WO2018137708A1 - 一种纳米炭-铁复合体系及其组合物、制备方法和用途 - Google Patents
一种纳米炭-铁复合体系及其组合物、制备方法和用途 Download PDFInfo
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Definitions
- the invention relates to the field of nano drugs, in particular to a nano carbon-iron composite system and a composition thereof, a preparation method thereof, and application in tumor treatment.
- Cancer is currently one of the number one killers of human life. There are 8 million new cancer patients worldwide each year, and China alone adds 3 million cases every year.
- the main means of current treatment is surgical removal of cancerous tissue, or chemotherapy to kill cancerous cells, or a combination of the two.
- surgical resection or chemotherapy is a more effective treatment for cancer.
- the current predicament of cancer treatment is: if the tumor cells metastasize to other sites after surgical resection, the chemotherapy drugs will not attack the normal cells and tumor cells in the same way, resulting in serious toxic side effects, even tumors.
- the tolerance of cells to chemotherapeutic drugs Multidrug resistance, MDR).
- RAS is The most common oncogene, which encodes a RAS protein, is a small G protein whose activity depends on binding to GTP.
- the mutated RAS protein loses the activity of hydrolyzing GTP, thereby activating genes downstream of the RAS pathway, leading to cell carcinogenesis.
- RAS-mutated tumor cells are able to increase intracellular iron content by up-regulating transferrin receptor 1 and down-regulating ferritin.
- Treatment of cells expressing RAS with this small molecule results in cell death through an "oxidative, non-apoptotic" mechanism.
- Dixon et al. recognized that this pattern of cell death is a new way of different from apoptosis, necrosis and autophagy, and named this iron-dependent death pattern as "iron repulsion". (Reference 1).
- iron death can be tested by compounds (eg, erastin, Ras selective lethal small molecule 3 and butyl sulfinine sulfoxide) Imine) or clinical drugs (eg, sulfasalazine, sorafenib, and artesunate) are induced.
- compounds eg, erastin, Ras selective lethal small molecule 3 and butyl sulfinine sulfoxide
- clinical drugs eg, sulfasalazine, sorafenib, and artesunate
- Activation of mitochondrial voltage-dependent anion channels and mitogen-activated protein kinases, up-regulation of endoplasmic reticulum stress and inhibition of cystine/glutamate reversal proteins are involved in the induction of ferroptosis.
- This process is characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS) from iron metabolism, and can be oxidized by iron chelators (eg, deferoxamine and deferoxamine mesylate) and lipid peroxidation.
- Inhibitors eg, ferrostatin, liproxstatin, and zileuton are inhibited.
- iron is the highest trace element in the human body. It is widely distributed in various organs and tissues of the human body and plays an important role in the process of DNA synthesis and electron transport and oxygen transport. Iron and the phenomenon of involvement in cell death in the event of brain damage and neurodegenerative diseases have long been noted. Iron deposition was found in the brains of patients with Parkinson's disease and Alzheimer's disease. Iron chelators for 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1, 2, 3, A neurotoxic model induced by 6-tetrahydropyridine (MPP+) or ⁇ -amyloid (A ⁇ ) can provide protection.
- 6-OHDA 6-hydroxydopamine
- a neurotoxic model induced by 6-tetrahydropyridine (MPP+) or ⁇ -amyloid (A ⁇ ) can provide protection.
- ferroptosis exists in various neurodegenerative diseases, and the ferroptosis of nerve cells can be regulated by drugs to control the occurrence and development of such nervous system diseases. Inducing ferroptosis to cells to induce apoptosis under the action of various inducers may be a new concept of tumor treatment.
- Macrophages are a group of cells with considerable plasticity and pluripotency. Under the influence of different microenvironments in vitro and in vivo, they show obvious functional differences, mainly manifested by the anti-inflammatory subspecies of M2 macrophages. Transformation of M1 macrophages of inflammatory subspecies. When the nano-iron oxide was co-cultured with macrophages, it was found that hydrogen peroxide increased by 11 times and hydroxyl radicals increased by 16 times, indicating that nano-iron oxide increased the production of ROS by macrophages, which increased the cytotoxicity of cancer cells.
- nano-iron oxide induces M1 macrophages
- macrophages were isolated from co-cultures and found to increase mRNA associated with pro-inflammatory M1-type responses, significantly upregulating M1-related TNF ⁇ and CD86 markers, associated with M2
- the CD206 and IL10 markers were significantly reduced.
- nano-iron oxide significantly inhibits the growth of subcutaneous adenocarcinoma in mice.
- intravenous injection of nano-iron oxide prior to intravenous injection of tumor cells prevents liver metastasis.
- Fluorescence activated cell sorting (FACS) and histopathological studies indicate that the observed inhibition of tumor growth is accompanied by an increase in pro-inflammatory M1 macrophages in tumor tissues (Reference 6).
- iron sucrose Sodium gluconate complexes such as Ferrlecit and iron glucan are absorbed parenterally by injection to treat severe iron deficiency, iron deficiency anemia, intestinal iron absorption, and even newer Ferumoxytol (Nano-iron oxide), etc., are aimed at the treatment of iron deficiency in patients with iron deficiency to solve the problem of anemia.
- These products are directly injected intravenously into the blood to regulate the content of iron in the human blood to solve the problem of iron deficiency.
- this type of iron does not enter cells, inducing ferroptosis.
- iron-containing preparations are of great significance for the development of anti-tumor drugs.
- the current research direction of cancer treatment is developing in two directions.
- One is intelligent, that is, intelligently recognizes normal cells and tumor cells; one is to reduce the side effects of anti-tumor drugs, reduce or even eliminate tolerance, and reduce the dose of chemotherapy.
- Years of research have shown that nanoparticles can cross the cell membrane and directly enter the nucleus to act on tumor cells.
- nanoparticles such as graphene, magnetic nanoparticles, carbon nanotubes, etc.
- a drug carrier to achieve precise drug delivery, as disclosed in Chinese patent CN105944110A, a nanocarbon quantum dot assisted drug delivery carrier system, using polyethylene Alcohol (PEG) acts as a cross-linker, and transferrin (Tf) is a targeting molecule that forms a targeted nanocarrier by covalent coupling.
- PEG polyethylene Alcohol
- Tf transferrin
- nanocarrier drugs have a very frustrating problem in both preparation and animal experiments: how to increase the drug carrying amount and release the drug accurately at the target.
- cells have a mechanism of actively excreting iron, resulting in a failure to produce a sufficiently high iron concentration in the cells, and thus cannot effectively cause iron death and proinflammatory macrophage polarization to induce cancer cell death. Therefore, it is urgent to find a suitable targeted nano drug-loading preparation, and prepare a drug carrier system with a suitable concentration of Fe preparation to increase the efficiency of iron entering the cell, thereby allowing the cells to reach a relatively high iron concentration in a short time. , induces iron death and pro-inflammatory macrophage polarization.
- the main mechanism of action in inducing cancer cell death is the polarization of pro-inflammatory macrophages, which produces a series of inflammatory factors and ROS, which in turn activates the activity of caspase-3, which ultimately leads to the death of cancer cells in apoptotic form. .
- Reference 5 Generation of hydrogen peroxide primary contributes to the induction of Fe(II)-dependentapoptosis in Jurkat cells by (-)-epigallocatechin gallate, Carcinogenesis, 25(9), 1567-1574, 2004;
- the present invention proposes a nano-carbon-loaded combination system for cells and animals in vitro. In the experiment, both showed good inhibition effects on solid tumors.
- the invention provides a nano carbon-iron composite system, which is characterized in that:
- the composite system is a composite structure formed by using an acid-treated nano-carbon as a carrier and a ferrous ion and/or an iron ion in an iron salt; the composite system has a particle diameter of 50 nm to 500 nm, preferably It is 90 nm to 300 nm, more preferably 100 nm to 250 nm; still more preferably 120 to 180 nm.
- the concentration of ferrous ions or/and iron ions is 1.36 to 13.6 mg/mL; preferably, the concentration of ferrous ions or/and iron ions is 1.5 to 8.33 mg/mL; more preferably, 2.73 ⁇ 5.46 mg / mL.
- the iron salt is selected from the group consisting of ferrous sulfate, iron sulfate, ferrous chloride, ferric chloride, ferrous gluconate, iron sucrose, ferric citrate, ferrous succinate, iron sorbitol, and ferrous fumarate. Any one or more of them; preferably, the iron salt is ferrous sulfate, iron sulfate, ferrous chloride or ferric chloride; more preferably, the iron salt is ferrous sulfate.
- the pH of the composite system is from 3.0 to 6.0; preferably, the pH of the composite system is from 3.5 to 4.5.
- the mass ratio of the nanocarbon to the iron element is 40:1 to 3:1; preferably 30:1 to 5:1; more preferably 18:1 to 6:1.
- the carbon nanocarbon has a carbon content of 86 to 98%, a hydrogen content of 0.5 to 2.5%, and an oxygen content of 1.0 to 10.0%; preferably, the carbon content is 94 to 97%, the hydrogen content is 0.7 to 1.0%, and oxygen. The content is 2.0 to 4.5%.
- the nano carbon includes at least one or more of nano carbon particles, carbon nanotubes, carbon quantum dots, graphene, fullerenes, nano carbon rods, and nano carbon fibers; preferably, the nano carbon is nano carbon particles. More preferably, the nanocarbon is nano carbon black C 40 .
- the content of the carboxyl group in the nanocarbon is from 0.01 mmol/g to 2.0 mmol/g; preferably from 0.01 mmol/g to 1.0 mmol/g, more preferably from 0.03 mmol/g to 0.7 mmol/g.
- the nano carbon and iron salt are a composite structure formed by combining various interactions such as electrostatic interaction, complexation and van der Waals force.
- the composite system further comprises sodium citrate, the mass ratio of the sodium citrate to the iron element in the iron salt is 0.1 to 3; preferably, the mass ratio of the sodium citrate to the iron element in the iron salt is 1-2;
- the sodium citrate forms a complex with ferrous ions and/or iron ions.
- the suspension system further comprises a suspending agent selected from one or more of methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, and dextran. ; preferably polyvinylpyrrolidone K 30 .
- Suspending agents can increase the viscosity of the dispersion medium to reduce the sedimentation rate of the particles or increase the hydrophilicity of the particles.
- the concentration of the suspending agent is from 10 to 40 mg/ml; preferably, the concentration is from 15 to 25 mg/ml.
- the invention also proposes a preparation method of a nano carbon-iron composite system, which is characterized in that method 1 or method 2 is adopted:
- the steps of method one include:
- step b) mixing the nanocarbon suspension obtained in step a) with an iron salt, and stirring under air to dissolve the iron salt completely to obtain a mixed liquid;
- the mixture obtained in the step b) is homogenized by a high-pressure homogenizer to obtain a homogeneous liquid, and the pH thereof is determined to be 3.0 to 6.0; preferably, the pH of the homogenizing solution is 3.5 to 4.5, that is, preferred;
- the homogenization pressure is 30 to 120 MPa; more preferably, the homogenization pressure is 90 MPa.
- the steps include:
- the acid-treated nano-carbon is uniformly dispersed in a physiological saline solution of the suspension, homogenized for 5 minutes to prepare a suspension, and the pH of the suspension is adjusted to 6.5-8.0 with sodium citrate, preferably , the pH is adjusted to 6.8 ⁇ 7.2; then homogenized by a high-pressure homogenizer to obtain a mixed solution, bottling spare; preferably, the homogenization pressure is 30 ⁇ 120MPa; more preferably, the homogenization pressure is 90MPa;
- step b) When the solid in step b) is dissolved, it is uniformly mixed with the mixture obtained in the step a), and the pH is determined to be 3.0 to 6.0; preferably, the pH is 3.5 to 4.5.
- the invention also provides a use for preparing a medicament for treating solid tumor based on the above nano carbon iron composite system; preferably, preparing a medicament for treating liver cancer, lung cancer, gastric cancer, colon cancer, breast cancer, cervical cancer, thyroid cancer or ovarian cancer Uses; further preferred, for use in the manufacture of a medicament for the treatment of breast cancer, cervical cancer and liver cancer.
- the present invention also provides a suspension for injection, comprising the nano carbon-iron composite system according to any one of the above, the nano carbon-iron composite system uniformly and stably dispersed in a mixture containing polyvinylpyrrolidone and lemon
- the polyvinylpyrrolidone is polyvinylpyrrolidone K 30 ; preferably, the concentration of ferrous ions or/and iron ions in the composite system is 1.36-13.6 mg/mL; Preferably, in the composite system, the concentration of ferrous ions or/and iron ions is 1.5-8.33 mg/mL; more preferably, 2.73-5.46 mg/mL.
- nano-carbon as a carrier to transport iron, the transport effect is better, because the cells have iron excretion mechanism, resulting in the inability to produce a sufficiently high iron concentration in the cells, thus unable to effectively cause iron death and pro-inflammatory macrophage It induces cancer cell death.
- the use of nano-carbon as a carrier can increase the efficiency of iron entry into cells, allowing cells to reach a relatively high iron concentration in a short period of time, producing iron death and inducing macrophage polarization, thereby promoting apoptosis of cancer cells.
- the preparation includes SMMC7721 liver cancer cells, A549 lung cancer cells, SGC-7901 gastric cancer cells, HCT116 colon cancer cells, MDA-MB-231 breast cancer cells, Hela cervical cancer cells, TPC-1 thyroid cancer cells, SKOV3 ovarian cancer cells. All solid tumors, including murine liver cancer H22 cells, have strong inhibitory effects, especially for breast cancer, cervical cancer and liver cancer.
- Figure 16 Hela tumor growth volume map of nano-carbon-ferrous sulfate composite system
- Figure 33 Tumor growth volume map of SMMC-7721 in nano-carbon-sucrose iron complex system group
- Figure 40 Volumetric growth of H22 tumors in a nanocarbon-ferrous sulfate composite system prepared by two methods
- Fig. 42 Tumor growth volume map of MDA-MB-231 in a nano-carbon-ferrous sulfate composite system prepared by two methods
- sample A is prepared according to method 1
- sample B is prepared according to method 2
- the above samples were combined with different iron salts with different raw materials.
- the structure and composition of the nano-carbon-iron composite system were studied by XPS and infrared spectroscopy respectively. It was found that the nano-carbon-iron composite system has A fairly consistent structure. Therefore, the present invention can be realized by various iron salts, and will not be enumerated one by one.
- the adsorption of iron on nano-carbon has little effect on the composition. It may be that Fe adsorbs on the surface of nano-carbon and causes a small amount of water to coordinate on Fe, resulting in a slight increase in O content. .
- composition C (at%) O(at%) Fe(at%) N (at%) Nano carbon 94.85 4.01 - 1.15 Nano-carbon-iron composite 92.06 6.06 0.89 1.24
- the nano-carbon iron composite has 1216 cm -1 , 1128 cm -1 , 640 cm -1 , 608 cm -1 , and 471 cm -1 peaks more than nano-carbon.
- there is a new absorption peak at 604 cm -1 and 443 cm -1 which indicates a Fe-O bond (1216 cm -1 , and 1128 cm -1 is an absorption peak of an iron citrate complex).
- the nano-carbon-iron composite is a composite of various interactions such as electrostatic interaction, complexation and van der Waals force.
- the nano-carbon used in the experiment has a certain amount of carboxyl groups on the surface after acid oxidation treatment, and the carboxyl group content is between 0.01 mmol/g and 0.10 mmol/g.
- the carboxyl group content is less than 0.03 mmol/g, the suspension system is stabilized, sedimentation is likely to occur, and a stable suspension cannot be formed.
- the carboxyl group is a hydrophilic group, the higher the content is favorable for the stability of the suspension system.
- increasing the content of polyvinylpyrrolidone K30 (PVPK30) can also improve the stability of the suspension system to some extent, it also significantly increases the viscosity of the system. Not conducive to injection administration.
- the carboxyl content is higher than 0.08mmol/g, and the color of the suspension becomes lighter (changing from black to light black), which is not conducive to the observation of the nano-carbon tracer effect. Therefore, it is reasonable to consider that the carboxyl group content is preferably in the range of 0.03 mmol/g to 0.08 mmol/g.
- both the stability of the formulation and the pharmacological efficacy require the particle size of the suspension. Since the capillary diameter of the tumor tissue is about 50 nm, and the lymphatic vessel pore diameter is about 150 nm, when the nano-carbon iron complex particle size is less than 50 nm, it is easy to enter the capillaries, which affects the concentration of iron in the blood; on the other hand, the macrophage pair The phagocytosis of the particles is selective. The larger the particle size, the easier the particles are phagocytosed by macrophages.
- the stability of the suspension becomes poor, and the sedimentation and aggregation tend to occur during the placement process, which cannot meet the stability requirements.
- the lymphatic vessel opening is around 150 nm, large-particle nano-carbon iron particles exceeding 300 nm are likely to block the lymphatic vessels, so that the nano-carbon iron particles that need to enter after the subsequent passage cannot pass through the lymphatic vessels, thereby weakening the tracer action and the therapeutic effect.
- the particle size range of the nano-carbon-iron composite is controlled to be between 90 nm and 300 nm; wherein, more preferably, the solution is from 100 nm to 250 nm; more preferably, the solution is 120 nm. Between 180nm.
- the sodium citrate added in the preparation process in addition to adjusting the pH value of the suspension, another larger use is as an anticoagulant to ensure that the suspension can have a certain fluidity after injection, so that the nano carbon-iron composite The active ingredient iron is transported into the cells.
- the complex contains ferrous iron and ferric iron, and is a main active ingredient which exerts an anticancer effect by "iron death” and macrophage M2 polarization to induce apoptosis by M1.
- the iron in the composite may be derived from ferrous sulfate, iron sulfate, ferrous chloride, ferric chloride, iron sucrose, ferrous succinate, ferrous gluconate, iron dextran, iron sorbate, ferrous fumarate, An organic or inorganic iron salt such as ammonium ferric citrate, a preferred solution is to use ferrous sulfate.
- nano-carbon has a large specific surface area and a large number of voids, has a large adsorption capacity, and van der Waals force, complexation and electrostatic interaction exist between the surface oxygen-containing group and the iron ion.
- the adsorption strength of carbon iron is moderate, and the nano-carbon iron complex is phagocytized by tumor macrophages, and the ferrous ion undergoes Fenton reaction. Therefore, the nano-carbon-iron composite is preferably a soluble divalent iron salt, and a more preferred solution is ferrous sulfate.
- the invention also includes a mass ratio range of nano carbon particles and ferrous sulfate, which is a key factor for the anti-cancer effect of the nano-carbon iron complex, and is mainly obtained by pharmacological experiments.
- High doses of iron are directly cytotoxic to tumor cells in vitro and in vivo, and therefore, a reasonable choice of iron dose is required.
- the mass ratio of nano-carbon to iron was 9.2:1, which had a higher inhibition rate on tumor cells. Therefore, the ratio of nano-carbon to iron in pharmacological experiments was 3:1 to 40:1. The results show that when the ratio of nano-carbon to iron is 5:1 ⁇ 30:1, the tumor has a high tumor inhibition rate, reaching 50-80%.
- the ratio of nano-carbon to iron is greater than 30:1, and the tumor inhibition rate is poor; while the ratio of nano-carbon to iron is less than 5:1, which is slightly toxic. Therefore, the preferred preferred ratio of mass ratio ranges from 5:1 to 30:1, and a more preferred embodiment is from 6:1 to 18:1;
- SMMC7721 liver cancer cells A549 lung cancer cells, SGC-7901 gastric cancer cells, HCT116 colon cancer cells, MDA-MB-231 breast cancer cells, Hela cervical cancer cells, TPC-1 thyroid cancer cells, SKOV3 ovarian cancer cells, and murine liver cancer H22 cell
- the cells were mixed with DMEM medium, RMPI1640 medium, fetal bovine serum (FBS), cell digestive trypsin, penicillin streptomycin mixture, phosphate buffer (PBS, pH 7.4).
- FBS fetal bovine serum
- PBS phosphate buffer
- mice Female, 4 to 6 weeks old, weighing 20 ⁇ 2 g. Free drinking water and eating during the experiment. The daily light was 12h, and 5 mice/cage were kept in separate air supply cages.
- mice Clean-grade inbred Kunming mice, female, 6-7 weeks old, weighing 20 ⁇ 2g. Free drinking water and eating during the experiment.
- the mice (5/cage) cages were ventilated with a central ventilation system for 12 hours of daily light.
- the cells grown in the log phase were collected, the cell suspension concentration was adjusted, 100 ⁇ L was added to each well, and the density of the cells to be tested was 1 ⁇ 10 3 to 10 4 cells/well (the edge cells were filled with sterile PBS).
- the cells growing in the log phase were collected, and the cell suspension concentration was adjusted. A 6-well plate was taken, and 1 mL of the cell suspension was added to each well to make the number of cells 3 ⁇ 10 4 .
- 5% CO 2 incubate at 37 ° C for 24 h, add a concentration gradient (containing nano carbon of 125, 62.5, 15.63, 3.91 ⁇ g / mL and iron ions of 13.65, 6.83, 1.71, 0.43 ⁇ g / mL) of nano-carbon iron solution, set 3 duplicate holes. It was then incubated for 48 h at 5% CO 2 at 37 °C.
- the cells were digested and counted, and a negative control group, a same concentration of nano-carbon control group, and the same concentration of iron control group were set.
- the cells in the logarithmic growth phase were collected, the cell suspension concentration was adjusted to 3 ⁇ 10 7 cells/mL, and the cells were inoculated subcutaneously into the right upper extremity of nude mice by 0.1 mL/cell (about 3 ⁇ 10 6 cells) to be inoculated.
- the tumor-bearing mice were randomly divided into a negative control group (0.9% sodium chloride injection), a nano carbon control group, an iron control group, and a nano-carbon iron suspension experiment.
- Group, cisplatin control group intraperitoneal injection, dose 5 mg / kg
- 8 nude mice Intratumoral injection of the above various drugs.
- the H22 tumor-bearing mice were extracted from the milky white thick ascites, the number of cells was adjusted to 3 ⁇ 10 7 cells/mL, and 0.1 mL of cell suspension was inoculated subcutaneously in the right upper limb of Kunming mice (about 3 ⁇ 10 6 cells).
- the tumor volume of the mice to be inoculated was 100 mm 3 on average, the tumor-bearing mice were randomly divided into negative control group (0.9% sodium chloride injection), nano carbon control group, iron control group, and nano-carbon-iron mixture.
- the suspension experimental group and the cisplatin control group (intraperitoneal injection, administered at a dose of 5 mg/kg), 8 mice per group. Intratumoral injection of the above various drugs.
- mice obtained a mouse model of cancer lymph node metastasis. Mice were treated when the tumor diameter was 6-8 mm with neither ulceration nor necrosis. The mice were randomly divided into 4 groups, 10 in each group, which were negative control group (0.9% sodium chloride injection), nano carbon control group, iron control group and nano-carbon iron suspension experimental group. Ten days after the inoculation, the mice were sacrificed, axillary lymph nodes were collected, weighed, fixed, and pathologically examined.
- mice The milky white ascites of H22 tumor-bearing mice was extracted, the number of cells was adjusted to 3 ⁇ 10 7 cells/mL, and 0.05 mL of cell suspension was inoculated subcutaneously into the left hind footpad of Kunming mice (about 1.5 ⁇ 10 6 cells). , obtained a mouse model of cancer lymph node metastasis. Mice were treated when the tumor diameter was 6-8 mm with neither ulceration nor necrosis. The mice were randomly divided into 4 groups, 10 in each group, which were negative control group (0.9% sodium chloride injection), nano carbon control group, iron control group and nano-carbon iron suspension experimental group. Ten days after the inoculation, the mice were sacrificed, axillary lymph nodes were collected, weighed, fixed, and pathologically examined.
- the cells growing in the log phase were collected, the cell suspension concentration was adjusted, and a 6-well plate was taken. After adding a cover glass to each well, 1 ml of the cell suspension was added to make 3 ⁇ 10 4 cells per well. Incubate with 5% CO2 at 37 °C for 24 h, add nano-carbon iron solution at a concentration of 125:13.65 ⁇ g/mL, and set up 3 replicate wells. It was then incubated for 48 h at 5% CO 2 at 37 °C. Each well was fixed with 1 ml of 4% paraformaldehyde solution for 30 min and stained with Prussian blue.
- mice were injected with 50 ul of drug on the footpad. After 10 minutes, the mice were sacrificed, and the axillary lymph nodes, common peroneal lymph nodes, and para-aortic lymph nodes were dissected, scored and photographed. The scoring criteria were: 1 point for all black stains in the lymph nodes, 0.5 points for some black stains, and 0 points for no black stains.
- nano-carbon, ferrous sulfate, ferrous gluconate, ferric citrate, iron sulphate and nano-carbon ferrous gluconate, nano-carbonic acid ferric citrate, nano-carbon sucrose iron have no inhibitory effect on the growth of 9 kinds of tumors.
- the inhibition rate of nano-carbon ferrous sulfate on 9 kinds of tumors reached more than 50%.
- the growth inhibition effect of nano-carbon ferrous sulfate at different mass ratios was compared. For example, as shown in Table 22, when the concentration of nano carbon is 25 mg/mL, the mass ratio of nano carbon-ferrous sulfate is 2:1 to 30:1, and the tumor inhibition rate is better, 50% to 80%.
- the mass ratio is 2:1, the toxicity is large, and the mass ratio is less than 5:1, which is slightly mild; while the mass ratio is greater than 30:1, the tumor growth inhibition effect is poor, so consider choosing 5:1 ⁇ 30 More preferably, it is 6:1 to 18:1.
- the nano-carbon-ferrous sulfate composite system prepared by the two methods has a good inhibitory effect on three kinds of cancer cells, and there is no significant difference between the two methods.
- Tumor inhibition rate (%) 80.93 81.73 80.06 76.17 73.10 68.13 52.14 31.23 26.87
- nano-carbon, ferrous sulfate, ferrous gluconate, ferric citrate, iron sucrose have no inhibitory effect on metastatic lymph nodes
- nano-carbon ferrous gluconate, nano-carbon citrate ferric amine has no inhibitory effect on lymph nodes
- nano-carbon Iron sucrose inhibited H22 and TPC-1 lymph node metastasis (P ⁇ 0.05)
- nano-carbon ferrous sulfate inhibited 9 lymph node metastasis.
- the three stations are all black-stained, but the black-stained lymph nodes are weaker than nano-carbon, while nano-carbon
- the traceability of the tube was poor, only the axillary lymph node was partially black-stained, the common lymph node and the para-aortic lymph node were not traced, and the carbon nanotube-ferrous sulfate was not traced. Therefore, nano-carbon was selected as the carrier.
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Abstract
本发明提出一种纳米炭-铁复合体系,所述复合体系以酸处理纳米炭为载体,与铁盐中的亚铁离子和/或铁离子相互作用形成的一种复合结构。基于本发明的纳米炭-铁复合体系的体外实验以及动物试验中,对肝癌、乳腺癌、宫颈癌在内的实体瘤表现出非常高效的抑制效果,具有良好的靶向性。据此,本发明还提出了一种基于所述纳米炭载铁复合体系的制备方法,并将其用在制备治疗实体肿瘤药物中的用途,以及基于纳米炭载铁复合体系的注射用悬浮液。
Description
本发明涉及纳米药物领域,具体涉及一种纳米炭-铁复合体系及其组合物、制备方法,以及在肿瘤治疗中的应用。
癌症目前是威胁人类生命的头号杀手之一,全球每年新增癌症病人达800万,仅中国每年就新增300万病例。对于实体瘤,当前治疗的主要手段是手术切除癌变组织,或者采用化疗的方式来杀死癌变细胞,或者二者联合施治。一般情况下,手术切除或者化疗,都是比较有效的癌症治疗手段。然而,目前癌症治疗面临的困境是:手术切除后如果肿瘤细胞转移至其他部位导致的复发,采用化疗后化疗药物无差别的对正常细胞和肿瘤细胞的攻击,从而引起的严重毒副作用,乃至肿瘤细胞对化疗药物形成的耐受性(Multidrug resistance,MDR)。
解决上述困境的方法之一是以PD-1,CAR-T等为代表的免疫细胞疗法,正在在全球范围内如火如荼的开展临床研究,有望成为攻克某种肿瘤的终极解决方案。然而,免疫细胞疗法,也正是因为其基因层面的精准性,目前看来,对一些基因具有异乎寻常的临床疗效;而对另外一些基因而言,则完全无效。使得具有更普适的肿瘤治疗方案,还需要全球科学家进行更多的研究与探索。
除了免疫细胞疗法,2012年,Dixon等在研究小分子弹性蛋白(erastin)杀死含有致癌基因RAS突变的肿瘤细胞的作用机制时被发现了一种新的铁依赖性的细胞凋亡:RAS是最常见的癌基因,它所编码的RAS蛋白是一种小G蛋白,其活性依赖与GTP的结合,突变的RAS蛋白丧失了水解GTP的活性,从而激活RAS通路下游相关基因,导致细胞癌变。RAS突变的肿瘤细胞能够通过上调转铁蛋白受体1和下调铁蛋白的作用增加细胞内的铁含量。用这个小分子处理表达有RAS的细胞,导致细胞通过一个“氧化性的、非凋亡的”机制死亡。在大量研究的基础上,Dixon等人认识到这种细胞死亡方式是一种不同于凋亡、坏死 和自噬的新方式,并将这种铁依赖的死亡方式命名为“铁死亡(Ferroptosis)”(参考文献1)。
进一步研究发现,细胞表面转铁蛋白受体和细胞内谷氨酰胺刺激代谢途径在死亡过程中起到了至关重要的作用。谷氨酰胺的抑制是铁死亡必不可少的部分,可减轻缺血再灌注引起的心脏损伤,这些暗示铁死亡是一种治疗相关疾病很有前景的方法(参考文献2)。
基于上述研究的基础上,“铁死亡”的作用机理获得确认后,2016年,美国的一个肿瘤实验室采用一种超细(直径小于10nm)的聚乙二醇包裹的硅纳米颗粒,这种颗粒通过靶向黑色素瘤的多肽进行功能化处理,这中硅纳米颗粒能够在饥饿状态的癌细胞和荷瘤小鼠中诱导一种称之为“铁死亡”的程序化细胞死亡方式。在进一步研究中,通过脂质活性氧检测(ROS)和使用铁螯合剂(DFO)的实验中证实硅纳米颗粒是通过铁死亡途径诱导细胞凋亡的(参考文献3)。
另外,2016年发表于Nature子刊的综述性文献《Ferroptosis:process and function》全面回顾总结了自2012年提出铁死亡后,有关铁死亡的研究结论,指出铁死亡的形态学特征是线粒体变小,线粒体膜密度增大,线粒体嵴减少或消失,外线粒体膜破裂。在癌细胞和某些正常细胞(例如肾小管细胞,神经元,成纤维细胞和T细胞),铁死亡可以被实验化合物(例如,erastin,Ras选择性致死小分子3和丁硫氨酸亚砜亚胺)或临床药物(例如,柳氮磺吡啶、索拉非尼和青蒿琥酯)诱导。线粒体电压依赖性阴离子通道和促分裂原活化蛋白激酶的激活,内质网应激的上调和胱氨酸/谷氨酸反转运蛋白的抑制参与ferroptosis的诱导。该过程具有脂质过氧化产物和来自铁代谢的致死活性氧(ROS)的积累的特征,并且可以被铁螯合剂(例如,去铁胺和去铁胺甲磺酸盐)和脂质过氧化抑制剂(例如,ferrostatin,liproxstatin和zileuton)抑制。
对于ferroptosis中直接引起细胞凋亡的ROS,其产生,目前比较合理的解释是由于细胞内铁浓度的增加,促进了细胞内Fenton(芬顿)反应的进行,从而形成了氧化性极强的ROS,并在细胞内形成积聚,从而导致细胞的凋亡(参考文献5)。
目前的研究表明,铁是人体中铁是人体内含量最高的微量元素,广泛分布在人体的各器官组织,在DNA的合成、电子传递氧运送等过程中起着重要的作用。 铁与在脑损伤和神经退行疾病发生时参与细胞死亡的现象很早之前就被人们注意到了。帕金森病和阿尔茨海默症患者的脑内均发现了铁的沉积,铁螯合剂对6-羟基多巴胺(6-OHDA)、1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPP+)或β-淀粉样蛋白(Aβ)引起的神经毒性模型可以起到保护作用。随着ferroptosis研究的进行,明确ferroptosis是否存在于各种神经退行性疾病中,可以通过药物调控神经细胞的ferroptosis,从而控制这类神经系统疾病的发生与发展。在各种诱导剂的作用下,诱发ferroptosis作用到细胞引起细胞凋亡,也许是一种全新的肿瘤治疗理念。
此外,研究还发现,细胞铁还能诱导巨噬细胞的功能性选择。巨噬细胞是一种具有相当可塑性和多能性的细胞群体,在体内外不同的微环境影响下,表现出明显的功能性差异,主要表现是抗炎性亚种的M2巨噬细胞到促炎性亚种的M1巨噬细胞的转化。在将纳米氧化铁与巨噬细胞共同培养时,发现过氧化氢增加11倍和羟基自由基增加16倍,表明纳米氧化铁通过巨噬细胞增加ROS的产生,其增加癌细胞的细胞毒性。为进一步确定纳米氧化铁是否诱导M1巨噬细胞,从共培养物中分离巨噬细胞,发现与促炎M1型反应相关的mRNA增加,显著上调M1相关的TNFα和CD86标记物,与M2相关的CD206和IL10标记物显著降低。在体内,纳米氧化铁显著抑制小鼠皮下腺癌的生长。此外,静脉注射肿瘤细胞之前静脉注射纳米氧化铁预防肝转移。荧光激活细胞分选术(FACS)和组织病理学研究表明,观察到的肿瘤生长抑制伴随着肿瘤组织中促炎性M1巨噬细胞的增加(参考文献6)。
在铁调节巨噬细胞的功能性选择的研究中,发现超顺磁性氧化铁纳米颗粒(SPION)中的铁诱导THP1细胞来源的M2巨噬细胞向高CD86+和肿瘤坏死因子(TNF-α+)巨噬细胞亚型的表型转移。M2巨噬细胞的这种表型转移伴随着细胞内的铁蛋白和组织蛋白酶L的水平上调,这是M1巨噬细胞的特征性标志(参考文献7)。
目前与铁相关的一些药品,已经获批上市的有铁蔗糖如
葡糖酸钠铁络合物如Ferrlecit、铁葡聚糖,通过注射的方式在胃肠外进行吸收,治疗严重的缺铁、缺铁性贫血、肠内铁吸收的问题,乃至较新的Ferumoxytol(纳米氧化铁)等,均是针对缺铁性患者对体内补充微量元素铁,以解决贫血的问题。这类 产品直接静脉注射进入血液,调节人体血液中的铁元素的含量,解决缺铁的问题。然而,由于细胞转铁蛋白的存在,这类铁并不能进入细胞,诱导ferroptosis。
此外,关于纳米铁的一些临床上的开发,譬如德国科学家最新申请的一份专利WO2015/007730A1,提出了一种抑制ferroptosis的抑制剂,有望解决ferroptosis引起的ROS应激神经功能性障碍疾病。
由此可见,含铁制剂对于抗肿瘤药物的开发有着重要意义。
当前的肿瘤治疗研究方向,在向两个方向发展,一个是智能化,即智能识别正常细胞与肿瘤细胞;一个是降低抗肿瘤药的毒副作用,降低甚至消除耐受性,降低化疗用药剂量。多年的研究表明,纳米微粒能跨越细胞膜的屏蔽作用,直接进入细胞核,作用于肿瘤细胞。利用纳米微粒,譬如石墨烯、磁性纳米颗粒、碳纳米管等,作为药物载体,实现精准给药,如中国专利 CN105944110A所揭示的,一种纳米碳量子点辅助给药载体系统,采用聚乙二醇(PEG)作为交联物,转铁蛋白(Tf)为靶向分子,通过共价偶联形成靶向纳米载体。
然而,一般的靶向纳米载体药,在制备与动物实验中均存在一个令人非常沮丧的问题:如何提高药物的携带量并将药物准确在靶点释放。通过纳米载体药来实现靶向给药,就必须首先提高药物的携带量,即纳米载体必须关联一定量的药物分子;其次,通过纳米载体进入体内到达靶点位置后,纳米载体携带的药物分子自动脱落,并达到一定的浓度,从而实现具有临床治疗作用。
此外,细胞有主动排出铁的机制,导致无法在细胞内产生足够高的铁浓度,因此无法有效的引起铁死亡和促炎巨噬细胞极化诱导癌细胞死亡。因此,急需寻找一种适合的靶向纳米载药制剂,搭载适宜浓度的Fe制剂,制备成药物载带体系,以提高铁进入细胞的效率,从而在短时间内让细胞达到比较高的铁浓度,诱导铁死亡和促炎巨噬细胞极化。在这两种作用机制中,诱导癌细胞死亡的主要作用机制是促炎巨噬细胞极化,产生一系列炎症因子及ROS,进而激活caspase-3的活性,最终导致癌细胞以凋亡形式死亡。
参考文献1:Ferroptosis:An Iron-DependentForm of Nonapoptotic Cell Death,Cell 149,1060–1072,May 25,2012;
参考文献2:Glutaminolysis and Transferrin Regulate Ferroptosis,Molecular Cell 59,298–308,July 16,2015;
参考文献3:Ultrasmall nanoparticles induce ferroptosis innutrient-deprived cancer cells and suppresstumour growth,Nature Technology,26Sep,2016;
参考文献4:Ferroptosis:process and function,Cell Death and Differentiation 23,369–379,2016;
参考文献5:Generation of hydrogenperoxide primarily contributes to the induction of Fe(Ⅱ)-dependentapoptosis in Jurkat cells by(-)-epigallocatechin gallate,Carcinogenesis,25(9),1567-1574,2004;
参考文献6:Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues,Nature NanoTechnology,Sep26,2016;
参考文献7:SPION primes THP1 derived M2 macrophages towards M1-likemacrophages,Biochemical and Biophysical Research Communications441,737–742,2013.
发明内容
针对目前肿瘤治疗上存在的问题,基于M1型巨噬细胞极化诱导癌细胞凋亡与纳米微粒的精准给药特性,本发明提出了一种纳米炭载铁的组合体系,在体外细胞以及动物实验中,均表现出了对实体肿瘤的良好抑制效果。
本发明提出了一种纳米炭-铁复合体系,其特征在于:
所述复合体系是以酸处理后的纳米炭为载体,与铁盐中的亚铁离子和/或铁离子形成的一种复合结构;所述复合体系粒径在50nm~500nm之间,优选的是90nm~300nm,进一步优选的是100nm~250nm;更优选的是120~180nm。
如上任一所述的复合体系,其特征在于:
所述铁盐中,亚铁离子或/和铁离子的浓度为1.36~13.6mg/mL;优选的,亚铁离子或/和铁离子的浓度为1.5~8.33mg/mL;更优选的,2.73~5.46mg/mL。
如上任一所述的复合体系,其特征在于:
所述铁盐选自硫酸亚铁、硫酸铁、氯化亚铁、三氯化铁、葡萄糖酸亚铁、蔗糖铁、柠檬酸铁胺、琥珀酸亚铁、山梨醇铁、富马酸亚铁中的任意一种或几种;优选的,所述铁盐为硫酸亚铁、硫酸铁、氯化亚铁或三氯化铁;更优选的,所述 铁盐是硫酸亚铁。
如上任一所述的复合体系,其特征在于:
所述复合体系的pH为3.0~6.0;优选的,所述复合体系的pH为3.5~4.5。
如上任一所述的复合体系,其特征在于:
所述纳米炭与铁元素的质量比为40:1~3:1;优选的是30:1~5:1;更优选的是18:1~6:1。
如上所述复合体系,其特征在于:
所述纳米炭中的碳含量为86~98%、氢含量为0.5~2.5%、氧含量为1.0~10.0%;优选的,碳含量为94~97%、氢含量为0.7~1.0%、氧含量为2.0~4.5%。
如上任一所述的复合体系,其特征在于:
所述纳米炭包括纳米炭颗粒、纳米碳管、碳量子点、石墨烯、富勒烯、纳米碳棒、纳米碳纤维中的至少一种或几种;优选的,所述纳米炭为纳米炭颗粒;更优选的,所述纳米炭为纳米炭黑C
40。
如上任一所述的复合体系,其特征在于:
所述纳米炭中的羧基含量为0.01mmol/g~2.0mmol/g;优选的是0.01mmol/g~1.0mmol/g,更优选的是0.03mmol/g~0.7mmol/g。
如上任一所述的复合体系,其特征在于:
所述纳米炭、铁盐是以静电作用、络合作用和范德华力等多种相互作用结合并形成的复合结构。
如上任一所述的复合体系,其特征在于:
所述复合体系中还包括柠檬酸钠,所述柠檬酸钠与铁盐中铁元素的质量比为0.1~3;优选的,柠檬酸钠与铁盐中铁元素的质量比为1~2;优选的,所述柠檬酸钠与亚铁离子和/或铁离子形成络合物。
如上任一所述的复合体系,其特征在于:
所述复合体系中还包括助悬剂,所述助悬剂选自甲基纤维素、羧甲基纤维素钠、羟丙基纤维素、聚乙烯吡咯烷酮、葡聚糖中的一种或多种;优选聚乙烯吡咯烷酮K
30。助悬剂能增加分散介质的黏度,以降低微粒的沉降速度或增加微粒亲水性的附加剂。
所述助悬剂的浓度为10~40mg/ml;优选的,浓度为15-25mg/ml。
本发明还提出了一种纳米炭-铁复合体系的制备方法,其特征在于,采用方法一或者方法二制备:
方法一的步骤包括:
a)将酸处理后的纳米炭均匀分散于助悬剂的生理盐水溶液中,制备成悬浮液;再用柠檬酸钠将上述悬浮液的pH值调至6.5-8.0;优选的,pH值调至6.8~7.2;
b)将步骤a)中获得的纳米炭悬浮液与铁盐混合,并在隔绝空气下搅拌至铁盐完全溶解,获得混合液;
c)将步骤b)中获得的混合液用高压均质机进行均质,获得均质液,测定其pH为3.0~6.0;优选的,均质液pH为3.5~4.5,即得;优选的,均质压力为30~120MPa;更优选的,均质压力为90MPa。
或采用方法二制备,步骤包括:
a)将酸处理后的纳米炭均匀分散于助悬剂的生理盐水溶液中,匀浆5分钟,制备成悬浮液,再用柠檬酸钠将上述悬浮液的pH值调至6.5-8.0,优选的,pH值调至6.8~7.2;然后用高压均质机进行均质,获得混合液,装瓶备用;优选的,均质压力为30~120MPa;更优选的,均质压力为90MPa;
b)将铁盐溶于生理盐水中,装瓶,冻干,充氮气密封保存获得铁盐固体;
c)使用时将步骤b)中的固体溶解后与a)步骤获得的混合液混合均匀,测定其pH为3.0~6.0;优选的,pH为3.5~4.5,即得。
本发明还提出了基于上述纳米炭铁复合体系,在制备治疗实体瘤药物中的用途;优选的,在制备治疗肝癌、肺癌、胃癌、结肠癌、乳腺癌、宫颈癌、甲状腺癌或卵巢癌药物中的用途;进一步优选的,在制备治疗乳腺癌、宫颈癌和肝癌药物中的用途。
本发明还提供了一种注射用悬浮液,其特征在于,包括如上任一所述的纳米炭-铁复合体系,所述纳米炭-铁复合体系均匀、稳定的分散在含有聚乙烯吡咯烷酮和柠檬酸钠的混合液中;优选的,所述的聚乙烯吡咯烷酮为聚乙烯吡咯烷酮K
30;优选的,所述复合体系中,亚铁离子或/和铁离子的浓度为1.36-13.6mg/mL;优选的,所述复合体系中,亚铁离子或/和铁离子的浓度为1.5-8.33mg/mL;更优选的,2.73-5.46mg/mL。
基于本发明所提出的纳米炭-铁复合体系,理论分析与体外细胞实验和动物实验均表明,本发明具有良好的效果:
1.形成了稳定的纳米炭-铁复合体系,具有良好的稳定性、生物适应性;
2.以纳米炭作为载体转运铁,转运效果较好,因为本身细胞有铁的排出机制,导致无法在细胞内产生足够高的铁浓度,因此无法有效的引起铁死亡和促炎巨噬细胞极化诱导癌细胞死亡。采用纳米炭作为载体之后,能够提高铁进入细胞的效率,在短时间内让细胞达到比较高的铁浓度,产生铁死亡和诱导巨噬细胞极化,从而促使癌细胞凋亡。
3.该制剂对包括SMMC7721肝癌细胞、A549肺癌细胞、SGC-7901胃癌细胞、HCT116结肠癌细胞、MDA-MB-231乳腺癌细胞、Hela宫颈癌细胞、TPC-1甲状腺癌细胞、SKOV3卵巢癌细胞、鼠源性肝癌H22细胞在内的一切实体瘤,均有很强的抑制作用,尤其是对乳腺癌、宫颈癌和肝癌治疗效果更好。
图1a酸处理纳米炭XPS谱
图1b酸处理纳米炭-铁复合体系XPS谱
图2纳米炭和纳米炭-铁复合体系红外光谱图
图3a1阴性对照组细胞铁离子普鲁士蓝染色图
图3b1纳米炭组细胞铁离子普鲁士蓝染色图
图3c1硫酸亚铁组细胞铁离子普鲁士蓝染色图
图3d1纳米炭-硫酸亚铁复合体系组细胞铁离子普鲁士蓝染色图
图3a2阴性对照组H22肿瘤铁离子普鲁士蓝染色图
图3b2纳米炭组H22肿瘤铁离子普鲁士蓝染色图
图3c2硫酸亚铁组H22肿瘤铁离子普鲁士蓝染色图
图3d2纳米炭-硫酸亚铁复合体系组H22肿瘤铁离子普鲁士蓝染色图
图4纳米炭-硫酸亚铁复合体系组H22肿瘤生长体积图
图5纳米炭-蔗糖铁复合体系体系组H22肿瘤生长体积图
图6纳米炭-葡萄糖酸亚铁复合体系组H22肿瘤生长体积图
图7纳米炭-柠檬酸铁胺复合体系组H22肿瘤生长体积图
图8纳米炭-硫酸亚铁复合体系组A549肿瘤生长体积图
图9纳米炭-蔗糖铁复合体系组A549肿瘤生长体积图
图10纳米炭-葡萄糖酸亚铁复合体系组A549肿瘤生长体积图
图11纳米炭-柠檬酸铁胺复合体系组A549肿瘤生长体积图
图12纳米炭-硫酸亚铁复合体系组HCT116肿瘤生长体积图
图13纳米炭-蔗糖铁复合体系组HCT116肿瘤生长体积图
图14纳米炭-葡萄糖酸亚铁复合体系组HCT116肿瘤生长体积图
图15纳米炭-柠檬酸铁胺复合体系组HCT116肿瘤生长体积图
图16纳米炭-硫酸亚铁复合体系组Hela肿瘤生长体积图
图17纳米炭-蔗糖铁复合体系组Hela肿瘤生长体积图
图18纳米炭-葡萄糖酸亚铁复合体系组Hela肿瘤生长体积图
图19纳米炭-柠檬酸铁胺复合体系组Hela肿瘤生长体积图
图20纳米炭-硫酸亚铁复合体系组MDA-MB-231肿瘤生长体积图
图21纳米炭-蔗糖铁复合体系组MDA-MB-231肿瘤生长体积图
图22纳米炭-葡萄糖酸亚铁复合体系组MDA-MB-231肿瘤生长体积图图23纳米炭-柠檬酸铁胺复合体系组MDA-MB-231肿瘤生长体积图图24纳米炭-硫酸亚铁复合体系组SGC-7901肿瘤生长体积图
图25纳米炭-蔗糖铁复合体系组SGC-7901肿瘤生长体积图
图26纳米炭-葡萄糖酸亚铁复合体系组SGC-7901肿瘤生长体积图
图27纳米炭-柠檬酸铁胺复合体系组SGC-7901肿瘤生长体积图
图28纳米炭-硫酸亚铁复合体系组SKOV3肿瘤生长体积图
图29纳米炭-蔗糖铁复合体系组SKOV3肿瘤生长体积图
图30纳米炭-葡萄糖酸亚铁复合体系组SKOV3肿瘤生长体积图
图31纳米炭-柠檬酸铁胺复合体系组SKOV3肿瘤生长体积图
图32纳米炭-硫酸亚铁复合体系组SMMC-7721肿瘤生长体积图
图33纳米炭-蔗糖铁复合体系组SMMC-7721肿瘤生长体积图
图34纳米炭-葡萄糖酸亚铁复合体系组SMMC-7721肿瘤生长体积图
图35纳米炭-柠檬酸铁胺复合体系组SMMC-7721肿瘤生长体积图
图36纳米炭-硫酸亚铁复合体系组TPC-1肿瘤生长体积图
图37纳米炭-蔗糖铁复合体系组TPC-1肿瘤生长体积图
图38纳米炭-葡萄糖酸亚铁复合体系组TPC-1肿瘤生长体积图
图39纳米炭-柠檬酸铁胺复合体系组TPC-1肿瘤生长体积图
图40两种方法制备的纳米炭-硫酸亚铁复合体系组H22肿瘤生长体积图
图41两种方法制备的纳米炭-硫酸亚铁复合体系组Hela肿瘤生长体积图
图42两种方法制备的纳米炭-硫酸亚铁复合体系组MDA-MB-231肿瘤生长体积图
图43纳米炭-硫酸亚铁复合体系组作用于Hela细胞的细胞存活率图
图44a纳米炭对小鼠淋巴结的示踪效果
图44b纳米碳管对小鼠淋巴结的示踪效果
图44c纳米炭-硫酸亚铁复合体系组对小鼠淋巴结的示踪效果
图44d纳米碳管-硫酸亚铁复合体系组对小鼠淋巴结的示踪效果
具体实施方法
一、纳米炭-铁复合体系的制备
制备了以下样品,每个样品的原料组合详见下表1-16:
1、纳米炭+硫酸亚铁
表1
2、纳米炭+氯化亚铁
表2
3、纳米炭+三氯化铁
表3
4、纳米炭+硫酸铁
表4
5、碳纳米管+七水硫酸亚铁
表5
6、石墨烯+七水硫酸亚铁
表6
7、碳量子点+七水硫酸亚铁
表7
8、富勒烯+七水硫酸亚铁
表8
9、活性炭+七水硫酸亚铁
表9
10、纳米炭+氢氧化铁
表10
11、纳米炭+蔗糖铁
表11
12、纳米炭+琥珀酸亚铁
表12
13、纳米炭+葡萄糖酸亚铁
表13
14、纳米炭+山梨醇铁
表14
15、纳米炭+富马酸亚铁
表15
16、纳米炭+柠檬酸铁铵
表16
每个样品具体的制备工艺如下(样品A按方法1制备,样品B按方法2制备)
1、纳米炭+硫酸亚铁
样品1A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.01mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品1B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.01mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品2A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品2B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二);使用时将组份一、二混合。
样品3A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末 250mg(羧基含量2.00mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品3B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量2.00mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品4A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末200mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁67.8mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品4B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末400mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁135.5mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品5A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末400mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁271.0mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品5B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末800mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁542.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品6
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末800mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁406.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品6B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末1600mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁813.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品7A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末1000mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁677.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品7B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末2000mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后 高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁1355.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品8A
100ml生理盐中加入2000mg PVPK30,室温下充分溶解后加入纳米炭粉末2500mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入500mg柠檬酸钠),然后加入七水硫酸亚铁1355mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品8B
100ml生理盐中加入2000mg PVPK30,室温下充分溶解后加入纳米炭粉末5000mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁2710mg固体溶于1000mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品9A
1000ml生理盐中加入20000mg PVPK30,室温下充分溶解后加入纳米炭粉末25000mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入5000mg柠檬酸钠),然后加入七水硫酸亚铁13550mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品9B
1000ml生理盐中加入20000mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁27100mg固体溶于10000mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品10A
10ml生理盐中加入100mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品10B
10ml生理盐中加入100mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品11A
10ml生理盐中加入400mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品11B
10ml生理盐中加入400mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品12A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径90nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质5次(压力120mpa),均质完成后收 集混悬液于西林瓶中,充氮气密封保存。
样品12B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径90nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质5次(压力120mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品13A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径120nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力110mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品13B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径120nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力110mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品14A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径180nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力80mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品14B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径180nm),充分搅拌分散均匀,加入柠檬酸 钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力80mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品15A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径300nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力60mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品15B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径300nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质3次(压力60mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
样品16A
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径500nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质2次(压力30mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品16B
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末500mg(羧基含量0.07mmol/g,粒径500nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),室温下充分混合均匀后高压均质2次(压力30mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存(组份一);取七水硫酸亚铁271.0mg固体溶于10mL生理盐水中,装瓶,冻干,充氮气密封保存(组份二):使用时将组份一、二混合。
2、纳米炭+氯化亚铁
样品17
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入氯化亚铁48.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品18
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入氯化亚铁96.9mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品19
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入氯化亚铁290.7mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
3、纳米炭+三氯化铁
样品20
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入三氯化铁65.9mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品21
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入三氯化铁131.8mg, 室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品22
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入三氯化铁395.4mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
4、纳米炭+硫酸铁
样品23
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入硫酸铁48.7mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品24
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入硫酸铁97.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品25
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭粉末250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入硫酸铁292.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
5、碳纳米管+七水硫酸亚铁
样品26
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入碳纳米管250mg (羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
6、石墨烯+七水硫酸亚铁
样品27
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入石墨烯250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
7、碳量子点+七水硫酸亚铁
样品28
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入碳量子点250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
8、富勒烯+七水硫酸亚铁
样品29
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入富勒烯250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
9、活性炭+七水硫酸亚铁
样品30
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入活性炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入七水硫酸亚铁135.5mg, 室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品31
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入氢氧化铁51.9mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品32
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入氢氧化铁155.7mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
11、纳米炭+蔗糖铁
样品33
9.3ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入蔗糖铁0.7mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品34
8.6ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入蔗糖铁1.4mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品35
5.8ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节 pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入蔗糖铁4.2mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
12、纳米炭+琥珀酸亚铁
样品36
9.75ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入琥珀酸亚铁0.25mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品37
9.5ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入琥珀酸亚铁0.5mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品38
8.5ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入琥珀酸亚铁1.5mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
13、纳米炭+葡萄糖酸亚铁
样品39
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入葡萄糖酸亚铁108.7mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品40
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入葡萄糖酸亚铁217.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品41
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入葡萄糖酸亚铁652.5mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
14、纳米炭+山梨醇铁
样品42
9.45ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入山梨醇铁0.55mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品43
8.9ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入山梨醇铁1.1mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品44
6.7ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入山梨醇铁3.3mL,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
15、纳米炭+富马酸亚铁
样品45
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入富马酸亚铁41.4mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品46
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入富马酸亚铁82.8mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品47
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入富马酸亚铁248.4mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
16、纳米炭+柠檬酸铁铵
样品48
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入柠檬酸铁铵118.9mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品49
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入柠檬酸铁铵237.9mg,室 温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
样品50
10ml生理盐中加入200mg PVPK30,室温下充分溶解后加入纳米炭250mg(羧基含量0.07mmol/g,粒径160nm),充分搅拌分散均匀,加入柠檬酸钠调节pH值至6.8~7.2之间(约加入50mg柠檬酸钠),然后加入柠檬酸铁铵713.7mg,室温下充分混合均匀后高压均质3次(压力90mpa),均质完成后收集混悬液于西林瓶中,充氮气密封保存。
对上述样品按照相似的工艺,以不同的原料与不同的铁盐进行组合,对纳米炭-铁复合体系的结构与成分分别用XPS谱和红外光谱进行研究,发现,纳米炭-铁复合体系具有相当一致的结构。因此多种铁盐均可实现本发明,不再一一列举。
如图1所示,对比纳米炭及复合体系的XPS谱可以看到,加入Fe使得纳米炭的O出现了新的峰,说明Fe与O产生了相互作用。其中纳米炭中与Fe相互作用的O的比例为52.5%;结合元素组成,结果表明多个O原子与同一个Fe发生了相互作用。纳米炭中O主要以C-O单键形式存在(C-OH或者C-O-C);C-O-C的难以完全电离成C-O-,仅-OH可能部分电离成-O-。因此Fe与O的相互作用中既有静电相互作用(Fe
2+/Fe
3+与-O-),也有Fe与O之间的络合作用,而且是多配位的相互作用。
元素分析图谱中可以看出,如表17所示,纳米炭吸附铁后对组成影响较小,可能是Fe吸附在纳米炭表面导致少量水配位在Fe上,导致了O含量略有升高。
表17纳米炭和纳米炭-铁复合物元素含量比较
组成 | C(at%) | O(at%) | Fe(at%) | N(at%) |
纳米炭 | 94.85 | 4.01 | - | 1.15 |
纳米炭-铁复合物 | 92.06 | 6.06 | 0.89 | 1.24 |
如图3所示的红外图谱,纳米炭铁复合物比纳米炭多出1216cm
-1,1128cm
-1,640cm
-1,608cm
-1,471cm
-1峰。一般在604cm
-1,443cm
-1有新吸收峰,预示有Fe-O键(1216cm
-1,1128cm
-1是柠檬酸铁络合物的吸收峰)。
综合XPS、元素分析、红外光谱分析结果,可以确定纳米炭-铁复合物是以,静电相互作用、络合作用和范德华力等多种相互作用结合并形成的复合物。
对于粒径以及浓度的选择,实验中所用的纳米炭是通过酸氧化处理后表面含 有一定量羧基,其羧基含量在0.01mmol/g~0.10mmol/g之间。当羧基含量低于0.03mmol/g,降低混悬液体系稳定,容易发生沉降,不能形成稳定的悬浮液。由于羧基为亲水性基团,含量越高有利于混悬体系稳定性,虽然提高聚乙烯吡咯烷酮K30(PVPK30)含量也能够一定程度提高混悬体系稳定性,但是同时也会显著增加体系粘度,不利于注射给药。羧基含量高于0.08mmol/g,混悬液颜色变浅(由黑色向浅黑色变化),不利于纳米炭示踪效果的观察。因此,综合考虑,羧基含量范围优选在0.03mmol/g~0.08mmol/g是合理的。
对于纳米炭-铁复合体系而言,制剂稳定性和药理疗效两方面都对混悬液粒径有所要求。由于肿瘤组织的毛细血管孔径在50nm左右,而淋巴管孔径在150nm左右,当纳米炭铁复合物粒径小于50nm,容易进入毛细血管,会影响到血液中铁的浓度;另一方面巨噬细胞对颗粒的吞噬有选择性,粒径越大颗粒越容易被巨噬细胞吞噬;当纳米炭粒径大于300nm,混悬液稳定性变差,放置过程容易发生沉降和聚集,无法达到稳定性要求,而且由于淋巴管开口在150nm左右,超过300nm的大颗粒纳米炭铁颗粒很有可能堵塞淋巴管,使后续后需要进入的纳米炭铁颗粒无法通过淋巴管,减弱示踪作用和治疗作用。因此,从制剂稳定性和药理疗效两方面考虑,将纳米炭-铁复合物粒径范围控制在90nm~300nm之间;其中,更优选的是方案是100nm~250nm;更优选的方案是120nm~180nm之间。
制备工艺中所加的柠檬酸钠,除了调节悬浮液的pH值外,另外一个更大的用途就是作为抗凝剂,保证注射后悬浮液能够具有一定的流动性,让纳米炭-铁复合物将有效成分铁转运到细胞内。
所述复合物中含有二价铁和三价铁,它是以“铁死亡”和巨噬细胞M2向M1极化诱导细胞凋亡发挥抗癌作用的主要活性成分。复合物中的铁可以来自于硫酸亚铁、硫酸铁、氯化亚铁、氯化铁、蔗糖铁、琥珀酸亚铁、葡萄糖酸亚铁、右旋糖酐铁、山梨醇铁、富马酸亚铁、柠檬酸铁铵等有机或无机铁盐,优选的方案是使用硫酸亚铁。
纳米炭-铁复合体系中,纳米炭有大的比表面积和大量的空隙,具有较大的吸附容量且表面的含氧基团和铁离子之间存在范德华力、络合作用和静电相互作用,炭铁吸附结合强度适中,纳米炭铁复合物被肿瘤的巨噬细胞吞噬后二价铁离子发生芬顿反应。因此,纳米炭-铁复合物优选可溶性的二价铁盐,更优选的方案是硫酸亚铁。
本发明还包括纳米炭颗粒和硫酸亚铁的质量比例范围,这是纳米炭铁复合物发挥抗癌药效的关键因素,主要通过药理实验得到。高剂量的铁对体外和体内肿瘤细胞具有直接的细胞毒性,因此,需要合理选择铁的剂量。经过多次实验证实,纳米炭和铁的质量比为9.2:1时对肿瘤细胞具有较高的抑制率,因此药理实验设计纳米炭和铁的比例为3:1~40:1。结果表明,当纳米炭和铁的比例为5:1~30:1时对肿瘤有较高的抑瘤率,达到50~80%。纳米炭和铁的比例大于30:1,抑瘤率差;而纳米炭和铁的比例小于5:1有轻微的毒性。因此,组合优选的质量比例范围是5:1~30:1,更优选的方案是6:1~18:1;。
在上述理论分析的指导下,对于上述样品,按照如下方式进行了细胞实验和动物实验:
1、实验材料:
1)细胞株
SMMC7721肝癌细胞、A549肺癌细胞、SGC-7901胃癌细胞、HCT116结肠癌细胞、MDA-MB-231乳腺癌细胞、Hela宫颈癌细胞、TPC-1甲状腺癌细胞、SKOV3卵巢癌细胞、鼠源性肝癌H22细胞
2)细胞培养基
细胞用DMEM培养基、RMPI1640培养基、胎牛血清(FBS)、细胞消化液胰酶、青霉素链霉素混合液、磷酸盐缓冲液(PBS,pH 7.4)
3)实验动物
BalB/c-nu小鼠,雌性,4~6周龄,体重20±2g。实验过程中自由饮水及进食。每日光照12h,小鼠5只/笼,采用独立送风隔离笼具饲养。
清洁级近交系昆明小鼠,雌性,6-7周龄,体重20±2g。实验过程中自由饮水及进食。每日光照12h,小鼠(5只/笼)笼均采用中央换气系统通气。
4)实验药品及主要仪器设备
纳米炭-铁混悬液(纳米炭:铁离子=9.2:1)、纳米炭混悬液、硫酸亚铁、葡萄糖酸亚铁、蔗糖铁、柠檬酸铁铵、顺铂注射液、0.9%氯化钠注射液、普鲁士蓝染色试剂盒、核固红染液、二甲苯、无水乙醇、盐酸、中性树胶、脱水机、包埋机、病理切片机、组织摊片机、高速离心机、鼓风干燥箱、恒温水浴锅、倒置荧光显微镜、生物光学显微镜、恒温培养箱、纯水仪、高压灭菌锅、超净工作台、酶标仪、电子天平
2、实验方法:
1)细胞实验
收集对数期生长的细胞,调整细胞悬液浓度,每孔加入100μL,铺板使待测细胞密度为1×10
3~10
4个/孔(边缘孔用无菌PBS填充)。5%CO2,37℃孵育24h,加入浓度梯度(含纳米炭为125、62.5、15.63、3.91μg/mL和铁离子为13.65、6.83、1.71、0.43μg/mL)的纳米炭铁溶液,设3个复孔。然后在5%CO2,37℃条件下孵育48h。每孔加入10μLCCK8溶液,继续培养2h。在酶标仪OD=450nm处测量各孔的吸光值。同时设阴性对照组、相同浓度纳米炭对照组和相同浓度铁剂对照组。
收集对数期生长的细胞,调整细胞悬液浓度,取6孔板,每孔加入1mL细胞悬液,使细胞数量为3×10
4个。5%CO
2,37℃孵育24h,加入浓度梯度(含纳米炭为125、62.5、15.63、3.91μg/mL和铁离子为13.65、6.83、1.71、0.43μg/mL)的纳米炭铁溶液,设3个复孔。然后在5%CO2,37℃条件下孵育48h。消化细胞并计数,同时设阴性对照组、相同浓度纳米炭对照组和相同浓度铁剂对照组。
2)抑制肿瘤生长实验
收集对数生长期的细胞,调整细胞悬液浓度为3×10
7个细胞/mL,将细胞接种于裸鼠右上肢皮下0.1mL/只(约含细胞数3×10
6个),待接种好的小鼠瘤体积平均达100mm
3时将荷瘤鼠随机分组,分别为阴性对照组(0.9%氯化钠注射液)、纳米炭对照组、铁剂对照组、纳米炭铁混悬液实验组、顺铂对照组(腹腔注射,给药剂量为5mg/kg),每组8只裸鼠。瘤内注射上述各种药物。记录肿瘤体积变化,体积计算公式为:体积=(长度×宽度
2)/2。
抽取H22荷瘤小鼠乳白色浓稠腹水,调整细胞数为3×10
7个细胞/mL,在昆明小鼠右上肢皮下接种0.1mL细胞悬液,(约含细胞数3×10
6个),待接种好的小鼠瘤体积平均达100mm
3时将荷瘤鼠随机分组,分别为阴性对照组(0.9%氯化钠注射液)、纳米炭对照组、铁剂对照组、纳米炭-铁混悬液实验组、顺铂对照组(腹腔注射,给药剂量为5mg/kg),每组8只小鼠。瘤内注射上述各种药物。记录肿瘤体积变化,体积计算公式为:体积=(长度×宽度
2)/2。
3)抑制淋巴结转移实验
收集对数生长期的细胞,调整细胞悬液浓度为3×10
7个细胞/mL,将细胞接种于裸鼠左后肢足垫皮下,接种体积0.05mL(约含细胞数1.5×10
6个),得到癌 淋巴结转移小鼠模型。当肿瘤直径达6~8mm、既无溃疡也无坏死时治疗小鼠。将小鼠随机分成4组,每组10只,分别为阴性对照组(0.9%氯化钠注射液)、纳米炭对照组、铁剂对照组、纳米炭铁混悬液实验组。接种后10天,处死小鼠,收集腘淋巴结,称重,固定,做病理学检查。
抽取H22荷瘤小鼠乳白色浓稠腹水,调整细胞数为3×10
7个细胞/mL,在昆明小鼠左后肢足垫皮下接种0.05mL细胞悬液(约含细胞数1.5×10
6个),得到癌淋巴结转移小鼠模型。当肿瘤直径达6~8mm、既无溃疡也无坏死时治疗小鼠。将小鼠随机分成4组,每组10只,分别为阴性对照组(0.9%氯化钠注射液)、纳米炭对照组、铁剂对照组、纳米炭铁混悬液实验组。接种后10天,处死小鼠,收集腘淋巴结,称重,固定,做病理学检查。
4)铁离子细胞内分布实验
收集对数期生长的细胞,调整细胞悬液浓度,取6孔板,每孔加入盖玻片后加入1ml细胞悬液,使每孔细胞达到3×10
4个。5%CO2,37℃孵育24h,加入浓度125:13.65μg/mL的纳米炭铁溶液,设3个复孔。然后在5%CO2,37℃条件下孵育48h。每孔加入1ml4%多聚甲醛溶液固定30min,普鲁士蓝染色。
H22皮下瘤实验中,3周观察结束后,取阴性组、纳米炭组、硫酸亚铁组及纳米炭硫酸亚铁组肿瘤固定,普鲁士蓝染色,观察肿瘤中铁离子。
5)小鼠淋巴结示踪实验
KM小鼠,足垫注射50ul药物,10min后,处死小鼠,解剖其腘窝淋巴结、髂总淋巴结、腹主动脉旁淋巴结,评分并拍照。评分标准为:淋巴结全部黑染为1分,部分黑染为0.5分,无黑染为0分。
3、实验结果
1)细胞结果:
同时考察了纳米炭与硫酸亚铁、葡萄糖酸亚铁、柠檬酸铁铵及蔗糖铁混合物对各种癌细胞的抑制作用,从结果上看,4种铁剂中纳米炭-硫酸亚铁混合物的抑制作用最强,对Hela细胞及SMMC-7721肝癌细胞、H22肝癌细胞的效果最好,细胞存活率为49.54%-61.26%,即抑制率为39.74%-50.46%。结果见表18~21。图43为纳米炭硫酸亚铁作用于Hela宫颈癌细胞48h后的细胞存活率,为49.54%。
表18纳米炭-硫酸亚铁作用于各种癌细胞后的细胞存活率
表19纳米炭-葡萄糖酸亚铁作用于各种癌细胞后的细胞存活率
表20纳米炭-蔗糖铁作用于各种癌细胞后的细胞存活率
表21纳米炭-柠檬酸铁铵作用于各种癌细胞后的细胞存活率
抑制肿瘤生长结果:
同时考察了纳米炭与硫酸亚铁、葡萄糖酸亚铁、柠檬酸铁铵及蔗糖铁混合物对各种癌细胞移植皮下瘤的抑制作用,结果显示,4种铁剂中纳米炭硫酸亚铁混合物的抑制作用最强,对各种癌细胞的抑瘤率为50~73%,其中对H22肝癌细胞的皮下瘤抑制作用最强,达到73%,结果见图4。4种铁剂对9种癌细胞皮下移植瘤的生长抑制作用见图4-图39。单独的纳米炭、硫酸亚铁、葡萄糖酸亚铁、柠檬酸铁胺、蔗糖铁及纳米炭葡萄糖酸亚铁、纳米炭柠檬酸铁胺、纳米炭蔗糖铁对9种肿瘤生长基本无抑制作用,而纳米炭硫酸亚铁对9种肿瘤的抑瘤率均达到50%以上。同时还比较了纳米炭硫酸亚铁不同质量比例时对肿瘤的生长抑制作用。举例说明,如表22所示,当纳米炭的浓度为25mg/mL时,纳米炭-硫酸亚铁的质量比例为2:1~30:1时抑瘤率较好,为50%~80%,但质量比例为2:1时毒性较大,质量比例小于5:1时有较轻微的毒性;而质量比例大于30:1时对肿瘤生长抑制作用较差,所以考虑选择5:1~30:1;更优选的是6:1~18:1。
由于纳米炭-硫酸亚铁复合体系的制备有两种方法,因此选取了H22肝癌细胞、Hela宫颈癌细胞和MDA-MB-231乳腺癌细胞对两种方法的疗效进行比较,结果见图40-42。两种方法制备的纳米炭-硫酸亚铁复合体系对3种癌细胞均有较好的抑制作用,且两种方法之间无显著性差异。
表22不同质量比例的纳米炭硫酸亚铁对H22肿瘤生长抑制作用
质量比例 | 2:1 | 5:1 | 6:1 | 12:1 | 18:1 | 24:1 | 30:1 | 35:1 | 40:1 |
抑瘤率(%) | 80.93 | 81.73 | 80.06 | 76.17 | 73.10 | 68.13 | 52.14 | 31.23 | 26.87 |
同时考察了纳米炭与硫酸亚铁、葡萄糖酸亚铁、柠檬酸铁铵及蔗糖铁混合物对各种癌细胞淋巴结转移的抑制作用,结果显示,4种铁剂中纳米炭硫酸亚铁混合物的抑制作用最强,转移淋巴结的重量明显减轻,转移率明显减少,对包括H22、A549、HCT、Hela、MDA-MB-231、SGC-7901、SKOV3、SMMC-7721、TPC-1在内的9种肿瘤细胞在动物体内的抑制作用,详细的动物实验对比结果见表23。单独的纳米炭、硫酸亚铁、葡萄糖酸亚铁、柠檬酸铁胺、蔗糖铁对转移淋巴结无抑制作用,纳米炭葡萄糖酸亚铁、纳米炭柠檬酸铁胺对淋巴结也无抑制作用,纳米炭蔗糖铁对H22、TPC-1淋巴结转移有抑制作用(P<0.05),而纳米炭硫酸亚铁对9种淋巴结转移均有抑制作用。
表23纳米炭铁作用于各种癌细胞淋巴结转移的淋巴结重量
注:“*”表示与阴性组相比P<0.05;“**”表示与阴性组相比P<0.01。
细胞实验中,阴性组(a1)、纳米炭组(b1)及硫酸亚铁组(c1)未见到被染色的铁离子,纳米炭-硫酸亚铁组(d1)可见较多被染色的铁离子。动物肿瘤中,阴性组(a2)及纳米炭组(b2)未观察到铁离子存在;硫酸亚铁组(c2)观察到极少铁离子;而纳米炭-硫酸亚铁组肿瘤(d2)中观察到大量铁离子存在。结果见图3。说明纳米炭-铁复合体系能有效的将铁转运到细胞内,增加细胞内铁的浓度。
为了筛选合适的载体,我们对纳米炭-硫酸亚铁及纳米碳管-硫酸亚铁对小鼠淋巴结的示踪性进行了比较。小鼠淋巴结示踪结果中,纳米炭、纳米炭-硫酸亚铁示踪结果比较好,而纳米碳管、纳米碳管-硫酸亚铁示踪结果差,结果见图44,评分结果见表24。纳米炭示踪结果很好,三站淋巴结全部黑染,纳米炭-硫酸亚铁示踪结果也比较好,三站淋巴结全部黑染,但黑染淋巴结的黑度弱于纳米炭,而纳米碳管示踪性差,只有腘窝淋巴结部分黑染,髂总淋巴结和腹主动脉旁淋巴结无示踪性,纳米碳管-硫酸亚铁无示踪性,因此选择纳米炭作为载体。
表24纳米炭、纳米碳管、纳米炭-硫酸亚铁、纳米碳管-硫酸亚铁示踪评分结果
需要说明的是:以上仅用以说明而非限制本发明的技术方案,尽管参照上述实施例对本发明进行了详细说明,本领域的普通技术人员应当理解:依然可以对本发明进行修改或者等同替换,而不脱离本发明的精神和范围的任何修改或局部替换,均应涵盖在本发明的权利要求范围当中。
Claims (16)
- 一种纳米炭-铁复合体系,其特征在于:所述复合体系是以酸处理后的纳米炭为载体,与铁盐中的亚铁离子和/或铁离子形成的一种复合结构;所述复合体系的粒径在50nm~500nm之间,优选的是90nm~300nm,进一步优选的是100nm~250nm;更优选的是120~180nm。
- 根据权利要求1所述的复合体系,其特征在于,所述铁盐中,亚铁离子或/和铁离子的浓度为1.36~13.6mg/mL;优选的,亚铁离子或/和铁离子的浓度为1.5~8.33mg/mL;更优选的,2.73~5.46mg/mL。
- 根据权利要求1或2所述的纳米炭-铁复合体系,其特征在于:所述复合体系的pH为3.0~6.0;优选的,所述复合体系的pH为3.5~4.5。
- 根据权利要求2所述的复合体系,其特征在于,所述铁盐选自硫酸亚铁、硫酸铁、氯化亚铁、三氯化铁、葡萄糖酸亚铁、蔗糖铁、柠檬酸铁胺、琥珀酸亚铁、山梨醇铁、富马酸亚铁中的任意一种或几种;优选的,所述铁盐为硫酸亚铁、硫酸铁、氯化亚铁或三氯化铁;更优选的,所述铁盐为硫酸亚铁。
- 根据权利要求1-2、4任一项所述的复合体系,其特征在于,所述纳米炭与铁盐中铁元素的质量比为40:1~3:1;优选的是30:1~5:1;更优选的是18:1~6:1。
- 根据权利要求1或2所述复合体系,其特征在于,其中,所述纳米炭中的碳含量为86~98%、氢含量为0.5~2.5%、氧含量为1.0~10.0%;优选的,碳含量为94~97%、氢含量为0.7~1.0%、氧含量为2.0~4.5%。
- 根据权利要求6所述复合体系,其特征在于,所述纳米炭包括纳米炭颗粒、纳米碳管、碳量子点、石墨烯、富勒烯、纳米碳棒、纳米碳纤维中的至少一种或几种;优选的,所述纳米炭为纳米炭颗粒;更优选的,所述纳米炭为纳米炭黑C40。
- 根据权利要求7所述的复合体系,其特征在于,所述纳米炭中的羧基含量为0.01mmol/g~2.0mmol/g;优选的是0.01mmol/g~1.0mmol/g,更优选的是0.03mmol/g~0.7mmol/g。
- 根据权利要求1或2所述的复合体系,其特征在于,所述纳米炭、铁盐是以静电作用、络合作用和范德华力等多种相互作用结合并形成的复合结构。
- 根据权利要求1或2所述的复合体系,其特征在于,所述复合体系中还包 括柠檬酸钠,所述柠檬酸钠与铁盐中铁元素的质量比为0.1~3;优选的,柠檬酸钠与铁盐中铁元素的质量比为1~2。
- 根据权利要求10所述的复合体系,其特征在于,所述柠檬酸钠与亚铁离子和/或铁离子形成络合物。
- 根据权利要求1或2所述的复合体系,其特征在于,所述复合体系中还包括助悬剂,所述助悬剂选自甲基纤维素、羧甲基纤维素钠、羟丙基纤维素、聚乙烯吡咯烷酮、葡聚糖中的一种或多种;优选聚乙烯吡咯烷酮K 30。
- 根据权利要求12所述的复合体系,其特征在于,所述助悬剂的浓度为10~40 mg/ml;优选的,浓度为15-25mg/ml。
- 一种纳米炭-铁复合体系的制备方法,其特征在于,采用方法一或者方法二制备:方法一的步骤包括:a)将酸处理后的纳米炭均匀分散于助悬剂的生理盐水溶液中,制备成悬浮液;再用柠檬酸钠将上述悬浮液的pH值调至6.5-8.0;优选的,pH值调至6.8~7.2;b)将步骤a)中获得的纳米炭悬浮液与铁盐混合,并在隔绝空气下搅拌至铁盐完全溶解,获得混合液;c)将步骤b)中获得的混合液用高压均质机进行均质,获得均质液,测定其pH为3.0~6.0;优选的,均质液pH为3.5~4.5,即得;优选的,均质压力为30~120MPa;更优选的,均质压力为90MPa;或采用方法二制备,步骤包括:b)将酸处理后的纳米炭均匀分散于助悬剂的生理盐水溶液中,匀浆5分钟,制备成悬浮液,再用柠檬酸钠将上述悬浮液的pH值调至6.5-8.0,优选的,pH值调至6.8~7.2;然后用高压均质机进行均质,获得混合液,装瓶备用;优选的,均质压力为30~120MPa;更优选的,均质压力为90MPa;b)将铁盐溶于生理盐水中,装瓶,冻干,充氮气密封保存获得铁盐固体;c)使用时将步骤b)中的固体溶解后与a)步骤获得的混合液混合均匀,测定其pH为3.0~6.0;优选的,pH为3.5~4.5,即得。
- 根据权利要求1或2所述的纳米炭-铁复合体系,在制备治疗实体瘤药物中的用途;优选的,在制备治疗肝癌、肺癌、胃癌、结肠癌、乳腺癌、宫颈癌、甲 状腺癌或卵巢癌药物中的用途;进一步优选的,在制备治疗乳腺癌、宫颈癌和肝癌药物中的用途。
- 一种注射用悬浮液,其特征在于,包括如上任一所述的纳米炭-铁复合体系,所述纳米炭-铁复合体系均匀、稳定的分散在含有聚乙烯吡咯烷酮和柠檬酸钠的混合液中;优选的,所述的聚乙烯吡咯烷酮为聚乙烯吡咯烷酮K 30;优选的,所述复合体系中,亚铁离子或/和铁离子的浓度为1.36-13.6mg/mL;优选的,所述复合体系中,亚铁离子或/和铁离子的浓度为1.5-8.33mg/mL;更优选的,2.73-5.46mg/mL。
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