CN112656944A - Preparation method and application of oleanolic acid nanogel - Google Patents
Preparation method and application of oleanolic acid nanogel Download PDFInfo
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Abstract
The invention discloses a preparation method and application of oleanolic acid nanogel. The nanogel is formed by self-assembling oleanolic acid in a mixed solvent of ethanol/DMSO and water. With the change of the ethanol/DMSO and oleanolic acid concentrations, different forms of assemblies were obtained, respectively: nanoparticles, nanogels and gels. Compared with free medicines, the nanogel has better anti-tumor activity, can improve the fibrosis degree of tumors and promote the infiltration of immune cells, and is used for chemotherapy and immunotherapy of tumors. The nanometer gel can be used as a medicine per se, and can also be used for encapsulating a photoacoustic sensitizer to obtain the oleanolic acid/sensitizer nanometer medicine for photoacoustic/chemotherapeutics of tumors. The nanogel has great application potential in the fields of drug delivery, tissue engineering, chemicals and the like.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to a preparation method and application of oleanolic acid nanogel.
Background
As the origin of drug development, natural drugs play an important role in medicinal chemistry. Pentacyclic triterpene Oleanolic Acid (OA) existing in natural plants has various biological activities such as antitumor effect, antioxidant effect, immunity enhancing effect, liver protecting effect and heart protecting effect, and has wide application prospect. Bag et al discovered in 2012 that Oleanolic Acid has a chiral rigid backbone and readily aggregates in solution, assembles into a fibrous structure through intermolecular hydrogen bonding, pi-pi stacking, and the aggregating properties of the triterpene backbone, and then forms a supramolecular organogel (B.G. Bag, K. Paul, Vesicular and fibrous Gels by Self-assembled of Nanosized Oleanolic Acid, Asian Journal of Organic Chemistry, 1 (2012) 150-. Although the oleanolic acid organogel is used for encapsulating adriamycin in the team, the treatment effect of the oleanolic acid organogel is not evaluated, and the organogel has high toxicity, so that the supermolecule gel generally applied to a drug carrier and a medical material is mainly hydrogel.
Recent statistical data of national cancer centers show that breast cancer is the first of female malignant tumor incidence and has become a major public health problem worldwide. Among the various types of human breast cancer, Triple Negative Breast Cancer (TNBC) is the most aggressive type. The tumor microenvironment (TEM) is composed of extracellular matrix (ECM), tumor-associated fibroblasts (CAFs), immune cells and the vascular system, which surround the tumor cells and protect them from invasion. Collagen produced by fibroblasts forms a physical barrier, blocking drug delivery and infiltration of cytotoxic T cells. Also, CAFs support the development of tumors by remodeling the ECM, the prognosis of TNBC is related to central fibrosis of the tumor, the higher the degree of fibrosis, the greater the propensity of tumors to metastasize to distant sites (Takai K, Le A, Weaver V M, et al. Targeting the cancer-associated fibrous as a metastatic in triple-negative rupture cancer [ J ]. Oncotarget, 2016, 7(50): 82889-82901.). Thus, targeting CAFs and reconstituting ECM may be an effective strategy to address the complex etiology of cancer.
Transforming growth factor-beta (TGF- β) is a key regulator for remodeling the ECM and controlling CAF activation, promoting the accumulation of hyperplasia and the growth rate of tumors. TGF-. beta.drives the expression of collagen in the ECM through the TGF-. beta./Smad signaling pathway. Research shows that the TGF-beta is blocked, the generation of collagen is reduced, and the blood vessel is normalized to improve the distribution of the medicine in breast cancer, increase the immune cell infiltration of tumor parts, and reverse the immunosuppressive environment of tumor. Oleanolic Acid has been shown to reduce Renal Fibrosis and collagen deposition in C57BL/6 mice by increasing nuclear transport of Nrf2 (ZHao D, Luan Z. Oleanolic Acid anchors Renal Fibrosis through TGF-. beta./compact Pathway in a Rat Model of Universal aqueous organization [ J ]. 2020: 1-8.). In addition, researchers find that oleanolic acid has strong binding capacity with TGF-beta 1 receptor and can be used as an antagonist of TGF-beta 1. This suggests that it is potential for OA to attenuate fibrosis through TGF-. beta./Smad signaling (Yoshimura H, Sugawara K, Saito M, et al. In vitro TGF-. beta.1 Antagonistic Activity of Ursolic and Olianolic Acids Isolated from clinical endendranthus sites [ J ]. Planta medicine, 2003, 69(7): 673. 675.).
Photo-induced photothermal therapy (PTT) and photodynamic therapy (PDT) have high specificity, i.e., after administration, a tumor site is irradiated with an external light source to generate heat or Reactive Oxygen Species (ROS), with little side effect on other tissues. However, PTT and PDT have the disadvantage of limited depth of light penetration, and thus PTT or PDT alone may not be effective in inhibiting tumors. The ultrasound is used as a mechanical wave, has good penetrating power in a living body, can reach deep tumors, and can be used for diagnosis and treatment of the tumors. Sonosensitizers produce ROS under ultrasound stimulation, causing oxidative damage to intracellular proteins or DNA, resulting in cell death (Zhu, Piao, Chen, et al, Nanoenzyme-amplified Cancer nanometer Therapy by catalysis carbon dioxide vapor oxygen no, 2018, 12: 3780-once 3795.).
Therefore, we obtained a nanogel by varying the ethanol ratio and the oleanolic acid concentration. The nanogel can effectively inhibit the proliferation of breast cancer cells, remodels the tumor microenvironment and improves the proportion of immune cells at the tumor part by reducing the fibrosis degree of the tumor, and can be used for chemotherapy and immunotherapy of the tumor. The nanogel can be used for encapsulating a photoacoustic sensitizer to obtain an OA/sensitizer nano-drug for photoacoustic/chemotherapy of tumors.
Disclosure of Invention
The invention aims to provide a preparation method and application of oleanolic acid nanogel. The invention takes oleanolic acid as a raw material, constructs a nanogel with the function of regulating and controlling the tumor microenvironment, and can be used for chemotherapy and immunotherapy of tumors. The nanometer gel can be used for encapsulating a photoacoustic sensitizer to obtain an OA/sensitizer nanometer drug for photoacoustic/chemotherapy of tumors.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing oleanolic acid nanogel comprises the following steps
(1) Dissolving oleanolic acid in ethanol or DMSO;
(2) and (2) dropwise adding the solution obtained in the step (1) into an aqueous solution in a vortex state, and standing at room temperature for 0.5-24 h to obtain the oleanolic acid nanogel.
The aqueous solution is selected from water, aqueous solution containing drug molecules, physiological saline solution, phosphate buffer solution, cell culture medium or mixed solution thereof.
The concentration of oleanolic acid in the nanogel is 0.1-5 mg/mL.
The volume concentration of the ethanol or DMSO in the nanogel is 10-100%.
Application of oleanolic acid nanogel in preparation of antitumor drugs is provided.
Application of oleanolic acid nanogel in resisting fibrosis is provided.
Use of oleanolic acid nanogels in immunotherapy.
Application of OA/sensitizer nano-drug in photoacoustic/chemotherapy.
The invention has the beneficial effects that:
firstly, the invention obtains assemblies with different structures by changing the proportion of ethanol and the concentration of oleanolic acid;
secondly, compared with free drugs and oleanolic acid nanoparticles, the nanogel obtained by the invention has greatly improved anti-tumor effect.
Thirdly, the nanogel obtained by the invention can regulate and control the microenvironment of the tumor, weaken the fibrosis degree of the tumor and promote the infiltration of toxic T cells, and can be used for chemotherapy and immunotherapy of the tumor.
Fourthly, the OA/sensitizer nano-drug obtained by the invention can be used for photoacoustic/chemotherapy of tumors and inhibiting growth of the tumors.
Drawings
FIG. 1 is an oleanolic acid gel prepared in example 1.
Fig. 2 is a graph showing the rheological results of oleanolic acid gel prepared in example 1 under frequency sweep.
FIG. 3 is OA-NG prepared in example 2.
FIG. 4 is a transmission electron micrograph of OA-NG prepared in example 2.
FIG. 5 is a transmission electron micrograph of OA-NP prepared from example 3.
FIG. 6 is a UV spectrum of ORM prepared in example 5.
FIG. 7 is a graph showing the effect of OA-NG on cytotoxicity of 4T1 in example 6.
FIG. 8 is a graph showing the effect of ORM on cytotoxicity of 4T1 in example 7.
FIG. 9 is a graph showing the effect of OA-NG on tumor volume in 4T1 tumor-bearing mice in example 8.
FIG. 10 is a graph of the effect of OA-NG on tumor fibrosis in example 8.
FIG. 11 is a graph showing the effect of OA-NG on immune cells at the tumor site of 4T 1-bearing mice in example 9.
Detailed Description
The present invention is further described below in conjunction with specific examples to assist those of ordinary skill in the art in further understanding the present invention, but are not intended to limit the invention in any way.
Example 1
mu.L of an ethanol solution containing 4 mg/mL of oleanolic acid was added dropwise to 500. mu.L of water in a vortex state, and left to stand at room temperature for 24 hours, and a jelly-like gel was observed to obtain an oleanolic acid gel, as shown in FIG. 1. The rheological properties of the gel were characterized by rheometers, as shown in fig. 2, G 'is an order of magnitude higher than G ″, and as the scanning frequency was varied, G' remained substantially constant, satisfying the rheological properties of the gel.
Example 2
Dripping 150 μ L ethanol solution containing 3.33mg/mL oleanolic acid into 850 μ L water under vortex state, standing at room temperature for 24 h, centrifuging to observe jelly-like gel to obtain oleanolic acid nanogel OA-NG, as shown in FIG. 3. The structure of oleanolic acid nanogel appears as a sphere with fuzzy edges, and part of small spheres are adhered (figure 4).
Example 3
Dripping 50 mu L of ethanol solution containing 10 mg/mL of oleanolic acid into 950 mu L of water in a vortex state, and standing at room temperature for 24 h to obtain the oleanolic acid nanoparticle OA-NP. Observation by transmission electron microscopy revealed that the nanoparticles were spherical with well-defined edges, with the spheres being uniformly distributed without sticking (fig. 5).
Example 4
Adding 400 μ L DMSO solution containing 5mg/mL oleanolic acid into 200 μ L water under vortex state, standing at room temperature for 24 hr, and observing jelly-like gel to obtain oleanolic acid organogel.
Example 5
Adding 150 μ L ethanol solution containing 3.33mg/mL oleanolic acid dropwise into 850 μ L aqueous solution containing Methylene Blue (MB) and Rose Bengal (RB) under vortex state, standing at room temperature for 24 h, centrifuging, and observing jelly-like gel to obtain nanometer gel ORM. The ultraviolet absorption of the sample was measured, and the result is shown in FIG. 6, in which RB has a characteristic absorption peak at 565nm, MB has an ultraviolet absorption peak at 667nm, and ORM has characteristic absorption peaks at 565nm and 667nm, indicating that OA successfully entrapped RB and MB.
Example 6
The anti-tumor activity of nanogel OA-NG in example 2 on murine breast cancer cell 4T1 was determined by the MTT method. As shown in FIG. 7, the cell survival rates of the OA-NP and OA-NG experimental groups were lower than that of free OA at the same OA (50. mu.g/mL) concentration, and the effect of OA-NG on the inhibition of proliferation of 4T1 cells was stronger than that of OA-NP.
Example 7
The antitumor activity of ORM against murine breast cancer cell 4T1 was measured by MTT assay. The results are shown in fig. 8, where the photoacoustic combination therapy of ORM is superior to the monotherapy, and the antitumor effect is greatly improved.
Example 8
A 4T1 mouse model of breast cancer in situ was used to validate the in vivo anti-tumor activity of the nanogels. One group of mice was treated with OA-NG (15 mg/kg) from example 2 by tail vein injection, and one group of mice was treated with OA-NP (15 mg/kg) from example 3 by tail vein injection; one group of mice was treated by tail vein injection of free OA (15 mg/kg); the other group was treated with intravenous PBS as a control. Tumor volumes were measured every other day, and as a result, as shown in FIG. 9, the volume of the tumors of the OA-NG group was significantly smaller than that of the free OA-NP group after 14 days of treatment. The results of sirius red staining of treated tumor tissues are shown in FIG. 10, where the expression of collagen in the OA-NG group is minimal, and the ability of OA-NG to inhibit tumor fibrosis is stronger than that of OA-NP.
Example 9
OA-NG (15 mg/kg) in example 2, OA-NP (15 mg/kg) in example 3, and free OA (15 mg/kg) were injected into a 4T1 mouse model of in situ breast cancer via caudal vein, PBS was injected intravenously as a control group, and 24 hours later, tumor tissue was taken out, and the content of infiltrating toxic T cells in the tumor was measured by flow cytometry. As shown in FIG. 11, the tumor site of mice treated with OA-NG was infiltrated with 14.6% of CD8+ T cells, the OA-NP group was infiltrated with 6.57% of CD8+ T cells, and the PBS group was infiltrated with only 1.26% of CD8+ T cells. OA-NG is most effective immunotherapeutically compared to OA-NP and free OA.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (8)
1. A preparation method of oleanolic acid nanogel is characterized by comprising the following steps: the method comprises the following steps:
(1) dissolving oleanolic acid in ethanol or DMSO;
(2) and (2) dropwise adding the solution obtained in the step (1) into an aqueous solution in a vortex state, and standing at room temperature for 0.5-24 h to obtain the oleanolic acid nanogel.
2. The method for preparing oleanolic acid nanogel according to claim 1, wherein the method comprises the following steps: the concentration of oleanolic acid in the nanogel is 0.1-5 mg/mL.
3. The method for preparing oleanolic acid nanogel according to claim 1, wherein the method comprises the following steps: the volume concentration of the ethanol or DMSO in the nanogel is 10-100%.
4. The method for preparing oleanolic acid nanogel according to claim 1, wherein the method comprises the following steps: the aqueous solution is selected from water, aqueous solution containing drug molecules, physiological saline solution, phosphate buffer solution, cell culture medium or mixed solution thereof.
5. An application of the oleanolic acid nanogel prepared by the method of claim 1 in preparing a medicine for resisting breast cancer.
6. An application of the oleanolic acid nanogel prepared by the method of claim 1 in preparing a medicine for regulating and controlling a tumor microenvironment.
7. An application of oleanolic acid nanogel prepared by the method of claim 1 in preparation of immunotherapy drugs.
8. An application of oleanolic acid nanogel prepared by the method of claim 1 as a drug carrier.
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