CN112826808B - Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof - Google Patents

Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof Download PDF

Info

Publication number
CN112826808B
CN112826808B CN202110064445.6A CN202110064445A CN112826808B CN 112826808 B CN112826808 B CN 112826808B CN 202110064445 A CN202110064445 A CN 202110064445A CN 112826808 B CN112826808 B CN 112826808B
Authority
CN
China
Prior art keywords
lipid
mix
cdn
preparation
neutral
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
CN202110064445.6A
Other languages
Chinese (zh)
Other versions
CN112826808A (en
Inventor
杨振军
于小桐
代宏
孙旭东
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.)
Peking University
Original Assignee
Peking 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 Peking University filed Critical Peking University
Priority to CN202110064445.6A priority Critical patent/CN112826808B/en
Publication of CN112826808A publication Critical patent/CN112826808A/en
Application granted granted Critical
Publication of CN112826808B publication Critical patent/CN112826808B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • 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
    • A61K47/54—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 organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nanotechnology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Diabetes (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Neurology (AREA)
  • General Physics & Mathematics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Neurosurgery (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Obesity (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Emergency Medicine (AREA)
  • Optics & Photonics (AREA)
  • Manufacturing & Machinery (AREA)

Abstract

The invention discloses a neutral/cationic mixed lipid nano preparation of cyclic dinucleotides (cDN) or analogues thereof and application thereof. The preparation consists of neutral cytidine lipid DNCA, cationic lipid CLD, PEG2000-DSPE, a metal salt/PBS buffer system and cyclic dinucleotide. The neutral/cationic mixed lipid nano preparation of the cyclic dinucleotide or the analogue thereof obtained after the entrapment by the method has the advantages of good stability, high delivery efficiency, strong cell-entering capacity, high bioactivity, low toxicity and the like, and has important potential application value in the aspects of anti-tumor immunity, anti-virus/bacterial infection and autoimmune disease treatment.

Description

Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof
Technical Field
The invention relates to a nano preparation of cyclic dinucleotide (cDN) or an analogue thereof encapsulated by a neutral/cationic mixed lipid material, and also relates to a preparation method and application of the preparation. The invention belongs to the technical field of biological medicines.
Background
To date, a total of four examples of cyclic dinucleotides (cDN) class of second messenger molecules, cyclic diguanylic acid (c-di-GMP), cyclic diguanylic acid (c-di-AMP) and 3', 3' -cyclic guanylylic acid (3 ',3' -cGAMP) derived from microorganisms, have been found; and 2',3' -cyclic guanosine adenylate (2 ',3' -cGAMP) found in mammals, which play an important regulatory role in the signal transduction system of living systems.
Chen topic group discovered in 2013 that its target is a key adaptor protein in the innate immune signaling pathway-interferon gene stimulating factor (STING). STING is activated and transported from the endoplasmic reticulum membrane to the golgi apparatus, while TANK-binding kinase 1 (TBK 1) in the cytoplasm is recruited to the golgi apparatus for autophosphorylation, and binds STING to phosphorylate it. Vesicles carrying STING-TBK1 complexes are then transferred to the perinuclear region and bind to IRF3, which is structurally conserved and positively charged, IRF3 is activated by TBK1 phosphorylation, subsequently dissociates from STING proteins, dimerizes, translocates into the nucleus to induce expression of Interferons (IFNs) and other cytokines. The STING protein has been proved to be closely related to the occurrence and development of various diseases such as tumors, pathogenic microorganism infection, autoimmunity and the like, and the cDN compound serving as an agonist thereof has become a potential drug for tumor immunotherapy after an immune checkpoint inhibitor, so that phase I clinical tests are currently carried out, and encouraging primary results are obtained. However, cDN compounds are easily degraded by phosphodiesterase, and are easy to randomly diffuse and clear after administration, and have difficulty in transmembrane due to negative charge, so that clinical application is limited.
Chemical structure modification and vector delivery are two effective strategies to address the above-mentioned problems, and Rp, Rp-2 ',3' -c-diAMPSS (ADU-S100) developed by Aduro Biotech is the first analog to enter clinical trial cDN, whose EC in THP-1 cells5041.5. mu. mol. L-1Significantly higher than 2',3' -cGAMP (EC)50252.5μmol·L-1) And ADU-S100 can obviously inhibit tumor growth and cancer cell metastasis by injection administration. Thereafter, Aduro Biotech and Nowa pharmaceutical company developed, in cooperation with ADU-S100, an optimally active ribose 3' -position bis-F-substituted cDN analog with an EC of50The value was 4.5. mu. mol. L-1Compared with ADU-S100, the improvement is 8 times. The chemical modification improves the stability of cDN moleculesThe affinity with a target STING, but still has the problems that negative charges are difficult to pass through a membrane, and the active concentration is far higher than the physiological concentration in vivo, so that the potential drug toxicity problem exists.
No cDN-class drugs currently undergoing clinical research relate to a carrier delivery technology, and a novel high-efficiency low-toxicity delivery carrier can effectively solve the problem which cannot be solved by chemical modification, so that the administration concentration of cDN molecules is further reduced while the stability of the molecules is ensured. Traditional drug carriers comprise two major types, namely cationic liposome and cationic polymer, can be combined with drugs through electrostatic interaction due to the existence of positive charges on the surface, but can be combined with negatively charged serum protein after intravenous injection, so that the targeting property is poor and the drugs cannot circulate in vivo for a long time; and can be combined with biological membranes in cells to destroy the normal membrane structure and generate stronger in vivo toxicity.
The inventor designs and synthesizes cytidine acetamide glycerol ether molecule DNCA (CN108059619A) in the early period, which can combine and entrap nucleic acid medicine (CN1084478807A) through hydrogen bond and pi-pi stacking effect. Combining DNCA and cationic lipid material CLD which is synthesized by previous design of the inventor and takes cystine as a framework, 3' -dipeptide-siRNA conjugate is transfected with high efficiency and has been successfully applied at a cellular level while reducing toxicity of the cationic lipid material (Mol Pharm,2019,16, 4920). Further optimizing the delivery system, reformulating and exploring the optimal DNCA/CLD prescription, adding PEG2000-DSPE and PBS/Ca2+The neutral/cation mixed lipid delivery system (Mix delivery system for short) with high efficiency and low toxicity is obtained; combining with the inventor's earlier designed novel one-pot phosphoramidite synthesis method (CN102199183B) of cyclic diguanylic acid and analogues thereof, the compound Rp, Rp-3', 3 '-di (2' -F) -cG with optimal activity and single configuration is synthesized and separatedsAsMP (EC of RAW-Lucia ISG cells50EC at 200nM in THP1-Dual ISG cells50159nM) and suboptimal compounds RpS-cdG (EC of RAW-Lucia ISG cells)50329nM), cDN/Mix preparation with high efficiency and low toxicity is innovatively designed. The modification method combining chemical modification and carrier obviously improves the in vivo and in vitro stability of cDN medicaments, and can realize better swelling at lower doseThe immunotherapy effect of tumors (including but not limited to melanoma and breast cancer), 5 mug of 3', 3' -cGAMP/Mix preparation for intratumoral administration to treat melanoma mice can realize 43% of mice long-term survival, 83% of mice long-term survival generate tumor-specific immune memory, and no tumor growth is seen after tumor vaccination; after being administrated by tail vein, 11% of breast cancer mice realize long-term survival, the preparation has better stability in systemic delivery, and the tumor inhibition effect is obviously better than that of the unencapsulated cDN. RpS-cdG and Rp, Rp-3 ',3' -di (2 ' -F) -cGsAsThe MP activity is further improved, and 22 percent and 12.5 percent of breast cancer mice can be completely cured by intratumoral administration of 0.1 mu g after the Mix entrapment. The nano preparation has the advantages of good stability, high delivery efficiency, strong cell-entering capability, low toxicity, good biological activity and the like, and has important potential application value in the aspects of anti-tumor immunity, anti-infection and autoimmune disease treatment.
Disclosure of Invention
The invention aims to provide a high-efficiency low-toxicity cyclic dinucleotide or cyclic dinucleotide analogue nano preparation encapsulated by neutral/cationic mixed lipid materials and a preparation method thereof, so as to improve the druggability of the drugs.
In order to achieve the purpose, the invention adopts the following technical means:
the neutral/cation mixed lipid nano preparation of cyclic dinucleotides (cDN) or analogues thereof is named as cDN/Mix, and the nano preparation consists of neutral cytidine lipid DNCA, cation lipid CLD, PEG2000-DSPE, a metal salt/PBS buffer system and cDN or analogues thereof.
Preferably, the structural formula of the neutral cytidine lipid DNCA is shown as the formula I:
Figure BDA0002903599030000031
the structural formula of the cationic lipid CLD is shown as a formula II:
Figure BDA0002903599030000032
the structural formula of the PEG2000-DSPE is shown as a formula III:
Figure BDA0002903599030000041
wherein, preferably, the metal salt is calcium salt, more preferably CaCl2。
Among them, it is preferable that cDN include 3', 3' -cGAMP, 2',3' -cGAMP, c-di-GMP and c-di-AMP.
Among them, preferably, the cDN analogues include, but are not limited to, RpS-cdG of formula IV and Rp, Rp-3 ',3' -di (2 ' -F) -cG of formula VsAsMP。
Figure BDA0002903599030000042
Preferably, the mixed lipid preparation is prepared by the following method: dissolving neutral cytidine lipid DNCA, cationic lipid CLD and PEG2000-DSPE in ethanol solution, dissolving cyclic dinucleotide or analogues thereof in DEPC water, mixing the substances by using prepared PBS containing metal salt, and performing ultrasonic treatment to obtain the mixed lipid preparation, namely cDN/Mix;
among them, preferred is DNCA: the CLD is as follows: cDN molar ratio 10:6:1, PEG2000-DSPE content 0.5 mol% of total lipid material, prepared to contain 0.1mM CaCl2And PBS (g) were mixed.
Furthermore, the invention also provides application of the mixed lipid preparation in preparation of drugs or reagents for resisting tumors, viruses, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, diabetes and the like independently or in combination with other drugs.
Among them, preferably, the tumor includes, but is not limited to, melanoma and breast cancer.
Wherein, preferably, the drug or the reagent contains unit preparations with different specifications, and the prevention or treatment of the diseases is carried out by one or more administration routes of oral administration, intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection and the like through clinically feasible administration routes.
Preferably, the combination drug comprises an anti-tumor drug and an anti-tumor monoclonal antibody.
Compared with the prior art, the invention has the beneficial effects that:
1. the modification method combining the neutral/cationic mixed entrapment and the chemical modification innovatively designs the high-efficiency and low-toxicity neutral/cationic mixed lipid nano preparation, realizes high-efficiency cell transfection and in-vivo delivery of the medicine, and realizes a better anti-tumor immunotherapy effect at a lower administration concentration and dosage. Taking c-di-GMP (cdG) as an example, the ability of the optimized formulation cdG/Mix to induce IFN-I was 50 times greater than that of the unencapsulated cdG. The preparation can achieve better immunotherapy effect in various tumors (including but not limited to melanoma and breast cancer) by lower dose administration, 43% of mice can survive for a long time by 5 mu g of intratumoral administration of the 3', 3' -cGAMP/Mix preparation for treating melanoma, 83% of mice which survive for a long time generate tumor specific immune memory, and no tumor growth occurs after tumor inoculation; after the tail vein administration of the breast cancer mice, 11 percent of the mice realize long-term survival, the preparation has better stability in systemic delivery, and the tumor inhibition effect is obviously better than that of the unencapsulated cdG. Synthesizing and separating to obtain the single configuration compound Rp, Rp-3 ',3' -di (2 ' -F) -cG with optimal activitysAsMP and a suboptimal compound RpS-cdG, the activities of which are further improved, and 22 percent and 12.5 percent of breast cancer mice can be completely cured by intratumoral administration of 0.1 mu g after the Mix loading. The preparation has the advantages of good stability, high delivery efficiency, strong cell-entering capability, high biological activity, low toxicity and the like, and has important potential application value in the aspects of anti-tumor immunity, anti-virus/bacterial infection and autoimmune disease treatment.
2. The basic group head of the neutral nucleoside lipid material can be combined with cDN drugs through hydrogen bond action and pi-pi stacking action, the dosage of the cationic lipid material is reduced after the neutral nucleoside lipid material is doped, the surface of the nanoparticle is electronegative, and the cationic lipid material is in a neutral stateThe charged particles or proteins are not easy to be adsorbed in the circulation process, and the in vivo application is more stable. In addition, preferred is PBS/Ca2+The preparation system can effectively improve the intracellular transport and in vivo delivery of cDN medicaments.
3. The one-kettle phosphoramidite method is a chemical synthesis method which can simply, efficiently and quickly carry out large-scale preparation of cDN medicines, and the product obtained by the method has the advantages of stable physicochemical property, good biological activity, good membrane permeability and the like, fully explores the advantages and rules of a composite modification mode for carrying out delivery of the medicines, and lays a foundation for industrial production and clinical application of cDN/Mix preparations.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) photograph of cdG/Mix lipid complex and empty vector;
FIG. 2 is an optimization process of cdG/Mix formulation;
wherein: A. optimization of the DNCA/CLD ratio in the preparation (cdG 1.4.4 μ M); B. optimizing Ca in a system at an optimal DNCA/CLD ratio2+Concentration; C. at optimal DNCA/CLD/Ca2+Performing PEG2000-DSPE proportion optimization under the condition; D. abbreviation of formulation in figure E; E. the ability of the various formulations described above to deliver 500nM cdG to induce IFN-I; the cdG dosing concentrations in panels B-E were all 500 nM;
FIG. 3 is an activity alignment of cdG and its analogs at the level of Mix entrapment;
wherein: A. different cdG and its analogues induced IFN-I production by THP-1Dual and RAW-Lucia ISG reporter cells (fold relative to PBS group) at 18h administration using the Mix delivery system; B. EC50 of the compound Rp, Rp-3 ',3' -di (2 ' -F) -cGSAsMP in THP-1Dual ISG cells; C. preference is given to compounds RpS-cdG and Rp, Rp-3 ',3' -di (2 ' -F) -cGsAsEC of MP in RAW-Lucia ISG cells50；
Fig. 4 shows the levels of the critical cytokine mRNA (n-3 or 4) in tumor tissues of mice administered with cDN/Mix intratumorally at different times at a dose of cDN of 5 μ g/mouse;
wherein: A. intratumoral Ifnb mRNA levels at different times; B. cxcl9mRNA levels within the tumor at different times; C. cxcl10mRNA levels within the tumor at different times; CD G/Mix, 3' -cGAMP/Mix and 2',3' -cGAMP/Mix formulations intratumorally administered 8h differential levels of key cytokine mRNA;
FIG. 5 shows the tumor growth inhibiting effect and in vivo safety results of cdG/Mix, 3' -cGAMP/Mix and 2',3' -cGAMP/Mix formulations in melanoma mice;
wherein: a dosing regimen of the cdn/Mix formulation for the treatment of melanoma-bearing mice; B. tumor growth curves for different dosing groups during treatment; C. long-term survival curves for mice in different dosing groups; D. growth curves after re-inoculation of tumors in long-term surviving mice; E. biochemical results of liver and kidney function and blood circulation of mice of different administration groups; F. the results of the immunohistochemistry of the liver of the mice in different administration groups; 0.0001. beta; p < 0.0001;
FIG. 6 shows the immunotherapeutic effect of different doses of cdG/Mix formulations administered intratumorally and tail vein, respectively, in breast cancer mice;
wherein: a dosing regimen for cdG/Mix for treating breast cancer tumor-bearing mice; B. tumor growth curves for each mouse in different dosing groups during treatment; C. mean tumor growth curves for different dosing groups of mice during treatment; D. long-term survival curves for mice in different dosing groups; E. average growth curve after re-inoculation of tumor in long-term surviving mice;
FIG. 7 shows the immunotherapeutic effect of different doses of RpS-cdG/Mix formulations administered intratumorally and tail vein, respectively, in breast cancer mice;
wherein: A. modeling and administration scheme for treating breast cancer tumor-bearing mice; B. tumor growth curves for different dosing groups during treatment; C. long-term survival curves for mice in different dosing groups (n-9-11);
FIG. 8 shows different doses of Rp, Rp-3 ',3' -di (2 ' -F) -cGsAsThe MP/Mix preparation has the immunotherapy effect in breast cancer mice after intratumoral injection and tail vein injection administration respectively;
wherein: A. modeling and administration scheme for treating breast cancer tumor-bearing mice; B. tumor growth curves for different dosing groups during treatment; C. long-term survival curves (n-9-11) for mice from different dosing groups.
Detailed Description
The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description of the specific embodiments proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 neutral/cationic Mixed lipid Material entrapping cDN-type drugs to form cDN/Mix lipid Complex
Figure BDA0002903599030000071
Mixing cDN medicines with lipid material at a molar ratio of DNCA (chemical formula I): CLD (formula II): cDN (formula IV/V) ═ 10:6:1, PEG2000-DSPE is added in 0.5 mol% of all lipid materials, and the solution environment of the preparation is 0.1mM CaCl2The PBS (1). cDN and its analogues were synthesized according to the methods of the literature (CN102199183A), DNCA was synthesized according to the methods of the literature (CN108059619A), CLD was synthesized according to the methods of the literature (New JChem,2014,38(10),4952-4962), PEG2000-DSPE was purchased from Yuye corporation, and PBS was purchased from Beijing Mey morning.
Briefly, lipid materials were dissolved in ethanol solution, cDN-class drugs were dissolved in DEPC water, and prepared to contain 0.1mM CaCl2Mixing the substances with PBS, and performing ultrasonic treatment at 26 deg.C for 30min at ultrasonic frequency of 150W and 40kHz to obtain nanometer lipid complex (cDN/Mix).
Using c-di-GMP (cdG for short) as an example of a ligand of STING protein, a fresh stock of lipids was prepared in ethanol. 74.26mg DNCA, 68.5mg CLD, 2.2mg PEG2000-DSPE were weighed and dissolved in 20mL ethanol to form a lipid stock solution, which was sonicated at 37 ℃ for 4 minutes to form a homogeneous lipid mixture (Mix). Subsequently, 20. mu.l of the above stock solution was added to 480. mu.l of a pre-formulated solution containing 0.1mM CaCl2In PBS, 500. mu.l of working lipid stock was formed. This amount of lipid was used to form cdG/Mix formulations with 10 nmol. Dissolving cdG medicinal dry powderDissolving in DEPC water to form 200 μ M stock solution, adding 50 μ l of the stock solution into 450 μ l of pre-prepared PBS/CaCl2Mu.l of working cdG stock was formed in solution, 500. mu.l of working lipid stock was mixed with 500. mu.l of working cdG stock, and after brief low speed centrifugation, lipid complexes cdG/Mix were formed by sonication at 26 ℃ for 10 min.
FIG. 1 is an exemplary Transmission Electron Micrograph (TEM) of cdG/Mix and empty vector without cdG prepared by these methods, showing cdG/Mix are uniformly dense spherical particles with a diameter of about 100 nm; dynamic Light Scattering (DLS) results showed that the particle surface charge was-9.37 mV, the diameter was 141.1nm, the polydispersity was 0.266, and the particle size and surface charge were stable over one week. Because the sample is dehydrated and dried before TEM shooting, the particles shrink due to water loss, and DLS measures the hydrated particle size of the nanoparticles, so that the particle size measured by the TEM method is smaller than that measured by the DLS method.
The final optimized formulation cdG/Mix achieved efficient delivery of such drugs (fig. 2A-C), with lower concentrations (500nM) inducing cells to produce IFN-i around 50 times more potent than the unencapsulated cdG (fig. 2D and E). Ca2+Is a key factor for improving the transfection capability of lipid materials, and the IFN-I inducing capability of the doped preparation is improved by about 2 times.
EXAMPLE 2 the reporter lines RAW-Lucia ISG and THP1-Dual ISG expressing luciferase were used to examine the ability of cdG and its analogues to induce IFN-I production in cells at the level of entrapment
1. Sample preparation: 3', 3' -cGAMP, Sp-3 ',3' -cGsAMP、Rp-3′,3′-cGsAMP、Sp-3′,3′-cGAsMP、Rp-3′,3′-cGAsMP、3′,3′-cGsAsMP、2′,3′-cGAMP、cdG、RpS-cdG、SpS-cdG、3′,3′-di(2′-F)-cGsAsMP-1、3′,3′-di(2′-F)-cGsAsMP-2、3′,3′-di(2′-F)-cGsAsMP-3、3′,3′-di(2′-F)-cGsAsMP-4. The above compound was synthesized according to the method of the literature (CN102199183A), in which 3', 3' -di (2 ' -F) -cGsAsMP-1、3′,3′-di(2′-F)-cGsAsMP-2、3′,3′-di(2′-F)-cGsAsMP-3、3′,3′-di(2′-F)-cGsAsMP-4 is isolated 3', 3' -di (2 ' -F) -cGsAs4 diastereomers of MP, 3', 3' -di (2 ' -F) -cGsAsMP-4 is the most active Rp, Rp-3 ',3' -di (2 ' -F) -cGsAsMP。
2. The method comprises the following steps:
RAW-Lucia ISG and THP1-Dual ISG cells were plated in 96-well plates at 10 ten thousand/well, and transfected after 18 hours of culture. cDN and modifications thereof were administered at a concentration of 500nM and cDN/Mix lipid complex was prepared as in example 1. Adding the preparation into a culture plate, feeding and culturing for 18h, taking 20 μ l of cell culture supernatant per well, transferring to a 96-well white enzyme label plate, adding 50 μ l of QUANTI-LucTM detection reagent per well, and detecting fluorescence intensity by using a micropore plate type chemiluminescence detector.
3. Results
The activity difference between diastereoisomers of phosphothio cDN is obvious, the activity of the Rp configuration modifier is better and is obviously higher than that of natural cDN, and the Sp configuration modifier has almost no activity (figure 3A). Screening to obtain the single configuration compound Rp, Rp-3 ',3' -di (2 ' -F) -cG with the best activitysAsMP (EC of RAW-Lucia ISG cells50EC at 200nM in THP1-Dual ISG cells50159nM) and suboptimal compounds RpS-cdG (EC of RAW-Lucia ISG cells)50329nM, fig. 3B and C).
Example 3 the ability of cDN/Mix formulations to induce immune cells in the tumor microenvironment of tumor-bearing mice to upregulate Ifnb, Cxcl9 and Cxcl10mRNA was investigated using the qPCR technique
1. Sample preparation: PBS, Mix lipid, cdG, 3' -cGAMP/Mix, 2',3' -cGAMP/Mix, cdG/Mix.
2. The method comprises the following steps:
constructing a melanoma mouse model: a highly invasive melanoma model was constructed using 6-8 week old female C57bl/6 mice, each of which was subcutaneously inoculated with 20 ten thousand B16F10 cells in the right axillary region and had a tumor volume of about 100mm3When the corresponding cDN/Mix lipid complex was prepared and injected intratumorally into the tumor as in example 1The dose of cDN was 5 μ g/mouse.
qPCR experiments: separately after 4/6/8/12h administration, mice were sacrificed and 100mg of tumor tissue was taken, minced, ground, soaked in 1ml TRIzol reagent for total RNA extraction, RNA was reverse transcribed into cDNA using reverse transcription kit, real-time fluorescent quantitative PCR was performed, and the ability of cDN/Mix to induce immune cells in the tumor microenvironment to up-regulate Ifnb, Cxcl9 and Cxcl10 mrnas was examined.
3. Results
The chemotactic inducing property of the chemotactic factor can promote the differentiation of immune cells and cause the protein extravasation in tissues, and is a key cytokine which plays a role in anti-tumor immunity in a tumor microenvironment. Chemokine receptor-3 (CXCR3) belongs to the group THThe 1 cell chemokine receptor, one of the receptors of the CXC chemokine subfamily, is expressed on activated T cells, NK cells, dendritic cells, macrophages and B cells, and is also expressed on some tumor cells and vascular endothelium. CXCL9, CXCL10 chemotactic activated T H1 the cells reach the tumor part to play a phagocytosis role, simultaneously up-regulate the secretion of CXCL9 and CXCL10, activate and chemotaxis NK cells and CD8+The T cells reach the tumor part to kill tumor. IFN-beta is a key protein downstream of cGAS-STING pathway, is universally expressed in macrophages, lymphocytes, dendritic cells, fibroblasts and plasmacytoid dendritic cells (pDCs), and has an important role in defense against microbial infection, regulation of autoimmune diseases and tumor immunity.
cdG/Mix significantly upregulated the expression of Ifnb, Cxcl9, and Cxcl10 mRNAs in the tumor microenvironment compared to the unencapsulated group. If Ifnb mRNA levels in the tumor microenvironment were significantly elevated at 4h of administration and were not detectable at later time points, immediate degradation of Ifnb mRNA could be relevant to avoid over-activation of T cells, leading to autoimmunity. Cxcl9 and Cxcl10mRNA levels in tumor tissues of 4h cdG groups are higher than those of cdG/Mix groups, the levels of 8h and 12h cdG/Mix groups are obviously higher than those of naked groups, and 12h Cxcl9mRNA is about 3 times and 150 times of that of non-encapsulated groups and PBS groups, which is probably caused by slow drug entry rate or slow-release effect of the drug after encapsulation. The 24h associated cytokine mRNA was essentially undetectable (FIGS. 4A-C).
cdG/Mix, 3' -cGAMP/Mix, and 2',3' -cGAMP/Mix formulations administered for 8h all significantly stimulated an increase in Cxcl9mRNA levels, with less difference in Cxcl10 and Ifnb mRNA levels, and no statistical difference in Cxcl9mRNA levels between the three native cDN/Mix formulations, compared to the naked group (FIG. 4D).
Example 4 examination of the immunotherapeutic Effect and in vivo safety of the 3', 3' -cGAMP/Mix, 2',3' -cGAMP/Mix, cdG/Mix formulations in melanoma mice
1. Sample preparation: PBS, Mix lipid, cdG, 3' -cGAMP/Mix, 2',3' -cGAMP/Mix, cdG/Mix, the corresponding cDN/Mix formulation was prepared as in example 1.
2. The method comprises the following steps:
a highly invasive melanoma model was constructed using 6-8 week old female C57bl/6 mice, each mouse was inoculated subcutaneously with 20 ten thousand B16F10 cells in the right underarm area, and administration was initiated 7 days later with rice-sized tumor protrusions reaching the surface of the mouse skin. cDN dose was 5 ug/mouse, and was administered intratumorally 5 times, the treatment schedule is shown in fig. 5A, and the tumor volume was measured at different administration groups during the treatment period (V ═ 0.5 xl × W)2) The drug effect difference of cDN/Mix group, naked group and PBS group is observed, and the tumor grows to 1500mm3The mice are sacrificed and blood is taken to carry out biochemical detection of liver and kidney function and blood and liver immunohistochemical (HE staining) experiments, and the liver and kidney toxicity of the medicine is detected.
The mice which can survive for a long time after the cDN/Mix preparation is administrated are taken as an experimental group, the mice which are not subjected to any experiment at the same week age are taken as a control group, and 2 groups of mice are all inoculated with 10 groups of mice in the right axillary area under the skin5B16F10 cells, without therapeutic intervention, the tumor volume growth of two groups of mice was recorded, and it was observed whether cDN/Mix-cured mice developed anti-tumor immune memory against melanoma cells.
3. Results
The results are shown in fig. 5B-F, 2',3' -cGAMP/Mix, 3' -cGAMP/Mix, and cdG/Mix formulations significantly inhibited tumor growth rate compared to the non-entrapped group, with no statistical difference in antitumor efficacy (fig. 5B), and long-term survival was achieved in 33.3%, 42.9%, and 16.7% of mice, respectively (fig. 5C), with 83% of the long-term survival mice showing no tumor growth and tumor-specific immune memory in the tumor re-challenge experiment (fig. 5D).
The biochemical results of the blood of the mice show that the difference of the liver and kidney function indexes of the mice compared with the control group has no statistical significance (figure 5E); the immunohistochemistry of liver shows no obvious drug-induced liver injury, and the preparation has good in vivo safety (FIG. 5F).
Example 5 examination of the immunotherapeutic Effect of cdG/Mix formulations at various doses and administration regimens in breast cancer mice
1. Sample preparation: PBS, Mix lipid, cdG/Mix, prepared cdG/Mix according to the method of example 1.
2. The method comprises the following steps:
a second pair of breast pads of 6-8 week-old female C57bl/6 mice are inoculated with 75 ten thousand EO771 cells of C57 bl/6-derived breast cancer cells subcutaneously to construct an in situ model of mouse breast cancer. Different dose groups are set, the effective antitumor agent amount of a smaller animal level is searched, an intravenous administration group is set, and the difference of drug effects of cdG/Mix different administration routes is observed. The groups were set as PBS group, Mix lipid material group, cdG/Mix 1. mu.g group, cdG/Mix 5. mu.g group, cdG/Mix 10. mu.g group (all 5 groups above were intratumoral administration), and cdG/Mix 10. mu.g tail vein administration group. 5 doses were administered, the treatment schedule is shown in fig. 6A, and the tumor volumes were measured for different groups of doses during the treatment period (V ═ 0.5 xl × W)2) Observing the difference of drug effects of different groups, and growing the tumor to 1500mm3Mice were sacrificed at time.
3. As a result:
cdG/Mix preparation can inhibit breast cancer growth effectively by intratumoral injection and tail vein injection, has better curative effect than PBS group and Mix lipid material group, and has anti-multiple tumor activity (fig. 6B and C). cdG/Mix groups with 1 μ g, 5 μ g, 10 μ g intratumoral and tail vein administration showed long-term survival in 30%, 22%, 20% and 11% of mice, respectively (FIG. 6D). Of these, tumor growth was not seen in 87.5% (7/8) of the long-lived mice in tumor re-challenge experiments, resulting in tumor-specific immunological memory (FIG. 6E). The lower dose (1 μ g) treatment group can achieve better tumor inhibition and long-term survival rate, and the preparation has better in vivo stability. Intravenous administration offers the possibility of treating non-superficial accessible tumors or hematologic tumors. The weight of the mice slowly increases during the treatment period, the weight loss is not obvious, the health condition of the mice is good, the weight loss caused by drug toxicity or excessive tumor burden is not seen, and the preparation safety is better.
Example 6 examination of Mix-entrapped RpS-cdG for tumor immunotherapy in breast cancer mice
1. Group setting: PBS, Mix lipid, cdG/Mix 1. mu.g, Rps-cdG/Mix 0.1. mu.g, Rps-cdG/Mix 1. mu.g (all 5 groups above are intratumoral), and RpS-cdG/Mix 2. mu.g were administered intravenously. The corresponding formulation was prepared according to the method of example 1.
2. The method comprises the following steps:
a second pair of breast pads of 6-8 week-old female C57bl/6 mice are inoculated with 75 ten thousand EO771 cells of C57 bl/6-derived breast cancer cells subcutaneously to construct an in situ model of mouse breast cancer. 5 doses were administered, the treatment schedule is shown in fig. 7A, and the tumor volumes were measured for different groups of doses during the treatment period (V ═ 0.5 xl × W)2) Observing the difference of drug effects of different groups, and growing the tumor to 1500mm3Mice were sacrificed at time.
3. As a result:
the low-dose RpS-cdG/Mix intratumoral and intravenous administration can obviously inhibit the growth of the mammary tumor of the mouse, and the effect is obviously better than that of the natural cdG. The very low dose of 0.1 μ g intratumorally administered was the best in inhibiting tumor growth, with 22% of tumor-bearing mice achieving long-term survival significantly better than 1 μ g intratumorally administered (fig. 7B and C). Intravenous administration of 2 μ g RpS-cdG/Mix significantly inhibited tumor growth, with a therapeutic effect comparable to intratumoral administration of 0.1 μ g, suggesting that RpS-cdG/Mix formulations had better stability in systemic administration (fig. 7B).
Example 7 examination of Mix entrapping Rp, Rp-3 ',3' -di (2 ' -F) -cGsAsAntitumor immunotherapy effect of MP in breast cancer mice
1. Group setting: PBS, Mix lipid, cdG/Mix 1. mu.g, Rp-3 ',3' -di (2 ' -F) -cGsAsMP 0.1μg、Rp,Rp-3′,3′-di(2′-F)-cGsAsMP 1. mu.g (all of the above 5 groups were intratumorally administered), Rp-3 ',3' -di (2 ' -F) -cGsAsMP 2. mu.g and 0.2. mu.g were administered intravenously. The corresponding formulation was prepared according to the method of example 1.
2. The method comprises the following steps:
a second pair of breast pads of 6-8 week-old female C57bl/6 mice are inoculated with 75 ten thousand EO771 cells of C57 bl/6-derived breast cancer cells subcutaneously to construct an in situ model of mouse breast cancer. 5 doses were administered, the treatment schedule is shown in fig. 8A, and the tumor volumes were measured for different groups of doses during the treatment period (V ═ 0.5 xl × W)2) Observing the difference of drug effects of different groups, and growing the tumor to 1500mm3Mice were sacrificed at time.
3. As a result:
low dose of Rp, Rp-3 ',3' -di (2 ' -F) -cGsAsBoth the MP/Mix intratumoral and intravenous administration can obviously inhibit the growth of the mammary tumor of the mouse, and the effect is obviously superior to that of natural cdG. The very low dose of 0.1 μ g intratumoral administration had the best activity to inhibit tumor growth, with 12.5% of tumor-bearing mice achieving long-term survival significantly better than 1 μ g intratumoral administration (fig. 8B and C). 2 μ gRp, Rp-3 ',3' -di (2 ' -F) -cGsAsThe intravenous administration of MP/Mix significantly inhibited tumor growth, with a therapeutic effect comparable to that of intratumoral administration of 0.1 μ g, suggesting that the formulation had better stability in systemic administration (fig. 8B).
The information shown and described in detail herein is sufficient to achieve the above-mentioned objects of the present invention, and therefore the preferred embodiments of the present invention represent the subject matter of the present invention, which is broadly encompassed by the present invention. The scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art, and that the scope of the present invention is therefore not limited by anything other than the appended claims, in which the singular form of an element used herein does not mean "one and only" one "unless explicitly so stated, but rather" one or more ". All structural, compositional, and functional equivalents to the elements of the above-described preferred embodiments and additional embodiments that are known to those of ordinary skill in the art are therefore incorporated herein by reference and are intended to be encompassed by the present claims.
Moreover, no apparatus or method is required to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. It will be apparent, however, to one skilled in the art that various changes and modifications in form, reagents and synthetic details may be made without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims (9)

1. A cyclic di-nucleotide (cDN) or its analogue neutral/cation mixed lipid nanometer preparation, named cDN/Mix, characterized in that, the preparation is composed of neutral cytidine lipid DNCA, cation lipid CLD, PEG2000-DSPE, metal salt/PBS buffer system and cyclic di-nucleotide or its analogue;
wherein the metal salt is a calcium salt;
wherein cDN includes 3', 3' -cGAMP, 2',3' -cGAMP, c-di-GMP, and c-di-AMP; the cDN analog comprises RpS-cdG shown in chemical formula IV and Rp, Rp-3 ',3' -di (2 ' -F) -cG shown in chemical formula VsAsMP；
Figure FDA0003496617450000011
2. The mixed lipid nano-preparation of claim 1, wherein the structural formula of the neutral cytidine lipid DNCA is shown in formula I:
Figure FDA0003496617450000012
the structural formula of the cationic lipid CLD is shown as a formula II:
Figure FDA0003496617450000013
the structural formula of the PEG2000-DSPE is shown as a formula III:
Figure FDA0003496617450000021
3. the mixed lipid nano-formulation of claim 1, wherein the metal salt is CaCl2。
4. The mixed lipid nano-preparation according to claim 1, which is prepared by the following method: dissolving neutral cytidine lipid DNCA, cationic lipid CLD and PEG2000-DSPE in ethanol solution, dissolving cyclic dinucleotide or analogues thereof in DEPC water, mixing the substances by using prepared PBS containing metal salt, and performing ultrasonic treatment to obtain the mixed lipid preparation, namely cDN/Mix.
5. The mixed lipid nano-formulation according to claim 4, wherein the ratio of DNCA: the CLD is as follows: cDN was mixed with 0.5 mol% of PEG2000-DSPE to the total amount of lipids at a molar ratio of 10:6:1, and PBS containing 0.1mM CaCl2 was prepared.
6. The use of the mixed lipid nanoparticle formulation of claim 1 in the preparation of an anti-tumor drug or agent, alone or in combination with other drugs.
7. The use of claim 6, wherein the tumor comprises melanoma and breast cancer.
8. The use according to claim 6, wherein the medicament or agent comprises unit preparations of different specifications, and the prevention or treatment of the disease is carried out by one or more administration routes of oral administration, intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection through clinically feasible administration routes.
9. The use according to claim 6, wherein the combination comprises an anti-neoplastic drug.
CN202110064445.6A 2021-01-18 2021-01-18 Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof Active CN112826808B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110064445.6A CN112826808B (en) 2021-01-18 2021-01-18 Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110064445.6A CN112826808B (en) 2021-01-18 2021-01-18 Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof

Publications (2)

Publication Number Publication Date
CN112826808A CN112826808A (en) 2021-05-25
CN112826808B true CN112826808B (en) 2022-03-25

Family

ID=75928728

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110064445.6A Active CN112826808B (en) 2021-01-18 2021-01-18 Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof

Country Status (1)

Country Link
CN (1) CN112826808B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113797327A (en) * 2021-09-24 2021-12-17 北京大学 Nucleic acid drug delivery carrier for carrying mRNA and preparation method and application thereof
CN114246829B (en) * 2021-12-31 2023-03-24 北京大学 Method for encapsulating antisense nucleic acid medicament, medicinal preparation prepared by method and application of medicinal preparation in liver cancer treatment
CN114903856A (en) * 2022-05-06 2022-08-16 四川大学华西医院 2 ', 3' -cGAMP-loaded liposome nano preparation and preparation method and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199183B (en) * 2010-03-26 2013-12-18 北京大学 C-di-GMP, analogues thereof and preparation method thereof
CN108059619B (en) * 2016-11-09 2019-10-25 北京大学 A kind of base acetamide glycerin ether molecule, chemical synthesis process and its application in field of gene
CN108478807B (en) * 2018-04-11 2021-03-23 北京制绘基因生物科技有限公司 Nucleic acid drug delivery system and application thereof

Also Published As

Publication number Publication date
CN112826808A (en) 2021-05-25

Similar Documents

Publication Publication Date Title
CN112826808B (en) Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof
Ye et al. Targeted delivery of chlorogenic acid by mannosylated liposomes to effectively promote the polarization of TAMs for the treatment of glioblastoma
CN110522918B (en) Targeting element and preparation method and application thereof
CN106667914B (en) Composition of targeted liposome-cyclic dinucleotide, preparation method and application of targeted liposome-cyclic dinucleotide in resisting tumors
Yuan et al. Hypoxia-modulatory nanomaterials to relieve tumor hypoxic microenvironment and enhance immunotherapy: Where do we stand?
Yang et al. A new concept of enhancing immuno-chemotherapeutic effects against B16F10 tumor via systemic administration by taking advantages of the limitation of EPR effect
EP2860193B1 (en) Polypeptide with function of targeted diagnosis and therapy of nasopharyngeal carcinoma, nanoparticles carrying same and use thereof
Dai et al. Nanomedicines modulating myeloid-derived suppressor cells for improving cancer immunotherapy
WO2017162055A1 (en) Application of cyclic dinucleotide cgamp-liposome for resisting tumours
CN110585131A (en) Chemotherapy drug co-loaded 1-methyltryptophan immune prodrug micelle, preparation method and application thereof
CN111514306B (en) Fullerene nano-particles for enhancing anti-tumor immunotherapy
Zheng et al. Lenvatinib-and vadimezan-loaded synthetic high-density lipoprotein for combinational immunochemotherapy of metastatic triple-negative breast cancer
Gao et al. Self-amplified ROS production from fatty acid oxidation enhanced tumor immunotherapy by atorvastatin/PD-L1 siRNA lipopeptide nanoplexes
Chen et al. Nanomedicines targeting activation of STING to reshape tumor immune microenvironment and enhance immunotherapeutic efficacy
JP2022075622A (en) Drug delivery carrier and pharmaceutical preparation for delivering many kinds of therapeutic agents in common using the same
CN110898231B (en) Functionalized Lalotxel liposome and preparation method and application thereof
Hu et al. The application of nanoparticles in immunotherapy for hepatocellular carcinoma
Fan et al. Polysialic acid self-assembled nanocomplexes for neutrophil-based immunotherapy to suppress lung metastasis of breast cancer
CN107823652B (en) Long-circulation self-assembly composite nano preparation, preparation method and application thereof
Yu et al. Novel formulation of c-di-GMP with cytidinyl/cationic lipid reverses T cell exhaustion and activates stronger anti-tumor immunity
CN114191539B (en) Exosome nano particle for compositely co-carrying small molecule nucleic acid and active protein, and preparation method and application thereof
US11260068B2 (en) Long-circulating liposome modified with c(RGD-ACP-K)
CN115919776A (en) Mixed lipid material nano preparation of pig spleen extract and application thereof
CN117045619B (en) Liposome-polymer nanoparticle loaded with melittin oncolytic peptide and adenosine A2AR receptor inhibitor, and preparation and application thereof
CN112979881B (en) Macromolecules, compositions and methods for enhancing anticancer drug uptake

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