An anti-tumor nano-drug loaded with natural antioxidants and a screening method for its loading ratio

Abstract

The present invention discloses an anti-tumor nano-drug loaded with natural antioxidants, belonging to the field of biomedicine. The natural antioxidant nano-drug of the present invention includes a nano-carrier and two or more natural antioxidants simultaneously loaded in the nano-carrier in a certain proportion. This nano-drug can effectively prolong the blood circulation time of the antioxidants, promote the enrichment of the antioxidants at the tumor tissue, and can deliver the natural antioxidants into the tumor cells in an optimal proportion, thereby producing a highly synergistic tumor suppression effect, enabling the natural antioxidant nano-drug to efficiently kill tumors and their drug-resistant tumor cells when the dosage is equivalent to that of chemotherapy drugs, and having a high tumor suppression effect on various tumor models, and also having an anti-multi-drug resistance effect. At the same time, this nano-drug has excellent biocompatibility and hardly causes any toxic and side effects to the body, and can solve the problem that chemotherapy drugs are prone to produce toxic and side effects. Therefore, the development of this natural antioxidant nano-drug can promote the application of natural antioxidants in clinical tumor treatment and provide a new treatment method for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to natural antioxidant nano-drugs. Technical Background

[0002] Natural antioxidants have certain anti-tumor activities, and have selective inhibitory effects on tumor cells and normal cells, and are an ideal anti-tumor drug. However, due to the low toxicity of these antioxidants themselves, short circulation time in vivo, and no tumor targeting, they only show certain tumor inhibitory effects at extremely high doses and have no practical clinical application value. At the same time, at high doses, they also inevitably bring certain side effects.

[0003] The combination strategy is an effective method to improve the efficacy of drugs. Physically mixing and combining multiple natural antioxidants can improve their inhibitory effect on tumor cells and reduce the single-drug dosage, but its dosage is still far higher than that of chemotherapy drugs; at the same time, it still has the disadvantages of short blood circulation time and no tumor targeting when used alone in vivo. As is well known, when drugs are combined, the combination ratio is crucial, which will affect the combination effect of the drugs. When natural antioxidants are physically mixed and combined, they cannot enter tumor cells at a fixed ratio in vivo. Therefore, the current combination of antioxidants cannot achieve a highly efficient and synergistic tumor inhibitory effect in vivo. Summary of the Invention

[0004] Aiming at the above problems, the present invention combines the advantages of nanotechnology and natural antioxidants, and develops a novel natural antioxidant nano-drug, which simultaneously loads two or more natural antioxidants onto a nanocarrier at a certain ratio. This nano-drug can effectively prolong the blood circulation time of the antioxidants, promote the enrichment of the antioxidants at the tumor tissue, and can deliver the natural antioxidants into tumor cells at a fixed ratio, thereby producing a highly efficient and synergistic tumor inhibitory effect, making the natural antioxidant nano-drug produce a therapeutic effect comparable to that of chemotherapy drugs. At the same time, this nano-drug has excellent biocompatibility and anti-multidrug resistance effects, hardly causing any toxic side effects to the body, effectively overcoming the problem that chemotherapy drugs are prone to produce toxic side effects, and providing a new option for tumor treatment.

[0005] The present invention includes the following technical solutions:

[0006] An anti-tumor nano-drug loaded with natural antioxidants, comprising a nanocarrier and two or more natural antioxidants simultaneously loaded onto the nanocarrier at a certain ratio. By simultaneous loading, various natural antioxidants can enter cells at a fixed ratio to achieve the best synergistic effect.

[0007] As an alternative, in the above-mentioned nano-drugs, the natural antioxidant is selected from lipoic acid (LA), vitamin C (VC), vitamin K3 (VK3), anthocyanin (ATC), thymol quinone (TQ), curcumin (Cur), genistein, and pharmaceutically acceptable derivatives thereof. The derivatives are pharmaceutically acceptable salts or pharmaceutically acceptable modified products obtained by non-substantially modifying the natural antioxidant without affecting the exertion of its core function (including but not limited to grafting functional groups onto the natural antioxidant molecule).

[0008] As an alternative, in the above-mentioned nano-drugs, the nano-carrier is selected from common nano-drug carriers such as liposomes, vesicles, micelles, etc.

[0009] As an alternative, in the above-mentioned nano-drugs, the natural antioxidant is a small molecule antioxidant; the molecular weight of the small molecule antioxidant is less than 1000.0 Da, and it can be effectively loaded into the nano-carrier.

[0010] As an alternative, in the above-mentioned nano-drugs, the molar ratio between the natural antioxidants is 1:100 to 100:1.

[0011] As an alternative, in the above-mentioned nano-drugs, the ratio between the natural antioxidants is the optimal ratio of antioxidant synergistic effect screened by in vitro methods.

[0012] As an alternative, in the above-mentioned nano-drugs, the natural antioxidants can be recycled and regenerated with each other inside cells.

[0013] As an alternative, in the above-mentioned nano-drugs, one of the natural antioxidants is lipoic acid and / or lipoic acid derivatives. Further, the remaining natural antioxidants are antioxidants that can be recycled with lipoic acid. During the process of the remaining natural antioxidants being recycled with lipoic acid, it can promote the production of reactive oxygen species (ROS). When these small molecule antioxidants exist alone in cells, they are easily metabolized quickly. The remaining natural antioxidants can generate ROS by losing electrons in cells and are oxidized themselves (in the oxidized state and unable to continue generating ROS); while the dihydrolipoic acid (DHLA) generated by the reduction of LA in cells is a strong reducing agent. DHLA can reduce these oxidized compounds and then can continue to generate ROS. At this time, DHLA is oxidized to LA (which can be reduced to DHLA again to achieve the LA cycle). At the same time, ROS can also be generated during the DHLA cycle. Thus, the mutual recycling of LA and other antioxidants is achieved. The mutual recycling of LA and other antioxidants slows down the metabolism of LA and the remaining natural antioxidants and reduces the amount of them excreted out of the cell. At the same time, it can promote the production of ROS to maintain a high level of natural antioxidants in the cell and play a stronger anti-tumor role.

[0014] As an alternative, in the above-mentioned nano-drugs, the natural antioxidant is LA and VC. Further, the molar ratio of VC to LA is 2:1.

[0015] As an alternative, in the above-mentioned nano-drugs, the natural antioxidants are LA, VC and VK3. Further, the molar ratio of LA, VC and VK3 is 2:1:1.

[0016] As an alternative, in the above-mentioned nano-drugs, the remaining natural antioxidants are VC and ATC. Further, the molar ratio of VC to ATC is 2:1.

[0017] As an alternative, in the above-mentioned nano-drugs, the remaining natural antioxidants are LA and TQ. Further, the molar ratio of VC to TQ is 1:1.

[0018] As an alternative, in the above-mentioned nano-drugs, the remaining natural antioxidants are LA and Cur. Further, the molar ratio of LA to Cur is 2:1.

[0019] As an alternative, the particle size of the above-mentioned nano-drugs is less than 500 nm, preferably 100 - 200 nm. The nano-drugs in this particle size range can be enriched at the tumor site through the enhanced permeability and retention effect (EPR effect) of solid tumor tissues to achieve passive targeting effect.

[0020] The present invention also discloses an in vitro screening method for the loading ratio of natural antioxidants in the above-mentioned nano-drugs, and the screening method includes the following steps:

[0021] (1) Mix two or more of the natural antioxidants in different ratios and load them onto the nano-carrier to prepare a series of natural antioxidant nano-drugs with different loading ratios;

[0022] (2) After co-culturing the nano-drugs with different loading ratios prepared in step (1) with tumor cells at different concentrations in vitro for a period of time, calculate the survival rate of the tumor cells, calculate the combination index (CI) through the cell survival rate, and select the loading ratio of the natural antioxidant corresponding to the nano-drug with the lowest CI value and a CI value less than 0.9 as the target ratio.

[0023] As an optional method, in the above in vitro screening method, it further includes step (3): After co-culturing the carrier mixture formed by mixing the nano-carriers separately loaded with each natural antioxidant according to the target ratio obtained in step (2) with tumor cells at different concentrations in vitro for a period of time, calculate the survival rate of the tumor cells, and calculate the combination index between the natural antioxidants in the carrier mixture and the nano-drug with the target ratio. If the combination index of the nano-drug with the target ratio is less than the combination index corresponding to the carrier mixture, it indicates that the target ratio has a better synergistic anti-tumor effect.

[0024] As an optional method, in the above in vitro screening method, the calculation method of the combination index (CI) is specifically as follows: According to a series of cell survival rates obtained in the co-culture experiment, draw a tumor inhibition rate-concentration curve for combined drug use, and obtain the concentrations D1, D2,..., Dn of each natural antioxidant corresponding to a certain specific tumor inhibition rate (such as 20%, 30%, 40%, 50%, 60%, 70%, 80%) when combined drug use reaches. Then, calculate the combination index (CI) according to the following formula:

[0025] CI = D1 / Dx 1 + D2 / Dx 2 +... + Dn / Dx n ;

[0026] where Dx 1 、Dx 2 、...、Dx nThey are the concentrations corresponding to achieving the specific tumor inhibition rate when using the corresponding natural antioxidants alone. Optionally, after separately co-culturing each natural antioxidant with tumor cells at different concentrations in vitro for a period of time, calculate the survival rate of the tumor cells, plot the tumor inhibition rate-concentration curve of the corresponding natural antioxidant, and then obtain the concentration Dx corresponding to achieving the specific tumor inhibition rate when using each natural antioxidant alone 1 、Dx 2 、…、Dx n ; CI ≤ 0.1 indicates a very strong synergistic effect (+++++), 0.1 < CI ≤ 0.3 indicates a strong synergistic effect (++++); 0.3 < CI ≤ 0.7 is a synergistic effect (+++); 0.7 < CI ≤ 0.85 is a moderate synergistic effect (++); 0.85 < CI ≤ 0.9 is a mild synergistic effect (+); 0.9 < CI ≤ 1.1 indicates approximate additivity, 1.1 < CI ≤ 1.2 indicates slight additive antagonism; 1.2 < CI ≤ 1.45 is a moderate antagonism.

[0027] The present invention also discloses an application of the above-mentioned nano-drug, characterized in that it is used for in vivo or in vitro anti-tumor effect testing and the preparation of clinical anti-tumor drugs.

[0028] The present invention also discloses an application of the above-mentioned nano-drug, characterized in that it is used for the preparation of anti-tumor drugs with anti-multidrug resistance effects.

[0029] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0030] Advantages of the present invention:

[0031] 1. The nano-drug of the present invention simultaneously loads different natural antioxidants in a fixed ratio in the carrier, can enable the natural antioxidants to enter the cells in a fixed ratio to enhance the anti-tumor activity of these antioxidants, and enables it to achieve excellent anti-tumor effects at a low dose (100 mg / kg). Developing this type of natural antioxidant nano-drug can promote the application of natural antioxidants in clinical tumor treatment.

[0032] 2. The anti-tumor effect of the nano-drug of the present invention is greatly improved compared with simple physical mixing, approaching or even exceeding the tumor cell toxicity of general chemotherapy drugs, enabling the natural antioxidant nano-drug to efficiently kill tumors and their drug-resistant tumor cells under the condition of a comparable dosage to chemotherapy drugs, and having a high tumor inhibition effect on various tumor models. The nano-drug has good in vivo biocompatibility and biological safety, and hardly causes toxic side effects to the body during the tumor treatment process, greatly improving the survival rate of animals. Description of the Drawings

[0033] Figure 1 Schematic diagram of the constructed nano-drug;

[0034] Figure 2 Toxicity evaluation of lipoic acid (LA) and sodium lipoate (LA-Na) on different cells in Example 1;

[0035] Figure 3 Toxicity of natural antioxidants (a-d) and lipid nano-drugs (e-h) loaded with natural antioxidants on U251 cells in Example 2;

[0036] Figure 4 Cytotoxicity graph (a) and synergy index (b) of lipid nano-drugs loaded with different ratios of VC / LA in Example 3;

[0037] Figure 5 Cytotoxicity (a), synergy index (b) and percentage of apoptosis (c) of different combinations in Example 3;

[0038] Figure 6 Cytotoxicity graph (a) and synergy index (b) of lipid nano-drugs loaded with different ratios of LA, VC and VK3 in Example 4;

[0039] Figure 7 Cytotoxicity graph (a) and synergy index (b) of lipid nano-drugs loaded with different ratios of VC / ATC in Example 5;

[0040] Figure 8 Cytotoxicity (a), synergy index (b) and percentage of apoptosis (c) of different combinations in Example 5;

[0041] Figure 9 Cytotoxicity graph of dendrimer-loaded LA / TQ vesicular nanoparticles in Example 6;

[0042] Figure 10 Cytotoxicity graph (a) and synergy index (b) of dendrimer nanoparticles loaded with different ratios of LA / TQ in Example 6;

[0043] Figure 11 Cytotoxicity (a), synergy index (b) and percentage of apoptosis (c) of different combinations in Example 6;

[0044] Figure 12 Cytotoxicity graph of micelles and micelle nanoparticles loaded with LA / Cur in Example 7;

[0045] Figure 13Cytotoxicity graphs (a) and synergy indices (b) of micelle nanoparticles loaded with different ratios of LA / Cur in Example 7; Figure 14 Cytotoxicity (a), synergy index (b) and percentage of apoptosis (c) of different combinations in Example 7;

[0046] Figure 15 Results of the anti-tumor mechanism study of 1@nanodrug 1 in Example 8: intracellular LA content (a), intracellular VC content (b), intracellular ROS production rate (c) and ROS content (d);

[0047] Figure 16 Cytotoxicity of 1@nanodrug 1 (a) and TMZ (b) against U251 and U251 / TR in Example 9;

[0048] Figure 17 Acute toxicity assessment of 1@nanodrug 1 in Example 10: changes in mouse body weight (a), weights of excised mouse tissues (b), renal function indicators (c) and liver function indicators (d);

[0049] Figure 18 Immunogenicity assessment of 1@nanodrug 1 in Example 10: white blood cell count (a) and cytokine (TNF-α and IL-6) detection (b);

[0050] Figure 19 Micronucleus test assessment of 1@nanodrug 1 in Example 10: micronucleus rate (a) and ratio of PCE / NCE (b).

[0051] Figure 20 Chromosome aberration assessment of 1@nanodrug 1 in Example 10: chromosome aberration rate;

[0052] Figure 21 Anti-tumor assessment of 1@nanodrug 1 against the U251 subcutaneous tumor model in Example 11: changes in mouse tumor volume (a), combination index Q of in vivo synergistic anti-tumor effect (b) and tumor inhibition rate (c);

[0053] Figure 22 Anti-tumor assessment of 1@nanodrug 1 against the U251 subcutaneous tumor model in Example 11: changes in mouse body weight (a) and survival rate (b);

[0054] Figure 23 Bone marrow suppression assessment of 1@nanodrug 1 against subcutaneous U251 tumor-bearing nude mice in Example 11: white blood cell count (a), platelet count (b) and red blood cell count (c);

[0055] Figure 24Antitumor evaluation of 1@nano drug 1 on U251 / TR subcutaneous tumor model in Example 11: changes in tumor volume (a), tumor inhibition rate (b), changes in mouse body weight (c), and survival rate (d) of mice. Detailed implementation manners

[0056] The above content of the present invention will be further described in detail through the specific implementation manners of the examples below. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Any modifications made without departing from the spirit and principles of the present invention, as well as equivalent replacements or improvements made according to the common general knowledge and conventional means in the art, shall be included within the protection scope of the present invention.

[0057] Example 1.

[0058] Preparation and toxicity evaluation of sodium lipoate

[0059] 1. Preparation of sodium lipoate:

[0060] Accurately weigh 1.03 g of lipoic acid (LA) into a 100 ml round-bottom flask. During stirring, gradually add 30 ml of sodium ethoxide dropwise through a constant-pressure dropping funnel. After the solution is added dropwise, continue stirring overnight, and a large amount of sodium lipoate precipitates in the solution. Filter to obtain sodium lipoate. Dry the obtained sodium lipoate in an oven for 24 hours to obtain pure sodium lipoate monomer (LA-Na).

[0061] 2. Antitumor activity evaluation of lipoic acid and sodium lipoate:

[0062] Select human glioma cells (U251), human colon cancer cells (HT29), and human breast cancer cells (MCF-7) in the logarithmic growth active phase, and inoculate them into 96-well plates respectively. After culturing for 24 h, add different concentrations of LA and LA-Na to them respectively, set different concentration gradients, set 5 parallel samples for each concentration, and set a control group. After continuing to culture for 48 h, aspirate the old medium, add 200 μl of medium containing 10% (v / v) MTT and continue to incubate for 2 h. Then aspirate the old medium again, add 150 μl of DMSO to each well, and shake on an oscillator for 2 min. Finally, measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The results are as Figure 2 shown. The experimental results show that preparing LA into sodium salt (LA-Na) does not affect its antitumor activity. LA and LA-Na have similar cytotoxicity to U251, HT29, and MCF-7 respectively.

[0063] Example 2:

[0064] Antitumor activity of natural antioxidant small molecule monomers

[0065] (1) Cytotoxicity assessment of natural antioxidant small molecule monomers: U251 cells in the logarithmic growth active phase were selected and inoculated into 96-well plates. After culturing for 24 h, different concentrations of lipoic acid (LA), vitamin C (Vitamin C, VC), vitamin K3 (Vitamin K3, VK3), and anthocyanin (Anthocyan, ATC) were added to them respectively. Five parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, the old medium was aspirated, 200 μl of medium containing 10% (v / v) MTT was added and incubated for another 2 h. Then the old medium was aspirated again, 150 μl of DMSO was added to each well, and it was shaken on a shaker for 2 min. Finally, the absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated. The results are as Figure 3 shown in a - d. The experimental results showed that lipoic acid, vitamin C, vitamin K3, and anthocyanin alone had low toxicity to tumor cells.

[0066] (2) Cytotoxicity assessment of natural antioxidant small molecules loaded on liposomes: LA, VC, VK3, and ATC were respectively loaded on liposomes to prepare lipid nano-drugs. Then U251 cells in the logarithmic growth active phase were selected and inoculated into 96-well plates. After culturing for 24 h, different concentrations of LA, VC, VK3, and ATC lipid nano-drugs were added to them respectively. Five parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, the old medium was aspirated, 200 μl of medium containing 10% (v / v) MTT was added and incubated for another 2 h. Then the old medium was aspirated again, 150 μl of DMSO was added to each well, and it was shaken on a shaker for 2 min. Finally, the absorbance at 490 nm was measured with an ELISA reader, and the cell survival rate was calculated. The results are as Figure 3 shown in e - h. The experimental results showed that after LA, VC, VK3, and ATC were individually loaded on liposomes, their anti-tumor activities were still low, and they were slightly worse than those of small molecule monomers in inhibiting tumor growth.

[0067] Example 3

[0068] In vitro anti-synergistic tumor assessment of liposome-loaded natural antioxidant nano-drug of lipoic acid and VC (nano-drug 1)

[0069] (1) Preparation of nano-drugs with different VC / LA loading amounts:

[0070] VC and LA were loaded on liposomes according to the following ratios (n VC :n LA = 2:1, 1:4, 1:1, 10:1, and 1:10) respectively to prepare 1@nano-drug 1, 2@nano-drug 1, 3@nano-drug 1, 4@nano-drug 1, and 5@nano-drug 1.

[0071] (2) Nano-drug synergistic anti-tumor:

[0072] Select U251 cells in the logarithmic growth active phase and inoculate them into 96-well plates respectively. After culturing for 24 h, add 1@ nano-drug 1, 2@ nano-drug 1, 3@ nano-drug 1, 4@ nano-drug 1 and 5@ nano-drug 1 into different wells respectively. Different concentration gradients are set for each nano-drug, 5 parallel samples are set for each concentration, and a control group is set. After continuing to culture for 48 h, rinse with PBS buffer (pH = 7.4), add 100 μl of medium containing 10% (v / v) MTT and continue to incubate for 2 h. Measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell survival rate. The results are as Figure 4 shown. Figure 4 a shows that nano-drugs 1 with different VC / LA loading amounts have different tumor suppression effects. The tumor suppression effect of 1@ nano-drug 1 (IC 5 V 0 C = 84.5 μM; IC 5 L 0 A = 42.3 μM; n VC :n LA ≈ 2:1) is better than that of 2@ nano-drug 1 (IC 5 V 0 C = 152.3 μM; IC 5 L 0 A = 609.2 μM), 3@ nano-drug 1 (IC 5 V 0 C = 224.9 μM; IC 5 L 0 A = 224.9 μM), 4@ nano-drug 1 (IC V C 50 = 545.6 μM; IC 5 L 0 A = 54.6 μM) and 5@ nano-drug 1 (IC 5 V 0 C = 330.8 μM; IC 5 L0 A = 3308 μM) is good. At the same time, under various inhibition rate conditions, the combination index (0.05 < CI < 0.4) of Nano-drug 1 is less than 1 ( Figure 4 b), indicating that the nano-drug has the best synergistic effect.

[0073] (3) Synergistic anti-tumor effect of different forms of materials

[0074] 1@ Nano-drug 1 and [VC liposome nano-drug + LA liposome nano-drug] (n VC :n LA ≈ 2:1) mixture cytotoxicity assessment: Select U251 cells in the logarithmic growth active phase and inoculate them into 96-well plates respectively. After culturing for 24 h, add 1@ Nano-drug 1 and [VC liposome nano-drug + LA liposome nano-drug] mixture into different wells respectively. Set different concentration gradients for each material, set 5 parallel samples for each concentration, and set a control group. After continuing to culture for 48 h, rinse with PBS buffer (pH = 7.4), add 100 μl of medium containing 10% (v / v) MTT and continue to incubate for 2 h. Measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The results are as Figure 5 shown. It can be seen from the analysis of the experimental results that 1@ Nano-drug 1 with VC / LA loaded on the same liposome in a fixed ratio has excellent anti-tumor activity ( Figure 5 a), which can produce a strong synergistic tumor suppression effect at a low dose ( Figure 5 b, 0.05 < CI < 0.4); while the nano-drug mixture [VC liposome nano-drug + LA liposome nano-drug] mixed in the same ratio has a poor tumor suppression effect ( Figure 5 a), and there is almost no synergistic inhibition effect ( Figure 5 b, 0.9 < CI < 1.5).

[0075] 1@ Nano-drug 1 and [VC liposome nano-drug + LA liposome nano-drug] (n VC :n LA≈2:1) Induced apoptosis of the mixture: U251 cells in the logarithmic growth active phase were selected and inoculated in a 6-well plate. After 24 h of culture, 1@nanodrug 1 and the mixture were added to different wells respectively. After incubation for 24 h, the medium and adherent cells were collected. The cell precipitate was collected by centrifugation and washed twice with PBS. Finally, according to the instructions of the AnnexinV-FITC / PI apoptosis detection kit, Annexin V-FITC and PI were added and incubated for 15 min, and then flow cytometry analysis was performed. Data processing was carried out by FlowJo. The experimental results showed that 1@nanodrug 1 had a stronger ability to induce apoptosis than the mixture, and its apoptosis rate (31.5%) was 5.5 times that of the mixture (5.7%) Figure 5 c).

[0076] When small molecule compounds VC and LA are used for combined tumor therapy, when they are simultaneously loaded into liposomes, the optimal synergy between the two can be achieved by regulating the ratio of VC and LA. At the same time, it was also verified that the optimal synergy can be achieved only when VC and LA are fixed and loaded into the carrier at the optimal ratio. It is speculated that mainly because only simultaneous loading can enable VC and LA to enter the cell at a fixed ratio, while free mixing of VC and LA cannot achieve this, so the best synergy effect cannot be achieved.

[0077] In this experiment, when lipoic acid was replaced with sodium lipoate, lipid nanoparticles with optimal synergistic tumor therapy could still be obtained by screening.

[0078] Example 4

[0079] External anti-synergistic tumor evaluation of liposome-loaded natural antioxidant nanodrugs (nanodrug 2) of LA, VC and VK3

[0080] (1) Preparation of nanodrugs with different LA, VC and VK3 loading amounts:

[0081] LA, VC and VK3 were loaded into liposomes according to the following ratios (n LA :n VC :n VK3 =1:1:1, 2:1:1, 2:2:1, 2:1:2 and 1:1:2) respectively to prepare 1@nanodrug 2, 2@nanodrug 2, 3@nanodrug 2, 4@nanodrug 2 and 5@nanodrug 2 respectively.

[0082] (2) Nanodrug synergistic anti-tumor:

[0083] Select U251 cells in the logarithmic growth active phase and inoculate them into 96-well plates respectively. After culturing for 24 h, add 1@nanodrug 2, 2@nanodrug 2, 3@nanodrug 2, 4@nanodrug 2, and 5@nanodrug 2 into different well plates respectively. Set different concentration gradients for each nanodrug, set 5 parallel samples for each concentration, and set a control group. After continuing to culture for 48 h, rinse with PBS buffer (pH = 7.4), add 100 μl of medium containing 10% (v / v) MTT and continue to incubate for 2 h. Measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The results are as Figure 6 shown. Figure 6 a shows that nanodrug 2 with different LA, VC, and VK loading amounts has different tumor suppression effects. Among them, the tumor suppression effect of 2@nanodrug 2 (IC 5 L 0 A = 44.2 μM; IC 5 V 0 C = 22.1 μM; IC 5 V 0 K3 = 22.1 μM; n LA :n VC :n VK3 = 2:1:1) is better than that of 1@nanodrug 2 (IC 5 L 0 A = 352.4 μM; IC 5 V 0 C = 352.4 μM; IC 5 V 0 K3 = 352.4 μM), 3@nanodrug 2 (IC 5 L 0 A = 224.6 μM; IC 5 V 0 C = 224.6 μM; IC 5 V 0 K3 = 112.3 μM), 4@nanodrug 2 (IC 5 L 0 A = 409.5 μM; IC5 V 0 C = 207.8 μM; IC 5 V 0 K3 = 409.5 μM) and 5@nanodrug 2 (IC 5 L 0 A = 68.9 μM; IC 5 V 0 C = 68.9 μM; IC 5 V 0 K3 = 137.8 μM) is better. At the same time, 2@nanodrug 2 has the smallest synergy index ( Figure 6 b, 0.25 < CI < 0.55) at various inhibition rates, indicating that this nanodrug has the best synergy effect.

[0084] Small molecule natural antioxidants LA, VC, and VK3 are used for combined cancer treatment. When they are simultaneously loaded into liposomes, the optimal synergy of the three can be achieved by regulating the ratio of LA, VC, and VK3. It is also verified that the optimal synergy can only be achieved when LA, VC, and VK3 are simultaneously loaded into the carrier in the optimal ratio. It is speculated that this is mainly because only simultaneous loading can enable LA, VC, and VK3 to enter the cells in a fixed ratio, while freely mixed LA, VC, and VK3 cannot achieve this, so the best synergy effect cannot be achieved.

[0085] In this experiment, when lipoic acid was replaced with sodium lipoate, lipid nanodrugs with optimal synergistic cancer treatment could still be obtained through screening.

[0086] Example 5

[0087] External anti-synergy cancer evaluation of liposome-loaded natural antioxidant nanodrug (nanodrug 3) of VC and anthocyan (ATC)

[0088] (1) Preparation of nanodrugs with different VC / ATC loading amounts:

[0089] VC and ATC were loaded into liposomes according to the following ratios (n VC :n ATC = 1:1, 2:1, 1:2, 5:1, and 1:5) respectively to prepare 1@nanodrug 3, 2@nanodrug 3, 3@nanodrug 3, 4@nanodrug 3, and 5@nanodrug 3.

[0090] (2) Nano-drug synergistic anti-tumor:

[0091] Select U251 cells in the logarithmic growth active phase and inoculate them into 96-well plates respectively. After culturing for 24 h, add 1@ Nano-drug 3, 2@ Nano-drug 3, 3@ Nano-drug 3, 4@ Nano-drug 3, and 5@ Nano-drug 3 into different wells respectively. Set different concentration gradients for each nano-drug, set 5 parallel samples for each concentration, and set a control group. After continuing to culture for 48 h, rinse with PBS buffer (pH = 7.4), add 100 μl of medium containing 10% (v / v) MTT and continue to incubate for 2 h. Measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The results are as Figure 7 shown. Figure 7 a shows that nano-drugs 3 with different VC and ATC loading amounts have different tumor suppression effects. Among them, the tumor suppression effect of 2@ Nano-drug 3 (IC 5 V 0 C = 121 μM; IC 5 A 0 TC = 60.5 μM; n VC :n ATC ≈ 2:1) is better than that of 1@ Nano-drug 3 (IC V C 50 = 704.8 μM; IC 5 A 0 TC = 704.8 μM), 3@ Nano-drug 3 (IC 5 V 0 C = 402.2 μM; IC 5 A 0 TC = 804.4 μM), 4@ Nano-drug 3 (IC 5 V 0 C = 1203 μM; IC 5 A 0 TC = 241 μM) and 5@ Nano-drug 3 (IC 5 V 0 C = 189 μM; IC 5 A 0 TC= 945 μM) is good. At the same time, compared with other nano-drugs, Nano-drug 2@3 has the smallest synergy index ( Figure 7 b, 0.2 < CI < 0.4) at each inhibition rate, indicating that this nano-drug has the best synergy effect.

[0092] (3) Synergistic anti-tumor effects of different forms of materials

[0093] Cytotoxicity evaluation of Nano-drug 2@3 and [VC liposome nano-drug + ATC liposome nano-drug] (n VC :n ATC ≈ 2:1) mixture: U251 cells in the logarithmic growth active phase were selected and inoculated into 96-well plates. After culturing for 24 h, Nano-drug 2@3 and [VC liposome nano-drug + ATC liposome nano-drug] mixture were added to different wells respectively. Different concentration gradients were set for each material, 5 parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, it was rinsed with PBS buffer (pH = 7.4), 100 μl of medium containing 10% (v / v) MTT was added and incubated for another 2 h, and the absorbance value at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell survival rate. The results are as Figure 8 shown in a, b. It can be seen from the analysis of the experimental results that Nano-drug 2@3 with VC / ATC loaded on the same liposome at the optimal ratio (0.2 < CI < 0.4) has better anti-tumor effects than the freely mixed [VC liposome nano-drug + ATC liposome nano-drug] (0.89 < CI < 1.4), and it has a strong synergistic tumor suppression effect.

[0094] Apoptosis induction of Nano-drug 2@3 and [VC liposome nano-drug + ATC liposome nano-drug] (n VC :n ATC ≈ 2:1) mixture: U251 cells in the logarithmic growth active phase were selected and inoculated into 6-well plates. After culturing for 24 h, Nano-drug 2@3 and [VC liposome nano-drug + ATC liposome nano-drug] mixture were added to different wells respectively. After incubating for 24 h, the medium and adherent cells were collected. The cell precipitate was collected by centrifugation and washed twice with PBS. Finally, according to the instructions of the AnnexinV-FITC / PI apoptosis detection kit, AnnexinV-FITC and PI were added and incubated for 15 min, and then flow cytometry analysis was performed. Data processing was carried out by FlowJo. The experimental results found that the apoptosis induction ability of the mixture was weak, and there were still a large number of normal cells (56.9%) after induction, and the apoptotic cells were only (33.7%). However, Nano-drug 2@3 had a stronger apoptosis induction ability than the mixture, and the apoptosis rate of the cells was (61.8%), which was 1.8 times that of the mixture ( Figure 8 c).

[0095] When small molecule compounds VC and ATC are used for combined cancer treatment and are simultaneously loaded into liposomes, the optimal synergy between the two can be achieved by regulating the ratio of VC and LPC. It has also been verified that the optimal synergy can only be achieved when VC and ATC are simultaneously loaded into the carrier in the optimal ratio. It is speculated that this is mainly because only when they are simultaneously loaded can VC and ATC enter the cells in a fixed ratio, while the free mixture of VC and ATC lipid nanoparticles cannot achieve this, so the best synergistic effect cannot be achieved.

[0096] In this experiment, when lipoic acid was replaced with sodium lipoate, lipid nanoparticles with optimal synergistic cancer treatment could still be obtained through screening.

[0097] Example 6

[0098] External anti-synergistic cancer evaluation of dendrimer-loaded natural antioxidant nanoparticles (nanoparticle 4) of lipoic acid and thymoquinone (TQ)

[0099] (1) Preparation of dendrimer nanoparticles loaded with LA and TQ respectively

[0100] Dissolve the dendrimer (5 mg) in 10 ml of water, and add the LA / TQ mother liquor (2 mg) dissolved in DMSO to it respectively. After shaking and dialysis, LA / TQ dendrimer nanoparticles are obtained.

[0101] (2) Cytotoxicity evaluation of LA / TQ dendrimer nanoparticles

[0102] Cytotoxicity evaluation of LA / TQ dendrimer nanoparticles: Select human non-small cell lung cancer (A549) cells in the logarithmic growth active phase and inoculate them in a 96-well plate. After culturing for 24 h, add different concentrations of pure dendrimer vesicle nanoparticles, LA dendrimer nanoparticles, and TQ dendrimer nanoparticles to them respectively. Set 5 parallel samples for each concentration and set a control group. After continuing to culture for 48 h, aspirate the old culture medium, add 200 μl of culture medium containing 10% (v / v) MTT and continue to incubate for 2 h. Then aspirate the old culture medium again, add 150 μl of DMSO to each well, and shake on an oscillator for 2 min. Finally, measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell survival rate. The results are as Figure 9 shown. The experimental results show that pure vesicle nanoparticles have no tumor inhibitory effect, while LA / TQ dendrimer nanoparticles show weak cytotoxicity to A549 cells.

[0103] (3) Preparation of nanoparticles with different LA / TQ loading amounts:

[0104] Mix LA and TQ in the following ratio (nLA :n TQ were respectively loaded into dendrimer nanoparticles at ratios of 1:1, 5:1, 1:5, 10:1 and 1:10, and 1@nanodrug 4, 2@nanodrug 4, 3@nanodrug 4, 4@nanodrug 4 and 5@nanodrug 4 were respectively prepared.

[0105] (4) Synergistic antitumor effect of nanodrugs:

[0106] A549 cells in the logarithmic growth active phase were selected and inoculated into 96-well plates. After 24 h of culture, 1@nanodrug 4, 2@nanodrug 4, 3@nanodrug 4, 4@nanodrug 4 and 5@nanodrug 4 were added to different wells respectively. Different concentration gradients were set for each nanodrug, 5 parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, they were rinsed with PBS buffer (pH = 7.4), 100 μl of medium containing 10% (v / v) MTT was added and incubated for another 2 h, and the absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell survival rate. The results are shown as Figure 10 follows. Figure 10 a shows that nanodrug 4 with different LA / PT loading amounts has different tumor suppression effects. Among them, the tumor suppression effect of 1@nanodrug 4 (IC 5 L 0 A = 57.1 μM; IC 5 T 0 Q = 51.7 μM; n LA :n TQ ≈1:1) is better than that of 2@nanodrug 4 (IC 5 L 0 A = 264.9 μM; IC 5 T 0 Q = 53 μM), 3@nanodrug 4 (IC 5 L 0 A = 133.9 μM; IC 5 T 0 Q = 669.5 μM), 4@nanodrug 4 (IC 5 L 0 A = 381.7 μM; IC 5 T 0Q = 38.2 μM) and 5@nanodrug 4 (IC 5 L 0 A = 74.7 μM; IC 5 T 0 Q = 747 μM) is better. At the same time, compared with other nanodrugs, 1@nanodrug 4 has the smallest combination index ( Figure 10 b, 0.1 < CI < 0.25) at each inhibition rate, indicating that this nanodrug has the best synergistic effect.

[0107] (5) Synergistic antitumor effect of different forms of materials

[0108] 1@nanodrug 4 and [LA dendrimer nanoparticles + TQ dendrimer nanoparticles] (n LA :n TQ ≈ 1:1) mixture cytotoxicity assessment: Select A549 cells in the logarithmic growth active phase and inoculate them into 96-well plates respectively. After culturing for 24 h, add 1@nanodrug 4 and [LA dendrimer nanoparticles + TQ dendrimer nanoparticles] mixture into different wells respectively. Set different concentration gradients for each material, set 5 parallel samples for each concentration, and set a control group. After continuing to culture for 48 h, rinse with PBS buffer (pH = 7.4), add 100 μl of medium containing 10% (v / v) MTT and continue to incubate for 2 h, measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The results are as Figure 11 shown. It can be seen from the analysis of the experimental results that 1@nanodrug 4 with LA / TQ loaded on the same liposome in the optimal ratio has better antitumor effect than the freely mixed [LA dendrimer nanoparticles + TQ dendrimer nanoparticles] ( Figure 11 a). And at each inhibition rate, the CI value of 1@nanodrug 4 (0.1 < CI < 0.25) is lower than that of the mixture (0.15 < CI < 0.45), and 1@nanodrug 4 shows better synergistic tumor suppression effect ( Figure 11 b).

[0109] 1@nanodrug 4 and [LA dendrimer nanoparticles + TQ dendrimer nanoparticles] (n LA :n PT≈1:1) Mixture-induced apoptosis: A549 cells in the logarithmic growth active phase were selected and seeded in a 6-well plate. After culturing for 24 h, 1@nano-drug 4 and [LA dendrimer nanoparticles + TQ dendrimer nanoparticles] mixture were added into different wells respectively. After incubation for 24 h, the medium and adherent cells were collected. The cell precipitate was collected by centrifugation and washed twice with PBS. Finally, according to the instructions of the AnnexinV-FITC / PI apoptosis detection kit, Annexin V-FITC and PI were added and incubated for 15 min, and then flow cytometry analysis was performed. Data processing was carried out by FlowJo. The experimental results showed that the mixture had a weak ability to induce apoptosis, and there were still a large number of normal cells (63.9%) after induction, and the apoptotic cells were only (31.5%). While 1@nano-drug 4 had a stronger ability to induce apoptosis than the mixture, and the apoptosis rate of cells was (62.3%), which was 1.97 times that of the mixture. Figure 11 c).

[0110] When the small molecule natural antioxidants LA and TQ are used for combined tumor therapy and are simultaneously loaded into dendrimers, the optimal synergy between the two can be achieved by regulating the ratio of LA and TQ. It was also verified that the optimal synergy can be achieved only when LA and TQ are fixed at the optimal ratio and simultaneously loaded into the carrier. It is speculated that this is mainly because only simultaneous loading can enable LA and TQ to enter the cells at a fixed ratio, while the freely mixed LA and TQ lipid nano-drugs cannot achieve this, so the best synergy effect cannot be achieved.

[0111] In this experiment, when lipoic acid was replaced with sodium lipoate, vesicle nano-drugs with optimal synergistic tumor therapy could still be obtained by screening.

[0112] Example 7

[0113] In vitro anti-synergistic tumor evaluation of micelle-loaded natural antioxidant nano-drugs (nano-drug 5) of lipoic acid (LA) and curcumin (Cur)

[0114] (1) Preparation of micelle nanoparticles loaded with LA and Cur respectively and cytotoxicity evaluation

[0115] Pluronic (5 mg) was dissolved in 10 ml of water, and at the same time, the LA / Cur mother liquor (2 mg) dissolved in DMSO was added respectively. After oscillation and dialysis, LA / Cur micelle nanoparticles with a particle size less than 200 nm were obtained.

[0116] (2) Cytotoxicity evaluation of LA and Cur micelle nanoparticles

[0117] Cytotoxicity evaluation of LA / Cur micelle nanoparticles: HT29 cells in the logarithmic growth active phase were selected and inoculated into 96-well plates. After culturing for 24 h, different concentrations of pure micelle nanoparticles, LA micelle nanoparticles, and Cur micelle nanoparticles were added to them respectively. Five parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, the old culture medium was aspirated, 200 μl of culture medium containing 10% (v / v) MTT was added and incubated for another 2 h. Then the old culture medium was aspirated again, 150 μl of DMSO was added to each well, and it was shaken on an oscillator for 2 min. Finally, the absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated. The results are as Figure 12 shown. The experimental results found that pure micelle nanoparticles had no tumor inhibitory effect, while LA micelle nanoparticles and Cur micelle nanoparticles had certain toxicity to HT29 cells.

[0118] (3) Preparation of nano-drugs with different LA / Cur loadings:

[0119] LA and Cur were loaded into micelle nanoparticles according to the following ratios (n LA :n Cur = 1:1, 2:1, 1:2, 10:1, and 1:10) respectively, and nano-drug 5@1, nano-drug 5@2, nano-drug 5@3, nano-drug 5@4, and nano-drug 5@5 were prepared respectively.

[0120] (4) Synergistic anti-tumor effect of nano-drugs:

[0121] HT29 cells in the logarithmic growth active phase were selected and inoculated into 96-well plates respectively. After culturing for 24 h, nano-drug 5@1, nano-drug 5@2, nano-drug 5@3, nano-drug 5@4, and nano-drug 5@5 were added to different wells respectively. Different concentration gradients were set for each nano-drug, five parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, it was rinsed with PBS buffer (pH = 7.4), 100 μl of culture medium containing 10% (v / v) MTT was added and incubated for another 2 h, and the absorbance at 490 nm was measured with an ELISA reader, and the cell survival rate was calculated. The results are as Figure 13 shown. Figure 13 a shows that nano-drugs 5 with different LA / Cur loadings had different tumor inhibitory effects. Among them, nano-drug 5@2 (IC 5 L 0 A = 57.6 μM; IC 5 C 0 ur = 28.8 μM; n LA :nCur ≈2:1) has a better tumor inhibitory effect than 1@nanodrug 5 (IC 5 L 0 A = 177.6 μM; IC 5 C 0 ur = 177.6 μM), 3@nanodrug 5 (IC 5 L 0 A = 72.2 μM; IC 5 C 0 ur = 144.4 μM), 4@nanodrug 5 (IC 5 L 0 A = 395.6 μM; IC 5 C 0 ur = 39.6 μM) and 5@nanodrug 5 (IC 5 L 0 A = 119 μM; IC 5 C 0 ur = 1190 μM). At the same time, compared with other nanodrugs, 2@nanodrug 5 has the smallest synergy index ( Figure 13 b, 0.2 < CI < 0.4) at various inhibition rates, indicating that this nanodrug has the best synergy effect.

[0122] (5) Synergistic antitumor effect of different forms of materials

[0123] Cytotoxicity evaluation of 2@nanodrug 5 and [LA micelle nanoparticles + Cur micelle nanoparticles] (n LA :n Cur ≈2:1) mixture: Select HT29 cells in the logarithmic growth active phase and inoculate them into 96-well plates respectively. After culturing for 24 h, add 2@nanodrug 5 and [LA micelle nanoparticles + Cur micelle nanoparticles] mixture into different wells respectively. Set different concentration gradients for each material, set 5 parallel samples for each concentration, and set a control group. After continuing to culture for 48 h, rinse with PBS buffer (pH = 7.4), add 100 μl of medium containing 10% (v / v) MTT and continue to incubate for 2 h, measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The results are as Figure 14As shown. From the analysis of experimental results, it can be seen that LA / Cur loaded on the same liposome 2@nanodrug 5 at the optimal ratio (0.2 < CI < 0.4) has more excellent anti-tumor effects than the free mixture of [LA micelle nanoparticles + Cur micelle nanoparticles] (0.6 < CI < 0.9) (14a), and it has a strong synergistic tumor suppression effect (14b).

[0124] 2@nanodrug 5 and [LA micelle nanoparticles + Cur micelle nanoparticles] (n LA :n Cur ≈2:1) mixture-induced apoptosis: Select HT29 cells in the logarithmic growth active phase and inoculate them in a 6-well plate. After culturing for 24 h, add 2@nanodrug 5 and (n LA :n Cur ≈2:1) mixture into different well plates respectively. After incubating for 24 h, collect the medium and adherent cells. Centrifuge to collect the cell precipitate and wash it twice with PBS. Finally, according to the instructions of the AnnexinV-FITC / PI apoptosis detection kit, add Annexin V-FITC and PI and incubate for 15 min, and then perform flow cytometry analysis. Data processing is carried out by FlowJo. The experimental results show that the mixture has a weak ability to induce apoptosis, and there are still a large number of normal cells (66.9%) after induction, and the apoptotic cells are only (29.2%). However, 2@nanodrug 5 has a stronger ability to induce apoptosis than the mixture, and the apoptosis rate of cells is (50.3%), which is 1.72 times that of the mixture ( Figure 14 c).

[0125] When the small molecule compounds LA and Cur are used for combined tumor treatment, loading them on micelles at the same time can achieve the optimal synergy between the two by regulating the ratio of LA and Cur. At the same time, it is also verified that the optimal synergy can be achieved only when LA and Cur are loaded in the carrier at the optimal ratio simultaneously. It is speculated that mainly because only simultaneous loading can enable LA and Cur to enter the cells at a fixed ratio, while the free mixture of LA and Cur micelle nanodrugs cannot achieve this, so the best synergistic effect cannot be achieved.

[0126] Example 8

[0127] Taking 1@nanodrug 1 loaded with VC / LA as an example, study its anti-tumor mechanism

[0128] (1) Detection of intracellular LA and VC contents: Determine the intracellular VC and LA concentrations by high performance liquid chromatography. Briefly, select U251 cells in the logarithmic growth active phase (5×10 4 cells / ml) and inoculate them in a 6-well plate, and incubate them at 37°C / 5% CO 2Cultivate below. After incubating for 24 h, incubate the cells with LA lipid nanoparticles, VC lipid nanoparticles, 1@nanodrug 1, and the mixture of [VC lipid nanoparticles + LA lipid nanoparticles] for 0, 10, 20, 30, 40, 60, 120, 180, 240, 300, and 360 min respectively. After incubation, wash the cells 3 times by centrifugation with PBS. Lyse the cell pellet with lysis buffer (containing 1 mM EDTA). Centrifuge the lysis buffer at 4 °C for 30 min (10000 rpm / min), then collect the supernatant and store it at -20 °C. Finally, detect VC / LA by high performance liquid chromatography. As Figure 15 As shown in a, b, when the individual VC and LA lipid nanoparticles act on the cells, after a period of time, the VC / LA in the cells is rapidly metabolized. However, when VC and LA are incubated simultaneously, the contents of VC and LA in the cells increase, indicating that VC and LA can recycle each other. In addition, compared with [VC lipid nanoparticles + LA lipid nanoparticles], 1@nanodrug 1 can maintain a relatively high level of VC and LA in the cells ( Figure 15 a, b), indicating that when VC and LA are loaded into the same lipid in the optimal ratio, the optimal mutual recycling of VC and LA can be achieved, thereby maintaining a high level of VC / LA in the cells and promoting its anti-tumor effect.

[0129] (2) Generation rate of reactive oxygen species (ROS): Stain the cells with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and detect the intracellular reactive oxygen species (ROS) with a reactive oxygen species detection kit. Briefly, select U251 cells in the logarithmic growth active phase (5×10 3 cells / ml) and inoculate them into a 96-well plate, and culture them at 37 °C / 5% CO 2 . After incubating for 24 h, in serum-free medium, incubate the cells with DCFH-DA (10 μM) for 30 min. After incubation, wash the cells 3 times with PBS, then incubate the cells with 1@nanodrug 1 and [VC lipid nanoparticles + LA lipid nanoparticles], and detect the fluorescence intensity of DCF (λ Em = 525 nm; λ Ex = 488 nm) in the cells by a fluorescence microplate reader at 0, 10, 20, 30, 40, 50, and 60 min after incubation. The change in the intracellular ROS concentration is positively correlated with the change in the DCF fluorescence intensity. Normalize the DCF fluorescence intensity of each group of cells at 0 min and set it as 100%. As Figure 15 shown in c, when VC and LA are simultaneously loaded into the same lipid body, this nanodrug can rapidly generate ROS, and the rate of generating ROS is 3.14 times that of [VC lipid nanoparticles + LA lipid nanoparticles]. The results show that when VC and LA are loaded into the same lipid body in the optimal ratio, the rate of generating ROS by the nanodrug can be effectively increased.

[0130] (3) Intracellular ROS detection: Briefly, U251 cells in the logarithmic growth active phase (5×10 3 cells / ml) were seeded in 96-well plates and cultured at 37 °C / 5% CO 2 . After incubation for 24 h, the cells were incubated with 1@nanodrug 1 and [VC lipid nanodrug + LA lipid nanodrug] for 6 h. After incubation, in serum-free medium, the cells were incubated with DCFH-DA (10 μM) for 30 min. After incubation, the cells were washed 3 times with PBS, and the fluorescence intensity of DCF in the cells (λ Em = 525 nm; λ Ex = 488 nm) was detected by a fluorescence microplate reader. Untreated cells were used as blank controls, and the fluorescence intensity of untreated cells was normalized to 100%. As Figure 15 shown in d, the amount of ROS generated intracellularly by the lipid nanodrug 1@nanodrug 1 loaded with both VC and LA was significantly higher than that of the [VC lipid nanodrug + LA lipid nanodrug] mixture, and the ROS level was 1.7 times that of the latter. Thus, it was shown that 1@nanodrug 1 could promote a large amount of ROS generation in cells, which should be the reason for its high cytotoxicity to tumor cells.

[0131] Through literature research and experimental studies, it was found that VC and LA could generate ROS intracellularly, thereby promoting apoptosis of tumor cells. When VC and LA were used in combination, they could recycle and regenerate each other, and when both were loaded in the same carrier at the optimal synergistic ratio, the maximum recycling and regeneration could be achieved, thereby efficiently generating ROS and promoting their optimal anti-tumor effect at low doses.

[0132] Example 9

[0133] Taking 1@nanodrug 1 loaded with VC / LA as an example, the cytotoxicity of the nanodrug to drug-resistant cells was evaluated

[0134] The in vitro cytotoxicity of 1@nanodrug 1 was evaluated by the MTT method. Briefly, U251 cells and U251 (U251 / TR) cells resistant to temozolomide (TMZ) (5×10 3 / well) were seeded in 96-well plates and cultured at 37 °C / 5% CO 2Incubate in a cell incubator until the cells are completely adherent. After 24 h of incubation, aspirate the old medium and add 200 μl of complete medium containing different concentrations of 1@nano-drug 1 and TMZ, respectively. Cells without adding any material serve as the blank group. After 72 h of cell incubation, aspirate the old medium and add 200 μl of medium containing 10% MTT (5 mg / ml) and incubate for 2 h. After incubation, remove the medium and add 150 μl of dimethyl sulfoxide to each well. The cytotoxicity of the material was evaluated by measuring the absorbance at 490 nm of each well plate using an enzyme-linked immunosorbent assay reader. The cell survival rate was calculated according to the following formula: Cell survival rate (%) = A 490 (sample) / A 490 (control) × 100%. IC 50 was calculated using Calcusyn software, and all experimental data were in good agreement with the calculated data. The experimental results showed that 1@nano-drug 1 had an undifferentiated inhibitory effect on U251 and its drug-resistant cells (U251 / TR). Its IC 5 V 0 C for U251 was 84.5 μM, while its IC 5 V 0 C for U251 / TR was 111.4 μM ( Figure 16 a). However, the first-line chemotherapy drug for glioblastoma, temozolomide (TMZ), had an IC 5 T 0 MZ for U251 of 34.2 μM, while its IC 5 T 0 MZ for U251 / TR was 438.6 μM ( Figure 16 b). The experimental results showed that 1@nano-drug 1 could kill tumor cells at a dose comparable to that of the chemotherapy drug (TMZ), and it had an undifferentiated inhibitory effect on both tumor cells and their drug-resistant tumor cells, showing anti-multidrug resistance.

[0135] Example 10

[0136] Taking 1@nano-drug 1 loaded with VC / LA as an example, the in vivo biocompatibility and biosafety of the nano-drug were evaluated

[0137] Acute toxicity experiment: Through the acute toxicity test, the in vivo biocompatibility / biosafety of 1@nanodrug 1 was evaluated. BALB / c mice weighing approximately 20 g were purchased and raised under standard conditions, allowing them free access to food and water. After observing for one week, the corresponding experiment was carried out. The mice were randomly divided into 3 groups, with 10 mice in each group (5 males and 5 females), and then 0.2 ml of 1@nanodrug 1 (1000 mg / kg) prepared with sterile normal saline was injected through the tail vein. At the same time, saline-treated and untreated (blank) mice were used as controls, and the body weight was recorded every 2 days within 2 weeks after injection. After the observation ended, 1000 μl of blood samples from the mice were collected and divided into two parts: 150 μl of blood was collected in a blood collection tube containing EDTA-K 2 ; 850 μl of blood was collected in a 1.5 ml EP tube. The blood collected in the EP tube was placed at room temperature for 30 min and then centrifuged for 15 min (3000 rpm / min), and centrifuged twice, and the supernatant was collected each time. The liver function indexes of the mice were detected by an automatic hematology analyzer: alanine aminotransferase, glutamate transaminase, alkaline phosphatase, total bilirubin, albumin, renal function indexes: blood urea nitrogen, creatinine and uric acid (UA), and white blood cell count: white blood cells, lymphocytes, monocytes and neutrophils. After the observation ended, no deaths occurred in the three groups of mice, and as Figure 17 shown in a, even at a dose of 1000 mg / kg, the body weight of the mice in the 1@nanodrug 1 group did not decrease, and the body weight increased slightly like that of the saline group and the blank group. Furthermore, the heart, liver, spleen, lungs and kidneys of the mice were weighed, and there was no significant difference in the weights of the various organs of the mice in the 1@nanodrug group compared with those of the saline group and the blank group ( Figure 17 b), indicating that the nanodrug did not cause significant toxic damage to the mice. By detecting the renal function indexes, the blood urea nitrogen, creatinine and uric acid of the mice in the 1@nanodrug 1 group were all within the normal range ( Figure 17 c), indicating that the nanodrug did not cause changes in renal function. By detecting the liver function indexes, the alanine aminotransferase, glutamate transaminase, alkaline phosphatase, total bilirubin, albumin of the mice in the 1@nanodrug 1 group were all within the normal range ( Figure 17 d), indicating that the nanodrug did not cause changes in liver function. At the same time, a complete blood analysis was carried out, and the white blood cell count of the mice in the 1@nanodrug 1 group: white blood cells, lymphocytes, monocytes and neutrophils were at normal levels ( Figure 18 a), indicating that the nanodrug did not cause a significant immune response in vivo. In summary, it shows that 1@nanodrug 1 has excellent biocompatibility and does not cause a significant immune response.

[0138] Immunogenicity reaction test: To evaluate whether 1@nanodrug 1 induces an immune response in vivo, it was further evaluated by detecting IL-6 and TNF-α in serum. Female BALB / c mice weighing approximately 20 g were purchased and raised under standard conditions with free access to food and water. Corresponding experiments were conducted after one week of observation. The mice were randomly divided into 3 groups of 3 mice each. One group of mice was injected with 0.2 ml of 1@nanodrug 1 prepared with sterile saline via the tail vein, and another group of mice was injected with 0.2 ml of sterile saline. Mice without any treatment were used as the blank control. After 24 h of administration, blood was collected by enucleating the eyeballs and the blood was collected in 1.5 ml EP tubes. After the blood was placed at room temperature for 30 min, it was centrifuged for 15 min (3000 rpm / min), and centrifuged twice, and the upper serum was collected each time. The cytokines in the serum were detected according to the instructions of the ELISA kits for mouse tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). All animal experiments were conducted in accordance with the guidelines and approved by the Animal Ethics Committee of Sichuan University. As Figure 18 shown in b, there were no significant differences in TNF-α and IL-6 in the serum of mice in the 1@nanodrug 1 group compared with the saline group and the blank group, indicating that the nanodrug did not significantly induce an immune response in the body.

[0139] Micronucleus assay: The micronucleus test is a genotoxicity test method for detecting chromosome or mitotic apparatus damage. Therefore, the genotoxicity of 1@nano-drug 1 was evaluated by the micronucleus test. BALB / c mice weighing approximately 20 g were purchased and raised under standard conditions so that they could freely access food and water. After observing for one week, the corresponding experiment was carried out. The mice were randomly divided into 3 groups, with 10 mice in each group (5 males and 5 females). One group of mice was injected with 0.2 ml of 1@nano-drug 1 (1000 mg / kg) prepared with sterile saline through the tail vein, and another group of mice was injected with 0.2 ml of sterile saline once a day for 2 days. The third group of mice was injected with cyclophosphamide (100 mg / kg) through the tail vein 24 h before the end of treatment. At the end of treatment, the mice were sacrificed by cervical dislocation, and the bone marrow cells of the mouse femurs were collected into a small amount (1 ml) of fetal bovine serum (FBS). The bone marrow suspension of the femur was aspirated with a 21G needle syringe and dropped onto a glass slide to prepare a smear. After the smear was dried, it was placed in methanol solution for cell fixation for 10 min, and then put into Giemsa stain (10%, v / v) for staining for 30 min. After staining, the smear was washed with clear water and then air-dried naturally. The prepared smear was placed under a microscope for observation, 2000 red blood cells (per animal) were recorded, and micronucleus scoring was performed on polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) to evaluate the genotoxicity of 1@nano-drug 1. Compared with the cyclophosphamide (positive control) group, the number of micronuclei formed in the mice (male and female) in the 1@nano-drug 1 and saline (negative control) groups was significantly reduced ( Figure 19 a) and the PCE / NCE ratio was significantly increased ( Figure 19 b, the normal range of PCE / NCE is 0.6 - 1.2. If the ratio < 0.1, it indicates that the formation of PCE is severely inhibited), indicating that 1@nano-drug 1 has almost no genotoxicity.

[0140] Chromosomal aberration: Chromosomal aberration refers to the changes in the number and structure of chromosomes in biological cells. Therefore, the chromosomal aberration (CA) method was used to determine the genotoxicity of 1@nano-drug 1. BALB / c mice weighing approximately 20 g were purchased and raised under standard conditions, allowing them to have free access to food and water. After observing for one week, corresponding experiments were carried out. The mice were randomly divided into 3 groups, with 10 mice in each group (5 males and 5 females). One group of mice was injected with 0.2 ml of 1@nano-drug 1 (1000 mg / kg) prepared with sterile normal saline through the tail vein, another group of mice was injected with 0.2 ml of sterile normal saline once a day for 2 days. The third group of mice was injected with cyclophosphamide (100 mg / kg) through the tail vein 24 h before the end of treatment. All animals were given colchicine (4 mg / kg) intraperitoneally 2 h before cervical dislocation to sacrifice the mice. The bone marrow cells of the mouse femurs were collected / rinsed with PBS (pH 6.8, 5 ml), and then centrifuged for 10 min (1000 rpm / min) to obtain cell pellets. The collected cell pellets were placed in a hypotonic solution of 0.075 M KCl and incubated at 37 °C for 30 min, and then fixed with an ice acetic acid / methanol fixative (glacial acetic acid / methanol, 1:3, v / v) for 1 min. After fixation, it was centrifuged for 10 min (1000 rpm / min) to obtain cell pellets. The collected cell pellets were fixed for another 20 min and then centrifuged (this step was repeated once). After centrifugation, the supernatant was removed, and finally, approximately 0.5 ml of cell suspension was obtained. The cell suspension was dropped onto pre-cooled (4 °C) glass slides, dried, and stained with Giemsa stain (10%, v / v) for 30 min. The frequency of chromosomal aberration was determined by scoring 100 well-dispersed metaphase cells (per animal), and this was used as an index of genotoxicity. Breaks of various chromosomes and chromatids, gaps of chromosomes and chromatids, deletions, loss of centromeres, and exchanges were recorded. The chromosomes of the cells of the mice in the 1@nano-drug 1 group and the normal saline (negative control) group remained in normal structure, while in the cyclophosphamide (positive control) group, the chromosomes of both female and male mice showed variations (terminal deletions of chromosomes, intrachromosomal gaps, and symmetrical interchromosomal exchanges). Chromosomal variation statistics were carried out, and the results showed that compared with the negative control, administration of 1@nano-drug 1 did not significantly increase the percentage of chromosomal variations in male and female mice, while in male and female mice treated with cyclophosphamide, the percentage of chromosomal variations increased significantly( Figure 20 ). In summary, it is shown that 1@nano-drug 1 has almost no genotoxicity and can be applied in vivo.

[0141] The above experimental results show that the natural antioxidant nano-drug has excellent biocompatibility and will not produce significant toxic and side effects even at high doses, showing potential for in vivo application.

[0142] Example 11

[0143] Taking the 1@ nanomedicine 1 loaded with VC / LA as an example, the in vivo synergistic antitumor effect of the nanomedicine was evaluated

[0144] Evaluation with subcutaneous U251 tumor model: A subcutaneous U251 tumor model was established in 4-week-old nude mice (about 20 g). When the tumor grew to ~50 mm 3 , the nude mice were randomly divided into 7 groups with 6 mice in each group, namely the normal saline group, temozolomide (TMZ, a first-line chemotherapy drug for glioma, 15 mg / kg), VC liposome (62.5 mg / kg), LA liposome (37.5 mg / kg), [VC+LA] (62.5+37.5 mg / kg), [VC liposome+LA liposome] (62.5+37.5 mg / kg), 1@ nanomedicine 1 (15 mg / kg) and 1@ nanomedicine 1 (100 mg / kg) groups. The above 7 groups of nude mice were administered via the tail vein every 2 days for a total of 11 times. During this period, the tumor volume and body weight of the mice were recorded. The experimental results are as Figure 21 shown in a. The VC liposome and LA liposome only showed a weak effect of inhibiting tumor growth, and the tumor inhibition rates were 11.5% and 8.6% respectively ( Figure 21 c), indicating that the tumor treatment effect of simple natural antioxidant nanomedicines is poor. When VC and LA are used in combination, the tumor inhibition effect of the direct mixture of natural antioxidants VC and LA ([VC+LA]) is extremely poor, and the tumor inhibition rate is only 2.6% ( Figure 21 c). This is mainly because small molecule antioxidants have a short blood circulation time in vivo and no tumor targeting, which leads to extremely poor curative effects. Even when used in combination, their in vivo tumor inhibition effect can hardly be improved. Although the curative effect of the nanoparticle mixture ([VC liposome+LA liposome]) is improved compared with [VC+LA], its inhibition rate can only reach 20.0% ( Figure 21 c). However, we found that the 1@ nanomedicine 1 loaded with VC and LA simultaneously has a good tumor inhibition effect. The curative effect of 1@ nanomedicine 1 at a dose of 15 mg / kg is close to that of the chemotherapy drug TMZ (15 mg / kg), and its inhibition rate can reach 40.0%. When the administration dose of 1@ nanomedicine 1 increases, the tumor inhibition rate also increases, showing a strong in vivo synergistic tumor inhibition effect ( Figure 21 b, Q>1). It is speculated that this is mainly because 1@ nanomedicine 1 can effectively extend the blood circulation time of antioxidants, and at the same time can deliver VC and LA into cells in a fixed ratio and recycle with each other in cells, thus achieving an efficient tumor inhibition effect. Although the chemotherapy drug TMZ has a certain tumor inhibition effect (inhibition rate of 42.5%), it causes certain toxic side effects during the tumor treatment process, resulting in a significant decrease in the body weight of the miceFigure 22 a), the median survival was only 34 days ( Figure 22 b). At the same time, it also caused a decrease in the number of white blood cells ( Figure 23 a) and platelets ( Figure 23 b), leading to severe myelosuppression. However, 1@nanodrug1 has excellent biocompatibility and hardly causes toxic side effects during tumor treatment ( Figure 22 and Figure 23 ), so it has the potential for clinical application.

[0145] Subcutaneous U251 / TR tumor model evaluation: A U251 / TR (temozolomide-resistant U251) tumor model was established subcutaneously in 4-week-old nude mice (about 20 g). When the tumor grew to ~50 mm 3 , the nude mice were randomly divided into 5 groups of 6 each, namely the normal saline group, temozolomide (TMZ, 15 mg / kg), temozolomide (TMZ, 50 mg / kg), 1@nanodrug1 (50 mg / kg), and 1@nanodrug1 (200 mg / kg) groups. The above 5 groups of nude mice were given intravenous tail vein injections every 2 days for a total of 11 times. During this period, the tumor volume and body weight of the mice were recorded. The experimental results are as Figure 24 shown in a,b. The U251 / TR tumor has strong resistance to TMZ. At the same dose, its inhibition rate on the U251 / TR tumor is only 10.5% (vs 42.5% for the U251 tumor). Although increasing the dosage of TMZ can increase the inhibition rate to a certain extent, it also increases the toxic side effects of the body. As Figure 24 shown in c, as the dosage of the chemotherapeutic drug increases, the decrease in the body weight of the mice increases and the mortality of the mice also increases. At the 50 mg / kg dose, its mortality is higher than that of the blank control group ( Figure 24 d). However, the 1@nanodrug1 group can achieve a tumor inhibition effect higher than that of TMZ at the 50 mg / kg dose ( Figure 24 a), with an inhibition rate of 65.8% ( Figure 24 b), and as the dosage increases, its tumor inhibition rate can reach 90.0% (100 mg / kg, Figure 24 b). 1@nanodrug1 shows an increasing tumor inhibition effect with the increase in dosage, but it still does not produce significant toxic side effects ( Figure 24 c,d). Even at the 100 mg / kg dose, the body weight of the mice tends to be normal, and the survival rate of the mice during the observation period is 100%. The experimental results show that 1@nanodrug1 not only has a non-selective inhibitory effect on tumor cells and their drug-resistant tumor cells during cell dormancy, but also has a non-selective inhibitory effect on tumors and their drug-resistant tumors, indicating its potential to inhibit drug-resistant tumors.

[0146] In a variety of tumor models, the 1@nanodrug 1 phase has shown excellent synergistic anti-tumor effects. Compared with chemotherapeutic drugs, it not only presents excellent tumor treatment effects but also avoids the toxic and side effects caused by chemotherapeutic drugs to the body. The above experimental results show that simply physically mixing antioxidants directly cannot achieve excellent synergistic effects, mainly because natural antioxidants have a short circulation time in the body and no tumor targeting. Preparing antioxidants into nanodrugs can promote their enrichment in tumor tissues, but when drugs are used in combination, the best synergistic tumor suppression effect still cannot be achieved. However, when we load mutually recyclable antioxidants in the optimal ratio into nanoparticles at the same time, it is possible to deliver antioxidants into tumor cells at a fixed optimal ratio, and the antioxidants can also recycle and regenerate with each other inside tumor cells, thereby amplifying the therapeutic efficacy of antioxidants and achieving the optimal synergistic tumor treatment effect. Therefore, the 1@nanodrug 1 has potential clinical development value. Through this method, the development of a variety of natural antioxidant nanodrugs can be realized.

[0147] The above in vivo synergistic anti-tumor experiments show that when the particle size of the natural antioxidant nanodrug does not exceed 500 nanometers, good in vivo synergistic anti-tumor effects can be achieved. When the particle size of the natural antioxidant nanodrug is between 100 and 200 nanometers, the in vivo synergistic anti-tumor effect is better. It is speculated that mainly because the nanodrugs in this particle size range can be enriched at the tumor site through the enhanced permeability and retention effect (EPR effect) of solid tumor tissues, achieving passive targeting effects.

[0148] Example 12

[0149] Referring to the experimental methods of Examples 2-5 and Examples 9 and 11, replacing vitamin C with its derivative ascorbyl palmitate therein gives similar in vitro and in vivo tumor suppression effects.

[0150] The above are only the preferred embodiments of the present invention, which are illustrative rather than restrictive to the present invention; those of ordinary skill in the art understand that many changes, modifications, and even equivalent changes can be made within the spirit and scope defined by the claims of the present invention, but all will fall within the protection scope of the present invention.

Claims

1. An anti-tumor nano-drug loaded with natural antioxidants, characterized in that, it includes a nano-carrier and two or more natural antioxidants loaded in the nano-carrier in a certain proportion. The natural antioxidants are selected from lipoic acid, vitamin C, vitamin K3, anthocyanin, thymol quinone, and curcumin. The natural antioxidants are the anti-tumor active ingredients of the anti-tumor nano-drug. The ratio between the natural antioxidants is a ratio obtained by an in vitro screening method with a synergistic anti-tumor effect. The in vitro screening method includes the following steps: (1) Mix two or more natural antioxidants in different proportions and load them into the nano-carrier to prepare a series of natural antioxidant nano-drugs with different loading ratios; (2) After co-culturing the nano-drugs with different loading ratios prepared in step (1) with tumor cells at different concentrations in vitro for a period of time, calculate the survival rate of the tumor cells, calculate the synergy index CI through the cell survival rate, and select the natural antioxidant loading ratio corresponding to the nano-drug with a CI value less than 0.9 as the target ratio.

2. The nano-drug according to claim 1, characterized in that, The specific calculation method of the synergy index CI is as follows: Based on a series of cell survival rates obtained from the co-culture experiment, draw the tumor inhibition rate-concentration curve for the combined drug use. According to the curve, obtain the concentrations D1, D2, …, Dn of each natural antioxidant corresponding to a certain specific tumor inhibition rate for the combined drug use. Then, calculate the synergy index CI according to the following formula: CI = D1 / Dx 1 + D2 / Dx 2 +…+ Dn / Dx n ; where Dx 1 、Dx 2 、…、Dx n are the concentrations corresponding to the specific tumor inhibition rate when the natural antioxidant nanodrugs are separately loaded 3. The nano-drug according to claim 1, characterized in that, The in vitro screening method further includes step (3): After co-culturing the carrier mixture formed by mixing the nano-carriers separately loaded with each natural antioxidant according to the target ratio obtained in step (2) with tumor cells at different concentrations in vitro for a period of time, calculate the survival rate of the tumor cells, and calculate the synergy index between the natural antioxidants in the carrier mixture and the nano-drug with the target ratio. If the synergy index of the nano-drug with the target ratio is less than the synergy index corresponding to the carrier mixture, it indicates that the target ratio has a better synergistic anti-tumor effect.

4. An anti-tumor nano-drug loaded with natural antioxidants, characterized in that, it includes a nano-carrier and two or more natural antioxidants loaded in the nano-carrier in a certain proportion. The natural antioxidants are selected from lipoic acid, vitamin C, vitamin K3, anthocyanin, thymol quinone, and curcumin. The natural antioxidants are the anti-tumor active ingredients of the anti-tumor nano-drug; there is a synergistic anti-tumor effect between the natural antioxidants. The natural antioxidants are lipoic acid and vitamin C, and the molar ratio of vitamin C to lipoic acid is 2:1; Or, the natural antioxidants are lipoic acid, vitamin C, and vitamin K3, and the molar ratio of lipoic acid, vitamin C, and vitamin K3 is 2:1:1; Or, the natural antioxidants are vitamin C and anthocyanin, and the molar ratio of vitamin C to anthocyanin is 2:1; Or, the natural antioxidants are lipoic acid and thymol quinone, and the molar ratio of lipoic acid to thymol quinone is 1:1 or 5:1 or 1:5, 10:1 or 1:10; Or, the natural antioxidants are lipoic acid and curcumin, and the molar ratio of lipoic acid to curcumin is 2:

1.

5. The nano-drug according to claim 4, characterized in that, The nano-carrier is selected from liposomes, vesicles, and micelles.

6. The nano-drug according to claim 4, wherein, the ratio between the natural antioxidants is the optimal ratio of antioxidant synergistic effect screened by an in vitro method.

7. The nano-drug according to claim 4, wherein, one of the natural antioxidants is lipoic acid and / or a pharmaceutically acceptable lipoate.

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