Application of combined use of mangosteen shell extract alpha-mangostin and adriamycin for treating breast tumor

Abstract

The invention discloses an application of combined use of mangosteen shell extract alpha-mangostin and adriamycin in treatment of breast tumor, which comprises the following steps: coating alpha-mangostin by using chitosan temperature-sensitive hydrogel as a carrier for subcutaneous injection, and doping the alpha-mangostin into the chitosan temperature-sensitive hydrogel by using the anti-fibrosis effect of the alpha-mangostin to prepare the anti-breast tumor drug. The doxorubicin nano-particles are slowly released around a tumor, the activity of CAF is inhibited, the expression of alpha-FAP protein and SMA-alpha protein of tumor-associated fibroblasts in a tumor microenvironment is regulated, and a compact extracellular matrix is loosened, so that the doxorubicin nano-particles for combined treatment can permeate into tumor tissues, and the effect of inhibiting tumor growth is improved; a polyamino acid nano material with glutathione responsiveness is used as a carrier to coat adriamycin and is used for tail vein injection, and adriamycin effectively permeates into tumor tissues so as to kill tumor cells. According to the combined treatment method, the bioavailability of indissolvable drugs is solved, and the anti-tumor effect is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of combining alpha-mangostin extracted from mangosteen shell and doxorubicin for treating breast tumors. Background Art

[0002] Malignant tumors are one of the major health problems facing the world, and their morbidity and mortality rates have remained high. Therefore, malignant tumors have become an important hot topic in current medical research and clinical practice. The existing traditional clinical cancer treatment methods include surgery, radiotherapy and chemotherapy. Traditional treatment modes do not have tumor-specific killing effects, can cause damage to normal cells or tissues, and are prone to serious toxic side effects.

[0003] In recent years, nanotechnology has gradually become an important direction in biomedical research, aiming to provide effective intervention means for the diagnosis and treatment of various diseases (including tumors). Nano drugs can improve the water solubility and bioavailability of drugs, prolong the circulation time of drugs in the blood, increase the half-life of drugs and the enrichment of drugs in tumor sites, and reduce the toxic and side effects on normal tissues and organs of the body. However, nano drugs often only reduce toxic and side effects without significantly improving the therapeutic effect. Traditional therapeutic drugs have poor solubility and low water solubility in physiological environments, resulting in insufficient solubility of drugs in the body, which in turn affects their absorption and thus their therapeutic effect. Summary of the invention

[0004] The technical solution of the present invention to solve the above technical problem is to provide an application of combining α-mangostin extracted from mangosteen shell and doxorubicin for treating breast tumors.

[0005] Furthermore, in the application, the purity of α-mangostin is greater than 98%, and the purity of doxorubicin is greater than 98%.

[0006] Furthermore, in the application, α-mangostin has the ability to inhibit the proliferation of fibroblasts, and is used to reduce the growth support of tumors by reducing the number of fibroblasts in the tumor microenvironment.

[0007] Furthermore, in the application, α-mangostin is used to regulate related proteins of tumor-associated fibroblasts.

[0008] Furthermore, in the application, chitosan thermosensitive hydrogel is used as a carrier to encapsulate α-mangostin.

[0009] Furthermore, in the application, a polyamino acid nanomaterial with glutathione responsiveness is used as a carrier to encapsulate the chemotherapy drug doxorubicin.

[0010] Furthermore, in the application, the concentration of α-mangostin is 50 mg / kg, and the concentration of doxorubicin is 3 mg / kg.

[0011] The technical solution of the present invention utilizes the anti-fibrosis effect of α-mangostin, incorporates it into chitosan thermosensitive hydrogel, and slowly releases it around the tumor, inhibits the activity of CAF, regulates the tumor microenvironment, loosens the dense extracellular matrix, and allows the nanoparticles loaded with chemotherapy drugs (doxorubicin) for combined treatment to penetrate into the tumor tissue, thereby improving the effect of inhibiting tumor growth. This combined treatment method solves the bioavailability of poorly soluble drugs, improves the anti-tumor effect, and can be widely used in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0013] Figure 1 This is a SEM image of the chitosan thermosensitive hydrogel of the present invention;

[0014] Figure 2 This is the in vivo degradation image of the chitosan thermosensitive hydrogel of the present invention;

[0015] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum image of the polyamino acid nanomaterial of the present invention;

[0016] Figure 4 This is an in vitro drug release image of the polyamino acid nanomaterial of the present invention;

[0017] Figure 5 This is an image of α-mangostin cell migration of the present invention;

[0018] Figure 6 This is an image of the activation effect of TGF-β on fibroblasts of the present invention;

[0019] Figure 7 This is an image of Western blotting of α-mangostin of the present invention on tumor-associated fibroblast-related proteins;

[0020] Figure 8 This is an image of the volume change of mouse tumor during treatment by the present invention;

[0021] Fig. 9 This is an image of the tumor weight of mice after the treatment of the present invention;

[0022] Fig.10This is the tumor tissue penetration image of mice of the present invention;

[0023] Fig.11 This is a Masson-stained image of mouse tumor tissue of the present invention;

[0024] Fig.12 This is an image of the weight change of mice during the treatment of the present invention;

[0025] Fig.13 The cytotoxicity of the thermosensitive hydrogel of the present invention;

[0026] Fig.14 The cytotoxicity of the polyamino acid nanomaterial of the present invention;

[0027] Fig.15 This is the hemolysis experiment image of the thermosensitive hydrogel of the present invention;

[0028] Fig.16 This is the image of the hemolysis experiment of the polyamino acid nanomaterial of the present invention. DETAILED DESCRIPTION

[0029] The present invention proposes an application of combining alpha-mangostin extracted from mangosteen shell and doxorubicin for treating breast tumors, aiming to solve the bioavailability of poorly soluble drugs and improve the anti-tumor effect.

[0030] α-mangostin, an extract from mangosteen shell, has anti-fibrotic effects. Studies have shown that α-mangostin effectively reduces the activity of CAFs through the TGF-β / Smad signaling pathway, changing it from an activated state to a resting state. However, due to its poor water solubility and stability, α-mangostin has low bioavailability and poor therapeutic effect. Chitosan thermosensitive hydrogel can slowly release drugs locally, increase drug enrichment at the tumor site, and improve drug bioavailability.

[0031] α-mangostin is encapsulated in chitosan thermosensitive hydrogel and slowly released to regulate the tumor microenvironment, inhibit the activity of CAFs, and reduce the production of collagen, so that subsequent nanomedicines can better penetrate deep into the tumor tissue, increase the enrichment of nanomedicines at the tumor site, kill tumor cells, and inhibit the growth and metastasis of cancer cells.

[0032] The present invention utilizes the anti-fibrosis effect of α-mangostin, incorporates it into chitosan thermosensitive hydrogel, and slowly releases it around the tumor, inhibits the activity of CAF, regulates the tumor microenvironment, loosens the dense extracellular matrix, and allows the nanoparticles loaded with chemotherapy drugs in the combined treatment to penetrate into the tumor tissue, thereby improving the effect of inhibiting tumor growth. This combined treatment method solves the bioavailability of poorly soluble drugs, improves the anti-tumor effect, and can be widely used in the field of biomedicine.

[0033] The application of the combined use of α-mangostin extract from mangosteen shell and doxorubicin for treating breast tumors proposed by the present invention will be described in the following specific examples:

[0034] In the technical solution of this embodiment, α-mangostin extracted from mangosteen shell and doxorubicin are used in combination for anti-breast tumor treatment.

[0035] Furthermore, in the application, the purity of α-mangostin is greater than 98%, and the purity of doxorubicin is greater than 98%.

[0036] Furthermore, in the application, α-mangostin inhibits the proliferation ability of fibroblasts.

[0037] Furthermore, in the application, α-mangostin regulates related proteins of tumor-associated fibroblasts.

[0038] Furthermore, in the application, the chitosan thermosensitive hydrogel coats α-mangostin, thereby improving the bioavailability and water solubility of α-mangostin, and slowly releasing it in the tumor microenvironment.

[0039] Furthermore, in the application, the glutathione-responsive polyamino acid nanomaterial coated with the chemotherapy drug doxorubicin inhibits the proliferation ability of breast cancer cells.

[0040] Furthermore, in the application, α-mangostin and doxorubicin are used in combination to inhibit breast tumor growth.

[0041] Furthermore, the breast tumor is obtained from a mixed tumor of fibroblast NIH3T3 cells and breast cancer cell 4T1 cells.

[0042] Furthermore, the α-mangostin regulates tumor-associated fibroblasts, reshapes the tumor microenvironment, and enhances the penetration of doxorubicin nanomedicine.

[0043] Furthermore, the effective concentration of α-mangostin is 50 mg / kg, and the effective concentration of doxorubicin is 3 mg / kg.

[0044] Verification test:

[0045] 1. Preparation method of chitosan thermosensitive hydrogel, in vivo degradation and preparation of hydrogel-encapsulated α-mangostin:

[0046] Preparation: Weigh chitosan and dissolve it in 1% acetic acid solution. Stir magnetically at room temperature for 3 hours until the chitosan powder is completely dissolved. Add P407, P188 and glycerol respectively, stir at room temperature, and place at 4°C overnight to dissolve. After adding the thermosensitive hydrogel to the EP tube, quickly put the EP tube into liquid nitrogen to break it. Freeze-dry the sample for 24 hours, dry and store the sample after freeze-drying, and take SEM photos (such as Figure 1As described above), it can be seen from the figure that the hydrogel has an obvious porous network structure, which is conducive to the loading and release of drugs.

[0047] In vivo degradation: 100 μL of the thermosensitive hydrogel was injected subcutaneously into the back of 7 Balb / c mice provided by Yisi Experimental Animal Technology Co., Ltd., and one mouse was killed every day. The degradation degree of the hydrogel was observed and photographed (e.g. Figure 2 As shown in the figure, it can be observed that after the hydrogel is implanted in the body, the hydrogel forms an obvious bulge under the skin on day 1. As time goes by, this bulge gradually shrinks, and is basically flat on the 5th day, and is almost completely degraded on the 7th day, indicating that the hydrogel has good biodegradability in the body. From the color changes around the skin, it can be seen that a mild inflammatory reaction to the implantation of common biomaterials occurs on day 1-2, and the redness and swelling gradually subside over time, indicating that the hydrogel has good biocompatibility and will not cause severe inflammatory reactions.

[0048] Hydrogel encapsulation of α-mangostin: Add α-mangostin to the prepared hydrogel and physically stir it, and after mixing, defoam it by centrifugation. Store it in a refrigerator at 4°C for later use.

[0049] 2. Preparation method of polyamino acid nanomaterials and polyamino acid nanomaterials loaded with DOX and their in vitro release:

[0050] Preparation of polyamino acid nanomaterials (NP): It is synthesized by one-step ring-opening polymerization of L-phenylalanine N-carboxylic anhydride (NCA) and L-cystine NCA using amino-terminal mPEG (mPEG-NH2) as a macromolecular initiator. Add mPEG-NH2 and toluene to the reaction bottle, seal it in an oil bath, connect it to liquid nitrogen and cool it to dry. Add dry N,N-dimethylformamide (DMF) to the reaction bottle, then add appropriate amounts of L-cystine NCA and L-phenylalanine NCA, and react at room temperature for 3 days. After the reaction is completed, add cold ether for precipitation, then dissolve the precipitated product with DMF, dialyze it using a 3500Da dialysis bag, and finally freeze-dry to obtain NP.

[0051] Preparation of polyamino acid nanomaterials encapsulated with DOX: Weigh 50 mg of nanomaterials and 20 mg of DOX-HCl and dissolve them in 8 mL and 2 mL of DMF, respectively, to obtain the corresponding solutions. Take 3 μL of triethylamine and add it to the DOX / DMF solution to neutralize the electrons. Mix the two solutions and stir them magnetically for 7 hours. Then add the mixture drop by drop into an equal volume of deionized water and stir magnetically overnight. The next day, transfer all the solutions to a 3500Da dialysis bag for 8 hours of dialysis to remove excess DMF and DOX. After dialysis, freeze in a refrigerator at -20°C, freeze-dry, and weigh for later use.

[0052] In vitro release: Dissolve the prepared samples in 5 mL of PBS buffer solution containing or not containing 10 mM GSH. Add the prepared sample solutions to 3500 Da dialysis bags, place the whole in a 50 mL centrifuge tube, and add 30 mL of the corresponding solvent. Fix the 50 mL centrifuge tube in a conical flask, place the conical flask in a 37°C 70 rpm shaker, take 2 mL of liquid at 1, 3, 6, 12, 24, 36, 48 and 60 h, and add 2 mL of the corresponding solution. Use a UV spectrophotometer to detect the content of the collected liquid and draw an image (such as Figure 4 As shown in the figure, under the condition of 10mM GSH, the release of DOX increased rapidly within the first 10h, reaching about 50%, and then the release rate gradually slowed down and reached about 70% at about 60h. Under the condition of PBS, the release of DOX reached about 30% at 60h. This shows that the nanoparticles are GSH-responsive, and the release rate increased significantly in the presence of GSH.

[0053] 3. Regulatory effect of α-mangostin on tumor-associated fibroblasts:

[0054] Cell migration: Take NIH3T3 cells with good growth status, culture them in an incubator at 37°C and 5% CO2 for 24 hours, digest them with trypsin for 3 minutes, terminate the process with DMEM culture medium, and count them. 5 Add a certain number of 3T3 cells to a 6-well plate, shake it crosswise to evenly distribute the cells at the bottom of the well plate, place it in an incubator, and culture it for 24 hours. After 24 hours, use a 10μL pipette tip and a ruler to draw lines, take the supernatant, wash it lightly 3 times with PBS, and add 2mL DMEM culture medium and different concentrations of α-mangostin to each well for culture. Take photos and record them at 0h and 24h (such as Figure 5 As shown in the figure, as the concentration of α-M increases, the migration ability of cells decreases significantly. This indicates that α-M has a concentration-dependent inhibitory effect on cell migration. At higher concentrations (3.0 μg / mL and above), α-M almost completely blocked the migration of cells.

[0055] Western blot of cells: 3T3 cells in good condition were placed at 2×10 5 Add 10 ng / mL of TGF-β to the wells that need to be treated with 3T3 cells for 12 h (eg Figure 6As shown in the figure, the 3T3 cells added with TGF-β become slender and spindle-shaped, which can prove that they have been activated into tumor-associated fibroblasts). Remove the supernatant, rinse with PBS, and add 2mL DMEM and 3.0μg / mL α-mangostin to the drug well for co-culture. Remove the supernatant, rinse with PBS, scrape the cells in the 6-well plate with a scraper under ice bath, add cell lysis solution, lyse for 10min, add the sample to the EP tube for centrifugation, and take the supernatant. Use BSA for protein quantification, add appropriate Loading buffer, 95℃ metal bath for 5min, and store at -20℃ for standby use. Prepare 10% SDS-PEG gel, load the sample, 90V voltage for 30min, and then adjust to 120V until the run is completed. Transfer the membrane, cut the protein gel, sandwich the sponge, filter paper, gel, methanol-activated PVDF membrane, filter paper and sponge in this way, 100mA current, transfer the membrane under ice bath for 2h. Block with blocking solution for 3 h, wash the membrane with TBST 5 times, incubate with primary antibody at 4°C overnight, wash the membrane with TBST 5 times, incubate with secondary antibody at 4°C for 4 h, wash the membrane with TBST 5 times again, add developer, and develop (such as Figure 7 As shown in the figure, α-SMA is a marker of tumor-associated fibroblasts. The results show that TGF-β can activate NIH3T3 cells into tumor-associated fibroblasts, and α-mangostin can inhibit the activation of tumor-associated fibroblasts and convert them into a quiescent state. It can be observed from the figure that the addition of TGF-β causes a significant expression of α-SMA protein, which is consistent with the above Figure 6 The results were consistent. In addition, the addition of α-mangostin decreased the expression of α-SMA protein, indicating that α-mangostin can inhibit the activity of CAF.

[0056] 4. Combined therapy in vivo experiments:

[0057] Model construction: 20 6-week-old Babl / c female mice weighing 18-19g were provided by Yisi Experimental Animal Technology Co., Ltd., and a total of 1.4 million cells of 3T3 and 4T1 cells in a ratio of 1:1 were injected into the second pair of mammary pads under the armpits. Seven days later, the mice developed tumors, indicating that the model was successfully established.

[0058] The mice were divided into 4 groups, 5 in each group, including control group (PBS), doxorubicin group (DOX), nanomaterial-coated doxorubicin group (NP / DOX), and nanomaterial-coated doxorubicin combined with α-mangostin group (NP / DOX+α-M). 3Treatment was started at 1:100 pm. On the first day, mice were subcutaneously injected with 100 μL thermosensitive hydrogel-encapsulated α-mangostin, and on the same day, 150 μL NP / DOX was injected into the tail vein. The treatment was repeated once every three days for a total of 4 times. After administration, the length (A, mm) and width (B, mm) of the tumor were measured with a vernier caliper every two days, and the volume (V, mm) of the tumor was calculated. 3 ), the formula is: V = A × B 2 ×0.5. (e.g. Figure 8 On the 14th day of treatment, the mice were uniformly killed, dissected, the tumors were separated, and weighed and recorded (as shown in Fig. 9 shown). Figure 8 , 9 The results were consistent. After 14 days of treatment in each group, the NP / DOX+α-M group had the best tumor inhibition effect.

[0059] Penetration of drugs in the tumor site: The isolated tumor was stored in 4% paraformaldehyde. The tumor was embedded in wax blocks and frozen and cut into white slices. The slices were soaked in xylene for 20 minutes for dewaxing, and then soaked in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 10 minutes respectively, and then soaked in distilled water for 2 minutes for hydration. The slices were placed in citric acid repair solution, heated in a microwave for 2 minutes and 30 seconds, cooled to room temperature, and rinsed with PBS 5 times. Under light-proof conditions, DAPI was used to stain the cell nucleus, soaked for 5 minutes, rinsed 3 times with PBS, and finally covered with glycerol. Store in a dark place and use a fluorescence microscope to record (such as Fig.10 shown). Fig.10 The image shows the penetration of nanomedicine into tumor tissue after tail vein administration of NP / DOX. The red intensity in the image represents the distribution of NP / DOX in tumor tissue. The NP / DOX group drug is enriched at the edge of the tumor, while the addition of α-M allows NP / DOX to better penetrate into the tumor tissue, indicating that α-M plays a role in regulating the tumor microenvironment and loosening the cell matrix, allowing nanomedicine to better penetrate into the tumor tissue.

[0060] Masson staining: First, dewax and hydrate the tumor sections, then use hematoxylin to stain the cell nuclei for 10 minutes, differentiate with 1% hydrochloric acid alcohol for 15 seconds, and rinse with distilled water for 10 minutes until it turns blue. Use Ponceau red to stain for 10 minutes and quickly rinse with distilled water. Treat the sections with phosphomolybdic acid for 5 minutes, counterstain with aniline blue solution for 5 minutes, and differentiate with 1% glacial acetic acid for 1 minute. Finally, put the sections in 95% alcohol, anhydrous ethanol, and xylene in turn for dehydration, dry, and seal. Observe and record with a microscope (such as Fig.11 Among them, the blue color is collagen, and the blue color in the α-mangostin group is significantly less, indicating that α-mangostin has the effect of regulating the tumor microenvironment. Fig.10 The NP / DOX chemotherapy drugs shown can better penetrate into the tumor tissue and exert a better anti-tumor effect.

[0061] 5. Safety testing of thermosensitive hydrogels and GSH-responsive nanomaterials:

[0062] MTT: Digest, terminate and count NIH3T3 cells in good growth condition. Inoculate 5000 cells / well in a 96-well plate and culture in a cell culture incubator at 37°C and 5% CO2 for 24 hours. Sterilize the gelled thermosensitive hydrogel with UV light, soak it in DMEM culture medium, and extract it at 0, 3, 6, 9 and 12 hours. Add 5 replicate wells of the extract obtained at each time to a 96-well plate and culture it in a cell culture incubator at 37°C and 5% CO2 for 12 hours. Add 20μL MTT solution to each well to a final concentration of 0.5mg / mL. Culture in a cell culture incubator at 37°C and 5% CO2 for 4 hours. Add 150μL DMSO to produce crystal violet. Finally, use an enzyme reader to measure the absorbance at 570nm (such as Fig.13 As shown in the figure, the survival rate of the hydrogel 12h extract is higher than 80%, indicating that the hydrogel has low cytotoxicity. Cell plating was performed as above, and different concentrations of nanomaterials were dissolved in PBS and added to 5 replicate wells. After culturing for 24h, MTT and DMSO were added in turn, and the absorbance was measured by a microplate reader (as shown in the figure). Fig.14 As shown in the figure, when the maximum concentration of the nanomaterial reaches 20 μg / mL, the cell survival rate is higher than 90%, indicating that the nanomaterial has low cytotoxicity.

[0063] Hemolysis experiment: Take 5 mL of healthy mouse blood, add 10 mL of PBS, mix and centrifuge at 4°C, 1200 rpm, 5 min. Remove the supernatant, wash the lower layer of red blood cells with PBS 6 times, and finally disperse the red blood cells in 45 mL PBS. Dissolve the thermosensitive hydrogel and nanomaterials in 0.6 mL PBS at different concentrations, and then add 0.2 mL of red blood cell dispersion. A Triton-100 positive control and a PBS negative control were set. Place the marked EP tube in a 37°C water bath for 2 hours, then centrifuge at 3000 rpm for 10 minutes, remove 200 μL of the supernatant and add it to a 96-well plate, use an enzyme reader to measure the absorbance at 540 nm, calculate, and take pictures (such as Fig.15 , 16 As shown in the figure, it can be seen that when the concentrations of the hydrogel and nanomaterials reach 1.25 mg / mL and 125 μg / mL respectively, the hemolysis percentage is less than 5%, indicating that the hydrogel and nanomaterials are relatively safe.

[0064] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention 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 by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. The application of combined use of α-mangostin extract from mangosteen shell and doxorubicin for treating breast tumors, characterized in that: include: Chitosan thermosensitive hydrogel was used as a carrier to encapsulate α-mangostin for subcutaneous injection to regulate α-FAP protein and SMA-α protein of tumor-associated fibroblasts; Glutathione-responsive polyamino acid nanomaterials are used as carriers to encapsulate doxorubicin for tail vein injection to kill tumor cells.

2. The use according to claim 1, characterized in that: In the application, the purity of α-mangostin is greater than 98.00%, and the purity of doxorubicin is greater than 98.00%.

3. The use according to claim 1, characterized in that: In the application, the concentration of α-mangostin is 50 mg / kg, and the concentration of doxorubicin is 3 mg / kg.

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