WO2023284582A1 - 一种用于低温热疗的药物制剂及其制备方法和用途 - Google Patents

一种用于低温热疗的药物制剂及其制备方法和用途 Download PDF

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WO2023284582A1
WO2023284582A1 PCT/CN2022/103774 CN2022103774W WO2023284582A1 WO 2023284582 A1 WO2023284582 A1 WO 2023284582A1 CN 2022103774 W CN2022103774 W CN 2022103774W WO 2023284582 A1 WO2023284582 A1 WO 2023284582A1
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carbon
nano
injection
ferrous sulfate
water
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PCT/CN2022/103774
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French (fr)
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唐小海
唐可欣
黄源芳
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四川瀛瑞医药科技有限公司
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Priority to EP22808564.3A priority Critical patent/EP4151238A1/en
Priority to JP2022572693A priority patent/JP7641992B2/ja
Publication of WO2023284582A1 publication Critical patent/WO2023284582A1/zh
Priority to US18/126,867 priority patent/US20230330135A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a pharmaceutical preparation for low-temperature hyperthermia and its preparation method and application.
  • Photothermal therapy is a new type of tumor treatment method developed recently. It has great application prospects due to its small trauma, obvious therapeutic effect, and lower toxic and side effects.
  • carbon nanotubes, graphene, carbon quantum dots, and fullerenes are currently hot research directions.
  • carbon atoms form covalent bonds with SP 2 hybridization, and extend to the surroundings to form a honeycomb six-membered ring structure.
  • the ⁇ electrons on the ring are enriched to form delocalized large ⁇ bonds. Therefore, SP 2 hybridization endows these carbon Materials have many special properties, including the ability to absorb NIR light. These carbon materials can quickly convert light energy into heat after absorbing NIR, killing tumor cells, and thus are ideal potential candidates for hyperthermia.
  • the surface modification is mainly to modify the surface group of carbon, which is beneficial to disperse in water, and at the same time improve the conversion efficiency of NIR during the thermotherapy process.
  • these carbon materials are a mixture of a certain particle size range, and it is difficult to control the consistency between batches in the process of refining and surface modification, resulting in differences in product properties between batches, affecting comprehensive evaluation, and hindering A later stage test was carried out.
  • the particle shape and size are difficult to control.
  • carbon nanotubes have a tubular structure, and the two ends of the preparation process need to be passivated, but usually the treatment may not be complete, and there are still some ends that are like needle points.
  • nano-carbon heating is that nano-carbon can absorb near-infrared rays (NIR) efficiently and convert them into heat, while body tissues have weak absorption of NIR (high transmittance), and less conversion into heat. Therefore, taking advantage of the above differences, using nano-carbon as a hyperthermia medium, after injecting into the cancer focus, the NIR of external irradiation can pass through the normal tissue on the surface, reach the cancer focus, heat the nano-carbon, increase the temperature of the tumor tissue, and produce hyperthermia. .
  • the temperature required for nano-carbon hyperthermia is relatively high, and it needs to be above 50°C to cause necrosis and coagulation of tumor cells, and it is easy to affect the damage of adjacent normal tissue cells.
  • the drug nano-carbon iron developed by us in the early stage is a nano-suspension preparation with nano-carbon as the carrier and ferrous ions as the active ingredient. It is an innovative anti-cancer drug. Its mechanism of action is: nano-carbon iron (CNSI-Fe) After local injection into the cancer tissue, it enters the cancer cell through the overexpressed iron channel on the cancer cell membrane. After a large amount of iron ions enter the cancer cells rich in hydrogen peroxide (H 2 O 2 ), Fenton reaction occurs with H 2 O 2 to generate a large number of hydroxyl radicals ( OH). OH has extremely strong oxidation properties.
  • Lipid-ROS lipid reactive oxygen species
  • the influencing factors of Fenton reaction are: pH value, H 2 O 2 dosage, Fe 2+ dosage, reaction time and reaction temperature. Under the condition that other conditions remain unchanged, increasing the reaction temperature can accelerate the reaction speed and enhance the reaction rate. After the nano-carbon iron is locally injected into the cancer tissue, it is irradiated with near-infrared to increase its temperature, which can enhance the Fenton reaction of iron ions.
  • the temperature of directly using nano-carbon preparations in the prior art for hyperthermia is usually higher than 50°C. If the temperature is higher than 50°C, the surrounding skin will often be burned. Therapeutic pharmaceutical preparations, and the preparations that do not burn the skin and are convenient for patients to use.
  • the present invention provides a new application of nano-carbon iron suspension injection, specifically adopting the following technical solutions:
  • the first aspect of the present invention provides the use of a nano-carbon-iron composite preparation in the preparation of tumor hyperthermia drugs used in conjunction with near-infrared light;
  • near-infrared light refers to the use of near-infrared light to heat tissues; further, the wavelength of the near-infrared light is 780-2600 nm; more preferably, the wavelength of the near-infrared light is 780-2600 nm 1400nm;
  • the nano-carbon-iron composite preparation includes nano-carbon suspension injection and ferrous sulfate for injection, and the mass ratio of the two is 20-100:3-300; preferably, the mass ratio of the two is 50:7.5 ⁇ 180; more preferably, the mass ratio of the two is 50:15 ⁇ 45;
  • the nano-carbon-iron composite preparation when the nano-carbon-iron composite preparation is in use, mix the nano-carbon suspension injection and ferrous sulfate for injection uniformly according to the above mass ratio, and measure its pH value to be 2-7; preferably, measure its pH value to be 3 ⁇ 5.5; that is;
  • the nano-carbon iron suspension injection contains nano-carbon, suspending agent, normal saline, pH regulator, sodium chloride and water for injection; further, every 1000mL nano-carbon suspension injection includes: Nano-carbon 20-100g, suspending agent 17-30g, pH regulator 2-4g, sodium chloride 8-10g, and the balance is water for injection; further preferably, every 1000mL nano-carbon suspension injection includes: Charcoal 50g, suspending agent 20g, pH adjuster 3g, sodium chloride 9g, the balance is water for injection;
  • the nano-carbon is any one or several of nano-carbon particles, carbon nanotubes, carbon quantum dots, graphene, fullerene, nano-carbon rods, and nano-carbon fibers; preferably, the nano-carbon It is nano-carbon particles or graphene; further preferably, the nano-carbon is carbon black;
  • the iron salt is selected from ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, ferrous gluconate, iron sucrose, ferric ammonium citrate, ferrous succinate, ferric sorbitol, rich Any one or several in ferrous malate; preferably, the iron salt is ferrous sulfate, ferric sulfate, ferrous chloride or ferric chloride; further preferably, the iron salt is ferrous sulfate ;
  • the suspending agent is any one or more of poloxamer, polyvinylpyrrolidone C30 (PVP C30), Tween-80; preferably, the suspending agent is poloxamer ;
  • the pH regulator is any one of sodium citrate, sodium acetate, sodium borate, sodium phosphate, sodium bicarbonate; preferably, the pH regulator is sodium citrate, and the citric acid Sodium forms a complex with ferrous and/or ferric ions in the ferric salt;
  • preparation method of the nano-carbon suspension injection comprises the following steps:
  • step 2) adding a formulated amount of pH regulator and suspending agent to the salt solution obtained in step 1) and stirring until completely dissolved to obtain an auxiliary material liquid;
  • step 3 adding a formula amount of pretreated nano-carbon to the auxiliary material liquid obtained in step 2) and stirring evenly, then adding the remaining amount of water for injection to obtain a constant volume solution;
  • the pretreatment includes degreasing the nano-carbon with ethyl acetate and then washing it with 8-15% v/v of HNO3 aqueous solution, wherein, the quality of nano-carbon: the volume of HNO3 aqueous solution is 1g: 3 ⁇ 5/ml, and then wash the nano-carbon with water to be nearly neutral, and then wash it with 0.08-0.15mol/L NaOH aqueous solution.
  • the mass of nano-carbon the volume of NaOH aqueous solution is 1g:3 ⁇ 5/ml, and then Washing the nano-carbon with water is nearly neutral; further, the quality of the nano-carbon: the volume of the HNO3 aqueous solution is 1g:4/ml; further, the quality of the nano-carbon described in step S1: the volume of the NaOH aqueous solution is 1g:4 /ml;
  • the HNO 3 is 10% v/v HNO 3 aqueous solution;
  • step 4 the homogenization speed is 18000rpm, and the homogenization time is 5min;
  • step 4 the homogenization pressure is 20000psi, and the homogenization times are 3 times;
  • the sterilization temperature is 121°C, and the sterilization time is 15 minutes;
  • ferrous sulfate for injection is lyophilized powder of ferrous sulfate for injection
  • the freeze-dried ferrous sulfate powder for injection is made of the following raw materials: ferrous sulfate heptahydrate, sulfuric acid and water for injection, wherein the mass ratio of ferrous sulfate heptahydrate: sulfuric acid: water for injection is 0.1-0.2g: 0.4-0.85 ⁇ g: 2g; preferably, the sulfuric acid is 1% sulfuric acid by mass;
  • the preparation method of described ferrous sulfate lyophilized powder for injection comprises the following steps:
  • the operation of the freeze-drying step specifically includes:
  • S51 Pre-freezing: quickly cool down to -10 ⁇ -5°C, and keep warm for 1 ⁇ 3h, then quickly cool down to -50 ⁇ -40°C, and keep warm for 1 ⁇ 3h; preferably, cool down to -5°C, keep warm for 2h; Preferably, quickly cool down to -45°C and keep it warm for 2 hours;
  • S52 Drying S521: After 2 ⁇ 4h, the temperature is raised from -50 ⁇ -40°C to -20 ⁇ -10°C, and then kept at -20 ⁇ -10°C, 80 ⁇ 120mtorr for 2 ⁇ 4h; preferably, after 3h Ramp up the temperature from -45°C to -15°C, and then keep it at -15°C and 100mtorr for 3h;
  • the invention discloses that nano-carbon-iron composite preparations can be used for low-temperature hyperthermia of tumors.
  • the pharmaceutical composition of the present invention can significantly reduce the dosage and reduce the occurrence of adverse reactions during low-temperature hyperthermia;
  • the thermal therapy temperature of the invented composite preparation is lower, it is not easy to damage the surrounding normal tissues, and it also reduces the adverse reactions during nano-carbon hyperthermia; and further proves that the nano-carbon-iron composite preparation has a synergistic effect with near-infrared irradiation.
  • the present invention Based on the means and technical effects of low-temperature hyperthermia, the present invention has extensive clinical significance.
  • Fig. 1 each group acts on the tumor volume growth curve of SMMC-7721 liver cancer xenograft tumor in nude mice;
  • Fig. 2 The tumor volume growth curves of each group acting on HCT-116 colon cancer xenograft tumor in nude mice;
  • Fig. 4 Waveform diagram of hydroxyl free radicals in SMMC-7721 liver cancer tumor tissue
  • Fig. 5 Inhibitory effect of each group on lymph node metastasis of H22 liver cancer cells.
  • Embodiment 1 Preparation of Nano-carbon Suspension Injection
  • nano carbon suspension injection every 1000mL nano carbon suspension injection comprises: nano carbon 50g, suspending agent 20g, pH adjusting agent 3g, sodium chloride 9g, the balance is water for injection, and its preparation method comprises The following steps:
  • step 1) Take the water for injection of 90% v/v of the above-mentioned formulation amount, add the sodium chloride of the formulation amount and stir until completely dissolved to obtain a saline solution; The suspension is stirred until it is completely dissolved to obtain an auxiliary material liquid; 3) to the auxiliary material liquid obtained in step 2), add a formula amount of pretreated nano-carbon and stir evenly, then add the remaining amount of water for injection to obtain a constant volume solution, said The pre-treatment includes washing with 10% v/v HNO3 aqueous solution after degreasing with ethyl acetate, wherein, the quality of nano-carbon: the volume of HNO3 aqueous solution is 1g:4/ml, and then the nano-carbon is washed with water to be nearly neutral, and then Adopt the NaOH aqueous solution washing of 0.10mol/L, wherein, nano-carbon quality: the volume of NaOH aqueous solution is 1g:4/ml, then wash nano-carbon with water and be nearly
  • Embodiment 2 preparation ferrous sulfate for injection
  • a freeze-dried powder of ferrous sulfate for injection is made from the following raw materials: ferrous sulfate heptahydrate, sulfuric acid and water for injection, wherein the mass ratio of ferrous sulfate heptahydrate: 1% sulfuric acid: water for injection is 0.149g: 0.4 ⁇ 0.85 ⁇ g: 2g; preferably, the sulfuric acid is sulfuric acid with a mass percentage content of 1%; its preparation method comprises the following steps:
  • S1 Take water for injection with 90% of the formula volume, take water for injection with 90% of the formula volume, continuously fill N 2 under the liquid surface during the preparation process, and control the nitrogen flow rate at 4.0-5.0m3/h;
  • S2 Then add sulfuric acid to adjust the pH to 2.4;
  • S3 take the formula amount of ferrous sulfate heptahydrate and fully stir to dissolve it;
  • S4 add the remaining sulfuric acid to adjust the pH to 2.8, use the remaining purified water to make the volume to the full amount, and stir evenly to obtain the ferrous sulfate liquid ;
  • S5 After filtering the obtained medicinal solution through a 0.45 ⁇ m microporous membrane, it can be obtained after filling, freeze-drying, vacuum plugging, and capping.
  • the operation of the freeze-drying step specifically includes:
  • S51 pre-freezing quickly cool down to -5°C, keep warm for 2 hours, then quickly cool down to -45°C, and keep warm for 2 hours;
  • S523 After 4 hours, ramp the temperature to 0°C to 20°C, and then Incubate at 100mtorr and 20°C for 4h.
  • Examples 3-22 choose different ratios of nano-carbon suspension injection, ferrous sulfate for injection and different near-infrared light for tumor hyperthermia
  • Example 3 50mg 21mg 780
  • Example 4 50mg 21mg 808
  • Example 5 50mg 21mg 1064
  • Example 6 50mg 21mg 1400
  • Example 7 50mg 21mg 2600
  • Example 8 50mg 15mg 1064
  • Example 9 50mg 45mg 808
  • Example 10 50mg 7.5mg 808
  • Example 11 50mg 90mg 808
  • Example 12 50mg 180mg 808
  • Example 13 50mg 3mg 808
  • Example 14 50mg 300mg 808 Example 15 20mg 3mg 808 Example 16 20mg 30mg 808 Example 17 20mg 150mg 808 Example 18 20mg 300mg 808 Example 19 100mg 3mg 808 Example 20 100mg 30mg 808 Example 21 100mg 150mg 808 Example 22 100mg 300mg 808
  • SMMC7721 liver cancer cells HCT116 colon cancer cells
  • MDA-MB-231 breast cancer cells mouse-derived liver cancer H22 cells
  • DMEM medium for cells RMPI1640 medium, fetal bovine serum (FBS), cell digestion liquid trypsin, penicillin-streptomycin mixed solution, phosphate buffered saline (PBS, pH 7.4)
  • mice Female, 5-7 weeks old, weighing 20 ⁇ 2g. Drinking water and food were free during the experiment. The light was 12 hours a day, and 5 mice/cage were raised in isolation cages with independent air supply.
  • mice SPF grade Balb/c mice, female, 4-6 weeks old, weighing 20 ⁇ 2g. Drinking water and food were free during the experiment. The light was 12 hours a day, and 5 mice/cage were raised in isolation cages with independent air supply.
  • Example 4 nano-carbon iron suspension injection, embodiment 8 nano-carbon iron suspension injection, nano-carbon suspension injection (concentration is the same as in Example 4, 8), 0.9% sodium chloride injection, air blast Drying oven, constant temperature water bath, biological optical microscope, constant temperature incubator, pure water instrument, high pressure sterilizer, ultra-clean workbench, electronic balance, 808nm near-infrared thermotherapy instrument, 1064nm laser thermotherapy instrument, infrared thermal imager, Electron Spin Resonance Spectrometer (ESR)
  • ESR Electron Spin Resonance Spectrometer
  • the cells in the logarithmic growth phase were collected, counted, and the cell density was adjusted to 30,000 cells/ml, and 1 ml was added to a 25 ⁇ 25 mm glass dish, incubated in 5% CO 2 at 37°C for 24 hours.
  • the cells were divided into negative group, near-infrared irradiation group, nano-carbon iron group, nano-carbon near-infrared irradiation group (37°C, 42°C, 45°C, 48°C, 50°C), nano-carbon-iron near-infrared irradiation group ( 37°C, 42°C, 45°C, 48°C, 50°C), three for each group.
  • the negative group and the near-infrared irradiation group were replaced with new culture medium, and the nano-carbon-iron group, nano-carbon near-infrared irradiation group and nano-carbon-iron near-infrared irradiation group were replaced with a culture solution containing nano-carbon or nano-carbon-iron.
  • the nano-carbon near-infrared irradiation group and the nano-carbon-iron near-infrared irradiation group were irradiated to the required temperature with a 808nm near-infrared hyperthermia device, and the temperature was maintained for 10 minutes.
  • the irradiation time of the near-infrared irradiation group was consistent with that of the test group with the longest irradiation time. After irradiation, continue to cultivate for 48h. The cells were digested with trypsin and counted to calculate the cell inhibition rate.
  • the tumor - bearing mice were randomly divided into negative control group, near-infrared irradiation group, nano-carbon iron group, nano-carbon near-infrared irradiation group, and nano-carbon-iron near-infrared irradiation group (using Example 4 nano carbon iron suspension injection).
  • the administration method is intratumoral injection, the administration volume is 50 ⁇ L/time, and the second administration is given after 2 days interval, and the administration is administered twice in total.
  • 10 minutes after intratumoral injection of drugs the nano-carbon near-infrared irradiation group and the nano-carbon-iron near-infrared irradiation group were irradiated with 808nm near-infrared rays, respectively, with a power density of 0.5W/cm 2 and an irradiation time of 30 minutes to maintain the temperature at 45°C. about.
  • the near-infrared irradiation group was irradiated for the same time.
  • the observation time is 21 days.
  • the tumor tissue was taken, and 0.9% sodium chloride injection was added to prepare a 10% homogenate, and a capture agent was added to detect hydroxyl radicals by ESR.
  • Extract milky white ascites from H22 liver cancer-bearing mice add it to normal saline, centrifuge and resuspend, adjust the number of cells to 3 ⁇ 10 7 cells/mL, inoculate subcutaneously on the left hind foot pad of Balb/c mice, and inoculate 50 ⁇ L each , to obtain a mouse model of cancer lymph node metastasis. Mice were treated when tumors reached 6-8 mm in diameter, neither ulcerated nor necrotic.
  • Mice are randomly divided into 4 groups, 7 in every group, respectively negative control group, nano-carbon iron group, near-infrared irradiation group, nano-carbon near-infrared irradiation group and nano-carbon-iron near-infrared irradiation group (using embodiment 8 nanometer Carbon iron suspension injection).
  • the administration method is intratumoral injection, the administration volume is 50 ⁇ L/time, and the second administration is given after 2 days interval, and the administration is administered twice in total.
  • the nano-carbon near-infrared irradiation group and the nano-carbon-iron near-infrared irradiation group were respectively irradiated with 1064nm near-infrared rays at the popliteal lymph nodes of the left hindlimb of the mice, with a power density of 0.5W/cm 2 and an irradiation time of 30 minutes. Keep its temperature at around 45°C.
  • the near-infrared irradiation group was irradiated for the same time. Mice were sacrificed 2 weeks after irradiation, and popliteal lymph nodes were collected and weighed.
  • the near-infrared irradiation group had no obvious killing effect on SMMC7721 liver cancer cells, HCT116 colon cancer cells, and MDA-MB-231 breast cancer cells, and the inhibition rates were all less than 10%.
  • the inhibitory rates of nano-carbon iron acting alone on SMMC7721 liver cancer cells, HCT116 colon cancer cells, and MDA-MB-231 breast cancer cells were 40.15 ⁇ 2.98%, 34.97 ⁇ 1.67%, and 32.85 ⁇ 3.07%, respectively.
  • the inhibition rates of nano-carbon near-infrared irradiation group and nano-carbon-iron near-infrared irradiation group on three kinds of cells are shown in Table 1-3.
  • the inhibition rate of nano-carbon iron is greatly increased by near-infrared irradiation, which is better than that of single-use nano-carbon iron and nano-carbon near-infrared irradiation group at the same temperature.
  • the inhibition rate of the nano-carbon near-infrared irradiation group on the three kinds of cells reached more than 90% when the temperature reached 50°C, while the inhibition rate of the nano-carbon-iron near-infrared irradiation group reached more than 90% when the temperature was 45°C.
  • the inhibition rates of subcutaneous xenograft tumors of three kinds of cells by nano-carbon iron were 48.57%, 52.03%, and 45.79%, respectively, and the inhibition rates of subcutaneous xenograft tumors of three kinds of cells by nano-carbon near-infrared irradiation were 53.81%, 48.06%, and 49.35%, respectively.
  • the inhibition rates of subcutaneous xenograft tumors of three kinds of cells by near-infrared irradiation of nano-carbon iron were 90.78%, 90.14%, and 93.3%, respectively. Compared with near-infrared irradiation of nano-carbon iron or nano-carbon iron alone, there are significant statistical differences.
  • the q values of nano-carbon iron near-infrared irradiation are all greater than 1, indicating that nano-carbon iron near-infrared irradiation has a synergistic effect, which can enhance the anticancer effect of nano-carbon iron and nano-carbon hyperthermia.
  • FIG. 4 the content of hydroxyl free radicals in the tumors of the animals in each group was compared.
  • Fig. 4a shows that the sample is not processed in any way
  • Fig. 4b and d show that the nano-carbon near-infrared irradiation and the hydroxyl radicals produced by near-infrared irradiation are not significant
  • the nano-carbon-iron group (Fig. 4c) and the nano-carbon-iron group are nearly Infrared irradiation group (Figure 4e) had the highest intratumoral hydroxyl free radical content, which was about twice that of nano-carbon iron group. It shows that after near-infrared irradiation of nano-carbon iron, it can effectively increase the temperature in the tumor, improve the efficiency of the Fenton reaction, and generate more hydroxyl radicals.
  • the near-infrared irradiation of nano-carbon iron has the strongest inhibitory effect on lymph node metastasis, and the weight of metastatic lymph nodes is the lightest. Compared with single nano-carbon iron or nano-carbon near-infrared irradiation, there are significant statistical differences (p ⁇ 0.05). According to King's formula calculation, the q value of nano-carbon iron near-infrared irradiation is greater than 1, indicating that nano-carbon iron near-infrared irradiation has a synergistic effect, which can enhance the effect of nano-carbon iron and nano-carbon hyperthermia on inhibiting lymph node metastasis.

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Abstract

一种纳米炭铁复合制剂在制备配合近红外光使用的肿瘤热疗药物中的用途，在低温热疗时，可以降低给药剂量，减少不良反应的发生；并且采用复合制剂进行热疗的温度较低，不易损伤周围正常组织，也降低纳米炭热疗时的不良反应；并进一步证明了纳米炭铁复合制剂与近红外照射具有协同效果。基于低温热疗的手段以及技术效果，具有广泛的临床意义。

Description

一种用于低温热疗的药物制剂及其制备方法和用途 技术领域
本发明属于医药技术领域，具体涉及一种用于低温热疗的药物制剂及其制备方法和用途。
背景技术
光热治疗是近期发展起来的一类新型肿瘤治疗方法，由于创伤小、治疗效果明显，毒副作用更低，因此有极大的应用前景。在热疗试剂的开发过程中，碳纳米管、石墨烯、碳量子点、富勒烯是目前研究中的热点方向。这些材料中碳原子以SP 2杂化形成共价键，并向周围延伸，形成蜂窝状六元环结构，环上π电子富集，形成离域大π键，因此SP 2杂化赋予这些碳材料很多特殊性质，其中包括可以吸收NIR光。这些碳材料在吸收NIR后可以迅速将光能转化为热量，杀死肿瘤细胞，因此是理想的热疗潜在候选材料。
经过长时间的科研数据积累，热疗试剂的开发取得了巨大的进步，其中有效提高热转化效率，表面修饰提高靶向性、毒性研究都取得了一定进展，但是在研究过程中也存在很多问题，限制了临床应用，主要存在以下缺陷：(1)制备工艺控制难度大。碳纳米管、石墨烯、富勒烯的粗品杂质较多，在体内可能会会产生毒副反应，亲水性差，不利于在水中分散，因此需要精制和表面修饰。精制过程主要除去杂质，提高纯度，降低杂质产生毒副作用的风险。表面修饰主要是对碳的表面基团修饰，有利于在水中分散，同时提高热疗过程对NIR的转化效率。但是在试剂开发过程中发现，这些碳材料是一定粒径范围的混合物，在精制和表面修饰过程中很难控制批次间的一致性，导致批次间产品性质有差异，影响综合评价，阻碍了后期试验开展。(2)颗粒形状和大小难以控制。上述材料中，碳纳米管为管状结构，在制备过程 两个端头需要钝化处理，但通常情况可能处理不彻底，还存在部分如同针尖一样的端头，在给药后，这些锋利的端头很容易刺破血管壁，然后进入细胞核，存在潜在的遗传毒性。(3)靶向性较差，精准度不够。局部给药后，碳纳米管、石墨烯、碳量子点、富勒烯的靶向性不好，它们在肿瘤周围富集的同时，部分会弥散到正常组织周围。在热疗中，弥散在正常组织周围的碳也会发热，导致正常组织灼伤。(4)成本较高。由于制备工艺复杂，涉及制备、精制、化学修饰等步骤，成本较高，限制其规模化生产和临床应用。在上述应用背景下，热疗制剂的应用一直仅限于前期探索阶段，未出现一种可安全稳定可控的进入临床应用阶段的试剂，可安全有效的供患者使用。
另外，纳米炭升温的原理是纳米炭能高效率地吸收近红外线(NIR)，转化为热量，而机体组织对NIR吸收弱(透过率高)，转化为热量的较少。因此，利用上述差异，使用纳米炭作为热疗介质，注射入癌灶后，外照射的NIR可以透过表层正常组织，到达癌灶，加热纳米炭，使肿瘤组织温度升高，产生热疗作用。但是，纳米炭热疗所需的温度较高，需要达到50℃以上，才能使肿瘤细胞发生坏死、凝固，且易影响邻近正常组织细胞受损。我们前期开发的药物纳米炭铁是以纳米炭为载体，以二价铁离子为有效成分的纳米混悬制剂，是一种抗癌创新药物，其作用机理是：纳米炭铁(CNSI-Fe)局部注射进入癌组织后，通过癌细胞膜上表达过多的铁通道进入癌细胞内。大量铁离子进入富含过氧化氢(H 2O 2)的癌细胞后与H 2O 2发生芬顿反应，产生大量的羟基自由基(·OH)，·OH具有极强的氧化性能，在细胞内与不饱和多脂肪酸(UPFAs)作用产生大量的极具破坏性脂质过氧化氢(L-OOH)，也就是脂质活性氧(Lipid-ROS)，Lipid-ROS能摧毁细胞器而导致细胞破坏，引发铁死亡(Ferroptosis)。
CNSI-Fe 2 ++H 2O 2→ROS(·OH)→L-ROS→Ferroptosis
芬顿反应的影响因素有：pH值、H 2O 2投加量、Fe 2+投加量、反应时 间和反应温度。在其余条件不变的情况下，提高反应温度，可使反应速度加快，反应速率增强。纳米炭铁局部注射入癌组织后，用近红外照射，使其温度升高，可增强铁离子的芬顿反应。但是目前直接使用现有技术中的纳米炭制剂进行热疗温度通常高于50℃，如果温度高于50℃，周围皮肤往往会被灼伤，因此本领域亟需一种能够充分发挥纳米炭作为热疗药物制剂，且不灼伤皮肤方便患者使用的制剂。
发明内容
为克服以上技术缺陷，本发明提供了一种纳米炭铁混悬注射液的新用途，具体采用以下技术方案：
本发明的第一方面提供了一种纳米炭铁复合制剂在制备配合近红外光使用的肿瘤热疗药物中的用途；
进一步的，所述配合近红外光使用是指采用近红外光加热组织；更进一步的，所述近红外光的波长为780～2600nm；更进一步优选的，所述近红外光的波长为780～1400nm；
进一步的，所述纳米炭铁复合制剂包括纳米炭混悬注射液以及注射用硫酸亚铁，且二者质量比为：20～100：3～300；优选的，二者质量比为50：7.5～180；进一步优选的，二者质量比为50：15～45；
进一步的，所述纳米炭铁复合制剂在使用时将纳米炭混悬注射液与注射用硫酸亚铁按上述质量比混合均匀，测定其pH值为2～7；优选的，测定其pH为3～5.5；即得；
进一步的，所述纳米炭铁混悬注射液含有纳米炭、助悬剂、生理盐水、pH调节剂，氯化钠以及注射用水；更进一步的，每1000mL纳米炭混悬注射液中，包括：纳米炭20～100g，助悬剂17～30g，pH调节剂2～4g，氯化钠8～10g，余量为注射用水；进一步优选的，每1000mL纳米炭混悬注射液中，包括：纳米炭50g，助悬剂20g，pH 调节剂3g，氯化钠9g，余量为注射用水；
更进一步的，所述纳米炭为纳米炭颗粒、纳米碳管、碳量子点、石墨烯、富勒烯、纳米碳棒、纳米碳纤维中的任意一种或几种；优选的，所述纳米炭为纳米炭颗粒或石墨烯；进一步优选的，所述纳米炭为炭黑；
更进一步的，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；进一步优选的，所述铁盐为硫酸亚铁；
更进一步的，所述助悬剂为泊洛沙姆、聚乙烯吡咯烷酮C30(PVP C30)、吐温-80中的任一种或几种；优选的，所述助悬剂为泊洛沙姆；
更进一步的，所述pH调节剂为柠檬酸钠、醋酸钠、硼酸钠、磷酸钠、碳酸氢钠中的任一种；优选的，所述pH调节剂为柠檬酸钠，且所述柠檬酸钠与铁盐中的亚铁离子和/或铁离子形成络合物；
更进一步的，所述纳米炭混悬注射液的制备方法包括以下步骤：
1)取上述配方量90％v/v的注射用水，加入配方量的氯化钠搅拌至完全溶解获得盐溶液；
2)向步骤1)获得的盐溶液中加入配方量的pH调节剂和助悬剂搅拌至完全溶解获得辅料液；
3)向步骤2)获得的辅料液中加入配方量的经前处理后的纳米炭并搅拌均匀然后加入余量注射用水定容获得定容液；
4)然后将3)获得的定容液以转速15000～20000rpm匀浆3～10min，再以15000～30000psi压力均质1～5次后，经过灌装、轧盖，然后在115～130℃下蒸汽灭菌10～30min，即得；
进一步的，步骤3)中，所述前处理包括将纳米炭经过乙酸乙酯 脱脂后先采用8～15％v/v的HNO 3水溶液洗涤，其中，纳米炭质量：HNO 3水溶液体积为1g:3～5/ml，然后再用水洗涤纳米炭近中性，然后再采用0.08～0.15mol/L的NaOH水溶液洗涤，其中，纳米炭质量：NaOH水溶液体积为1g:3～5/ml，然后再用水洗涤纳米炭近中性；更进一步的，所述的纳米炭质量：HNO 3水溶液体积为1g:4/ml；更进一步的，步骤S1所述的纳米炭质量：NaOH水溶液体积为1g:4/ml；优选的，所述HNO 3为10％v/v的HNO 3水溶液；
进一步的，步骤4)中，所述的匀浆转速为18000rpm，匀浆时间为5min；
进一步的，步骤4)中，所述的均质压力20000psi，均质次数为3次；
进一步的，步骤4)中，所述的灭菌温度为121℃，灭菌时间为15min；
进一步的，所述注射用硫酸亚铁为注射用硫酸亚铁冻干粉；
更进一步的，所述注射用硫酸亚铁冻干粉由以下原料制成：七水硫酸亚铁、硫酸以及注射用水，其中七水硫酸亚铁：硫酸：注射用水质量比为0.1～0.2g：0.4～0.85μg：2g；优选的，所述硫酸为质量百分含量1％的硫酸；
更进一步的，所述注射用硫酸亚铁冻干粉的制备方法包括以下步骤：
S1：取配方量90％的注射用水，制备过程中在液面下持续充入N 2，氮气流速控制4.0～5.0m 3/h；
S2：然后加入硫酸调节pH至2.4～2.8；优选调节pH至2.4；
S3：取配方量的七水硫酸亚铁充分搅拌使之溶解；
S4：加入余量硫酸调节pH至2.8～3.0，用余量纯化水定容至全量，并搅拌均匀得硫酸亚铁药液；优选调节pH至2.8；
S5：将所得药液经微孔滤膜过滤后，再灌装、冻干、真空压塞、轧盖后，即得；优选的，所述微孔滤膜孔径为0.45μm；
进一步的，S5中，所述冻干步骤的操作具体包括：
S51预冻：快速降温至-10～-5℃，并保温1～3h，然后再快速降温至-50～-40℃，并保温1～3h；优选的，降温至-5℃、保温2h；优选的，快速降温至-45℃，并保温2h；
S52干燥：S521：经过2～4h坡度升温从-50～-40℃升至-20～-10℃，然后在-20～-10℃、80～120mtorr下保温2～4h；优选的，经过3h坡度升温从-45℃升至-15℃，然后在-15℃、100mtorr下保温3h；
S522：经过1～3h再坡度升温至-20～-10℃至-10～0℃，然后在80～120mtorr、-10～0℃下保温1～3h；优选的，经过2h再坡度升温至-15℃～-5℃，然后在100mtorr、-5℃下保温2h；
S523：经过3～6h坡度升温至0℃～20℃，然后在80～120mtorr、20℃下保温3～6h；优选的，经过4h坡度升温至0℃～20℃，然后在100mtorr、20℃下保温4h。
有益效果
本发明公开了纳米炭铁复合制剂可以用于肿瘤低温热疗，相对于纳米炭制剂，本发明药物组合物在低温热疗时，可以显著降低给药剂量，减少不良反应的发生；并且采用本发明复合制剂进行热疗的温度较低，不易损伤周围正常组织，也降低纳米炭热疗时的不良反应；并进一步证明了纳米炭铁复合制剂与近红外照射具有协同效果。基于低温热疗的手段以及技术效果，本发明具有广泛的临床意义。
附图说明
图1各组作用于SMMC-7721肝癌裸鼠异种移植瘤的肿瘤体积 生长曲线；
图2各组作用于HCT-116结肠癌裸鼠异种移植瘤的肿瘤体积生长曲线；
图3各组作用于MDA-MB-231乳腺癌裸鼠异种移植瘤的肿瘤体积生长曲线；
图4 SMMC-7721肝癌肿瘤组织的羟基自由基波形图；
图5各组对H22肝癌细胞淋巴结转移的抑制作用。
具体实施例方式
(一)实施例
实施例1：制备纳米炭混悬注射液
一种纳米炭混悬注射液，每1000mL纳米炭混悬注射液中，包括：纳米炭50g，助悬剂20g，pH调节剂3g，氯化钠9g，余量为注射用水，其制备方法包括以下步骤：
1)取上述配方量90％v/v的注射用水，加入配方量的氯化钠搅拌至完全溶解获得盐溶液；2)向步骤1)获得的盐溶液中加入配方量的pH调节剂和助悬剂搅拌至完全溶解获得辅料液；3)向步骤2)获得的辅料液中加入配方量的经前处理后的纳米炭并搅拌均匀然后加入余量注射用水定容获得定容液，所述前处理包括经过乙酸乙酯脱脂后先采用10％v/v的HNO 3水溶液洗涤，其中，纳米炭质量：HNO 3水溶液体积为1g:4/ml，然后用水洗涤纳米炭近中性，然后再采用0.10mol/L的NaOH水溶液洗涤，其中，纳米炭质量：NaOH水溶液体积为1g:4/ml，然后用水洗涤纳米炭近中性；4)然后将3)获得的定容液以转速18000rpm匀浆5min，再以20000psi压力均质3次后，经过灌装、轧盖，然后在121℃下蒸汽灭菌15min，即得。
实施例2：制备注射用硫酸亚铁
一种注射用硫酸亚铁冻干粉由以下原料制成：七水硫酸亚铁、硫酸以及注射用水，其中七水硫酸亚铁：质量百分1％硫酸：注射用水质量比为0.149g：0.4～0.85μg：2g；优选的，所述硫酸为质量百分含量1％的硫酸；其制备方法包括以下步骤：
S1：取配方量90％的注射用水，取配方量90％的注射用水，制备过程中在液面下持续充入N 2，氮气流速控制4.0～5.0m3/h；S2：然后加入硫酸调节pH至2.4；S3：取配方量的七水硫酸亚铁充分搅拌使之溶解；S4：加入余量硫酸调节pH至2.8，用余量纯化水定容至全量，并搅拌均匀得硫酸亚铁药液；S5：将所得药液经0.45μm微孔滤膜过滤后，再灌装、冻干、真空压塞、轧盖后即得，其中，所述冻干步骤的操作具体包括：
S51预冻：快速降温至-5℃，并保温2h，然后再快速降温至-45℃，并保温2h；S52干燥：S521：经过3h坡度升温从-45℃升至-15℃，然后在-15℃、100mtorr下保温3h；S522：经过2h再坡度升温至-15℃至-5℃，然后在100mtorr、-5℃下保温2h；S523：经过4h坡度升温至0℃～20℃，然后在100mtorr、20℃下保温4h。
实施例3～22选择不同配比的纳米炭混悬注射液和注射用硫酸亚铁以及不同近红外光进行肿瘤热疗
  纳米炭混悬注射液 注射用硫酸亚铁 红外波长(nm)
实施例3 50mg 21mg 780
实施例4 50mg 21mg 808
实施例5 50mg 21mg 1064
实施例6 50mg 21mg 1400
实施例7 50mg 21mg 2600
实施例8 50mg 15mg 1064
实施例9 50mg 45mg 808
实施例10 50mg 7.5mg 808
实施例11 50mg 90mg 808
实施例12 50mg 180mg 808
实施例13 50mg 3mg 808
实施例14 50mg 300mg 808
实施例15 20mg 3mg 808
实施例16 20mg 30mg 808
实施例17 20mg 150mg 808
实施例18 20mg 300mg 808
实施例19 100mg 3mg 808
实施例20 100mg 30mg 808
实施例21 100mg 150mg 808
实施例22 100mg 300mg 808
(二)实验例
1、实验材料：
1)细胞株
SMMC7721肝癌细胞、HCT116结肠癌细胞、MDA-MB-231乳腺癌细胞、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，5-7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，采用独立送风隔离笼具饲养。
SPF级Balb/c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。
4)实验药品及主要仪器设备
实施例4纳米炭铁混悬注射液、实施例8纳米炭铁混悬注射液、 纳米炭混悬注射液(浓度与实施例4、8均相同)、0.9％氯化钠注射液、鼓风干燥箱、恒温水浴锅、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、电子天平、808nm近红外热疗仪、1064nm激光热疗仪、红外热像仪、电子自旋共振波谱仪(ESR)
2、实验方法：
1)细胞实验
收集对数生长期的细胞，计数，调节细胞密度为30000个/ml，取1ml加入到25×25mm的玻璃皿中，5％CO 2，37℃孵育24h。将细胞分组，分别为阴性组、近红外照射组、纳米炭铁组、纳米炭近红外照射组(37℃、42℃、45℃、48℃、50℃)、纳米炭铁近红外照射组(37℃、42℃、45℃、48℃、50℃)，每组做3个。阴性组和近红外照射组换上新的培养液，纳米炭铁组、纳米炭近红外照射组和纳米炭铁近红外照射组换上含有纳米炭或纳米炭铁的培养液。纳米炭近红外照射组和纳米炭铁近红外照射组用808nm近红外热疗仪分别照射至所需温度，并使温度维持10min。近红外照射组的照射时间与试验组照射时间最长组保持一致。照射完后，继续培养48h。细胞用胰酶消化并计数，计算细胞抑制率。
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、近红外照射组、纳米炭铁组、纳米炭近红外照射组以及纳米炭铁近红外照射组(采用实施例4纳米炭铁混悬注射液)。给药方式为瘤内注射，给药体积为50μL/次，间隔2天后第二次给药，共给药2次。瘤内注射药物后10min，纳米炭近红外照射组、纳米炭铁近 红外照射组分别用808nm近红外线照射肿瘤，功率密度为0.5W/cm 2，照射时间为30min，使其温度维持在45℃左右。近红外照射组照射相同的时间。每周测量小鼠肿瘤体积，体积计算公式为：体积＝(长度×宽度 2)/2。观察时间为21天。
在近红外照射后24h，取肿瘤组织，加入0.9％氯化钠注射液，制备成10％的匀浆液，加入捕获剂，用ESR检测羟基自由基。
3)抑制淋巴结转移实验
抽取H22肝癌荷瘤小鼠的乳白色腹水，加入生理盐水中，离心重悬，调整细胞数为3×10 7个/mL，接种于Balb/c小鼠的左后肢足垫皮下，每只接种50μL，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。将小鼠随机分为4组，每组7只，分别为阴性对照组、纳米炭铁组、近红外照射组、纳米炭近红外照射组以及纳米炭铁近红外照射组(采用实施例8纳米炭铁混悬注射液)。给药方式为瘤内注射，给药体积为50μL/次，间隔2天后第二次给药，共给药2次。瘤内注射药物后10min，纳米炭近红外照射组、纳米炭铁近红外照射组分别用1064nm近红外线照射小鼠左后肢腘窝淋巴结处，功率密度为0.5W/cm 2，照射时间为30min，使其温度维持在45℃左右。近红外照射组照射相同的时间。照射后2周处死小鼠，收集腘窝淋巴结并称重。
3、实验结果：
1)细胞实验
近红外照射组对SMMC7721肝癌细胞、HCT116结肠癌细胞、MDA-MB-231乳腺癌细胞均无明显杀伤作用，抑制率均小于10％。纳米炭铁单独作用于SMMC7721肝癌细胞、HCT116结肠癌细胞、MDA-MB-231乳腺癌细胞的抑制率分别为40.15±2.98％、 34.97±1.67％、32.85±3.07％。纳米炭近红外照射组和纳米炭铁近红外照射组对3种细胞的抑制率见表1-3。温度为37℃和40℃时对细胞基本无影响，随着温度的上升，抑制率逐渐增加。温度≥42℃时，纳米炭铁用近红外照射的抑制率大大增加，优于单用纳米炭铁和相同温度下的纳米炭近红外照射组。纳米炭近红外照射组在温度达到50℃时对3种细胞的抑制率达到90％以上，而纳米炭铁近红外照射组在温度为45℃时抑制率即达到90％以上。
表1纳米炭近红外照射组和纳米炭铁近红外照射组对SMMC7721肝癌细胞的抑制率
Figure PCTCN2022103774-appb-000001
表2纳米炭近红外照射组和纳米炭铁近红外照射组对HCT116结肠癌细胞的抑制率
Figure PCTCN2022103774-appb-000002
表3纳米炭近红外照射组和纳米炭铁近红外照射组对MDA-MB-231乳腺癌细胞的抑制率
Figure PCTCN2022103774-appb-000003
Figure PCTCN2022103774-appb-000004
2)抑制肿瘤生长实验
考察了纳米炭铁、纳米炭近红外照射、纳米炭铁近红外照射及近红外照射对SMMC7721肝癌细胞、HCT116结肠癌细胞、MDA-MB-231乳腺癌细胞裸鼠异种皮下移植瘤的生长抑制作用(图1-3)。结果显示，观察结束时，单独近红外照射对三种细胞的皮下移植瘤均无明显的抑制作用。纳米炭铁对三种细胞的皮下移植瘤抑制率分别为48.57％、52.03％、45.79％，纳米炭近红外照射对三种细胞的皮下移植瘤抑制率分别为53.81％、48.06％、49.35％，纳米炭铁近红外照射对三种细胞的皮下移植瘤抑制率分别为90.78％、90.14％、93.3％。纳米炭铁近红外照射与单独的纳米炭铁或纳米炭近红外照射相比，均有显著的统计学差异。根据金氏公式法计算，纳米炭铁近红外照射的q值均大于1，表明纳米炭铁近红外照射具有协同作用，可以增强纳米炭铁及纳米炭热疗的抗癌效果。
同时比较了各组动物肿瘤内的羟基自由基含量(图4)。结果分别显示：图4a表示不使用任何方式处理样品；图4b、d表示纳米炭近红外照射及近红外照射产生的羟基自由基不显著，而纳米炭铁组(图4c)以及纳米炭铁近红外照射组(图4e)的肿瘤内羟基自由基含量最高，约为纳米炭铁组的2倍。说明纳米炭铁近红外照射后，能有效升高肿瘤内温度，提高芬顿反应的效率，产生更多的羟基自由基。
3)抑制淋巴结转移实验
考察了纳米炭铁、纳米炭近红外照射、纳米炭铁近红外照射及近红外照射对H22肝癌细胞淋巴结转移的抑制作用(图5)。结果显示，与阴性组相比(“*”为p＜0.05，“**”为p＜0.01)，近红外照射 组的淋巴结重量无明显变化；纳米炭铁组、纳米炭近红外照射组及纳米炭铁及红外照射组的淋巴结重量均显著性降低(p＜0.01)。纳米炭铁近红外照射对淋巴结转移的抑制作用最强，转移淋巴结的重量最轻，与单独的纳米炭铁或纳米炭近红外照射相比，均有显著的统计学差异(p＜0.05)。根据金氏公式法计算，纳米炭铁近红外照射的q值大于1，表明纳米炭铁近红外照射具有协同作用，可以增强纳米炭铁及纳米炭热疗的抑制淋巴结转移效果。
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 (15)

  1. 一种纳米炭铁复合制剂在制备配合近红外光使用的肿瘤热疗药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述配合近红外光使用是指采用近红外光加热组织。
  3. 根据权利要求1所述的用途，其特征在于，所述近红外光的波长为780～2600nm。
  4. 根据权利要求1～3任一项所述的用途，其特征在于，所述纳米炭铁复合制剂包括纳米炭混悬注射液以及注射用硫酸亚铁，且二者质量比为：20～100∶3～300；优选的，二者质量比为50∶7.5～180。
  5. 根据权利要求4所述的用途，其特征在于，所述纳米炭铁复合制剂在使用时将纳米炭混悬注射液与注射用硫酸亚铁混合均匀，测定其pH值为2～7；优选的，测定其pH为3-5.5。
  6. 根据权利要求4所述的用途，其特征在于，所述纳米炭混悬注射液含有纳米炭、助悬剂、生理盐水、pH调节剂，氯化钠以及注射用水；更进一步的，每1000mL纳米炭混悬注射液中，包括：纳米炭20～100g，助悬剂17～30g，pH调节剂2～4g，氯化钠8～10g，余量为注射用水。
  7. 根据权利要求6所述的用途，其特征在于，所述纳米炭为纳米炭颗粒、纳米碳管、碳量子点、石墨烯、富勒烯、纳米碳棒、纳米碳纤维中的任意一种或几种。
  8. 根据权利要求6所述的用途，其特征在于，所述助悬剂为泊洛沙姆、聚乙烯吡咯烷酮C30(即PVP C30)、吐温-80中的任一种或几种。
  9. 根据权利要求6所述的用途，其特征在于，所述pH调节剂为柠檬酸钠、醋酸钠、硼酸钠、磷酸钠、碳酸氢钠中的任一种。
  10. 根据权利要求4所述的用途，其特征在于，所述注射用硫酸亚铁为注射用硫酸亚铁冻干粉；优选的，所述注射用硫酸亚铁冻干粉 由以下原料制成：七水硫酸亚铁、硫酸以及注射用水，其中七水硫酸亚铁∶硫酸∶注射用水质量比为0.1～0.2g∶0.4～0.85μg∶2g。
  11. 根据权利要求10所述的用途，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。
  12. 根据权利要求6～9任一项所述的用途，其特征在于，纳米炭混悬注射液的制备方法包括以下步骤：
    1)取上述配方量90％v/v的注射用水，加入配方量的氯化钠搅拌至完全溶解获得盐溶液；
    2)向步骤1)获得的盐溶液中加入配方量的pH调节剂和助悬剂搅拌至完全溶解获得辅料液；
    3)向步骤2)获得的辅料液中加入配方量的经前处理后的纳米炭并搅拌均匀然后加入余量注射用水定容获得定容液；
    4)然后将3)获得的定容液以转速15000～20000rpm匀浆3～10min，再以15000～30000psi压力均质1～5次后，经过灌装、轧盖，然后在115～130℃下蒸汽灭菌10～30min，即得。
  13. 根据权利要求12所述的用途，其特征在于，步骤3)中，所述前处理包括将纳米炭经过乙酸乙酯脱脂后先采用8～15％v/v的HNO 3水溶液洗涤，其中，纳米炭质量：HNO 3水溶液体积为1g∶3～5/ml，然后再用水洗涤纳米炭近中性，然后再采用0.08～0.15mol/L的NaOH水溶液洗涤，其中，纳米炭质量：NaOH水溶液体积为1g∶3～5/ml，然后再用水洗涤纳米炭近中性。
  14. 根据权利要求10～11任一项所述的用途，其特征在于，所述注射用硫酸亚铁冻干粉的制备方法包括以下步骤：
    S1：取配方量90％的注射用水，制备全程中，在液面下持续充入N 2，氮气流速控制4.0～5.0m 3/h；
    S2：然后加入硫酸调节pH至2.4～2.8；
    S3：取配方量的七水硫酸亚铁充分搅拌使之溶解；
    S4：加入余量硫酸调节pH至2.8～3.0，用余量纯化水定容至全量，并搅拌均匀得硫酸亚铁药液；
    S5：将所得药液经微孔滤膜过滤后，再灌装、冻干、真空压塞、轧盖后，即得。
  15. 根据权利要求14所述的用途，其特征在于，所述注射用硫酸亚铁冻干粉的制备方法S5中，所述冻干步骤的操作具体包括：
    S51预冻：快速降温至-10～-5℃，并保温1～3h，然后再快速降温至-50～-40℃，并保温1～3h；
    S52干燥：S521：经过2～4h坡度升温从-50～-40℃升至-20～-10℃，然后在-20～-10℃、80～120mtorr下保温2～4h；
    S522：经过1～3h再坡度升温至-20～-10℃至-10～0℃，然后在80～120mtorr、-10～0℃下保温1～3h；
    S523：经过3～6h坡度升温至0℃～20℃，然后在80～120mtorr、20℃下保温3～6h。
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