A bioorthogonal Janus microsphere and its preparation method and application

Abstract

The invention discloses a bio-orthogonal Janus microsphere and a preparation method and application thereof, belonging to the field of biomedical technology, and the method comprises: step one, preparing a cyclooctene-modified adriamycin prodrug; step two, preparing a pregel solution A and a pregel solution B; the pregel solution A comprises a hydrogel precursor solution and a cyclooctene-modified adriamycin prodrug; the pregel solution B comprises a hydrogel precursor solution and tetrazine-modified indocyanine green; step three, respectively injecting the pregel solution A and the pregel solution B into two channels of a θ-type capillary, and generating Janus droplets under the action of a high-voltage electric field by microfluidic electrospraying technology, collecting and solidifying with a collecting liquid, and obtaining the bio-orthogonal Janus microsphere. The Janus microsphere prepared by the present invention has sufficient drug loading, reduces the adverse reactions of the drug, can be directly injected into the patient's tumor site, and continuously releases drugs as a drug library to continuously inhibit tumor growth and prevent tumor metastasis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and relates to a tumor post-operative drug, and in particular to a bioorthogonal Janus microsphere and a preparation method and application thereof. Background Art

[0002] Bioorthogonal chemistry is a booming technology that has revolutionized interdisciplinary research and opened up new avenues for understanding, exploring, and manipulating biological systems with high precision and selectivity. To date, bioorthogonal reactions have successfully achieved precise biomolecule labeling, antibody-drug conjugate synthesis, prodrug activation, and other applications in cancer therapy. However, bioorthogonal reactions remain a challenge in terms of selectivity for cancer cells due to nonspecific reactions leading to off-target effects and adverse reactions. In addition, the limited bioavailability of drugs, coupled with defects in drug delivery methods, leads to insufficient drug concentrations at the tumor site, thereby reducing the therapeutic effect. And some substrates involved in bioorthogonal reactions, including transition metals, may exhibit toxicity or immunogenicity in biological systems. At the same time, the complexity and challenges of designing and synthesizing bioorthogonal prodrugs and drug delivery systems have hindered their widespread clinical application. Therefore, it is of great significance to develop a delivery system that can effectively achieve drug loading and bioorthogonal activation, as well as convenient delivery.

[0003] Microfluidics, known for its high precision and controllability, provides a versatile method for processing fluids in microchannels to prepare simple or multi-component microcarriers. This technology is widely used in the biomedical field, including drug and cell delivery, cell culture, and tissue engineering. In recent years, microfluidics has attracted extensive research in the fields of oncology, tissue engineering, and other medical applications, providing an effective option for the manufacture of precisely controlled multifunctional drug carriers. However, the application of microfluidics to develop bioorthogonal drug-carrying and activation carriers is still in its infancy. How to integrate bioorthogonal prodrug molecules and activators into microfluidic carriers to achieve multiple functions and effectively achieve synergistic treatment of tumors remains a challenge. Summary of the invention

[0004] The purpose of the present invention is to provide a bioorthogonal Janus microsphere and its preparation method and application in view of the deficiencies in the prior art. The bioorthogonal prodrug and the photothermal activator are respectively loaded on both sides of the Janus microsphere by microfluidic technology, and the combination of photothermal therapy and bioorthogonal chemotherapy is gradually realized. The Janus microsphere can be directly implanted into the tumor resection site after surgery to inhibit tumor recurrence and metastasis. This kind of bioorthogonal Janus microsphere has the advantages of simple preparation method, good material biocompatibility, clear drug structure, low cost, and high drug loading rate. At the same time, it can also avoid drug burst release and conceal drug toxicity and side effects, etc., and can be applied to the field of local efficient drug delivery and combined treatment of tumors.

[0005] To achieve the above-mentioned purpose, the present invention provides a method for preparing bio-orthogonal Janus microspheres, which has the following characteristics: comprising the following steps: step 1, preparing cyclooctene-modified doxorubicin prodrug by ester exchange reaction between trans-cyclooctene p-nitrobenzyl ester and doxorubicin hydrochloride; step 2, preparing pregel solution A and pregel solution B; pregel solution A comprises a hydrogel precursor solution and cyclooctene-modified doxorubicin prodrug; pregel solution B comprises a hydrogel precursor solution and tetrazine-modified indocyanine green; the hydrogel precursor is a compound that can be solidified into a hydrogel, such as methacryloyl hyaluronic acid, sodium alginate and methacryloyl gelatin; step 3, building a microfluidic device including a θ-type capillary, injecting the pregel solution A and the pregel solution B into two channels of the θ-type capillary respectively, generating Janus droplets under the action of a high voltage electric field by microfluidic electrospraying technology, collecting and solidifying with a collecting liquid, and obtaining bio-orthogonal Janus microspheres.

[0006] Furthermore, the present invention provides a method for preparing bioorthogonal Janus microspheres, which may also have the following characteristics: wherein in step 2, the hydrogel precursor solutions of the pregel solution A and the pregel solution B are both mixed solutions containing sodium alginate and methacryloyl gelatin; in step 3, the collection solution is a solution containing Ca 2+ The solution is cured by ultraviolet light.

[0007] Furthermore, the present invention provides a method for preparing bioorthogonal Janus microspheres, which may also have the following characteristics: wherein, in step three, the collection liquid is a calcium chloride solution with a mass concentration of 2 wt%; and the ultraviolet light intensity is 5 W / cm2.

[0008] Furthermore, the present invention provides a method for preparing bioorthogonal Janus microspheres, which may also have the following characteristics: wherein, in step 3, the diameter of the θ-type capillary is 200 μm; the flow rate of the injection of the pregel solution A and the pregel solution B is 2 mL h -1 ; The voltage of the high voltage electric field is 4kV.

[0009] Furthermore, the present invention provides a method for preparing bioorthogonal Janus microspheres, which may also have the following characteristics: wherein, in step one, the method for preparing the cyclooctene-modified doxorubicin prodrug is specifically as follows: dissolving trans-cyclooctene p-nitrobenzyl ester in N,N-dimethylformamide; subsequently adding N,N-diisopropylethylamine and doxorubicin hydrochloride; stirring the mixed solution in the dark to react; and obtaining the cyclooctene-modified doxorubicin prodrug through post-treatment.

[0010] Furthermore, the present invention provides a method for preparing bioorthogonal Janus microspheres, which may also have the following characteristics: wherein the amount ratio of trans-cyclooctene p-nitrobenzyl ester, N,N-dimethylformamide, N,N-diisopropylethylamine and doxorubicin hydrochloride is 0.01-0.05mmol:1mL:0.1-0.5mmol:0.01-0.05mmol, preferably 0.025mmol:1mL:0.25mmol:0.03mmol; the stirring reaction temperature is 25-35°C, and the stirring reaction time is 2-4 days.

[0011] Furthermore, the present invention provides a method for preparing bioorthogonal Janus microspheres, which may also have the following characteristics: wherein, in the method for preparing the cyclooctene-modified doxorubicin prodrug, post-treatment includes: adding pure water to the liquid after the reaction to obtain a mixed solution; extracting the mixed solution with ethyl acetate for multiple times, and combining the obtained organic phases; washing the obtained organic phase with saturated NaHCO3, distilled water and saturated NaCl solution for multiple times in sequence; then drying the organic phase with anhydrous Na2SO4; evaporating the solvent from the dried organic phase under reduced pressure to obtain a red crude product; purifying the crude product by column chromatography, the solvents being dichloromethane and methanol, and finally obtaining the cyclooctene-modified doxorubicin prodrug.

[0012] Furthermore, the present invention provides a method for preparing bioorthogonal Janus microspheres, which may also have the following characteristics: wherein, in step 2, in the pregel solution A, the mass ratio of sodium alginate, methacryloyl gelatin and cyclooctene-modified doxorubicin prodrug is 1-3:4-6:0.05-0.5, preferably 2:5:0.1; in the pregel solution B, the mass ratio of sodium alginate, methacryloyl gelatin and tetrazine-modified indocyanine green is 0.5-2:4-6:0.05-0.5, preferably 1:5:0.1.

[0013] The present invention also provides bioorthogonal Janus microspheres prepared by the preparation method.

[0014] The present invention also provides the use of the bioorthogonal Janus microspheres in preparing drugs for treating tumors after surgery.

[0015] The beneficial effects of the present invention are: the present invention provides a bio-orthogonal Janus microsphere and its preparation method and application. The Janus microsphere drug carrier prepared by the present invention has sufficient drug loading capacity, reduces the adverse reactions of the drug, can be directly injected into the patient's tumor site, and continuously releases drugs as a drug library to continuously inhibit tumor growth and prevent tumor metastasis. The preparation method of the present invention is simple, and the prepared drug carrier material has the characteristics of good biocompatibility, clear structure, low cost, high drug loading rate, etc. Specifically:

[0016] 1. The doxorubicin prodrug used in the present invention is used as both a prodrug molecule and a bioorthogonal reaction reagent for bioorthogonal chemotherapy activation. The tetrazine-modified indocyanine green (Tz-ICG) used in the present invention is used as both a photothermal reagent and a bioorthogonal reaction reagent for doxorubicin prodrug activation. Compared with the prior art, the present invention simplifies the production process, increases the functionality of the drug, and reduces the toxicity of the drug and the material.

[0017] Second, the method of the present invention has low production cost, the prepared material has good biocompatibility, and the method is simple and easy to operate, does not require very high technical requirements, and is easy to regulate the hydrogel and microfluidic chip.

[0018] 3. The bioorthogonal Janus microspheres prepared by the present invention have a high drug loading rate and can also avoid sudden drug release and conceal drug toxicity and side effects.

[0019] 4. The bioorthogonal Janus microspheres prepared by the present invention are implantable drug carriers that can be directly implanted into the site of tumor resection after surgery. They can be used for local drug delivery to the tumor, kill residual tumor cells through photothermal therapy combined with bioorthogonal sustained-release chemotherapy, inhibit tumor recurrence and metastasis, achieve long-term drug accumulation in lesions and sustained drug release, can be administered autonomously, and have strong clinical practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the synthesis of cyclooctene-modified doxorubicin prodrug;

[0021] Figure 2 The nuclear magnetic resonance spectroscopy characterization of the cyclooctene-modified doxorubicin prodrug prepared in step 1 of Example 1;

[0022] Figure 3 is a diagram of the microfluidic device constructed in step three of the present invention;

[0023] Figure 4 The optical microscope, particle size distribution and scanning electron microscope images of the Janus microspheres prepared in Example 1 and Comparative Examples 1 to 3 are shown;

[0024] Figure 5 is the photothermal temperature rise curve of the Janus microspheres prepared in Example 1 and Comparative Examples 1 to 3;

[0025] Figure 6 It is the photothermal imaging image of Janus microspheres prepared in Example 1 and Comparative Examples 1 to 3;

[0026] Figure 7 is the live-dead staining picture of tumor cells in different treatment groups;

[0027] Figure 8Schematic diagram of the bioorthogonal reaction between cyclooctene-modified doxorubicin prodrug and tetrazine-modified indocyanine green;

[0028] Fig. 9 This is a diagram showing the effects of different treatment groups in inhibiting tumor recurrence and metastasis after surgery. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments.

[0030] Example 1

[0031] This embodiment provides a bioorthogonal Janus microsphere, and the preparation method thereof comprises the following steps:

[0032] Step 1: Preparation of cyclooctene-modified doxorubicin prodrug (TCO-DOX), the reaction process is as follows: Figure 1 As shown, the specific method includes the following steps:

[0033] (1) The compound trans-cyclooctene p-nitrobenzyl ester ((2E)-TCO-PNB, 15 mg, 0.05 mmol) was dissolved in 2 mL of N,N-dimethylformamide (DMF). DIPEA (64.5 mg, 0.50 mmol) and doxorubicin hydrochloride (35 mg, 0.06 mmol) were added to the solution. The mixed solution was stirred at 30° C. in the dark for 3 days. Subsequently, 10 mL of water was added to the solution to obtain a mixed solution, and the mixed solution was extracted 4 times with ethyl acetate (50 mL), and the obtained organic phases were finally combined.

[0034] (2) The organic phase obtained in step (1) was washed three times with saturated NaHCO3 (50 mL), distilled water (50 mL) and saturated NaCl solution (50 mL) respectively, and then the organic phase was dried over anhydrous Na2SO4 for 2 hours.

[0035] (3) The dried organic phase was evaporated under reduced pressure to obtain a red crude product, which was purified by column chromatography using dichloromethane (CH2Cl2) and methanol (MeOH) as solvent (the volume ratio of CH2Cl2 to MeOH was 98:2) to finally obtain TCO-DOX (20 mg, yield 57.5%).

[0036] TCO-DOX was characterized by nuclear magnetic resonance spectroscopy. Figure 2 As shown, H NMR spectrum 1 HNMR (400 MHz, CDCl3, ppm) results confirmed the successful synthesis of TCO-DOX.

[0037] Step 2: prepare pregel solution A and pregel solution B.

[0038] Pregel solution A includes sodium alginate (SA, 2.0 wt%), methacryloyl gelatin (GelMA, 5.0 wt%) and TCO-DOX (0.1 wt%). Pregel solution B includes sodium alginate (SA, 1.0 wt%), methacryloyl gelatin (GelMA, 5.0 wt%) and tetrazine-modified indocyanine green (Tz-ICG, 0.1 wt%).

[0039] Step 3: Prepare bioorthogonal Janus microspheres loaded with dual drugs.

[0040] Build a microfluidic device, such as Figure 3 As shown, the device includes two injection needles, two circular capillaries, a θ-type capillary and a glass slide. The two circular capillaries and the θ-type capillary are fixed on the glass slide. The two circular capillaries are respectively connected to the two channels of the θ-type capillary, and the two injection needles are respectively connected to the two circular capillaries.

[0041] Pregel solution A and pregel solution B were respectively pumped into the solution at a rate of 0.2 mL h -1 The flow rate was injected into each channel of the θ-type capillary, and then Janus droplets were generated under the action of high voltage electric field (4kV) and gravity by microfluidic electrospray technology. The droplets were collected with calcium chloride solution (CaCl2, 2.0wt%) and solidified into Janus microspheres, and the microspheres were further solidified with ultraviolet light. The microspheres obtained in this way are bioorthogonal Janus microspheres loaded with dual drugs, denoted as JM ICG / DOX .

[0042] Of course, the sodium alginate and methacrylated gelatin in the pregel solution A and the pregel solution B of this embodiment can also be other hydrogel precursors that can be cured into hydrogels, such as methacrylated hyaluronic acid, or sodium alginate, methacrylated gelatin, etc., and the curing method is changed accordingly. For example, if the hydrogel precursor in the pregel solution A and the pregel solution B is only methacrylated gelatin, the collected liquid is oil.

[0043] Comparative Example 1

[0044] This comparative example provides a Janus microsphere loaded with Tz-ICG, and the preparation method thereof comprises the following steps:

[0045] Step 1: prepare pregel solution B and pregel solution C.

[0046] Pregel solution B includes sodium alginate (SA, 1.0 wt%), methacryloyl gelatin (GelMA, 5.0 wt%) and tetrazine-modified indocyanine green (Tz-ICG, 0.1 wt%). Pregel solution C includes sodium alginate (SA, 2.0 wt%) and methacryloyl gelatin (GelMA, 5.0 wt%).

[0047] Step 2: Prepare Janus microspheres loaded with Tz-ICG.

[0048] The same microfluidic device as in Example 1 was constructed.

[0049] Pregel solution B and pregel solution C were respectively pumped into the flask at 0.2 mL h -1 The flow rate was injected into each channel of the θ-type capillary, and then Janus droplets were generated under the action of high voltage electric field (4kV) and gravity by microfluidic electrospray technology. The droplets were collected with calcium chloride solution (CaCl2, 2.0wt%) and solidified into Janus microspheres, and the microspheres were further cured with ultraviolet light. The microspheres obtained in this way are Janus microspheres loaded with Tz-ICG, denoted as JM ICG .

[0050] Comparative Example 2

[0051] This comparative example provides a Janus microsphere loaded with TCO-DOX, and the preparation method thereof comprises the following steps:

[0052] Step 1: Prepare cyclooctene-modified doxorubicin prodrug (TCO-DOX) in the same manner as in Example 1.

[0053] Step 2: prepare pregel solution A and pregel solution D.

[0054] Pregel solution A includes sodium alginate (SA, 2.0 wt%), methacryloyl gelatin (GelMA, 5.0 wt%) and TCO-DOX (0.1 wt%). Pregel solution D includes sodium alginate (SA, 1.0 wt%) and methacryloyl gelatin (GelMA, 5.0 wt%).

[0055] Step 3: Prepare Janus microspheres loaded with TCO-DOX.

[0056] The same microfluidic device as in Example 1 was constructed.

[0057] Pregel solution A and pregel solution D were respectively pumped into the flask at 0.2 mL h -1The flow rate was injected into each channel of the θ-type capillary, and then Janus droplets were generated under the action of high voltage electric field (4kV) and gravity by microfluidic electrospray technology. The droplets were collected with calcium chloride solution (CaCl2, 2.0wt%) and solidified into Janus microspheres, and the microspheres were further cured with ultraviolet light. The microspheres obtained in this way are Janus microspheres loaded with Tz-ICG, denoted as JM DOX .

[0058] Comparative Example 3

[0059] This comparative example provides a drug-free Janus microsphere, and the preparation method thereof comprises the following steps:

[0060] Step 1: prepare pregel solution C and pregel solution D.

[0061] Pregel solution C includes sodium alginate (SA, 2.0 wt%) and methacrylated gelatin (GelMA, 5.0 wt%). Pregel solution D includes sodium alginate (SA, 1.0 wt%) and methacrylated gelatin (GelMA, 5.0 wt%).

[0062] Step 2: Prepare drug-free Janus microspheres.

[0063] The same microfluidic device as in Example 1 was constructed.

[0064] Pregel solution C and pregel solution D were respectively pumped into the flask at 0.2 mL h -1 The flow rate was injected into each channel of the θ-type capillary, and then Janus droplets were generated under the action of high voltage electric field (4kV) and gravity by microfluidic electrospray technology. The droplets were collected with calcium chloride solution (CaCl2, 2.0wt%) and solidified into Janus microspheres, and the microspheres were further cured with ultraviolet light. The microspheres obtained in this way are Janus microspheres loaded with Tz-ICG, denoted as JM B .

[0065] Figure 4 The optical microscopy images, corresponding particle size distribution and scanning electron microscopy images of four types of Janus microspheres are shown in Figure 1. Figure 4 It can be seen that the morphology of various Janus microspheres is regular and the particle size distribution is uniform, which proves the successful preparation of various Janus microspheres.

[0066] Photothermal effect of bioorthogonal Janus microspheres: 808 nm laser irradiation of JM B , JM DOX , JM ICG , JM ICG / DOX The particles were kept for 300s and the temperature changes were recorded using an infrared thermal imager (FLIR E5-XT). Figure 5 As shown in a, the photothermal curve shows that with the extension of 808nm laser irradiation time, the JM loaded with Tz-ICG ICG and JM ICG / DOX The temperature of JM increased significantly. After 5 minutes of near-infrared light irradiation, ICG The temperature rose by 53.6℃, ​​JM ICG / DOX The temperature rose by 54.7°C. In contrast, under the same conditions, the microparticles without Tz-ICG loading (JM B and JM DOX ) can be neglected. In addition, the effects of different power intensities (0, 0.3, 0.6 and 1.0 W cm -2 ) 808nm laser irradiation JM ICG / DOX , the results are as follows Figure 5 As shown in (b), with the increase of laser power density, JM ICG / DOX The photothermal temperature change is significantly increased. In addition, the thermal imaging image ( Figure 6 ) can directly reveal the above-mentioned light-induced JM ICG and JM ICG / DOX Photothermal heating process. In order to detect JM ICG / DOX Photothermal stability, using near-infrared light (1.0Wcm -2 ) for 100 s (laser on), followed by natural cooling for 420 s (laser off). This on / off cycle was repeated 5 times. Figure 5 As shown in c, after five repeated near-infrared “on-off” cycles, JM ICG / DOX The temperature increase ability of JM ICG / DOX photothermal stability.

[0067] The bioorthogonal Janus microspheres of the present invention can be used for postoperative treatment of tumors. The verification experiment is as follows:

[0068] In vitro tumor cell killing assay: 5×10 4 4T1 cells were seeded into each well of a 24-well plate and cultured for 12 h to allow the cells to adhere. Then various Janus microspheres were added and cultured for 4 h. DOX and JM ICG / DOX The TCO-DOX content in the treatment group was 30 μg. ICG and JM ICG / DOX The treated group was irradiated with near-infrared laser for 5 minutes, then incubated for 4 hours, and then the microspheres were removed and incubated for another 20 hours. The remaining groups were incubated in the dark for 24 hours. Subsequently, the live / dead staining method was used to study the cytotoxicity of Janus microspheres on cancer cells. Calcein and PI were added to each well for staining and observed under a fluorescence microscope. The results are shown in Figure 7 As shown, JM B and JMICG No effect on cell proliferation in the absence of light, JM DOX It has a slight inhibitory effect on cancer cells. ICG / DOX Treatment showed stronger cytotoxicity to cancer cells, which was due to the ICG / DOX The slowly released TCO-DOX and Tz-ICG undergo a bioorthogonal reaction to activate the efficacy of DOX. Figure 8 In addition, JM ICG It showed stronger cell killing performance under light. ICG / DOX The treatment group achieved the best killing effect on 4T1 cells by simultaneously performing dual orthogonal drug activation and photothermal therapy under light.

[0069] In vivo postoperative tumor treatment: 4T1 cells (4×10 6 When the tumor grows to about 300 mm 3 , 90% of the tumor was removed surgically, and the remaining tumor volume was about 30mm 3 The postoperative residual tumor model was established. Bioorthogonal Janus microspheres (TCO-DOX, 200 μg / mouse) were implanted at the tumor resection site. The mice were randomly divided into seven groups (n=6): saline group, DOX·HCl administration group, JM DOX (No light), JM ICG (No light), JM ICG / DOX (No light), JM ICG (Light) and JM ICG / DOX (light exposure). After 2 weeks, the mice were killed, and the tumors and lungs were removed. The tumors were fixed in 4% (v / v) paraformaldehyde solution. The lung tissues were placed in Bouin's to observe the tumor metastasis nodules. Each tissue was cut into 5 μm thick sections for hematoxylin (TUNEL) and eosin (H&E) staining. The experimental results are shown in Fig. 9 As shown, a is the postoperative tumor photo, b is the tumor size of each group, c is the tumor weight of each group, d is the H&E staining of tumor tissue of each group, e is the TUNEL staining of tumor tissue of each group, f is the lung photo of each group, and g is the H&E staining of lung of each group. It can be seen that compared with other experimental groups, JM ICG / DOX Significantly inhibited tumor growth and lung metastasis.

[0070] The present invention uses microfluidic technology to load bioorthogonal prodrugs and photothermal activators on both sides of Janus microspheres, and gradually realizes the combination of photothermal therapy and bioorthogonal chemotherapy. The Janus microspheres can be directly implanted into the patient's tumor resection site after surgery, and used as a drug reservoir for sustained release of drugs, thereby inhibiting the growth of residual tumors after surgery and distal metastasis. Such bioorthogonal Janus microspheres have the advantages of simple preparation, good material biocompatibility, low cost, high drug loading rate, and concealed drug toxicity and side effects, and are expected to be promoted and applied in tumor treatment.

[0071] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operating procedures used herein are reagents, materials and conventional procedures widely used in the corresponding fields.

[0072] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of bioorthogonal Janus microspheres in the preparation of postoperative therapeutic drugs for breast cancer, characterized in that: The method for preparing bioorthogonal Janus microspheres comprises the following steps: Step 1: preparing a cyclooctene-modified doxorubicin prodrug by trans-cyclooctene p-nitrobenzyl ester and doxorubicin hydrochloride through an ester exchange reaction; Step 2, preparing pregel solution A and pregel solution B; The pre-gel solution A includes a hydrogel precursor solution and a cyclooctene-modified doxorubicin prodrug; Pre-gel solution B includes hydrogel precursor solution and tetrazine-modified indocyanine green; Step 3: construct a microfluidic device including a θ-type capillary, inject the pregel solution A and the pregel solution B into two channels of the θ-type capillary respectively, generate Janus droplets under the action of a high voltage electric field by microfluidic electrospraying technology, collect and solidify with a collection liquid, and obtain bioorthogonal Janus microspheres; The hydrogel precursor solutions of the pregel solution A and the pregel solution B are both mixed solutions containing sodium alginate and methacrylated gelatin; the collected solution is a solution containing Ca 2+ The solution is cured by ultraviolet light.

2. The use according to claim 1, characterized in that: in, In step 3, the collected solution is a calcium chloride solution with a mass concentration of 2 wt%; the ultraviolet light intensity is 5 W / cm 2 .

3. The use according to claim 1, characterized in that: in, In step 3, the diameter of the θ-shaped capillary is 200 μm; the flow rate of the pregel solution A and the pregel solution B is 2 mL h −1 ; The voltage of the high voltage electric field is 4 kV.

4. The use according to claim 1, characterized in that: in, In step 1, the preparation method of the cyclooctene-modified doxorubicin prodrug is specifically as follows: dissolving trans-cyclooctene p-nitrobenzyl ester in N,N-dimethylformamide; subsequently adding N,N-diisopropylethylamine and doxorubicin hydrochloride; stirring the mixed solution in the dark to react; and obtaining the cyclooctene-modified doxorubicin prodrug through post-treatment.

5. The use according to claim 4, characterized in that: in, The amount ratio of the trans-cyclooctene p-nitrobenzyl ester, N,N-dimethylformamide, N,N-diisopropylethylamine and doxorubicin hydrochloride is 0.01-0.05 mmol: 1 mL: 0.1-0.5 mmol: 0.01-0.05 mmol; The stirring reaction temperature is 25~35°C, and the stirring reaction time is 2~4 days.

6. The use according to claim 4, Features: Wherein, in the preparation method of the cyclooctene-modified doxorubicin prodrug, the post-treatment comprises: Adding pure water to the liquid after the reaction to obtain a mixed solution; extracting the mixed solution with ethyl acetate for multiple times, and combining the obtained organic phases; The obtained organic phase was washed with saturated NaHCO3, distilled water and saturated NaCl solution for multiple times; the organic phase was then dried over anhydrous Na2SO4; The dried organic phase is evaporated under reduced pressure to obtain a red crude product; the crude product is purified by column chromatography using dichloromethane and methanol as solvents to finally obtain a cyclooctene-modified doxorubicin prodrug.

7. The use according to claim 1, characterized in that: in, In step 2, in the pre-gel solution A, the mass ratio of sodium alginate, methacryloyl gelatin and cyclooctene-modified doxorubicin prodrug is 1-3:4-6:0.05-0.5; In the pre-gel solution B, the mass ratio of sodium alginate, methacryloyl gelatin and tetrazine-modified indocyanine green is 0.5-2:4-6:0.05-0.5.

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