Carrier-free self-assembled nanodrug and preparation method and application thereof

Abstract

The application discloses a carrier-free self-assembled nanodrug as well as a preparation method and application thereof. The nanodrug is a nanoparticle obtained by co-assembly of a water-soluble drug and a fat-soluble drug. The water-soluble drug is epigallocatechin gallate, and the fat-soluble drug is homoharringtonine. The preparation method of the nanodrug is simple, easy to synthesize and capable of realizing batch preparation. The two drugs are self-assembled into the nanoparticle without using a carrier, thereby reducing the toxicity risk and having good biocompatibility. The obtained nanodrug has higher bioavailability than a single drug. After the two drugs are combined, the nanodrug can play a synergistic anti-tumor effect through different mechanisms and improve the anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the field of drug nanotechnology, specifically to a carrier-free self-assembled nanomedicine, its preparation method, and its application. Background Technology

[0002] Breast cancer is a disease in which mammary duct epithelial cells, under the influence of various internal and external carcinogenic factors, lose their normal characteristics and proliferate abnormally, exceeding the limits of self-repair and becoming cancerous. It is one of the most common malignant tumors in women. Currently, surgery, chemotherapy, and radiotherapy remain common treatments for cancer. Chemotherapy uses chemical drugs to inhibit the proliferation, invasion, and metastasis of cancer cells, ultimately killing them. It is the primary treatment for most solid tumors and hematologic malignancies. The chemical drugs used in chemotherapy are called chemotherapy drugs.

[0003] Studies have found that natural products not only have synergistic anti-tumor effects but can also improve patients' quality of life by reducing the side effects of radiotherapy and chemotherapy. They demonstrate effectiveness either by altering the course of cancer development or by blocking processes such as cell differentiation, cell growth, angiogenesis, apoptosis, and metastasis. This has led to an increasing number of chemotherapy drugs prepared from natural products. However, due to the emergence of chemotherapy drug resistance and dose-dependent toxic side effects, single-drug therapy can no longer meet clinical needs, thus necessitating the use of combination chemotherapy drugs to address this problem.

[0004] While the combined use of multiple chemotherapy drugs can reduce the toxic side effects and the possibility of drug resistance caused by monotherapy, it also presents challenges such as frequent dosing and difficulty in determining the dosage distribution. Chemotherapy drug co-delivery systems offer an effective solution to these problems. These systems encapsulate two or more chemotherapy drugs in a single carrier, leveraging the synergistic effect between the drugs to enhance their tumor-killing effect. Compared to monotherapy and combination therapy, co-delivery systems offer the following advantages: 1) Two or more chemotherapy drugs exert a synergistic effect, producing a stronger anti-tumor effect at a smaller dose, reducing toxic side effects, and avoiding the development of drug resistance; 2) The timing and location of drug release can be controlled to coordinate the pharmacokinetics and pharmacodynamics of different drugs; 3) The frequency of dosing is reduced, improving patient compliance; 4) A single dosing dose is administered, avoiding the uncertainty of dosage distribution when administering multiple drugs.

[0005] Currently, a series of nanomedicine delivery systems have been established, including polymer micelles, nanoparticles, nanotubes, and nanogels. However, most of these nanomedicine delivery systems require a carrier, and the introduction of nanocarriers complicates the preparation process of nanoformulations. Furthermore, the introduced carrier materials are often difficult to degrade, which can lead to additional toxic effects. Therefore, the search for novel, safe, simple, and efficient antitumor nanoformulations has become a current research hotspot. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a carrier-free self-assembled nanomedicine, thereby solving the technical problems mentioned above. Most current nanomedicine delivery systems require the use of carriers, and the introduction of nanocarriers leads to complex preparation processes for nano-formulations. Furthermore, the introduced carrier materials are often difficult to degrade, which can result in additional toxic effects.

[0007] To achieve this objective, the present invention adopts the following technical solution: A first aspect of the present invention is to provide a carrier-free self-assembled nanomedicine, wherein the nanomedicine is nanoparticles obtained by co-assembling a water-soluble drug and a lipid-soluble drug, wherein the water-soluble drug is epigallocatechin gallate and the lipid-soluble drug is homoharringtonine.

[0008] In one alternative embodiment, the nanoparticles are spherical in shape and have an average particle size of 240.67 nm.

[0009] In one optional embodiment, the mass ratio of epigallocatechin gallate to fat-soluble homoharringtonine is (1-3):1.

[0010] A second aspect of the present invention is to provide a method for preparing carrier-free self-assembled nanomedicines, comprising the following steps: (1) Dissolve the base of homoharringtonine in dimethyl sulfoxide to obtain the first solution; (2) Dissolve epigallocatechin gallate in deionized water to obtain a second solution; (3) Add the first solution to the second solution to obtain a mixed solution; (4) The mixed solution is vortexed and heated in a water bath at 37°C for 72 hours under light-protected conditions. It is then centrifuged at 15000 r for 30 minutes, the precipitate is washed repeatedly, and then deionized water is added to reconstitute it to obtain carrier-free self-assembled nanoparticle drugs.

[0011] In an alternative embodiment, in step (3), 20-40 μL of the first solution is added to the second solution every 15 seconds.

[0012] A third aspect of the present invention is to provide the application of carrier-free self-assembled nanomedicines in the preparation of antitumor drugs.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation method is simple, easy to synthesize, and can be mass-produced.

[0014] (2) The two drugs self-assemble to form nanoparticles without the need for a carrier, reducing the risk of toxicity and exhibiting good biocompatibility.

[0015] (3) The obtained nano-formulation has higher bioavailability than single drugs. The combination of the two drugs can play a synergistic anti-tumor role through different mechanisms, thereby improving the anti-tumor effect. Attached Figure Description

[0016] Figure 1 This is a morphological structure diagram of the nanomedicine prepared in Example 1 of the present invention; Figure 2 This is a diagram showing the particle size distribution of the nanomedicine prepared in Example 1 of this invention; Figure 3 This is a potential diagram of the nanomedicine prepared in Example 1 of the present invention; Figure 4 The ultraviolet spectrum of the nanomedicine prepared in Example 1 of this invention; Figure 5 The infrared spectrum of the nanomedicine prepared in Example 1 of this invention; Figure 6 The results of cytotoxicity tests are shown for the nanomedicine prepared in Example 1 of this invention. Figure 7 The image below shows a scratch test performed on the nanomedicine prepared in Example 1 of this invention. Figure 8 Microscopic image below of the scratch test performed on the epigallocatechin gallate ester of the present invention; Figure 9 Microscopic image below of the scratch test performed on the homoharringtonine alkaloid of the present invention; Figure 10 This is a diagram showing the inhibitory effect of the nanomedicine prepared in Example 1 of the present invention on cell migration; Figure 11 The image below shows a microscopic view of the cell viability staining experiment performed on the nanomedicine prepared in Example 1 of this invention. Figure 12 The effect of the nanomedicine prepared in Example 1 of this invention on the activity of tumor cells. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0018] Among the raw materials used in the following examples, homoharringtonine (Shanghai Titan Technology Co., Ltd.), CAS No.: 26833-87-4; epigallocatechin gallate (Shanghai Titan Technology Co., Ltd.), CAS No.: 989-51-5.

[0019] Example 1 A method for preparing carrier-free self-assembled nanomedicines includes the following steps: Step 1: Dissolve 1g of homoharringtonine (HHT) in dimethyl sulfoxide to obtain the first solution.

[0020] Step 2: Dissolve 1g of epigallocatechin gallate (EGCG) in deionized water to obtain a second solution.

[0021] Step 3: Take 100 μL of the first solution and add it dropwise to 100 μL of the second solution at a rate of 15 s / 20 μL (20 μL every 15 s) to obtain the third solution.

[0022] Step 4: Gently vortex the third solution until well mixed, then heat in a 37°C water bath for 3 days in the dark.

[0023] Step 5: Add 1 ml of deionized water to break the polarity, centrifuge at 15000 r for 30 min, wash the precipitate three times, and redissolve it with deionized water to obtain the self-assembled nanomedicine (HE).

[0024] Example 2 A method for preparing carrier-free self-assembled nanomedicines includes the following steps: Step 1: Dissolve 1g of homoharringtonine base in dimethyl sulfoxide to obtain the first solution.

[0025] Step 2: Dissolve 2g of epigallocatechin gallate in deionized water to obtain a second solution.

[0026] Step 3: Take 100 μL of the first solution and add it dropwise to 100 μL of the second solution at a rate of 10 s / 20 μL (20 μL every 10 s) to obtain the third solution.

[0027] Step 4: Gently vortex the third solution until well mixed, then heat in a 37°C water bath for 3 days in the dark.

[0028] Step 5: Add 1 ml of deionized water to break the polarity, centrifuge at 20000 r for 45 min, wash the precipitate three times, and redissolve it with deionized water to obtain the self-assembled nanomedicine.

[0029] Example 3 A method for preparing carrier-free self-assembled nanomedicines includes the following steps: Step 1: Dissolve 1g of homoharringtonine base in dimethyl sulfoxide to obtain the first solution.

[0030] Step 2: Dissolve 3g of epigallocatechin gallate in deionized water to obtain a second solution.

[0031] Step 3: Take 100 μL of the first solution and add it dropwise to 100 μL of the second solution at a rate of 20 μL / 20s (20 μL every 20s) to obtain the third solution.

[0032] Step 4: Gently vortex the third solution until well mixed, then heat in a 37°C water bath for 3 days in the dark.

[0033] Step 5: Add 1 ml of deionized water to break the polarity, centrifuge at 25000 r for 20 min, wash the precipitate three times, add deionized water to redissolve, and you can obtain the self-assembled nanomedicine.

[0034] I. Transmission Electron Microscopy Characterization The nanomedicine prepared in Example 1 of this invention was uniformly dispersed in water, and 10 μL was dropped onto a copper grid. After the water was evaporated, the grid containing the sample was placed in a transmission electron microscope, and the microstructure of the nanoparticles was observed. The results are as follows: Figure 1 As shown, Figure 1 As can be seen, the nanoparticles formed by the self-assembly of lipid-soluble homoharringtonine and water-soluble epigallocatechin gallate are nanospheres with a particle size of 240.67±23.53 nm. Figure 1 b is the particle size distribution diagram of nanoparticles formed by the self-assembly of lipid-soluble homoharringtonine alkaloid and water-soluble epigallocatechin gallate.

[0035] II. Detection of water, particle size, and zeta potential After the nanomedicine prepared in Example 1 was uniformly dispersed in water, the water content, particle size, and potential of the nanoparticles were measured using a nanoparticle size analyzer. The results are as follows: Figure 2 and Figure 3 As shown.

[0036] from Figure 2It can be seen that HHT and EGCG form spherical nanoparticles with a diameter of 284.4 ± 15.64 nm; from Figure 3 The HE potential distribution is -15.97±4.64 mV, which is much lower than that of single-drug HHT and EGCG.

[0037] III. Ultraviolet Spectroscopic Characterization The nanomedicine prepared in Example 1 of this invention was uniformly dispersed in water, and a full-wavelength scan was performed using an ultraviolet spectrometer. The results are as follows: Figure 4 As shown. From Figure 4 As can be seen, homoharringtonine (HHT) has an absorption peak at 288 nm, and epigallocatechin gallate (EGCG) has an absorption peak at 273 nm. After the two self-assemble to form nanomedicine (HE), the absorption peaks of both changed position. This is due to the π-π conjugation effect between homoharringtonine and epigallocatechin gallate, resulting in an absorption peak at 281 nm. This indicates that homoharringtonine and epigallocatechin gallate successfully self-assembled to synthesize nanoparticles.

[0038] IV. Infrared Characterization The surface functional groups of EGCG, HHT and its self-assembled complex HE were detected and analyzed by FT-IR infrared spectroscopy. The wavenumber range was 4000~400 cm-1. The samples were prepared by KBr pellet method.

[0039] The results are as follows Figure 5As shown, EGCG exhibits a broad and strong absorption peak in the 3600-3200 cm⁻¹ range, corresponding to the stretching vibration of the phenolic hydroxyl group (-OH). An aromatic ring C=C stretching vibration peak is observed near 1600 cm⁻¹, an aromatic skeleton vibration near 1510 cm⁻¹, and a COH bending vibration near 1270 cm⁻¹. These peaks collectively reflect the abundant phenolic hydroxyl structure in EGCG. HHT shows a moderate-intensity absorption peak near 3000 cm⁻¹, belonging to the aromatic ring CH stretching vibration, and may also contain weak stretching absorptions of some hydroxyl groups (-OH) or amides (NH). An aliphatic CH (-CH₂, -CH₃) stretching vibration is observed near 2930 cm⁻¹, an amide carbonyl C=O stretching vibration is observed at 1650 cm⁻¹, and a CN bending vibration is observed at 1450 cm⁻¹. This reflects that HHT possesses a carbonyl group, an aromatic ring, and a small amount of hydroxyl structures, enabling it to form hydrogen bonds or π-π stacking. In contrast, the infrared spectrum of the self-assembled complex HE shows a broadening and redshift of the -OH absorption peak at 3400 cm⁻¹, indicating that EGCG and HHT may form a stable complex through hydrogen bonding or electrostatic interactions. Simultaneously, the intensity of the aromatic CH peak near 3000 cm⁻¹ decreases, suggesting that the hydrogen atoms on the aromatic ring of HHT are shielded by EGCG or partially participate in the interaction. The overlapping and shifting of peaks in the 1650 cm⁻¹ (C=O) and 1600 cm⁻¹ (C=C) regions suggest that the electron cloud density changes are caused by π-π stacking or hydrogen bonding coordination between HHT and EGCG. These results indicate that the formation of the self-assembled complex HE involves the self-assembly of EGCG and HHT through intermolecular hydrogen bonding, aromatic ring π-π interactions, and hydrophobic interactions, with intermolecular interactions leading to the shift and intensity changes of the original characteristic peaks.

[0040] V. Cytotoxicity Test The cytotoxicity of HE synthesized in Example 1 against breast cancer cells was evaluated using the CCK8 assay.

[0041] 4T1 (breast cancer cells) (1.5 × 10⁻⁶) were seeded in 96-well plates. 4Cells / well), 100 μL of culture medium was added to each well; the culture plate was placed in an incubator at 37℃ with 5% CO2 and cultured for 24 h. Cell growth was observed under a microscope. When the cell adhesion rate reached 60-70%, the supernatant was discarded, and 100 μL of different concentrations of HE, HHT, and EGCG solutions (concentrations of 62.5, 31.25, 15.625, 7.8125, and 3.90625 ng / mL, respectively) were added to each well. A control group (10% FBS RPMI-1640 medium + cells) and a blank group (10% FBS RPMI-1640 medium) were also set up. All groups were incubated at 37℃ with 5% CO2. After 24 h of incubation, the culture medium was removed from all groups, and then 100 μL of the prepared 10% HHT solution was added to each well. Prepare CCK-8 culture medium and aspirate thoroughly. Incubate the culture plate in an incubator for 1 hour. After incubation, measure the absorbance at 450 nm using a microplate reader. Cell viability is calculated using the following formula:

[0042] Where As is the absorbance of the experimental group, Ab is the absorbance of the blank group, and Ac is the absorbance of the control group.

[0043] Each group was tested in triplicate, and the average value was taken. The cytotoxicity of EGCG, HE, and HHT to cells was evaluated based on the obtained cell viability (CV%).

[0044] The results are as follows Figure 6 As shown, compared with the control group, EGCG, HHT, and HE all exhibited a dose-dependent decrease in cell viability. EGCG had a weak effect on cell viability within the range of 3.91–62.5 ng / mL, with cell survival generally maintained above 90%, suggesting low cytotoxicity to 4T1 cells. In contrast, cell viability in the HHT-treated group gradually decreased with increasing concentration, dropping to approximately 40% at 62.5 ng / mL. HE showed significantly enhanced cell inhibition at all corresponding concentrations, exhibiting stronger cytotoxic activity and a more pronounced dose-dependent effect compared to EGCG and HHT. It caused a decrease in cell viability to approximately 40% at 15.625 ng / mL, and at the highest concentration of 62.5 ng / mL, cell viability was only about 20%–25%. These results indicate that the HHT-EGCG self-assembled complex HE has a significant synergistic antitumor effect, significantly stronger than the single-agent drugs at the same dosage.

[0045] VI. Scratch Test 4T1 (breast cancer cells) (40 × 10⁻⁶ cells) were seeded in 12-well plates. 4Cells / well), 1000 μL of culture medium was added to each well; the culture plate was placed in an incubator at 37°C with 5% CO2 for 24 h. Cell growth was observed under a microscope. When the cell adhesion rate reached 90-100%, a 200 μL pipette tip was held perpendicular to the bottom of the plate, and a single, even, and straight stroke was made on the cell layer. The old culture medium was discarded, and the cells were gently rinsed 2-3 times with sterile PBS to remove cell debris. The plate was then placed under a microscope, the stroke location was marked, and at least three different fields of view were captured at 0 h under a 10x objective lens. According to the experimental design, different concentrations of HHT, EGCG, and HE solutions were added to each well (the concentrations for the three groups were 31.25, 15.625, and 7.8125 ng / mL, respectively), and a control group (10% FBS RPMI-1640 medium + cells) was set up. All groups were incubated at 37°C with 5% CO2 for 24 h and 48 h. Observe the speed at which the scratches close and take photographs at the same location using a microscope. The results are as follows: Figure 7-9 As shown. The widths of three vertical scratches were measured to assess cell migration. The results are as follows. Figure 10 As shown.

[0046]

[0047] in : 0-hour scratch width. The width of the scratch after t hours.

[0048] from Figure 10 As shown in Figure a, at 24 hours, the control group exhibited active cell migration and significant scratch healing. With increasing drug concentration, the scratch healing rates in the HE, HHT, and EGCG groups all decreased to varying degrees. However, the EGCG group only showed a slight inhibitory effect on migration, while the HE group showed the most significant inhibitory effect on cell migration at the same concentration. Figure 10 As shown in b, at 48 hours, the scratches in the control group were nearly completely healed; the EGCG group maintained a weak inhibitory effect, the HHT group showed moderate migration inhibition, while the healing rate in the HE group had decreased to approximately 22% at 31.25 ng / ml, indicating the most significant inhibition. These results are consistent with the trends in cytotoxicity and cell viability assays, confirming that HE not only has a stronger cytotoxic effect but also significantly inhibits the migration ability of tumor cells.

[0049] VII. Cell Viability Staining 4T1 (breast cancer cells) (1.5 × 10⁻⁶) were seeded in 96-well plates. 4Cells / well), 100 μL of culture medium was added to each well; the culture plate was placed in an incubator at 37℃ with 5% CO2 for 24 h, and the cell growth status was observed under a microscope. When the cell adhesion rate reached 70-80%, the supernatant was discarded, and 100 μL of different concentrations of HE, EGCG, and HHT solutions were added to each well (the concentrations of all groups were 31.25, 15.625, and 7.8125 ng / mL, respectively). A control group (10% FBSRPMI-1640 medium + cells) was also set up. All groups were placed in an incubator at 37℃ with 5% CO2 for 24 h. After the culture medium was removed from all groups, Calcein AM and PI dyes were added to each well to stain the cells. The culture plate was incubated in an incubator for 0.5 h, and then the stained cells were imaged using a confocal microscope. Green fluorescence indicated live cells, and red fluorescence indicated dead cells. The results are shown below. Figure 11 As shown.

[0050] The cell necrosis rate was statistically analyzed based on the confocal microscope image below, and the results are as follows: Figure 12 As shown, the HE, HHT, and EGCG groups all exhibited some degree of cell death, with a dose-dependent trend increasing with concentration. Comparative results showed that, under the same concentration conditions, the EGCG group showed less change in cell death rate, indicating only mild killing; the cell death rate in the HHT group gradually increased with increasing dose; while the HE-induced cell necrosis rate was significantly higher than that in the HHT and EGCG groups, indicating that HE can more effectively induce 4T1 cell death, and its cytotoxic effect is significantly better than that of single drugs. This result is consistent with the trends in cytotoxicity and scratch assays, confirming that HE has a stronger ability to inhibit tumor cell activity and induce cell death.

[0051] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0052] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A carrier-free self-assembled nanomedicine, characterized in that, The nanomedicine is nanoparticles obtained by co-assembling a water-soluble drug and a lipid-soluble drug. The water-soluble drug is epigallocatechin gallate, and the lipid-soluble drug is homoharringtonine. The mass ratio of epigallocatechin gallate to homoharringtonine is (1-3):

1.

2. The carrier-free self-assembled nanomedicine according to claim 1, characterized in that, The nanoparticles are spherical in shape with an average particle size of 240.67 nm.

3. A method for preparing a carrier-free self-assembled nanomedicine according to claim 1, characterized in that, The steps include the following: (1) Dissolve the base of homoharringtonine in dimethyl sulfoxide to obtain the first solution; (2) Dissolve epigallocatechin gallate in deionized water to obtain a second solution; (3) Add the first solution to the second solution to obtain a mixed solution; (4) The mixed solution is vortexed and heated in a water bath at 37°C for 72 h under light-protected conditions. It is then centrifuged at 15000 r for 30 min, the precipitate is washed repeatedly, and then deionized water is added to reconstitute it to obtain carrier-free self-assembled nanomedicine.

4. The method for preparing carrier-free self-assembled nanomedicines according to claim 3, characterized in that, In step (3), 20-40 μL of the first solution is added to the second solution every 15 seconds.

5. The application of the carrier-free self-assembled nanomedicine according to any one of claims 1-2 in the preparation of anti-breast cancer drugs.

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