CN114699371B - Transferrin-mediated single-wall carbon nano Guan Tao sevoflurane U liposome complex and preparation method and application thereof - Google Patents
Transferrin-mediated single-wall carbon nano Guan Tao sevoflurane U liposome complex and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome compound, a preparation method and application thereof, wherein the compound is mainly a carbon nano tube liposome compound formed by peach heptaketone U, single-walled carbon nano tubes, lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino and distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin. The invention combines the high-efficiency cell uptake capacity of the carbon nano tube and the high drug carrying capacity of the liposome, is convenient for the low-concentration single-wall carbon nano tube to send high-concentration drugs into target cells, reduces the dosage of the single-wall carbon nano tube, obviously reduces the related toxicity caused by the single-wall carbon nano tube, can promote the drugs to enter tumor cells to reach deeper parts of the tumor more efficiently by utilizing the penetrating capacity of the single-wall carbon nano tube, can inhibit the growth of the tumor cells more efficiently, and can expand the application range of the carbon nano tube and the liposome as drug delivery systems.
Description
Technical Field
The invention belongs to the technical field of medicine production, in particular to the liposome field, and particularly relates to a transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound, a preparation method thereof and application thereof in preparing antitumor drugs.
Background
Prostate cancer (PCa) is the second most common cancer in men, and most prostate cancer patients develop Castration Resistant Prostate Cancer (CRPC) later, currently lacking effective treatments for CRPC. The herba Begoniae Laciniatae is a common Tibetan medicine, is a dry mature fruit of radix Begoniae Laciniatae Sinopodophyllum hexandrum of the genus Begonia of the family berberidaceae, has effects of regulating menstruation and promoting blood circulation, and is mainly used for treating amenorrhea due to blood stasis, dystocia, stillbirth and placenta. Has long history as a Tibetan medicine for learning. Modern chemical component researches show that the isopentenyl flavonoid compound has strong anti-tumor activity as a representative active component in the lobelia. Early researches found that the prenylated flavonoid compound tao-sevoflurane U extracted from Nelumbo nucifera can play an anti-prostate tumor role by inhibiting PC-3 cell proliferation, metastasis and induction of apoptosis. But the clinical use of tao' er sevoflurane U is limited due to its extremely low water solubility and poor pharmacokinetic behavior.
Nanoparticles, dendrimers, liposomes, nanomicelles, carbon Nanotubes (CNTs), and the like are all nano-carriers with a certain application prospect at present. CNTs show great attractive potential in the fields of drug delivery and targeted therapy due to the large specific surface area, excellent transmembrane property and easy modification of tube walls. CNTs can be used as drug carriers to covalently bind drug molecules and then transport the drug throughout the body. Moreover, CNTs are shaped so that they can enter different cells by passive diffusion or endocytosis, and have extremely strong ability to penetrate cell barriers. However, the limited surface area for direct binding to CNTs results in limited drug carrying capacity, and therefore high concentrations of CNTs can deliver sufficient doses of drug.
The liposome has great advantages in this respect, not only has higher drug carrying capacity, but also has good biocompatibility and biodegradability, can control the release rate of the drug, and improves the pharmacokinetic and pharmacodynamic characteristics of the drug. However, conventional liposomes are easily phagocytosed by blood and reticuloendothelial macrophages as drug carriers, resulting in an excessively short circulation time in vivo. In order to overcome the main obstacle, after the liposome is modified by polyethylene glycol (PEG), the surface hydrophilicity and the steric hindrance effect of the liposome can be changed to play a long circulation role, so that the physical stability and the biological stability of the liposome are improved. However, the steric hindrance prevents the liposome from entering the tumor cells, so that the therapeutic effect of the carried medicine is reduced.
The transferrin and transferrin receptor have important roles in human body, and the affinity of transferrin and transferrin receptor on the surface of tumor cells is 10-100 times of that of normal cell surface, so that the development of a compound which uses transferrin as a targeting functional group to be connected with liposome and enters the tumor cells through transferrin-mediated transport, and effectively realizes the anti-tumor targeting therapeutic role has important significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome compound, and a preparation method and application thereof, which can effectively solve the application problem of the sevoflurane U in clinical treatment of prostate tumors.
In order to achieve the above object, one of the technical solutions of the present invention is:
a transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome compound is mainly a carbon nano tube liposome compound formed by peach heptanone U, single-walled carbon nano tubes, lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino and distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin.
The liposome is formed by inserting single-walled carbon nanotubes into spherical liposome, and has a sugar-gourd shape and a particle size of 250-280 nm.
One of the technical schemes of the invention is as follows: a method for preparing transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome complex comprises wrapping sevoflurane U in liposome, and co-modifying the liposome with single-walled carbon nano tube and tumor cell specific ligand transferrin.
The preparation method comprises the steps of preparing Cheng Tao sevoflurane U liposome from sevoflurane U, and then mixing the Cheng Tao sevoflurane U liposome with carboxylated single-walled carbon nanotubes to prepare a single-walled carbon nano Guan Tao sevoflurane U liposome compound; and then mixing the single-walled carbon nano Guan Tao heptaketone U liposome complex with distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle to prepare the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome complex.
Still further, the method comprises the steps of:
(1) Preparation of single-walled carbon nano Guan Tao heptanone U liposome complex:
mixing 48-72 mg of lecithin, 16-24 mg of cholesterol and 16-24 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino, adding organic solvent chloroform (6.4-9.6 mL) and performing ultrasonic dissolution; dissolving 4.8-7.2 mg of tao er sevoflurane U in absolute ethyl alcohol (1.6-2.4 mL), adding into the mixture, and performing reduced pressure rotary evaporation to form a film; then adding PBS solution (8-12 mL), rotating for hydration, and performing ultrasonic treatment by a probe under ice bath to obtain a suspension of the U liposome of the peach sevoflurane;
dissolving 80-120mg Pluronic F127 in PBS (8-12 mL), adding 8-12 mg carboxylated single-walled carbon nanotube under-ice-bath probe for ultrasonic treatment to obtain carboxylated single-walled carbon nanotube suspension; then 8-12 mg of EDC and 8-12 mg of NHS are added in turn for ultrasonic mixing and activation; finally adding the suspension of the U-shaped liposome of the peach sevoflurane, stirring, centrifuging, collecting precipitate, adding PBS (8-12 mL) for redispersion, and obtaining the suspension of the U-shaped liposome complex of the peach sevoflurane, namely the single-wall carbon nano Guan Tao;
(2) Preparation of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle:
weighing 16-24 mg distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin, dissolving in organic solvent chloroform (4-6 mL), evaporating under reduced pressure to form a film, removing the organic solvent, drying, adding PBS solution (8-12 mL) for rotary hydration, and preparing distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle;
(3) Preparation of transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome complex:
mixing the single-walled carbon nano Guan Tao heptaketone U liposome composite suspension with distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle, reacting and incubating, and removing free distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin after the reaction to obtain the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome composite.
The temperature of the reduced pressure rotary evaporation in the step (1) is 45 ℃ and the time is 1h; the temperature of the rotary hydration is 55 ℃ and the time is 2 hours; the ultrasonic power of the probe is 260-300 w, and the time is 3-5 min; the activation time is 30min; stirring at room temperature for 3h; the centrifugation speed is 10000g and the time is 30min; the pH of the PBS solution was 6.5.
The temperature of reduced pressure evaporation in the step (2) is 45 ℃, the temperature of drying is 50 ℃, the temperature of rotary hydration is 55 ℃ and the time is 2h.
The temperature of the reaction incubation in step (3) was 37℃for 1h.
One of the technical schemes of the invention is as follows: application of transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome complex in preparing tumor treating medicine is provided.
The tumor is a prostate tumor.
The invention has the beneficial effects that:
the transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound can improve the bioavailability of the medicine and achieve the purposes of enhancing the medicine effect and targeting. The invention utilizes the double layered structure of liposome to wrap the indissoluble medicine in the lipid double layered structure, enhances the water solubility of the medicine, can better control the medicine release, is beneficial to improving the in vivo absorption of the peach sevoflurane U and expands the application of the peach sevoflurane U in the field of biological pharmacy. The invention adopts a biological coupling strategy to realize the design of complex liposome, and utilizes single-wall carbon nano tube and tumor cell specific ligand transferrin to co-modify the liposome. The transferrin is modified on the surface of the U-shaped liposome of the tao er sevoflurane by adopting a post-insertion method, so that the active targeting capability of the drug-loaded liposome can be improved, and the selectivity of the drug to tumor cells can be improved. The drug-loaded liposome is attached to the carbon nano tube through a covalent bond to form a transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound. The novel method combines the high-efficiency cell uptake capacity of the carbon nanotubes and the high drug carrying capacity of the liposome, is convenient for the low-concentration single-wall carbon nanotubes to send high-concentration drugs into target cells, reduces the dosage of the single-wall carbon nanotubes, obviously reduces the related toxicity caused by the single-wall carbon nanotubes, can promote the drugs to enter tumor cells to reach deeper parts of the tumor more efficiently by utilizing the penetrating capacity of the single-wall carbon nanotubes, inhibits the growth of the tumor cells more efficiently, and can expand the application range of the carbon nanotubes and the liposome as drug delivery systems. This will have a beneficial effect on the clinical use of carbon nanotubes and liposomes as drug carriers. The experimental study on the growth inhibition effect of the transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound on DU145 prostatic cancer cells proves that the liposome compound prepared by the invention has stronger killing power on tumor cells. The quantitative uptake test proves that the transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound is easier to be identified and absorbed by DU145 prostatic cancer cells than a free medicament, and has better active targeting. The in vitro release degree experiment proves that the liposome compound has obvious slow release effect. The preparation method is simple, and has strong targeting property, low toxicity and good effect.
Drawings
FIG. 1 is a graph showing particle size distribution of transferrin-mediated single wall carbon nano Guan Tao heptanone U liposome complexes.
FIG. 2 is a transmission electron microscopy image of transferrin-mediated single wall carbon nano Guan Tao heptanone U liposome complex.
FIG. 3 shows the results of an experiment of the inhibitory activity of transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome complex on tumor cells.
FIG. 4 shows the results of an experiment for uptake of tumor cells by transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome complex.
FIG. 5 shows the results of in vitro release experiments of transferrin-mediated single wall carbon nano Guan Tao heptanone U liposome complex.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples.
EXAMPLE 1 transferrin-mediated preparation of Single wall carbon nano Guan Tao heptanone U liposome complexes
(1) Preparation of single-walled carbon nano Guan Tao heptanone U liposome complex:
48mg of lecithin, 16mg of cholesterol and 16mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino (DSPE-PEG 2000-Amine) are placed in a 250mL round bottom flask, and 6.4mL of organic solvent chloroform is added for ultrasonic dissolution (400W) for 3min; dissolving 4.8mg of tao er's heptanone U in 1.6mL of absolute ethyl alcohol, adding the solution into the round-bottomed flask, performing rotary evaporation at 45 ℃ under reduced pressure for 1h, and removing the organic solvent to form a uniform film; then 8mL of PBS solution with pH of 6.5 is added, the mixture is rotationally hydrated for 2 hours at 55 ℃, the probe is subjected to ultrasonic treatment for 4 minutes under ice bath, the power is 300W, the ultrasonic treatment is stopped for 3s, and the suspension of the U liposome of the peach sevoflurane is obtained;
80mg Pluronic F127 is dissolved in 8mL of PBS solution with pH of 6.5, 8mg of carboxylated single-walled carbon nanotubes are added, the probe is subjected to ultrasonic treatment for 4min under ice bath, the power is 260W, and carboxylated single-walled carbon nanotube suspension is obtained; then 8mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is added and mixed for 30min by ultrasound (400W), 8mg of N-hydroxysulfosuccinimide (NHS) is added and mixed for 30min by ultrasound (400W), and the mixture is activated; finally adding the suspension of the U-shaped liposome of the tao-sevoflurane, stirring the mixture at room temperature for 3 hours, centrifuging 10000g for 30 minutes, collecting precipitate, adding 8mL of PBS solution with pH of 6.5 for redispersion, and obtaining the suspension of the U-shaped liposome complex of the single-walled carbon nano Guan Tao-sevoflurane;
(2) Preparation of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin (DSPE-PEG-TF) micelles:
weighing 16mg of DSPE-PEG-TF, dissolving in 4mL of organic solvent chloroform, evaporating under reduced pressure at 45 ℃ in a 150mL round bottom flask to form a film, removing chloroform, placing in a vacuum dryer at 50 ℃ overnight, fully drying, adding 8mL of PBS solution with pH of 6.5, and carrying out rotary hydration at 55 ℃ for 2h to prepare DSPE-PEG-TF micelle;
(3) Preparation of transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome complex:
mixing the 4mL single-walled carbon nano Guan Tao heptaketone U liposome composite suspension with 4mL DSPE-PEG-TF micelle, reacting and incubating for 1h at 37 ℃ under a constant temperature oscillator (150 r/min), and removing free DSPE-PEG-TF by passing the reacted solution through a CL-4B agarose gel column (1 cm multiplied by 20 cm) to obtain the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome composite.
EXAMPLE 2 transferrin-mediated preparation of Single wall carbon nano Guan Tao heptanone U liposome complexes
(1) Preparation of single-walled carbon nano Guan Tao heptanone U liposome complex:
60mg of lecithin, 20mg of cholesterol, 20mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino (DSPE-PEG 2000-Amine) are placed in a 250mL round bottom flask, and 8mL of organic solvent chloroform is added for ultrasonic dissolution (400W) for 3min; 6mg of the tao-sevoflurane U is dissolved in 2mL of absolute ethyl alcohol, added into the round-bottom flask, decompressed and distilled for 1h at 45 ℃, and the organic solvent is removed to form a uniform film; then 10mL PBS solution with pH of 6.5 is added, the mixture is rotationally hydrated for 2 hours at 55 ℃, the probe is subjected to ultrasonic treatment for 4 minutes under ice bath, the power is 300W, the ultrasonic treatment is stopped for 3s, and the suspension of the U liposome of the peach sevoflurane is obtained;
dissolving 100mg Pluronic F127 in 10mL PBS (phosphate buffer solution) with pH of 6.5, adding 10mg carboxylated single-walled carbon nanotubes, and performing ultrasonic treatment for 4min with a probe under ice bath and power of 260W to obtain carboxylated single-walled carbon nanotube suspension; then 10mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is added and mixed for 30min with ultrasound (400W), 10mg of N-hydroxysulfosuccinimide (NHS) is added and mixed for 30min with ultrasound (400W) and activated; finally adding the suspension of the U-shaped liposome of the tao-sevoflurane, stirring the mixture at room temperature for 3 hours, centrifuging 10000g for 30 minutes, collecting precipitate, adding 10mL of PBS (phosphate buffer solution) with pH of 6.5 for redispersion, and obtaining the suspension of the U-shaped liposome complex of the single-walled carbon nano Guan Tao-sevoflurane;
(2) Preparation of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin (DSPE-PEG-TF) micelles:
weighing 20mg of DSPE-PEG-TF, dissolving in 5mL of organic solvent chloroform, evaporating under reduced pressure at 45 ℃ in a 150mL round bottom flask to form a film, removing chloroform, placing in a vacuum dryer at 50 ℃ overnight, fully drying, adding 10mL of PBS solution with pH of 6.5, and carrying out rotary hydration at 55 ℃ for 2h to prepare DSPE-PEG-TF micelle;
(3) Preparation of transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome complex:
mixing the 5mL single-walled carbon nano Guan Tao heptanone U liposome composite suspension with 5mL DSPE-PEG-TF micelle, reacting and incubating for 1h at 37 ℃ under a constant temperature oscillator (150 r/min), and removing free DSPE-PEG-TF by passing the reacted solution through a CL-4B agarose gel column (1 cm multiplied by 20 cm) to obtain the transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome composite.
EXAMPLE 3 transferrin-mediated preparation of Single wall carbon nano Guan Tao heptanone U liposome complexes
(1) Preparation of single-walled carbon nano Guan Tao heptanone U liposome complex:
72mg of lecithin, 24mg of cholesterol and 24mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino (DSPE-PEG 2000-Amine) are placed in a 250mL round bottom flask, and 9.6mL of organic solvent chloroform is added for ultrasonic dissolution (400W) for 3min; 7.2mg of tao er sevoflurane U is dissolved in 2.4mL of absolute ethyl alcohol, and then added into the round-bottom flask, and the mixture is subjected to rotary evaporation at 45 ℃ under reduced pressure for 1h, and organic solvent is removed to form a uniform film; then 12mL PBS solution with pH of 6.5 is added, the mixture is rotationally hydrated for 2 hours at 55 ℃, the probe is subjected to ultrasonic treatment for 4 minutes under ice bath, the power is 300W, the ultrasonic treatment is stopped for 3s, and the suspension of the U liposome of the peach sevoflurane is obtained;
dissolving 120mg Pluronic F127 in 12mL PBS (phosphate buffer solution) with pH of 6.5, adding 12mg carboxylated single-walled carbon nanotubes, and performing ultrasonic treatment for 4min with a probe under ice bath and power of 260W to obtain carboxylated single-walled carbon nanotube suspension; then adding 12mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and mixing for 30min by ultrasonic (400W), adding 12mg of N-hydroxysulfosuccinimide (NHS) and mixing by ultrasonic (400W), and activating for 30min; finally adding the suspension of the U-shaped liposome of the tao-sevoflurane, stirring the mixture at room temperature for 3 hours, centrifuging 10000g for 30 minutes, collecting precipitate, adding 12mL of PBS (phosphate buffer solution) with pH of 6.5 for redispersion, and obtaining the suspension of the U-shaped liposome complex of the single-walled carbon nano Guan Tao-sevoflurane;
(2) Preparation of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin (DSPE-PEG-TF) micelles:
weighing 24mg of DSPE-PEG-TF, dissolving in 6mL of organic solvent chloroform, evaporating under reduced pressure at 45 ℃ in a 150mL round bottom flask to form a film, removing chloroform, placing in a vacuum dryer at 50 ℃ overnight, fully drying, adding 12mL of PBS solution with pH of 6.5, and carrying out rotary hydration at 55 ℃ for 2h to prepare DSPE-PEG-TF micelle;
(3) Preparation of transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome complex:
mixing the 6mL single-walled carbon nano Guan Tao heptanone U liposome composite suspension with 6mL DSPE-PEG-TF micelle, reacting and incubating for 1h at 37 ℃ under a constant temperature oscillator (150 r/min), and removing free DSPE-PEG-TF by passing the reacted solution through a CL-4B agarose gel column (1 cm multiplied by 20 cm) to obtain the transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome composite.
EXAMPLE 4 transferrin-mediated preparation of Single wall carbon nano Guan Tao heptanone U liposome complexes
(1) Preparation of single-walled carbon nano Guan Tao heptanone U liposome complex:
54mg of lecithin, 18mg of cholesterol and 18mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino (DSPE-PEG 2000-Amine) are placed in a 250mL round bottom flask, 7.2mL of organic solvent chloroform is added for ultrasonic dissolution (400W) for 3min; after 5.4mg of tao-sevoflurane U was dissolved in 1.8mL of absolute ethanol, the mixture was added to the above round-bottomed flask, and the mixture was distilled under reduced pressure at 45℃for 1 hour to remove the organic solvent, thereby obtaining a uniform film; then 9mL PBS solution with pH of 6.5 is added, the mixture is rotationally hydrated for 2 hours at 55 ℃, the probe is subjected to ultrasonic treatment for 4 minutes under ice bath, the power is 300W, the ultrasonic treatment is stopped for 3s, and the suspension of the U liposome of the peach sevoflurane is obtained;
dissolving 90mg Pluronic F127 in 9mL PBS (phosphate buffer solution) with pH of 6.5, adding 9mg carboxylated single-walled carbon nanotubes, and performing ultrasonic treatment for 4min with a probe under ice bath and power of 260W to obtain carboxylated single-walled carbon nanotube suspension; then 9mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is added and mixed for 30min by ultrasound (400W), 9mg of N-hydroxysulfosuccinimide (NHS) is added and mixed for 30min by ultrasound (400W), and the mixture is activated; finally adding the suspension of the U-shaped liposome of the tao-sevoflurane, stirring the mixture at room temperature for 3 hours, centrifuging 10000g for 30 minutes, collecting precipitate, adding 9mL of PBS solution with pH of 6.5 for redispersion, and obtaining the suspension of the U-shaped liposome complex of the single-walled carbon nano Guan Tao-sevoflurane;
(2) Preparation of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin (DSPE-PEG-TF) micelles:
18mg of DSPE-PEG-TF is weighed and dissolved in 4.5mL of organic solvent chloroform, the chloroform is removed by reduced pressure evaporation at 45 ℃ in a 150mL round bottom flask, the mixture is placed in a vacuum drier at 50 ℃ overnight, 9mL of PBS solution with pH of 6.5 is added for full drying, and the mixture is subjected to rotary hydration at 55 ℃ for 2 hours to prepare DSPE-PEG-TF micelle;
(3) Preparation of transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome complex:
mixing the 4.5mL single-walled carbon nano Guan Tao sevoflurane U liposome composite suspension with 4.5mL DSPE-PEG-TF micelle, reacting and incubating for 1h at 37 ℃ under a constant temperature oscillator (150 r/min), and removing free DSPE-PEG-TF from the reacted solution through a CL-4B agarose gel column (1 cm multiplied by 20 cm) to obtain the transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome composite.
Comparative experiments
According to the preparation method of the example 1 (the mass ratio of lecithin to the tao er's heptanone U is 20:1), different proportions of lecithin and single-wall carbon nanotubes are selected to prepare a single-wall carbon nano Guan Tao er's heptanone U liposome compound, and the encapsulation efficiency and the drug loading rate are measured. The mass ratio of lecithin to single-walled carbon nanotubes is 2:1, 4:1 and 6:1 respectively. The results are shown in Table 1.
TABLE 1
The preparation method of example 1 (mass ratio of lecithin to single-walled carbon nanotubes is 6:1), the single-wall carbon nano Guan Tao sevoflurane U liposome compound is prepared by selecting lecithin and sevoflurane U in different proportions, and the encapsulation efficiency and the drug loading rate are measured. The mass ratio of lecithin to tao er's heptanone U is 20:1, 15:1 and 10:1 respectively. The results are shown in Table 2.
TABLE 2
As can be seen from tables 1 and 2, the encapsulation efficiency and the drug loading rate of the single-walled carbon nano Guan Tao heptaketone U liposome complex are in inverse proportion to the addition amount of the single-walled carbon nano tubes within a certain range, and when the ratio of lecithin to the single-walled carbon nano tubes is 6:1 (w/w), the encapsulation efficiency and the drug loading rate are ideal, namely 85.52% and 2.81%, respectively. Fixing the ratio, when the mass ratio of the lecithin to the peach sevoflurane U is 10:1, 15:1, 20:1 and 3 respectively, the encapsulation rate of the single-walled carbon nano Guan Tao-child sevoflurane U liposome compound obtained by different ratios is almost the same, and the encapsulation rate is about 82% -85%; and when the mass ratio of the lecithin to the tao-sevoflurane U is 10:1, the better drug loading rate is 5.11%, and the mass ratio of the lecithin to the tao-sevoflurane U is increased by nearly 1.8 times when compared with the mass ratio of the lecithin to the tao-sevoflurane U which is 15:1 and 20:1. Therefore, the preparation process of the single-walled carbon nano Guan Tao heptaketone U liposome compound is determined to be that the mass ratio of lecithin to the single-walled carbon nano tube is 6:1, and the encapsulation efficiency and the drug loading rate are both optimal when the mass ratio of the lecithin to the heptaketone U is 10:1.
Verification experiment:
1. measurement of particle size:
the particle size and the potential of the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome compound prepared in example 1 are measured by using a laser particle size analyzer, the detection result is shown in figure 1, the particle size of the liposome compound is uniformly distributed, the particle size is 280nm, and the polydispersity index (PDI) is 0.246.
2. Electron microscope morphological investigation
Taking a proper amount of transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound suspension prepared in the embodiment 2, diluting, dripping on a copper screen covered with a support film, dyeing for 4min by using a 2% phosphotungstic acid solution by mass fraction, naturally drying, and observing the morphology by using a transmission electron microscope. As shown in FIG. 2, the liposome was spheroid, and most of the single-walled carbon nanotubes were intercalated into the spheroid liposome and formed into a sugarcoated haws.
3. Determination of the inhibitory Activity of the Liposome Complex of the present invention on tumor cell growth
The transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound prepared in the example 3 is added into tumor cells for culture, and the survival rate of the tumor cells is measured.
DU145 prostate cancer cells (supplied from Shanghai cell Bank) were inoculated into 1640 medium (containing 10% fetal bovine serum, 100. Mu.g.mL) -1 Streptomycin, 100 U.mL -1 Penicillin), at 37 ℃, 5% CO 2 Culturing under saturated humidity condition, and passaging every other day. DU145 cells in the logarithmic growth phase were collected and prepared as single cell suspensions and seeded in 96-well plates at a cell density of 5000 cells/well. After 24h incubation, 200 μl of the example 3 preparation samples (transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome complex) or corresponding concentrations of drug (tao heptaketone U) solution with different concentration gradients (5, 10, 20, 30, 40, 50 μΜ) were added to 96-well plates (4 complex wells) and incubated for 24 and 48h, respectively. Before the termination of the culture for 4 hours, the 96-well plate was removed, and 20. Mu.L of MTT solution (5 mg. Multidot.mL) was added to each well -1 ) On a flat-plate oscillator, the speed of the flat-plate oscillator is 100 r.min -1 Gently shake for 3min to mix the MTT solution thoroughly. After termination of the culture, 2500 r.min using a plate centrifuge -1 The crystals of formazan were separated from the culture broth by centrifugation for 10min, and the culture broth was carefully aspirated along the wall using a pipette. 150 μl DMSO was added per void, shaken for 2min, and each well was examined at 490nm using an microplate readerOD, calculated cytostatic ratio, SPSS 17.0 calculated median lethal concentration (IC 50 ). Cell inhibition (%) = (OD control group-OD drug group)/(OD control group-OD blank) ×100%.
As shown in the experimental result in figure 3, the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome compound has an inhibition effect on DU145 cell proliferation, and the antiproliferation capability of a preparation group on prostate tumor cells is superior to that of a drug group along with the increase of the administration concentration, so that the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome compound has a stronger killing effect on the prostate tumor cells, can be effectively used for treating the prostate cancer, and has a stronger practical value.
4. Experiment of uptake of the Liposome Complex of the present invention into tumor cells
The transferrin-mediated single-wall carbon nano Guan Tao heptanone U liposome compound prepared in the example 4 is added into tumor cells for culture, and the drug intake in the cells is measured.
DU145 prostate cancer cells (supplied from Shanghai cell Bank) were inoculated into 1640 medium (containing 10% fetal bovine serum, 100. Mu.g.mL) -1 Streptomycin, 100 U.mL -1 Penicillin), at 37 ℃, 5% CO 2 Culturing under saturated humidity condition, and passaging every other day. DU145 cells in the logarithmic growth phase were collected and prepared into single cell suspensions, which were inoculated into 12-well plates for cultivation for 24 hours. When the cells were cultured until the cell fusion reached 80%, the medium in each well was discarded, and washed 3 times with 1mL of PBS solution. The final concentration of the sevoflurane U in each group is 10, 20, 30 and 40 mu M respectively by adding the sevoflurane U prepared by 1640 culture medium and the single-walled carbon nano Guan Tao sevoflurane U liposome compound mediated by transferrin. Incubating for 1h in a cell culture box, discarding the culture medium, adding a cell lysate to fully lyse cells, collecting cell suspension for treatment, and performing HPLC (high performance liquid chromatography) sample injection to determine the content of the drug. As shown in the experimental result in FIG. 4, DU145 cells have significantly lower uptake of the tao-heptanone U than the transferrin-mediated single-wall carbon nano Guan Tao-heptanone U liposome complex, which proves that the transferrin-mediated single-wall carbon nano Guan Tao-heptanone U liposome complex has stronger cell targeting uptake capacity and resistanceThe tumor effect is better.
5. In vitro Release test of Liposome Complex according to the present invention
2mL of the transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome compound suspension prepared in the example 1 is filled into a dialysis bag, the dialysis bag is put into 50mL of PBS solution (containing 1% Tween) as a release medium for release degree investigation, the drug content in the release medium is measured by a spectrophotometry at different time points, and an in-vitro accumulated release curve is drawn. The result is shown in fig. 5, and it can be seen from the graph that the transferrin-mediated single-wall carbon nano Guan Tao heptaketone U liposome compound prepared by the invention has obvious slow release effect.
Claims (9)
1. A transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome complex, which is characterized in that the complex is mainly a carbon nano tube liposome complex formed by peach heptanone U, single-walled carbon nano tubes, lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino group, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin; the preparation method comprises the steps of firstly preparing Cheng Tao sevoflurane U liposome from the sevoflurane U, and then mixing the Cheng Tao sevoflurane U liposome with carboxylated single-walled carbon nanotubes to prepare a single-walled carbon nano Guan Tao sevoflurane U liposome compound; and then mixing the single-walled carbon nano Guan Tao heptaketone U liposome complex with distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle to prepare the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome complex.
2. The transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome complex according to claim 1, wherein the liposome is a spherical liposome in which single-walled carbon nanotubes are inserted, and has a sugar gourd shape and a particle size of 250-280 nm.
3. A method for preparing a transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome complex according to claim 1, wherein the method comprises the steps of preparing Cheng Tao heptanone U liposome from heptanone U, and then mixing with carboxylated single-walled carbon nanotubes to prepare single-walled carbon nano Guan Tao heptanone U liposome complex; and then mixing the single-walled carbon nano Guan Tao heptaketone U liposome complex with distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle to prepare the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome complex.
4. A method of preparation according to claim 3, comprising the steps of:
(1) Preparation of single-walled carbon nano Guan Tao heptanone U liposome complex:
mixing 48-72 mg of lecithin, 16-24 mg of cholesterol and 16-24 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino, and adding an organic solvent chloroform for ultrasonic dissolution; dissolving 4.8-7.2 mg of tao-sevoflurane U in absolute ethyl alcohol, adding the absolute ethyl alcohol into the mixture, and performing reduced pressure rotary evaporation to form a film; then adding PBS solution for rotary hydration, and performing ultrasonic treatment by a probe under ice bath to obtain a suspension of the U liposome of the peach sevoflurane;
dissolving 80~120mg Pluronic F127 in PBS solution, adding 8-12 mg of carboxylated single-walled carbon nanotube under-ice-bath probe for ultrasonic treatment to obtain carboxylated single-walled carbon nanotube suspension; then 8-12 mg of EDC and 8-12 mg of NHS are sequentially added for ultrasonic mixing and activation; finally adding the suspension of the U-shaped liposome of the peach sevoflurane, stirring, centrifuging, collecting precipitate, adding PBS solution for redispersion to obtain suspension of the U-shaped liposome complex of the peach sevoflurane Guan Tao-nanometer single-wall carbon;
(2) Preparation of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle:
weighing 16-24 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin, dissolving in chloroform serving as an organic solvent, evaporating under reduced pressure to form a film, removing the organic solvent, drying, adding PBS (phosphate buffer solution) for rotary hydration, and preparing distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle;
(3) Preparation of transferrin-mediated single-walled carbon nano Guan Tao sevoflurane U liposome complex:
mixing the single-walled carbon nano Guan Tao heptaketone U liposome composite suspension with distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin micelle, reacting and incubating, and removing free distearoyl phosphatidylethanolamine-polyethylene glycol 2000-transferrin after the reaction to obtain the transferrin-mediated single-walled carbon nano Guan Tao heptaketone U liposome composite.
5. The method according to claim 4, wherein the temperature of the reduced pressure rotary evaporation in the step (1) is 45 ℃ for 1 hour; the temperature of the rotary hydration is 55 ℃ and the time is 2 hours; the ultrasonic power of the probe is 260-300 w, and the time is 3-5 min; the activation time is 30min; stirring at room temperature for 3h; the centrifugation speed is 10000g and the time is 30min; the pH of the PBS solution was 6.5.
6. The process according to claim 4, wherein the reduced pressure evaporation in the step (2) is carried out at a temperature of 45℃and a drying temperature of 50℃and a spin-hydration temperature of 55℃for 2 hours.
7. The method according to claim 4, wherein the reaction is performed at 37℃for 1 hour in step (3).
8. Use of a transferrin-mediated single-walled carbon nano Guan Tao heptanone U liposome complex according to claim 1 in the preparation of a medicament for treating a tumor.
9. The use according to claim 8, wherein the tumor is a prostate tumor.
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