A responsive nano-micelle for enhancing deep tumor penetration and its preparation method and application

Abstract

The present invention discloses a responsive nano-micelle for enhancing deep tumor penetration, a preparation method and an application thereof, and belongs to the field of biomedicine technology. The nano-micelle is obtained by co-assembling gambogic acid and nano-materials. The preparation method comprises the following steps: dissolving the nano-material and gambogic acid in dimethyl sulfoxide, preparing a mixed solution after ultrasonic oscillation, then dropping it into ultrapure water at a uniform speed while stirring to react, dialyzing and centrifuging after the reaction, collecting the supernatant, and obtaining the responsive nano-micelle. The present invention also discloses the application of the nano-micelle in the preparation of anti-tumor drugs. The raw materials in the present invention are widely available and the synthesis method is simple. In addition, the present invention can realize the release of drugs inside the tumor and ensure the safety of medication.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a responsive nano-micelle for enhancing deep tumor penetration, and a preparation method and application thereof. Background Art

[0002] The treatment of breast cancer, especially triple-negative breast cancer, usually relies on cytotoxic chemotherapy drugs. However, these drugs can cause toxic side effects, which limit the patient's quality of life and treatment effect. In order to overcome the toxic side effects, researchers have turned to the development of anti-tumor nanodrugs with polymer carriers, which use nano-scale carriers such as liposomes, polymer nanoparticles or nanoemulsions to encapsulate anticancer drugs. Nanodrugs can be delivered to tumor tissues more accurately through passive or active targeting strategies, reducing damage to healthy tissues. Although anti-tumor nanodrugs with polymer carriers can reduce drug toxic side effects, their efficacy has not been significantly improved compared to the original drugs, mainly because the low permeability in solid tumors limits the effectiveness of nanodrug therapy.

[0003] Studies have found that the positively charged surface of nanocarriers helps to enhance deep penetration into tumor tissues. This effect is partly due to the charge adsorption between tumor cells and the positively charged surface of nanocarriers, which induces transcytosis and relies on caveolin-mediated endocytosis and exocytosis mechanisms to achieve transcellular delivery. However, the existing technology still has a large lack of technology for maintaining the negative charge or neutral state of nanocarriers in the blood circulation, while being able to quickly take on a positive charge after entering the tumor tissue. Since nanomedicines are still affected by the normal pH interstitial environment after leaking out of tumor microvessels, traditional acid-responsive charge reversal strategies cannot achieve charge reversal, and it is difficult to effectively diffuse into acidic areas, resulting in their main accumulation around tumor blood vessels, and failing to effectively solve the problem of nanomedicine penetration in tumor tissues.

[0004] Therefore, how to provide an anti-tumor drug with good deep penetration ability and capable of releasing the drug inside cells is a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a responsive nano-micelle for enhancing deep tumor penetration and a preparation method and application thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A responsive nano-micelle for enhancing deep tumor penetration, wherein the nano-micelle is co-assembled from gambogic acid and nano-materials.

[0008] Preferably, the mass ratio of the nanomaterial to gambogic acid is 8:0.5.

[0009] Preferably, the gambogic acid is gambogic acid labeled with Cy5.5 fluorescent dye.

[0010] Preferably, the method for preparing the nanomaterial comprises the following steps:

[0011] (1) adding NH2-SS-NH2 and triethylamine to a DMF solution of Fmoc-PEG-NHS, removing the solvent after the reaction is complete, dissolving in dichloromethane, washing with water, drying, and then adding icy ether to precipitate, filtering and drying the obtained precipitate, and then dissolving it in toluene with lactide, glycolide and catalyst stannous octoate to react, and after the reaction is completed, concentrating under reduced pressure to precipitate, filtering and drying the obtained precipitate, and then dissolving it in dichloromethane, adding piperidine to react, and after the reaction is completed, washing, drying, and concentrating under reduced pressure to precipitate, and then filtering and drying to obtain NH2-PEG-SS-PLGA;

[0012] (2) Adding EDC and NHS to the dichloromethane solution of Boc-Glu-OtBu to dissolve it completely and then activate it, then adding the NH2-PEG-SS-PLGA and triethylamine to react, after the reaction is completed, a precipitate is precipitated, and then it is filtered and dried, and then dissolved in dichloromethane, and trifluoroacetic acid is added to react, after the reaction is completed, it is washed, dried, and concentrated under reduced pressure to precipitate, and then filtered and dried to obtain PLGA-SS-PEG-Glu, i.e., the nanomaterial.

[0013] More preferably, the method for preparing the nanomaterial specifically comprises the following steps:

[0014] (1) Weigh 2.0 g of Fmoc-PEG-NHS (fluorenylmethoxycarbonyl-polyethylene glycol-succinimide ester) and dissolve it in 10 mL of DMF, add NH2-SS-NH2 (amino-disulfide bond-amino) (10.0 eq.) and triethylamine (2.0 eq.) and react at room temperature for 6 h after complete dissolution, then remove the solvent by vacuum rotary evaporation, redissolve it in 10 mL of dichloromethane and wash with water to remove excess NH2-SS-NH2, then dry it over anhydrous sodium sulfate, then add it to a large amount of icy ether to precipitate, collect the precipitate by filtration, and obtain the Fmoc-NH-PEG-SS-NH2 product after vacuum drying;

[0015] (2) Weigh 1.0 g of Fmoc-PEG-SS-NH2, 0.64 g of lactide, 0.51 g of glycolide and catalyst stannous octoate and dissolve them in 10 mL of anhydrous toluene. React at 130° C. for 12 h. Concentrate the reaction solution under reduced pressure and add it to a large amount of icy ether to precipitate. Collect the precipitate by filtration and dry it in vacuum to obtain Fmoc-NH-PEG-SS-PLGA product.

[0016] (3) Weigh 1.0 g of Fmoc-NH2-PEG-SS-PLGA and dissolve it in 10 mL of dichloromethane. Add 3 mL of piperidine to dissolve it completely. React at room temperature for 2 h. Wash it twice with 10% citric acid and once with pure water. Dry it with anhydrous sodium sulfate. After concentrating under reduced pressure, add it to a large amount of icy ether to precipitate. Collect the precipitate by filtration and dry it in vacuum to obtain the NH2-PEG-SS-PLGA product.

[0017] (4) Weigh 0.1 g of Boc-Glu-OtBu (Boc-L-glutamic acid-1-tert-butyl ester) and dissolve it in 10 mL of dichloromethane. Add EDC (1.0 eq.) and NHS (2.0 eq.) to dissolve completely. After activation at room temperature for 1 h, add NH2-PEG-SS-PLGA (0.5 eq.) and triethylamine (2.0 eq.). React at room temperature for 4 h. Then, add it to a large amount of anhydrous ether to precipitate. Collect the precipitate and dry it in vacuum to obtain PLGA-SS-PEG-Glu-OtBu(-Boc) product.

[0018] (5) Weigh 1.0 g of PLGA-SS-PEG-Glu-OtBu(-Boc) and dissolve it in 10 mL of dichloromethane. Add 3 mL of trifluoroacetic acid to dissolve it completely. After reacting at room temperature for 0.5 h, wash it twice with a saturated NaHCO3 aqueous solution and twice with pure water, and then dry it with anhydrous sodium sulfate. After concentrating under reduced pressure, add it to a large amount of icy ether to precipitate. Collect the precipitate by filtration and dry it in vacuum to obtain the PLGA-SS-PEG-Glu product.

[0019] Preferably, the particle size of the nano micelles is 150 nm.

[0020] Beneficial effects: The present invention provides a nano drug based on GGT enzyme response, which exhibits good long-circulation characteristics in the blood circulation, but when it approaches the tumor blood vessels with high expression of GGT enzyme, it will undergo charge reversal. This reversal process promotes the transcytosis of the nano drug, helps to improve the deep penetration ability of the drug, and ultimately achieves the release of the drug in the cell, giving it excellent anti-tumor properties.

[0021] A method for preparing responsive nanomicelles for enhancing deep tumor penetration comprises the following steps:

[0022] The nanomaterial and gambogic acid are dissolved in dimethyl sulfoxide, and a mixed solution is prepared after ultrasonic oscillation, and then added dropwise to ultrapure water at a uniform speed while stirring to react. After the reaction is completed, the supernatant is collected by dialysis and centrifugation to obtain the responsive nanomicelles.

[0023] Preferably, the volume ratio of the mixed solution to ultrapure water is 1:4.

[0024] Preferably, the centrifugal speed is 3000 rpm and the time is 15 min.

[0025] Preferably, the dialysis is performed in ultrapure water for 24 hours, and the dialysate is replaced every 2-4 hours.

[0026] Application of responsive nanomicelles for enhancing deep tumor penetration in the preparation of anti-tumor drugs.

[0027] Preferably, the tumor is triple-negative breast cancer.

[0028] Compared with the prior art, the present invention has the following advantages and technical effects:

[0029] The raw materials in the present invention are widely available, and the polylactic acid-glycolic acid copolymer and polyethylene glycol used can be industrially produced, and the synthesis method is simple. In addition, the nanomaterial synthesized by the present invention is an amphiphilic block copolymer, which has amphiphilicity, biocompatibility and degradability. The micelles prepared using the nanomaterial have good stability, which is conducive to drug encapsulation and delivery. In addition, the nano micelles obtained by the present invention can respond to the GGT enzyme, so that the material is positively charged, which is conducive to the transcytosis of the material by the cell and enhances the deep penetration of the nano drug; at the same time, the hydrophilic end and the hydrophobic end are connected by a disulfide bond. Under the reducing conditions formed by the high concentration of glutathione in the tumor cells, the disulfide bond breaks, causing the hydrophilic shell polyethylene glycol segment to fall off, and the wrapped drug core is exposed, so that the drug is released inside the tumor, and the safety of the drug is ensured. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0031] Figure 1 The H NMR spectra of the two intermediate monomers required for preparing nanomicelles in Examples 2 and 3;

[0032] Among them, A is NH2-PEG-SS-PLGA, B is PLGA-SS-PEG-Glu;

[0033] Figure 2The Tyndall effect diagram of the responsive empty and drug-loaded nanomicelles prepared in Examples 2 and 3;

[0034] Among them, from left to right are ultrapure water, empty micelles and drug-loaded micelles;

[0035] Figure 3 Transmission electron microscopy images and particle size distribution diagrams of the responsive empty and drug-loaded nanomicelles prepared in Examples 2 and 3;

[0036] Wherein, A is the transmission electron microscopy image of Example 2, B is the transmission electron microscopy image of Example 3, C is the transmission electron microscopy particle size distribution image of Example 2, and D is the transmission electron microscopy particle size distribution image of Example 3;

[0037] Figure 4 The hydrated particle size diagram and Zeta potential diagram of the responsive empty and drug-loaded nanomicelles prepared in Examples 2 and 3;

[0038] Among them, A is the hydrated particle size diagram, and B is the Zeta potential diagram;

[0039] Figure 5 The penetration of the Cy5.5-labeled responsive drug-loaded micelles obtained in Example 4 into 4T1 triple-negative breast cancer tumors;

[0040] Among them, A is the permeation situation at different time points and the permeation situation diagram of the tumor profile, and B is the histogram of the permeation situation at different time points. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] The room temperature described in the embodiments of the present invention is 25±2°C.

[0044] The raw materials in the present invention are all purchased from commercial sources.

[0045] Among them, Fmoc-PEG-NHS was purchased from Xi'an Ruixi Biotechnology Co., Ltd.;

[0046] NH2-SS-NH2 was purchased from Xi’an Ruixi Biotechnology Co., Ltd.;

[0047] Boc-Glu-OtBu was purchased from MCE;

[0048] Cy5.5 fluorescent dye-labeled gambogic acid was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0049] Example 1

[0050] A method for preparing a nanomaterial comprises the following steps:

[0051] (1) Weigh 2.0 g of Fmoc-PEG-NHS and dissolve it in 10 mL of DMF. Add NH2-SS-NH2 (10.0 eq.) and triethylamine (2.0 eq.) and react at room temperature for 6 h after complete dissolution. Then remove the solvent by vacuum rotary evaporation. Redissolve it in 10 mL of dichloromethane and wash with water to remove excess NH2-SS-NH2. Then dry it over anhydrous sodium sulfate. Then add it to a large amount of icy ether to precipitate. Collect the precipitate by filtration and dry it in vacuum to obtain Fmoc-NH-PEG-SS-NH2 product.

[0052] (2) Weigh 1.0 g of Fmoc-PEG-SS-NH2, 0.64 g of lactide, 0.51 g of glycolide and catalyst stannous octoate and dissolve them in 10 mL of anhydrous toluene. React at 130° C. for 12 h. Concentrate the reaction solution under reduced pressure and add it to a large amount of icy ether to precipitate. Collect the precipitate by filtration and dry it in vacuum to obtain Fmoc-NH-PEG-SS-PLGA product.

[0053] (3) Weigh 1.0 g of Fmoc-NH2-PEG-SS-PLGA and dissolve it in 10 mL of dichloromethane. Add 3 mL of piperidine to dissolve it completely. React at room temperature for 2 h. Wash it twice with 10% citric acid and once with pure water. Dry it with anhydrous sodium sulfate. After concentrating under reduced pressure, add it to a large amount of icy ether to precipitate. Collect the precipitate by filtration and dry it in vacuum to obtain the NH2-PEG-SS-PLGA product.

[0054] (4) Weigh 0.1 g of Boc-Glu-OtBu and dissolve it in 10 mL of dichloromethane. Add EDC (1.0 eq.) and NHS (2.0 eq.) to dissolve it completely. After activation at room temperature for 1 h, add NH2-PEG-SS-PLGA (0.5 eq.) and triethylamine (2.0 eq.). React at room temperature for 4 h. Then add it to a large amount of anhydrous ether to precipitate. Collect the precipitate and dry it in vacuum to obtain PLGA-SS-PEG-Glu-OtBu(-Boc) product.

[0055] (5) Weigh 1.0 g of PLGA-SS-PEG-Glu-OtBu(-Boc) and dissolve it in 10 mL of dichloromethane. Add 3 mL of trifluoroacetic acid to dissolve it completely. After reacting at room temperature for 0.5 h, wash it twice with a saturated NaHCO3 aqueous solution and twice with pure water, and then dry it with anhydrous sodium sulfate. After vacuum concentration, add it to a large amount of icy ether to precipitate. Collect the precipitate by filtration and dry it in vacuum to obtain the PLGA-SS-PEG-Glu product, which is the dual-responsive nanomaterial.

[0056] Example 2

[0057] A responsive nanomicelle for enhancing deep tumor penetration is a responsive empty nanomicelle, and a preparation method thereof comprises the following steps:

[0058] 8 mg of the dual-responsive nanomaterial obtained in Example 1 was weighed and dissolved in 1 mL of DMSO, and a DMSO solution of the dual-responsive nanomaterial was obtained by ultrasonic treatment for 5 min. After the solution (1 mL) was collected using a 1 mL syringe, it was added dropwise to a round-bottom flask containing 4 mL of ultrapure water at a speed of 20 μL / min by a microinjection pump, and the round-bottom flask was placed on a magnetic stirrer and stirred at a speed of 800 rpm during the addition. After the injection is completed, the obtained mixed solution is placed in a dialysis bag and dialyzed in 1000 mL of ultrapure water for 24 h, and the dialysate is replaced once every 2-4 h. After the dialysis is completed, it is centrifuged at a low speed of 3000 rpm for 15 min to remove the free dual-responsive nanomaterial, and the supernatant is collected to obtain responsive empty nano micelles.

[0059] Example 3

[0060] A responsive nano-micelle for enhancing deep tumor penetration is a responsive drug-loaded garcinia acid nano-micelle, and a preparation method thereof comprises the following steps:

[0061] 8mg of the dual-responsive nanomaterial obtained in Example 1 and 0.5mg of gambogic acid were dissolved in 1mL DMSO, and a mixed solution was obtained after ultrasonic treatment for 5min. After the mixed solution (1mL) was collected using a 1mL syringe, a microinjection pump was used to drop into a round-bottom flask containing 4mL of ultrapure water at a speed of 20μL / min, and the round-bottom flask was placed on a magnetic stirrer and stirred at a speed of 800rpm during the addition. After the injection is completed, the resulting mixture is placed in a dialysis bag and dialyzed in 1000mL of ultrapure water for 24h, and the dialysate is replaced once every 2-4h. After the dialysis is completed, the free gambogic acid is removed by low-speed centrifugation at a speed of 3000rpm for 15min, and the supernatant is collected to obtain responsive drug-loaded gambogic acid nano-micelles.

[0062] Example 4

[0063] A responsive nano-micelle for enhancing deep tumor penetration is a Cy5.5-labeled responsive drug-loaded gambogic acid nano-micelle, and the preparation method thereof comprises the following steps:

[0064] 8mg of the dual-responsive nanomaterial obtained in Example 1 and 0.5mg of Cy5.5-labeled gambogic acid were dissolved in 1mL DMSO, and a mixed solution was obtained after ultrasonic treatment for 5min. After the mixed solution (1mL) was collected by a 1mL syringe, a microinjection pump was used to drop into a round-bottom flask containing 4mL of ultrapure water at a speed of 20μL / min, and the round-bottom flask was placed on a magnetic stirrer and stirred at a speed of 800rpm during the addition. After the injection is completed, the resulting mixture is placed in a dialysis bag and dialyzed in 1000mL of ultrapure water for 24h, and the dialysate is replaced once every 2-4h. After the dialysis is completed, the free Cy5.5-labeled gambogic acid is removed by low-speed centrifugation at a speed of 3000rpm for 15min, and the supernatant is collected to obtain Cy5.5-labeled responsive drug-loaded gambogic acid nano-micelles.

[0065] Effect verification

[0066] 1. Characterization of intermediates and micellar Tyndall effect

[0067] Use nuclear magnetic proton spectroscopy to detect monomeric substances of nanomaterials, such as Figure 1 As shown, A is the precursor substance NH2-PEG-SS-PLGA of the nanomaterial monomer, and B is the responsive nanomaterial monomer PLGA-SS-PEG-Glu obtained in Example 1. Figure 2 As shown, when a beam of light is irradiated on the nanoformulation, a bright light path is presented, indicating that the empty and drug-loaded nanomicelles exhibit an obvious Tyndall effect, confirming the formation of nanomicelles.

[0068] 2. Morphological characteristics

[0069] The morphological characteristics of the responsive empty and drug-loaded micelles prepared in Examples 2 and 3 were detected by transmission electron microscopy. The prepared empty and drug-loaded nanomicelles were diluted 5 times, 10 μL was aspirated and dropped onto a 200-mesh copper mesh, and then naturally dried. Then, negatively stained with 0.2% phosphotungstic acid, dried, and then the morphology was observed using a transmission electron microscope. The transmission electron microscope images and particle size distribution diagrams of the two are shown in FIG. Figure 3 As shown, wherein A is the transmission electron micrograph of Example 2, B is the transmission electron micrograph of Example 3, C is the transmission electron micrograph of the particle size distribution of Example 2, and D is the transmission electron micrograph of the particle size distribution of Example 3; Figure 3 It can be seen that the nanomicelles are round under a transmission electron microscope, with particle sizes of about 40-80 nm, spherical particles, and relatively uniform dispersion.

[0070] 3. Hydration Particle Size and Charge Detection

[0071] The hydrated particle size and charge of the responsive empty and drug-loaded nanomicelles prepared in Examples 2 and 3 were detected using a Malvern laser particle size scanner. Figure 4 As shown, the hydrated particle sizes of the responsive empty nanomicelles obtained in Example 2 and the responsive drug-loaded nanomicelles obtained in Example 3 are 110 nm and 155 nm, respectively, and the charges are -16 mV and -18 mV, respectively. This indicates that the nanomicelles obtained in the present invention have a small particle size, can be enriched at the tumor site, and have good stability and dispersibility.

[0072] 4. Deep penetration evaluation

[0073] The tumor retention of the Cy5.5-labeled responsive drug-loaded nanomicelles obtained in Example 4 was evaluated using a near-infrared imager. A certain amount of Cy5.5-labeled nanomicelles was injected into the tail vein of mice, and the fluorescence distribution in the mice was observed at different time points. Figure 5 As shown, about 12 hours after injection, the tumor fluorescence uptake reached a peak. After 96 hours, the tumor was removed and cut open for infrared imaging. The fluorescence intensity in the middle of the tumor was still strong, indicating that the Cy5.5-labeled responsive nanomicelles were able to penetrate deeply and enhance the retention time.

[0074] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A responsive nanomicelle for enhancing deep tumor penetration, characterized in that: The nano micelles are obtained by co-assembling gamboge acid and nanomaterials; The preparation method of the nano material comprises the following steps: (1) Add NH2-SS-NH2 and triethylamine to a DMF solution of Fmoc-PEG-NHS. After the reaction is complete, remove the solvent, dissolve in dichloromethane, wash with water, dry, and then add icy ether to precipitate. After filtering and drying the precipitate, dissolve it in toluene with lactide, glycolide and stannous octoate to react. After the reaction is completed, concentrate under reduced pressure to precipitate. After filtering and drying, the precipitate is dissolved in dichloromethane, dried, and then dissolved in dichloromethane. Add piperidine to react. After the reaction is completed, wash with water, dry, and concentrate under reduced pressure to precipitate. Then filter and dry to obtain NH2-PEG-SS-PLGA. (2) Adding EDC and NHS to the dichloromethane solution of Boc-Glu-OtBu to dissolve completely for activation, then adding the NH2-PEG-SS-PLGA and triethylamine to react, after the reaction is completed, a precipitate is precipitated, and then filtered and dried, and then dissolved in dichloromethane, and trifluoroacetic acid is added to react, after the reaction is completed, the precipitate is washed with water, dried, and concentrated under reduced pressure to precipitate, and then filtered and dried to obtain PLGA-SS-PEG-Glu, i.e., the nanomaterial; The particle size of the nano micelle is 150 nm.

2. The responsive nanomicelle for enhancing deep tumor penetration according to claim 1, characterized in that: The mass ratio of the nano material to gambogic acid is 8:0.

5.

3. The responsive nanomicelle for enhancing deep tumor penetration according to claim 1, characterized in that: The gambogic acid is gambogic acid labeled with Cy5.5 fluorescent dye.

4. A method for preparing a responsive nanomicelle for enhancing deep tumor penetration according to any one of claims 1 to 3, characterized in that: The following steps are involved: The nanomaterial and gambogic acid are dissolved in dimethyl sulfoxide, and a mixed solution is prepared after ultrasonic oscillation, and then added dropwise to ultrapure water at a uniform speed while stirring for reaction. After the reaction is completed, the supernatant is collected by dialysis and centrifugation to obtain the responsive nanomicelles.

5. The method for preparing a responsive nano-micelle for enhancing deep tumor penetration according to claim 4, characterized in that: The volume ratio of the mixed solution to ultrapure water is 1:

4.

6. The method for preparing a responsive nanomicelle for enhancing deep tumor penetration according to claim 4, characterized in that: The centrifugal speed is 3000 rpm and the time is 15 min.

7. Use of a responsive nano-micelle for enhancing deep tumor penetration as claimed in any one of claims 1 to 3 in the preparation of anti-tumor drugs; The tumor is triple-negative breast cancer.

Images