CN115120561A - Combined drug metal organic hybrid nano assembly and application thereof - Google Patents

Combined drug metal organic hybrid nano assembly and application thereof Download PDF

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CN115120561A
CN115120561A CN202210769044.5A CN202210769044A CN115120561A CN 115120561 A CN115120561 A CN 115120561A CN 202210769044 A CN202210769044 A CN 202210769044A CN 115120561 A CN115120561 A CN 115120561A
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莫然
张经纬
王广基
董鹤
洪晓丹
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China Pharmaceutical University
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Abstract

The invention discloses a composite medicament metal organic hybrid nano assembly and application thereof, belonging to the technical field of biological medicines. The hybrid nano-assembly is formed by ionic interaction of a combined drug and metal ions; the combination drug is the combination of an antiviral drug and an anti-inflammatory and liver-protecting drug, or the combination of a chemotherapeutic drug and a chemotherapeutic sensitizer; the metalThe ions being selected from Mg 2+ 、Ca 2+ 、ZrO 2+ 、HfO 2+ 、Mn 2+ 、Fe 2+ 、Fe 3+ 、Cu 2+ 、Cu + 、Zn 2+ 、Pt 2+ 、Pt 4+ 、Pt 6+ 、Gd 3+ 、GdO + 、Ag + . According to the invention, the hybrid nano-assembly is prepared by simply mixing the combined medicament serving as an organic component and metal ions, so that the in vivo half-life period of the medicament can be prolonged, the concentration of the medicament at a focus part can be improved, a fixed medicament proportion can be maintained, and synchronous pharmacokinetics and tissue distribution are realized, thereby enhancing the synergistic treatment effect of the combined medicament.

Description

Combined drug metal organic hybrid nano assembly and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a combined medicine metal organic hybrid nano assembly and application thereof.
Background
Clinical practice and exploratory studies over the last decade have shown that a single treatment modality is not effective in treating some refractory diseases that are physiologically complex. Thus, treatment of refractory diseases has shifted from single therapy to combination therapy, increasing the efficiency of available treatment regimens. Combination therapy is a widely used disease treatment strategy that increases the potential therapeutic gains by combining existing disease modifying therapies or new drugs with acceptable theoretical grounds, good safety and efficacy profiles. Combination therapy can alter different biological pathways, reduce the effective therapeutic dose required, reduce drug resistance, and reduce the overall cost of treatment.
Chronic hepatitis b is a chronic disease caused by hepatitis b virus infection, and cannot be cured at present. Persistent hepatitis b virus infection can cause liver inflammation, leading to liver fibrosis and cirrhosis, and ultimately leading to liver cancer, which can pose a life risk. The nucleoside antiviral drugs widely used in clinic can effectively inhibit the replication of the virus for a long time and inhibit the exacerbation of hepatitis B, but are ineffective to chronic inflammation generated by virus invasion. Clinically, taking antiviral drugs and liver-protecting drugs at intervals is not an ideal combined administration mode and still needs to be improved. Co-delivery of antiviral and anti-inflammatory hepatoprotective drug combinations for combination therapy of chronic hepatitis b may ameliorate the deficiencies of existing therapeutic strategies.
Malignant tumors are a serious disease that seriously threatens human health and life, and tumor stem-like cells (CSCs) are a heterogeneous group of cells with self-renewal and self-differentiation ability in tumor tissues. Research has shown that CSCs are important factors leading to tumor chemotherapy resistance, recurrence and metastasis. In order to overcome tumor resistance, a combined treatment strategy of a chemosensitizer and a chemotherapeutic drug utilizes the chemosensitizer to reduce the chemoresistance of the drug-resistant tumor, and shows enhanced treatment effect.
The realization of combination therapy requires the effective integration of multiple therapeutic agents in a single carrier, rather than simple mixing, to produce a favorable synergistic therapeutic effect and maintain the specificity and persistence of the combination therapy. The rapidly developing biomaterials and nanotechnology provide a research basis for combined drug co-delivery, making it possible to co-load several drugs into one delivery vehicle by simple physical adsorption or chemical interaction without loss of drug activity. The nano-carrier can prolong the half-life period of the medicine in vivo, is beneficial to delivering the medicine to a focus part, enhances the treatment effect and reduces the toxic and side effect. The two drugs are loaded in one nano carrier together, so that different drugs are consistent in space and time in the treatment process, and have synchronous pharmacokinetics and biological distribution, thereby achieving a synergistic treatment effect. Although some nano-carriers (including liposomes, polymer nanoparticles, dendrimers and inorganic nanoparticles) are developed for drug combination delivery at present, the application of the nano-carriers in drug combination delivery is limited by the problems of low drug loading rate, complex synthesis/preparation method and the like in practice.
Therefore, there is still a need to develop a carrier platform which has a simple preparation method and high drug loading rate and can load different combination drugs for co-delivery of the combination drugs.
Disclosure of Invention
The invention aims to provide a hybrid nano assembly formed by co-assembling a combined drug and metal ions.
The invention also aims to provide the application of the hybrid nano assembly in preparing medicines for treating chronic hepatitis B or tumors.
In order to achieve the purpose, the invention adopts the following technical scheme:
the combined drug metal-organic hybrid nano assembly is formed by the interaction of the combined drug and metal ions through positive and negative ions;
the combination drug is the combination of an antiviral drug and an anti-inflammatory and liver-protecting drug, or the combination of a chemotherapeutic drug and a chemotherapeutic sensitizer;
the metal ion is selected from Mg 2+ 、Ca 2+ 、ZrO 2+ 、HfO 2+ 、Mn 2+ 、Fe 2+ 、Fe 3+ 、Cu 2+ 、Cu + 、Zn 2+ 、Pt 2+ 、Pt 4+ 、Pt 6+ 、Gd 3+ 、GdO + Or Ag + ；
The drug molecules of the antiviral drug, the anti-inflammatory and liver-protecting drug, the chemotherapeutic drug and the chemotherapeutic sensitizer contain phosphonic acid group-PO 3 H 2 Or phosphoric acid group-OPO 3 H 2 Or modified with phosphoric acid groups-OPO 3 H 2 。
In some embodiments, the antiviral drug is selected from tenofovir, entecavir, lamivudine, telbivudine, or adefovir.
In some embodiments, the anti-inflammatory and hepatoprotective agent is selected from glycyrrhizic acid, glycyrrhetinic acid, magnesium isoglycyrrhizinate, ammonium glycyrrhizinate, diammonium glycyrrhizinate, silymarin, bicyclol, tiopronin, or penicillamine.
In some embodiments, the chemotherapeutic is selected from camptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, topotecan, vinblastine, vincristine, vinorelbine, vindesine, cytarabine, gemcitabine, ancitabine, 5-fluorouracil, fluorodeoxyuridylic acid, 6-mercaptopurine, methotrexate, doxorubicin, epirubicin, doxorubicin, daunorubicin, mitoxantrone, paclitaxel, docetaxel, nimustine, teniposide, etoposide, aminoglutethimide, or melphalan.
In some embodiments, the chemosensitizer is selected from all-trans retinoic acid, 1, 3-cis retinoic acid, 9-cis retinoic acid, retinol, retinal, vismodegib, arsenic trioxide, arsenic sulfide, gallopamil, quinidine, verapamil, chlorpromazine, cyclosporin a, reserpine, digoxin, amiodarone, progesterone, flavanone, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valsalvapor, bicoda, efolidar, azaquinad, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopag, fumonisin C, lapatinib, rosuvastatin, sulfasalazine, or febuxostat.
In some embodiments, the drug molecules of the antiviral drugs, the anti-inflammatory and hepatoprotective drugs, the chemotherapeutic sensitizers and the modified phosphate-OPO 3 H 2 With responsive linking arms therebetween.
In some embodiments, the combination drug metal-organic hybrid nano-assembly further comprises a pharmaceutically acceptable adjuvant.
In some embodiments, the excipient is selected from polyethylene glycol, polyvinylpyrrolidone, poloxamer, polyvinyl alcohol, polyoxazoline, vitamin E polyethylene glycol succinate, cholesterol, soy lecithin, Igepal CO-520, or cetyltrimethylammonium bromide.
The invention also provides application of the composite medicament metal-organic hybrid nano-assembly in preparing a medicament for treating chronic hepatitis B, so as to be used for antiviral, anti-inflammatory and liver-protecting combined treatment of chronic hepatitis B.
The invention also provides application of the composite medicament metal organic hybrid nano-assembly in preparing medicaments for treating tumors, which is used for treating common tumors or dry-type related drug-resistant tumors.
The metal organic hybrid nano assembly is nanoparticles formed by assembling organic molecules and metal ions through ionic interaction, and the hybrid nano assembly is prepared by simply mixing the combined medicament serving as an organic component and the metal ions, and has the advantages of high medicament loading rate, simplicity in preparation and the like. Because the organic molecules of phosphoric acid and metal ions have strong acting force, a hybrid nano assembly is easy to form. In order to co-load the combination drug inIn the hybrid nano-assembly, the required drug has phosphonic acid group (-PO) 3 H 2 ) Or a phosphate group (-OPO) 3 H 2 ) Or by chemical modification of the phosphate group (-OPO) 3 H 2 ) So that the hybrid nano-assembly can be assembled with metal ions to form a hybrid nano-assembly of the combined drug. As shown in fig. 1.
The combined medicament is loaded in the nano assembly, so that the half-life period of the medicament in vivo can be prolonged, the concentration of the medicament at a focus position can be improved, a fixed medicament proportion can be maintained, and synchronous pharmacokinetics and tissue distribution can be realized, thereby enhancing the synergistic treatment effect of the combined medicament. Meanwhile, under the stimulation of a focus microenvironment physiological signal (such as high expression phosphatase, hypoxia, acid and high oxidation/reduction), the chemically modified phosphate group is degraded in response and falls off, so that the prototype drug is released, and the activity of the drug is ensured to be exerted.
Drawings
FIG. 1 is the preparation of the combined drug hybrid nano-assembly and the responsive drug release principle thereof.
FIG. 2 is a Transmission Electron Microscope (TEM) and high angle annular dark field scanning transmission electron microscope (HAADF-STEM) characterization of TFV/GAP/NA (Zr) in example 1.
FIG. 3 is a TEM representation of AFV/SMP/NA (Fe) from example 1.
FIG. 4 is a graph of the in vitro release of TFV/GAP/NA (Zr), AFV/GAP/NA (Mg), AFV/SMP/NA (Fe), and TFV/SMP/NA (Cu) from example 1.
FIG. 5 is the in vitro safety of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 6 is the in vitro anti-inflammatory activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) of example 1.
FIG. 7 is a graph of the in vitro antiviral activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) of example 1.
FIG. 8 is the in vivo drug liver tissue distribution of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 9 is the in vivo antiviral activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) of example 1.
FIG. 10 is the in vivo anti-inflammatory activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) of example 1.
FIG. 11 is a TEM characterization of LAMP/SMP/NA (Hf) in example 2.
FIG. 12 is a graph of the in vitro release of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 13 is the in vitro safety of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 14 is a graph of the in vitro anti-inflammatory activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 15 is a graph of the in vitro antiviral activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 16 is the in vivo drug liver tissue distribution of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 17 is a graph of the in vivo antiviral activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 18 is a graph of the in vivo anti-inflammatory activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 19 is a TEM and HAADF-STEM characterization of r-ATRAP/FdUMP/NA (Hf) in example 3.
FIG. 20 is a graph showing the in vitro responsive release of r-ATRAP/FdUMP/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) in example 3.
FIG. 21 is a graph of the in vitro responsive release of r-ATRAP/FdUMP/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) from example 3.
FIG. 22 is a graph of the in vitro anti-tumor activity of r-ATRAP/FdUMP/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) in example 3.
FIG. 23 is a graph of the in vivo antitumor activity of r-ATRAP/FdUMP/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) in example 3.
FIG. 24 is a TEM characterization of r-VERP/r-CPTP/NA (Hf) in example 4.
FIG. 25 is a graph of the in vitro responsive release of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) from example 4.
FIG. 26 is a graph showing the in vitro responsive release of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) from example 4.
FIG. 27 is a graph of the in vitro anti-tumor activity of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) of example 4.
FIG. 28 is a graph of the in vivo anti-tumor activity of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) of example 4.
Detailed Description
The invention is described in further detail below with reference to the figures and the examples, but the invention should not be construed as being limited thereto. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples were carried out according to the conventional conditions in the art.
Example 1
Combined drug hybrid nano-assembly for combined treatment of chronic hepatitis B
Modification of antiviral drug and anti-inflammatory and liver-protecting drug
The antiviral drugs entecavir, lamivudine or telbivudine (1.9mmol) are dissolved in 5mL trimethyl phosphate, added into a nitrogen protection reaction tube, and phosphorus oxychloride (0.33mL,3.8mmol) is added into the solution drop by drop, and reacted for 16h at 4 ℃. After the reaction is finished, 10mL of water is added into the reaction liquid to quench the reaction, the reaction liquid is stirred for 30min, the reaction liquid is extracted for 10 times by ethyl acetate, the water phase is collected and is mixed with methanol for spin drying, and an oily product is obtained. And (4) purifying by column chromatography to obtain phosphorylated entecavir, lamivudine or telbivudine products.
Taking a 250mL reaction bottle for anhydrous drying and replacing nitrogen for protection, dissolving phosphorus oxychloride (2mL,14.6mmol) in 60mL anhydrous tetrahydrofuran and adding the solution into the reaction bottle, dissolving anti-inflammatory and liver-protecting drugs of glycyrrhetinic acid, silymarin or bicyclol (6.45mmol) in a mixed solution of 5.1mL anhydrous pyridine and 30mL anhydrous tetrahydrofuran, dropwise adding the mixed solution under the ice bath condition, keeping the temperature at 0 ℃ for reaction for 30min, and transferring the mixed solution to the room temperature for reaction for 2 h. After the reaction, the solvent was removed by rotary evaporation under reduced pressure from the reaction mixture, and the oily residue was dissolved in 100mL of methylene chloride, washed with 1M hydrochloric acid solution, washed with water, washed with saturated brine, and the organic phase was collected and dried over anhydrous sodium sulfate. Filtering, removing solvent by rotary evaporation to obtain oily crude product, and purifying by column chromatography to obtain phosphorylated glycyrrhetinic acid, silymarin or bicyclol product.
Preparation of hybrid nano assembly of combined drug
The antiviral drug and the anti-inflammatory drug are mixed with metal ions in an aqueous solution together to form the combined drug hybrid nano assembly.
Dissolving Tenofovir (TFV), Adefovir (AFV) or phosphorylated Entecavir (ETVP) in 2% F-68 aqueous solution to prepare 21mM solution A, and dissolving phosphorylated Glycyrrhetinic Acid (GAP) or phosphorylated Silymarin (SMP) in tetrahydrofuran to prepare 18mM solution B. 1mL of solution A and 0.2mL of solution B are jointly dropped into 1mL of 18mM zirconium oxychloride 2% F-68 aqueous solution, and after stirring for 15min, the combined drug hybrid nano-assembly TFV/GAP/NA (Zr), AFV/GAP/NA (Zr), ETVP/GAP/NA (Zr), TFV/SMP/NA (Zr), AFV/SMP/NA (Zr) or ETVP/SMP/NA (Zr) is prepared. And (3) placing the nano assembly solution into a dialysis bag, and dialyzing in deionized water overnight to remove residual small molecule drugs and organic solvents. After dialysis, the solution was added to a centrifuge tube, centrifuged at high speed, the supernatant was discarded, and washed twice with deionized water (resuspension + washing). Finally, the nano assemblies were suspended in physiological saline, and the particle size and polydispersity index (PDI) of the assemblies were characterized using a particle size analyzer, with the results shown in table 1. TFV/GAP/NA (Zr) is selected to be used for carrying out morphology characterization through a Transmission Electron Microscope (TEM) and element mapping characterization through a high-angle annular dark field scanning transmission electron microscope (HAADF-STEM), and the result is shown in figure 2.
Table 1 characterization of particle size of combination drug hybrid nano-assemblies
Figure BDA0003723255290000061
Dissolving TFV, AFV or ETVP in 3mL water to obtain 0.3mM solution, adding to 70mL chloroform, adding 1.82g hexadecyltrimethylammonium bromide and 3mL n-hexanol, dissolving GAP or SMP in 1mL tetrahydrofuran to obtain 0.2mM solution, adding to the above solution, stirring at 35 deg.C for 30min, adding 1.6mL MgCl with concentration of 0.5M 2 (or CaCl) 2 、CuCl 2 、FeCl 3 ) Aqueous solution to form hybrid nano-assembly TFV/GAP/NA (Mg), AFV/GAP/NA (Mg), ETVP/GAP-NA (Ca), TFV/SMP/NA (Cu), AFV/SMP/NA (Fe), ETVP/SMP/NA (Fe), stirring at room temperature for 12h, continuously centrifuging and resuspending and washing three times. Finally, the hybrid nano-assemblies were resuspended in physiological saline and the particle size and PDI of the assemblies were characterized by a particle size analyzer, with the results shown in table 2. AFV/SMP/NA (Fe) was selected for morphology characterization by TEM (FIG. 3).
Table 2 characterization of particle size of combination drug hybrid nano-assemblies
Figure BDA0003723255290000062
Drug release of combined drug hybrid nano-assembly
In order to simulate the drug release condition of the liver part, 1mL of combined drug hybrid nano assembly (TFV/GAP/NA (Zr), AFV/GAP/NA (Mg), AFV/SMP/NA (Fe) or TFV/SMP/NA (Cu)) is added into a dialysis bag, 1U/mL of phosphatase (simulated hepatitis B hyperphosphatemia environment) is added at the same time, the dialysis bag is soaked in 40mL of PBS release medium containing Tween 80 after being sealed, sampling is carried out at fixed time points, the corresponding drug concentration is measured by LC-MS/MS, and the drug release amount is calculated. The drug release profile is shown in figure 4. Under the condition of simulating high phosphatase level of the liver, phosphate groups are hydrolyzed by phosphatase, and antiviral drugs and anti-inflammatory and liver-protecting drugs in the hybrid nano-assembly can be quickly released.
In-vitro safety and anti-inflammatory activity determination of combined drug hybrid nano-assembly
1. In vitro safety study of combination drug hybrid nano-assemblies
RAW264.7 cells and L-02 cells were separately added at 4X 10 cells per well 4 The density of individual cells was seeded in a 96-well plate and after 24h of culture, the medium was aspirated. Adding serum-free culture medium containing hybrid nano-assembly (TFV/GAP/NA (Zr) or AFV/SMP/NA (Fe)) with different drug concentrations, incubating for 24h and 48h in a cell culture box respectively, and detecting cell activity by using a CCK-8 detection reagent. Cell viability results are shown in FIG. 5, where the hybrid nano-assemblies of different drug concentrations were incubated for 24h and 48hAfter h, the cell activity is not obviously reduced compared with that of a blank group, which indicates that the combined drug hybrid nano-assembly is biologically safe.
2. In vitro anti-inflammatory activity study of combination drug hybrid nano-assemblies
RAW264.7 cells were plated at 4X 10 cells per well 4 The density of individual cells was seeded in 96-well plates and after 24h of culture, the medium was aspirated. Serum-free medium containing hybrid nano-assemblies (TFV/GAP/NA (Zr) or AFV/SMP/NA (Fe), GAP or SMP is 40 mu M and LPS (1 mu g/mL) is added, after 24h of incubation in an incubator, cell supernatant is collected, after reasonable dilution, the level of inflammatory factors is determined by TNF-alpha, IL-1 beta and IL-6ELISA kits, and the in vitro anti-inflammatory activity of GAP is examined. In vitro anti-inflammatory results are shown in fig. 6, the level of inflammatory factors is obviously increased after LPS is added into cells, and the level of inflammatory factors is obviously reduced after different combined drug hybrid nano-assemblies are added, which indicates that the combined drug hybrid nano-assemblies have good anti-inflammatory activity.
Fifthly, in-vitro antiviral activity determination of combined drug hybrid nano assembly
HepG2.2.15 cells at 4X 10 per well 4 The density of individual cells was seeded in a 96-well plate and after 24h of culture, the medium was aspirated. Adding new culture medium containing hybrid nano assembly (TFV/GAP/NA (Zr) or AFV/SMP/NA (Fe)), incubating in incubator every 3 days to change new culture medium containing drug, observing cell state at 3, 6 and 9 days, respectively, sucking supernatant, and analyzing HBV DNA determination. HBV DNA was extracted using the universal genomic DNA extraction kit and virus copy number detection was performed with Taqman probe in CFX96 Real-time PCR detection system (Bio-Rad). The in vitro antiviral results are shown in fig. 7, which shows that the combined drug hybrid nano-assembly has good and continuous inhibition effect on hepatitis B virus.
Six, in vivo pharmacokinetic investigation of combined drug hybrid nano-assembly
C57BL/6 mice were randomly grouped into 10 mice per group, the tail vein was injected with the combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3mg/kg) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3mg/kg)) and the corresponding free combination drug (TFV + GAP or AFV + SMP), five mice per group were bled at predetermined interval time points, five mice were bled at adjacent interval time points, and samples were analyzed with LC-MS/MS system to determine the amount of drug after treatment. Pharmacokinetic parameters were calculated using a non-compartmental model of Phoenix WinNonlin kinetic software, and the results are shown in tables 3 and 4, the pharmacokinetic parameters of the hybrid nano-assembly preparation group were significantly improved compared to the corresponding free drug group, and the half-life of the drug in vivo was significantly prolonged.
TABLE 3 pharmacokinetic parameters of TFV/GAP/NA (Zr) and free (TFV + GAP)
Figure BDA0003723255290000081
TABLE 4 pharmacokinetic parameters of AFV/SMP/NA (Fe) and free (AFV + SMP)
Figure BDA0003723255290000082
Seventhly, investigation of liver tissue drug distribution of combined drug hybrid nano assembly
Randomly grouping 35 mice C57BL/6 according to each group, injecting a combined drug hybrid nano assembly preparation (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3mg/kg) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3mg/kg)) and free combined drugs (TFV + GAP or AFV + SMP) with the same concentration at the preset time point, collecting livers after five mice in each group die, weighing 0.1g of liver tissues after washing, and determining the drug concentration in the livers by LC-MS/MS after grinding treatment. As a result, as shown in fig. 8, the hybrid nano-assembly preparation group can significantly promote the accumulation of the drug in the liver compared to the corresponding free drug.
Eighthly, in vivo antiviral activity investigation of combined drug hybrid nano-assembly
HBV gene-transfected C57BL/6 mice were randomly grouped into 5 mice per group, and the tail vein was injected with a combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3mg/kg) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3mg/kg)), once every three days for 15 days. Mice were bled from the orbit and sera isolated at 3, 6, 9, 12, 15 days after the first dose, respectively, for HBV DNA extraction and viral copy number detection with Taqman probes in the CFX96 Real-time PCR detection system (Bio-Rad). On day 15, mice were sacrificed to collect livers, 0.1g of liver tissue was weighed, homogenized, centrifuged to take the supernatant, and the number of HBV copies in the livers was determined by PCR. The results are shown in fig. 9, the continuous administration of the hybrid nano-assembly preparation can significantly inhibit the HBV levels in blood and liver, indicating that these combined drug hybrid nano-assemblies have the optimal sustained inhibitory effect on hepatitis b virus.
Nine, in vivo anti-inflammatory activity investigation of combined drug hybrid nano-assembly
C57BL/6 mice were randomly grouped into 6 mice per group and the tail vein was injected with a combination drug hybrid nano-assembly formulation (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3mg/kg) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3mg/kg)), once every three days for 15 consecutive days. After the last administration for 2 hours, the mice in the administration group are injected with 0.2 percent of carbon tetrachloride/corn oil solution in the abdominal cavity, and the administration dosage of the carbon tetrachloride is 32mg/kg, so as to induce an acute liver injury model. After 20 hours of carbon tetrachloride injection, all mice were bled from the orbit, and then the mice were sacrificed and liver tissue was collected. The collected blood samples were centrifuged and the supernatant was diluted appropriately and assayed for inflammatory factor levels using TNF- α, IL-1 β, and IL-6ELISA kits. 0.1g of liver tissue is weighed, homogenized, diluted appropriately and measured for inflammatory factor levels using TNF-alpha, IL-1 beta, and IL-6ELISA kits. The results are shown in fig. 10, after the liver injury is induced by carbon tetrachloride, the level of inflammatory factors in blood and liver is obviously increased, and the pre-administration of the hybrid nano-assembly preparation can obviously reduce the level of inflammatory factors in blood and liver, which indicates that the hybrid nano-assembly of the combination drugs has the optimal anti-inflammatory and liver-protecting effects.
Example 2
Combined drug hybrid nano-assembly for combined treatment of chronic hepatitis B
Preparation of hybrid nano assembly of first and second combined medicaments
The antiviral drug and the anti-inflammatory drug are mixed and assembled with metal ions in the aqueous solution to form the combined drug hybrid nano assembly.
Dissolving TFV, ETVP or phosphorylated Lamivudine (LAMP) in 2% PVP aqueous solution to prepare 21mM solution A, dissolving GAP or SMP in tetrahydrofuran to prepare 18mM solution B, adding 1mL of solution A and 0.2mL of solution B into 1mL of 18mM hafnium tetrachloride 2% PVP aqueous solution dropwise, stirring for 15min to obtain a combined drug hybrid nano-assembly TFV/GAP/NA (Hf), ETVP/GAP/NA (Hf), LAMP/GAP/NA (Hf), TFV/SMP/NA (Hf), ETVP/SMP/NA (Hf) or SMP/NA (Hf), placing the assembly solution in a dialysis bag, and dialyzing in deionized water overnight to remove residual small molecular drugs. The solution in the dialysis bag was centrifuged at high speed and the white assembly nanoparticles were deposited at the bottom of the centrifuge tube. The supernatant was discarded, and the precipitate was washed with deionized water and centrifuged twice. Finally, the combined drug hybrid nano-assemblies were resuspended in physiological saline and the particle size of the assemblies was characterized with a potential particle size analyzer, the results are shown in table 5. LAMP/SMP/na (hf) was selected for topographic characterization by TEM (fig. 11).
Table 5 particle size characterization of combination drug hybrid nano-assemblies
Figure BDA0003723255290000101
Drug release of combined drug hybrid nano-assembly
1mL of the combination drug hybrid nano-assembly (TFV/GAP/NA (Hf)) or ETVP/SMP/NA (Hf)) is added into a dialysis bag, 1U/mL of phosphatase (simulating hepatitis B hyperphosphatase environment) is added at the same time, the dialysis bag is soaked in 40mL of PBS release medium containing Tween 80 after being sealed, sampling is carried out at fixed time points, corresponding drug concentration is determined by LC-MS/MS, and the drug release amount is calculated. The drug release profile is shown in figure 12. Under the condition of simulating high phosphatase level of the liver, the antiviral drug and the anti-inflammatory and liver-protecting drug in the hybrid nano assembly can be gradually released.
Third, the in vitro safety and anti-inflammatory activity of the combined drug hybrid nano assembly are determined
1. In vitro safety study of combination drug hybrid nano-assemblies
RAW264.7 cells and L-02 cells were separately added at 4X 10 cells per well 4 The density of individual cells was seeded in 96-well plates and after 24h of culture, the medium was aspirated. Serum-free culture media containing hybrid nano-assemblies (TFV/GAP/NA (Hf)) or ETVP/SMP/NA (Hf)) with different drug concentrations are added, the cells are respectively incubated for 24h and 48h in a cell culture box, and the cell activity is detected by using a CCK-8 detection reagent. The cell survival rate results are shown in fig. 13, after incubation for 24h and 48h, the cell activities of the hybrid nano-assemblies with different drug concentrations were not significantly reduced compared with the blank group, which indicates that the hybrid nano-assemblies with the combination drugs are biologically safe.
2. In vitro anti-inflammatory activity study of combination drug hybrid nano-assemblies
RAW264.7 cells were plated at 4X 10 cells per well 4 The density of individual cells was seeded in 96-well plates and after 24h of culture, the medium was aspirated. Serum-free medium containing hybrid nano-assemblies (TFV/GAP/NA (Hf) or ETVP/SMP/NA (Hf), GAP or SMP being 40 μ M and LPS (1 μ g/mL) was added, after 24h incubation in an incubator, cell supernatants were collected, after appropriate dilution, inflammatory factor levels were determined using TNF- α, IL-1 β, IL-6ELISA kits, and the anti-inflammatory activity of GAP in vitro was examined. In vitro anti-inflammatory results are shown in fig. 14, the level of inflammatory factors is obviously increased after LPS is added into cells, and the level of inflammatory factors is obviously reduced after different combined drug hybrid nano-assemblies are added, which indicates that the combined drug hybrid nano-assemblies have optimal anti-inflammatory activity.
In-vitro antiviral activity determination of combined drug hybrid nano-assembly
HepG2.2.15 cells were plated at 4X 10 cells per well 4 The density of individual cells was seeded in a 96-well plate and after 24h of culture, the medium was aspirated. Adding new culture medium containing hybrid nano assembly (TFV/GAP/NA (Hf) or ETVP/SMP/NA (Hf), TFV or ETV is 154 muM), incubating the culture medium containing new drug every 3 days in the incubator, observing cell state at 3, 6 and 9 days, sucking supernatant, and analyzing HBV DNA determination. HBV DNA was extracted using a universal genomic DNA extraction kit and tested in CFX96 Real-time PCViral copy number detection was performed with Taqman probes in the R detection System (Bio-Rad). In vitro antiviral results are shown in fig. 15, which shows that the combined drug hybrid nano-assembly has optimal continuous inhibition effect on hepatitis B virus.
In vivo pharmacokinetics investigation of combined drug hybrid nano-assemblies
C57BL/6 mice were randomly grouped into 10 mice per group, the tail vein was injected with the combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3mg/kg) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3mg/kg)) and the corresponding free combination drug (TFV + GAP or ETV + SMP), five mice per group were bled at predetermined interval time points, five mice were bled at adjacent interval time points, and samples were analyzed with LC-MS/MS system to determine the amount of drug after treatment. Pharmacokinetic parameters were calculated using a non-compartmental model of Phoenix WinNonlin kinetic software, and the results are shown in tables 6 and 7, the pharmacokinetic parameters of the hybrid nano-assembly preparation group were significantly improved compared to the corresponding free drug group, and the half-life of the drug in vivo was significantly prolonged.
TABLE 6 pharmacokinetic parameters of TFV/GAP/NA (Hf) and free (TFV + GAP)
Figure BDA0003723255290000111
TABLE 7 pharmacokinetic parameters of ETVP/SMP/NA (Hf) and free (ETV + SMP)
Figure BDA0003723255290000112
Figure BDA0003723255290000121
Sixthly, investigation of liver tissue drug distribution of combined drug hybrid nano assembly
Randomly grouping 35 mice of C57BL/6 according to each group, injecting a combination drug hybrid nano assembly preparation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3mg/kg) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3mg/kg)) and free combination drugs (TFV + GAP or ETVP + SMP) with the same concentration at the tail vein, collecting livers after five mice in each group are sacrificed, weighing 0.1g of liver tissues after washing, and determining drug concentration in the livers by LC-MS/MS after grinding treatment. The results are shown in fig. 16, the hybrid nano-assembly formulation group can significantly promote drug accumulation in the liver compared to the corresponding free drug.
Seventh, investigation of in vivo antiviral Activity of combination drug hybrid Nano Assembly
HBV gene-transfected C57BL/6 mice were randomly grouped into 5 mice per group, and the tail vein was injected with a combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3mg/kg) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3mg/kg)), once every three days for 15 days. Mice were bled from the orbit and serum was isolated at 3, 6, 9, 12, 15 days after the first dose, HBV DNA extraction was performed, and virus copy number detection was performed with Taqman probe in CFX96 Real-time PCR detection system (Bio-Rad). On day 15, mice were sacrificed to collect livers, 0.1g of liver tissue was weighed, homogenized, centrifuged to take the supernatant, and the number of HBV copies in the livers was determined by PCR. The results are shown in fig. 17, the continuous administration of the hybrid nano-assembly preparation can significantly inhibit the HBV levels in blood and liver, indicating that these combined drug hybrid nano-assemblies have the optimal sustained inhibitory effect on hepatitis b virus.
Eighthly, in vivo anti-inflammatory activity investigation of combined drug hybrid nano-assembly
C57BL/6 mice were randomized into groups of 6 mice per group and the tail vein was injected with a combination drug hybrid nanoassembly formulation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3mg/kg) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3mg/kg)), once every three days for 15 consecutive days. After the last administration for 2 hours, the mice in the administration group are injected with 0.2 percent of carbon tetrachloride/corn oil solution in the abdominal cavity, and the administration dose of the carbon tetrachloride is 32mg/kg, so as to induce the acute liver injury model. After 20 hours of carbon tetrachloride injection, all mice were bled from the orbit, and then the mice were sacrificed and liver tissue was collected. The collected blood samples were centrifuged and the supernatant was diluted appropriately and the levels of inflammatory factors were measured using TNF-. alpha.IL-1. beta., and IL-6ELISA kits. 0.1g of liver tissue was weighed, homogenized, diluted appropriately and measured for inflammatory factor levels using TNF-. alpha.IL-1. beta., and IL-6ELISA kits. The results are shown in fig. 18, after the liver injury induced by carbon tetrachloride, the level of inflammatory factors in blood and liver is obviously increased, and the pre-administration of the hybrid nano-assembly preparation can obviously reduce the level of inflammatory factors in blood and liver, which indicates that the hybrid nano-assembly of the combination drugs has the optimal anti-inflammatory and liver-protecting effects.
Example 3
Combined drug hybrid nano-assembly for drug-resistant tumor combination therapy
Modification of chemotherapeutic drugs and chemosensitizers/differentiation inducers
Modification of chemotherapeutic drugs
5-fluoro-2' -deoxyuridine, camptothecin, doxorubicin or paclitaxel (1.9mmol) was dissolved in 5mL trimethyl phosphate, added to a nitrogen-protected reaction tube, and phosphorus oxychloride (0.33mL,3.8mmol) was added dropwise to the solution and reacted at 4 ℃ for 16 h. After the reaction is finished, 10mL of water is added into the reaction liquid to quench the reaction, the reaction liquid is stirred for 30min, the reaction liquid is extracted for 10 times by ethyl acetate, the water phase is collected and is mixed with methanol for spin drying, and an oily product is obtained. And (4) performing column chromatography to obtain a phosphorylation product.
Phosphorylated hypoxia-responsive differentiation inducer synthesis
All-trans retinoic acid, 1, 3-cis retinoic acid, or 9-cis retinoic acid (10mmol) was dissolved in a mixed solution of 100mL of methylene chloride and 6mL of dimethyl sulfoxide, and 4- (4- (hydroxymethyl) phenyl) diazenyl) phenol (3.16g,10mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (2.4g,12mmol), 1-hydroxybenzotriazole (1.62g,12mmol), and N, N-diisopropylethylamine (5mL) were added thereto and reacted at room temperature overnight. After the reaction is finished, washing the reaction solution with water, washing with saturated salt water, drying with anhydrous sodium sulfate, filtering, spin-drying the organic phase to obtain a crude product, and performing column chromatography to obtain the azobenzene modified product.
Phosphorus oxychloride (0.2mL,2.3mmol) was dissolved in 2mL of anhydrous THF, added to a nitrogen-protected reaction tube and placed under a 0 ℃ ice-water mixed bath, and a mixed solution of azobenzene-modified differentiation inducer (0.67mmol) dissolved in anhydrous pyridine (0.53mL,6.5mmol) and 3mL of anhydrous THF was added dropwise to the above reaction solution and reacted at 0 ℃ for 3 hours. After the reaction is finished, the reaction liquid is dried in a spinning mode, residues are dissolved in ethyl acetate, an organic phase is washed by water, a dilute hydrochloric acid solution is washed by saturated salt water, anhydrous sodium sulfate is dried, and the organic phase is dried in a spinning mode after filtration to obtain a crude product. Column chromatography gave phosphorylated hypoxia-responsive product.
Preparation of hybrid nano assembly of combined drug
Fluorodeoxyuridylate (FdUMP), active oxygen-responsive phosphorylated camptothecin (r-CPTP) or phosphorylated doxorubicin (r-DOXP) was dissolved in 5mL of 2% TPGS solution to prepare a 15mM solution, and the pH was adjusted to weak alkalinity (pH 8) with sodium hydroxide solution. Phosphorylated hypoxia-responsive all-trans retinoic acid (r-ATRAP) or 1, 3-cis retinoic acid (r-CRAP) is dissolved in 1mL of ethanol to prepare 76mM solution, 2mL of 3mM hafnium tetrachloride 2% TPGS solution is added dropwise to the solution, and after stirring for 15min, a combined drug hybrid nano-assembly (r-ATRAP/FdUMP/NA (Hf), r-ATRAP/r-CPTP/NA (Hf), r-ATRAP/r-DOXP/NA (Hf), r-CRAP/FdUMP/NA (Hf), r-CRAP/r-CPTP/NA (Hf) or r-CRAP/r-DOXP/NA (Hf)) is formed. The assembly solution was placed in a dialysis bag and dialyzed overnight in deionized water to remove residual small molecule drug. After washing by continuous centrifugation and washing by rinsing and suspending three times, the resulting suspension was rinsed and suspended in physiological saline, and the particle size of the assembly was characterized by a potential particle size analyzer, and the results are shown in Table 8. r-ATRAP/FdUMP/NA (Hf) was chosen for morphology characterization by TEM and element mapping characterization by HAADF-STEM, and the results are shown in FIG. 19.
Table 8 particle size characterization of combination drug hybrid nano-assemblies
Figure BDA0003723255290000141
Drug release of combined drug hybrid nano-assembly
1mL of the combination drug hybrid nano-assembly (r-ATRAP/FdUMP/NA (Hf)) or r-CRAP/r-CPTP/NA (Hf)) was added to the dialysis bag, 10mM sodium dithionate (simulating a low oxygen environment) was added at the same time, the dialysis bag was sealed and immersed in 40mL of PBS release medium containing Tween 80, samples were taken at fixed time points, the corresponding drug concentrations were determined by HPLC, and the drug release amounts were calculated. The drug release profile is shown in figure 20. Under the condition of simulating tumor hypoxia, the hypoxia-responsive drug in the assembly is released rapidly, and the chemotherapeutic drug is released slowly.
1mL of the combination drug hybrid nano-assembly (r-ATRAP/FdUMP/NA (Hf)) or r-CRAP/r-CPTP/NA (Hf)) was added to the dialysis bag along with 10mM sodium dithionate, the dialysis bag was sealed and soaked in 40mL of PBS release medium containing Tween 80, and sampling was performed at fixed time points. And after 12h of release, adding hydrogen peroxide into the dialysis bag, putting the dialysis bag into a release medium for continuous release, sampling at a fixed time point, measuring the corresponding drug concentration by using HPLC (high performance liquid chromatography), and calculating the drug release amount. The drug release profile is shown in figure 21. Under the condition of simulating tumor hypoxia, the low-oxygen response medicine in the assembly is released quickly, the chemotherapeutic medicine is released slowly, and the release of the chemotherapeutic medicine is accelerated obviously by adding hydrogen peroxide to simulate the active oxygen condition.
In-vitro cytotoxicity determination of combined drug hybrid nano assembly on drug-resistant tumor cells
4T1 CSCs at 5X 10 per well 3 The density of each cell is inoculated in a 96-hole ultra-low adhesion culture plate, hybrid nano assemblies (r-ATRAP/FdUMP/NA (Hf)) or r-CRAP/r-CPTP/NA (Hf)) containing different drug concentrations are added, the mixture is placed in a low-oxygen culture box for culture for 48 hours, and the cell activity is detected by using a CCK-8 detection reagent. The cell survival rate results are shown in figure 22, and through simulation calculation, the IC50 values of r-ATRAP/FdUMP/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) are 0.4274 and 0.3510 mu M respectively, which shows that the combined drug hybrid nano-assembly reverses the drug resistance of tumor stem-like cells and has obvious killing effect.
Fifthly, determination of in vivo anti-tumor activity of combined drug hybrid nano assembly
4T1 CSCs were harvested and resuspended in a 1:1 volume ratio of PBS to Matrigel mixture at 5X 10 5 The density of each cell/cell is inoculated under the left breast pad of a Balb/c mouse with a depilatory abdomen, and a drug-resistant tumor mouse model is constructed. Randomly grouping the tumor-bearing mice into groups of six mice, and injecting the combined drug hybrid sodium into tail veinThe rice husk assembly preparation (r-ATRAP/FdUMP/NA (Hf) (FdUMP 3.33mg/kg, r-ATRAP 24mg/kg) or r-CRAP/r-CPTP/NA (Hf) (r-CPTP 3.33mg/kg, r-CRAP 24mg/kg)) was administered once every two days for 14 consecutive days. The long diameter and the short diameter of the tumor are measured every two days, the tumor volume is calculated, and the change of the tumor volume is detected. The results are shown in fig. 23, the combination drug hybrid nano-assembly preparation group has the best tumor inhibition effect.
Example 4
Combined drug hybrid nano-assembly for drug-resistant tumor combination therapy
Preparation of hybrid nano assembly of first and second combined medicaments
r-CPTP, r-DOXP or taxol phosphorylated correspondingly with active oxygen (r-PTXP) was dissolved in 5mL of 2% TPGS solution to prepare a solution having a concentration of 15mM, and the pH was adjusted to weak alkalinity (pH 8) with sodium hydroxide solution. Phosphorylated hypoxia-responsive verapamil (r-VERP) or cyclosporin A (r-CSAP) was dissolved in 1mL of ethanol to prepare a 76mM solution, and 2mL of a 3mM hafnium tetrachloride 2% TPGS solution was co-added dropwise to the above solution, followed by stirring for 15min to form a composite drug hybrid nano-assembly (r-VERP/r-CPTP/NA (Hf), r-VERP/r-DOXP/NA (Hf), r-VERP/r-PTXP/NA (Hf), r-CSAP/r-CPTP/NA (Hf), r-CSAP/r-DOXP/NA (Hf) or r-CSAP/r-PTXP/NA (Hf)). The assembly solution was placed in a dialysis bag and dialyzed overnight in deionized water to remove residual small molecule drug. After washing by continuous centrifugation and washing by rinsing and suspending three times, the resulting suspension was rinsed and suspended in physiological saline, and the particle size of the assembly was characterized by an electric potential particle size analyzer, and the results are shown in Table 13. The r-VERP/r-CPTP/NA (Hf) was selected for morphology characterization by TEM and the results are shown in FIG. 24.
Table 13 characterization of particle size of combination drug hybrid nano-assemblies
Figure BDA0003723255290000151
Drug release of combined drug hybrid nano-assembly
1mL of the combination drug hybrid nano-assembly (r-VERP/r-CPTP/NA (Hf)) or r-CSAP/r-DOXP/NA (Hf)) was added to a dialysis bag, 10mM sodium dithionate (simulating a low-oxygen environment) was added at the same time, the dialysis bag was soaked in 40mL of PBS release medium containing Tween 80 after being sealed, samples were taken at fixed time points, the corresponding drug concentrations were determined by HPLC, and the drug release amounts were calculated. The drug release profile is shown in figure 25. Under the condition of simulating tumor hypoxia, the hypoxia-responsive drug in the assembly is released rapidly, and the chemotherapeutic drug is released slowly.
1mL of the combination drug hybrid nano-assembly (r-VERP/r-CPTP/NA (Hf)) or r-CSAP/r-DOXP/NA (Hf)) was added to the dialysis bag, 10mM sodium dithionate (simulating a low oxygen environment) was added at the same time, the dialysis bag was soaked in 40mL of PBS release medium containing Tween 80 after being sealed, and samples were taken at fixed time points. And after 12h of release, adding hydrogen peroxide into the dialysis bag, putting the dialysis bag into a release medium for continuous release, sampling at a fixed time point, measuring the corresponding drug concentration by using HPLC (high performance liquid chromatography), and calculating the drug release amount. The drug release profile is shown in figure 26. Under the condition of simulating tumor hypoxia, the low-oxygen response medicine in the assembly is released quickly, the chemotherapeutic medicine is released slowly, and the release of the chemotherapeutic medicine is accelerated obviously by adding hydrogen peroxide to simulate the active oxygen condition.
Third, in vitro cytotoxicity determination of drug-resistant tumor cells by using combined drug hybrid nano-assembly
4T1 CSCs at 5X 10 per well 3 The density of each cell is inoculated in a 96-hole ultra-low adhesion culture plate, hybrid nano-assemblies (r-VERP/r-CPTP/NA (Hf)) or r-CSAP/r-DOXP/NA (Hf)) containing different drug concentrations are added, the mixture is placed in a low-oxygen incubator for culturing for 48 hours, and the cell activity is detected by using a CCK-8 detection reagent. The cell survival rate results are shown in FIG. 27, and through simulation calculation, the IC50 values of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) are 0.5293 and 0.1731 mu M respectively, which shows that the combined drug hybrid nano-assembly reverses the drug resistance of tumor stem-like cells and has obvious killing effect.
In vivo anti-tumor activity determination of combined drug hybrid nano assembly
4T1 CSCs were harvested and resuspended in a 1:1 volume ratio of PBS to Matrigel mixture at 5X 10 5 The density of each cell/cell is inoculated under the left breast pad of Balb/c mouse with unhaired abdomen to construct toleranceMouse model of drug tumor. Tumor-bearing mice were randomly grouped, six mice per group, and the tail vein was injected with a combination drug hybrid nano-assembly preparation (r-VERP/r-CPTP/NA (Hf) (r-CPTP 4mg/kg, r-VERP 24mg/kg) or r-CSAP/r-DOXP/NA (Hf) (r-DOXP 4mg/kg, r-CSAP 24mg/kg)), once every two days for 14 days. The long diameter and the short diameter of the tumor are measured every two days, the tumor volume is calculated, and the change of the tumor volume is detected. The results are shown in fig. 28, where the combination drug hybrid nano-assembly preparation group had the optimal tumor suppression effect.
In addition to the above, other embodiments of the present invention are also possible. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims (10)

1. The composite medicine metal organic hybrid nano assembly is characterized in that: formed by ionic interaction of the combined medicine and metal ions;
the combination drug is the combination of an antiviral drug and an anti-inflammatory and liver-protecting drug, or the combination of a chemotherapeutic drug and a chemotherapeutic sensitizer;
the metal ion is selected from Mg 2+ 、Ca 2+ 、ZrO 2+ 、HfO 2+ 、Mn 2+ 、Fe 2+ 、Fe 3+ 、Cu 2+ 、Cu + 、Zn 2+ 、Pt 2+ 、Pt 4+ 、Pt 6+ 、Gd 3+ 、GdO + 、Ag + ；
The drug molecules of the antiviral drug, the anti-inflammatory and liver-protecting drug, the chemotherapeutic drug and the chemotherapeutic sensitizer contain phosphonic acid group-PO 3 H 2 Or phosphoric acid group-OPO 3 H 2 Or chemically modified to contain a phosphate group-OPO 3 H 2 。
2. The combination drug metal-organic hybrid nano-assembly of claim 1, characterized in that: the antiviral drug is selected from tenofovir, entecavir, lamivudine, telbivudine or adefovir.
3. The combination drug metal-organic hybrid nano-assembly of claim 1, characterized in that: the anti-inflammatory and hepatoprotective agent is selected from glycyrrhizic acid, glycyrrhetinic acid, magnesium isoglycyrrhizinate, ammonium glycyrrhizinate, diammonium glycyrrhizinate, silymarin, bicyclol, tiopronin or penicillamine.
4. The combination drug metal-organic hybrid nano-assembly of claim 1, characterized in that: the chemotherapeutic drug is selected from camptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, topotecan, vinblastine, vincristine, vinorelbine, vindesine, cytarabine, gemcitabine, ancitabine, 5-fluorouracil, fluorodeoxyuridylic acid, 6-mercaptopurine, methotrexate, doxorubicin, epirubicin, doxorubicin, daunorubicin, mitoxantrone, paclitaxel, docetaxel, nimustine, teniposide, etoposide, aminoglutethimide or melphalan.
5. The combinatorial drug metal-organic hybrid nanoassembly according to claim 1, characterized in that: the chemosensitizer is selected from all-trans retinoic acid, 1, 3-cis retinoic acid, 9-cis retinoic acid, retinol, retinal, vismodegib, arsenic trioxide, arsenic sulfide, gallopamil, quinidine, verapamil, chlorpromazine, cyclosporine A, reserpine, digoxin, amiodarone, progesterone, wood flavone, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valcephradine, bilactadine, eticride, carfluquinad, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopag, fumonisin C, lapatinib, novobiocin, sulfasalazine or febuxostat.
6. The combination drug metal-organic hybrid nano-assembly of claim 1, characterized in that: the drug molecules of the antiviral drug, the anti-inflammatory and liver-protecting drug, the chemotherapeutic drug and the chemotherapeutic sensitizer and the modified phosphate group-OPO 3 H 2 Therein is provided withA responsive linker arm.
7. The combination drug metal-organic hybrid nano-assembly of claim 1, characterized in that: the combined medicine metal-organic hybrid nano assembly also contains pharmaceutically acceptable auxiliary materials.
8. The combination drug metal-organic hybrid nano-assembly of claim 7, characterized in that: the pharmaceutically acceptable adjuvants are selected from polyethylene glycol, polyvinylpyrrolidone, poloxamer, polyvinyl alcohol, polyoxazoline, vitamin E polyethylene glycol succinate, cholesterol, soybean lecithin, Igepal CO-520 or cetyl trimethyl ammonium bromide.
9. Use of the combination drug metal organic hybrid nano-assembly of claim 1 in the preparation of a chronic hepatitis b treatment drug.
10. The use of the combination drug metal organic hybrid nano-assembly of claim 1 in the preparation of a drug for treating tumor.
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