Nano pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention discloses a nano pharmaceutical composition as well as a preparation method and application thereof, and belongs to the field of pharmaceutical preparations. The nano-drug composition disclosed by the invention comprises active substances of the nano-drug composition, wherein the active substances comprise a natural alkaloid compound and a tumor cell apoptosis inducing factor; the natural alkaloid medicine comprises piperlongumine, and the tumor cell apoptosis inducing factor comprises TRAIL; the mask further comprises hyaluronate and PAMAM (Polyamidoamine). The preparation method comprises the following steps: S1, dissolving piperlongumine in an acetone solution, and adding a PAMAM solution to obtain a PP compound; s2, the PP compound and a TRAIL solution are mixed, and a PPT compound is obtained; and S3, mixing the PPT compound with a hyaluronic acid solution to obtain the HPPT. The nano-drug composition disclosed by the invention shows a remarkable synergistic effect in inhibiting breast cancer cells and tissue species.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a nano-drug composition, a preparation method thereof, and an application thereof. Background Art

[0002] As a major threat to the health of women globally, breast cancer has a high incidence and mortality rate. According to the data of the World Health Organization in 2020, the age-standardized incidence rate of breast cancer globally reached 47.8%, and the mortality rate was 13.6%. Currently, common treatment methods for breast cancer include surgery, chemotherapy, radiotherapy, endocrine and hormone therapies, and targeted therapy, etc. Surgery is often the first option. Although chemotherapy and radiotherapy after surgery help control tumors, radiotherapy may bring side effects. Especially for triple-negative breast cancer (TNBC), which is highly invasive, prone to recurrence, and lacks clear targets, after chemotherapy, problems such as cancer recurrence, drug resistance, and toxicity to non-target tissues may be faced. Therefore, it is crucial to develop new breast cancer treatment drugs with high efficiency and low toxicity. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this reason, the present invention provides a nano-drug composition, which has the effect of inhibiting the growth of tumor cells and tumor tissues in the treatment of breast cancer and has good application prospects.

[0004] The present invention also provides a preparation method of the above nano-drug composition.

[0005] The present invention also provides an application of the above drug composition in the preparation of a medicament for treating breast cancer (especially triple-negative breast cancer).

[0006] According to one aspect of the present invention, a nano-drug composition is provided, wherein the active substances of the nano-drug composition include natural alkaloid drugs and tumor cell apoptosis inducing factors; the nano-drug composition further includes hyaluronate and / or PAMAM;

[0007] The natural alkaloid drugs include piperlongumine, and the tumor cell apoptosis inducing factors include TRAIL.

[0008] The nano-drug composition according to the first aspect embodiment of the present invention has at least the following beneficial effects:

[0009] The direct killing effect of piperlongumine in the present invention synergizes with the apoptosis induction mechanism of TRAIL: Piperlongumine (PLG) causes an increase in oxidative stress in tumor cells by generating reactive oxygen species (ROS), disrupts the antioxidant defense system of the cells, and directly leads to the death of cancer cells, enabling TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) to bind to death receptors on the surface of tumor cells, initiate the endogenous apoptosis pathway, and selectively induce apoptosis of tumor cells. The combination of the two significantly enhances the anti-tumor effect and overcomes the drug resistance problem that may exist with a single drug.

[0010] Hyaluronate (HA) precisely delivers piperlongumine (PLG) and TRAIL to the tumor site by interacting with various receptors or molecules in the tumor microenvironment, reducing the toxic effect of the drug on normal cells and ensuring that the drug can exert its maximum efficacy at the tumor site; PAMAM dendrimer, as a nanocarrier, can form a stable carrier complex with HA through electrostatic interaction, which not only increases the water solubility and bioavailability of the drug, but also ensures the continuous release of the drug in tumor cells by regulating the drug release rate, enhancing its anti-tumor effect.

[0011] In the present invention, PLG destroys the antioxidant system of tumor cells by generating reactive oxygen species (ROS), weakening its defense ability; TRAIL triggers the extrinsic apoptosis pathway by activating death receptors (DR4 / DR5). The combination of the two forms a "defense-breaking - apoptosis-promoting" cascade effect. HPPT significantly enhances its anti-cancer effect by inhibiting cell activity, inducing apoptosis, and hindering the growth of tumor tissues.

[0012] According to some embodiments of the present invention, the ratio of piperlongumine to TRAIL is 1.25 - 5 (w / w).

[0013] According to some embodiments of the present invention, the mass ratio of piperlongumine to TRAIL is 1.25:1 to 5:1 (w / w).

[0014] According to some embodiments of the present invention, the nano-drug composition further includes a complex formed by electrostatic interaction between hyaluronate and PAMAM.

[0015] In the present invention, PAMAM dendrimer is a multi-layer branched nanostructure with many amino (-NH2) groups, which have a positive charge at an appropriate pH value. In the present invention, through electrostatic interaction, PLG, TRAIL, and HA can stably form a complex through PAMAM dendrimer, which not only improves the bioavailability of the drug, but also enhances the therapeutic effect by ensuring the stability of the drug in vivo and prolonging its circulation time in vivo.

[0016] The HPPT nano-drug composition of the present invention is spherical or nearly spherical. PLG and PAMAM form a hydrophobic core through hydrophobic interaction to encapsulate piperlongumine amide; the TRAIL plasmid is electrostatically adsorbed on the surface of PAMAM; HA is encapsulated on the outer layer of the complex through electrostatic or hydrogen bond interaction to form a hydrophilic shell layer.

[0017] According to some embodiments of the present invention, the molecular weight of the hyaluronate is 91KDa to 175KDa.

[0018] Low-molecular-weight HA may trigger a pro-inflammatory response, while high-molecular-weight HA has poor penetrability. Selecting HA with a specific molecular weight can balance the targeting efficiency and the tumor tissue penetration ability, and more effectively improve the interaction with various receptors or molecules.

[0019] According to some embodiments of the present invention, the volume ratio of hyaluronate to PAMAM in the carrier complex is 1:1 to 1:10 (v / v).

[0020] According to some embodiments of the present invention, when the drug composition is administered, the dosage concentration of piperlongumine amide is 1.25 to 20 μg / mL.

[0021] Preferably, when the drug composition is administered, the dosage concentration of piperlongumine amide is 1.25 to 20 μg / mL.

[0022] According to some embodiments of the present invention, the dosage concentration of TRAIL is 2 to 4 mg / mL.

[0023] According to the second aspect of the present invention, a preparation method of the above-mentioned drug composition is provided, including the following steps:

[0024] S1. Dissolve piperlongumine amide in an acetone solution, add a PAMAM solution to obtain a PP complex;

[0025] S2. Mix the PP complex and a TRAIL solution to obtain a PPT complex;

[0026] S3. Mix the PPT complex and a hyaluronic acid solution to obtain the drug composition HPPT.

[0027] According to the preparation method of the embodiment of the second aspect of the present invention, it has at least the following beneficial effects:

[0028] During the preparation process of the present invention, the uniform distribution of each component is ensured, and the action mechanism of each component is effectively retained, ensuring the activity and stability of the final drug complex. At the same time, this complexing process avoids complex chemical cross-linking reactions and reduces the occurrence of side reactions. The preparation method of the present invention does not require complex equipment and reaction conditions, so the preparation method of the present invention has a low production cost. The simple and efficient preparation method can greatly improve the production efficiency and reduce the complexity of operation.

[0029] According to some embodiments of the present invention, in step S1, the volume ratio of the piperlongumine solution to the PAMAM solution is 1:10 - 1:3.

[0030] According to some embodiments of the present invention, in step S2, the volume ratio of the PP complex to the TRAIL solution is 10:1 - 1:1.

[0031] According to some embodiments of the present invention, in step S3, the volume ratio of the PPT complex to the hyaluronic acid solution is 1:1 - 1:10.

[0032] Preferably, in step S3, the volume ratio of the PPT complex to the hyaluronic acid solution is 1:9.

[0033] According to some embodiments of the present invention, in step S1, the concentration of the acetone solution is AR grade > 99.5% (v / v).

[0034] According to some embodiments of the present invention, in step S1, the concentration of the piperlongumine solution is 0.5 - 5 mg / mL.

[0035] According to some embodiments of the present invention, in step S1, the solvent of the PAMAM solution is water.

[0036] According to some embodiments of the present invention, in step S1, the concentration of the PAMAM solution is 0.1 - 10 mg / mL.

[0037] According to some embodiments of the present invention, in step S2, the solvent of the TRAIL solution is water.

[0038] According to some embodiments of the present invention, in step S3, the solvent of the hyaluronic acid solution is water.

[0039] According to some embodiments of the present invention, in step S3, the concentration of the hyaluronic acid solution is 1 - 10 mg / mL.

[0040] According to some embodiments of the present invention, in step S1, the mixing and stirring time is 12 - 48 h.

[0041] According to some embodiments of the present invention, in step S2, the time of the mixing and stirring is 1 to 60 min.

[0042] Preferably, in step S2, the time of the mixing and stirring is 5 to 15 min.

[0043] According to some embodiments of the present invention, in step S3, the time of the mixing and stirring is 5 to 60 min.

[0044] Preferably, in step S3, the time of the mixing and stirring is 15 to 30 min.

[0045] According to some embodiments of the present invention, in steps S1, S2, and S3, the speed of the mixing and stirring is 200 to 500 rpm.

[0046] The targeted drug for treating breast cancer according to the third aspect embodiment of the present invention includes the above-mentioned nano-drug composition.

[0047] According to some embodiments of the present invention, the breast cancer is triple-negative breast cancer.

[0048] According to some embodiments of the present invention, the dosage form of the targeted drug is an injection.

[0049] According to some embodiments of the present invention, the targeted drug further includes pharmaceutical excipients.

[0050] Unless otherwise specified, the "about" in the present invention actually means that the allowable error is within the range of ±2%, for example, about 100 is actually 100 ± 2% × 100.

[0051] Other features and advantages of the present invention will be described in the subsequent description, and some of them will be obvious from the description or understood by implementing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, wherein:

[0053] Figure 1 It is a result graph of the influence of the nano-drug composition on the survival rate of 4T1 breast cancer cells for 24 h in Test Example 1 of the present invention;

[0054] Figure 2 It is a result graph of the influence of the nano-drug composition on the survival rate of 4T1 breast cancer cells for 48 h in Test Example 1 of the present invention;

[0055] Figure 3 It is a result graph of the influence of the nano-drug composition on the apoptosis rate of 4T1 breast cancer cells in Test Example 1 of the present invention;

[0056] Figure 4 This is the result graph of the effect of the nano-drug composition on the tumor weight of breast cancer-bearing mice in Test Example 1 of the present invention;

[0057] Figure 5 This is the result graph of the effect of the nano-drug composition on the tumor volume of breast cancer-bearing mice in Test Example 1 of the present invention. Detailed implementation manners

[0058] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0059] In the description of the present invention, the description of reference terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0060] The reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0061] The reagents used in the present invention:

[0062] Piperlongumine (PLG): CAS No. 20069-09-4, Merck & Co., Inc., purity ≥ 97% (HPLC);

[0063] Sodium Hyaluronate 100k-1 (HA), CAS No.: 9067-32-7, molecular weight range: 91KDa - 175KDa, manufacturer: Lifecore.

[0064] Aqueous solution of PAMAM Dendrimer G4-NH2 (CAS No.: 155773-72-1): Aldrich catalog number: 41,244-9, manufacturer: Dendritech, USA; concentration: 9.92% (w / w).

[0065] Acetone solution: CAS: 67 - 64 - 1, Merck, purity ≥ 99.5% (AR).

[0066] Example 1

[0067] This example provides a nano - drug composition, and the active ingredients of the drug composition are piperlongumine, TRAIL, sodium hyaluronate with a molecular weight of 91KDa - 175KDa, and PAMAM G4 - NH2.

[0068] This example also provides a preparation method of the drug composition. The specific process is as follows:

[0069] 1) Preparation of mother liquor: Dissolve piperlongumine in acetone solution and dissolve sodium hyaluronate in water to obtain a mother liquor of HA with a concentration of 1mg / mL and a mother liquor of PLG with a concentration of 5mg / mL; an aqueous solution of PAMAM G4 - NH2 with a concentration of 9.92% (w / w); TRAIL plasmid is extracted from Escherichia coli using GeneStar plasmid extraction kit, with a concentration of 2mg / mL.

[0070] 2) PP complex (PAMAM / PLG): Slowly drip 1mL of acetone solution of PLG into 10mL of 5mg / mL aqueous solution of PAMAM, stir slowly overnight, and evaporate the acetone solution to obtain the PP complex;

[0071] 3) PPT complex (PAMAM / PLG / TRAIL): Drop 100μL of TRAIL plasmid into the drug preparation bottle containing a stir bar and 1mL of PP complex and stir for 10min;

[0072] 4) HPPT: Slowly drip 100μL of PPT complex into the drug preparation bottle containing a stir bar and 900μL of 1mg / mL HA, stir the mixture at a constant speed of 250rpm in a rotary stirrer for 30min, and then let it stand for 30min to achieve uniform mixing of each component and obtain the HPPT nano - drug composition.

[0073] Test example

[0074] Drug preparation used in the test example:

[0075] HP (HA / PAMAM): Slowly add 100μL of PAMAM to 900μL of 1mg / mL HA in a drug preparation bottle containing a stir bar. Subsequently, stir the mixture at a constant speed in a rotary stirrer for 30min to ensure uniform mixing of the components.

[0076] PT complex (PLG / TARIL): 100 μL of TRAIL plasmid was dropped into an acetone solution containing a stir bar and 1 mL of PLG, and the mixture was slowly stirred overnight to obtain the PT complex.

[0077] HPP (HA / PAMAM / PLG): 100 μL of the PP complex was dropped into a drug preparation bottle containing a stir bar and 900 μL of 1 mg / mL HA. The mixture was stirred at a constant speed in a rotary stirrer for 30 min to ensure thorough mixing of all components.

[0078] HPT (HA / PAMAM / TARIL): 100 μL of the PT complex was added to a drug preparation bottle containing a stir bar and 900 μL of 1 mg / mL HA. The mixture was stirred at a constant speed in a rotary stirrer for 30 min to ensure thorough mixing of all components.

[0079] The criterion for successful preparation of each nanodrug is that when a laser pen is used to irradiate the drug solution, an obvious Tyndall effect can be observed.

[0080] Test Example 1. Effect of the drug composition on the activity of mouse breast cancer cells 4T1

[0081] 1. Experimental materials

[0082] (1) Experimental cells: Mouse breast cancer cells (4T1) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0083] 2. Experimental methods

[0084] (1) Culture and treatment of mouse breast cancer cells (4T1)

[0085] Mouse breast cancer cells (4T1) were cultured in RPMI-1640 culture medium containing 10% fetal bovine serum and 1% double antibody (100 U / mL penicillin, 100 mg / L streptomycin), and passaged in a 37 °C, 5% CO2 incubator. Adherent cells were digested with 0.25% trypsin and passaged twice a week. The experimental cells were all in the logarithmic growth phase.

[0086] (2) Detection of the effect of different drugs on the proliferation of 4T1 cells by the CCK-8 method

[0087] The breast cancer cells 4T1 were seeded at a density of 1×10 6Cells were seeded into 96-well plates at a density of

[0088] 3. Experimental Results

[0089] Determined by the CCK-8 assay, the results showed that with the increase of PLG concentration, the cell viability of each treatment group showed a downward trend. Especially after treatment with 20 μg / mL PLG for 24 hours, the cell viability was lower than 50%. And compared with the simple PLG group, the PPT, HPT groups and the HPPT group showed higher cytotoxicity, demonstrating the targeting effect of HA and the delivery effect of the HA / PAMAM vector. After treatment with the TRAIL plasmid alone for 24 hours, the cell viability was still higher than 90%, but after targeted delivery in combination with PLG, the cell viability decreased to 30% (PLG concentration was 20 μg / mL). And at the same PLG concentration, the HPPT group showed stronger toxicity than the PPT, HPT and PLG groups, indicating that the combined treatment had a synergistic enhanced cytotoxic effect. In addition, the 48-hour treatment group showed lower cell viability than the 24-hour treatment group, suggesting that the extension of the treatment time might enhance the cytotoxic effect, and the results were as Figure 1 and Figure 2 shown.

[0090] Test Example 2 Effect of Drugs on Apoptosis of Mouse Breast Cancer Cells 4T1

[0091] 1. Experimental Method

[0092] 4T1 cells were seeded at a density of 1×10 6Cells were inoculated at a density of cells / well in a 6-well plate. After culturing for 24 hours, a medium containing the drug or normal saline was added, and the cells were cultured in an incubator for another 24 hours. Then the cells were washed 3 times with PBS, and about 1-10 million cells from each sample were collected into a 1.5 ml centrifuge tube. The supernatant was discarded after centrifugation. The cell pellet was resuspended in 0.8-1 ml of cell staining buffer. 5 μL of Hoechst staining solution and 5 μL of propidium iodide (PI) staining solution were added, mixed well, and incubated on ice or at 4 °C for 20-30 min. Finally, flow cytometry was used to detect the red fluorescence and blue fluorescence to detect cell apoptosis.

[0093] 2. Experimental results

[0094] As follows Figure 3 As shown, the apoptosis rate of the untreated group was the lowest, and almost no apoptosis occurred. The apoptosis rate of the group treated with HA-PAMAM alone was not significantly increased either. After treatment with PLG alone, the percentage of apoptotic cells increased by more than 60% compared with the control group, showing a certain pro-apoptotic effect. However, on the basis of combined use of HA-PAMAM, when PLG (HPP group) or TRAIL (HPT group) was added, the apoptosis rate was further increased. This enhancement effect was most significant in the HPPT group treated with HA+PAMAM+PLG+TRAIL, and the proportion of apoptotic cells reached the highest value of 78%, indicating that the combined treatment of PLG and TRAIL has a synergistic effect in promoting cell apoptosis.

[0095] Test Example 3 Effect of the drug on the tumor size of tumor-bearing mice

[0096] 1. Experimental mice: Balb / c mice at 4-6 weeks of age were purchased from Zhuhai Bestong Biological Research Co., Ltd.

[0097] 2. Experimental method

[0098] Balb / c mice at 4-6 weeks of age, weighing 16-20 g, were randomly grouped after being raised in a pathogen-free environment for one week. Single-cell suspensions were prepared from 4T1 cells in the logarithmic growth phase at a concentration of 5×10 7 cells / mL, and then 100 μL of the cell suspension was inoculated subcutaneously into the abdominal side of the mice. When the tumor volume expanded to 50 mm 3 3, tail vein injection was used for drug administration, and the change in tumor volume was measured using a vernier caliper during the period. The formula for calculating the tumor volume is: V = (a × b 2) / 2. Where V is the tumor volume, a is the major axis of the tumor, and b is the minor axis of the tumor. The mice were divided into 6 groups, with 6 mice in each group: The administration group 1 was injected with 100 μL of normal saline, the administration group 2 was injected with 100 μL of HP, the administration group 3 was injected with 100 μL of PLG, the administration group 4 was injected with 100 μL of HPP, the administration group 5 was injected with 100 μL of HPT, and the administration group 6 was injected with 100 μL of HPPT. The drug was administered once every two days. After three weeks of drug administration, the mice were sacrificed, and their subcutaneous tumor tissues were removed and weighed.

[0099] 3. Experimental Results

[0100] As Figure 4 The tumor volume data showed that compared with the administration group 1, the tumor volumes of each treatment group decreased to varying degrees. Among them, the HPPT group had the most significant tumor inhibitory effect, indicating that the combination therapy had a synergistic enhanced anti-tumor effect. Over time, especially on the 9th and 11th days, the tumor volume of the HPPT group was significantly lower than that of other treatment groups, showing the best treatment effect. In addition, according to Figure 5 The results of weighing the mouse tumor tissues also showed that the tumor weight of the HPPT group was significantly lower than that of other groups, further confirming the effectiveness of the combination therapy. These results suggest that the combined application of HA, PAMAM, PLG, and TRAIL may be used as an effective anti-tumor treatment strategy, and its mechanism may involve enhancing tumor cell apoptosis and inhibiting tumor growth.

[0101] In summary, the present invention shows a significant synergistic effect in inhibiting breast cancer cells and tissues by using piperlongumine in combination with hyaluronic acid, polyamidoamine dendrimer, and TRAIL. HPPT achieves combined therapy with multiple targets by inhibiting cell activity, inducing apoptosis, hindering the growth of tumor tissues, targeted delivery, and the stability and release control of drug carriers, significantly enhancing its anti-breast cancer effect.

[0102] The above has described the embodiments of the present invention in detail with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the knowledge scope of those of ordinary skill in the art to which the present invention pertains, various changes can be made without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A nano-drug composition, characterized in that, The active substances of the nano-drug composition include natural alkaloid compounds and tumor cell apoptosis inducing factors; the nano-drug composition also includes hyaluronate and / or PAMAM; The natural alkaloid drug includes piperlongumine, and the tumor cell apoptosis inducing factor includes TRAIL plasmid.

2. The nano-drug composition according to claim 1, wherein The mass ratio of piperlongumine to TRAIL is 1.25:1 to 5:1 (w / w).

3. The nano-drug composition according to claim 1, wherein The molecular weight of the hyaluronate is 91KDa to 175KDa.

4. The nano-drug composition according to claim 1, wherein The PAMAM is a fourth-generation PAMAM dendrimer polymer with an amino end group.

5. The nano-drug composition according to claim 1, characterized in that, The volume ratio of the hyaluronate to PAMAM is 1:1 to 1:10 (v / v).

6. The nano-drug composition according to claim 1, characterized in that, The nano-drug composition is a liquid preparation; Preferably, the concentration of piperlongumine in the nano-drug composition is 1.25 to 20 μg / mL; Preferably, the concentration of TRAIL in the nano-drug composition is 2 to 4 μg / mL.

7. A method for preparing a nano-drug composition according to any one of claims 1 to 6, characterized in that, Comprising the following steps: S1. Dissolve piperlongumine in an acetone solution, add a PAMAM solution to obtain a PP complex; S2. Mix the PP complex and a TRAIL solution to obtain a PPT complex; S3. Mix the PPT complex and a hyaluronic acid solution to obtain the drug composition HPPT.

8. The preparation method according to claim 7, characterized in that, In step S1, the volume ratio of the piperlongumine solution to the PAMAM solution is 1:10 - 1:

3.

9. The preparation method according to claim 7, wherein In step S2, the volume ratio of the PP complex to the TRAIL solution is 10:1 to 1:1; preferably, in step S3, the volume ratio of the PPT complex to the hyaluronic acid solution is 1:1 to 1:

10.

10. Use of the nano-drug composition according to any one of claims 1 to 7 or the nano-drug composition prepared by the preparation method according to any one of claims 8 to 9 in the preparation of a drug for preventing and / or treating breast cancer.

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