WO2019206030A1 - 一种抗肿瘤纳米药物 - Google Patents

一种抗肿瘤纳米药物 Download PDF

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WO2019206030A1
WO2019206030A1 PCT/CN2019/083390 CN2019083390W WO2019206030A1 WO 2019206030 A1 WO2019206030 A1 WO 2019206030A1 CN 2019083390 W CN2019083390 W CN 2019083390W WO 2019206030 A1 WO2019206030 A1 WO 2019206030A1
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lipoic acid
nanomedicine
antitumor
tumor
cells
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PCT/CN2019/083390
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English (en)
French (fr)
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张仕勇
廖春燕
陈英
代鑫
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四川大学
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Priority to EP19791524.2A priority Critical patent/EP3795151A4/en
Priority to US17/050,776 priority patent/US20210093607A1/en
Publication of WO2019206030A1 publication Critical patent/WO2019206030A1/zh

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Definitions

  • the invention belongs to the field of biological materials, and particularly relates to an anti-tumor nano drug.
  • Cancer is a common and multiple major disease that seriously endangers human health, and its mortality rate ranks second in the overall mortality rate of human diseases.
  • Chemotherapy is an important means of cancer treatment.
  • traditional chemotherapy drugs are prone to cause toxic and side effects such as impaired liver and kidney function, bone marrow suppression, and reduced human immunity. Therefore, the development of new chemotherapy drugs with anti-tumor activity but no toxicity or low toxicity to normal tissues. Can solve the problems faced by traditional chemotherapy drugs well.
  • LA (R)-(+)-lipoic acid [(R)-(+)-Lipoic Acid, abbreviated as LA] is a kind of B vitamins synthesized in the mitochondria of lipoic acid synthase, which has stable blood sugar, strengthen liver function, Relieve fatigue, beauty and anti-aging effects.
  • large doses of LA also have a certain anti-tumor effect, and are not toxic to normal cells at the same dose, so LA has excellent application prospects as a natural anti-tumor active substance.
  • the small molecule LA has both hydrophilicity and lipophilicity, it can reach any part after entering the body and is easily removed quickly, resulting in a large dosage and poor curative effect.
  • Combination therapy can improve the therapeutic effect of LA, but when combined with other small molecule drugs, various drugs cannot enter the cell at a predetermined ratio, and it is difficult to achieve the effect of "1+1 greater than 2".
  • the present invention has developed a novel anti-tumor nano drug for the above problems.
  • the nano drug prepares (R)-(+)-lipoic acid as a lipoic acid polymer loaded on a nano drug carrier or directly prepares a lipoic acid polymer into nanoparticles, which significantly increases (R)-(+)-
  • the anti-tumor effect of lipoic acid monomer avoids the problem of large side effects of traditional chemotherapy drugs.
  • the anti-tumor active ingredient lipoic acid multimer can also be used in combination with other substances having anti-tumor activity, and enter the cell at a predetermined ratio to achieve a synergistic anti-tumor effect of "1+1 greater than 2", further improving the nano drug. Efficacy.
  • An anti-tumor nano drug whose anti-tumor activity component mainly exists in the form of a lipoic acid multimer.
  • the nano drug may be in the form of a liposome, a dendrimer, a polymer micelle, a vesicle, an aggregate, or the like.
  • the lipoic acid multimer contains an R-type chiral structure.
  • the lipoic acid multimer is mainly composed of (R)-(+)-lipoic acid or a pharmaceutically acceptable salt thereof.
  • the lipoic acid multimer is constructed from (R)-(+)-lipoic acid or a pharmaceutically acceptable salt thereof.
  • the structural formula of the lipoic acid multimer is as follows:
  • n ⁇ 2 and R is a hydroxyl group or a functional group linked by an ester bond/amide bond or an O - M + structure (M + is a metal ion).
  • the lipoic acid multimer is present in at least one of a chain polymer, a micelle, a vesicle, and an aggregate.
  • the lipoic acid multimer is prepared by lipoic acid itself or by template molecule assisted preparation.
  • the lipoic acid multimer can be used in combination with other substances having antitumor activity.
  • the lipoic acid multimer can synergize with other substances having antitumor activity to resist tumor, and has a "1+1 greater than 2" effect.
  • the other substance having antitumor activity is any substance capable of producing a synergistic antitumor effect with the lipoic acid multimer.
  • traditional chemotherapy small molecule drugs such as camptothecin, hydroxyurea, pilocarpine, doxorubicin hydrochloride, gemcitabine hydrochloride, cytarabine, etc.
  • natural anti-tumor active substances such as anthocyanins, resveratrol , curcumin, tomato, tea polyphenols, astaxanthin, etc.
  • the lipoic acid polymer may be physically or chemically bonded to the nanocarrier.
  • the nano drug carrier is a liposome, a dendrimer, a polymer micelle, a vesicle, an aggregate, a lipoic acid polymer, or the like.
  • the lipoic acid multimer also functions as a nanocarrier.
  • the lipoic acid chain polymer, micelles, vesicles or aggregates can be used directly for anti-tumor.
  • the lipoic acid multimer may be physically or chemically bonded to the nano drug carrier, and the nano drug carrier is loaded with other anti-tumor active ingredients, and the two are synergistically resistant.
  • the tumor; or the lipoic acid multimer is directly used to load other substances having antitumor activity, and the two synergistically resist tumor.
  • the lipoic acid multimer is physically supported in a dendrimer nanocarrier for antitumor.
  • the lipoic acid multimer is physically contained in a liposome nanocarrier for antitumor.
  • the lipoic acid multimer is physically encapsulated in a liposome nanocarrier, while the liposome is loaded with a hydrophobic natural antitumor active substance, curcumin.
  • the lipoic acid multimer is physically contained in a liposome nanocarrier, and the liposome is loaded with a hydrophilic chemotherapeutic drug, gemcitabine hydrochloride.
  • the lipoic acid multimer is physically contained in the liposome nanocarrier, while the liposome is loaded with a hydrophilic natural antitumor active substance, resveratrol.
  • the polymer in the form of a lipoic acid chain polymer, a micelle, a vesicle, an aggregate or the like is loaded by a physical entrapment or a chemical bonding. Tumor active substance.
  • the lipoic acid micelle multimer is covalently linked to a hydrophilic chemotherapeutic drug, cytarabine, and a physiologically hydrophobic natural antitumor active substance, anthocyanin.
  • the lipoic acid vesicle polymer is physically loaded with a hydrophilic chemotherapeutic drug, hydroxyurea.
  • the lipoic acid vesicle multimer is covalently linked to the hydrophilic chemotherapeutic drug cytarabine, and the hydrophilic chemotherapeutic drug-hydroxyurea is physically loaded.
  • LA is chemically linked with other substances having antitumor activity, and the resulting linker is constructed into a chain polymer, a micelle, a vesicle, an aggregate, and the like. Multimer.
  • LA directly covalently links the hydrophilic drug-hydroxyurea for direct formation of micelles, and further forms stable nanoparticles by lipoic acid crosslinking.
  • LA and other substances having antitumor activity are physically mixed to form a polymer in the form of a chain polymer, a micelle, a vesicle, an aggregate or the like.
  • LA directly forms aggregates with the chemotherapeutic drug-camptothecin and the natural antitumor active substance anthocyanin, and further forms a stable nanoparticle by cross-linking with lipoic acid.
  • the invention also provides the use of a lipoic acid multimer which is useful for the preparation of the antitumor nanomedicine described above.
  • the nano drug of the invention uses non-toxic lipoic acid multimer as an anti-tumor active ingredient, avoids the toxic side effects caused by the traditional chemotherapy drugs, and overcomes the inherent defects of the small molecule drugs, and is not easily removed after entering the blood circulation.
  • the targeting is good, so the antitumor activity can be significantly improved compared with the lipoic acid monomer.
  • the lipoic acid multimer When used in combination with other substances having antitumor activity, it can also enter the cells at a predetermined ratio. Compared with the simple mixing of several drugs, the ratio of the combined drugs can be controlled to reach "1+1 greater than 2". The synergistic anti-tumor effect further enhances the efficacy of nanomedicine.
  • Figure 1 is a schematic view of a lipoic acid multimer in Example 1;
  • Example 2 is a graph showing the characterization results of lipoic acid micelles, vesicles and aggregates in Example 1, wherein a) is a DLS size map, and b) and c) are lipoic acid vesicles and lipoic acid aggregates respectively containing fluorescent pink. Fluorescence intensity change diagram of fluorescent pink B before and after Triton X-100 was added after passing through the gel column;
  • FIG. 3 is a graph showing the results of anti-tumor mechanism of lipoic acid micelles in Example 2, wherein a) is a toxicity test result of lipoic acid micelles on human colon cancer cells (SW480), and b) is a cell mitochondria after different materials are applied. Relative to the O 2 -. level, c) the percentage loss of mitochondrial membrane potential after action of different materials, and d) the relative amount of Caspase-3 activation in cells after treatment with different materials;
  • Example 4 is a cytotoxicity test result of lipoic acid micelles and lipoic acid monomers in Example 3;
  • Example 6 is a test result of normal cytotoxicity of lipoic acid micelles in Example 5, wherein a) is a toxicity test result on human renal epithelial cells (293T), and b) is a toxicity test result on mouse fibroblasts (3T3). ;
  • Figure 7 is a hemolysis and coagulation performance evaluation of lipoic acid micelles in Example 5, wherein a) is hemolysis rate, and b) is coagulability;
  • Figure 8 is a graph showing changes in body weight of mice in Example 5.
  • HGF human gingival fibroblasts
  • HGF human gingival fibroblasts
  • Figure 11 is a diagram showing the toxicity evaluation of the nano drug prepared by the lipoic acid micelle and the lipoic acid monomer in Example 7 co-loaded with the curcumin in the liposome, wherein a) is cytotoxicity, and b) is a synergistic index;
  • Example 12 is a result of covalently linked cytarabine and its combined anti-tumor study of lipoic acid micelles in Example 8, wherein a) is a cytotoxicity test result, and b) is a synergistic index;
  • Figure 13 is a diagram showing the results of the covalent attachment of hydroxyurea with lipoic acid in Example 9 and preparation of the nanomedicine and its combined antitumor research, wherein a) is cytotoxicity and b) is a synergistic index;
  • Example 14 is a result of directly preparing a nanoparticle of lipoic acid, anthocyanin and lycopene in Example 10 and a combined antitumor effect thereof, wherein a) is cytotoxicity, and b) is a synergistic index;
  • Figure 15 is a graph showing the physical loading of thiourea of lipoic acid vesicles in Example 11 and the results of the combined antitumor studies thereof, wherein a) is cytotoxicity, and b) is a synergistic index;
  • Figure 16 is a synergistic antitumor result of hydroxyurea and lipoic acid vesicles in Example 12, wherein a) is the relative O 2 -. level in the mitochondria after the material is applied to the cells, b) is the percentage loss of the mitochondrial membrane potential, c) Relative percentage of activated caspase-3;
  • Figure 17 is an in vivo anti-tumor assessment of the nanomedicine in Example 13, wherein a) is the change in tumor volume in mice, b) is the change in body weight of the mouse, c) is the survival rate of the mouse, and d) is synergistic anti-tumor in vivo. Joint index (Q);
  • Figure 18 is a schematic view showing the structure of the antitumor nano drug of the present invention.
  • the lipoic acid multimer can exist as lipoic acid micelles, lipoic acid vesicles, and lipoic acid aggregates, as shown in FIG.
  • lipoic acid 100 mg was added to 50 ml of deionized water, and 1 M aqueous NaOH solution was added dropwise with stirring until the lipoic acid was completely dissolved, and then the solution was neutralized with 1 M HCl solution, and finally the solution was lyophilized to obtain a pale yellow color.
  • Sodium lipoic acid powder 41.2 mg (0.2 mol) of sodium lipoate was weighed, dissolved in 1 ml of deionized water, and ultrasonically prepared to obtain nanoparticles having a size of about 15 nm. The obtained nanoparticles were self-crosslinked by 365 nm ultraviolet light to initiate lipoic acid disulfide bond reaction, and the reaction was carried out for 2.5 h. After dialysis for 48 h, cross-linked lipoic acid micelles (c-LAMs) having a size of about 15 nm were obtained. As shown in Figure 2a.
  • the above vesicle structure was determined by the fluorescent peach B leak test. Specifically, as follows: 50 ⁇ l of the super amphiphilic mother liquor was added to 5 ml of a 0.5 mg/ml aqueous solution of Fluorescent Red B under ultrasonic conditions to prepare a nanoparticle solution containing fluorescent peach B. The resulting solution was separated by a gel column to collect a ribbon having a Tydal phenomenon. 2 ml of this collection solution was taken, 40 ⁇ l of 10% Triton X-100 solution was added thereto, and the change in fluorescence intensity of the fluorescent peach B before and after the addition of the nanoparticles to Triton X-100 was monitored. As shown in Fig.
  • the fluorescence intensity of the fluorescent peach B was very low before the addition of Triton X-100, and the fluorescence intensity was high after the addition. This is mainly due to the fact that the nanoparticles contain a large amount of hydrophilic fluorescent pink B.
  • the fluorescent pink B is quenched by fluorescence aggregation at a high concentration, so the fluorescence intensity is very low, and the addition of Triton X-100 destroys the structure of the nanometer.
  • the encapsulated fluorescent peach B is released to release fluorescence. Prove that it is a vesicle.
  • the vesicle nanoparticles prepared above were self-crosslinked by a lipoic acid disulfide bond initiated by 365 nm ultraviolet light. After 2.5 h of reaction, the pH of the solution was adjusted to be alkaline, and dichloromethane was extracted to remove 1,4,7- in the solution. Triazadecane, then adjust the pH of the solution back to neutral again, and after 48 hours of dialysis, finally obtain cross-linked Lipoic acid vesicles (c-LAVs) with a size of about 220 nm, as shown in Figure 2b. Shown.
  • lipoic acid 41.2 mg was dissolved in 1 ml of DMF and shaken on a shaker for 2 h to obtain a 0.2 M lipoic acid mother liquor. 50 ⁇ l of this mother liquor was added to 5 ml of deionized water under ultrasonic conditions to prepare lipoic acid nanoparticles having a size of about 80 nm. The obtained nanoparticles were self-crosslinked by a 365 nm ultraviolet light to induce lipoic acid disulfide bonds, and the reaction was carried out for 2.5 hours. After dialysis, cross-linked Lipoic Acid Nanoparticles (c-LANPs) having a size of about 75 nm were obtained. ), the results are shown in Figure 2a.
  • c-LANPs cross-linked Lipoic Acid Nanoparticles
  • the above aggregate structure was determined by the Fluorescence Pink B leak test. Specifically, as follows: 50 ⁇ l of lipoic acid mother liquor was added to 5 ml of a 0.5 mg/ml aqueous solution of Fluorescent Red B under ultrasonic conditions to prepare a nanoparticle solution containing fluorescent peach B. The resulting solution was separated by a gel column to collect a solution having a Tydal phenomenon. 2 ml of this collection solution was taken, 40 ⁇ l of 10% Triton X-100 solution was added thereto, and the change in fluorescence intensity of the fluorescent peach B before and after the addition of the nanoparticles to Triton X-100 was monitored. As shown in Fig. 2d, it was found that there was no significant change in the fluorescence intensity of the fluorescent peach B before and after the destruction of the nanoparticle structure. Combined with the large size, the nanoparticles were judged to exist as aggregates.
  • c-LAMs cross-linked lipoic acid micelles
  • SW480 Human colon cancer cells in the logarithmic growth phase were selected and inoculated into 96-well plates. After 24 hours of culture, different concentrations of LA and c-LAMs were added to them, and different concentration gradients were set. Parallel samples and set the control group. After continuing to culture for 48 hours, after removing the medium, 100 ⁇ l of medium containing 10% (v/v) MTT was added and incubation was continued for 2 hours, then the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and it was shaken on a shaker. At 2 min, the absorbance at 490 nm was measured by a microplate reader, and the cell survival rate was calculated. The results are shown in Fig. 3a. The results showed that c-LAMs had certain cytotoxicity to tumor cells SW480 and had better antitumor activity relative to LA.
  • LA (R)-(+)-lipoic acid
  • GSH glutathione
  • TrxR thioredoxinreductase
  • DHLA Dihydrolipoic acid
  • LA can directly oxidize the sulfhydryl group of the protein on the mitochondrial permeablity transition pore (mPTP)
  • mPTP mitochondrial permeablity transition pore
  • DHLA can also indirectly oxidize the sulfhydryl group of the protein on mPTP through the generated superoxide anion radical (O 2 - ⁇ ).
  • AIF Apoptosis Inducing Factor
  • cytochrome released from mitochondria C can stimulate the expression of proapoptotic proteins (Caspase-9, Caspase-3) and induce apoptosis.
  • the superoxide anion radical (O 2 - ⁇ ) produced by DHLA can also reduce the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL, and further induce the expression of proapoptotic protein (Caspase-9, Caspase-3). The amount is increased to induce apoptosis.
  • c-LAMs are constructed by LA, on the one hand, they have the same disulfide bond structure; on the other hand, intracellular reduced glutathione (GSH) and thioredoxin reductase (Thioredoxinreductase, Under the action of TrxR), the reduction product of c-LAMs and LA is also dihydrolipoic acid.
  • GSH glutathione
  • TrxR thioredoxin reductase
  • c-LAMs can produce superoxide anion in the mitochondria of cells, stimulate the opening of mitochondrial membrane permeability transition pores, and cause membrane potential to decrease, further releasing cytochrome C in cytoplasm in mitochondria, thereby regulating Caspase-3 dependence.
  • the apoptotic pathway induces apoptosis in cells, and the anti-tumor activity is enhanced relative to LA.
  • SW480 cells with active logarithmic growth phase were prepared and prepared into cell suspensions and added to 6-well plates at approximately 5 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM c-LAMs and LA were added, respectively, and a blank control group without any material was placed. After incubation for 4 h, the old medium was removed, 1 ml of fluorescamine dye with a concentration of 50 ⁇ M was added, and after incubation for 2 h, cysteine was added to give a final concentration of 200 ⁇ M. After incubation for 0.5 h, the old medium was aspirated and Krebs was used.
  • Mitochondrial membrane potential detection SW480 cells in the logarithmic growth phase were prepared and prepared into cell suspensions and added to a 6-well plate, with about 1 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM LA and c-LAMs were added, respectively, and a blank control group without any material was placed. After incubation for 24 h, the old medium was aspirated, and each group of cells was collected and centrifuged to obtain pure cells. Add 1 ml of mitochondrial membrane potential detection kit (JC-1) working solution to the collected cells, mix well and incubate for 20 min in the incubator, then centrifuge at 4 °C to remove the mitochondrial membrane potential detection kit (JC-1).
  • JC-1 mitochondrial membrane potential detection kit
  • SW480 cells with active logarithmic growth phase were prepared and prepared into cell suspensions and added to 6-well plates at approximately 1 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM c-LAMs and LA were added, respectively, and a blank control group without any material was placed. After 4 h of incubation, the old medium was removed and the cells were collected. The collected cells were centrifuged at 1200 rpm for 5 min at 4 ° C, and the supernatant was carefully aspirated. The cells were washed twice with cold PBS and the supernatant was aspirated as much as possible.
  • a cell lysate containing 1 mM phenylmethylsulfonyl fluoride (PMSF, protease inhibitor) was added to the collected cells, placed on ice for 30 min, and vortexed once every 5 min. After centrifugation at 12000 rpm for 60 min at 4 ° C, the supernatant was quickly collected in another clean centrifuge tube, 20 ⁇ l of the supernatant was taken, and the cytochrome C content in the cytoplasm was analyzed by western blot. The results indicate that c-LAMs can induce cytochrome C release into the cytoplasm more strongly than LA.
  • PMSF phenylmethylsulfonyl fluoride
  • Caspase-3 viability assay SW480 cells with active logarithmic growth phase were prepared and prepared into cell suspensions and added to 6-well plates at approximately 1 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM c-LAMs and LA were added, respectively, and a blank control group without any material was placed. After 4 h of incubation, the old medium was removed and the cells were collected. The collected cells were centrifuged at 1200 rpm for 5 min at 4 ° C, and the supernatant was carefully aspirated. The cells were washed twice with cold PBS and the supernatant was aspirated as much as possible.
  • the lipoic acid micelles were replaced with lipoic acid vesicles and aggregates, respectively, and the same results were obtained.
  • lipoic acid micelles were selected to evaluate the antitumor activity of lipoic acid multimers and LA.
  • HepG2 Human hepatoma cells (HepG2) in the logarithmic growth phase were selected and inoculated into 96-well plates. After 24 hours of culture, c-LAMs and LA were added respectively. Two materials were set with different concentration gradients, 5 parallel samples were set for each concentration, and a control group was set. After 72 hours of culture, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min. After using the microplate reader to measure the absorbance at 490 nm, the cell survival rate was calculated. The experimental results are shown in Figure 4.
  • the c-LAMs have better antitumor activity than LA.
  • the reason may be that c-LAMs enter the cells in the form of nanoparticles. Compared with LA, the intracellular concentration is higher, and thus exhibits a better antitumor effect.
  • the lipoic acid micelles were replaced with lipoic acid vesicles or lipoic acid aggregates, and the same results were obtained.
  • the lipoic acid multimer and LA were separately loaded in the liposome for evaluation of antitumor activity.
  • lipoic acid micelles c-LAMs
  • LA lipoic acid micelles
  • c-LAMs Dissolve 10 mg of c-LAMs and 10 mg of LA in 20 ml of deionized water, respectively, and then add 10 mg of liposome. After ultrasonication for 2 min, a clear and transparent solution was obtained. The liposome extruder was passed through a 400 nm polycarbonate membrane for 3 times each. The 2KD dialysis bags were separately loaded, and lyophilized for 48 hours, and then lyophilized to obtain two kinds of nano drugs.
  • the physical load of c-LAMs is nano drug I in the liposome
  • LA is nano drug II in the liposome.
  • Nanomedicines I and II Human hepatoma cells (HepG2) in the logarithmic growth phase were selected and inoculated into 96-well plates. After 24 hours of culture, nanomedicines I and II were added. Two materials were set with different concentration gradients, 5 parallel samples were set for each concentration, and a control group was set. After 72 hours of culture, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min. After that, the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate. The experimental results are shown in Figure 5. Nanomedicine I has better antitumor activity than nanomedicine II.
  • the lipoic acid micelles were replaced with lipoic acid vesicles or lipoic acid aggregates, and the same results were obtained.
  • lipoic acid micelles c-LAMs
  • the toxicity of lipoic acid multimers to normal cells/organisms was evaluated.
  • c-LAMs Human renal epithelial cells (293T) and mouse fibroblasts (3T3) in logarithmic growth phase were selected and inoculated into 96-well plates. After 24 hours of culture, c-LAMs were added to set different concentration gradients. Five concentrations were set for each concentration and a control group was set. After culture for 48 hours, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min. After that, the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate. The experimental results are shown in Figure 6. The c-LAMs were not toxic to normal cells 293T and 3T3 at a certain concentration.
  • Hemolysis evaluation blood was collected from the orbital fossa, 3 ml of fresh blood of normal mice was collected, and 12 ml of physiological saline was added, and red blood cells were collected by centrifugation at 4 ° C, and the collected red blood cells were washed 3 times with physiological saline to prepare 10% (v/v). Red blood cell suspension.
  • Hemolysis ratio% (A sample -A negative )/(A positive -A negative ) ⁇ 100 (1)
  • a sample is the absorbance of the material group
  • a negative is the absorbance of the negative control group
  • a positive is the absorbance of the positive control group.
  • the experimental results are shown in Figure 7a.
  • the hemolysis rate is also increased.
  • the hemolysis rate is still less than 5% (less than 5% is considered normal), so the description of c- LAMs are hemolytic.
  • Coagulation evaluation Configure a red blood cell suspension with a concentration of 2%, take 50 ⁇ l of cell suspension in a 6-well plate, add different concentrations of c-LAMs solution, and make the final concentration of 30mg/ml, 20mg/ml, 10mg respectively. /ml, 5mg/ml and 1mg/ml. After standing at room temperature for 2 h, an inverted fluorescence microscope was used to observe whether there was coagulation. The experimental results are shown in Fig. 7b, and c-LAMs have no obvious coagulation even at high concentrations.
  • mice 15 four-week-old BALB/c mice (20 g/mouse) were randomly divided into 3 groups, 5 in each group, which were blank control group, saline group and c-LAMs group.
  • a large dose of c-LAMs 200 mg/kg was injected into the c-LAMs group through the tail vein, and an equal volume of physiological saline was injected into the saline group, and the blank control group did not perform any treatment.
  • the body weight change of the mice was monitored once every 2 days. After two weeks of administration, the blood of the mice was collected by blood collection from the orbits, and blood samples of each group were subjected to blood routine examination and liver and kidney function tests. The experimental results are shown in Fig. 8. After the large dose of the c-LAMs group, the body weight of the mice remained normal, and the blood and liver and kidney functions of the mice were normal, which was consistent with the results of the saline group and the blank group.
  • c-LAMs have almost no toxic effects on normal cells and the body, and also have good biocompatibility.
  • the lipoic acid micelles were replaced with lipoic acid vesicles or lipoic acid aggregates, and the same results were obtained.
  • c-LAMs lipoic acid micelles
  • HGF Human gingival fibroblasts
  • c-LAMs were added to set different concentration gradients. 5 parallel samples and set the control group.
  • the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min. After that, the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate.
  • the experimental results are shown in Figure 9.
  • c-LAMs are not toxic to normal cells at higher doses, while Gem ⁇ HCl and DOX ⁇ HCl kill normal cells at very low doses.
  • c-LAMs, Gem ⁇ HCl and DOX ⁇ HCl were physically loaded into the liposome to prepare three nano drugs, namely nano drug III, nano drug IV and nano drug V, and evaluated for toxicity to normal cells. :
  • HGF Human gingival fibroblasts with active logarithmic growth were selected and seeded in 96-well plates. After 24 hours of culture, nano-drug III, nano-drug IV and nano-drug V were added. Set different concentration gradients, set 5 parallel samples for each concentration, and set the control group. After culture for 48 hours, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min. After that, the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate. The experimental results are shown in Fig. 10. Nanomedicine III is not toxic to normal cells at high concentrations, while nanomedicines IV and V are highly toxic to normal cells even at lower concentrations.
  • the lipoic acid micelles were replaced with lipoic acid vesicles or lipoic acid aggregates, and the same results were obtained.
  • c-LAMs and LA were separately loaded into liposomes with the hydrophobic natural anti-tumor active curcumin, and two nanomedicines VI and VII were prepared and subjected to synergistic anti-tumor evaluation.
  • c-LAMs and 5 mg of LA were dissolved in 20 ml of deionized water, respectively, and 10 mg of liposome was added thereto. After ultrasonication for 2 min, a clear transparent solution was obtained, and the liposome extruder was passed through a 400 nm polycarbonate membrane for 3 times. Liposomes containing c-LAMs and LA, respectively. 5 mg of curcumin was dissolved in 200 ⁇ l of dimethyl sulfoxide (DMSO), and the liposome obtained above was added separately, shaken in a water bath at 60 ° C for 20 min, and then placed in a 2KD dialysis bag, dialysis for 48 hours, and lyophilized to obtain two nano drugs. . Among them, c-LAMs and curcumin are physically loaded in the liposome for the nano drug VI, and LA and curcumin are physically loaded in the liposome to be the nano drug VII.
  • DMSO dimethyl sulfoxide
  • HepG2 Human hepatoma cells (HepG2) in a logarithmic growth phase were selected and seeded in 96-well plates. After 24 hours of culture, c-LAMs, LA, curcumin, and nanomedicines VI and VII prepared in this example were added, respectively. Set different concentration gradients, set 5 parallel samples for each concentration, and set the control group. After 72 hours of culture, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min.
  • the Coordination Index (CI) of the two nanomedicines was calculated by the Chou-Talalay joint index formula.
  • CI ⁇ 1 indicates synergy
  • CI>1 indicates antagonistic.
  • the experimental results are shown in Figure 11.
  • the combination of the two drugs has a better synergistic anti-tumor effect
  • the nano-drug VI has a better synergistic effect than VII.
  • the possible reason is that c-LAMs have better anti-tumor effect than LA.
  • anti-tumor shows a better synergistic effect.
  • c-LAMs covalently linked the hydrophilic chemotherapy drug cytarabine and performed synergistic anti-tumor evaluation.
  • SW480 cells in the logarithmic growth phase were selected and seeded in 96-well plates. After 24 hours of culture, cytarabine, c-LAMs, LA, cytarabine + c-LAMs, cytarabine + LA and The nanomedicine prepared in this embodiment. Set different concentration gradients, set 5 parallel samples for each concentration, and set the control group. After culture for 48 hours, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min.
  • the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate.
  • the CI values of cytarabine + c-LAMs, cytarabine + LA and the nanomedicine prepared in this example were calculated.
  • the experimental results are shown in Fig. 12.
  • the combination of the two drugs can increase the toxicity of the drug, and cytarabine + c-LAMs and cytarabine + LA have certain synergistic effects, but the nano drug synergy prepared in this embodiment
  • the anti-tumor effect is the best, mainly because the two drugs in the nano drug can enter the cell at a predetermined ratio, thus showing a better synergistic effect.
  • Lipoic acid and the chemotherapeutic drug hydroxyurea form a small molecule by covalent attachment, and then nanoparticle is prepared by coprecipitation method, and the nano drug is further obtained by disulfide polymerization of lipoic acid, and synergistic anti-tumor evaluation is performed.
  • HepG2 cells in the logarithmic growth phase were selected and seeded in 96-well plates.
  • LA, hydroxyurea, LA+hydroxyurea and nanomedicine prepared in this example were added. Set different concentration gradients, set 5 parallel samples for each concentration, and set the control group.
  • the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min. After that, the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate.
  • the CI values of LA + hydroxyurea and the nanomedicine prepared in this example were calculated.
  • the experimental results are shown in Fig. 13.
  • the LA+hydroxyurea and the nanomedicine prepared in the present embodiment all have a synergistic antitumor effect, but the nanomedicine prepared in this embodiment is more effective, mainly because the two drugs in the nano drug can A predetermined proportion of cells are introduced, thereby exhibiting a better synergistic effect.
  • Lipoic acid forms nanoparticles directly with natural anti-tumor active substances, tomato and anthocyanins, and lipoic acid is further polymerized to form nano-drugs for synergistic anti-tumor research.
  • HepG2 cells in the logarithmic growth phase were selected and seeded in 96-well plates. After 24 hours of culture, tomato, anthocyanin, lipoic acid + lycopene + anthocyanin and nanomedicine prepared in this example were added. Set different concentration gradients, set 5 parallel samples for each concentration, and set the control group. After 72 hours of culture, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min. After that, the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate.
  • the CI values of lipoic acid + lycopene + anthocyanin and the nanomedicine prepared in this example were calculated.
  • the experimental results are shown in Fig. 14.
  • the lipoic acid + lycopene + anthocyanin and the nano-drug prepared in this example all have a certain synergistic anti-tumor effect, but the nano-medicine prepared in this embodiment is better, mainly due to the nanometer.
  • the three drugs in the drug can enter the cells at a predetermined ratio, thereby exhibiting a better synergistic effect.
  • Lipoic acid vesicles physically load the hydrophilic chemotherapeutic drug hydroxyurea to prepare nanomedicines and conduct synergistic anti-tumor studies.
  • SW480 cells in the logarithmic growth phase were selected and seeded in 96-well plates. After 24 hours of culture, hydroxyurea, LA, c-LAVs, hydroxyurea + LA, hydroxyurea + c-LAVs and nanometers prepared in this example were added. drug. Set different concentration gradients, set 5 parallel samples for each concentration, and set the control group. After culture for 48 hours, the old medium was aspirated, 100 ⁇ l of medium containing 10% (v/v) MTT was added to each well, and after incubation for 2 hours, the old medium was aspirated, 150 ⁇ l of DMSO was added to each well, and the shaker was shaken for 2 min.
  • the absorbance at 490 nm was measured with a microplate reader to calculate the cell survival rate.
  • the CI values of hydroxyurea + LA, hydroxyurea + c-LAVs and the nanomedicine prepared in this example were calculated.
  • the experimental results are shown in Fig. 15.
  • the hydroxyurea+LA, hydroxyurea+c-LAVs and the nanomedicine prepared in the present embodiment all have a synergistic antitumor effect, but the nanomedicine prepared in this embodiment has a better effect, mainly Since the two drugs in the nano drug can enter the cell at a predetermined ratio, a better synergistic effect is exhibited.
  • SW480 cells with active logarithmic growth phase were prepared and prepared into cell suspensions and added to 6-well plates at approximately 5 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM hydroxyurea, c-LAVs and the nanomedicine of this example were added, respectively, and a blank control group without any material was placed. After incubation for 4 h, the old medium was removed, 1 ml of fluorescamine dye with a concentration of 50 ⁇ M was added, and after incubation for 2 h, cysteine was added to give a final concentration of 200 ⁇ M. After incubation for 0.5 h, the old medium was aspirated and Krebs was used.
  • Mitochondrial membrane potential detection SW480 cells in the logarithmic growth phase were prepared and prepared into cell suspensions and added to a 6-well plate, with about 1 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM hydroxyurea, c-LAVs and the nanomedicine of this example were added, respectively, and a blank control group without any material was placed. After incubation for 24 h, the old medium was aspirated, and each group of cells was collected and centrifuged to obtain pure cells. Add 1 ml of mitochondrial membrane potential detection kit (JC-1) working solution to the collected cells, mix well and incubate for 20 min in the incubator, then centrifuge at 4 °C to remove the mitochondrial membrane potential detection kit (JC-1).
  • JC-1 mitochondrial membrane potential detection kit
  • the solution was washed twice with JC-1 staining buffer (1x) on ice, and finally dispersed in 500 ⁇ l of the staining buffer to obtain a cell suspension, and the change in cell membrane potential was measured by flow cytometry.
  • the hydroxyurea, c-LAVs and the nanomedicine of the present example all caused a decrease in the membrane potential of the mitochondria, and the nanomedicine of the present example was able to induce a decrease in the mitochondrial membrane potential more strongly at the same concentration.
  • SW480 cells with active logarithmic growth phase were prepared and prepared into cell suspensions and added to 6-well plates at approximately 1 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM hydroxyurea, c-LAVs and the nanomedicine of this example were added, respectively, and a blank control group without any material was placed. After 4 h of incubation, the old medium was removed and the cells were collected. The collected cells were centrifuged at 1200 rpm for 5 min at 4 ° C, and the supernatant was carefully aspirated. The cells were washed twice with cold PBS and the supernatant was aspirated as much as possible.
  • Caspase-3 viability assay SW480 cells with active logarithmic growth phase were prepared and prepared into cell suspensions and added to 6-well plates at approximately 1 ⁇ 10 5 cells per well. After incubation for 24 h, 1 mM hydroxyurea, c-LAVs and the nanomedicine of this example were added, respectively, and a blank control group without any material was placed. After 4 h of incubation, the old medium was removed and the cells were collected. The collected cells were centrifuged at 1200 rpm for 5 min at 4 ° C, and the supernatant was carefully aspirated. The cells were washed twice with cold PBS and the supernatant was aspirated as much as possible.
  • a cell lysate containing 1 mM phenylmethylsulfonyl fluoride (PMSF, protease inhibitor) was added to the collected cells, placed on ice for 30 min, and vortexed once every 5 min. After centrifugation at 12000 rpm for 60 min at 4 ° C, the supernatant was quickly collected in another clean centrifuge tube, 20 ⁇ l of the supernatant was taken, and the Caspase-3 viability assay kit was used for detection, and the absorbance at A405 nm or A400 nm was measured by a microplate reader. The value of the relative activity of Caspase-3 was calculated according to the absorbance ratio of the cells of the material group and the blank control group.
  • PMSF phenylmethylsulfonyl fluoride
  • hydroxyurea can produce ROS in the mitochondria with c-LAVs, and induce mitochondrial membrane potential to decrease, which promotes the release of cytochrome C from the mitochondria into the cytoplasm, thereby regulating the caspase-3-dependent apoptotic pathway.
  • nano-drugs prepared by loading hydroxyurea in c-LAVs as an example, the synergistic anti-tumor effect of nanomedicine was evaluated.
  • the SW480 tumor model was established subcutaneously in 4 weeks old nude mice (about 20 g). When the tumor grew to 100 mm 3 , the nude mice were randomly divided into 7 groups, 5 in each group, respectively, the lipoic acid group (LA, 10 mg/kg).
  • the lipoic acid vesicle group has obvious tumor inhibition effect relative to the lipoic acid group, indicating that lipoic acid is prepared into a vesicle form, which can effectively promote the enrichment of the drug at the tumor tissue, thereby increasing sulfur.
  • hydroxyurea group showed that hydroxyurea monomer has better tumor inhibition effect, mainly because it is a small molecule chemotherapy drug, which can inhibit tumor growth at a lower concentration; hydroxyurea + lipoic acid group and The hydroxyurea + lipoic acid vesicle group has a good tumor inhibiting effect, mainly due to the combination of hydroxyurea and LA and c-LAVs, showing a better combined anti-tumor effect, and the latter has better anti-tumor effect.
  • the nano-drugs prepared in this example have the best tumor inhibition effect, mainly due to the passive targeting of nanoparticles to tumor tissues and hydroxyurea And c-LAVs can enter the cell at a predetermined ratio.
  • Example 1 a mixture of (R)-(+)-lipoic acid and (S)-(-)-lipoic acid was used instead of (R)-(+)-lipoic acid as a raw material to successfully produce an R-containing chirality.
  • a series of lipoic acid multimers such as micelles, vesicles, and aggregates.
  • the anti-tumor effect of the obtained lipoic acid multimer and the anti-tumor nano drug obtained by the combination with the traditional drug was evaluated by referring to the methods of Examples 2-13, and the results showed that the hybrid lipoic acid obtained in the present example was more.
  • the polymer and the lipoic acid multimer constructed entirely from (R)-(+)-lipoic acid in Example 1 have substantially the same beneficial effects, that is, both of them can significantly enhance the antitumor activity relative to the lipoic acid monomer. .
  • the combination of the two and several traditional drugs can achieve a synergistic anti-tumor effect of "1+1 greater than 2".
  • Example 1 0.1 equivalent of dithiothreitol (DTT) was used instead of ultraviolet light to initiate disulfide ring-opening polymerization. After 12 hours of reaction and 48 hours of dialysis, micelles and vesicles doped with a small amount of DTT were successfully prepared. , agglomerates and a series of hybrid lipoic acid multimers. At the same time, the anti-tumor effect of the obtained lipoic acid multimer and the anti-tumor nano drug obtained by the combination with the traditional drug was evaluated by referring to the methods of Examples 2-13, and the results showed that the hybrid lipoic acid obtained in the present example was more.
  • DTT dithiothreitol
  • the polymer and the lipoic acid multimer constructed entirely from (R)-(+)-lipoic acid in Example 1 have substantially the same beneficial effects, that is, both of them can significantly enhance the antitumor activity relative to the lipoic acid monomer. .
  • the combination of the two and several traditional drugs can achieve a synergistic anti-tumor effect of "1+1 greater than 2".

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Abstract

一种抗肿瘤纳米药物、制备方法及其应用，纳米药物主要以硫辛酸多聚体为活性成分。该纳米药物可以降低毒副作用，并且还可与其他具有抗肿瘤活性的物质联合使用。

Description

一种抗肿瘤纳米药物 技术领域
本发明属于生物材料领域，具体涉及一种抗肿瘤纳米药物。
技术背景
癌症是一类严重危害人类健康的常见、多发性重大疾病，其死亡率占人类疾病总体死亡率的第二位。化疗是癌症治疗的重要手段，然而传统化疗药物易引起肝肾功能受损、骨髓抑制、人体免疫力降低等毒副作用，因此开发具有抗肿瘤活性但对正常组织无毒或低毒的新型化疗药物能很好地解决传统化疗药物面临的问题。
(R)-(+)-硫辛酸[(R)-(+)-Lipoic Acid，简称LA]是硫辛酸合成酶在线粒体内合成的一类B族维生素，其具有稳定血糖、强化肝功、缓解疲劳、美容养颜以及抗衰老等功效。此外，研究发现大剂量的LA还具有一定的抗肿瘤作用，而在相同剂量下对正常细胞无毒，因此LA作为天然抗肿瘤活性物质具有极好的应用前景。但由于小分子LA兼具亲水性和亲脂性，进入体内后可到达任意部位且易被快速清除，导致其用量大、疗效差。联合用药可提高LA的治疗效果，但与其他小分子药物联用时，各种药物无法以预定比例入胞，难以达到“1+1大于2”的效果。
发明内容
本发明针对上述问题，开发了一种新型抗肿瘤纳米药物。该纳米药物将(R)-(+)-硫辛酸制备成硫辛酸多聚体负载于纳米药物载体或直接将硫辛酸多聚体制备成纳米粒子,在显著提高(R)-(+)-硫辛酸单体抗肿瘤效果的同时避免了传统化疗药物毒副作用大的问题。此外，该抗肿瘤活性成分硫辛酸多聚体还可与其他具有抗肿瘤活性的物质联合使用，以预定比例入胞，达到“1+1大于2”的协同抗肿瘤效果，进一步提高了纳米药物的疗效。
本发明通过以下技术方案来实现：
一种抗肿瘤纳米药物，其发挥抗肿瘤活性的成分主要以硫辛酸多聚体形式存在。
作为可选方式，该纳米药物存在的形式可为脂质体、树状分子、聚合物胶束、囊泡、聚集体等。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体中含有R型手性结构。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体主要由(R)-(+)-硫辛酸或其药学上可接受的盐构筑。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体由(R)-(+)-硫辛酸或其药 学上可接受的盐构筑。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体的结构式如下：
Figure PCTCN2019083390-appb-000001
其中，n≥2，R为羟基或通过酯键/酰胺键连接的官能基团或为O -M +结构(M +为金属离子)。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体的存在形式为链状聚合物、胶束、囊泡、聚集体中的至少一种。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体由硫辛酸自身或通过模板分子辅助制备得到。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体能够与其它具有抗肿瘤活性的物质联合使用。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体能够与其它具有抗肿瘤活性的物质协同抗肿瘤，起到“1+1大于2”的效果。
作为可选方式，在上述抗肿瘤纳米药物中，所述其它具有抗肿瘤活性的物质为任何能与所述硫辛酸多聚体产生协同抗肿瘤作用的物质。具体包括：传统化疗小分子药物，如喜树碱、羟基脲、匹杉琼、盐酸阿霉素、盐酸吉西他滨、阿糖胞苷等；天然具有抗肿瘤活性物质，如花青素、白藜芦醇、姜黄素、番茄素、茶多酚、虾青素等。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体可物理负载或通过化学成键连接于纳米载体中。所述纳米药物载体为脂质体、树状分子、聚合物胶束、囊泡、聚集体、硫辛酸多聚体等。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体同时还起到纳米载体的作用。所述硫辛酸链状聚合物、胶束、囊泡或聚集体可直接用于抗肿瘤。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体可物理负载或通过化学成键连接于纳米药物载体上，同时纳米药物载体负载其他抗肿瘤活性成分，二者协同抗肿瘤；或所述硫辛酸多聚体直接用于负载其他具有抗肿瘤活性的物质，二者协同抗肿瘤。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体物理负载在树状分子纳米载体中，用于抗肿瘤。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体物理包载于脂质体纳米载体中，用于抗肿瘤。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体物理包载于脂质体纳米载体中，同时脂质体负载疏水的天然抗肿瘤活性物质－姜黄素。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体物理包载于脂质体纳米载体中，同时脂质体负载亲水的化疗药物－盐酸吉西他滨。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸多聚体物理包载于脂质体纳米载体中，同时脂质体负载亲水的天然抗肿瘤活性物质－白藜芦醇。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸链状聚合物、胶束、囊泡、聚集体等形式的多聚体通过物理包载或化学成键的方式负载其它具有抗肿瘤活性的物质。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸胶束多聚体共价接连亲水化疗药物－阿糖胞苷，同时物理负载疏水的天然抗肿瘤活性物质－花青素。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸囊泡多聚体物理负载亲水化疗药物－羟基脲。
作为可选方式，在上述抗肿瘤纳米药物中，所述硫辛酸囊泡多聚体共价接连亲水化疗药物－阿糖胞苷，同时物理负载亲水化疗药物－羟基脲。
作为可选方式，在上述抗肿瘤纳米药物中，先将LA与其它具有抗肿瘤活性的物质进行化学连接，再将所得连接物构筑成链状聚合物、胶束、囊泡、聚集体等形式的多聚体。
作为可选方式，在上述抗肿瘤纳米药物中，LA直接共价接连亲水药物－羟基脲，用于直接形成胶束，进一步通过硫辛酸交联形成稳定纳米粒子。
作为可选方式，在上述抗肿瘤纳米药物中，将LA与其它具有抗肿瘤活性的物质物理混合共同构筑成链状聚合物、胶束、囊泡、聚集体等形式的多聚体。
作为可选方式，在上述抗肿瘤纳米药物中，LA直接与化疗药物－喜树碱和天然抗肿瘤活性物质－花青素形成聚集体，进一步通过硫辛酸交联形成稳定纳米粒子。
本发明还提供了一种硫辛酸多聚体的应用，其特征在于，将其用于制备上述的抗肿瘤纳米药物。
本说明书中公开的所有特征，或公开的所有方法或过程中的步骤，除了互相排斥的特征和/或步骤以外，均可以以任何方式组合。
本发明的有益效果
本发明所述纳米药物选用无毒的硫辛酸多聚体作为抗肿瘤活性成分，避免了传统化疗药物带来的毒副作用，同时克服了小分子药物的固有缺陷，进入血液循环后不易被清除，靶向性好，因此与硫辛酸单体相比，能够明显提高抗肿瘤活性。当硫辛酸多聚体与其它具有抗肿瘤活性的物质联合使用时，还能以预定比例入胞，与几种药物简单混合相比，联合用药的比例可控，达到“1+1大于2”的协同抗肿瘤效果，进一步提高纳米药物的疗效。
附图说明
图1为实施例1中硫辛酸多聚体示意图；
图2为实施例1中硫辛酸胶束、囊泡及聚集体的表征结果图，其中a)为DLS尺寸图，b)和c)分别为硫辛酸囊泡和硫辛酸聚集体包载荧光桃红B过凝胶柱后，Triton X-100加入前后荧光桃红B的荧光强度变化图；
图3为实施例2中硫辛酸胶束的抗肿瘤机制研究结果图，其中a)为硫辛酸胶束对人结肠癌细胞(SW480)的毒性检测结果，b)为不同材料作用后细胞线粒体内相对O 2 -.水平，c)为不同材料作用后线粒体膜电位损失百分比，d)为不同材料作用后细胞内相对Caspase-3激活量；
图4为实施例3中硫辛酸胶束和硫辛酸单体的细胞毒性检测结果；
图5为实施例4中纳米药物I和纳米药物Ⅱ的细胞毒性检测结果；
图6为实施例5中硫辛酸胶束对正常细胞毒性检测结果，其中a)为对人肾上皮细胞(293T)的毒性检测结果，b)为对鼠成纤维细胞(3T3)的毒性检测结果；
图7为实施例5中硫辛酸胶束的溶血和凝血性能评估，其中a)为溶血率，b)为凝血性；
图8为实施例5中小鼠体重变化；
图9为实施例6中硫辛酸胶束和传统化疗药物分子对人牙龈成纤维细胞(HGF)的毒性评估，其中a)为硫辛酸胶束，b)为盐酸吉西他滨和盐酸阿霉素；
图10为实施例6中三种纳米药物对人牙龈成纤维细胞(HGF)的毒性评估，其中a)为纳米药物Ⅲ，b)为纳米药物IV和纳米药物V；
图11为实施例7中硫辛酸胶束和硫辛酸单体分别与姜黄素共同负载于脂质体中，制备得到的纳米药物毒性评估，其中a)为细胞毒性，b)为协同指数；
图12为实施例8中硫辛酸胶束共价接连阿糖胞苷及其联合抗肿瘤研究结果，其中a)为细胞毒性检测结果，b)为协同指数；
图13为实施例9中硫辛酸共价接连羟基脲并制备成纳米药物及其联合抗肿瘤研究结 果，其中a)为细胞毒性，b)为协同指数；
图14为实施例10中硫辛酸、花青素和番茄素直接制备成纳米粒子及其联合抗肿瘤研究结果，其中a)为细胞毒性，b)为协同指数；
图15为实施例11中硫辛酸囊泡物理负载羟基脲及其联合抗肿瘤研究结果，其中a)为细胞毒性，b)为协同指数；
图16为实施例12中羟基脲和硫辛酸囊泡协同抗肿瘤结果，其中a)为材料作用细胞后线粒体内相对O 2 -.水平，b)为细胞线粒体膜电位的损失百分比，c)为相对激活caspase-3的百分比；
图17为实施例13中纳米药物的体内抗肿瘤评估，其中a)为小鼠肿瘤体积变化，b)为小鼠体重变化，c)为小鼠存活率，d)为药物体内协同抗肿瘤的联合指数(Q)；
图18为本发明所述抗肿瘤纳米药物的结构示意图。
具体实施方式
以下通过实施例的具体实施方式再对本发明的上述内容作进一步的详细说明。但不应当将此理解为本发明上述主题的范围仅限于以下的实例。在不脱离本发明的精神和原则之内做的任何修改，以及根据本领域普通技术知识和惯用手段做出的等同替换或者改进，均应包括在本发明的保护范围内。以下实施例中所用原料均可以从市场上购得，实施例中所涉及的硫辛酸单体，如无特殊说明均为(R)-(+)-硫辛酸。实施例中所涉及的百分比，如无特殊说明均为重量百分比。
实施例1
硫辛酸多聚体的制备。
硫辛酸多聚体可以硫辛酸胶束、硫辛酸囊泡和硫辛酸聚集体形式存在，如图1所示。
1、硫辛酸胶束(Cross-linked lipoic acid micelles,c-LAMs)的制备：
将100mg硫辛酸(LA)加入50ml去离子水中，搅拌下逐滴加入1M的NaOH水溶液直至硫辛酸完全溶解，再用1M的HCl溶液滴至溶液中性，最后将溶液冻干，得到淡黄色的硫辛酸钠粉末。称取41.2mg(0.2mol)硫辛酸钠，溶解在1ml去离子水中，超声后制备得到尺寸约为15nm的纳米粒子。将上述所得纳米粒子通过365nm紫外光照引发硫辛酸二硫键自交联，反应2.5h，透析48h后得到尺寸约15nm的交联硫辛酸胶束(Cross-linked lipoic acid micelles,c-LAMs)，如图2a所示。
2、硫辛酸囊泡(Cross-linked Lipoic acid vesicles,c-LAVs)制备：
将122.6mg硫辛酸(0.6mmol)和25.8mg模板分子1,4,7-三氮杂壬烷(0.2mmol)溶 于1ml N,N-二甲基甲酰胺(DMF)，制备得到浓度为0.2M的前驱液，将其放置在振荡器上振荡2h，形成超两亲分子母液。取50μl母液在超声条件下加入到5ml去离子水中制备得到由硫辛酸和1,4,7-三氮杂壬烷构筑的囊泡纳米粒子。DLS检测其尺寸在220nm左右。
以上囊泡结构通过荧光桃红B泄漏实验确定。具体如下：取50μl超两亲分子母液在超声条件下加入到5ml浓度为0.5mg/ml的荧光桃红B水溶液中，制备得到含荧光桃红B的纳米粒子溶液。所得溶液通过凝胶柱分离收集有丁达尔现象的色带。取2ml该收集液，向其中加入40μl 10％的Triton X-100溶液，监测纳米粒子加入Triton X-100前后荧光桃红B的荧光强度变化。如图2c所示，发现Triton X-100加入前，荧光桃红B荧光强度很低，而加入后荧光强度很高。这主要是由于纳米粒子包载了大量的亲水荧光桃红B，荧光桃红B在高浓度时发生荧光聚集淬灭，因此其荧光强度很低，而Triton X-100的加入破坏了纳米的结构，使得包载的荧光桃红B释放出来，从而释放出荧光。证明其为囊泡。
将以上制备的囊泡纳米粒子通过365nm紫外光照引发硫辛酸二硫键自交联，反应2.5h后，调节溶液pH值至碱性，二氯甲烷萃取，除去溶液中的1,4,7-三氮杂壬烷，之后再次将溶液pH值调回中性，透析48h后，最终得到尺寸约为220nm的交联硫辛酸囊泡(Cross-linked Lipoic acid vesicles,c-LAVs)，如图2b所示。
3、硫辛酸聚集体(Cross-linked Lipoic Acid Nanoparticles,c-LANPs)制备：
将41.2mg硫辛酸溶于1ml DMF中，在振荡器上振荡2h后得到0.2M的硫辛酸母液。取50μl该母液在超声条件下加入到5ml去离子水中制备得到尺寸约为80nm的硫辛酸纳米粒子。将上述所得纳米粒子通过365nm紫外光照引发硫辛酸二硫键自交联，反应2.5h，透析后得到尺寸约75nm左右的交联硫辛酸聚集体纳米粒子(Cross-linked Lipoic Acid Nanoparticles,c-LANPs)，结果如图2a所示。
以上聚集体结构通过荧光桃红B泄漏实验确定。具体如下：取50μl硫辛酸母液在超声条件下加入到5ml浓度为0.5mg/ml的荧光桃红B水溶液中，制备得到含荧光桃红B的纳米粒子溶液。所得溶液通过凝胶柱分离收集有丁达尔现象的溶液。取2ml该收集液，向其中加入40μl 10％的Triton X-100溶液，监测纳米粒子加入Triton X-100前后荧光桃红B的荧光强度变化。如图2d所示，发现纳米粒子结构破坏前后荧光桃红B荧光强度无明显变化，结合其尺寸较大的特点，判断该纳米粒子以聚集体形式存在。
实施例2
硫辛酸多聚体抗肿瘤机制研究。
选择交联硫辛酸胶束(c-LAMs)为例，研究硫辛酸多聚体的抗肿瘤活性机制。
1、细胞毒性评估：
选择处于对数生长活跃期的人结肠癌细胞(SW480)，接种于96孔板，培养24h后，再分别向其中加入不同浓度的LA和c-LAMs，设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。继续培养48h后，移去培养基后，加入100μl含有10％(v/v)MTT的培养基继续孵育2h后吸去旧的培养基，再每孔加入150μl的DMSO，并在振荡器上振荡2min，最后用酶标仪测定490nm处的吸光值，计算细胞存活率，其结果如图3a所示。实验结果发现，c-LAMs对肿瘤细胞SW480有一定的细胞毒性，且相对于LA有更好的抗肿瘤活性。
2、抗肿瘤机制研究：
研究表明(R)-(+)-硫辛酸(LA)进入肿瘤细胞后，在还原型谷胱甘肽(Glutathione，GSH)和硫氧还蛋白还原酶(Thioredoxinreductase，TrxR)的作用下部分还原为二氢硫辛酸(Dihydrolipoic acid，DHLA)。其中LA可直接氧化线粒体膜渗透性转换孔(mitochondrial permeablity transition pore,mPTP)上蛋白的巯基，DHLA通过产生的超氧阴离子自由基(O 2 -·)也可间接氧化mPTP上蛋白的巯基，二者共同作用造成线粒体膜通透性改变，导致细胞凋亡诱导因子(Apoptosis Inducing Factor,AIF)从线粒体内释放，进入细胞核引起核内DNA凝集，诱导细胞凋亡；同时从线粒体内释放的细胞色素C，可刺激促凋亡蛋白(Caspase-9,Caspase-3)表达量升高，诱导细胞凋亡。此外，DHLA产生的超氧阴离子自由基(O 2 -·)还可降低抗凋亡蛋白Bcl-2和Bcl-XL的表达量，进一步引起促凋亡蛋白(Caspase-9,Caspase-3)表达量升高，诱导细胞凋亡。
由于c-LAMs是由LA构筑，一方面，二者具有相同的二硫键结构；另一方面，在细胞内还原型谷胱甘肽(Glutathione，GSH)和硫氧还蛋白还原酶(Thioredoxinreductase，TrxR)作用下，c-LAMs与LA的还原产物同样为二氢硫辛酸。基于相同的胞内转化方式，本发明依照LA抗肿瘤机制的检查节点，设计了一系列实验验证c-LAMs的抗肿瘤机理。研究结果表明，c-LAMs能在细胞线粒体内产生超氧阴离子，刺激线粒体膜渗透性转换孔开放从而引起膜电位降低，进一步释放线粒体内的细胞色素C于细胞质内，从而调控Caspase-3依赖的细胞凋亡途径，诱导细胞发生凋亡，且抗肿瘤活性相对于LA有一定的增强。具体实验如下：
线粒体内超氧阴离子(O 2 -.)测定：取对数生长活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约5×10 5个。孵育24h后，分别加入1mM的c-LAMs和LA，并设置不加任何材料的空白对照组。孵育4h后，移除旧的培养基，加入1ml浓度为 50μM的荧光胺染料，孵育2h后加入半胱氨酸使其最终浓度为200μM，孵育0.5h后，吸除旧的培养基，用Krebs缓冲液清洗3次，再加入线粒体红染料进行线粒体染色30min后，采用37℃的培养基洗3次。最后通过激光共聚焦检测荧光胺在440-480nm处的发射来定量O 2 -.，检测线粒体红染料在590-650nm处的发射来定位细胞内的线粒体。实验结果如图3b所示，c-LAMs和LA都能诱导细胞线粒体内产生O 2 -.，且c-LAMs相对于LA在相同浓度下能更强地诱导产生O 2 -.。
线粒体膜电位检测：取对数生长期活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约1×10 5个。孵育24h后，分别加入1mM的LA和c-LAMs，并设置不加任何材料的空白对照组。孵育24h后，吸除旧的培养基，收集各组细胞并离心得到纯细胞。在收集到的细胞内加入1ml线粒体膜电位检测试剂盒(JC-1)工作液，充分混匀后培养箱内孵育20min，然后4℃离心，除去线粒体膜电位检测试剂盒(JC-1)工作液，在冰上用JC-1染色缓冲液(1x)洗涤2次，最后加入500μl染色缓冲液分散得到细胞悬浮液，用流式细胞仪检测细胞膜电位的变化。结果如图3c所示，c-LAMs和LA都能引起线粒体的膜电位降低，且c-LAMs相对于LA在相同浓度下能更强地诱导线粒体膜电位降低。
细胞色素C释放检测：取对数生长活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约1×10 5个。孵育24h后，分别加入1mM的c-LAMs和LA，并设置不加任何材料的空白对照组。孵育4h后，移除旧的培养基，收集细胞。将收集到的细胞在4℃条件下1200rpm离心5min，小心吸除上清液。再用冷的PBS洗涤细胞2次，每次尽可能吸尽上清液。在收集得到的细胞内加入100μl含1mM苯甲基磺酰氟(PMSF，蛋白酶抑制剂)的细胞裂解液，置于冰上30min，每5min涡旋振荡一次。于4℃条件下12000rpm离心60min，快速收集上清液于另一干净离心管中，取20μl上清液，采用western blot分析细胞质内细胞色素C的含量。结果表明c-LAMs相对于LA能更强地诱导细胞色素C释放到细胞质内。
Caspase-3活力检测：取对数生长活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约1×10 5个。孵育24h后，分别加入1mM的c-LAMs和LA，并设置不加任何材料的空白对照组。孵育4h后，移除旧的培养基，收集细胞。将收集到的细胞在4℃条件下1200rpm离心5min，小心吸除上清液。再用冷的PBS洗涤细胞2次，每次尽可能吸尽上清液。在收集得到的细胞内加入100μl含1mM苯甲基磺酰氟(PMSF，蛋白酶抑制剂)的细胞裂解液，置于冰上30min，每5min涡旋振荡一次。于4℃条件下12000rpm离心60min，快速收集上清液于另一干净离心管中，取20μl上清液，用Caspase-3活力检测 试剂盒进行检测，通过酶标仪测定A405nm或A400nm处的吸光值，根据材料组细胞与空白对照组细胞的吸光度比值，计算相对的Caspase-3的活性程度。实验结果如图3d所示，c-LAMs和LA都能刺激Caspase-3相对活力升高，且c-LAMs比LA能更强地激活Caspase-3诱导细胞凋亡。
在上述抗肿瘤机制研究方法中分别将硫辛酸胶束换成硫辛酸囊泡和聚集体，得到了相同的结果。
实施例3
硫辛酸多聚体与LA抗肿瘤活性评估。
选择硫辛酸胶束(c-LAMs)为例，评估硫辛酸多聚体与LA的抗肿瘤活性。
选择处于对数生长活跃期的人肝癌细胞(HepG2)，分别接种于96孔板，培养24h后，分别加入c-LAMs和LA。两种材料设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养72h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率，实验结果如图4所示，c-LAMs比LA具有更优的抗肿瘤活性，原因可能是c-LAMs以纳米粒子形式进入细胞，相对于LA，细胞内局部浓度较高，因此表现出较好的抗肿瘤效果。
在本实施例中，将所述的硫辛酸胶束换成硫辛酸囊泡或硫辛酸聚集体，得到了相同的结果。
实施例4
硫辛酸多聚体与LA分别负载于脂质体内，进行抗肿瘤活性评估。
选择硫辛酸胶束(c-LAMs)为例，与LA分别物理负载于脂质体内，制备得到两种纳米药物I和II，并进行抗肿瘤活性评估。
1、纳米药物I和II的制备：
将10mg c-LAMs和10mg LA分别溶于20ml去离子水中，再各自加入10mg脂质体，超声2min后得到澄清透明溶液，脂质体挤压器过400nm聚碳酯膜，各3次，再分别装入2KD透析袋，透析48h后冻干得到两种纳米药物。c-LAMs物理负载在脂质体内为纳米药物I，LA物理负载在脂质体内为纳米药物II。
2、纳米药物I和II的抗肿瘤效果评估：
选择处于对数生长活跃期的人肝癌细胞(HepG2)，分别接种于96孔板，培养24h后，分别加入纳米药物I和II。两种材料设置不同浓度梯度，每个浓度设置5个平行样，并 设置对照组。培养72h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。实验结果如图5所示，纳米药物I比纳米药物II有更好的抗肿瘤活性。
在本实施例中，将所述的硫辛酸胶束换成硫辛酸囊泡或硫辛酸聚集体，得到了相同的结果。
实施例5
硫辛酸多聚体对正常细胞/机体毒性评估。
以硫辛酸胶束(c-LAMs)为例，评估硫辛酸多聚体对正常细胞/机体毒性。
正常细胞毒性：选择对数生长期的人肾上皮细胞(293T)和小鼠成纤维细胞(3T3)，分别接种于96孔板，培养24h后，分别加入c-LAMs，设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养48h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。实验结果如图6所示，c-LAMs在一定浓度条件下对正常细胞293T和3T3无毒。
溶血评估：通过眼窝取血，收集正常小鼠新鲜血液3ml，并加入12ml生理盐水，4℃离心收集红细胞，将收集到的红细胞用生理盐水洗涤3次，制备成10％(v/v)的红细胞悬浮液。取50μl 10％红细胞悬浮液于1.5ml EP管中，共取7组，其中5组分别加入950μl不同浓度的c-LAMs生理盐水溶液，使其最终为30mg/ml、20mg/ml、10mg/ml、5mg/ml和1mg/ml，另外2组各自加入950μl生理盐水和去离子水，分别作为阴性对照组作为阳性对照组。将上述样本在培养箱中孵育2h后离心，每组取150μl上清液于96孔板中，通过酶标仪检测452nm处的吸光值，用公式(1)计算溶血率。
Hemolysis ratio％＝(A sample-A negative)/(A positive-A negative)×100    (1)
其中A sample为材料组的吸光值，A negative为阴性对照组的吸光值，A positive为阳性对照组的吸光值。实验结果如图7a所示，当材料浓度升高，溶血率也相应升高，直到c-LAMs达到饱和浓度时，溶血率仍低于5％(小于5％视为正常)，因此说明c-LAMs无溶血性。
凝血评估：配置浓度为2％的红细胞悬浮液，分别取50μl细胞悬浮液于6孔板中，分别加入不同浓度的c-LAMs溶液，使其最终浓度分别为30mg/ml、20mg/ml、10mg/ml、5mg/ml和1mg/ml。室温放置2h后，倒置荧光显微镜观察是否有凝血现象。实验结果如图7b所示，c-LAMs即使在高浓度的情况下，也无明显的凝血现象。
小鼠急毒性评估：15只四周龄的BALB/c小鼠(20g/只)，随机分为3组，每组5只，分别为空白对照组、生理盐水组以及c-LAMs组。通过尾静脉向c-LAMs组注射大剂量的c-LAMs(200mg/kg),同时向生理盐水组注射等体积的生理盐水，空白对照组不做任何处理。每2天监测1次小鼠的体重变化，给药两周后，通过眼窝取血收集小鼠血液，各组血液样本进行血常规检查和肝肾功能检查。实验结果如图8所示，c-LAMs组在大剂量给药后,小鼠体重仍保持正常，且小鼠血液和肝肾功能正常，与生理盐水组和空白组结果一致。
通过上述实验可证明c-LAMs对正常细胞及机体几乎无毒性作用，同时也有很好的生物相容性。
在本实施例中，将所述的硫辛酸胶束换成硫辛酸囊泡或硫辛酸聚集体，得到了相同的结果。
实施例6
硫辛酸多聚体和传统化疗药物对正常细胞的毒性评估。
选择硫辛酸胶束(c-LAMs)为例，对比传统化疗药物盐酸吉西他滨(Gem·HCl)和盐酸阿霉素(DOX·HCl)对正常细胞的毒性。
1、c-LAMs、Gem·HCl和DOX·HCl对正常细胞的毒性评估：
选择对数生长期活跃期的人牙龈成纤维细胞(HGF)，接种于96孔板，培养24h后，分别加入c-LAMs、Gem·HCl和DOX·HCl，设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养48h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。实验结果如图9所示，c-LAMs在较高剂量下对正常细胞无毒，而Gem·HCl和DOX·HCl在很低剂量下就能杀死正常细胞。
2、c-LAMs、Gem·HCl和DOX·HCl分别物理负载于脂质体内，制备成三种纳米药物，分别为纳米药物III、纳米药物IV和纳米药物V，并评估其对正常细胞的毒性：
选择对数生长活跃期的人牙龈成纤维细胞(HGF)，接种于96孔板，培养24h后，分别加入纳米药物III、纳米药物IV和纳米药物V。设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养48h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。实验结果如图10所示，纳米药物III在高浓度时对正常细胞无毒，而纳米药物IV和V即使在较低浓度时对正常细胞的毒性都很大。
在本实施例中，将所述的硫辛酸胶束换成硫辛酸囊泡或硫辛酸聚集体，得到了相同的结果。
实施例7
c-LAMs和LA分别与疏水天然抗肿瘤活性物质姜黄素共同负载于脂质体中，制备得到两种纳米药物VI和VII，并进行协同抗肿瘤评估。
1、纳米药物VI和VII的制备：
将5mg c-LAMs和5mg LA分别溶于20ml去离子水中，再各自加入10mg脂质体，超声2min后得到澄清透明溶液，脂质体挤压器过400nm聚碳酯膜，各3次，得到分别包载c-LAMs和LA的脂质体。将5mg姜黄素溶于200μl二甲基亚砜(DMSO)，再分别加入上述得到的脂质体，60℃水浴中振摇20min后装入2KD透析袋，透析48h后冻干得到两种纳米药物。其中c-LAMs和姜黄素物理负载于脂质体内为纳米药物VI，LA和姜黄素物理负载于脂质体内为纳米药物VII。
2、纳米药物VI和VII的联合抗肿瘤研究：
选择处于对数生长活跃期的人肝癌细胞(HepG2)，接种于96孔板，培养24h后，分别加入c-LAMs、LA、姜黄素、以及本实施例制备的纳米药物VI和VII。设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养72h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。通过Chou-Talalay联合指数公式计算出两种纳米药物的协同指数(Combination Index,CI)，当CI<1表示协同，CI＝1表示相加，CI>1表示拮抗。实验结果如图11所示，两种药物联用有较好的协同抗肿瘤效果，且纳米药物VI比VII有更好的协同效果，可能原因是c-LAMs比LA有更好的抗肿瘤效果，从而与姜黄素联合抗肿瘤表现出更好的协同效果。
实施例8
c-LAMs共价接连亲水化疗药物阿糖胞苷，并进行协同抗肿瘤评估。
1、纳米药物的制备：
取适量c-LAMs水溶液，加入1.2当量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)和1.2当量N-羟基琥珀酰亚胺(NHS)，待反应2h后，加入1当量的阿糖胞苷，继续反应24h后将反应液装入2KD透析袋透析48h，透析液冻干得到纳米药物。
2、纳米药物联合抗肿瘤研究：
选择处于对数生长活跃期的SW480细胞，接种于96孔板，培养24h后，分别加入阿 糖胞苷、c-LAMs、LA、阿糖胞苷+c-LAMs、阿糖胞苷+LA和本实施例制备的纳米药物。设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养48h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。计算阿糖胞苷+c-LAMs、阿糖胞苷+LA和本实施例制备的纳米药物的CI值。实验结果如图12所示，两药联用能增加药物的毒性，其中阿糖胞苷+c-LAMs、阿糖胞苷+LA都有一定的协同功效，但本实施例制备的纳米药物协同抗肿瘤效果最好，主要是由于纳米药物中两药能以预定比例入胞，从而表现出较好的协同效果。
实施例9
硫辛酸与化疗药物羟基脲通过共价接连形成小分子单体，再通过共沉淀法制备成纳米粒子，进一步通过硫辛酸的二硫聚合得到纳米药物，并进行协同抗肿瘤评估。
1、小分子单体的制备：
将200mg硫辛酸，138mg N-羟基丁二酰亚胺(HOSU)，247mg二环己基碳二亚胺(DCC)溶于7ml四氢呋喃中，该混合体系在氮气保护下室温搅拌6h，过滤除去沉淀物，滤液减压浓缩得到淡黄色粉末，该固体直接和76mg羟基脲一起溶于8ml干燥的DMF，同时加入0.25ml三乙胺，氮气保护下室温搅拌48h，减压除去DMF，剩余固体通过柱层析(CH 2Cl 2:MeOH＝10:1)纯化得到目标小分子。
2、纳米药物的制备：
称取20mg上述小分子单体溶于200μl二甲基亚砜(DMSO)中，超声条件下将其滴加到20ml去离子水中，直至形成澄清透明的体系，365nm紫外光照引发硫辛酸二硫键自交联，反应2.5h，透析48h后，透析液冻干得到纳米药物。
3、联合抗肿瘤研究：
选择处于对数生长活跃期的HepG2细胞，接种于96孔板，培养24h后，分别加入LA、羟基脲、LA+羟基脲和本实施例制备的纳米药物。设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养72h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。计算LA+羟基脲和本实施例制备的纳米药物的CI值。实验结果如图13所示，LA+羟基脲和本实施例制备的纳米药物都具有一定的协同抗肿瘤效果，但本实施例制备的纳米药物效果更好，主要是由于纳米药物中两药能以预定比例入胞，从而表现出较好的协同效果。
实施例10
硫辛酸直接与天然抗肿瘤活性物质番茄素、花青素形成纳米粒子，硫辛酸进一步聚合形成纳米药物，进行协同抗肿瘤研究。
1、纳米药物的制备：
称取8.26mg LA、3mg番茄素和1mg花青素溶于200μl DMSO中，超声条件下将其滴加到20ml去离子水中，直至形成澄清透明的体系，365nm紫外光照引发硫辛酸二硫键自交联，反应2.5h，透析48h后，透析液冻干得到纳米药物。
2、联合抗肿瘤研究：
选择处于对数生长活跃期的HepG2细胞，接种于96孔板，培养24h后，分别加入番茄素、花青素、硫辛酸+番茄素+花青素和本实施例制备的纳米药物。设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养72h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。计算硫辛酸+番茄素+花青素和本实施例制备的纳米药物的CI值。实验结果如图14所示，硫辛酸+番茄素+花青素和本实施例制备的纳米药物都具有一定的协同抗肿瘤效果，但本实施例制备的纳米药物效果更好，主要是由于纳米药物中三药能以预定比例入胞，从而表现出较好的协同效果。
实施例11
硫辛酸囊泡(c-LAVs)物理负载亲水化疗药物羟基脲，制备得到纳米药物，并进行协同抗肿瘤研究。
1、纳米药物制备：
向含有过饱和羟基脲的c-LAVs溶液中，调节溶液pH值至碱性，使囊泡溶胀后置于37℃恒温摇床中振荡过夜，再将溶液pH值调回中性，装入2KD的透析袋中透析48h，透析液冻干得到纳米药物。
2、联合抗肿瘤研究：
选择处于对数生长活跃期的SW480细胞，接种于96孔板，培养24h后，分别加入羟基脲、LA、c-LAVs、羟基脲+LA、羟基脲+c-LAVs和本实施例制备的纳米药物。设置不同浓度梯度，每个浓度设置5个平行样，并设置对照组。培养48h后，吸去旧的培养基，每孔加入100μl含有10％(v/v)MTT的培养基，孵育2h后吸去旧的培养基，每孔再加入150μl DMSO，振荡器上振荡2min后用酶标仪测定490nm处的吸光值，计算细胞存活率。计算羟基脲+LA、羟基脲+c-LAVs和本实施例制备的纳米药物的CI值。实验结果如图15 所示，羟基脲+LA、羟基脲+c-LAVs和本实施例制备的纳米药物都具有一定的协同抗肿瘤效果，但本实施例制备的纳米药物效果更好，主要是由于纳米药物中两药能以预定比例入胞，从而表现出较好的协同效果。
实施例12
以c-LAVs包载羟基脲制备得到的纳米药物为例，研究其协同抗肿瘤机理。
线粒体内超氧阴离子(O 2 -.)测定：取对数生长活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约5×10 5个。孵育24h后，分别加入1mM的羟基脲、c-LAVs和本实施例的纳米药物，并设置不加任何材料的空白对照组。孵育4h后，移除旧的培养基，加入1ml浓度为50μM的荧光胺染料，孵育2h后加入半胱氨酸使其最终浓度为200μM，孵育0.5h后，吸除旧的培养基，用Krebs缓冲液清洗3次，再加入线粒体红染料进行线粒体染色30min后，采用37℃的培养基洗3次。最后通过激光共聚焦检测荧光胺在440-480nm处的发射来定量O 2 -.，检测线粒体红染料在590-650nm处的发射来定位细胞内的线粒体。实验结果如图16a所示，羟基脲、c-LAVs和本实施例的纳米药物都能诱导细胞线粒体内产生O 2 -.，本实施例的纳米药物在相同浓度下能更强地诱导产生O 2 -.。
线粒体膜电位检测：取对数生长期活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约1×10 5个。孵育24h后，分别加入1mM的羟基脲、c-LAVs和本实施例的纳米药物，并设置不加任何材料的空白对照组。孵育24h后，吸除旧的培养基，收集各组细胞并离心得到纯细胞。在收集到的细胞内加入1ml线粒体膜电位检测试剂盒(JC-1)工作液，充分混匀后培养箱内孵育20min，然后4℃离心，除去线粒体膜电位检测试剂盒(JC-1)工作液，在冰上用JC-1染色缓冲液(1x)洗涤2次，最后加入500μl染色缓冲液分散得到细胞悬浮液，用流式细胞仪检测细胞膜电位的变化。结果如图16b所示，羟基脲、c-LAVs和本实施例的纳米药物都能引起线粒体的膜电位降低，且本实施例的纳米药物在相同浓度下能更强地诱导线粒体膜电位降低。
细胞色素C释放检测：取对数生长活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约1×10 5个。孵育24h后，分别加入1mM的羟基脲、c-LAVs和本实施例的纳米药物，并设置不加任何材料的空白对照组。孵育4h后，移除旧的培养基，收集细胞。将收集到的细胞在4℃条件下1200rpm离心5min，小心吸除上清液。再用冷的PBS洗涤细胞2次，每次尽可能吸尽上清液。在收集得到的细胞内加入100μl含1mM苯甲基磺酰氟(PMSF，蛋白酶抑制剂)的细胞裂解液，置于冰上30min，每5min涡旋振荡一次。于4℃条件下12000rpm离心60min，快速收集上清液于另一干净离心管中，取20μl上清 液，采用western blot分析细胞质内细胞色素C的含量。结果表明本实施例的纳米药物能更强地诱导细胞色素C释放到细胞质内。
Caspase-3活力检测：取对数生长活跃期的SW480细胞，制备成细胞悬浮液并加入6孔板中，每孔细胞约1×10 5个。孵育24h后，分别加入1mM的羟基脲、c-LAVs和本实施例的纳米药物，并设置不加任何材料的空白对照组。孵育4h后，移除旧的培养基，收集细胞。将收集到的细胞在4℃条件下1200rpm离心5min，小心吸除上清液。再用冷的PBS洗涤细胞2次，每次尽可能吸尽上清液。在收集得到的细胞内加入100μl含1mM苯甲基磺酰氟(PMSF，蛋白酶抑制剂)的细胞裂解液，置于冰上30min，每5min涡旋振荡一次。于4℃条件下12000rpm离心60min，快速收集上清液于另一干净离心管中，取20μl上清液，用Caspase-3活力检测试剂盒进行检测，通过酶标仪测定A405nm或A400nm处的吸光值，根据材料组细胞与空白对照组细胞的吸光度比值，计算相对的Caspase-3的活性程度。实验结果如图16c所示，羟基脲、c-LAVs和本实施例的纳米药物都能刺激Caspase-3相对活力升高，且本实施例的纳米药物能更强地激活Caspase-3诱导细胞凋亡。
实验结果证明，羟基脲能与c-LAVs在线粒体内产生ROS，同时诱导线粒体膜电位降低，促使细胞色素C从线粒体内释放到细胞质内，进而调控Caspase-3依赖的凋亡途径。
实施例13
以c-LAVs包载羟基脲制备得到的纳米药物为例，评估纳米药物体内协同抗肿瘤效果。
在4周龄裸鼠(约20g)皮下建立SW480肿瘤模型，待肿瘤长至100mm 3时，将裸鼠随机分成7组，每组5只，分别为硫辛酸组(LA，10mg/kg),硫辛酸囊泡组(c-LAVs，10mg/ml)，羟基脲组(Hydrea,6mg/ml)，羟基脲+硫辛酸组(Hydrea+LA，Hydrea：6mg/ml；LA：10mg/ml)，羟基脲+硫辛酸囊泡组(Hydrea+c-LAVs，Hydrea：6mg/ml；c-LAVs：10mg/ml)以及硫辛酸囊泡包载羟基脲的纳米药物组(纳米药物，Hydrea：c-LAVs＝1:1.67，Hydrea:6mg/ml，c-LAVs：10mg/ml)，同时设置生理盐水组(saline)作为空白对照组。上述7组裸鼠，每3天尾静脉给药一次，共给药8次，期间记录小鼠的肿瘤体积以及体重。根据图17所示实验结果得到，硫辛酸囊泡组相对于硫辛酸组有明显的肿瘤抑制效果，说明硫辛酸制备成囊泡形式，能有效促进药物在肿瘤组织处的富集，进而提高硫辛酸的疗效；羟基脲组显示羟基脲单体具有较好的肿瘤抑制效果，主要是由于其为小分子化疗药物，能在较低浓度下较好地抑制肿瘤增长；羟基脲+硫辛酸组和羟基脲+硫辛酸囊泡组均有较好的抑制肿瘤效果，主要是由于羟基脲与LA和c-LAVs联用，表现出较好的联合抗肿瘤效果，且后者抗肿瘤效果更好，主要是由于c-LAVs相对于LA有更高的肿瘤富集量；本实 施例中制备的纳米药物的肿瘤抑制效果最好，主要是由于纳米粒子能被动靶向于肿瘤组织处，且羟基脲和c-LAVs能以预定比例入胞。
实施例14
在实施例1中用(R)-(+)-硫辛酸与(S)-(-)-硫辛酸的混合物代替(R)-(+)-硫辛酸作为原料成功制得含R型手性结构的胶束、囊泡、聚集体等一系列硫辛酸多聚体。同时参照实施例2-13的方法对所得的硫辛酸多聚体及其与传统药物联用获得的抗肿瘤纳米药物的抗肿瘤效果进行评价，结果显示，本实施例所得的杂化硫辛酸多聚体与实施例1中完全由(R)-(+)-硫辛酸构筑的硫辛酸多聚体具有基本一致的有益效果，即两者相对于硫辛酸单体，都能够明显提高抗肿瘤活性。两者与几种传统药物联用，都能够达到“1+1大于2”的协同抗肿瘤效果。
实施例15
在实施例1中用0.1当量的二硫苏糖醇(Dithiothreitol，DTT)代替紫外光照引发二硫键开环聚合，经反应12h，透析48h后成功制得掺杂少量DTT的胶束、囊泡、聚集体等一系列杂化硫辛酸多聚体。同时参照实施例2-13的方法对所得的硫辛酸多聚体及其与传统药物联用获得的抗肿瘤纳米药物的抗肿瘤效果进行评价，结果显示，本实施例所得的杂化硫辛酸多聚体与实施例1中完全由(R)-(+)-硫辛酸构筑的硫辛酸多聚体具有基本一致的有益效果，即两者相对于硫辛酸单体，都能够明显提高抗肿瘤活性。两者与几种传统药物联用，都能够达到“1+1大于2”的协同抗肿瘤效果。
以上所述仅为本发明的优选实施例，对本发明而言仅是说明性的，而非限制性的；本领域普通技术人员理解，在本发明权利要求所限定的精神和范围内可对其进行许多改变，修改，甚至等效变更，但都将落入本发明的保护范围。

Claims (10)

  1. 一种抗肿瘤纳米药物，其特征在于，其发挥抗肿瘤活性的成分主要以硫辛酸多聚体形式存在。
  2. 根据权利要求1所述的纳米药物，其特征在于，所述硫辛酸多聚体的存在形式为链状聚合物、胶束、囊泡、聚集体中的至少一种。
  3. 根据权利要求1所述的纳米药物，其特征在于，所述硫辛酸多聚体能够与其它具有抗肿瘤活性的物质联合使用。
  4. 根据权利要求1所述的纳米药物，其特征在于，所述硫辛酸多聚体能够与其它具有抗肿瘤活性的物质协同抗肿瘤，起到“1+1大于2”的效果。
  5. 根据权利要求4所述的纳米药物，其特征在于，所述其它具有抗肿瘤活性的物质为任何能与硫辛酸多聚体产生协同抗肿瘤作用的物质。
  6. 根据权利要求4所述的纳米药物，其特征在于，所述其它具有抗肿瘤活性的物质具体为传统化疗药物，如喜树碱、羟基脲、匹杉琼、盐酸阿霉素、盐酸吉西他滨、阿糖胞苷，或天然具有抗肿瘤活性物质，如花青素、白藜芦醇、姜黄素、番茄素、茶多酚、虾青素中的至少一种。
  7. 根据权利要求1所述的纳米药物，其特征在于，所述硫辛酸多聚体负载于纳米药物载体中。
  8. 根据权利要求7所述的纳米药物，其特征在于，所述纳米药物载体具体为脂质体、树状分子、聚合物胶束、囊泡或硫辛酸多聚体。
  9. 根据权利要求1所述的纳米药物，其特征在于，所述硫辛酸多聚体同时还起到纳米药物载体的作用。
  10. 一种硫辛酸多聚体的应用，其特征在于，将其用于制备如权利要求1所述的抗肿瘤纳米药物。
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