Metal-sulfoconic acid nano-complex, and preparation method and application thereof

Abstract

The application discloses a metal-sulfoacid nanocomposite, a preparation method and application thereof, and belongs to the field of biological medicines. The metal-sulfoacid nanocomposite is prepared by compounding sulfoacid and / or a sulfoacid derivative and Fenton-type metal ions to form stable composite particles. The composite particles can be dissociated into dihydrosulfoacid and release Fenton-type metal under a tumor cell microenvironment, and the two can synergistically and efficiently resist tumors in tumor cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an anti-tumor nano-drug. BACKGROUND

[0002] Cancer is a major disease that seriously endangers human health. Chemotherapy is an important means of treating cancer, but the serious side effects of chemotherapy, such as liver and kidney damage, bone marrow suppression, and cardiotoxicity, often bring great pain to patients. Chemodynamic therapy (CDT) is a new type of tumor treatment technology that takes the weakly acidic microenvironment of the tumor lesion as the reaction condition, promotes the Fenton or Fenton-like reaction of overexpressed hydrogen peroxide (H2O2) and transition metals to produce hydroxyl radicals (·OH), and induces the increase of intracellular reactive oxygen species (ROS) to kill tumor cells. However, due to the limited endogenous H2O2 content in tumor cells and the easy metabolic loss of transition metals, the amount of exogenous metal required for traditional chemodynamic therapy to achieve good efficacy is very high, which is easy to cause oxidative damage to normal tissues. Ways to enhance CDT therapy include adding substances that can increase endogenous H2O2, or combining with acoustic, photodynamic, photothermal, etc. to induce more H2O2 production, but the addition of substances that can increase endogenous H2O2 or the induction of H2O2 production by external force will inevitably cause secondary damage to the human body, and photodynamic and photothermal methods are only suitable for superficial tumors and have poor effect on deep solid tumors; further, the strategy of co-loading multiple reagents increases the difficulty of preparation of the preparation, and also makes it more difficult to evaluate the safety and effectiveness of various components of the drug in complex biological systems, hindering the clinical translation of such drugs. Therefore, so far, how to develop an anti-tumor drug based on chemodynamic therapy with low metal usage, simple system and easy preparation is still a problem to be solved. SUMMARY

[0003] To solve the above problems, the application provides a metal-lipoic acid nano-complex, which is formed by combining lipoic acid and / or lipoic acid derivatives with Fenton-type metal ions to form stable composite particles, the metal-lipoic acid nano-complex can dissociate to generate lipoic acid monomers and release Fenton-type metal ions under a cell microenvironment, the lipoic acid monomers and Fenton-type metal ions can undergo Fenton or Fenton-like reactions with H2O2 in the environment, so that LA / DHLA and oxidized metal ions / reduced metal ions are recycled, and the metabolic loss of metal ions is delayed. When the composite particles enter tumor tissues, the cross-linked lipoic acid particles are degraded into reduced lipoic acid (DHLA) under the action of glutathione (GSH) and thioredoxin reductase (TrxR) overexpressed in tumor cells, and Fenton-type metal ions are released; and because LA / DHLA has a lower redox potential than the Fenton-type metal redox pair, DHLA can make the oxidized metal ions after the Fenton / Fenton-like response in the cell return to the reduced state, thereby recycling LA / DHLA and oxidized metal ions / reduced metal ions, delaying the metabolic loss of Fenton-type metal, and thus persistently and efficiently generating hydroxyl radicals ·OH; in addition, the LA carrier itself can increase the production of H2O2 in tumor cells, providing more substrates for the Fenton / Fenton-like response. The nano-complex can greatly reduce the amount of metal, and does not need to add exogenous substances that can produce H2O2, solving the main problem existing in the current nano-materials that rely on metal delivery to achieve ROS elevation.

[0004] The application comprises the following technical solutions:

[0005] A metal-lipoic acid nano-complex comprises lipoic acid particles and Fenton-type metal ions combined therewith, the Fenton-type metal ions are metal ions capable of participating in Fenton reaction or Fenton-like reaction. The composite particles can release lipoic acid and / or lipoic acid derivatives and Fenton-type metal ions under the tumor cell microenvironment, the lipoic acid and / or lipoic acid derivatives can be converted into DHLA under the action of thioredoxin reductase (TrxR) and glutathione (GSH) overexpressed in tumor cells, reducing the Fenton-type metal ions and accelerating the generation of hydroxyl radicals (·OH), thereby increasing the tumor cell killing induced by lipid peroxidation of Fenton-type metal ions. The lipoic acid particles themselves can increase the production of H2O2 in tumor cells, providing more substrates for the Fenton / Fenton-like reaction and further promoting the generation of ROS.

[0006] As an option, in the metal-lipoic acid nano-complexes described above, the lipoic acid particles are formed from a raw material containing lipoic acid and / or lipoic acid derivatives. The formation process can include various modifications or modifications to the nanoparticles. The lipoic acid derivatives include lipoic acid salts or modifications obtained by non-substantial modifications to lipoic acid that do not affect the core function (including but not limited to grafting functional groups on lipoic acid molecules).

[0007] Lipoic acid or lipoic acid derivatives such as lipoic acid potassium salt, sodium salt or non-substantial modifications thereof can be converted into DHLA under the action of thioredoxin and glutathione (GSH) overexpressed in tumor cells, thereby achieving a synergistic anti-tumor effect with metal ions having redox properties. In addition to the above-mentioned lipoic acid potassium salt, sodium salt, the lipoic acid derivative can be a lipoic acid monomer grafted with different targeting groups, such as glucosamine which can target glucose receptors, folate which can target folate receptors on the surface of cancer cells, etc. These targeting groups endow the nano-complex with more functions, such as high blood-brain barrier penetration ability, targeting tumor cells, etc.

[0008] As an option, in the metal-lipoic acid nano-complexes described above, the Fenton-type metal ions are combined with the lipoic acid particles by coordination. The Fenton-type metal ions can be stably combined with the lipoic acid particles by coordination, and can remain stable in blood and normal cells, while in the microenvironment of high glutathione (GSH) concentration or acidic in tumor cells or tissues, the coordination ability decreases, and disassembly occurs, thereby releasing lipoic acid and / or lipoic acid derivatives and Fenton-type metal ions.

[0009] As an option, in the metal-lipoic acid nano-complexes described above, the Fenton-type metal ions are coordinated with the sulfhydryl and / or carboxyl groups in the lipoic acid particles. Coordination with sulfhydryl has redox sensitivity, and coordination with carboxyl has pH sensitivity.

[0010] As an option, in the metal-lipoic acid nano-complexes described above, the Fenton-type metal ions are metal ions that can participate in Fenton reaction or Fenton-like reaction.

[0011] As an option, in the metal-lipoic acid nano-complexes described above, the Fenton-type metal ions are one or more of Fe ions, Cu ions, Mn ions, Co ions, Cd ions, Ni ions, and Zn ions. The main role of the Fenton-type metal ions is to undergo Fenton reaction or Fenton-like reaction with intracellular overexpressed hydrogen peroxide, thereby inducing the production of hydroxyl radicals, thereby killing tumor cells.

[0012] As an option, in the above metal-lipoic acid nano-complex, the lipoic acid particles are any one of micelles, vesicles or disordered aggregates formed by self-assembly of lipoic acid and / or lipoic acid derivatives, for different occasions. For example, micelles have smaller size and better penetration, while vesicles have high drug loading capacity and can additionally load other drugs.

[0013] As an option, in the above metal-lipoic acid nano-complex, the lipoic acid particles are spontaneously assembled in water by hydrophobic disulfide five-membered rings and hydrophilic carboxyl groups in hydrophilic-hydrophobic interaction.

[0014] As an option, in the above metal-lipoic acid nano-complex, the lipoic acid particles include a hydrophilic carboxyl shell. The hydrophilic carboxyl shell makes the nano-complex surface negatively charged when circulating in the blood, which is beneficial for long circulation in vivo. When entering the tumor tissue, the negatively charged carboxylate is converted to uncharged carboxyl due to the micro-acidity of the tumor microenvironment, which is beneficial for endocytosis. This structure is beneficial for the stability of the material in the blood and normal tissue environment, improving the biological safety of the material.

[0015] As an option, in the above metal-lipoic acid nano-complex, part of the disulfide five-membered rings in the core of the lipoic acid particles are broken, and part of the mercapto groups are coordinated with Fenton-type metal ions by coordination. The Fenton-type metal ions are located in the core of the lipoic acid particles, avoiding their release into the blood and normal tissues during delivery, which is more beneficial for ensuring the biological safety and targeted delivery effect of the material. The anti-tumor nano-drug can specifically respond to ROS production in tumor cells. Under the action of GSH highly expressed in tumor cells, the disulfide bond in the lipoic acid aggregate is broken, the hydrophobicity of the core is weakened after breaking, the stability of the aggregate formed by hydrophobic interaction is weakened, and the aggregate is disassembled, and the mercapto groups coordinated with Fenton-type metal are consumed, thereby releasing lipoic acid and Fenton-type metal ions. In the process of blood circulation in the human body, the anti-tumor nano-drug cannot disassemble and release lipoic acid and Fenton-type metal ions due to the low content of GSH and H2O2 in normal tissues, so it cannot promote ROS production, and has high biological safety.

[0016] As an option, in the above metal-lipoic acid nano-complex, part of the disulfide bonds in the five-membered rings of the lipoic acid particles are first broken, and then the obtained mercapto groups are cross-linked to stabilize the lipoic acid particles. The lipoic acid particles are cross-linked inside. The lipoic acid nano-complex is very stable in normal blood and normal tissues with normal pH and low GSH concentration, but it is easily broken and disassembled in tumor tissues and cell environments with lower pH and high GSH concentration.

[0017] As an option, in the above metal-lipoic acid nano-complex, the particle size of the anti-tumor nano-drug is in the range of 10-150 nm. Therefore, the nanoparticles in this particle size range have good EPR effect and can be well enriched in tumor tissues, and more effectively enter tumor cells.

[0018] As an option, in the above metal-lipoic acid nano-complex, the lipoic acid can be racemic lipoic acid or non-racemic lipoic acid, preferably R-(+)-ɑ-lipoic acid, R-(+)-ɑ is an endogenous vitamin B widely present in mitochondrial coenzyme. In addition to being a health care product for antioxidant and hypoglycemic, more and more studies have shown that lipoic acid plays an anti-tumor role by inhibiting aerobic glycolysis, inducing apoptosis and promoting ROS elevation.

[0019] The application also provides a preparation method of the above metal-lipoic acid nano-complex, characterized in that it comprises the following steps: first, preparing lipoic acid particles through self-assembly, then adding a solution containing the Fenton-type metal ions to make the Fenton-type metal ions coordinate with the lipoic acid particles to form metal-lipoic acid complex particles.

[0020] Or first, mixing the constituent units of the lipoic acid particles with the solution of the Fenton-type metal ions to make them form a chelate through coordination, and then preparing metal-lipoic acid complex particles through self-assembly.

[0021] As an option, in the above preparation method, a cross-linking step is further included to cross-link part of the sulfydryl groups in the lipoic acid particles to form disulfide bonds, thereby improving the stability of the lipoic acid particles.

[0022] In the above preparation method, the following steps are included:

[0023] 1) preparing lipoic acid particles;

[0024] 2) breaking the disulfide bonds of the disulfide five-membered rings in the lipoic acid particles to form sulfydryl groups;

[0025] 3) adding a solution containing Fenton-type metal ions to make the Fenton-type metal ions coordinate and chelate with the sulfydryl groups to form metal-lipoic acid complex particles.

[0026] As an option, in the above preparation method, a cross-linking step is further included to cross-link part of the sulfydryl groups in the lipoic acid particles to form disulfide bonds, thereby improving the stability of the lipoic acid particles. Further, the cross-linking method can be oxygen cross-linking, photo cross-linking, mechanical stress cross-linking, catalytic cross-linking or appropriate pH cross-linking.

[0027] As an option, in the above preparation method, the lipophilic-hydrophilic interaction self-assembly of lipoic acid is used to prepare lipoic acid particles.

[0028] As an option, in the above preparation method, the method of breaking the disulfide bond is one or more of ultraviolet light breaking, ultrasonic breaking, thermal breaking, mechanical stress breaking, and base catalysis breaking.

[0029] As an option, in the above preparation method, a low concentration of Fenton-type metal ions is added dropwise to the lipoic acid particle solution in step 3). The low concentration of Fenton-type metal ions is added dropwise, which is conducive to the stable coordination of the metal ions inside the nanoparticles without coordination chelation with the peripheral carboxyl groups. The molar ratio of the Fenton-type metal ions to the lipoic acid particles is preferably 1:5-20, and further preferably 1:13.5. If the metal ions are added in excess, the surface charge of the lipoic acid and metal ion chelate decreases, and the nanocomposite is prone to agglomeration. If the amount of metal ions added is insufficient, the tumor treatment effect is reduced.

[0030] As an option, in the above preparation method, the Fenton-type metal ions are added dropwise, which can be added before or after the cross-linking of the lipoic acid particles, and is preferably added before the cross-linking.

[0031] As an option, in the above preparation method, the following steps are included:

[0032] 1) Dissolve lipoic acid in an acetone solution, and add the obtained lipoic acid-acetone solution dropwise to water to obtain a lipoic acid-acetone-water mixed solution, and remove the acetone by rotary evaporation to allow the lipoic acid to self-assemble in water to form a lipoic acid aggregate;

[0033] 2) irradiate the lipoic acid aggregate with 365 nm ultraviolet light for 4 hours to break the disulfide bond;

[0034] 3) add a solution containing metal ions dropwise to the solution obtained in step 2);

[0035] 4) cross-link by passing air and dialysis for 24 hours.

[0036] All the features disclosed in this specification, or all the steps of any method or process disclosed in this specification, can be combined in any combination, except where mutually exclusive.

[0037] The beneficial effects of the present application are:

[0038] 1. Better therapeutic effect. When the metal-lipoic acid nano-complex enters the tumor cells, the cross-linked lipoic acid is degraded into DHLA under the action of excess GSH and TrxR in the tumor cells, and Fenton metal ions are released. Because the LA / DHLA has a lower redox potential (-0.32V) than the transition metal ion redox pair, DHLA can make the oxidized metal ions after the Fenton / Fenton-like reaction in the cell return to the reduced state, thereby making LA / DHLA and oxidized metal ions / reduced metal ions circulate with each other, delaying the metabolic loss of the metal and further increasing the ROS content in the cell; in addition, LA increases the generation of ROS in tumor cells through its degradation product DHLA, which directly induces tumor cell apoptosis on the one hand, and provides more substrates for Fenton / Fenton-like response on the other hand, thereby persistently and efficiently killing tumor cells. Taking iron as an example, DHLA reduces Fe 3+ to Fe 2+ , accelerates the generation of ·OH in the Fenton reaction, and induces lipid peroxidation to kill tumor cells.

[0039] 2. Simple system, make the most of it. The nano-system is only constructed by transition metal ions and B vitamin lipoic acid, which not only has a simple system and low raw material cost, but also makes it easy to determine its in-vivo metabolic mechanism, and has great potential for clinical transformation.

[0040] 3. Low side effects, differential toxicity. The anti-tumor nano-drug described in the present application can specifically respond to ROS production in tumor cells. Under the action of high expression of GSH in tumor cells, the disulfide bond in the lipoic acid aggregate is broken, the hydrophobicity of the inner core is weakened after the disulfide bond is broken, the particle stability formed by hydrophilic and hydrophobic interaction is weakened, and disassembly occurs, releasing lipoic acid and metal ions; on the contrary, in the process of blood circulation in the human body, due to the low content of GSH and H2O2 in normal tissues, it cannot disassemble, and the nano-drug cannot play the role of promoting ROS production, and has high biological safety. Through the characterization of its particle size, storage stability, dialysis stability, and blood stability, it is determined that the nano-particle has good stability. Through cell experiments, it is obtained that the nano-particle has strong tumor cell-specific killing effect, and has low toxicity to normal cells. Taking lipoic acid iron nano-complex as an example, the IC 50 (50% of tumor cells are induced to apoptosis corresponding to the drug concentration) of lipoic acid iron nano-complex for tumor cell line MCF-7 is about 5 times the IC 50 of 293T cell line, which shows that the material has small toxicity to normal cells and large toxicity to tumor cell lines. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a structural schematic diagram of the three cross-linked lipoic acid particles described in Example 1 of the present application.

[0042] Figure 2 The electron microscope photos of the metal-lipoic acid aggregate material, iron lipoate nanoparticles, described in Example 2 of the present application: (left) iron-lipoate nanoparticles, (right) copper-lipoate nanoparticles;

[0043] Figure 3 The results of the cytotoxicity test of the metal-lipoate nanoparticles Cu@cLANs (left) and Fe@cLANs (right) described in Example 6 of the present application on three tumor cells (U251, MCF-7, HT29);

[0044] Figure 4 The results of the cytotoxicity test of the lipoate monomer LA (left), Fenton metal Fe (middle), and Cu (right) described in Example 6 of the present application on three tumor cells (U251, MCF-7, HT29);

[0045] Figure 5 The ultraviolet absorption spectrum of each group in the solution experiment for verifying the anti-tumor mechanism in vitro described in Example 7 of the present application;

[0046] Figure 6 The fluorescence pictures taken by laser confocal after the cells were co-incubated with DCFH-DA in the cell experiment for verifying the anti-tumor mechanism in vitro described in Example 7 of the present application;

[0047] Figure 7 The curves of the intracellular ROS, metal ions, and DHLA contents over time after the cLANs and Cu@cLANs were co-incubated with cells described in Example 7 of the present application;

[0048] Figure 8 The results of the cytotoxicity experiment of Fe@cLANs on three normal cells described in Example 8 of the present application;

[0049] Figure 9 The results of the hemolysis and coagulation characterization experiment of Fe@cLANs described in Example 8 of the present application;

[0050] Figure 10 The weight change curve of the mice after a large dose of Fe@cLANs was administered described in Example 9 of the present application;

[0051] Figure 11 The results of the in vivo anti-tumor effect evaluation of Cu@cLANs described in Example 10 of the present application, tumor volume change (left), mouse weight change (middle), and mouse survival cycle (right);

[0052] Figure 12Tumor volume change (left), mouse weight change (middle), and mouse survival period (right) for the results of the in vivo anti-tumor effect evaluation of the Fe@cLANs described in Example 11 of the present application.

[0053] Figure 13 Schematic diagram of the anti-tumor mechanism of the metal-lipoic acid nanoparticles described in the present application. DETAILED DESCRIPTION

[0054] The above description of the present application is further illustrated in detail by the specific embodiments of the examples below. However, this should not be understood as limiting the scope of the above subject matter of the present application to only the following examples. Any modifications made without departing from the spirit and principles of the present application, and equivalent replacements or improvements made according to the ordinary technical knowledge and means in the art, should be included in the protection scope of the present application.

[0055] Example 1 Preparation of crosslinked lipoic acid nanoparticles (cLANs)

[0056] 1. Sodium lipoate: 100 mg of lipoic acid (LA) was added to 50 mL of deionized water, and 0.1 M aqueous NaOH was added dropwise under stirring until the lipoic acid was completely dissolved. Then, 0.1 M HCl was added dropwise until the solution was neutral. Finally, the solution was freeze-dried to obtain a light yellow sodium lipoate powder.

[0057] 2. Lipoic acid micelles: 41.2 mg (0.2 mol) of sodium lipoate was weighed and dissolved in 1 mL of deionized water. After ultrasonic treatment, nanoparticles with a size of about 15 nm were prepared. The above obtained nanoparticles were irradiated by 365 nm ultraviolet light to initiate the self-crosslinking of the disulfide bond of lipoic acid. After 2.5 h of reaction and 48 h of dialysis, crosslinked lipoic acid micelle nanoparticles with a size of about 15 nm were obtained.

[0058] 3. Lipoic acid vesicles: 100 mg of sodium lipoate was dissolved in 10 mL of deionized water and dispersed by ultrasonic treatment. An additional 100 ul (1:9 of hexadecane and n-butyl acetate solvent) was added dropwise to obtain lipoic acid vesicles. The vesicles were irradiated by 365 nm ultraviolet light for 4 hours and dialyzed for 48 h. Dynamic light scattering (DLS) measurement showed that the nanoparticle size was about 30 nm and had high stability.

[0059] 4. Lipoic acid aggregates: 200 mg lipoic acid was dissolved in 10 mL acetone, and the solution was uniformly dispersed by ultrasonic treatment. The solution was slowly dripped into 100 mL water, and mixed by ultrasonic treatment. The acetone was removed by rotary evaporation at 40 °C for 30 min, and the lipoic acid was assembled into a milky white aggregate in water by slowly cooling. The concentration of the lipoic acid aggregate was about 2 mg / mL. The aggregate was irradiated with 365 nm ultraviolet light for 2 h, and then air was introduced to cross-link the sulfhydryl groups. The aggregate was dialyzed. Dynamic light scattering (DLS) measurement showed that the particle size of the nanoparticles was about 90 nm, and the nanoparticles had high stability.

[0060] The schematic diagram of the lipoic acid nanoparticles prepared in the above examples 2, 3 and 4 is shown in Figure 1 .

[0061] Example 2

[0062] Any one of the lipoic acid nanoparticles prepared in example 1 was taken, and an aqueous ferric chloride solution was added dropwise to the nanoparticle solution to prepare iron-lipoic acid nanoparticles (Fe@cLANs). The final concentration of the lipoic acid nanoparticles was 2 mg / mL, and the final concentration of the ferric chloride was 0.1 mg / mL. The obtained electron microscope image of the aggregate is shown in Figure 2 .

[0063] Any one of the lipoic acid nanoparticles prepared in example 1 was taken, and an aqueous copper chloride solution was added dropwise to the nanoparticle solution to finally prepare copper-lipoic acid nanoparticles (Cu@cLANs). The final concentration of the lipoic acid nanoparticles was 2 mg / mL, and the final concentration of the copper chloride was 0.1 mg / mL. The obtained electron microscope image of the aggregate is shown in Figure 2 .

[0064] Example 3

[0065] 200 mg lipoic acid was dissolved in 10 mL acetone, and the acetone was removed by rotary evaporation to obtain uncross-linked lipoic acid aggregates. The aggregates were heated at 70 °C to break the disulfide bonds, and then an aqueous solution of 0.5 mg / mL copper chloride was added dropwise. DTT was added to cross-link the disulfide bonds, and the reaction was performed for 1 h. The lipoic acid copper nanoparticles were obtained after dialysis for 24 h. The size of the lipoic acid copper nanoparticles was about 50 nm.

[0066] Example 4

[0067] 100 mg lipoic acid was dissolved in an acetone solution, and an aqueous solution of 0.1 mg / mL ferric chloride was added dropwise. After ultrasonic dispersion, the acetone was removed by rotary evaporation. The lipoic acid was intertwined with each other by hydrophobic interaction to obtain uncross-linked lipoic acid iron nanoparticles.

[0068] It is verified by experiments that the uncrosslinked Fe@cLANs in Example 4 and the crosslinked Fe@cLANs in Example 2 are compared, and material characterization proves that the crosslinked and uncrosslinked nanoparticles have good stability, but 5 mL of 2 mg / mL uncrosslinked lipoic acid nanoparticles can at most chelate 0.25 mg of Fe ions, and under the same conditions, the crosslinked lipoic acid nanoparticles can chelate more Fe ions.

[0069] Example 5: Verification of in vitro anti-tumor ability of metal lipoic acid nanoparticles:

[0070] Cancer cells in the logarithmic growth phase were selected, inoculated in a 96-well plate, and cultured for 24 h, then different concentrations of metal lipoic acid nanoparticles were added respectively, different concentration gradients were set, 5 parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, the culture medium was removed, 100 μL of culture medium containing 10% (v / v) MTT was added, incubated for 2 h, the old culture medium was removed, 100 μL of DMSO was added to each well, shaken for 2 min, and finally the absorbance at 490 nm was determined by an enzyme marker, and the cell survival rate was calculated, and the results are shown in Figure 3 It is found that Cu@cLANs have high cytotoxicity to various tumor cells. Similarly, Fe@cLANs have high cytotoxicity to various tumor cells, as shown in Figure 3 .

[0071] Further, the anti-tumor ability of lipoic acid and metal ions alone was verified, as shown in Figure 4 , lipoic acid nanoparticles and free Fe ions and Cu ions exhibit weak cytotoxicity to tumor cells. This indicates that after lipoic acid is combined with metal ions, its anti-tumor ability is further enhanced, and lipoic acid and Fenton metal have strong synergistic effect.

[0072] Example 6: Verification of anti-tumor mechanism of metal lipoic acid nanoparticles:

[0073] Taking copper-lipoic acid nanoparticles Cu@cLANs as an example, the anti-tumor mechanism of the composite material was verified by solution experiments and in vitro cell experiments.

[0074] Solution experiment: methylene blue (MB) is a compound often used to test the redox state, which is a benzene ring connected with a nitrogen sulfur chromogenic group with a lone pair of electrons, and has different molecular structures in oxidation and reduction environments, so it can also present different colors. Reduced MB is dark blue, while oxidized MB is colorless.

[0075] As mentioned earlier, under the action of GSH in cells, the disulfide bond of Cu@cLANs can be opened, releasing DHLA and Cu2+ Cu is then reduced to Cu 2+ Cu is then reduced to Cu + Fenton reaction occurs in the presence of excess H2O2 to generate a large amount of ·OH. To simulate this process, GSH (10 mM) and H2O2 (100 μM) were added to the solution of Cu@cLANs, respectively, and then the stock solution of MB was added dropwise to make the concentration of MB 10 μM. The control groups were Cu@cLANs solution, H2O2 solution, Cu@cLANs + GSH solution, respectively, and the concentrations of each compound were consistent with the experimental group. Then the stock solution of MB was added dropwise to the three control group solutions to make the concentration of MB 10 μM. The pH of the above four mixed solutions was adjusted to 7.4, and then placed in a 37°C water bath for two hours. After the reaction was completed, UV absorption measurement was performed on all the above solutions, as shown in Figure 5 Fig. 6, it can be clearly found that after the addition of GSH and H2O2, the peak at 668 nm in the mixed solution almost disappeared, that is, the absorption peak of reduced MB disappeared. This indicates that after the addition of GSH and H2O2, a large amount of ·OH is generated in the Cu@cLANs solution, which oxidizes MB and changes its structure, resulting in the disappearance of its absorption peak.

[0076] Cell experiment:

[0077] For example, copper and ubiquinol nanocomposites were used to detect intracellular ROS using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, λ ex = 488 nm, λ em = 525 nm). U251 cells (5 x 10 4 / well) were seeded in a Φ = 35 mm glass bottom culture dish and incubated at 37°C / 5% CO2 to allow the cells to adhere completely. After 24 h of incubation, the old culture medium was removed and Cu@cLANs material working solution was added. The cLANs, Cu ion groups and blank groups were set as controls. After 24 h of incubation, the material working solution was washed away, and the residual material was washed with PBS. Then DCFH-DA working solution was added for incubation for 30 min, and the DCFH-DA working solution was washed away. PBS was used for washing 3 times, and a small amount of PBS was left in the culture dish. Cell image acquisition was performed by laser confocal microscopy, and the results are shown in Figure 6 Fig. 7. After co-incubation of the experimental group Cu@cLANs with cells, strong ROS was generated in the cells.

[0078] The mechanism of ROS production by the nanoparticles in circulation was characterized by detecting the ROS production ability, metal ion content, and DHLA content in the cells over time. The mechanism of circulating metal ions means that the metal ion and DHLA content in the cells should be maintained at a high level, and the ROS production as an effect should also exhibit a continuously rising level. The specific experimental operation is as follows: ① Characterization of the change in ROS production ability in the cells over time: set up a separate copper ion group, a cLANs group, and a Cu@cLANs group, incubate them with tumor cells for different times, and then incubate them with a DCFH-DA working solution. The ROS content in the cells is detected by a fluorescence microplate reader or a flow cytometer, and a curve graph of the change in ROS content in the cells over time is drawn. ② Characterization of the change in metal ion / DHLA content in the cells over time: set up a free copper ion group, a cLANs group, and a Cu@cLANs group, incubate them with cells for different times, collect the cells and lyse them, and then test the metal and DHLA content in the cells by ICP-MS and HPLC, respectively. A curve graph of the change in metal / DHLA content in the cells over time is drawn, and the trend of the change in metal / DHLA content in the cells over time in the nanoparticle group and the free metal ion group is compared to verify the circulation of the material in the cells. The change in ROS content over time is as shown in Figure 7 the left, the change in relative Cu element content over time is as shown in Figure 7 the middle, and the change in relative DHLA content over time is as shown in Figure 7 the right.

[0079] Example 7 In vitro biocompatibility detection

[0080] 1. Cytotoxicity of normal cells

[0081] Normal cells in the active logarithmic growth phase were selected, inoculated in a 96-well plate, and cultured for 24 h. Then different concentrations of metal thioctic acid nanoparticles were added to them, different concentration gradients were set, 5 parallel samples were set for each concentration, and a control group was set. After continuing to culture for 48 h, the culture medium was removed, 100 μL of culture medium containing 10% (v / v) MTT was added for incubation for 2 h, the old culture medium was removed, 100 μL of DMSO was added to each well, shaken for 2 min, and finally the absorbance at 490 nm was measured by a microplate reader to calculate the cell survival rate, and the results are as shown in Figure 8 The experimental results show that the cytotoxicity of Fe@cLANs normal cells is lower than that of tumor cells, and the IC 50 of Fe@cLANs on normal cells is about 10 μg / mL, and the cytotoxicity of Fe@cLANs on normal cells is about 50 μg / mL.

[0082] 2. Hemolysis and coagulation experiment

[0083] The hemolysis coagulation test was used to evaluate the blood compatibility of the material. Taking Fe@cLANs as an example, the blood of Kunming mice was collected by orbital bleeding, and then centrifuged and washed with physiological saline to prepare a 10% (v / v) red blood cell (RBC) suspension. 950 μL of Fe@cLANs physiological saline solution with different concentrations (25, 20, 10, 5 and 1 mg / mL) was taken, and 50 μL of 10% (v / v) red blood cell suspension was added, and incubated in a 37°C water bath shaker for 2 h. Physiological saline was used as a negative control, and distilled water was used as a positive control. After incubation, the blood cells were removed by centrifugation at 1200 rpm / min for 10 min. After taking a photo, the supernatant was transferred to a 96-well plate, and the absorbance of the supernatant in the plate was detected at 542 nm. The hemolysis rate was calculated as follows: hemolysis rate % = (A sample -A negative ) / (A positive -A negative ) x 100%, where A sample is the absorbance value of the sample; A positive and A negative are the absorbance values of 50 μL of 10% (v / v) red blood cell suspension in 950 μL of distilled water and 950 μL of physiological saline, respectively.

[0084] The blood clotting experiment used the same method to collect blood and prepare a 2% (v / v) red blood cell (RBC) suspension. In a 24-well plate, 950 μL of Fe@cLANs physiological saline solution with different concentrations (25, 20, 10, 5 and 1 mg / mL) was added, followed by 50 μL of 2% (v / v) red blood cell suspension, and incubated at room temperature for 2 h. After 2 h of incubation, the blood clotting of red blood cells was observed by inverted microscope, and the experimental results were recorded by microscope magnification of 400 times. The 2% (v / v) red blood cell suspension was added to physiological saline, and this group was used as a negative control.

[0085] The results of the hemolysis coagulation experiment are shown in Figure 9 At a high concentration of 20 mg / mL, no obvious hemolysis and coagulation of mouse blood occurred. Cu@cLANs also had good biological safety.

[0086] Example 8: In vivo biocompatibility detection

[0087] Acute toxicity test was used to further evaluate the in vivo biocompatibility of Fe@cLANs. The acute toxicity test was divided into two steps: ① LD 50 test; ② physicochemical analysis of mice at the maximal tolerable dose (MTD). The physicochemical analysis included the following aspects: physiological state of mice (body weight, state), whole blood, blood biochemical analysis and histopathological examination of organs. According to the LD 50The test principle, set as follows: the dosage (768, 960, 1200, 1500, 1875 mg / kg) is administered.

[0088] 50 Kunming mice, half male and half female, are divided into 5 groups, 5 male and 5 female in each group. After the approximate lethal dose is explored in advance, each mouse is injected with 100 μL of Fe@cLANs nanoparticles of a given dose for the evaluation of subsequent mouse death. The death is input into the LD 50 Calculation software is used to output the 95% confidence interval of the LD 50 The acute toxicity test results are shown in Table 1 below.

[0089] Table 1. Acute toxicity test results

[0090]

[0091] After the completion of the acute toxicity test, a single large dose of Fe@cLANs is administered for testing. The experimental process is as follows: 18 female Kunming mice are randomly divided into three groups (dose group, control group, blank group), and a single tail vein injection of 100 μL of a large dose (800 mg / kg) of Fe@cLANs nanoparticles is performed. Saline is used as the control group, and untreated mice are used as the blank group. The body weight of the mice is recorded every 2 days after administration for a total of 14 days. After 14 days, some of the mice blood is collected from the orbit into a blood collection tube for whole blood analysis; the blood of the other mice is directly collected into a 1.5 mL centrifuge tube, and after being placed at room temperature for 30 min, the supernatant is collected by centrifugation at 3000 rpm / min to obtain serum, which is used for mouse liver and kidney function analysis.

[0092] The experimental results prove that the LD 50 of the mice is between 1000-1200 mg / kg, and after a large dose of tail vein administration, the body weight of the mice does not decrease significantly within 14 days, and the blood biochemical analysis does not show significant abnormalities, and the whole blood analysis does not show significant abnormalities. The body weight is shown in Table 2, and the blood biochemical analysis and whole blood analysis are shown in Table 3. Figure 10

[0093] Table 2. Whole blood indicators of acute toxicity in mice

[0094]

[0095] Table 3. Detection results of liver and kidney function of blood biochemical analysis of acute toxicity in mice

[0096]

[0097]

[0098] Example 9

[0099] ​The in vivo antitumor effect of Cu@cLANs was studied by establishing a subcutaneous mammary cancer doxorubicin-resistant cell (MCF-7ADR) tumor model in nude mice. MCF-7ADR cells were cultured at a concentration of 1×10⁻⁶ cells. 7 A certain number of Bab / c nude mice were inoculated into the anterior axillae. Seven days after modeling, when the tumors grew to 50 mm... 3 In this study, Cu@cLANs and various control groups were administered via tail vein injection to mice at a concentration of 5 mg / kg. The dosage for each control group was converted to an equivalent amount of the compound Cu or LA. Six mice were in each group. Administration was repeated every two days for 18 days, and changes in tumor volume and body weight were recorded. Figure 13 As shown on the left, the doxorubicin group showed a weaker inhibitory effect, with a tumor inhibition rate of only 40.8%, while the Cu@cLANs group achieved a tumor inhibition rate of 60.7%. Therefore, Cu@cLANs exhibits better in vivo antitumor effects. Compared to the control group and the chemotherapy drug group, Figure 11 The weight change curves in mice showed no significant abnormal changes in weight during treatment, demonstrating the good biocompatibility of the nanoparticles. The prolonged survival further proves the good therapeutic effect and low biotoxicity of Cu@cLANs.

[0100] Example 10

[0101] The in vivo antitumor effect of Fe@cLANs was studied by establishing a subcutaneous glioma cell (U251) tumor model in nude mice. Five 5-week-old female BALB / c nude mice were subcutaneously injected with 100 μL of U251 cells in PBS suspension (5 × 10⁻⁶). 5 A tumor model was established. As the tumor grew, it was dissected, and necrotic tissue and blood vessels were removed. Tumors with good quality were selected and surgically cut into 2mm pieces. 3 Fragments were inserted subcutaneously into new nude mice (24 female BALB / c nude mice) using a puncture needle. Seven days later, when the tumor volume increased to ~50 mm... 3 The antibody-mediated tumor assay was performed. Twenty-four female BALB / c nude mice were randomly divided into four groups: a blank control group (saline), TMZ (temozolomide, 10 mg / kg), and Fe@cLANs (50 mg / kg, 100 mg / kg), with six mice in each group. Administered the drugs every two days for 18 days. Tumor volume and body weight changes were recorded simultaneously with drug administration. The tumor volume and body weight at the start of administration were recorded as 100%. The formula for calculating mouse tumor volume was: volume in mm². 3 = 1 / 2 × length (mm) × width (mm). Experimental results are as follows: Figure 12As shown in the left, the tumor inhibition effect of TMZ group is weak, only 41.6% of inhibition rate, while the inhibition effect of Fe@cLANs of 50mg / kg and 100mg / kg is stronger, 65.8% and 87.3% respectively, which shows that Fe@cLANs greatly inhibits the increase of tumor volume. During the treatment, the body weight of mice (middle) does not show obvious abnormal change, which reflects the good biocompatibility of the nanoparticles. Figure 12 As shown in the right, the survival cycle statistics of Fe@cLANs group further proves that the iron-lipoic acid composite particles have good treatment effect and low biological toxicity. Figure 12

[0102] Example 11

[0103] Under the condition of nitrogen, lipoic acid (LA, 1.0g, 5mmol), 1-ethyl-3(3- dimethylpropylamine) carbodiimide (1.15g, 6.0mmol) and 4-dimethylamino pyridine (0.74g, 6mmol) were dissolved in 60ml of dichloromethane, after stirring for 2h, polyethylene glycol 400 (PEG400, 4.0g, 6mmol) was added, and then stirring at room temperature for 24h to obtain lipoic acid derivative LA-PEG400 grafted with PEG400. 10mg of LA-PEG400 was weighed and dissolved in 3mL of deionized water to prepare nanoparticles by ultrasonic. Then, an aqueous ferric chloride solution was added dropwise to the above nanoparticle solution to prepare iron-lipoic acid polyethylene glycol 400 nanoparticles (Fe@cLA-PEG400), wherein the final concentration of nanoparticles was 2mg / mL, and the final concentration of ferric chloride was 0.1mg / mL.

[0104] The obtained Fe@cLA-PEG400 was labeled with coumarin (C6#Fe@cLA-PEG400) for macrophage uptake experiment. Murine macrophages (RAW264.7) in the active logarithmic growth phase were inoculated in a 6-well plate, incubated in fresh serum-free medium for 2h, and then C6#Fe@cLA-PEG400 was added for incubation for 1h, 3h and 6h respectively. After incubation, the culture medium was removed and washed with PBS for 3 times to remove the materials not entered into the cells. Finally, the cells were collected and subjected to intracellular fluorescence intensity quantitative analysis by cell flow cytometry. The results show that only a small amount of nanoparticle drugs enter the cells after 6h of action with the cells, indicating that the nanoparticle drugs can effectively escape phagocytosis and have long circulation ability.

[0105] ​Referring to Embodiment 5, the in vitro anti-tumor effect of Fe@cLA-PEG400 was evaluated using a U251 tumor cell model; referring to Embodiment 10, the in vivo anti-tumor effect of Fe@cLA-PEG400 was evaluated using a U251 subcutaneous tumor model. The experimental results showed that Fe@cLA-PEG400 exhibited a better in vivo tumor inhibition effect than Fe@cLANs in Embodiment 10.

[0106] Embodiment 12

[0107] To the solution of the cross-linked thioctic acid aggregate-iron nanocomposite Fe@cLANs prepared in Embodiment 2, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added, and after stirring for 2 h, 10% molar amount of glucosamine (GlcN) was added. After 48 h of reaction, the obtained nanoparticles were dialyzed to prepare the glucosamine-modified nanodrug GlcN / Fe@cLANs with tumor targeting. Referring to the experimental methods described in Embodiments 7 and 8, the safety evaluation was performed, and the results showed that the glucosamine-modified nanodrug exhibited excellent biological safety. Further referring to Embodiments 5, 6, 9 and 10, the efficacy evaluation was performed, and the results showed that the targeted nanodrug exhibited a better therapeutic efficacy than the non-targeted nanodrug.

[0108] The above only describes the preferred application examples of the present application, which are only illustrative but not limiting; those skilled in the art understand that many changes, modifications and even equivalent changes can be made to the present application within the spirit and scope defined by the claims of the present application, and all shall fall within the protection scope of the present application.

Claims

1. A metal-lipoic acid nanoparticle complex, characterized in that, The lipoic acid particle and the Fenton-type metal ion combined therewith, the Fenton-type metal ion being a metal ion capable of participating in Fenton reaction or Fenton-like reaction; the lipoic acid particle being any one of micelles, vesicles or disordered aggregates formed by self-assembly of lipoic acid and / or lipoic acid derivatives.

2. The metal-lipoic acid nanoparticle complex of claim 1, wherein, The lipoic acid particle is formed by raw materials containing lipoic acid and / or lipoic acid derivatives.

3. The metal-lipoic acid nanoparticle complex of claim 1, wherein, The Fenton-type metal ion is combined with the lipoic acid particle by coordination or hydrophobic-hydrophilic interaction.

4. The metal-lipoic acid nanoparticle complex of claim 1, wherein, The Fenton-type metal ion is coordinated with the sulfydryl and / or carboxyl in the lipoic acid particle.

5. The metal-lipoic acid nanoparticle complex of claim 1, wherein, Part of the disulfide five-membered ring in the core of the lipoic acid particle is broken, and part of the sulfydryl is coordinated with the Fenton-type metal ion by coordination.

6. The metal-lipoic acid nanoparticle complex of claim 1, wherein, The lipoic acid particle is internally crosslinked.

7. A method for preparing the metal-lipoic acid nanocomposite according to claim 1, characterized in that, It comprises the following steps: first, prepare the lipoic acid particle by self-assembly, then add a solution containing the Fenton-type metal ion, so that the Fenton-type metal ion is stably combined with the lipoic acid particle to form a metal-lipoic acid composite particle; or first mix the constituent units of the lipoic acid particle with the solution of the Fenton-type metal ion, then prepare the metal-lipoic acid composite particle by self-assembly.

8. The method for preparing the metal-lipoic acid nanocomposite according to claim 7, characterized in that, It also comprises a crosslinking step, in which part of the sulfydryl in the metal-lipoic acid composite particle is crosslinked with each other to form disulfide bond, thereby improving its stability.

9. Use of the metal-lipoic acid nano-complex of claim 1, wherein, It is used for preparing an antitumor drug.

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