CN115120561B - Combined medicine metal organic hybridization nano assembly and application thereof - Google Patents

Combined medicine metal organic hybridization nano assembly and application thereof Download PDF

Info

Publication number
CN115120561B
CN115120561B CN202210769044.5A CN202210769044A CN115120561B CN 115120561 B CN115120561 B CN 115120561B CN 202210769044 A CN202210769044 A CN 202210769044A CN 115120561 B CN115120561 B CN 115120561B
Authority
CN
China
Prior art keywords
drug
medicine
assembly
hybrid nano
combined
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210769044.5A
Other languages
Chinese (zh)
Other versions
CN115120561A (en
Inventor
莫然
张经纬
王广基
董鹤
洪晓丹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Pharmaceutical University
Original Assignee
China Pharmaceutical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by China Pharmaceutical University filed Critical China Pharmaceutical University
Priority to CN202210769044.5A priority Critical patent/CN115120561B/en
Publication of CN115120561A publication Critical patent/CN115120561A/en
Application granted granted Critical
Publication of CN115120561B publication Critical patent/CN115120561B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses a metal organic hybrid nano-assembly of a combined drug and application thereof, belonging to the technical field of biological medicine. The hybrid nano-assembly is formed by ionic interaction between a combined drug and metal ions; the combined medicine is a combination of an antiviral medicine and an anti-inflammatory liver-protecting medicine or a combination of a chemotherapeutic medicine 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 hybrid nano-assembly is prepared by simply mixing the organic components and the metal ions, so that the in-vivo half-life of the drug can be prolonged, the concentration of the drug at a focus part can be improved, the fixed drug proportion can be maintained, and synchronous pharmacokinetic and tissue distribution can be realized, thereby enhancing the synergistic treatment effect of the combined drug.

Description

Combined medicine metal organic hybridization nano assembly and application thereof
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a metal organic hybrid nano-assembly of a combined medicine and application thereof.
Background
Clinical practice and exploratory studies in the last decade have shown that a single treatment modality is not effective in treating some physiologically complex refractory diseases. Thus, the methods of treatment for refractory diseases have shifted from monotherapy to combination therapy, increasing the efficiency of available treatment regimens. Combination therapy is a widely used disease treatment strategy that increases the possible therapeutic gain by combining existing disease-modifying therapies or new drugs, with acceptable theoretical basis, good safety and efficacy profiles. Combination therapy may alter different biological pathways, reduce the effective therapeutic dose required, reduce resistance, and reduce the overall cost of treatment.
Chronic hepatitis b is a chronic disease caused by infection with hepatitis b virus, and is currently incurable. Persistent hepatitis b virus infection causes liver inflammation, resulting in liver fibrosis and cirrhosis, and eventually, liver cancer is a life hazard. The nucleoside antiviral drugs widely used in clinic can effectively inhibit the replication of viruses for a long time to inhibit the exacerbation of hepatitis B, but are ineffective to treat chronic inflammation caused by virus invasion. Clinically, the intermittent administration of antiviral drugs and liver protection drugs is not an ideal combined administration mode, and needs to be improved. The co-delivery of antiviral and anti-inflammatory liver-protecting drug combinations for the combination treatment of chronic hepatitis b may ameliorate the deficiencies of existing therapeutic strategies.
Malignant tumors are a serious disease that threatens human health and life, and tumor stem-like cells (CSCs) are a heterogeneous cell with self-renewal and self-differentiation capabilities in tumor tissues. Studies have shown that CSCs are important factors in causing tumor chemotherapy resistance, recurrence and metastasis. In order to overcome tumor resistance, a combination treatment strategy of chemosensitizer and chemotherapeutic drug utilizes chemosensitizer to reduce the chemoresistance of resistant tumor, and shows enhanced therapeutic effect.
The realization of combination therapies requires the efficient integration of multiple therapeutic agents in a single carrier rather than simple mixing to produce beneficial synergistic therapeutic effects and maintain the specificity and persistence of the combination therapy. The rapidly evolving biomaterials and nanotechnology provide a research basis for co-delivery of co-drugs, making it possible to co-load several drugs into one delivery vehicle by simple physical adsorption or chemical interactions without loss of drug activity. The nano carrier can prolong the in vivo half-life of the medicine, is favorable for delivering the medicine to the focus part, enhances the treatment effect and reduces the toxic and side effects. The two medicaments are loaded in the same nano carrier, so that different medicaments are consistent in space and time in the treatment process, and synchronous pharmacokinetics and biological distribution are realized, and a synergistic treatment effect can be achieved. Although some nanocarriers (including liposomes, polymer nanoparticles, dendrimers, and inorganic nanoparticles) are currently developed for drug combination delivery, the problems of low drug loading rate, complex synthesis/preparation methods, and the like, which exist in practice, limit the application of the nanocarriers in combination drug delivery.
Therefore, there is still a need to develop a carrier platform with simple preparation method, high drug loading rate and capable of loading different combination drugs for co-delivery of the combination drugs.
Disclosure of Invention
The invention aims to provide a hybrid nano-assembly which is co-assembled by a combined drug and metal ions.
Another object of the present invention is to provide an application of the above hybrid nano-assembly in preparing a drug for treating chronic hepatitis b or tumor.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the metal organic hybrid nano-assembly of the combined medicine is formed by interaction of positive and negative ions of the combined medicine and metal ions;
the combined medicine is the combination of antiviral medicine and anti-inflammatory liver-protecting medicine or the combination of chemotherapeutic medicine and 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 medicine molecules of the antiviral medicine, the anti-inflammatory liver-protecting medicine, the chemotherapeutic medicine and the chemotherapeutic sensitizer contain phosphonic acid groups-PO 3 H 2 Or a phosphate group-OPO 3 H 2 Or modified with phosphate 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 liver protecting agent is selected from glycyrrhizic acid, glycyrrhetinic acid, magnesium isoglycyrrhetate, ammonium glycyrrhetate, diammonium glycyrrhetate, silymarin, bicyclo-ethanol, tiopronin, or penicillamine.
In some embodiments, the chemotherapeutic agent is selected from the group consisting of 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, and melphalan.
In some embodiments, the chemosensitizer is selected from all-trans retinoic acid, 1, 3-cis retinoic acid, 9-cis retinoic acid, retinol, retinal, vitamin d, arsenic trioxide, arsenic sulfide, gossypol, quinidine, verapamil, chlorpromazine, cyclosporin a, reserpine, digoxin, amiodarone, progesterone, genistein, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valpuidad, brinodad, exendid, oxazolidad, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopa, fumagillin C, lapatinib, novobiocin, sulfasalazine, or febuxostat.
In some embodiments, the drug molecule of the antiviral drug, anti-inflammatory liver protecting drug, chemotherapeutic sensitizer, and modified phosphate group-OPO 3 H 2 With a responsive linkage arm therebetween.
In some embodiments, the combination drug metal-organic hybrid nano-assembly further comprises a pharmaceutically acceptable adjuvant.
In some embodiments, the adjuvant 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 metal organic hybrid nano-assembly of the combined medicine in preparing a medicine for treating chronic hepatitis B, so as to be used for antiviral, anti-inflammatory and liver-protecting combined treatment of the chronic hepatitis B.
The invention also provides application of the metal organic hybrid nano-assembly of the combined medicine in preparing tumor treatment medicines, so as to treat common tumors or dry related drug-resistant tumors.
The metal organic hybrid nano assembly is a nano particle formed by assembling organic molecules and metal ions through ionic interaction, and the preparation method provided by the invention has the advantages of high drug loading capacity, simplicity in preparation and the like by taking the combined drug as an organic component and simply mixing the organic component and the metal ions. Because the phosphoric acid organic molecules and metal ions have strong acting force, the hybridized nano assembly is easy to form. In order to co-load the combination drug in the hybrid nano-assembly, it is desirable that the drug bear a phosphonic acid group (-PO) 3 H 2 ) Or a phosphate group (-OPO) 3 H 2 ) Or by chemically modifying the phosphate group (-OPO) 3 H 2 ) So that the hybrid nano-assembly can be assembled with metal ions to form the hybrid nano-assembly of the combined drug. As shown in fig. 1.
The combined medicine is co-carried in the nanometer assembly, so that the in vivo half-life period of the medicine can be prolonged, the concentration of the medicine at a focus part can be improved, the fixed medicine proportion can be maintained, and synchronous pharmacokinetics and tissue distribution can be realized, so that the synergistic treatment effect of the combined medicine is enhanced. Meanwhile, under the stimulation of focus microenvironment physiological signals (such as high-expression phosphatase, hypoxia, acid and high oxidation/reduction), the chemically modified phosphate group is subjected to responsive degradation to fall off, so that the proto-drug is released, and the activity of the drug is ensured.
Drawings
FIG. 1 is a schematic illustration of the preparation of hybrid drug combinations and their principle of responsive drug release.
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 characterization of AFV/SMP/NA (Fe) in example 1.
FIG. 4 shows in vitro drug delivery of TFV/GAP/NA (Zr), AFV/GAP/NA (Mg), AFV/SMP/NA (Fe) and TFV/SMP/NA (Cu) in example 1.
FIG. 5 shows the in vitro safety of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 6 shows the in vitro anti-inflammatory activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 7 shows in vitro antiviral activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 8 shows in vivo drug liver tissue distribution of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 9 shows in vivo antiviral activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 10 shows the in vivo anti-inflammatory activity of TFV/GAP/NA (Zr) and AFV/SMP/NA (Fe) in example 1.
FIG. 11 is a TEM characterization of LAMP/SMP/NA (Hf) in example 2.
FIG. 12 shows in vitro drug delivery of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) in example 2.
FIG. 13 is the in vitro safety profile of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 14 shows the in vitro anti-inflammatory activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 15 shows the in vitro antiviral activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 16 shows in vivo drug liver tissue distribution of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) in example 2.
FIG. 17 shows in vivo antiviral activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) in example 2.
FIG. 18 shows the in vivo anti-inflammatory activity of TFV/GAP/NA (Hf) and ETVP/SMP/NA (Hf) of example 2.
FIG. 19 is TEM and HAADF-STEM characterization of r-ATRAP/Fdump/NA (Hf) of example 3.
FIG. 20 is an in vitro responsive drug delivery of r-ATRAP/Fdump/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) in example 3.
FIG. 21 is an in vitro responsive drug delivery of r-ATRAP/Fdump/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) in example 3.
FIG. 22 shows the in vitro antitumor activity of r-ATRAP/Fdump/NA (Hf) and r-CRAP/r-CPTP/NA (Hf) in example 3.
FIG. 23 shows 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 an in vitro responsive drug delivery of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) in example 4.
FIG. 26 is an in vitro responsive drug delivery of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) in example 4.
FIG. 27 shows the in vitro antitumor Activity of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) in example 4.
FIG. 28 shows the in vivo antitumor activity of r-VERP/r-CPTP/NA (Hf) and r-CSAP/r-DOXP/NA (Hf) in example 4.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
Example 1
Combined drug hybridization nano assembly for chronic hepatitis B combined treatment
1. Modification of antiviral and anti-inflammatory liver-protecting drugs
Entecavir, lamivudine or telbivudine (1.9 mmol) as antiviral agent was dissolved in 5mL trimethyl phosphate, and added to the nitrogen-protected reaction tube, phosphorus oxychloride (0.33 mL,3.8 mmol) was added dropwise to the above solution, and reacted at 4℃for 16h. After the reaction, 10mL of water was added to the reaction solution to quench the reaction, the mixture was stirred for 30min, the reaction solution was extracted 10 times with ethyl acetate, the aqueous phase was collected and mixed with methanol to spin dry, and an oily product was obtained. Purifying by column chromatography to obtain phosphorylated entecavir, lamivudine or telbivudine product.
Taking a 250mL reaction bottle, carrying out anhydrous drying and replacing nitrogen protection, dissolving phosphorus oxychloride (2 mL,14.6 mmol) in 60mL anhydrous tetrahydrofuran, adding the solution into the reaction bottle, adding a mixed solution of anti-inflammatory liver-protecting medicament glycyrrhetinic acid, silymarin or dicyclo alcohol (6.45 mmol) in 5.1mL anhydrous pyridine and 30mL anhydrous tetrahydrofuran dropwise under ice bath condition, keeping the temperature of 0 ℃ for reaction for 30min, and transferring to room temperature for reaction for 2h. After the reaction, the solvent was removed by rotary evaporation under reduced pressure, and the oily residue was dissolved in 100mL of methylene chloride, washed with 1M hydrochloric acid solution, washed with water, saturated brine, and the organic phase was collected and dried over anhydrous sodium sulfate. Filtering, removing solvent by rotary evaporation of organic phase to obtain oily crude product, and purifying by column chromatography to obtain phosphorylated glycyrrhetinic acid, silymarin or bicyclo-ethanol product.
2. Preparation of hybrid nano-assembly of combined medicine
The antiviral drug and the anti-inflammatory drug are mixed and assembled together with metal ions in aqueous solution to form the combined drug hybridized nano-assembly.
Tenofovir (TFV), adefovir (AFV) or phosphorylated Entecavir (ETVP) in 2% aqueous F-68 solution was formulated as 21mM solution A, and phosphorylated Glycyrrhetinic Acid (GAP) or phosphorylated Silymarin (SMP) in tetrahydrofuran was formulated as 18mM solution B. 1mL of the solution A and 0.2mL of the solution B are added dropwise into 1mL of 18mM zirconium oxychloride 2%F-68 water solution together, 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. The nano-assembly solution is placed in a dialysis bag and dialyzed 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 (re-suspension + washing) twice with deionized water. Finally, the nano-assemblies were resuspended in physiological saline and the particle size and polydispersity index (PDI) of the assemblies were characterized using a particle size analyzer, and the results are shown in table 1. TFV/GAP/NA (Zr) was selected for morphology characterization by Transmission Electron Microscopy (TEM) and for elemental mapping characterization by high angle annular dark field scanning transmission electron microscopy (HAADF-STEM), the results of which are shown in FIG. 2.
Table 1 particle size characterization of combination drug hybrid nano-assemblies
TFV, AFV or ETVP is dissolved in 3mL of water to prepare a solution of 0.3mM, added into 70mL of chloroform, 1.82g of cetyltrimethylammonium bromide and 3mL of n-hexanol are added, GAP or SMP is dissolved in 1mL of tetrahydrofuran to prepare a solution of 0.2mM, added into the solution, stirred for 30min at 35 ℃, and 1.6mL of MgCl with the concentration of 0.5M is added 2 (or CaCl) 2 、CuCl 2 、FeCl 3 ) The aqueous solution forms a hybrid nano-assembly body TFV/GAP/NA (Mg), AFV/GAP/NA (Mg), ETVP/GAP/NA (Ca), TFV/SMP/NA (Cu), AFV/SMP/NA (Fe) and ETVP/SMP/NA (Fe) of the combined medicament, and the mixed medicament is continuously stirred for 12 hours at room temperature, and is continuously centrifuged and resuspended for 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, the results of which are shown in table 2. AFV/SMP/NA (Fe) was selected for morphology characterization by TEM (FIG. 3).
Table 2 particle size characterization of combination drug hybrid nano-assemblies
3. Drug release of combination drug hybrid nano-assemblies
To simulate the drug release conditions at the liver site, 1mL of the combined drug hybrid nano-assembly (TFV/GAP/NA (Zr), AFV/GAP/NA (Mg), AFV/SMP/NA (Fe) or TFV/SMP/NA (Cu)) was added to the dialysis bag, while 1U/mL of phosphatase (simulating the hepatitis b high phosphatase environment) was added, the dialysis bag was sealed and immersed in 40mL of PBS release medium containing tween 80, sampled at fixed time points, the corresponding drug concentration was measured with LC-MS/MS, and the drug release amount was calculated. The drug release profile is shown in figure 4. Under the condition of simulating the liver high phosphatase level, the phosphatase hydrolyzes the phosphate group, and the antiviral drugs and the anti-inflammatory liver-protecting drugs in the hybrid nano-assembly can be quickly released.
4. In vitro safety and anti-inflammatory Activity assays for hybrid nanocomposites of combination drugs
1. In vitro safety investigation of hybrid nano-assemblies of combination drugs
RAW264.7 cells and L-02 cells were each used in 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)) with different drug concentrations was added, and incubated in a cell incubator for 24h and 48h, respectively, and the cell activity was detected with CCK-8 detection reagent. The cell viability results are shown in fig. 5, and after the hybrid nano-assemblies with different drug concentrations are incubated for 24 hours and 48 hours, the cell activity is not obviously reduced compared with that of the blank group, which indicates that the hybrid nano-assemblies of the combined drugs are all biosafety.
2. In vitro anti-inflammatory activity investigation of hybrid nano-assembly of combined drug
RAW264.7 cells were plated at 4X 10 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 40. Mu.M) and LPS (1. Mu.g/mL) was added, after 24h incubation in an incubator, cell supernatants were collected, and after reasonable dilution, inflammatory factor levels were measured with TNF-alpha, IL-1. Beta. And IL-6ELISA kits to examine the in vitro anti-inflammatory activity of GAP. The in vitro anti-inflammatory results are shown in FIG. 6, where cells were addedThe level of inflammatory factors is obviously increased after LPS, and the level of inflammatory factors is obviously reduced after different combined drug hybridized nano-assemblies are added, which indicates that the combined drug hybridized nano-assemblies have good anti-inflammatory activity.
5. In vitro antiviral activity assay for hybrid nano-assemblies of combination drugs
HepG2.2.15 cells were used 4X 10 per well 4 The density of individual cells was seeded in 96-well plates and after 24h of culture, the medium was aspirated. Adding new culture medium containing hybridized nano assembly (TFV/GAP/NA (Zr) or AFV/SMP/NA (Fe)), incubating in incubator every 3 days, changing new culture medium containing medicine, observing cell state at 3, 6, 9 days, and sucking supernatant for HBV DNA assay. HBV DNA was extracted using a universal genomic DNA extraction kit and virus copy number detection was performed with Taqman probes in a CFX96 Real-time PCR detection system (Bio-Rad). The in vitro antiviral results are shown in figure 7, which shows that the hybrid nano-assemblies of the combination drugs have good continuous inhibition effect on hepatitis B virus.
6. In vivo pharmacokinetic investigation of combination drug hybrid nano-assemblies
C57BL/6 mice were randomly grouped into 10 groups of 10 mice each, tail vein injection combined drug hybrid nano-assembly formulations (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3 mg/kg)) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3 mg/kg)) and the corresponding free combined drug (tfv+gap or afv+smp), at predetermined interval time points, five mice per group were bled from the orbits, and the other five mice were bled at adjacent interval time points, and analysis was performed with LC-MS/MS system after sample treatment to determine the amount of drug. The pharmacokinetic parameters were calculated using a non-compartmental model of Phoenix WinNonlin kinetic software, and the results are shown in tables 3 and 4, wherein each parameter of the pharmacokinetics of the hybrid nano-assembly preparation group is obviously improved compared with the corresponding free drug group, and the in vivo half-life of the drug is obviously prolonged.
TABLE 3 pharmacokinetic parameters of TFV/GAP/NA (Zr) and free (TFV+GAP)
TABLE 4 pharmacokinetic parameters of AFV/SMP/NA (Fe) and free (AFV+SMP)
7. Liver tissue drug distribution investigation of combined drug hybrid nano-assembly
C57BL/6 mice were randomly grouped into 35 groups of each group, a combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3 mg/kg)) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3 mg/kg)) and a free combination drug (tfv+gap or afv+smp) corresponding to the same concentration were injected into the tail vein, and at a predetermined time point, five mice were sacrificed in each group, livers were collected, 0.1g of liver tissues were weighed after washing, and after grinding treatment, drug concentration in the livers was measured by LC-MS/MS. As shown in fig. 8, the hybrid nano-assembly formulation group can significantly promote drug accumulation in the liver compared to the corresponding free drug.
8. In vivo antiviral activity investigation of hybrid nano-assembly of combined drug
The HBV gene transfected C57BL/6 mice were randomly grouped into 5 groups, and the combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3 mg/kg) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3 mg/kg)) was administered once every three days for 15 consecutive days. At days 3, 6, 9, 12, 15 after the first dose, respectively, the mice orbits were bled and serum isolated, HBV DNA extraction was performed, and virus copy number detection was performed using 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 collect supernatant, and HBV copy number in livers was determined by PCR method. As shown in FIG. 9, the continuous administration of the hybrid nano-assembly preparation can obviously inhibit HBV levels in blood and liver, which indicates that the hybrid nano-assemblies of the combined medicines have optimal continuous inhibition effect on hepatitis B virus.
9. Investigation of in vivo anti-inflammatory Activity of hybrid nano-Assembly of Combined drug
C57BL/6 mice were randomly grouped into 6 groups, and tail vein injection of the combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Zr) (TFV 6mg/kg, GAP 3 mg/kg) or AFV/SMP/NA (Fe) (AFV 6mg/kg, SMP3 mg/kg)) was administered once every three days for 15 consecutive days. After 2 hours from the last administration, mice in the administration group were intraperitoneally injected with 0.2% carbon tetrachloride/corn oil solution at a carbon tetrachloride administration dose of 32mg/kg to induce an acute liver injury model. 20 hours after carbon tetrachloride injection, all mice were bled from the orbit, and the mice were sacrificed and liver tissue was collected. The collected blood samples were centrifuged and the supernatant serum was taken, after appropriate dilution, and inflammatory factor levels were measured using TNF- α, IL-1 β, and IL-6ELISA kits. After homogenizing liver tissue in 0.1g, TNF-alpha, IL-1 beta and IL-6ELISA kit are used to measure the level of inflammatory factor. As shown in fig. 10, after the liver injury is induced by carbon tetrachloride, the inflammatory factor level in blood and liver is obviously increased, and the pre-administration of the hybrid nano-assembly preparation obviously reduces the inflammatory factor level in blood and liver, which indicates that the hybrid nano-assembly of the combined medicines has the optimal anti-inflammatory and liver-protecting effects.
Example 2
Combined drug hybridization nano assembly for chronic hepatitis B combined treatment
1. Preparation of hybrid nano-assembly of combined medicine
The antiviral drug and the anti-inflammatory drug are mixed and assembled together with metal ions in aqueous solution to form the combined drug hybridized nano-assembly.
TFV, ETVP or phosphorylated Lafumipdine (LAMP) is dissolved in 2% PVP aqueous solution to prepare 21mM A solution, GAP or SMP is dissolved in tetrahydrofuran to prepare 18mM B solution, 1mL of A solution and 0.2mL of B solution are jointly dripped into 1mL of 18mM hafnium tetrachloride 2% PVP aqueous solution, and after stirring for 15min, the 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 LAMP/SMP/NA (Hf) is prepared, the assembly solution is placed in a dialysis bag, and residual small molecule drugs are removed by dialysis in deionized water overnight. The solution in the dialysis bag is centrifuged at a high speed and the white assembly nanoparticles are deposited on the bottom of the centrifuge tube. The supernatant was discarded, the precipitate was washed with deionized water and centrifuged twice. Finally, the hybrid nano-assembly of the combined drug was resuspended in physiological saline and the particle size of the assembly was characterized by a potentiometric particle size analyzer, and the results are shown in table 5. LAMP/SMP/NA (Hf) was chosen for morphology characterization by TEM (FIG. 11).
Table 5 particle size characterization of combination drug hybrid nano-assemblies
2. Drug release of combination drug hybrid nano-assemblies
1mL of the combined drug hybrid nano-assembly (TFV/GAP/NA (Hf) or ETVP/SMP/NA (Hf)) was added to a dialysis bag, 1U/mL of phosphatase (simulating the hepatitis B high phosphatase environment) was added at the same time, the dialysis bag was sealed and immersed in 40mL of PBS release medium containing Tween 80, sampling was performed at a fixed time point, the corresponding drug concentration was measured by LC-MS/MS, and the drug release amount was calculated. The drug release profile is shown in figure 12. Antiviral drugs and anti-inflammatory liver-protecting drugs in the hybrid nano-assembly can be gradually released under the condition of simulating the liver high phosphatase level.
3. In vitro safety and anti-inflammatory Activity assays for hybrid nanocomposites of combination drugs
1. In vitro safety investigation of hybrid nano-assemblies of combination drugs
RAW264.7 cells and L-02 cells were each used in 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 hybridized nano-assemblies (TFV/GAP/NA (Hf) or ETVP/SMP/NA (Hf)) with different drug concentrations was added, and incubated in a cell incubator for 24h and 48h, respectively, and cell activity was detected with CCK-8 detection reagent. The cell viability results are shown in fig. 13, and after the hybrid nano-assemblies with different drug concentrations are incubated for 24h and 48h, the cell activity is not obviously reduced compared with that of the blank group, which indicates that the hybrid nano-assemblies of the combined drugs are all biosafety.
2. In vitro anti-inflammatory activity investigation of hybrid nano-assembly of combined drug
RAW264.7 cells were plated at 4X 10 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 40. Mu.M) and LPS (1. Mu.g/mL) was added, after 24h incubation in an incubator, cell supernatants were collected, and after reasonable dilution, inflammatory factor levels were measured using TNF-. Alpha., IL-1. Beta., IL-6ELISA kit to examine the in vitro anti-inflammatory activity of GAP. 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 hybridized nano-assemblies are added, so that the combined drug hybridized nano-assemblies have optimal anti-inflammatory activity.
4. In vitro antiviral activity assay for hybrid nano-assemblies of combination drugs
HepG2.2.15 cells were used 4X 10 per well 4 The density of individual cells was seeded in 96-well plates and after 24h of culture, the medium was aspirated. Adding new culture medium containing hybridized nano assembly (TFV/GAP/NA (Hf) or ETVP/SMP/NA (Hf), TFV or ETV being 154 μm), incubating in incubator every 3 days for new culture medium containing medicine, observing cell state at 3, 6, 9 days respectively, and sucking supernatant for HBV DNA assay analysis. HBV DNA was extracted using a universal genomic DNA extraction kit and virus copy number detection was performed with Taqman probes in a CFX96 Real-time PCR detection system (Bio-Rad). The in vitro antiviral results are shown in figure 15, which shows that the hybrid nano-assemblies of the combination drugs have the optimal sustained inhibition effect on hepatitis B virus.
5. In vivo pharmacokinetic investigation of combination drug hybrid nano-assemblies
The C57BL/6 mice were randomly grouped into 10 groups of 10 mice each, and the tail vein injection combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3 mg/kg)) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3 mg/kg)) and the corresponding free combination drug (tfv+gap or etv+smp), and at predetermined interval time points, the orbitals of five mice of each group were bled, and the other five mice were bled at adjacent interval time points, and analysis was performed by LC-MS/MS system after sample treatment to determine the amount of drug. The pharmacokinetic parameters were calculated using a non-compartmental model of Phoenix WinNonlin kinetic software, and the results are shown in tables 6 and 7, wherein each parameter of the pharmacokinetics of the hybrid nano-assembly preparation group is obviously improved compared with the corresponding free drug group, and the in vivo half-life of the drug is obviously prolonged.
TABLE 6 pharmacokinetic parameters of TFV/GAP/NA (Hf) and free (TFV+GAP)
TABLE 7 pharmacokinetic parameters of ETVP/SMP/NA (Hf) and free (ETV+SMP)
6. Liver tissue drug distribution investigation of combined drug hybrid nano-assembly
C57BL/6 mice were randomly grouped into 35 groups of each group, a combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3 mg/kg)) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3 mg/kg)) and a free combination drug (tfv+gap or etvp+smp) corresponding to the same concentration were injected into the tail vein, and at a predetermined time point, five mice were sacrificed in each group, livers were collected, 0.1g of liver tissues were weighed after washing, and after grinding treatment, drug concentration in the livers was measured by LC-MS/MS. As shown in fig. 16, the hybrid nano-assembly formulation group can significantly promote drug accumulation in the liver compared to the corresponding free drug.
7. In vivo antiviral activity investigation of hybrid nano-assembly of combined drug
HBV gene transfected C57BL/6 mice were randomly grouped into 5 groups, and the combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3 mg/kg) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3 mg/kg)) was administered once every three days for 15 consecutive days. At days 3, 6, 9, 12, 15 after the first dose, respectively, the mice orbits were bled and serum isolated, HBV DNA extraction was performed, and virus copy number detection was performed using 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 collect supernatant, and HBV copy number in livers was determined by PCR method. As shown in FIG. 17, the continuous administration of the hybrid nano-assembly preparation can obviously inhibit HBV levels in blood and liver, which indicates that the hybrid nano-assemblies of the combined medicaments have the optimal continuous inhibition effect on hepatitis B virus.
8. Investigation of in vivo anti-inflammatory Activity of hybrid nano-Assembly of Combined drug
The C57BL/6 mice were randomly grouped into 6 groups, and the tail vein injection combination drug hybrid nano-assembly preparation (TFV/GAP/NA (Hf) (TFV 6mg/kg, GAP 3 mg/kg) or ETVP/SMP/NA (Hf) (ETVP 6mg/kg, SMP3 mg/kg)) was administered once every three days for 15 consecutive days. After 2 hours from the last administration, mice in the administration group were intraperitoneally injected with 0.2% carbon tetrachloride/corn oil solution at a carbon tetrachloride administration dose of 32mg/kg to induce an acute liver injury model. 20 hours after carbon tetrachloride injection, all mice were bled from the orbit, and the mice were sacrificed and liver tissue was collected. The collected blood samples were centrifuged and the supernatant serum was taken, after appropriate dilution, and inflammatory factor levels were measured using TNF- α, IL-1 β, and IL-6ELISA kits. After homogenizing liver tissue in 0.1g, TNF-alpha, IL-1 beta and IL-6ELISA kit are used to measure the level of inflammatory factor. As shown in fig. 18, after the liver injury is induced by carbon tetrachloride, the inflammatory factor level in blood and liver is obviously increased, and the pre-administration of the hybrid nano-assembly preparation obviously reduces the inflammatory factor level in blood and liver, which indicates that the hybrid nano-assembly of the combined medicines has the optimal anti-inflammatory and liver-protecting effects.
Example 3
Combined drug hybridized nano assembly for drug-resistant tumor combined treatment
1. Modification of chemotherapeutic drugs and chemosensitizer/differentiation inducer
Chemotherapeutic drug modification
5-fluoro-2' -deoxyuridine, camptothecin, doxorubicin or taxol (1.9 mmol) were dissolved in 5mL trimethyl phosphate, added to a nitrogen-protected reaction tube, phosphorus oxychloride (0.33 mL,3.8 mmol) was added dropwise to the above solution, and reacted at 4℃for 16h. After the reaction, 10mL of water was added to the reaction solution to quench the reaction, the mixture was stirred for 30min, the reaction solution was extracted 10 times with ethyl acetate, the aqueous phase was collected and mixed with methanol to spin dry, and an oily product was obtained. Column chromatography gives the phosphorylated product.
Phosphorylated hypoxia responsive differentiation inducer synthesis
All-trans retinoic acid, 1, 3-cis retinoic acid or 9-cis retinoic acid (10 mmol) was dissolved in a mixed solution of 100mL of methylene chloride and 6mL of dimethyl sulfoxide, 4- (4- (hydroxymethyl) phenyl) diazenyl) phenol (3.16 g,10 mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (2.4 g,12 mmol), 1-hydroxybenzotriazole (1.62 g,12 mmol), N, N-diisopropylethylamine (5 mL) was added and reacted overnight at room temperature. After the reaction is finished, the reaction liquid is washed with water, saturated brine is washed with water, anhydrous sodium sulfate is dried, an organic phase is dried by spin to obtain a crude product, and the azobenzene modified product is obtained by column chromatography.
Phosphorus oxychloride (0.2 mL,2.3 mmol) was dissolved in 2mL of anhydrous THF, added to a nitrogen-protected reaction tube and placed under an ice-water mixed bath at 0 ℃, and a mixed solution of azobenzene-modified differentiation inducer (0.67 mmol) dissolved in anhydrous pyridine (0.53 mL,6.5 mmol) and 3mL of anhydrous THF was added dropwise to the above reaction solution to react at 0℃for 3 hours. After the reaction, the reaction solution is dried by spin, the residue is dissolved in ethyl acetate, the organic phase is washed by water, diluted hydrochloric acid solution is washed by saturated saline water, the organic phase is dried by anhydrous sodium sulfate and filtered, and the crude product is obtained by spin drying. Column chromatography gives phosphorylated hypoxia response product.
2. Preparation of hybrid nano-assembly of combined medicine
Fluorodeoxyuridylic acid (FdUMP), active oxygen responsive phosphorylated camptothecin (r-CPTP) or phosphorylated doxorubicin (r-DOXP) was dissolved in 5ml of a 2% TPGS solution to prepare a 15mM solution, and the pH was adjusted to weak alkaline (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 a 76mM solution, and 2mL of 3mM hafnium tetrachloride 2% TPGS solution is added into the solution in a dropwise manner, 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 three times by continuous centrifugation, the assembly was suspended in physiological saline, and the particle size of the assembly was characterized by an potentiometric particle size analyzer, and the results are shown in Table 8. The r-ATRAP/FdUMP/NA (Hf) was selected 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
3. Drug release of combination drug hybrid nano-assemblies
1mL of the combined drug hybrid nano-assembly (r-ATRAP/FdUMP/NA (Hf) or r-CRAP/r-CPTP/NA (Hf)) was added to a dialysis bag, 10mM sodium dithionate (simulating a hypoxia 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 measured 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 drugs in the assembly are released rapidly, and the chemotherapeutic drugs are 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, 10mM sodium dithionate was added at the same time, and the dialysis bag was sealed and immersed in 40mL of Tween 80-containing PBS release medium, and samples were taken at fixed time points. After 12h of release, hydrogen peroxide is added into the dialysis bag, the dialysis bag is placed into a release medium for continuous release, sampling is carried out at a fixed time point, the concentration of the corresponding medicine is measured by HPLC, and the release amount of the medicine is calculated. The drug release profile is shown in figure 21. Under the condition of simulating tumor hypoxia, the drugs with hypoxia response in the assembly are released rapidly, the chemotherapeutic drugs are released slowly, and the hydrogen peroxide is added to simulate the condition of active oxygen, so that the release of the chemotherapeutic drugs is obviously accelerated.
4. In vitro cytotoxicity assay of combination drug hybrid nano-assemblies on drug-resistant tumor cells
4T1 CSCs were used 5X 10 per well 3 The density of individual cells was inoculated into 96-well ultra-low adhesion plates, hybrid nano-assemblies (r-ATRAP/FdUMP/NA (Hf) or r-CRAP/r-CPTP/NA (Hf)) containing different drug concentrations were added, and the cells were incubated in a hypoxia incubator for 48 hours, and the cell activity was detected using CCK-8 detection reagents. The cell viability results are shown in FIG. 22, and 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 indicate that the hybrid nano-assemblies of the combined drugs reverse the drug resistance of tumor stem-like cells and have obvious killing effect.
5. In vivo antitumor activity assay of hybrid nano-assemblies of combination drugs
4T1 CSCs were collected and resuspended in a 1:1 volume ratio of PBS and Matrigel mixed solution at 5X 10 5 The density of individual cells/cells was inoculated under the left breast pad of the abdominal dehaired Balb/c mice to construct a drug resistant tumor mouse model. Tumor-bearing mice were randomly grouped into six groups, and the combination drug hybrid nano-assembly formulations (r-ATRAP/FdUMP/NA (Hf) (FdUMP 3.33mg/kg, r-ATRAP 24 mg/kg) or r-CRAP/r-CPTP/NA (Hf) (r-CPTP 3.33mg/kg, r-CRAP 24 mg/kg)) were administered by tail vein injection once every two days for 14 consecutive days. Tumor length and diameter were measured every two days, tumor volume was calculated, and tumor volume changes were detected. The results are shown in fig. 23, and the combined drug hybrid nano-assembly preparation group has the best tumor inhibition effect.
Example 4
Combined drug hybridized nano assembly for drug-resistant tumor combined treatment
1. Preparation of hybrid nano-assembly of combined medicine
The corresponding phosphorylated paclitaxel (r-PTXP) of r-CPTP, r-DOXP or active oxygen was dissolved in 5ml of 2% TPGS solution to prepare a solution with a concentration of 15mM, and pH was adjusted to be weakly alkaline (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 added dropwise thereto together, followed by stirring for 15min to form a combination 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 three times by continuous centrifugation, the assembly was suspended in physiological saline, and the particle size was characterized by an potentiometric particle size analyzer, and the results are shown in Table 13. The morphology characterization was performed by TEM with r-VERP/r-CPTP/NA (Hf) and the results are shown in FIG. 24.
Table 13 particle size characterization of hybrid nano-assemblies of combination drugs
2. Drug release of combination drug hybrid nano-assemblies
1mL of the combined 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 hypoxia 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 measured 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 drugs in the assembly are released rapidly, and the chemotherapeutic drugs are 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 hypoxic environment) was added at the same time, and the dialysis bag was sealed and immersed in 40mL of PBS release medium containing Tween 80, and samples were taken at fixed time points. After 12h of release, hydrogen peroxide is added into the dialysis bag, the dialysis bag is placed into a release medium for continuous release, sampling is carried out at a fixed time point, the concentration of the corresponding medicine is measured by HPLC, and the release amount of the medicine is calculated. The drug release profile is shown in figure 26. Under the condition of simulating tumor hypoxia, the drugs with hypoxia response in the assembly are released rapidly, the chemotherapeutic drugs are released slowly, and the hydrogen peroxide is added to simulate the condition of active oxygen, so that the release of the chemotherapeutic drugs is obviously accelerated.
3. In vitro cytotoxicity assay of combination drug hybrid nano-assemblies on drug-resistant tumor cells
4T1 CSCs were used 5X 10 per well 3 The density of individual cells was inoculated into 96-well ultra-low adhesion plates, hybrid nano-assemblies (r-VERP/r-CPTP/NA (Hf) or r-CSAP/r-DOXP/NA (Hf)) containing different drug concentrations were added, placed in a hypoxia incubator for 48h, and cell activity was detected using CCK-8 detection reagents. The cell viability results are shown in FIG. 27, and 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 indicate that the hybrid nano-assemblies of the combined drugs reverse the drug resistance of tumor stem-like cells and have obvious killing effect.
4. In vivo antitumor activity assay of hybrid nano-assemblies of combination drugs
4T1 CSCs were collected and resuspended in a 1:1 volume ratio of PBS and Matrigel mixed solution at 5X 10 5 The density of individual cells/cells was inoculated under the left breast pad of the abdominal dehaired Balb/c mice to construct a drug resistant tumor mouse model. Tumor-bearing mice were randomly grouped into six groups, and the combination drug hybrid nano-assembly formulations (r-VERP/r-CPTP/NA (Hf) (r-CPTP 4mg/kg, r-VERP 24 mg/kg) or r-CSAP/r-DOXP/NA (Hf) (r-DOXP 4mg/kg, r-CSAP 24 mg/kg)) were administered once every two days for 14 consecutive days. Tumor length and diameter were measured every two days, tumor volume was calculated, and tumor volume changes were detected. The results are shown in fig. 28, where the combination drug hybrid nano-assembly formulation group had the optimal tumor suppression effect.
In addition to the implementations described above, other implementations of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.

Claims (4)

1. The metal organic hybrid nano-assembly of the combined medicine is characterized in that: is formed by ionic interaction between a combination drug and metal ions;
the combined medicine is a combination of an antiviral medicine and an anti-inflammatory liver-protecting medicine;
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 medicine molecule of the antiviral medicine and the anti-inflammatory liver-protecting medicine contains phosphonic acid group-PO 3 H 2 Or a phosphate group-OPO 3 H 2 Or after chemical modification contains phosphate group-OPO 3 H 2 ；
The antiviral drug is selected from tenofovir, entecavir, lamivudine, telbivudine or adefovir;
the anti-inflammatory and liver-protecting medicine is selected from glycyrrhizic acid, glycyrrhetinic acid, magnesium isoglycyrrhetate, ammonium glycyrrhizate, diammonium glycyrrhizate, silymarin, dicyclo-alcohol, tiopronin or penicillamine;
the medicine molecules of the antiviral medicine and the anti-inflammatory liver-protecting medicine and the modified phosphate group-OPO 3 H 2 With a responsive linkage arm therebetween.
2. The combination drug metal-organic hybrid nano-assembly according to claim 1, wherein: the metal organic hybrid nano-assembly of the combined medicine also contains pharmaceutically acceptable auxiliary materials.
3. The combination drug metal-organic hybrid nano-assembly according to claim 2, wherein: the pharmaceutically acceptable auxiliary materials are selected from polyethylene glycol, polyvinylpyrrolidone, poloxamer, polyvinyl alcohol, polyoxazoline, vitamin E polyethylene glycol succinate, cholesterol, soybean lecithin, igepal CO-520 or hexadecyl trimethyl ammonium bromide.
4. Use of the combination drug metal organic hybrid nano-assembly of claim 1 in the preparation of a medicament for the treatment of chronic hepatitis b.
CN202210769044.5A 2022-06-30 2022-06-30 Combined medicine metal organic hybridization nano assembly and application thereof Active CN115120561B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210769044.5A CN115120561B (en) 2022-06-30 2022-06-30 Combined medicine metal organic hybridization nano assembly and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210769044.5A CN115120561B (en) 2022-06-30 2022-06-30 Combined medicine metal organic hybridization nano assembly and application thereof

Publications (2)

Publication Number Publication Date
CN115120561A CN115120561A (en) 2022-09-30
CN115120561B true CN115120561B (en) 2023-12-19

Family

ID=83381318

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210769044.5A Active CN115120561B (en) 2022-06-30 2022-06-30 Combined medicine metal organic hybridization nano assembly and application thereof

Country Status (1)

Country Link
CN (1) CN115120561B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1518983A (en) * 2003-01-22 2004-08-11 赖庆莲 Coordinated modication combination for anti hepatitis B virus
CN1895671A (en) * 2005-12-08 2007-01-17 淮北市辉克药业有限公司 Compound preparation for treating viral liver disease
CN113952463A (en) * 2021-09-30 2022-01-21 宿州学院 Nano diagnosis and treatment agent and preparation method and application thereof
CN114588271A (en) * 2022-03-15 2022-06-07 北京理工大学 Metal-drug full-activity nano-drug and preparation method and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011520769A (en) * 2007-09-21 2011-07-21 サイトイミューン サイエンシズ インコーポレイテッド Nanotherapeutic colloidal metal compositions and methods

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1518983A (en) * 2003-01-22 2004-08-11 赖庆莲 Coordinated modication combination for anti hepatitis B virus
CN1895671A (en) * 2005-12-08 2007-01-17 淮北市辉克药业有限公司 Compound preparation for treating viral liver disease
CN113952463A (en) * 2021-09-30 2022-01-21 宿州学院 Nano diagnosis and treatment agent and preparation method and application thereof
CN114588271A (en) * 2022-03-15 2022-06-07 北京理工大学 Metal-drug full-activity nano-drug and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Nanotechnology approaches for global infectious diseases;Ameya R. Kirtane 等;《Nature Nanotechnology》;第369-390页 *
金类配合物的抗肿瘤活性研究进展;祁欢欢等;《药学进展》;第37卷(第6期);265-273 *

Also Published As

Publication number Publication date
CN115120561A (en) 2022-09-30

Similar Documents

Publication Publication Date Title
CN104225609B (en) A kind of inflammation targeted neutrophil leucocyte delivery system and its application
Yi et al. Cytotoxic effect of novel Flammulina velutipes sterols and its oral bioavailability via mixed micellar nanoformulation
CN1390143A (en) Amphiphilic prodrugs
Zhu et al. Preparation, characterization, and anti-tumor property of podophyllotoxin-loaded solid lipid nanoparticles
WO2015058664A1 (en) Use of icariin in preparing medicine for preventing or treating decrease in blood cells
CN105999299A (en) Small-molecular micelle drug-loaded nano-system, as well as preparation method and application thereof
CA2449856C (en) Pharmaceutical and diagnostic compositions containing nanoparticles useful for treating targeted tissues and cells
CN110772645A (en) Functionalized cell-penetrating peptide modified drug delivery system
CN110665009B (en) Nanometer gemcitabine for promoting normalization of tumor blood vessels and application thereof
CN111001006A (en) Curbitacin B and oxidation response antitumor prodrug co-carried bionic nanoparticle
CN112516329B (en) Self-assembled combined drug carrier based on macromolecule prodrug and application thereof
Luo et al. MPEG-PCL nanomicelles platform for synergistic metformin and chrysin delivery to breast cancer in mice
CN115120561B (en) Combined medicine metal organic hybridization nano assembly and application thereof
CN111793147B (en) Modified chitosan, double-response nano-carrier drug, and preparation method and application thereof
CN104478890A (en) All-trans retinoic acid-camptothecin anticancer drug conjugate as well as preparation method and application thereof
CN106317067B (en) A kind of antitumor medicine conjugate, preparation method, preparation and application
CN105770912B (en) Load medicine ATP sensitive liposome with tumour near-infrared fluorescent image displaying function and preparation method thereof
JP2019508388A (en) Use of fullerenes / metalfullerenes in the manufacture of a medicament for treating myelosuppression
CN104045823A (en) Glycyrrhetinic acid derivative, and preparation method and application thereof
CN108619528A (en) A kind of cyclodextrin-mesoporous silicon multifunctional nano load medicine particle
CN112741828B (en) Drug combination and preparation method and application thereof
EP1568379A1 (en) Medicinal preparation having chemotherapeutic encapsulated therein
Hong et al. Tumor microenvironment responded naturally extracted FOF1-ATPase loaded chromatophores for antitumor therapy
CN107693487B (en) Anthracycline antitumor antibiotic compound micelle and preparation method and application thereof
CN108888628B (en) Application of ginsenoside GRh2 in preparing anti-toxoplasma gondii compound preparation and medicine thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant