CN112891557A - ICG-beta-cyclodextrin drug delivery system and preparation method and application thereof - Google Patents

ICG-beta-cyclodextrin drug delivery system and preparation method and application thereof Download PDF

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CN112891557A
CN112891557A CN202110147566.7A CN202110147566A CN112891557A CN 112891557 A CN112891557 A CN 112891557A CN 202110147566 A CN202110147566 A CN 202110147566A CN 112891557 A CN112891557 A CN 112891557A
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icg
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cyclodextrin
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柯阳
王承潇
贺永胜
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Abstract

The characteristic of ICG on the specific marker of the liver cancer is utilized to serve as a targeting guide substance for liver cancer treatment, cyclodextrin is used as a carrier, insoluble anticancer drugs are included by utilizing the cavity effect of the cyclodextrin, and the ICG-cyclodextrin supermolecule system-based liver cancer targeting drug carrying system is constructed by connecting the cyclodextrin and the carrier through a covalent bond (ICG-CD). The treatment effect can be enhanced by preparing drug-loaded complexes respectively encapsulating different drugs and simultaneously administering the two complexes for combined treatment.

Description

ICG-beta-cyclodextrin drug delivery system and preparation method and application thereof
Technical Field
The invention belongs to the technical field of pharmaceutical compositions, and particularly relates to an ICG-beta-cyclodextrin drug delivery system, and a preparation method and application thereof.
Background
Liver cancer has 6 th and 4 th morbidity and mortality of all tumors in the global range, is more serious in the epidemic situation of China, has 4 th morbidity and 2 nd mortality, and causes huge burden to the society. The treatment method and curative effect of liver cancer are limited, which are important reasons for restricting the improvement of liver cancer prognosis. Currently, liver cancer targeting drugs include Sorafenib (Sorafenib), Regorafenib (Regorafenib), ranvatinib (Lenvatinib), and Cabozantinib (Cabozantinib). Their main mechanisms of action are: liver cancer often has abnormal expression and activation of multiple tyrosine kinase pathways compared with surrounding normal liver tissues, and the medicaments inhibit the tyrosine kinase pathways, so that the liver cancer is inhibited, and the surrounding normal liver tissues are not inhibited or slightly inhibited. However, liver cancer often has target drug resistance, resulting in unsatisfactory curative effect, and a new drug for treating liver cancer is urgently needed to be developed, so as to improve the survival rate of liver cancer patients.
Disclosure of Invention
The invention provides an ICG-beta-cyclodextrin drug delivery system and a preparation method and application thereof, and the specific technical scheme is as follows:
an ICG-beta-cyclodextrin drug delivery system comprises ICG-beta-cyclodextrin, and the structure of the ICG-beta-cyclodextrin drug delivery system is shown as the formula I:
Figure DEST_PATH_IMAGE002
an ICG-beta-cyclodextrin drug delivery system is prepared by the following method:
(1) preparation of carboxy-modified ICG: adding 1, 1, 2-trimethylbenzindole and ethyl iodide into acetonitrile, heating, refluxing, concentrating in vacuum to obtain a residue, adding diethyl ether into the residue to obtain a solid, washing the solid to obtain a compound 1, heating the compound 1 and glutaraldehyde dinitrile hydrochloride in acetic anhydride, cooling, and washing a product to obtain a compound 2;
adding 1, 1, 2-trimethylbenzindole and 6-iodohexanoic acid into acetonitrile, heating and refluxing, concentrating in vacuum to obtain a residue, adding diethyl ether into the residue to obtain a solid, and washing the solid to obtain a compound 3;
reacting the compound 2 with the compound 3 in pyridine, removing the solvent, purifying the residue by silica gel chromatography, and eluting to obtain a compound 4, wherein the compound 4 is a carboxyl modified ICG carrier and is marked as ICG-COOH;
(2) preparation of amino-modified cyclodextrins: firstly, preparing mono- (6-oxo-6-p-toluenesulfonyl) -beta-cyclodextrin, and then preparing ethylenediamine-beta-cyclodextrin, which is marked as 2N-beta-CD;
(3) preparing a supramolecular drug carrier: dissolving ICG-COOH in dimethyl sulfoxide, adding 3 times of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-hydroxysuccinimide in molar weight, adding 2N-beta-CD dissolved in dimethyl sulfoxide into the reaction solution, dialyzing, and freeze-drying the obtained sample to obtain the ICG-beta-cyclodextrin drug-loading system.
The invention also provides a pharmaceutical composition, and the application of the ICG-beta-cyclodextrin as a pharmaceutical active ingredient in preparing targeted liver cancer treatment drugs.
The invention also provides a pharmaceutical composition, and the targeted liver cancer treatment drug also comprises sorafenib and oxaliplatin.
Indocyanine Green (ICG) is approved by the FDA in the united states for use as a clinical near-infrared photocoagulant, and ICG is widely used clinically for assisted diagnosis of liver function, cardiac output, and retinal vasculature. Excitation wavelength: 760nm, emission wavelength: 830nm, which is a good photosensitizer, can absorb light energy and convert the light energy into heat energy or generate singlet oxygen to play a role in killing tumors under the irradiation of near infrared light.
ICG is a water soluble molecule with a molecular weight of 775, has double lipophilic and hydrophilic properties, is combined with serum protein (albumin and beta-lipoprotein) in blood, is taken by liver, is secreted into bile in a free form, is discharged out of body through intestine and excrement, does not participate in liver and intestine circulation and biochemical transformation, is not excreted from kidney, and has no toxic or side effect. ICG is applied to clinical liver cancer fluorescence imaging in recent years, and can accurately mark the position of liver cancer under a fluorescence laparoscope, and the specific mechanism is as follows: after intravenous injection, ICG is combined with serum protein, ICG entering liver blood sinus is selectively taken up by liver cell through endothelial cell, then enters bile in free form, and is combined with protein in bile, and when excited by light wave with wavelength of 750-810nm, infrared light with wavelength of about 830nm can be released, so that the fluorescent material has fluorescent characteristic. In normal liver tissues, indocyanine green can be completely excreted into biliary tracts in only a few hours, then enters cystic ducts and common bile ducts through various levels of bile ducts in the liver and finally enters intestinal tracts, the whole process has no intermediate metabolism and does not undergo enterohepatic circulation, and fluorescence on the surface of the liver and the biliary tracts disappears immediately.
However, when the liver is cancerated to cause damage to liver cells and capillary bile ducts, and the secretion and excretion functions of the liver cells and the capillary bile duct cells in the damaged liver tissue are disturbed, the ICG is retained in a pathological tissue, so that the ICG is accumulated in a liver cancer tissue, can be retained in the liver cancer for 1-2 weeks, and the fluorescence is delayed to disappear, so that the pathological tissue of the liver cancer is specifically marked, the pathological tissue of the liver cancer is caused to form strong fluorescence contrast with a normal tissue, and the position and the size of a liver lesion are displayed in real time, so that the liver cancer surgical system has a high clinical application value.
Cyclodextrins (CDs) are a class of cyclic oligosaccharides with structures as shown in FIG. 1, wherein β -CD and its derivatives are the most commonly used ones, and the structures are similar to a truncated cone with wide mouth and narrow bottom, hollow inside, 0.79 nm deep, 0.60-0.65nm in diameter, so that the formed cavity can be just filled with other molecules. The hydroxyl groups of the glucose units of the cyclodextrin face outward at the pores at both ends, while the methine protons are located in the cavities, whose structure is such that the cyclodextrin has a hydrophilic exterior and a hydrophobic cavity. It can form water-soluble inclusion complexes with small molecules and most compounds. In addition, the cyclodextrin has no obvious conformational change in the inclusion process, so that the host-guest inclusion complex of the cyclodextrin can exist in a solid state and can also be formed in water and certain organic solvents. This property is that cyclodextrins can be used to study the very important water-increasing effects in organisms. Thus, various size-matched guests can be encapsulated into the cavity by host-guest interactions under aqueous conditions.
The excellent properties of cyclodextrins, which exhibit good water solubility as well as biocompatibility and non-toxicity to biological systems, further facilitate extensive research into the use of cyclodextrins in the biomedical field.
Cyclodextrin is the macrocyclic compound with the critical performance in the earliest research, and the most important property is inclusion of organic matters, and the combination is based on non-covalent bond interaction. The following are generally concerned: (1) van der waals forces between the cyclodextrin and the guest; (2) the hydroxyl groups of the cyclodextrin associate with hydrogen bonds between guest molecules: (3) release of high energy water molecules and release of tensile energy (conformational strain energy) in the cyclodextrin cavity; (4) the water increasing effect of cyclodextrin molecule cavity; (5) the guest molecule must match the geometry (size and spatial configuration) of the cyclodextrin cavity.
A large number of researches show that the combined therapy can kill tumor cells through multiple mechanisms compared with single-drug therapy, and reduce the risk of drug resistance generation of tumors while enhancing the treatment effect. It is noteworthy, however, that the efficacy of the combination therapy was much lower than expected in many phase III experiments. This is mainly because in combination therapy of multiple drugs, the different drugs exert the maximum therapeutic effect only at a specific ratio. However, in the administration process of the traditional combination therapy, due to the pharmacokinetic and tissue distribution difference among different drugs, the drugs can not be ensured to reach proper proportion and concentration at the focus part after entering the human body, and the treatment effect is greatly limited. Therefore, the research on a drug delivery system, which accurately delivers a plurality of drugs into tumors in a specific ratio to play a synergistic effect, is the key to enhance the effect of the combination of the drugs on HCC.
The characteristic of ICG on the specific marker of the liver cancer is utilized in the invention to be used as a target guiding substance for treating the liver cancer, the cyclodextrin is used as a carrier, the cavity function of the cyclodextrin is utilized to include the insoluble anticancer drug, and the cyclodextrin and the carrier are connected by covalent bonds (ICG-CD) to construct a liver cancer target drug-carrying system based on an ICG-cyclodextrin supermolecule system. The treatment effect can be enhanced by preparing drug-loaded complexes respectively encapsulating different drugs and simultaneously administering the two complexes for combined treatment.
Drawings
FIG. 1. structure of beta-cyclodextrin;
FIG. 2 is a high performance liquid chromatography of OS-ICG-beta-CD: a: an ICG- β -CD vector; b: oxaliplatin; c: sorafenib;
FIG. 3 is a standard curve of OXA;
FIG. 4 is a standard curve of SOR;
FIG. 5 in vitro release of OXA and SOR in different formulations;
FIG. 6. Small animal Living body imaging System detects the distribution of OS-ICG-beta-CD in vivo;
FIG. 7. OS-ICG- β -CD targeting anti-tumor effect;
FIG. 8. effect of OS-ICG- β -CD on cell activity;
FIG. 9 graph of drug uptake by cells.
Detailed Description
Example (b):
in the study, sorafenib, oxaliplatin and ICG- β -CD were weighed out accurately in a certain ratio, and all of them were dissolved in a mixed solvent of ethanol/water (volume ratio =1:1:5), and stirred at room temperature in the dark. After most of ethanol in the reaction solution is removed by rotary evaporation, the reaction solution is filtered by a microporous membrane to remove unreacted drugs. And (3) spin-drying the filtrate, and then putting the filtrate into a vacuum drying oven for 12 hours to obtain an ICG-cyclodextrin supermolecular system carrying indissolvable targeting liver cancer treatment drug clathrate: oxaliplatin-sorafenib ICG- β -cyclodextrin. Meanwhile, in vivo pharmacokinetic experiments, targeted anti-liver cancer activity and cell in vitro experiments are carried out to verify the treatment effect.
1. Preparation method of ICG-beta-cyclodextrin
Preparation of carboxy-modified ICG: adding 1, 1, 2-trimethylbenzindole and ethyl iodide into acetonitrile, heating, refluxing, concentrating in vacuum to obtain a residue, adding diethyl ether into the residue to obtain a solid, washing the solid to obtain a compound 1, heating the compound 1 and glutaraldehyde dinitrile hydrochloride in acetic anhydride, cooling, and washing a product to obtain a compound 2;
adding 1, 1, 2-trimethylbenzindole and 6-iodohexanoic acid into acetonitrile, heating and refluxing, concentrating in vacuum to obtain a residue, adding diethyl ether into the residue to obtain a solid, and washing the solid to obtain a compound 3;
reacting the compound 2 with the compound 3 in pyridine, removing the solvent, purifying the residue by silica gel chromatography, and eluting to obtain a compound 4, wherein the compound 4 is a carboxyl modified ICG carrier and is marked as ICG-COOH;
preparation of amino-modified cyclodextrins: firstly, preparing mono- (6-oxo-6-p-toluenesulfonyl) -beta-cyclodextrin, and then preparing ethylenediamine-beta-cyclodextrin, which is marked as 2N-beta-CD;
preparing a supramolecular drug carrier: dissolving ICG-COOH in dimethyl sulfoxide, adding 3 times of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-hydroxysuccinimide in molar weight, adding 2N-beta-CD dissolved in dimethyl sulfoxide into the reaction solution, dialyzing, and freeze-drying the obtained sample to obtain the supramolecular drug carrier.
2. ICG-beta-cyclodextrin
After preparation, the structural formula of the compound is shown as a formula I, and the structural formula is expressed by ICG-beta-CD in the following.
Figure DEST_PATH_IMAGE004
3. Preparation and characterization of liver-targeting oxaliplatin-sorafenib ICG-beta-cyclodextrin drug delivery system
3.1 the preparation method comprises the following steps:
OXA-ICG-beta-CD: oxaliplatin and ICG- β -CD were accurately weighed in a certain ratio, and all of them were dissolved in a mixed solvent of ethanol and water (volume ratio =1:5), and stirred at room temperature in the dark. After most of ethanol in the reaction solution is removed by rotary evaporation, the reaction solution is filtered by a microporous membrane to remove the unreacted oxaliplatin. And (4) spin-drying the filtrate, and then putting the filtrate into a vacuum drying oven for 12 hours to obtain the OXA-ICG-beta-CD clathrate compound.
SOR-ICG-beta-CD: sorafenib and ICG- β -CD were accurately weighed in a certain ratio, and all of them were dissolved in a mixed solvent of ethanol and water (volume ratio =1:5), and stirred at room temperature in the dark. After most of ethanol in the reaction solution is removed by rotary evaporation, the reaction solution is filtered by a microporous filter membrane to remove unreacted sorafenib. And spin-drying the filtrate, and then putting the filtrate into a vacuum drying oven for 12 hours to obtain the SOR-ICG-beta-CD clathrate compound.
OS-ICG- β -CD: sorafenib and ICG- β -CD were accurately weighed in a certain ratio, and all of them were dissolved in a mixed solvent of ethanol and water (volume ratio =1:5), and stirred at room temperature in the dark. After most of ethanol in the reaction solution is removed by rotary evaporation, the reaction solution is filtered by a microporous filter membrane to remove unreacted sorafenib. And spin-drying the filtrate, and then putting the filtrate into a vacuum drying oven for 12 hours to obtain the SOR-ICG-beta-CD clathrate compound. The SOR-ICG-beta-CD clathrate compound and oxaliplatin are dissolved in a mixed solvent of ethanol/water (volume ratio =1:5) according to a certain ratio, and stirred at room temperature in a dark place. After most of ethanol in the reaction solution is removed by rotary evaporation, the reaction solution is filtered by a microporous membrane to remove the unreacted oxaliplatin. And (4) spin-drying the filtrate, and then putting the filtrate into a vacuum drying oven for 12 hours to obtain the OS-ICG-beta-CD clathrate compound.
Free OXA-SOR: namely a mixed solution of oxaliplatin and sorafenib.
3.2 establishment of method for determining contents of oxaliplatin and Sorafenib in OS-ICG-beta-CD
3.2.1 inspection of specificity of chromatographic conditions
Chromatographic conditions
A chromatographic column: a C18 column; mobile phase: methanol-water (10: 90, v/v); flow rate: 1.0 mL/min; detection wavelength: 250 nm; sample introduction amount: 20 μ L, column temperature: at 25 ℃. The results are shown in FIG. 2.
3.2.2 preparation of Standard Curve:
establishment of oxaliplatin standard curve
Preparing an oxaliplatin standard solution: accurately weighing 1 mg of oxaliplatin, placing the oxaliplatin into a 25 mL volumetric flask, adding a mobile phase for dissolving, diluting to a scale mark, and shaking up to obtain the oxaliplatin standard solution with the concentration of 40 mu g/mL.
Oxaliplatin solutions were then prepared in different concentration gradients. Precisely measuring the oxaliplatin standard solution by 3.5 mL, 3.0 mL, 2.5 mL, 2.0 mL, 1.5 mL, 1.0 mL and 0.5 mL respectively in a 5 mL volumetric flask, adding the mobile phase for dissolving and diluting to a scale mark, and shaking uniformly to obtain oxaliplatin solutions with the concentrations of 28 mug/mL, 24 mug/mL, 20 mug/mL, 16 mug/mL, 12 mug/mL, 8 mug/mL and 4 mug/mL respectively. HPLC detection, the peak area (A) was recorded and a standard curve with peak area (A) on the ordinate and oxaliplatin concentration (C) on the abscissa was plotted (FIG. 3).
Table 1 HPLC determination of peak area for oxaliplatin solutions at different concentrations (n = 3)
Figure DEST_PATH_IMAGE006
Establishment of Sorafenib standard curve
Accurately weighing 4.0 mg of sorafenib, placing the sorafenib in a 100 mL volumetric flask, adding absolute methanol to dilute the sorafenib to a scale mark, shaking up, and preparing the sorafenib standard solution with the concentration of 40 mug/mL.
Then preparing sorafenib solutions with different concentration gradients: diluting with mobile phase to obtain standard application solution (temporarily diluting) with concentration of 0.05. mu.g/mL, 0.10. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL, 1.0. mu.g/mL, 2.5. mu.g/mL, 5.0. mu.g/mL and 10. mu.g/mL. HPLC detection, the peak area (A) was recorded and a standard curve with peak area (A) on the ordinate and sorafenib concentration (C) on the abscissa was plotted (FIG. 4).
Table 2 HPLC determination of peak area for oxaliplatin solutions at different concentrations (n = 3)
Figure DEST_PATH_IMAGE008
3.4 encapsulation efficiency determination:
the encapsulation efficiency of OS-ICG-beta-CD is determined by removing unencapsulated free drug by centrifugation-dialysis, which is carried out as follows: taking a proper amount of OS-ICG-beta-CD solution, centrifuging for 5 min at the rotating speed of 5000 rpm, and filtering the obtained supernatant by using a filter membrane of 0.22 mu m so as to remove the non-coated SOR. The supernatant was then placed in a dialysis bag (molecular weight cut-off 8000- + 14000 Da) and dialyzed overnight against PBS buffer (0.01M, pH 7.4) to remove free OXA. The encapsulated OXA and SOR contents of the purified OS-ICG- β -CD were determined by HPLC using the method described in the standard curve plotting the purified OS-ICG- β -CD. The encapsulation efficiency (DEE) and the drug loading capacity (DLE) of the drug are respectively calculated according to the following formulas:
DEE (%) = (encapsulated drug/amount of drug administered in drug platform) × 100%
DLE (%) = (encapsulated drug in drug-loaded platform/(encapsulated drug in drug-loaded platform + drug-loaded platform)) × 100%
TABLE 3 encapsulation efficiency and molar ratio of OXA to SOR in OS-ICG- β -CD
Figure DEST_PATH_IMAGE010
3.5 OS-ICG-. beta. -CD in vitro Release assay:
the in vitro release rate of OXA and SOR in OS-ICG-beta-CD is determined by dialysis, which comprises the following steps: first, 2mL of the sample solution was added to a dialysis bag (molecular weight cut-off 8000- "14000 Da") and then the bag was placed in 20 mL of PBS solution (0.01M, pH 7.4) containing 0.05% Tween 80 (v/v) and shaken at 100 rpm at 37 ℃. Taking 1 mL of release medium outside the dialysis bag at 0.5h, 1h, 2h, 4h, 6 h, 8h, 12h, 24 h and 48 h, and simultaneously supplementing blank release medium with the same temperature and volume outside the dialysis bag. The sample solution was filtered through a 0.45 μm filter and the OXA and SOR contents were determined by HPLC as plotted against the OXA and SOR standard curves. All measurements were done in triplicate (fig. 5).
4. In vivo evaluation of OS-ICG-beta-CD
ICG-beta-cyclodextrin utilizes the targeting effect of ICG, and compared with the conventional administration mode, the ICG-beta-cyclodextrin has toxic and side effects on normal liver tissue cells, and has liquid medicine concentration and toxic and side effect data on normal organs in a liquid medicine path.
4.1 pharmacokinetics
ICG-beta-cyclodextrin is used as a drug loading platform to load sorafenib serving as a drug for targeted therapy of liver cancer, and the sorafenib is injected into a mouse body in a tail vein administration mode (sorafenib drugs are used for administration by an intragastric administration method), so that the toxic and side effects of the ICG-beta-cyclodextrin on normal liver tissue cells, and the concentration and the toxic and side effects of the ICG-beta-cyclodextrin on normal organs in a liquid medicine path are verified. And (3) adopting small animal in-vivo imaging to investigate the tissue distribution of the ICG-beta-CD drug-carrying system in the animal body.
4 weeks old ICR mice, weight 18-22g, male and female half, each group of 10. After administration for 0.5h, 1h, 2h, 4h and 8h, respectively, mice were anesthetized with isoflurane and scanned with an IVIS Spectrum near-infrared fluorescence imager, setting the excitation wavelength at 760nm and the emission wavelength at 830 nm. The relevant images were analyzed using Living Image software to detect the fluorescence signal of the liver (fig. 6).
4.2 ICG-beta-cyclodextrin medicine carrying system with high curative effect.
ICG-beta-cyclodextrin is used as a drug loading platform, and the concentration, duration and concentration change rule of the drug solution in the liver cancer cells, and whether the drug can be administered in deep liver cancer cells or not, namely the drug administration depth is improved.
Grouping experiments:
(1) tumor in situ
(2) In situ transplantation tumor + oxaliplatin
(3) In situ transplanted tumor + Sorafenib
(4) Orthotopic transplantation tumor + OS-ICG-beta-CD
Subcutaneous tumorigenesis of nude mice: in vitro culture of human hepatoma cell Hep3B, collecting cells in logarithmic phase to prepare suspension, inoculating to nude mice subcutaneously on back, injecting 0.2ml of cell suspension containing 2 × 10 cells into each nude mouse6Continuing conventional breeding until the tumor diameter reaches 1.0cm, anesthetizing nude mouse, taking out tumor under aseptic condition, and shearing tumor tissue into 1mm with ophthalmic scissors3And (5) placing the small tumor mass in a serum-free DMEM medium for later use.
Establishment of in situ transplanted tumor: under the aseptic condition, a nude mouse is anesthetized, the liver is exposed through the median transverse incision of the abdomen, 1 tunnel with the depth of 1-2 mm is made by a 10ml syringe needle, tumor tissue fragments are transplanted into the tunnel, and the whole abdomen is closed after the hemostasis is pressed by gelatin sponge. Sequentially sewing the subcutaneous layer and the cortical layer of the mouse, unbinding the mouse, moving to a mouse cage for placing padding, enabling the mouse cage to lie on the side, and covering the cotton.
The next day, mice were randomly grouped and given food, water. The day following inoculation, dosing was started. Mice were randomized into four groups of 6 mice each. During the administration period, the naked mouse was observed about diet, drinking water, body weight, coat gloss, and loss.
The administration concentration and the administration mode are as follows: ICG-. beta. -CD and the prepared complex were formulated to a concentration of 30. mu.g/mL, and 0.2mL was injected into mice via tail vein injection, and the size of mouse graft tumor was examined to observe the therapeutic effect (FIG. 7).
5. In vitro evaluation of OS-ICG-beta-CD
The part is mainly used for researching the in-vitro antitumor activity of the liver targeting oxaliplatin and sorafenib co-loaded ICG-beta-CD. Human liver cancer HepG2 cells are taken as research objects, and the activity inhibition effect of OS-ICG-beta-CD on tumor cells is examined by a CCK-8 method. And the uptake curves of the cells for the different drugs were analyzed by HPLC.
Cell culture: HepG2 was cultured in a cell culture box containing 5% CO2 at 37 ℃ in a high-glucose DMEM medium containing 10% fetal bovine serum.
5.1 CCK-8 method for detecting in vitro cytotoxicity of OS-ICG-beta-CD
Grouping experiments:
(1) HEPG2 cells
(2) HEPG2 cells + ICG-beta-CD
(3) HEPG2 cells + Sorafenib
(4) HEPG2 cells + oxaliplatin
(5) HEPG2 cells + Sorafenib
(6) HEPG2 cells + OS-ICG-beta-CD
HepG2 cells were first seeded (approximately 5X 10)3One/well) to 96-well plates and cultured for 24 h. Then certain different pharmaceutical preparations were added, 5 multiple wells per group. After the sample is added and incubated for 48 hours, the cell activity is detected by a CCK-8 method.
The specific operation is as follows: first, the original medium in the 96-well plate was removed, 200. mu.L of fresh medium containing 10% (v/v) CCK-8 reagent was added, and incubated at 37 ℃ for 2 h. Then, the medium containing CCK-8 was removed and washed 3 times with sterile PBS solution. The absorbance value (A) of each well at a wavelength of 450nm was measured using a microplate reader, and the corresponding cell viability was calculated from the measured absorbance (FIG. 8).
5.2 in vitro uptake drug assay
Experiment grouping
(1) HEPG2 cells + Sorafenib
(2) HEPG2 cells + oxaliplatin
(3) HEPG2 cells + oxaliplatin + sorafenib
(4) HEPG2 cells + OS-ICG-beta-CD
HepG2 cells were seeded (about 5X 10)3One/well) to 96-well plates and cultured for 24 h. Then adding certain different pharmaceutical preparations, wherein each group comprises 5 multiple wells, collecting cells at 0.5h, 1h, 2h, 4h, 6 h, 8h, 12h, 24 h, 48 h and 72h respectively, preparing homogenate, centrifuging to obtain supernatant, filtering the sample solution with 0.45 μm filter membrane, and determining the content of OXA and SOR by HPLC method according to the method drawn by OXA and SOR standard curve. All measurements were done in triplicate. A graph of drug uptake by cells was prepared (fig. 9).
6 results
From the data, the administration method has obvious advantages compared with the conventional administration method by adopting the OS-ICG-beta-CD, has lower toxic and side effects on normal liver tissue cells and on normal organs in a liquid medicine path compared with the conventional administration method, realizes administration on deep liver cancer cells, and improves the administration depth. In vitro evaluation, the ICG-beta-CD has better killing effect on human liver cancer HepG2 cells, and the cell uptake rate can be maintained at a higher level for a longer time.

Claims (5)

1. An ICG-beta-cyclodextrin drug delivery system is characterized by comprising ICG-beta-cyclodextrin, the structure of which is shown in formula I:
Figure DEST_PATH_IMAGE001
。
2. an ICG- β -cyclodextrin drug delivery system of claim 1, prepared by the method comprising:
(1) preparation of carboxy-modified ICG: adding 1, 1, 2-trimethylbenzindole and ethyl iodide into acetonitrile, heating, refluxing, concentrating in vacuum to obtain a residue, adding diethyl ether into the residue to obtain a solid, washing the solid to obtain a compound 1, heating the compound 1 and glutaraldehyde dinitrile hydrochloride in acetic anhydride, cooling, and washing a product to obtain a compound 2;
adding 1, 1, 2-trimethylbenzindole and 6-iodohexanoic acid into acetonitrile, heating and refluxing, concentrating in vacuum to obtain a residue, adding diethyl ether into the residue to obtain a solid, and washing the solid to obtain a compound 3;
reacting the compound 2 with the compound 3 in pyridine, removing the solvent, purifying the residue by silica gel chromatography, and eluting to obtain a compound 4, wherein the compound 4 is a carboxyl modified ICG carrier and is marked as ICG-COOH;
(2) preparation of amino-modified cyclodextrins: firstly, preparing mono- (6-oxo-6-p-toluenesulfonyl) -beta-cyclodextrin, and then preparing ethylenediamine-beta-cyclodextrin, which is marked as 2N-beta-CD;
(3) preparing a supramolecular drug carrier: dissolving ICG-COOH in dimethyl sulfoxide, adding 3 times of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-hydroxysuccinimide in molar weight, adding 2N-beta-CD dissolved in dimethyl sulfoxide into the reaction solution, dialyzing, and freeze-drying the obtained sample to obtain the ICG-beta-cyclodextrin drug-loading system.
3. A pharmaceutical composition, characterized by the use of ICG- β -cyclodextrin of claim 1 as a pharmaceutically active ingredient in the preparation of a targeted liver cancer therapeutic drug.
4. The pharmaceutical composition of claim 3, wherein the targeted liver cancer therapeutic further comprises sorafenib and oxaliplatin.
5. A pharmaceutical composition according to claim 4, prepared by the following process:
accurately weighing sorafenib and ICG-beta-CD in a certain proportion, completely dissolving the sorafenib and the ICG-beta-CD in a mixed solvent of ethanol and water (volume ratio =1:5), and stirring the mixture at room temperature in a dark place; after most of ethanol in the reaction solution is removed by rotary evaporation, filtering the reaction solution by using a microporous filter membrane to remove unreacted sorafenib; spin-drying the filtrate, and then putting the filtrate into a vacuum drying oven for 12 hours to obtain an SOR-ICG-beta-CD clathrate compound; dissolving the SOR-ICG-beta-CD clathrate and oxaliplatin in a certain ratio in a mixed solvent of ethanol/water (volume ratio =1:5), and stirring at room temperature in a dark place; removing most of ethanol in the reaction solution by rotary evaporation, and filtering the reaction solution by using a microporous filter membrane to remove unreacted oxaliplatin; and (4) spin-drying the filtrate, and then putting the filtrate into a vacuum drying oven for 12 hours to obtain the OS-ICG-beta-CD clathrate compound.
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