A targeted microwave-responsive nanobody and its preparation method and application

Abstract

The present invention discloses a targeted microwave-responsive nanobody and a preparation method and application thereof. The targeted microwave-responsive nanobody is based on a metal organic framework, on which paclitaxel and ionic liquid are adsorbed, and on which the surface of the base framework is modified with polydopamine, on which fucoidan is connected. The metal organic framework is used, the advantages of inorganic materials and organic compounds are combined, and the surface is modified with fucoidan to target P-selectin to achieve multiple targeting. The inner core is loaded with ionic liquid as a sensitizing material to enhance the microwave response capability, thereby providing a unique solution for local microwave treatment combined with chemotherapy for prostate cancer, and therefore has good potential.

Description

Technical Field

[0001] The present invention relates to the technical field of disease treatment, and in particular to a targeted microwave-responsive nanobody for treating prostate cancer, and a preparation method and application thereof. Background Art

[0002] Although paclitaxel chemotherapy can benefit prostate cancer patients in terms of prolonging survival and improving quality of life, there are obvious adverse drug reactions and drug resistance, which ultimately lead to treatment failure and poor prognosis.

[0003] With the rapid development of nanomedicine, nano-encapsulation and functional modification of special groups and small molecule drugs can significantly improve drug stability, prolong circulation time, and enhance targeted delivery capabilities. Combined with exogenous physical stimulation (such as near-infrared light and ultrasound) in the target area, precise controlled release can be achieved, which can expand the therapeutic effect, improve prognosis, and provide a new treatment approach for malignant tumors. However, existing treatment methods have the defects of insufficient targeting and weak response to exogenous stimulation. Therefore, finding functionally modified nanomaterials, precise and efficient target sites, and excellent external stimulation pathways is an important research direction for exploring nanomedicine treatment of prostate cancer.

[0004] Metal-organic framework (MOF) materials are composed of metal ions and organic ligand bridges. Due to the advantages of both inorganic complexes and organic coordination compounds, they can achieve high stability, ideal drug loading, strong modification ability and biosafety in one functional integration, and are expected to become a new generation of ideal delivery carriers. Secondly, organisms express P-selectin through activated endothelial cells and platelets, mediating the adhesion of tumor cells to vascular endothelium and the binding of platelets to tumor cells, and can be closely involved in the entire process of tumor occurrence and development. Therefore, P-selectin can be used as an excellent endogenous target to achieve simultaneous targeting of primary tumor cell lesions and their distant metastases. In addition, ultrasound-mediated microwave thermal therapy (MTT) and microwave dynamic therapy (MDT) are emerging highly selective treatment methods that use the highly enriched characteristics of microwave-sensitized nanomaterials under the guidance of ultrasound imaging to achieve target area thermal therapy and further produce reactive oxygen species to promote tumor necrosis and apoptosis. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the existing treatment methods for prostate cancer have the problems of insufficient targeting and weak response to exogenous stimulation.

[0006] The first object of the present invention is to provide a targeted microwave-responsive nanobody, which uses a metal organic framework as a basic framework, on which paclitaxel and an ionic liquid are adsorbed, and on which the surface of the basic framework is modified with polydopamine, on which fucoidan is connected.

[0007] Using metal-organic frameworks, combining the advantages of inorganic materials and organic compounds, the surface is modified with fucoidan to target P-selectin and achieve multiple tumor targeting. The inner core is loaded with ionic liquid as a sensitizing material to enhance microwave responsiveness, thus providing a unique solution for paclitaxel chemotherapy of prostate cancer, and therefore has good potential for the treatment of prostate cancer.

[0008] As a possible design, the metal organic framework uses zirconium and / or manganese as inorganic elements.

[0009] As a possible design, the ionic liquid is 1-butyl-3-methylimidazolium-L-lactate.

[0010] The second object of the present invention is to provide a method for preparing a targeted microwave-responsive nanobody, comprising:

[0011] preparing an organic metal framework and preparing a solution;

[0012] The solution, the ionic liquid and paclitaxel are mixed, reacted at -5 to 1°C and then dried to obtain a powder;

[0013] The powder and dopamine hydrochloride solution are mixed and incubated, and then centrifuged to obtain a precipitate, carbodiimide, N-hydroxysuccinimide and fucoidan are added to the precipitate to react in a buffer solution for 25 to 30 minutes, and the precipitate is centrifuged to obtain the targeted microwave-responsive nanobody.

[0014] The preparation method is simple and successfully synthesizes targeted microwave-responsive nanobodies, the required raw materials are easily available, and no special equipment is required. The targeted microwave controlled-release drug-loaded nanoprobes prepared by the method have uniform size, regular morphology, controllable particle size, and high stability.

[0015] As a possible design, preparing the organic metal framework and making it into a solution specifically includes:

[0016] Dissolve zirconium chloride, terephthalic acid and polyvinyl pyrrolidone in a solvent in a mass ratio of 6:4:3 to form a mixed solution;

[0017] Add manganese chloride tetrahydrate to the mixed solution and react at 110-120° C. and 1-2 MPa for 9-11 hours;

[0018] After the reaction is completed, centrifugation and washing are performed in sequence, and vacuum drying is performed at 55-65°C.

[0019] As a possible design, the mass ratio of the ionic liquid to paclitaxel is 0.5:2.

[0020] As a possible design, the mass ratio of carbodiimide, N-hydroxysuccinimide and fucoidan is 1:1:1.

[0021] As a possible design, the solution, ionic liquid and paclitaxel are mixed and then subjected to ultrasonic reaction in an ice water bath with an ultrasonic intensity of 40%.

[0022] As a possible design, after the reaction is completed, centrifugation and washing are carried out in sequence, and drying is performed at 55-65° C. The reagent used for washing is dimethylformamide and / or anhydrous ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0024] Figure 1 Schematic diagram of the structure of the targeted microwave-responsive nanobody in an embodiment of the present invention;

[0025] Figure 2 It is a transmission electron microscopy image of the targeted microwave-responsive nanobody in an embodiment of the present invention;

[0026] Figure 3 This is a particle size distribution diagram of the targeted microwave-responsive nanobody in an embodiment of the present invention;

[0027] Figure 4 This is a graph showing the size stability results of targeted microwave-responsive nanobodies in an embodiment of the present invention;

[0028] Figure 5 This is a graph showing the size stability results of targeted microwave-responsive nanobodies in an embodiment of the present invention;

[0029] Figure 6 This is a graph showing the size stability results of targeted microwave-responsive nanobodies in an embodiment of the present invention;

[0030] Figure 7 This is a graph showing the cytotoxicity results of targeted microwave-responsive nanobodies in an embodiment of the present invention;

[0031] Figure 8 This is a microwave heating diagram of the targeted microwave-responsive nanobody in an embodiment of the present invention;

[0032] Fig. 9This is a result diagram of the targeted microwave-responsive nanobody producing active oxygen in an embodiment of the present invention;

[0033] Fig.10 This is a graph showing the in vitro drug release curve of the targeted microwave-responsive nanobody in an embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0035] Example 1

[0036] The preparation of targeted microwave-responsive nanobodies comprises the following steps:

[0037] S1. Preparation of organic metal framework and solution

[0038] 13.4 mg of zirconium chloride, terephthalic acid and polyvinyl pyrrolidone were dissolved in 13.2 mL of dimethylformamide in a certain mass ratio (6:4:3) to form solution A. The obtained solution A was then ultrasonically treated and mixed, and 5.2 mg of manganese chloride tetrahydrate was added to form solution B, which was transferred to an autoclave (pressure of 1.5 MPa), placed in a preheated oven at 120°C for 10 h, and cooled to room temperature with water. The obtained reactants were collected, washed twice with dimethylformamide, washed twice with anhydrous ethanol, and vacuum dried at 60°C to obtain white powder C, which was dissolved in anhydrous ethanol (at a concentration of 10 mg / mL) to obtain solution D.

[0039] S2. Add 1 mL of 1-butyl-3-methylimidazole-L-lactate (at a concentration of 0.5 mg / mL) and 2 mg of paclitaxel (at a concentration of 1 mg / mL) to solution D, ultrasonically treat for 10 minutes (40% intensity, total time 2-3 h) in an ice bath, and vacuum dry at 60°C for 3 h to obtain a brown powder E.

[0040] S3. Powder E and dopamine hydrochloride solution were rotated and incubated in phosphate buffer (pH 8.5) at room temperature for 3 hours to produce solution F; solution F was ultracentrifuged (1000g, 5min), rinsed with deionized water, the supernatant was discarded, the precipitate was taken, 1 mg of carbodiimide, 1 mg of N-hydroxysuccinimide and 1 mg of fucoidan were added and resuspended in phosphate buffer (pH 8.5), and magnetically stirred for 30 minutes to obtain solution G; solution G was ultracentrifuged (1000g, 5min), rinsed with deionized water to obtain targeted microwave-responsive nanobodies.

[0041] The following experiments were performed on the targeted microwave-responsive nanobody prepared in Example 1.

[0042] Characterization experiment: The successfully prepared targeted microwave-responsive nanobody was diluted 100 times, and the particle size and potential of the nanobody were measured using dynamic light scattering. Figure 1-3 As shown by Figure 2 It can be seen that the targeted microwave-responsive nanobody has a regular shape and is square. Figure 3 It can be seen that the particle size distribution of the targeted microwave-responsive nanobody is uniform. Figure 1 In: 1 represents fucoidan, 2 represents Zr 4+ ion, 3 represents Mn 2+ ion, 4 represents terephthalic acid, 5 represents antitumor drugs, 6 represents ionic liquid, and 7 represents polydopamine.

[0043] Stability test: The diluted nanobody suspension was placed at 37°C for 12 hours, and the particle size, dispersion coefficient and potential of the nanobody were measured at different time points of 4 hours, 8 hours and 12 hours to observe the stability of the material. Figure 4-6 As shown by Figure 4 It can be seen that the targeted microwave-responsive nanobody has no significant change in particle size when placed in deionized water at 37°C for 12 hours, indicating good stability. Figure 5 It can be seen that the targeted microwave-responsive nanobody has no significant change in dispersion coefficient when placed in deionized water at 37°C for 12 hours, indicating good stability. Figure 6 It can be seen that the targeted microwave-responsive nanobody has no obvious change in potential when placed in deionized water at 37°C for 12 hours, indicating good stability.

[0044] Safety experiment: Human vascular endothelial cells were used and plated in 96-well plates at 104 cells / well and cultured for 24 hours until the cells adhered to the wall. Different concentrations of targeted microwave-responsive nanobodies (1, 2, 3, 4, 5 mg / mL) were co-cultured with the cells for 12 hours, stained with a CCK-8 kit, and the absorbance (OD) at 450nm was detected by an enzyme reader. The cell viability was calculated according to the following formula: Cell survival rate (%) = [(ODtest-ODblank) / (ODcontrol-ODblank)] × 100%. The results are as follows Figure 7 As shown by Figure 7 It can be seen that at different concentrations of 1, 2, 3, 4, and 5 mg / mL, the targeted microwave-responsive nanobodies were co-cultured with vascular endothelial cells for 12 hours, and there was no significant loss of cell viability in each group, indicating good biosafety.

[0045] Microwave thermal effect experiment: 5 mg of targeted microwave-responsive nanobody was dispersed in 1 mL of deionized water and heated at 1.5 W / cm 2The microwave irradiation was carried out for 5 minutes, and deionized water was used as the control group, which was given microwave irradiation under the same conditions. The heating effect was monitored by infrared thermal imaging and the heating curves of the two groups were recorded. Figure 8 As shown, targeted microwave-responsive nanobodies have significant heating performance under microwave irradiation.

[0046] Microwave kinetic effect experiment: The active oxygen components produced in the targeted microwave-responsive nanobody were detected using the terephthalic acid oxidation assay. A mixture of 10 μL of nanobody with different concentrations (1, 2, 3, 4, 5 mg / mL) and 100 μL of H2O2 was dispersed in 200 μL of terephthalic acid solution (1 mg / mL) for 20 minutes. The experimental group was irradiated with microwaves for 5 minutes (1.5 W / cm, 2450 MHz). The fluorescence intensity of each group was then recorded by fluorescence spectrophotometry (ex / em=315 / 435 nm). The results are shown in the figure. Fig. 9 As shown, targeted microwave-responsive nanobodies have obvious kinetic effects under microwave irradiation.

[0047] Drug release experiment: 0.4 mL of targeted microwave-responsive nanobody suspension (5 mg / mL) was added to a sealed dialysis bag and dialyzed with weakly acidic PBS buffer (pH = 5.5) containing 10% fetal bovine serum at 37°C. The bag was placed on a shaker and shaken continuously to simulate physiological conditions. At 2, 4, 8, 12, 24, and 48 h, 0.5 mL of solution was taken from the dialysis bag, and the absorption peak at 227 nm was observed using an ultraviolet spectrophotometer to calculate the amount of paclitaxel drug released. The experimental groups were irradiated with microwaves for 5 min at 4, 8, and 24 h, respectively, and then the solution was taken for analysis. The results are shown in the figure. Fig.10 As shown by Fig.10 It can be seen that the targeted microwave-responsive nanobody has a more prominent drug release ability under a weak acid environment (pH 5.5) after three intermittent in vitro microwave irradiations (indicated by the arrows in the figure) compared with the group without microwave irradiation.

[0048] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A targeted microwave-responsive nanobody, characterized in that: A metal organic framework is used as the basic framework, paclitaxel and ionic liquid are adsorbed on the basic framework, the surface of the basic framework is modified with polydopamine, and fucoidan is connected to the polydopamine; The preparation method of the targeted microwave-responsive nanobody comprises: preparing an organic metal framework and preparing a solution; The solution, the ionic liquid and paclitaxel are mixed, reacted at -5 to 1°C and then dried to obtain a powder; The powder and dopamine hydrochloride solution are mixed and incubated, and then centrifuged to obtain a precipitate, carbodiimide, N-hydroxysuccinimide and fucoidan are added to the precipitate to react in a buffer solution for 25 to 30 minutes, and the precipitate is centrifuged to obtain the targeted microwave-responsive nanobody; The method of preparing an organic metal framework and preparing a solution specifically includes: Dissolve zirconium chloride, terephthalic acid and polyvinyl pyrrolidone in a solvent in a mass ratio of 6:4:3 to form a mixed solution; Add manganese chloride tetrahydrate to the mixed solution and react at 110-120° C. and 1-2 MPa for 9-11 hours; After the reaction is completed, centrifugation and washing are performed in sequence, and vacuum drying is performed at 55-65°C; After the reaction is completed, centrifugation and washing are performed in sequence, and drying is performed at 55-65° C. The reagent used for washing is dimethylformamide and / or anhydrous ethanol.

2. The targeted microwave-responsive nanobody according to claim 1, characterized in that: The metal organic framework has zirconium and / or manganese as inorganic elements.

3. The targeted microwave-responsive nanobody according to claim 1, characterized in that: The ionic liquid is 1-butyl-3-methylimidazole-L-lactate.

4. The targeted microwave-responsive nanobody according to claim 1, characterized in that: The mass ratio of the ionic liquid to paclitaxel is 0.5:

2.

5. The targeted microwave-responsive nanobody according to claim 1, characterized in that: The mass ratio of the carbodiimide, N-hydroxysuccinimide and fucoidan is 1:1:

1.

6. The targeted microwave-responsive nanobody according to claim 1, characterized in that: The solution, ionic liquid and paclitaxel are mixed and then subjected to ultrasonic reaction in an ice water bath with an ultrasonic intensity of 40%.

7. Use of the targeted microwave-responsive nanobody according to any one of claims 1 to 6 in a drug for treating prostate cancer.

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