of Fe3O4 magnetic nanoparticles through in-situ polymerization to obtain Fe3O4@PDA nanoparticles. The Fe3O4@PDA nanoparticles are then washed and dispersed in a mixed solution of ethanol, deionized water, and ammonia. MPS is added to react and produce Fe3O4@PDA-MPS nanoparticles. The Fe3O4@PDA-MPS nanoparticles are then dispersed in acetonitrile, continuously stirred, and sonicated. Under argon protection, MAAc, a crosslinking agent, and an initiator are added, and the mixture is heated to reflux to obtain Fe3O4@PDA@PMAAc nanoparticles. Step S3: Add the Fe3O4@PDA@PMAAc nanoparticles to CaCl2 solution and stir, then add Na2HPO4 solution and continue stirring to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles, which is a cluster of microrobots that can simultaneously target and deliver multiple drugs. 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 Fe3O4 magnetic nanoparticles to dopamine hydrochloride is 1:0.2-0.

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

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

4. The method for preparing a microrobot cluster capable of simultaneously targeting and delivering multiple drugs as described in claim 2 or 3, characterized in that: Includes the following steps: Step S1: Prepare Fe3O4 magnetic nanoparticles; Step S2: A PDA layer is coated onto the surface of Fe3O4 magnetic nanoparticles through in-situ polymerization to obtain Fe3O4@PDA nanoparticles. The Fe3O4@PDA nanoparticles are then washed and dispersed in a mixed solution of ethanol, deionized water, and ammonia. MPS is added to react and produce Fe3O4@PDA-MPS nanoparticles. These Fe3O4@PDA-MPS nanoparticles are then dispersed in acetonitrile, continuously stirred, and sonicated. Under argon protection, MAAc, a crosslinking agent, and an initiator are added, and the mixture is heated to reflux to obtain Fe3O4@PDA@PMAAc nanoparticles. Step S3: The Fe3O4@PDA@PMAAc nanoparticles are added to a CaCl2 solution and stirred, then a Na2HPO4 solution is added and stirring is continued to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles, which are microrobot clusters capable of simultaneously targeting and delivering multiple drugs. In step S2, the Fe3O4@PDA nanoparticles are prepared by the following steps: Fe3O4 magnetic nanoparticles are dispersed in an alkaline buffer solution, stirred and sonicated, then dopamine hydrochloride is added and the reaction is continued in an ice-water bath to obtain Fe3O4@PDA nanoparticles; the mass ratio of Fe3O4 magnetic nanoparticles to dopamine hydrochloride is 1:0.2-0.

6.

5. The method for preparing a microrobot cluster capable of simultaneously targeting and delivering multiple drugs according to claim 4, characterized in that: Step S1 includes: adding sodium acetate and ethylene glycol to ferric chloride hexahydrate and stirring until homogeneous; then adding polyethylene glycol and sonicating the solution; finally placing the resulting solution into a reaction vessel and reacting at 100-300℃ for 8-16 h; after the reaction is completed, cooling to room temperature and washing to obtain Fe3O4 magnetic nanoparticles; the mass ratio of ferric chloride hexahydrate to sodium acetate is 1:2-3.

6. The method for preparing a microrobot swarm capable of simultaneously targeting and delivering multiple drugs according to claim 5, characterized in that: In step S2, the amount of MAAc used is 0.05-0.2 mL per milligram of Fe3O4@PDA-MPS nanoparticles; the mass ratio of Fe3O4@PDA-MPS nanoparticles to MBA is 1:0.005-0.01; the initiator is AIBN, and the mass ratio of Fe3O4@PDA-MPS nanoparticles to AIBN is 1:0.001-0.015; In step S3, the amount of CaCl2 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.002-0.006 mmol; the amount of Na2HPO4 used per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.004-0.01 mmol.

7. The method for preparing a microrobot swarm capable of simultaneously targeting and delivering multiple drugs according to claim 4, characterized in that: Step S3 includes: adding multiple drugs to a 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 stirring, and washing to obtain Fe3O4@PDA@PMAAc@CaP nanoparticles, thus forming a microrobot cluster capable of simultaneously targeting and delivering multiple drugs; the multiple drugs include doxorubicin, glucose oxidase, and MnCl2.

8. The method for preparing a microrobot cluster capable of simultaneously targeting and delivering multiple drugs according to claim 7, characterized in that: The dosage of doxorubicin per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg, the dosage of glucose oxidase per milligram of Fe3O4@PDA@PMAAc nanoparticles is 0.1-0.2 mg, and the dosage of MnCl2 per milligram of Fe3O4@PDA@PMAAc nanoparticles is 1-2 mmol.

9. The use of the microrobot cluster capable of simultaneously targeting and delivering multiple drugs as described in any one of claims 1 to 3 in the preparation of targeted drug delivery.

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