A tumor microenvironment-based emodin-loaded mpeg-plga nanoparticle, and a preparation method and application thereof

Abstract

The application discloses a kind of Baicalein-loaded mPEG-PLGA nanoparticles based on tumor microenvironment and its preparation method and application.The method is: ultrasonic dispersion preparation Baicalein uniform solution;Then mPEG-PLGA is prepared;Emulsion evaporation method is prepared Baicalein-loaded mPEG-PLGA nanoparticles (PMs-Ba);The nanoparticles can be accumulated in tumor site, improve tumor microenvironment, so as to enhance the sensitivity of breast cancer to chemotherapeutic drugs.The method is simple to operate, provides a new idea for traditional Chinese medicine to be used as adjuvant therapy, and the method has great application potential in early cancer clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, specifically relating to a tumor microenvironment-loaded mPEG-PLGA nanoparticle, its preparation method, and its application. Background Technology

[0002] Poly(D,L-lactide-co-glycolide) (PLGA) is approved by the U.S. Food and Drug Administration for drug delivery due to its excellent biocompatibility, biodegradability, controllable particle size and drug release, easily modifiable surface properties, and targeting capabilities. In recent years, PLGA-based polymeric nanoparticles have been widely used for targeted drug delivery. Among them, methoxypoly(ethylene glycol)-Poly(L-lactide-co-glycolide), mPEG-PLGA, is one of the most common biodegradable PLGA polymers used for drug delivery. mPEG-PLGA consists of two chemically distinct homopolymer blocks, with the mPEG portion being hydrophilic and the PLGA portion being hydrophobic. Due to its unique structure, it can self-assemble with drugs in water to form polymer micelles. Studies have shown that compared to traditional PLGA nanoparticles, mPEG-PLGA nanoparticle compositions have a longer residence time in the bloodstream.

[0003] In recent years, the tumor microenvironment (TME) has attracted considerable attention. In fact, the tumor microenvironment is extremely complex. Besides tumor cells, it contains stromal cells (including fibroblasts, immune and inflammatory cells, vascular endothelial cells, adipocytes, glial cells, smooth muscle cells, etc.) and the extracellular matrix, which can be divided into the tumor immune microenvironment, dominated by immune cells, and the tumor stromal microenvironment, dominated by fibroblasts. The tumor microenvironment possesses biological characteristics such as hypoxia, low pH, interstitial hypertension, inflammatory response, and immunosuppression, and is considered an important factor in tumor proliferation, invasion, migration, adhesion, and angiogenesis.

[0004] In the tumor immune microenvironment, lymphocytes infiltrating the tumor mediate an immunosuppressive tumor microenvironment, helping tumor cells achieve immune escape and promoting the malignant development of the tumor. Tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) play a major role in this process.

[0005] Within tumors, fibroblasts, vascular endothelial cells, and stromal cells constitute the tumor stromal microenvironment. Tumor-associated fibroblasts (CAFs) can release various cytokines that promote tumor cell growth and tumor angiogenesis. The tumor stromal microenvironment, together with the tumor immune microenvironment, contributes to tumor development and metastasis.

[0006] Baicalein is a flavonoid compound extracted from the root of Scutellaria baicalensis. Its chemical formula is C15H10O5, and its molecular weight is 270.24 g / mol. Baicalein appears as yellow needle-like crystals in ethanol and is soluble in methanol, ethanol, acetone, ethyl acetate, and hot glacial acetic acid. Baicalein possesses various pharmacological effects, including anti-inflammatory, antioxidant, antiviral, cardiovascular protective, anti-fibrotic, and anticancer properties.

[0007] Existing literature reports that baicalenin inhibits the proliferation and growth of cancer cells by regulating certain targets. For example, one article mentions that baicalenin inhibits the proliferation and growth of drug-resistant gastric cancer cells by regulating the expression of Notch1 and IncRNAAK022798 (Hang Qun. Baicalenin inhibits the proliferation and growth of drug-resistant gastric cancer cells SGC7901DDP by regulating the expression of Notch1 and IncRNAAK022798 [D]. Anhui Medical University, 2016.). However, baicalenin has low water solubility, which limits its clinical application. Furthermore, there are currently no reports on the effects of baicalenin on the tumor microenvironment, despite its excellent anti-fibrotic effect. Summary of the Invention

[0008] This invention aims to address the technical problems in existing technologies, such as low efficiency in tumor blood flow and the delivery of oxygen, drugs, and immune cells caused by the tumor microenvironment. Therefore, the purpose of this invention is to propose a tumor microenvironment-based mPEG-PLGA nanoparticle loaded with baicalensis, its preparation method, and its application. Specifically, it constructs a method for preparing tumor microenvironment-based mPEG-PLGA nanoparticles loaded with baicalensis (PMs-Ba) and applies it to the biomedical field. The design principle of a tumor microenvironment-targeting mPEG-PLGA nanoparticle loaded with the traditional Chinese medicine baicalensis is based on the fact that baicalensis has the effect of inhibiting the activation of cancer-associated fibroblasts. After loading it onto mPEG-PLGA nanoparticles, it can improve the tumor stromal microenvironment, thereby improving the tumor immune microenvironment and enhancing the sensitivity of breast cancer to chemotherapy. This invention, based on the anti-fibrotic effect of baicalensis, uses it as an adjuvant therapy for tumors to enhance the anti-tumor effect of drugs.

[0009] This invention, based on the unique tumor microenvironment, utilizes the low toxicity of the traditional Chinese medicine baicalein and its good biocompatibility with mPEG-PLGA to load baicalein onto mPEG-PLGA nanoparticles (PMs-Ba) to improve its water solubility and enhance its stability. Injection of these nanoparticles into tumor-bearing mice revealed their ability to accumulate at the tumor site, improve the tumor microenvironment, and enhance the sensitivity of breast cancer to chemotherapy drugs. This method is simple to operate and provides a basis for research on the use of traditional Chinese medicine to increase the sensitivity of breast cancer to chemotherapy.

[0010] This invention discloses a tumor microenvironment-loaded mPEG-PLGA nanoparticle, its preparation method, and its applications. These nanoparticles can accumulate at the tumor site, improving the tumor microenvironment and thus enhancing the sensitivity of breast cancer to chemotherapy drugs. The method is simple to operate and provides a new approach for the use of traditional Chinese medicine in adjuvant therapy. This method has great application potential in the clinical treatment of early-stage cancer.

[0011] This invention discloses a method for preparing mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment, comprising the following steps:

[0012] (1) Preparation of a homogeneous solution of baicalenin by ultrasonic dispersion: Add baicalenin to an organic solvent, sonicate, and collect a homogeneous baicalenin solution.

[0013] (2) Preparation of mPEG-PLGA: Dissolve PLGA-COOH (carboxyl-terminated poly(D,L-lactide-CO-glycolic acid)) in an organic solvent, add carboxyl activator and stir to react, then add PEG-NH2 (methoxy polyethylene glycolamine), continue stirring, and after the reaction is completed, wash and dry to obtain mPEG-PLGA.

[0014] (3) Preparation of mPEG-PLGA nanoparticles loaded with baicalenin by emulsion evaporation method: The baicalenin solution prepared in step (1) was added to the mPEG-PLGA solution and sonicated. Then, bovine serum albumin solution was added and sonicated again. After stirring, centrifugation, and freeze-drying, mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment were obtained.

[0015] Further, in step (1), the mass ratio of scutellaria baicalensis to the volume ratio of the organic solvent is 10 mg: (1-2) mL; the ultrasonic treatment is ultrasonic treatment in an ice bath for 1-10 min.

[0016] Further, the specific steps of step (1) are as follows: 5-20 mg of baicalein is added to 1-4 mL of organic solvent, and then the solution is placed in an ice bath and sonicated for 1-10 min; finally, a homogeneous brownish-yellow solution of baicalein is collected and stored at room temperature for further use. The organic solvent used to prepare the homogeneous solution of baicalein includes one or more of acetone, methanol, and dichloromethane.

[0017] Further, the carboxyl activator in step (2) includes one or more of dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0018] Furthermore, the organic solvent in step (2) includes one or more of dichloromethane, tetrahydrofuran, acetone, and ethyl acetate.

[0019] Further, in step (2), the mass ratio of PLGA-COOH to the volume of the organic solvent is 10 mg: (1-3) mL; the stirring time for adding the carboxyl activator is 2-4 h; the mass ratio of PEG-NH2 to PLGA-COOH is (1-2):1; and the stirring time is 12-36 h.

[0020] Further, the specific steps of step (2) are as follows: dissolve 10-40 mg of PLGA-COOH in an organic solvent, add a carboxyl activator, react for 2-4 h, add 10-40 mg of PEG-NH2, continue stirring for 12-36 h, and then wash and dry it.

[0021] Further, in step (3), the mass ratio of baicalensis in the baicalensis solution to mPEG-PLGA in the mPEG-PLGA solution is 1:(1-2); the sonication time for adding the baicalensis solution to the mPEG-PLGA solution is 1-3 min; the mass ratio of bovine serum albumin in the bovine serum albumin solution to mPEG-PLGA in the mPEG-PLGA solution is (1-2):1; the sonication time is 0.5-1.5 min; and the stirring time is 12-24 h.

[0022] Further, the specific process of step (3) is as follows: add scutellaria baicalensis solution to 1 mL mPEG-PLGA solution and sonicate for 1-3 min, then add 3-6 mL bovine serum albumin solution and continue sonicating for 0.5-1.5 min, then stir overnight, centrifuge, and freeze dry.

[0023] Further, in step (3), the concentration of the mPEG-PLGA solution is 10-40 mg / mL, and the solvent of the mPEG-PLGA solution is acetone, methanol, or dichloromethane; the concentration of the bovine serum albumin solution in step (3) is 5-15 mg / mL, and the solvent of the bovine serum albumin solution is deionized water or a phosphate buffer solution with a pH of 7.4.

[0024] Furthermore, in step (3), the concentration of the mPEG-PLGA solution is 10-20 mg / mL.

[0025] Furthermore, the power of the ultrasound in step (3) is 80-120W.

[0026] The present invention provides a preparation method for preparing mPEG-PLGA nanoparticles loaded with scutellarin based on the tumor microenvironment.

[0027] The present invention also provides the application of the mPEG-PLGA nanoparticles loaded with scutellarin based on the tumor microenvironment in the preparation of adjuvant chemotherapy drugs.

[0028] Furthermore, the mPEG-PLGA nanoparticles loaded with baicalensis based on the tumor microenvironment exhibit uniform shape and pore size, good thermal stability, excellent biocompatibility, large drug loading capacity, and slow drug release rate. Drug loading significantly increases the water solubility and stability of baicalensis. After entering the body, the nanoparticles accumulate in tumor tissue, improving the tumor microenvironment and thus enhancing the sensitivity of breast cancer to chemotherapy.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The mPEG-PLGA nanoparticles loaded with scutellaria baicalensis based on the tumor microenvironment prepared in this invention have uniform pore size, good biocompatibility and slow drug release rate.

[0031] (2) The mPEG-PLGA nanoparticles loaded with scutellarin based on the tumor microenvironment prepared in this invention can be enriched in the tumor site.

[0032] (3) The present invention provides a tumor microenvironment-loaded mPEG-PLGA nanoparticle based on baicalenin. Compared with the prior art which only focuses on the effect of baicalenin on mRNA expression in the tumor stroma microenvironment, loading baicalenin onto mPEG-PLGA nanoparticles can better improve the tumor microenvironment.

[0033] (4) The mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment prepared in this invention can accumulate at the tumor site, improve the tumor microenvironment, and thus enhance the sensitivity of breast cancer to chemotherapy drugs. The preparation method of this invention is simple to operate and provides a new idea for the use of traditional Chinese medicine in adjuvant therapy. This method has great application potential in the clinical treatment of early-stage cancer. Attached Figure Description

[0034] Figure 1 Scanning electron microscope image of mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 1.

[0035] Figure 2 This is a drug release diagram of the mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 1.

[0036] Figure 3 The infrared spectrum of the mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 1.

[0037] Figure 4 Cytotoxicity diagram of mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 1.

[0038] Figure 5 The in vivo distribution map of the mPEG-PLGA nanoparticles loaded with scutellarin based on the tumor microenvironment prepared in Example 2.

[0039] Figure 6 The figure shows the effect of mPEG-PLGA nanoparticles loaded with scutellarin based on the tumor microenvironment prepared in Example 2 on mRNA expression in the tumor stroma microenvironment. Detailed Implementation

[0040] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0041] Example 1

[0042] (1) Add 10 mg of baicalein to 2 mL of acetone, and then place the solution in an ice-water bath and sonicate it at 80 W for 1 min; finally, a uniform brownish-yellow solution of baicalein is collected and stored at room temperature for further use.

[0043] (2) Dissolve 10 mg of PLGA-COOH in 1 mL of acetone, add 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stir for 2 h, add 10 mg of PEG-NH2, continue stirring for 12 h, then wash and dry to obtain mPEG-PLGA.

[0044] (3) Add the baicalein solution obtained in step (1) to 1 mL of mPEG-PLGA solution with a concentration of 10 mg / mL (solvent is acetone) and sonicate at 80 W for 1 min. Then add 3 mL of bovine serum albumin solution (concentration of 5 mg / mL, solvent is deionized water) and continue to sonicate at 80 W for 0.5 min. Then stir for 12 h, centrifuge, and freeze dry to obtain mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment.

[0045] Example 2

[0046] (1) Add 10 mg of baicalein to 1.5 mL of methanol, and then place the solution in an ice-water bath and sonicate it at 80 W for 5.5 min; finally, a uniform brownish-yellow solution of baicalein was collected and stored at room temperature for further use.

[0047] (2) Dissolve 25 mg of PLGA-COOH in 8.3 mL of tetrahydrofuran, add 12.5 mg of dicyclohexylcarbodiimide, stir for 3 h, add 50 mg of PEG-NH2, continue stirring for 24 h, then wash and dry to obtain mPEG-PLGA.

[0048] (3) Add the baicalein solution obtained in step (1) to 1 mL of mPEG-PLGA solution with a concentration of 20 mg / mL (solvent is methanol) and sonicate at 100 W for 2 min. Then add 2 mL of bovine serum albumin solution (concentration of 10 mg / mL, solvent is phosphate buffer solution with pH of 7.4) and continue to sonicate at 100 W for 1 min. Then stir for 18 h, centrifuge, and freeze dry to obtain mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment.

[0049] Example 3

[0050] (1) Add 10 mg of baicalein to 1 mL of dichloromethane, and then place the solution in an ice-water bath and sonicate it at 80 W for 10 min; finally, a uniform brownish-yellow solution of baicalein is collected and stored at room temperature for further use.

[0051] (2) Dissolve 40 mg of PLGA-COOH in 8 mL of dichloromethane, add 20 mg of N,N'-diisopropylcarbodiimide, stir for 4 h, add 40 mg of PEG-NH2, continue stirring for 36 h, then wash and dry to obtain mPEG-PLGA.

[0052] (3) Add the baicalein solution obtained in step (1) to 1 mL of mPEG-PLGA solution with a concentration of 15 mg / mL (solvent is dichloromethane) and sonicate at 120 W for 3 min. Then add 2 mL of bovine serum albumin solution (concentration of 15 mg / mL, solvent is deionized water) and continue to sonicate at 120 W for 1.5 min. Then stir for 24 h, centrifuge, and freeze dry to obtain mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment.

[0053] Figure 1 This is a scanning electron microscope image of the mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 1. Figure 1 It can be seen that the mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment prepared in Example 1 are uniform spherical with a size of 100 nm.

[0054] Drug release capability: The in vitro release profile of baicalenin in the drug delivery system was detected using dialysis. In short, release was carried out in phosphate-buffered saline (PBS, pH 7.4 and 5.5). The tumor microenvironment-based mPEG-PLGA nanoparticles loaded with baicalenin, prepared in Example 1, were dispersed at a final concentration of 10 mg / mL in PBS at pH 7.4 and 5.5, respectively. These nanoparticles were then encapsulated in dialysis bags (molecular weight cutoff [MWCO] 10000 kDa) and immersed in 100 mL of PBS at pH 7.4 and pH 5.5, respectively, with stirring at 37°C and 100 rpm. At predetermined sampling times (2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h), 3 mL samples were collected, and equal volumes of fresh PBS at pH 7.4 and pH 5.5 were added, respectively. The extracted sample was then filtered and analyzed at 380 nm using a UVevis spectrophotometer.

[0055] Figure 2 This is a drug release diagram of the tumor microenvironment-loaded mPEG-PLGA nanoparticles based on baicalensis extract prepared in Example 1. Figure 2 It is known that scutellarin is more easily released from mPEG-PLGA nanoparticles under acidic conditions.

[0056] Figure 3The infrared spectrum of the mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 1 is shown below. Figure 3 It can be seen that the mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment prepared in Example 1 have the characteristic absorption peaks of mPEG-PLGA and baicalenin, indicating that baicalenin is completely encapsulated in the mPEG-PLGA nanoparticles.

[0057] Cell viability assay: Cell viability was determined using the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method. Cells in good logarithmic growth phase were seeded into 96-well plates, with four parallel wells per experiment. After cell attachment, the culture medium was discarded, and serum-free medium containing mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment was added at different concentrations (0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL). The cells were incubated together in a cell culture incubator. After 24 h, 48 h, and 72 h, the culture medium was removed, and the cells were cultured at 37 °C for 4 hours with thiazolyl blue solution (0.5 mg / mL, deionized water). Then, 100 μL of dimethyl sulfoxide was used to dissolve formazan crystals, and the absorbance was measured at 490 nm using a microplate reader (Cytation5, Biotek, USA).

[0058] Figure 4 The cytotoxicity diagram of the mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 1 is shown. Figure 4 It can be seen that the mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment prepared in Example 1 do not affect the proliferation of tumor cells.

[0059] In vivo distribution: The fluorescently labeled mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment (50 mg / kg) prepared in Example 1 were injected into mice, and the in vivo distribution of the mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment was observed using a small animal in vivo imaging system.

[0060] Figure 5 The in vivo distribution map of mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment prepared in Example 2 is shown. Figure 5 It can be seen that the mPEG-PLGA nanoparticles loaded with scutellarin based on the tumor microenvironment prepared in Example 2 can be enriched in mouse tumor sites.

[0061] Effects on mRNA in the tumor microenvironment: The mPEG-PLGA nanoparticles loaded with baicalein based on the tumor microenvironment prepared in Example 2 were injected into mice at 30 mg / kg. After one week, the tumor tissues of the mice were taken for immunohistochemical analysis.

[0062] Figure 6 This figure shows the effect of baicalein-loaded mPEG-PLGA nanoparticles prepared in Example 2 on mRNA expression in the tumor stromal microenvironment. Figure 6 It can be seen that, compared with the existing technology which only affects the expression of mRNA in the tumor stroma microenvironment by baicalensis, the mPEG-PLGA nanoparticles loaded with baicalensis based on the tumor microenvironment prepared in Example 2 of this invention can better downregulate the expression of anti-inflammatory factors in the tumor microenvironment, indicating that it can better improve the immune microenvironment of the tumor.

[0063] Efficacy of combined treatment: The mPEG-PLGA nanoparticles loaded with baicalensis based on the tumor microenvironment prepared in Example 2 were injected into mice at 30 mg / kg for one week. Then, mPEG-PLGA nanoparticles loaded with doxorubicin (PMs-ADM) loaded with the anticancer drug were injected at 1 mg / kg. The tumor tissue of the mice was photographed.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment, characterized in that, Includes the following steps: (1) Preparation of a homogeneous solution of baicalenin by ultrasonic dispersion: Add baicalenin to an organic solvent, sonicate in an ice bath for 1-10 min, and collect a homogeneous baicalenin solution, wherein the mass ratio of baicalenin to the volume of the organic solvent is 10 mg: (1-2) mL, and the organic solvent is acetone, methanol or dichloromethane. (2) Preparation of mPEG-PLGA: Dissolve PLGA-COOH in an organic solvent, add a carboxyl activator and stir for 2-4 h, then add PEG-NH2 and continue stirring for 12-36 h, wash and dry to obtain mPEG-PLGA, wherein the mass ratio of PLGA-COOH to the volume of the organic solvent is 10 mg:(1-3) mL, the mass ratio of PEG-NH2 to PLGA-COOH is (1-2):1, the carboxyl activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the organic solvent is dichloromethane; (3) Preparation of mPEG-PLGA nanoparticles loaded with baicalenin by emulsion evaporation: The baicalenin solution from step (1) was added to the mPEG-PLGA solution, and sonicated at 80-120W for 1-3 min. Then, bovine serum albumin solution was added and sonicated for another 0.5-1.5 min. The mixture was stirred for 12-24 h and then lyophilized by centrifugation to obtain nanoparticles, wherein: The mass ratio of scutellarin to mPEG-PLGA is 1:(1-2); The mass ratio of bovine serum albumin to mPEG-PLGA was (1-2):1; The concentration of mPEG-PLGA solution is 10-20 mg / mL; The concentration of bovine serum albumin solution is 5-15 mg / mL.

2. The mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment were prepared by the preparation method according to claim 1.

3. The application of the mPEG-PLGA nanoparticles loaded with baicalenin based on the tumor microenvironment as described in claim 2 in the preparation of adjuvant chemotherapy drugs.

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