Patent Application
20230330135
The present disclosure belongs to the technical field of medicines, and particularly relates to a carbon nanoparticles suspension injection-Fe mixture for low-temperature hyperthermia as well as preparation method.
Photothermal therapy is a new tumor therapy developed recently, and it has great application prospects because of its small trauma, obvious therapeutic effect and lower toxic and side effects. In the development process of reagents for hyperthermia, carbon nanotubes, graphene, carbon quantum dots and fullerene are the hot directions in the current researches. The carbon atoms in these materials form covalent bonds by SP2 hybridization and extend around to form a foveolate six-membered ring structure π electrons are enriched on the ring to form delocalized π bonds. Therefore, the SP2 hybridization gives these carbon materials many special properties, including absorbing NIR light. After absorbing NIR, these carbon materials can quickly convert light energy into heat to kill tumor cells, so that they are ideal potential candidate materials for hyperthermia.
After long-term accumulation of scientific research data, great progress has been obtained in the development of reagents for hyperthermia, including researches on the effective improvement of heat conversion efficiency, the surface modification for improving targeting ability and toxicity. However, there are also many problems limiting the clinical application in the research process. The main defects are as follows: (1) The preparation process is difficult to control. The crude products of carbon nanotubes, graphene and fullerene contain a lot of impurities, and toxic and side effects may be induced in vivo; the poor hydrophilicity is not conducive to dispersion in water, and thus refining and surface modification are required. In the refining process, impurities are mainly removed to improve purity and reduce the risk of toxic and side effects caused by impurities. In the surface modification, surface groups of carbon are mainly modified, which is conducive to the dispersion in water and improves the conversion efficiency of NIR during hyperthermia. However, it is found in the process of reagent development that these carbon materials are mixtures with a certain range of particle sizes, and it is difficult to control the consistency between batches in the refining and surface modification process, thereby leading to differences in product properties between batches, affecting the comprehensive evaluation, and hindering the implementation of later tests. (2) The shape and size of particles are difficult to control. In these materials, the carbon nanotubes are in a tubular structure, both ends need to be passivated during the preparation, but some needle-tip-like ends still exist usually due to incomplete treatment; after administration, these sharp ends tend to pierce blood vessel walls and then enter nucleuses, so that there is potential genetic toxicity. (3) The targeting ability is poor and the accuracy is insufficient. After local administration, owing to poor targeting ability, the carbon nanotubes, graphene, carbon quantum dots and fullerene are enriched around tumors, and some of them will diffuse to normal tissues. In the hyperthermia, the carbon diffused around the normal tissues will also become hot to result in burns to the normal tissues. (4) The cost is high. Because the complex preparation process involves the steps of preparation, refining and chemical modification, the cost is too high to limit its large-scale production and clinical applications. Under the above-mentioned application background, the application of preparations for hyperthermia has been limited to an early exploration stage, and there is no safe, stable and controllable reagent entering at a clinical application stage to be safely and effectively used by patients.
In addition, the heating principle of carbon nanoparticles is that carbon nanoparticles can efficiently absorb near-infrared ray (NIR) and convert NIR into heat, but body tissues have weak absorption of NIR (high transmittance), and less NIR is converted into heat. Therefore, taking advantage of the above differences, carbon nanoparticles are used as a medium of hyperthermia; after carbon nanoparticles are injected into a cancer focus, the externally irradiated NIR can reach the cancer focus through a surface normal tissue and heat the carbon nanoparticles, thereby raising the temperature of the tumor tissue to produce an effect of hyperthermia. However, the hyperthermia using carbon nanoparticles requires a temperature as high as 50° C. or above to result in necrosis and coagulation of tumor cells, and the higher temperature may easily damage cells of adjacent normal tissues. Carbon nanoparticles suspension injection-Fe, a previously developed drug, is a nanosuspension taking carbon nanoparticles as a carrier and ferrous ions as an effective component, and is an innovative anti-cancer drug. Its mechanism of action is as follows: after being locally injected into a cancer tissue, the carbon nanoparticles suspension injection-Fe (CNSI-Fe) enters cancer cells through over-expressed iron channels on cancer cell membranes. A large number of iron ions enter the cancer cells rich in hydrogen peroxide (H2O2) and then have a Fenton reaction with the H2O2 to produce a plenty of hydroxyl radicals (·OH); the ·OH with extremely strong oxidation properties reacts with unsaturated fatty acids (UPFAs) in the cells to produce a large number of highly destructive lipid hydrogen peroxide (L—OOH), that is, lipid reactive oxygen species (lipid-ROS), and the lipid-ROS can destroy organelles to lead to cell damage, thereby resulting in ferroptosis.
The influencing factors of the Fenton reaction include a pH value, an H2O2 dosage, an Fe2+ dosage, a reaction time and a reaction temperature. When the other conditions remain unchanged, increasing the reaction temperature can speed up the reaction and enhance the reaction rate. After CNSI-Fe is locally injected into a cancer tissue, near-infrared irradiation can elevate its temperature to enhance the Fenton reaction of iron ions. However, at present, the temperature of hyperthermia directly using a carbon nanoparticles preparation in the prior art is usually higher than 50° C., and the surrounding skin will often be burned if the temperature is higher than 50° C. Therefore, there is an urgent need in the field for a preparation that can give full play to carbon nanoparticles as a drug preparation for hyperthermia, without burning the skin and with convenience for patients to use.
The present disclosure provides a new application of a CNSI-Fe, and the following technical solutions are specifically adopted:
The first aspect of the present disclosure provides an application of a CNSI-Fe mixture in preparing a tumor hyperthermia drug used in combination with near-infrared light.
Further, the “used in combination with near-infrared light” refers to heating a tissue using near-infrared light; still further, a wavelength of the near-infrared light is 780-2,600 nm; and still further preferably, the wavelength of the near-infrared light is 780-1,400 nm.
Further, the CNSI-Fe mixture includes a carbon nanoparticles suspension injection (CNSI) and ferrous sulfate for injection, wherein a mass ratio of the CNSI to the ferrous sulfate for injection is 20-100:3-300; preferably, the mass ratio is 50:7.5-180; and more preferably, the mass ratio is 50:15-45.
Further, when the CNSI-Fe mixture is used, the CNSI and the ferrous sulfate for injection are uniformly mixed according to the above-mentioned mass ratio, and a pH value of the mixture is determined to be 2-7; preferably, the pH value is determined to be 3-5.5.
Further, the CNSI-Fe includes carbon nanoparticles, a suspending agent, saline, a pH adjuster, sodium chloride and water for injection; still further, each 1,000 mL of CNSI includes 20-100 g of carbon nanoparticles, 17-30 g of suspending agent, 2-4 g of pH adjuster, 8-10 g of sodium chloride and a remaining amount of water for injection; and more preferably, each 1,000 mL of CNSI includes 50 g of carbon nanoparticles, 20 g of suspending agent, 3 g of pH adjuster, 9 g of sodium chloride and a remaining amount of water for injection.
Still further, the carbon nanoparticles are any one or some of carbon nanoparticles, carbon nanotubes, carbon quantum dots, graphene, fullerenes, carbon nanorods and carbon nanofibers; preferably, the carbon nanoparticles are carbon nanoparticles or graphene; and more preferably, the carbon nanoparticles are carbon black.
Still further, the ferric salt is selected from any one or some of ferrous sulfate, ferric sulfate, ferrous chloride, ferric trichloride, ferrous gluconate, ferric sucrose, ferric ammonium citrate, ferrous succinate, ferric sorbitol and ferrous fumarate; preferably, the ferric salt is ferrous sulfate, ferric sulfate, ferrous chloride or ferric chloride; and more preferably, the ferric salt is ferrous sulfate.
Still further, the suspending agent is any one or some of poloxamer, polyvinylpyrrolidone C30 (PVPC30) and Tween-80; and preferably, the suspending agent is poloxamer.
Still further, the pH adjuster is any one of sodium citrate, sodium acetate, sodium borate, sodium phosphate and sodium bicarbonate; preferably the pH adjuster is sodium citrate, and the sodium citrate forms a complex compound with ferrous and/or ferric ions in the ferric salts.
Still further, a preparation method of the CNSI includes the following steps:
Further, the pretreatment in step 3) includes the following steps: firstly washing the carbon nanoparticles degreased by ethyl acetate using a 8-15%v/v HNO3 aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the HNO3 aqueous solution is 1 g : 3-5 /ml; then washing the carbon nanoparticles with water to be approximately neutral; then washing the carbon nanoparticles with a 0.08-0.15 mol/L NaOH aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the NaOH aqueous solution is 1 g : 3-5 /ml; and washing the carbon nanoparticles with water to be approximately neutral; still further, the mass-to-volume ratio of the carbon nanoparticles to the HNO3 aqueous solution in S1 is 1 g:4 /ml; and preferably, the HNO3 is a 10%v/v HNO3 aqueous solution.
Further, in step 4), the homogenization rate is 18,000 rpm, and the homogenization time is 5 min.
Further, in step 4), the homogenization pressure is 20,000 psi, and the homogenization is performed for 3 times.
Further, in step 4), the sterilization temperature is 121° C., and the sterilization time is 115 min.
Further, the ferrous sulfate for injection is a lyophilized powder of ferrous sulfate for injection.
Still further, the lyophilized powder of ferrous sulfate for injection is prepared from the following raw materials: ferrous sulfate heptahydrate, sulfuric acid and water for injection, wherein a mass ratio of ferrous sulfate heptahydrate : sulfuric acid : water for injection is 0.1-0.2 g : 0.4-0.85 µg : 2 g; and preferably, the sulfuric acid is 1% (by weight) sulfuric acid.
Still further, a preparation method of the lyophilized powder of ferrous sulfate for injection includes the following steps:
Further, the lyophilization step in S5 includes the following specific operations:
The present disclosure discloses a CNSI-Fe mixture which can be used for the low-temperature hyperthermia of tumor. Compared with carbon nanoparticles preparations, when the pharmaceutical composition is used for low-temperature hyperthermia, the administration dosage can be significantly decreased so as to reduce the occurrence of adverse reactions; the hyperthermia using the mixture of the present disclosure has a lower temperature, so that the surrounding normal tissues are not liable to be damaged, and the adverse reactions during the hyperthermia using carbon nanoparticles can also be reduced. Moreover, it is further proved that CNSI-Fe mixture and the near-infrared irradiation are synergic. Based on the means and technical effects of low-temperature hyperthermia, the present disclosure has extensive clinical significance.
For a CNSI, each 1,000 mL of CNSI included 50 g of carbon nanoparticles, 20 g of suspending agent, 3 g of pH adjuster, 9 g of sodium chloride and a remaining amount of water for injection; and a preparation method included the following steps:
1) 90% v/v of the formulation amount of water for injection was taken, the formulation amount of sodium chloride was added, and the mixture was stirred until the mixture was completely dissolved to obtain a salt solution; 2) the formulation amounts of pH adjuster and suspending agent were added to the salt solution obtained in step 1), and the mixture was stirred until the mixture was completely dissolved to obtain an excipient solution; 3) the formulation amount of pretreated carbon nanoparticles was added to the excipient solution obtained in step 2), the mixture was stirred evenly, and then the remaining amount of water for injection was added to a constant volume to obtain a constant volume solution, wherein the pretreatment included the following steps: firstly the carbon nanoparticles degreased by ethyl acetate was washed using a 10%v/v HNO3 aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the HNO3 aqueous solution was 1 g : 4 /ml, and then the carbon nanoparticles were washed with water to be approximately neutral; then the carbon nanoparticles were washed with a 0.10 mol/L NaOH aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the NaOH aqueous solution was 1 g : 4 /ml, and then the carbon nanoparticles were washed with water to be approximately neutral; and 4) the constant volume solution obtained in step 3) was homogenized at a rate of 18,000 rpm for 5 min and then at a pressure of 20,000 psi 3 times, then filled and capped, and finally sterilized by steam at 121° C. for 15 min to obtain the CNSI.
A lyophilized powder of ferrous sulfate for injection was prepared from the following raw materials: ferrous sulfate heptahydrate, sulfuric acid and water for injection, wherein a mass ratio of ferrous sulfate heptahydrate : sulfuric acid : water for injection was 0.149 g : 0.4-0.85 µg : 2 g; preferably, the sulfuric acid was 1% (by weight) sulfuric acid. A preparation method included the following steps:
S1: 90% of the formulation amount of water for injection was taken, N2 was continuously filled below the liquid level during the preparation, and a nitrogen flow rate was controlled at 4.0-5.0 m3/h; S2: sulfuric acid was then added to adjust the pH value to 2.4; S3: the formulation amount of ferrous sulfate heptahydrate was taken and fully stirred for dissolution; S4: the remaining amount of sulfuric acid was added to adjust the pH value to 2.8, the remaining amount of purified water was added to fix the volume to a full volume, and the mixture was stirred evenly to obtain a ferrous sulfate solution; and S5: the obtained solution was filtered with a 0.45 µm microporous filter membrane, filled, lyophilized, vacuum plugged and capped to obtain the lyophilized powder; wherein the lyophilization step included the following specific operations:
S51 pre-freezing: the temperature was rapidly lowered to -5° C. and kept for 2 h, and then the temperature was rapidly lowered to -45° C. and kept for 2 h; S52 drying: S521: the temperature was gradually elevated from -45° C. to -15° C. within 3 h and kept at -15° C. and 100 mtorr for 3 h; S522: the temperature was gradually elevated to (-15 to -5)°C within 2 h and kept at -5° C. and 100 mtorr for 2 h; and S523: the temperature was gradually elevated to 0-20° C. within 4 h and kept at 20° C. and 100 mtorr for 4 h.
In examples 3-22, the CNSI and the ferrous sulfate for injection in different proportions and near-infrared light of different wavelengths were selected for hyperthermia of tumor.
SMMC7721 liver cancer cells, HCT116 colon cancer cells, MDA-MB-231 breast cancer cells, and mouse derived liver cancer H22 cells
DMEM cell medium, RMPI1640 medium, fetal bovine serum (FBS), trypsin for cell digestive solution, penicillin-streptomycin mixture, and phosphate buffer solution (PBS, pH 7.4)
BalB/c-nu mice, male, 5-7 weeks old, and weighing 20±2 g. The mice could drink and eat freely during the experiments. The mice were irradiated by light for 12 h every day, and fed in isolated cages with separate air supply, with 5 mice in each cage.
SPF-class Balb/c mice, male, 4-6 weeks old, and weighing 20±2 g. The mice could drink and eat freely during the experiments. The mice were irradiated by light for 12 h every day, and fed in isolated cages with separate air supply, with 5 mice in each cage.
CNSI-Fe for example 4, CNSI-Fe for example 8, CNSI (with a concentration the same as those used in examples 4 and 8), 0.9% sodium chloride injection, blast drying oven, thermostatic water bath kettle, biological optical microscope, thermostatic incubator, water purifier, autoclave, ultra-clean workbench, electronic balance, 808 nm near-infrared hyperthermia instrument, 1,064 nm laser hyperthermia instrument, infrared thermograph, and electron spin resonance (ESR) spectrometer
Cells in a logarithmic growth phase were collected and counted, a cell density was adjusted to 30,000 cells/ml, and 1 ml was added to a 25×25 mm glass dish and incubated with 5% CO2 at 37° C. for 24 h. The cells were divided into a negative group, a near-infrared irradiation group, a CNSI-Fe group, a CNSI + near-infrared irradiation group (37° C., 42° C., 45° C., 48° C. and 50° C.), and a CNSI-Fe + near-infrared irradiation group (37° C., 42° C., 45° C., 48° C. and 50° C.), with 3 duplicates for each group. The negative group and the near-infrared irradiation group were replaced with new culture solutions, and the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group were replaced with culture solutions containing CNSI or CNSI-Fe. The CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group were irradiated to the required temperatures using the 808 nm near-infrared hyperthermia instrument, respectively, and the temperatures were maintained for 10 min. The irradiation time of the near-infrared radiation group was consistent with the longest irradiation time of the test group. After irradiation, the cells were cultured for another 48 h. The cells were digested with trypsin and counted, and a cell inhibition rate was calculated.
Cells in a logarithmic growth phase were collected, a concentration of the cell suspension was adjusted to 3×107 cells/mL, and the cells were inoculated subcutaneously in the upper right limb of each nude mouse at 0.1 mL/mouse (including about 3×106 cells); when an average tumor volume of the inoculated mice reached 100 mm3, and the tumor bearing mice were randomly divided into a negative control group, a CNSI-Fe group, a CNSI + near-infrared irradiation group and a CNSI-Fe + near-infrared irradiation group (adopting the CNSI-Fe used in example 4). The method of administration was intratumoral injection, and a volume of administration was 50 µL/time; the second administration was given after an interval of 2 days, and the mice were administrated twice in total. At 10 min after the intratumoral injection, tumors were irradiated by 808 nm near-infrared ray in the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group, respectively, wherein a power density was 0.5 W/cm2, an irradiation time was 30 min, and a temperature was maintained at about 45° C. The near-infrared radiation group was irradiated for the same period of time. The tumor volume of the mice were measured every week, and the volume was calculated by the following formula: volume=(length × width2)/2. The observation lasted for 21 days.
At 24 h after the near-infrared irradiation, the tumor tissue was taken from each mouse, 0.9% sodium chloride injection was added to prepare a 10% homogenate, a capture agent was added, and hydroxyl radicals were detected with ESR.
Milky white ascites was extracted from each tumor bearing mouse with H22 liver cancer and added to normal saline, and the mixture was centrifugated and re-suspended; the number of cells was adjusted to 3×107 cells/mL, the cells were inoculated subcutaneously on the foot pad of the left hind limb of each Balb/c mouse, and each mouse was inoculated with 50 µL to obtain a mouse model of cancer lymph node metastasis. The mice were treated when a tumor diameter reached 6-8 mm and there was neither ulcer nor necrosis. The mice were randomly divided into 5 groups, with 7 mice in each group, that is, a negative control group, a CNSI-Fe group, a near-infrared irradiation group, a CNSI + near-infrared irradiation group and a CNSI-Fe + near-infrared irradiation group (adopting the CNSI-Fe used in example 8). The method of administration was intratumoral injection, and a volume of administration was 50 µL/time; the second administration was given after an interval of 2 days, and the mice were administrated twice in total. At 10 min after the intratumoral injection of drugs, each mouse was irradiated in the position of axillary lymph nodes in the left hind limb by 1,064 nm near-infrared ray in the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group, respectively, wherein a power density was 0.5 W/cm2, an irradiation time was 30 min, and a temperature was maintained at about 45° C. The near-infrared radiation group was irradiated for the same period of time. At 2 weeks after the irradiation, the mice were killed, and the axillary lymph nodes were collected and weighed.
In the near-infrared irradiation group, there was no obvious killing effect on the SMMC7721 liver cancer cells, the HCT116 colon cancer cells and the MDA-MB-231 breast cancer cells, and the inhibition rates were all less than 10%. The inhibition rates of the CNSI-Fe acting alone to the SMMC7721 liver cancer cells, the HCT116 colon cancer cells and the MDA-MB-231 breast cancer cells were 40.15±2.98%, 34.97±1.67% and 32.85±3.07%, respectively. The inhibition rates to the three kinds of cells in the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group are as shown in Tables 1-3. When the temperature was 37° C. and 40° C., there was basically no effect on the cells, and the inhibition rates gradually increased as the temperature was elevated. When the temperature was ≥ 42° C., the inhibition rate in the CNSI-Fe + near-infrared irradiation group was greatly increased and was better than those in the CNSI-Fe group and the CNSI + near-infrared irradiation group at the same temperature. In the CNSI + near-infrared irradiation group, when the temperature reached 50° C., the inhibition rate to the three kinds of cells reached above 90%; but in the CNSI-Fe + near-infrared irradiation group, the inhibition rate to the three kinds of cells reached above 90% when the temperature was 45° C.
The inhibitory effects of CNSI-Fe, CNSI + near-infrared irradiation, CNSI-Fe + near-infrared irradiation, and near-infrared irradiation on the growth of subcutaneous xenograft tumor of the nude mice with SMMC7721 liver cancer cells, HCT116 colon cancer cells and MDA-MB-231 breast cancer cells were observed (
At the same time, the content of hydroxyl radicals in tumors of animals was compared among the groups (
The inhibitory effects of the CNSI-Fe, CNSI + near-infrared irradiation, CNSI-Fe + near-infrared irradiation, and near-infrared irradiation on the lymph node metastasis of H22 liver cancer cells were tested (
It should be stated that the above-mentioned embodiments are only used for explaining, rather than limiting, the technical solutions of the present disclosure. Although the present disclosure is explained in detail by reference to the above-mentioned embodiments, those of ordinary skill in the art should understand that modifications or equivalent substitutions may be made to the present disclosure still, and any modification or local substitution without deviating from the spirit and scope of the present disclosure should fall into the scope of the claims of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110791901.7 | Jul 2021 | CN | national |
This application is a bypass continuation application of PCT Application No.: PCT/CN2022/103774, filed Jul. 5, 2022, and to Chinese Pat. Application No. 2021107919017, filed Jul. 13, 2021, the contents of which is incorporated herein in the entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/103774 | Jul 2022 | WO |
| Child | 18126867 | US |
