WO2019158060A1 - 聚唾液酸与阳离子化合物形成的聚离子复合物及其制备方法和应用 - Google Patents

聚唾液酸与阳离子化合物形成的聚离子复合物及其制备方法和应用 Download PDF

Info

Publication number
WO2019158060A1
WO2019158060A1 PCT/CN2019/074902 CN2019074902W WO2019158060A1 WO 2019158060 A1 WO2019158060 A1 WO 2019158060A1 CN 2019074902 W CN2019074902 W CN 2019074902W WO 2019158060 A1 WO2019158060 A1 WO 2019158060A1
Authority
WO
WIPO (PCT)
Prior art keywords
tumor
group
doxorubicin
solution
psa
Prior art date
Application number
PCT/CN2019/074902
Other languages
English (en)
French (fr)
Inventor
邓意辉
骆翔
胡玲
刘铭琦
李聪
邱秋钧
苏钰清
郑煌亮
刘梦阳
刘欣荣
宋艳志
Original Assignee
沈阳药科大学
广州制高点医药科技有限公司
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 沈阳药科大学, 广州制高点医药科技有限公司 filed Critical 沈阳药科大学
Publication of WO2019158060A1 publication Critical patent/WO2019158060A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the technical field of medicine and relates to a polyionic complex formed by polysialic acid (PSA) and a cationic compound, a preparation method and application thereof.
  • PSA polysialic acid
  • Ehrlich a well-known German hematologist and founder of immunology, proposed the concept of "magic bullet" targeted drug delivery. Under the inspiration of this concept, medical researchers from all over the world are mentally searching for tumor-targeted drugs, and have obtained many corresponding research results.
  • nano-carrier size affects the cellular uptake mechanism and endocytic pathway (Patel B., et al.Particle engineering to enhance or lessen particle uptake by alveolar macrophages and to influence the therapeutic outcome European Journal of Pharmaceutics and Biopharmaceutics 89 (2015) 163–174), nano-carriers with small particle size (less than 100 nm) can directly enter the lymph nodes, and nanoparticles larger than 200 nm indirectly enter the lymph nodes by mononuclear uptake (Xiang SD, et al.
  • cationic nanocarriers can be phagocytic or dendritic cells faster and more than negatively charged or neutral nanocarriers (Dendritic cell) , DC) uptake; after entering the cell, the negatively charged or neutral nanocarriers are localized in the lysosome, while the positively charged nanoparticles show the ability of the lysosome to escape, mainly localized in the area around the nucleus, causing different immunity response.
  • the amount of aggregation in the tumor increases with the increase of molecular mass and area under curve (AUC), indicating that the EPR effect has molecular mass and cycle time dependence (Maeda H. Macromolecular therapeutics in cancer treatment: The EPR effect and beyond [J]. Journal of Controlled Release, 2012, 164(2): 138-144.).
  • AUC area under curve
  • the discovery and characteristics of the EPR effect provide a biological basis for the construction of a tumor-targeted drug delivery system. The researchers applied it to targeted therapy of tumors and established a technique for PEGylated nanocarriers, which greatly extended the cycle of the carrier. Time increases the ability to target.
  • Recombinant uricases are used in adult gout patients who are unable to adapt or can not tolerate the pain of conventional treatment, once every two weeks (8 mg each time), even if pegloticase is modified with two mPEG with a molecular weight of 10,000 Daltons ( The amide bond) caused 92% of patients to develop anti-pegloticase antibodies, could not continue to use, and even faced the risk of being delisted. We must also face extremely bad realities. In 1984, only 0.2% of healthy people had anti-PEG antibodies. In 2009, this figure reached 25%, while hepatitis C patients had a higher proportion of anti-PEG antibodies, reaching 44%. The current clinical use of pegylated interferon has decreased efficacy and increased side effects.
  • BIND-014 is a PEGylated PLA/PLGA docetaxel nanoparticle based on "long circulation of escape immune response and controlled drug release, and can be actively targeted” compared to conventional docetaxel, after more than 24 hours, BIND-014 plasma drug concentration is still at least 100 times higher, and the intratumoral drug concentration is 10 times higher, showing excellent tumor growth inhibition in multiple tumor models.
  • One of the possible reasons for the failure of BIND-014 is the anti-PEG antibody present in the human body, which is also related to the release rate of the drug, the degree of release, and the complex reaction of the immune system.
  • lung cancer cells can remotely regulate osteoblasts, that is, lung cancer cells and osteoblasts in the bones of the legs can promote each other remotely.
  • SiglecF-expressing neutrophils play a mediating role in mouse lung cancer models (Engblom C, Pfirschke C, Zilionis R, et al.
  • TDDS targeted drug delivery systems
  • targets targets
  • 1 blood barrier For example, the degradation of nano drug carriers by various enzymes in the blood circulation, conditioning and phagocytosis and clearance of the mononuclear phagocyte system (MPS), the accumulation of nano drug carriers caused by the adsorption of plasma proteins, and the filtration of glomeruli Overworked. 2 tumor tissue barrier.
  • MPS mononuclear phagocyte system
  • the dense extracellular matrix (ECM) and the interstitial fluid pressure (IFP) in the matrix surrounding the tumor make it difficult for the nano drug carrier to pass through the tumor stroma and contact the tumor cells, which greatly weakens the construction of the tumor cells.
  • the process of nano drug carriers entering tumor cells has a transmembrane barrier that must have suitable surface characteristics and dimensions to smoothly cross the cell membrane into the cell. 4 Transmune barriers within tumor cells.
  • the vast majority of nanomedicine carriers enter the tumor cells by endocytic pathway, and then enter the endosomes, which in turn enter the lysosomes. Endosomes and lysosomes are the most important barriers for carrier transport in cells because their internal acidic environment (pH 4.0-6.0) and enzymes tend to degrade the carrier and its entrapped drug.
  • the neutrophils are inevitably “damaged” during the process of separation and purification, in vitro incubation and reinfusion, leading to the body's immune system.
  • the "damaged” neutrophils are quickly removed.
  • the immune system should be placed at the top of the list.
  • immunopharmaceutics Immunopharmaceutics or Immunopharmacy.
  • the theory of immunology perfecting the construction of pharmacy-related theories, guiding prescription design, preparation process, quality control and rational application.
  • immunopharmacy is the basic theory, method, technique and means of applying immunology to study a theoretical discipline in the design of pharmaceutical products. Because the immune system is extremely complex, it can be said that it is “a whole body”, and various factors can affect the immune system, including various indicators of pharmacy, such as material type, drug type, dose, particle size, shape, and charge. Sex, drug loading, etc.
  • Immunopharmaceuticals have made us realize that when designing drug carriers, we must pay attention to the following questions: 1. Whether the materials used are endogenous substances, or can be metabolized and do not cause (or cause less) immune reactions; Disease-associated immune cell changes, such as L-selectin on the neutrophil membrane of tumors and inflammation (careful selection of E-selectin and P-selectin, because of the need to circulate to the lesion site to be able to target binding); Increase the metabolic burden of immune cells (such as the connection of drugs and carrier materials need to avoid amide bonds); 4, the use of a normal mouse model of the immune system for the evaluation of nanocarriers, and animal species; 5, to consider the issue of cell cycle, For example, doxorubicin is a cell non-periodic specific drug, PEGylated liposome, nanoparticle, etc., at higher doses, because it does not selectively damage/kill immune cells, it does not produce anti-PEG antibodies, and cannot The ABC problem was found; however, topot
  • Nano-graphene oxide a potential multifunctional platform for cancer therapy [J]. Advanced Healthcare Materials, 2013, 2(8): 1072-1090.) are materials that are not metabolizable or difficult to metabolize, and none of them are suitable as targeted drug carriers for injection.
  • the percentage of various white blood cells is: 50-70% of neutrophils; 1-4% of eosinophils; 0 ⁇ 1% of basophils; 20-40 of lymphocytes %; monocytes are 1-7% (3 to 8%), and the cells having phagocytic ability are neutrophils and monocytes.
  • the most prevalent neutrophils are derived from bone marrow hematopoietic stem cells, which enter the blood or tissues after differentiation and development in the bone marrow.
  • the ratio of distribution in bone marrow, blood and connective tissue is 28:1:25 (about 52%, 2%, 46%, there are also literatures that the neutrophils in the bone marrow are 100 times more neutrophils in the blood).
  • the neutrophil's blood circulation time (the time in the blood) is about 6-8 hours, then leaves the blood, enters the tissue through the blood vessel wall, and does not return to the blood after entering the tissue.
  • neutrophils are the first line of defense in the blood, the first-in-command soldiers in charge, and mononuclear phagocytic cells (macrophages) deal with casualties.
  • the nanocarriers enter the body, the earliest response is neutrophils.
  • NPS Neutrophil phagocytic system
  • neutrophils can be used to target tumors and sites of inflammation, similar to “ride a ride.”
  • neutrophils neutrophils start to cause phagocytosis, causing a disorder of cell membranes, causing respiratory bursts, increasing oxygen consumption by cells, producing a large amount of peroxides and superoxides, also known as In the short period of "oxygen burst”; re-perfusion tissue regained oxygen supply, there is also an “oxygen burst”), and the designed nanocarriers cannot damage neutrophils in a short time.
  • paclitaxel nanoparticle Abraxane is a typical representative
  • the present invention selects sialic acid substances for related research, and unexpectedly finds that the polysialic acid-drug polyion complex is far superior to common injections and covalently coupled nanoparticles (Note: covalent conjugate formation) Nanoparticles, called “covalently coupled nanoparticles").
  • Sialic acid also known as sugar acid, is a class of nine-carbon monosaccharides that are mainly linked as short-chain residues to the ends of glycoproteins, glycolipids and oligosaccharides by ⁇ -glycosidic bonds.
  • SA Sialic acid
  • red blood cells and vascular endothelial cells are highly sialylated. Studies have shown that the lifespan of red blood cells after sialidase treatment has been reduced from 120 days to just a few hours.
  • many pathogens use SA to "dress" themselves to mask their own epitopes, inhibit complement activation pathways of complement, reduce immunogenicity and successfully escape the host immune system.
  • Polysialic acid is a homopolymer of a plurality of SA monomers linked by ⁇ -2,8 and/or ⁇ -2,9, wherein the PSA linked by ⁇ -2,8 is non-immunogenic and biodegradable.
  • PSA can give the modified molecule a longer blood circulation time, and current research has found that it has a good effect on the modification of protein molecules.
  • PSA does not appear to affect their binding to the corresponding receptors while eliminating the immunogenicity and antigenicity of the modified proteins/polypeptides.
  • PSA is mainly used for the modification of protein peptide drugs, and the PSA is oxidized to obtain an aldehyde group, and the amino group of the protein polypeptide is linked by a Schiff base reaction (N-terminal polysialylation application number: 201310022631.9 application date: 2007-07-25 ).
  • a Schiff base reaction N-terminal polysialylation application number: 201310022631.9 application date: 2007-07-25 .
  • PSA can not only play the role of immune camouflage, but also “ride the windmill”, combine with Selectin and Siglec on the surface of cells such as neutrophils, and utilize the unique deformation of neutrophils to pass through the function of blood vessel wall. Release the drug in the tissue to achieve targeted drug delivery and improve efficacy.
  • the technical problem solved by the present invention is to overcome the defects of the prior art, and based on the theory of "immunization pharmacy", a large number of negatively charged groups contained in the PSA molecule are used to form a stable polyion complex with a cationic compound (Polyion complexes, PICs). ).
  • the polyion complex can bind to sialic acid receptors (Selectin, Siglec) on the surface of neutrophils, "find” to neutrophils, and use neutrophils as a drug carrier, while fully considering immune cells. Functional features that result in superior pharmacological activity.
  • the polyion complex of the present invention comprises polysialic acid PSA and a cationic compound, and the main factors affecting the performance and efficacy of the polyion complex include the mass ratio of PSA to the drug, and the degree of polymerization (average molecular weight) of PSA.
  • the mass ratio of PSA to cationic compound is from 2:1 to 50:1, preferably from 5:1 to 30:1, more preferably from 5:1 to 20:1; the polyionic complex formed includes one or more Cationic compound.
  • the concentration of the cationic compound is 1 to 10 mg ⁇ mL -1
  • the mass ratio of the polysialic acid to the cationic compound is 5:1 to 50:1
  • the PSA concentration cannot be greater than 50 mg ⁇ mL -1 , otherwise the viscosity of the preparation is too large to be A stable polyion complex is prepared.
  • the PSA polymerization degree of the present invention is 2-600, preferably 2-270; the average molecular weight is 600-100,000 Daltons (600 Da to 100 kDa); the preferred molecular weight is 3000-8000 Daltons (3000 Da to 80 kDa), Preferably, it is from 5,000 to 50,000 Daltons (5000 Da to 50 kDa).
  • the cationic compound includes anti-tumor, anti-inflammatory, antibiotic compounds (aminoglycosides such as streptomycin, gentamicin, kanamycin, sisomicin, tobramycin, amikacin) , netilmicin; macrolides, such as erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin, dirithromycin, fluoroerythromycin; tetracyclines, such as chlortetracycline, Oxytetracycline, tetracycline, methicillin, doxycycline, dimethylaminotetracycline and its derivatives amicycline compounds).
  • antibiotic compounds aminoglycosides such as streptomycin, gentamicin, kanamycin, sisomicin, tobramycin, amikacin
  • macrolides such as erythromycin, azithromycin, clarithromycin, roxithromycin, telithromycin, dirithromycin, fluoroerythromycin
  • the antitumor drug is selected from one or more of the following drugs: anthracyclines such as doxorubicin, epirubicin, pirarubicin and idarubicin; Antitumor drugs, such as pilocarpine maleate, mitoxantrone hydrochloride; camptothecin antitumor drugs, such as topotecan, irinotecan, exenotecan (exatecan), aminocamptothecin; Antineoplastic agents such as gefitinib, imatinib, nilotinib, sunitinib, lapatinib, tofacitinib, crizotinib, massetinib, emtricidine Entectinib, ibrutinib, afatinib, flumatinib, erlotinib, neratinib, erlotinib Alectinib), Apatinib, Talazoparib,
  • alkaloids are also classified into azole alkaloids, decane alkaloids, piperidine alkaloids, quinoline and quinolone alkaloids, acridone alkaloids, quinazoline alkaloids. , imidazole alkaloids, isoquinoline alkaloids, terpenoid alkaloids, guanidine and xanthine alkaloids, macrocyclic alkaloids, terpenoid alkaloids, terpenoid alkaloids, organic amine alkaloids, such as Vinblastine, vincristine, vinorelbine, berberine, berbamine, etc.
  • Metformin may prevent multidrug-resistant breast cancer https://www.medicalnewstoday.com/articles/320278.php , Qianjintoine (Han Li. Hydrochloride can reverse the multidrug resistance of tumors Role and mechanism. 2013.PhD Thesis. Zhengzhou University.), Han Fangjia (Ye Switzerlanduang; Sun Aizheng. Reversal of anti-drug effect of tetrandrine on human cancer cells of doxorubicin or vincristine-resistant strains. China Chinese Journal of Traditional Chinese Medicine, 1996, 21.6: 369-371.) and Clinical observation of verapamil combined with antitumor drugs in treating malignant pleural effusion.
  • the invention further provides a method for preparing the polyion complex (PICs) comprising:
  • solution A concentration: 0.1 mg/ml to 10 mg/ml
  • glucose, trehalose, sucrose, lactose, mannitol, sorbitol, xylitol, glycerin, sodium chloride and other substances can be used to adjust the osmotic pressure to meet the injection requirements. Freeze drying or spray drying can also be carried out to obtain a solid product.
  • excipients such as EDTA (disodium salt, calcium sodium salt), poloxamer, dextran, polyvinylpyrrolidone, polysaccharides (such as ginseng polysaccharide, astragalus polysaccharide, lentinan) and pH adjuster can be added to the prescription. Wait.
  • EDTA sodium salt, calcium sodium salt
  • poloxamer dextran
  • polyvinylpyrrolidone polysaccharides
  • polysaccharides such as ginseng polysaccharide, astragalus polysaccharide, lentinan
  • pH adjuster can be added to the prescription. Wait.
  • the cationic drug is an antitumor drug, especially an anthracycline such as doxorubicin, epirubicin, pirarubicin or idarubicin
  • the mass ratio between PSA and cationic drug is 5 /1 ⁇ 20/1
  • the average molecular weight of PSA is 20,000 Daltons (20 kDa) to 50,000 Daltons (50 kDa)
  • the degree of polymerization is 70-170.
  • the complex of the invention is prepared by the following method:
  • Covalent conjugates of PSA and cationic drugs used in comparative tests were prepared by the following methods:
  • the invention has the characteristics of simple operation, controllable quality and low cost, and has practical application value; 4 unexpectedly found that the toxicity of the PSA-cisplatin polyion complex is greatly reduced, which is much lower than that of the cisplatin solution group; It was found that the inhibition rate of PSA-mitoxantrone polyion complex was greater than that of mitoxantrone liposomes.
  • Figure 1 is a transmission electron microscope (TEM) image of a polyionic complex and covalently coupled nanoparticles
  • Figure 2 is a representation of a polysialic acid-doxorubicin covalent conjugate
  • A polysialic acid infrared spectrum B: polysialic acid nuclear magnetic spectrum C: polysialic acid-doxorubicin covalent conjugate infrared spectrum D: polysialic acid-doxorubicin covalent conjugate nuclear magnetic spectrum;
  • Figure 3 is an in vitro release experiment of polyionic complexes and covalently coupled nanoparticles
  • Figure 4 is a concentration gradient centrifugation enrichment of peripheral blood neutrophils
  • Figure 5 shows the purity of neutrophils by flow cytometry
  • Figure 6 shows the effect of the CCK8 method on the neutrophil activity of doxorubicin preparations
  • Figure 7 is a view of the intake of neutrophils to doxorubicin preparations
  • Figure 8 is a view showing the release behavior of neutrophils after ingestion of doxorubicin preparation
  • Figure 9 is the pharmacokinetic behavior of doxorubicin in Wistar rats.
  • Figure 10 shows the fluorescence imaging and tissue distribution of doxorubicin preparation S180 tumor-bearing Kunming mice
  • Figure 11 shows the fluorescence imaging and tissue distribution of doxorubicin preparation A549 tumor-bearing nude mice
  • Figure 12 is a flow cytometry analysis of in vitro uptake of doxorubicin preparation by A549 lung cancer cells
  • Figure 13 is a laser confocal microscopy analysis of the transport behavior of doxorubicin preparations in A549 lung cancer cells;
  • Figure 14 is a tumor growth curve of S180 tumor-bearing Kunming mice
  • Figure 15 shows the tumor inhibition rate of doxorubicin preparation against S180 tumor-bearing Kunming mice
  • Figure 16 shows the changes in tumor weight of S180 tumor-bearing Kunming mice
  • Figure 17 shows the survival rate of S180 tumor-bearing Kunming mice
  • Figure 18 shows the tumor shedding phenomenon of S180 tumor-bearing Kunming mice
  • Figure 19 is a tumor inhibition index of doxorubicin preparation against S180 tumor-bearing Kunming mice
  • Figure 20 is a S180 tumor-bearing Kunming mouse tumor and important tissue sections
  • Figure 21 is a photograph of an isolated tumor of A549 tumor-bearing nude mice.
  • Figure 22 is a tumor growth curve of A549 tumor-bearing nude mice
  • Figure 23 is a graph showing the tumor inhibition rate of doxorubicin preparation against A549 tumor-bearing nude mice.
  • Figure 24 shows the change in tumor weight of A549 tumor-bearing nude mice
  • Figure 25 is a tumor inhibition index of doxorubicin preparation against A549 tumor-bearing nude mice
  • Figure 26 is a tumor and important tissue section of A549 tumor-bearing nude mice
  • Figure 27 shows the tumor inhibition rate of S180 for different drug polyion complexes and corresponding drug solutions.
  • the degree of polymerization of the PSA used is 2, 3, 4, 5, 6, 10, 16, 32, 100, 130-170, 200-270, respectively, and the corresponding molecular weights are 644.50, 957.73, 1270.97, 1584.21, 1897.45, respectively.
  • the average molecular weight is 3000, 5000, 16000, 30,000, 4 to 50,000 and 6 to 80,000 Daltons.
  • PSAs with degree of polymerization 2, 3, 4, 5, 6 were purchased from Nacalai, USA; PSAs with a degree of polymerization of 7 to 10 were purchased from Nacalai tesque, Japan; PSA (average molecular weight 11.0 kDa, polydispersity coefficient (pd) 1.17; average Molecular weight 22.7kDa, polydispersity index (pd) 1.34; average molecular weight 39.0kDa, polydispersity coefficient (pd) 1.40), from Camida, Ireland; average molecular weight 30kDa purchased from British carbosynth company (Cabosons Chemical Technology (Suzhou) Limited Company); the remaining molecular weight PSA is obtained or self-made from Jiangnan University.
  • N.D. means that it cannot be detected.
  • the stability of the preparation is greater than 6 months; when doxorubicin When the concentration is 1 to 10 mg ⁇ mL -1 , the mass ratio of polysialic acid to doxorubicin is 5:1 to 50:1, preferably 5:1 to 20:1, more preferably 5:1 to 10: 1.
  • the PSA concentration cannot be greater than 50 mg ⁇ mL -1 , otherwise the viscosity of the preparation is too large to prepare a stable polyion complex.
  • the average molecular weight is 644.50, 957.73, 1270.97, 1584.21, 1897.45 and the average molecular weight is 3000, 5000, 10000, 20000, 30000, 4 to 50,000.
  • the results show that when the average molecular weight is less than 3000, the stability of the preparation is less than 1 month (the average particle size is greater than 230 nm, it is difficult to use filtration sterilization)
  • the average molecular weight is 3,000 to 10,000, the stability of the preparation is less than 5 months (average particle diameter is more than 200 nm); when the average molecular weight is 10,000 to 80,000 Daltons, the average particle diameter is 60-200 nm, and the stability of the preparation is more than 6 Months; when the average molecular weight is 20,000 to 50,000 Daltons, the average particle size is 80-160 nm, and the formulation stability is greater than 6 months.
  • the PSA has a molecular weight of from 10,000 to 80,000 Daltons, preferably from 20,000 to 50,000 Daltons.
  • the reaction solution was diluted twice with re-distilled water, transferred to a dialysis bag having a molecular weight cut-off of 1 kDa, and dialyzed against 1000 volumes of re-distilled water for 24 hours, and the dialysate was changed every 4 hours.
  • the suspension in the dialysis bag was granulated through a 0.8 ⁇ m microporous membrane to obtain polysialic acid doxorubicin covalently coupled nanoparticles (PSANPs) in a yield of about 75%.
  • PSANPs polysialic acid doxorubicin covalently coupled nanoparticles
  • N.D. means that it cannot be detected.
  • the stability of the obtained conjugate nanoparticle preparation is more than 6 months, but The average particle size is larger than the particle size of the polysianic acid doxorubicin polyion complex; when the concentration of doxorubicin is 1 to 10 mg ⁇ mL -1 , the mass ratio of polysialic acid to doxorubicin is 5 : 1 ⁇ 50: 1, PSA concentration can not be greater than 50mg ⁇ mL -1 , otherwise the viscosity of the preparation is too large to prepare a stable polyion complex.
  • Example 3 15 test samples the polyionic complex prepared by the composition of the PSA having an average molecular weight of 30,000 Daltons in Examples 1 and 2 and a degree of polymerization of 100 and a mass ratio of polysialic acid to doxorubicin of 5:1 was used as Example 3 15 test samples.
  • V 0 is the volume of the released medium
  • C n is the concentration at the nth sampling
  • V is the sampling volume
  • M t is the total drug concentration
  • the doxorubicin solution group was rapidly released under neutral and slightly acidic conditions; the covalently coupled nanoparticles were not released under neutral and slightly acidic conditions; the polyion complexes were slow under both pH conditions. Release ability, but under weakly acidic conditions, its release is accelerated and pH sensitive.
  • the neutrophils were isolated and purified by the mouse neutrophil isolation kit (LZS1100) from Tianjin Haoyang Biological Products Co., Ltd. The whole process samples, reagents and experimental environment were all under 20 ⁇ 2 °C. get on.
  • LZS1100 mouse neutrophil isolation kit
  • the amount of heparin added is 10 IU ⁇ mL -1 whole blood.
  • the FITC-conjugated monoclonal anti-mouse Ly-6G/Ly-6C (Gr-1) antibody was added at 250 ng ⁇ mL -1 , and cultured in a 5% CO 2 incubator at 37 ° C for 0.5 h. The cells were collected, centrifuged at 5000 rpm for 3 min, and the supernatant was removed. The cells were resuspended in PBS, centrifuged at 5000 rpm for 3 min, the supernatant was discarded, and the cells were redispersed by adding 200 ⁇ L of PBS. The fluorescence intensity of the samples was measured on a flow cytometer.
  • the culture plate After taking out the culture plate, replace 100 ⁇ L of the culture solution containing the sterile doxorubicin solution, the polyion complex and the covalently coupled nanoparticles (the concentration of doxorubicin is 5 mg ⁇ mL -1 ) in 5% CO 2 , The cells were cultured for 0.5 h in a 37 ° C incubator, and the cells were collected, centrifuged at 5000 rpm for 3 min, and the supernatant was removed. The cells were resuspended in PBS, centrifuged at 5000 rpm for 3 min, and the supernatant was discarded.
  • One group of neutrophils was redispersed in 200 ⁇ L PBS, and the fluorescence intensity of the samples was measured on a flow cytometer. 1 ⁇ 10 4 cells were collected from each sample and detected by PE channel. Data was analyzed using FlowJo 7.6.1 software.
  • another group of neutrophils was treated by adding 500 ⁇ L of cell lysate, and then centrifuged at 5000 rpm for 3 minutes. 200 ⁇ L of the supernatant was added to a 96-well plate, and the fluorescence was detected by excitation at 470 nm and emission at 580 nm. Doxorubicin content. The results are shown in Figure 7.
  • Example 7 Release behavior of neutrophils after ingestion of doxorubicin preparation
  • the procedure for neutrophil uptake of the doxorubicin formulation is the same as in Example 4.
  • the neutrophils of the ingested preparation were redispersed into a cell suspension (1 ⁇ 10 6 /mL) with PBS, and the cell suspension was dispensed into a sterile 1.5 mL centrifuge tube at 5% CO 2 , 37 ° C.
  • the cells were cultured in the incubator, and the cells were collected at 1, 2, 4, 8, 12, and 24 hours, respectively, and centrifuged at 5000 rpm for 3 minutes, and the supernatant and the lower cell pellet were separated.
  • the supernatant was directly stimulated by a microplate reader at 470 nm and emitted at 580 nm for fluorescence detection to determine the extracellular doxorubicin content.
  • the lower cell pellet was treated by adding 500 ⁇ L of cell lysate, and then centrifuged at 5000 rpm for 3 minutes. 200 ⁇ L of the supernatant was added to a 96-well plate, and the fluorescence was detected by excitation at 470 nm and emission at 580 nm to determine the intracellular doxorubicin. content.
  • the results are shown in Figure 8.
  • the release of neutrophils indicates that the doxorubicin solution group can also be partially taken up by neutrophils, but the neutrophils are killed by the doxorubicin solution and the doxorubicin is rapidly released; Both polyion complexes and covalently coupled nanoparticles can be slowly released from neutrophils.
  • the results indicate that neutrophils can carry the polyion complexes with covalently coupled nanoparticles for a longer period of time, and the organics will deliver the formulation to sites of tumor release, inflammation, and other release chemokines (CXCL1).
  • the plasma was separated by centrifugation at 5000 rpm for 10 min, 100 ⁇ L of plasma was taken, 900 ⁇ L of methanol/water (volume ratio 50/50) was added, vortexed for 5 minutes, centrifuged at 10,000 rpm for 5 minutes, and 200 ⁇ L of the supernatant was excited by a microplate reader.
  • the doxorubicin concentration was measured at 470 nm and at 580 nm. The results are shown in Figure 9.
  • the covalently coupled nanoparticles and polyion complexes all exhibit a certain long cycle effect. Among them, the covalently coupled nanoparticles have better stability and the best circulation in the body; the polyion complex removal rate is slightly faster than the covalently coupled nanoparticles, and the polyion complex may be partially disintegrated in the body and rapidly cleared.
  • the preserved S180 cell cryopreservation tube was taken out in liquid nitrogen and rapidly resuscitated in water at 37 °C.
  • the resuscitated S180 cell suspension was inoculated into the peritoneal cavity of Kunming mice (0.6 mL/mouse). After 6 to 7 days, the milky white viscous ascites was taken under aseptic conditions and counted under an inverted microscope. When the tumor cell activity was greater than 95%, the physiological saline was diluted into a cell suspension, and the dilution factor was adjusted to make the suspension tumor. The number of cells was 1.8 ⁇ 10 7 cells ⁇ mL -1 .
  • the S180 cell suspension was inoculated into the subcutaneous tissue of the right anterior armpit of the mouse with 75% alcohol disinfection. Each mouse was inoculated with 0.2 mL, and a total of 12 were inoculated.
  • the mice were randomly divided into 4 groups, namely, control group, doxorubicin solution, polyion complex and covalently coupled nanoparticles, 3 in each group. Each group of mice began to perform in vivo imaging after the tumor volume reached 1000 mm 3 (about 10 to 12 days after inoculation). Intravenous injection was performed at 1, 4, 8 and 24 hours, respectively, at a dose of 5 mg ⁇ kg -1 of doxorubicin. The mice were sacrificed by cervical dislocation after 24 hours, and the tumor and other major organs were isolated for isolated organs. Imaging. The results are shown in Figure 10.
  • the distribution in the medium increased by 114% and 163%; however, the distribution of polyion complexes and covalently coupled nanoparticles was reduced by 74% and 60% in the kidney, respectively, in the heart. The amount is reduced by 78% and 51%.
  • the positively charged amino group of doxorubicin is surrounded by a large number of negatively charged carboxyl groups of polysialic acid; the amino group of doxorubicin in the covalently coupled nanoparticles passes through the amide bond and the carboxyl group of PSA Linked, therefore, the amino sugar structure of doxorubicin in the two preparations could not be combined with the myocardial cell membrane, effectively reducing the accumulation of free doxorubicin in the heart.
  • polyionic complexes and covalently coupled nanoparticles use NPS pathway and EPR effect to target tumors in vivo, but neutrophils are highly susceptible to liver and lung after phagocytosis due to their ability to digest foreign bodies.
  • the clearance of macrophages in the liver leads to an increase in the amount of distribution in the liver and lungs.
  • the cryopreserved tube of the preserved A549 cells was taken out in liquid nitrogen and rapidly resuscitated in water at 37 °C.
  • the resuscitated A549 cell suspension was cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 ° C under 5% CO 2 . After 2 to 3 passages, count under an inverted microscope. When the tumor cell activity is greater than 95%, dilute with physiological saline to form a cell suspension, adjust the dilution factor, and make the number of tumor cells of the suspension 1 ⁇ 10 7 cells. ⁇ mL -1 .
  • mice The right back of the nude mice was sterilized with 75% alcohol, and the A549 cell suspension was inoculated into the subcutaneous tissue of the right back of the mouse. Each mouse was inoculated with 0.1 mL, and a total of 12 were inoculated. On days 18-20 after tumor-bearing, mice were randomly divided into 4 groups of 3 animals each. Intravenous injection was performed at 1, 4, 8 and 24 hours, respectively, at a dose of 5 mg ⁇ kg -1 of doxorubicin. The mice were sacrificed by cervical dislocation after 24 hours, and the tumor and other major organs were isolated for isolated organs. Imaging. The results are shown in Figure 11.
  • the distribution of polyionic complexes and covalently coupled nanoparticles increased by 54% and 87%, respectively, compared with doxorubicin solution; polyion complexes and covalently coupled nanoparticles were compared with doxorubicin solution in the liver, respectively.
  • the distribution in the medium increased by 53% and 49%; the distribution of polyionic complexes and covalently coupled nanoparticles in the heart was similar to that of doxorubicin; the polyion complexes were more flexible than the covalently coupled nanoparticles.
  • the distribution of specific solution in the kidney increased by 75% and 73%; and the distribution of polyion complexes and covalently coupled nanoparticles was significantly less than that of doxorubicin in the lungs of A549 tumor-bearing nude mice, respectively.
  • the distribution of S180 tumor-bearing Kunming mouse lungs indicates that the S180-bearing Kunming mouse lung macrophages have different scavenging ability to the preparation; however, the liver macrophages of the two animal models have similar scavenging ability to the preparation.
  • the probable cause is that liver macrophages are not dependent on external factors for formulation clearance, while macrophage clearance preparations in the lung may depend on other factors.
  • Kunming mice have a complete immune system
  • S180 tumors can induce strong inflammatory response in Kunming mice, prompting the neutrophils in the body to rapidly phagocytose the preparation; while A549 tumors have weaker induction of inflammation in nude mice, effective activation
  • the ability of neutrophils to decrease and neutrophil uptake are less, so neutrophil-mediated preparations are less taken up by macrophages.
  • the Kupffer cells in the liver can directly and efficiently ingest the preparation, so the liver has similar intake of the preparation.
  • Example 11 In vitro uptake and intracellular transport of doxorubicin preparations in A549 lung cancer cells
  • Collect A549 cells in logarithmic growth phase wash them once with PBS, gently pipette the cells with appropriate amount of fresh medium, and make them into a cell suspension, and prepare a cell suspension with a concentration of 2.5 ⁇ 10 5 /mL.
  • the density of 5 ⁇ 10 5 /well was inoculated into a 6-well culture plate, and 2 mL of the culture system was added to each well, and cultured at 37 ° C in a 5% CO 2 incubator for 24 hours.
  • the culture plate After taking out the culture plate, replace 100 ⁇ L of the culture solution containing the sterile doxorubicin solution, the polyion complex and the covalently coupled nanoparticles (the concentration of doxorubicin is 5 mg ⁇ mL -1 ) in 5% CO 2 ,
  • the cells were cultured for 4 hours in a 37 ° C incubator, and the cells were collected, centrifuged at 1500 rpm for 5 min, and the supernatant was removed.
  • the cells were resuspended in PBS, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were redispersed by adding 200 ⁇ L of PBS.
  • the fluorescence intensity of the sample was measured on a flow cytometer. 1 ⁇ 10 4 cells were collected from each sample and detected by PE channel. .
  • the data was analyzed using FlowJo 7.6.1 software to obtain the mean fluorescence intensity.
  • the experimental results show that the doxorubicin solution rapidly diffuses into the A549 cells in a molecular form, so the intake is the largest; the polyion complex and the covalently coupled nanoparticles are mediated by the sialic acid ligand on the surface of the carrier, and are actively taken up by A549 cells.
  • the intake was significantly greater than the control group.
  • A549 cells in the logarithmic growth phase were taken, and the medium in the culture dish was carefully aspirated, washed once with PBS, fresh medium was added, and the cells were gently pipetted to become a cell suspension.
  • the clean cell slides were immersed in 75% ethanol for 5 min, washed three times with sterile PBS, and washed once with cell culture.
  • the coverslips were placed in a twelve-well plate, and the cells were seeded and placed in a 5% CO 2 incubator at 37 ° C overnight to make it approximately 50% to 80% full.
  • the medium was changed to a medium containing doxorubicin, which was a culture solution containing doxorubicin solution, polyion complex and covalently coupled nanoparticles (bore doxorubicin concentration of 5 mg/mL).
  • doxorubicin a culture solution containing doxorubicin solution, polyion complex and covalently coupled nanoparticles (bore doxorubicin concentration of 5 mg/mL).
  • the experimental results show that the doxorubicin solution rapidly diffuses into the A549 cells in a molecular form and enters the nucleus. It binds to the DNA and appears red. When it is superimposed with the blue dye of the nucleus, it shows a distinct purple nucleus.
  • the polyion complex is A549 cells. After ingestion, enter the cell's inclusion bodies and lysosomes. As the pH gradually decreases, the doxorubicin in the polyion complex accelerates and releases, spreads rapidly, and is evenly distributed throughout the cell, but the nuclear region is more flexible.
  • the distribution of stars was less than that of the solution group, and it was light purple when superimposed with the blue dye of the nucleus; the covalently coupled nanoparticles were ingested by A549 cells, and showed obvious dot-like distribution in the cytoplasm, indicating aggregation in the inclusion bodies. In lysosomes, there is almost no distribution of doxorubicin in the nucleus.
  • the nucleus region only shows the blue color of the nuclear dye, indicating that the covalently coupled nanoparticles enter the A549 cells through the endocytic cytosolic pathway, due to doxorubicin and The amide bond between polysialic acids is not easily broken, resulting in the inability of the doxorubicin molecule in the nanoparticles to be effectively released and failing to enter the nucleus. Inhibition.
  • cytopharmaceutics it is necessary to use the theory of "cytopharmaceutics" to design and study cell targeting and cell carriers, especially for the factors that influence the results of in vitro cell experiments, such as different prescription compositions and physical and chemical properties of the preparations.
  • the preserved S180 cell cryopreservation tube was taken out in liquid nitrogen and rapidly resuscitated in water at 37 °C.
  • the resuscitated S180 cell suspension was inoculated into the peritoneal cavity of Kunming mice (0.6 mL/mouse). After 6 to 7 days, the milky white viscous ascites was taken under aseptic conditions and counted under an inverted microscope. When the tumor cell activity was greater than 95%, the physiological saline was diluted into a cell suspension, and the dilution factor was adjusted to make the suspension tumor. The number of cells was 1.8 ⁇ 10 7 cells ⁇ mL -1 .
  • the S180 cell suspension was inoculated into the subcutaneous tissue of the right anterior armpit of the mouse with 75% alcohol disinfection. Each mouse was inoculated with 0.2 mL, and a total of 24 cells were inoculated. On the third day after tumor-bearing, the mice were randomly divided into 4 groups, namely, control group, doxorubicin solution, polyion complex and covalently coupled nanoparticles, 6 in each group. Each group of mice was administered with tail vein injection after the tumor volume reached 100 mm 3 (day 3 after inoculation), once every 3 days for 5 times (3, 6, 9, 12 and 15 after inoculation).
  • the tumor inhibition rate was: polyion complex > doxorubicin solution > covalently coupled nanoparticles.
  • Doxorubicin solution has poor tumor targeting, non-specific toxicity to normal tissues, low tumor inhibition rate, small body mass and short median survival time; polyion complexes and covalently coupled nanoparticles can Efficient targeting of S180 tumors, but covalently coupled nanoparticles into the cells can not quickly release doxorubicin molecules, resulting in a tumor inhibition rate less than the polyion complex and doxorubicin solution.
  • the polyion complex enters the cell, it can release doxorubicin rapidly, inhibit tumor growth, and have the best tumor inhibition effect.
  • Tumor weight, survival analysis and median survival time showed that the order of toxicity of the three preparation groups was: doxorubicin solution>covalently coupled nanoparticles>polyion complex. Because the cytotoxicity of doxorubicin preparation is relatively large and has a strong inhibitory effect on tumors, it has a good anti-tumor effect in this test, and also causes non-specific damage of experimental animals. It is directly reflected by the body weight of the mouse. Therefore, in order to better reflect the effectiveness and targeting of the preparation, taking into account the inhibition of the tumor cells by the preparation and the non-specific damage to the body, we propose a new evaluation index - "Tumor-inhibition index".
  • TI index ) can be divided into two types of “mass inhibition index” or "volume inhibition index”, namely "tumor-bearing animal mass / tumor mass” or “tumor-bearing animal volume / tumor volume”, can also be used.
  • the quality of the tumor-bearing animal/tumor volume, the quality of the tumor-bearing animal/tumor volume, the quality of the tumor-bearing animal/tumor mass, and the larger the tumor inhibition index the better the overall therapeutic effect. Since the body mass of mice is relatively easy to obtain, we chose to use the quality tumor suppressor index for calculation and analysis.
  • the tumor inhibition index of each group we found that the polyion complex is much larger than the other groups, while the doxorubicin solution and the covalently coupled nanoparticles have a certain tumor inhibition rate, but the tumor inhibition index is similar to the control group. It shows that the damage caused by the non-specific toxicity of the doxorubicin solution and the covalently coupled nanoparticles counteracts the anti-tumor effect.
  • the tumor suppressor index organically combines tumor growth with quality of life (QOL), provides more information for objective evaluation of anti-tumor drug efficacy, gains more thoughts and revelations, and fully reflects its Important value in the anti-tumor experiment process.
  • QOL quality of life
  • the tumors in the polyion complex group showed shedding, indicating that the polyion complex can not only effectively target tumor cells, but also act on the tumor microenvironment. Killing cells around the tumor, causing tumors to fall off. This "shedding” is associated with "selectin” expressed on vascular endothelial cells at the tumor site and that determines tumor metastasis and invasion.
  • SA Sialic acid
  • Cytotoxicity experiments have shown that high concentrations of the formulation produce cytotoxicity to neutrophils.
  • neutrophils produce more differentiation due to stimulation by cytokines.
  • Neutrophils with high expression of SiglecF can efficiently ingest the preparation, and this neutrophil is most helpful for tumor growth, so it kills it. It is also a strategy to effectively inhibit tumors; at the same time, relatively ingested neutrophils play a complete physiological function because they are not damaged, and can be delivered to the target site under the guidance of cytokines.
  • the drug in the covalently coupled nanoparticle is coupled with the PSA and cannot be released in the cell, which leads to the drug not entering the cell nucleus; 2.
  • the polyion complex can be efficiently and quickly targeted and integrated in the cell. L-selectin and SiglecF on the surface of granulocytes are taken up by neutrophils before disintegration of the preparation, and then delivered to the tumor target site under the guidance of cytokines using neutrophils as a vector.
  • Freshly stripped heart, liver, spleen, lung, kidney, and tumor tissues were washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde for more than 24 hours. After the fixation, the tissue was taken out from the fixative and used in a fume hood. The scalpel will smooth the tissue.
  • the trimmed tissue block is placed in an embedding cassette, the embedding cassette is placed in distilled water, the tissue is washed at a lower flow rate, and rinsed for 12-24 hours.
  • each tissue block was immersed in the completely melted paraffin (60 ° C incubator) for 1 h, and then the tissue block was transferred to a new paraffin liquid for secondary dipping for 1 h.
  • the wax-impregnated tissue is placed in an embedding machine for embedding.
  • the melted wax is first placed in the embedding frame.
  • the tissue is taken out from the dehydration box, and the embedding frame is placed in the embedding frame and the corresponding label is attached.
  • the wax block was placed on a paraffin slicer and sliced continuously, the slice thickness was 5 ⁇ m, the slice was floated on a spreader, the tissue was flattened at 40 ° C warm water, the tissue was picked up with an anti-offset slide, and baked in an oven at 60 ° C. . After the water is baked and the wax is baked, the slides are taken out and stored at room temperature for use.
  • tissue sections were placed in a solution of xylene I and xylene II for 20 min, 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol were immersed for 5 min, and finally rinsed with distilled water for 10 min.
  • the slices were stained with hematoxylin dye solution for 8 min, rinsed with distilled water to remove the color, and then differentiated with 1% hydrochloric acid alcohol for 20 s, and finally rinsed with distilled water to return to blue.
  • the sections were stained for 3 min in Yihong dye solution, and then rinsed with distilled water for 20 s.
  • the sections were immersed in 95% alcohol I and 95% alcohol II for 5 min, then immersed in absolute ethanol I and absolute ethanol II for 5 min, then soaked in xylene I and xylene II for 5 min, and then dehydrated and transparent.
  • the slices were taken out from the xylene and dried, and the tablets were sealed with a neutral gum.
  • Doxorubicin solution group local necrosis of tumor cells, powdering; multiple myocardial ruptures, even cell lysis (arrow); local necrosis (arrow) in the spleen, atrophy of the renal tubules (arrow); covalently coupled nanoparticles Group: local necrosis of tumor cells; myocardial cells intact; polyion complex group: tumor cells showed multiple focal necrosis, even cell lysis (arrow), showing large area of pigmentation; myocardial cells intact.
  • the cryopreserved tube of the preserved A549 cells was taken out in liquid nitrogen and rapidly resuscitated in water at 37 °C.
  • the resuscitated A549 cell suspension was cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 ° C under 5% CO 2 . After 2-3 passages, count under an inverted microscope. When the tumor cell activity is greater than 95%, dilute with physiological saline to form a cell suspension, and adjust the dilution factor so that the number of tumor cells in the suspension is 1 ⁇ 10 7 cells. ⁇ mL -1 .
  • mice After disinfection with 75% alcohol, A549 cell suspension was inoculated into the subcutaneous tissue of the right back of the mouse, and each mouse was inoculated with 0.1 mL, and a total of 24 cells were inoculated.
  • the mice On the 9th day after tumor-bearing, the mice were randomly divided into 4 groups, namely, control group, doxorubicin solution, polyion complex and covalently coupled nanoparticles, 6 in each group.
  • Each group of nude mice began to be administered intravenously after the tumor volume reached 100 mm 3 (about 9 days after inoculation), once every 3 days for 5 times (9, 14, 19 and 24 days after inoculation).
  • the single administration dose of each group was 5 mg doxorubicin ⁇ kg -1 , and the control group 5% Glu was administered 10 mL ⁇ kg -1 .
  • Data such as tumor volume, body mass, and death events were recorded throughout the trial. The results are shown in Figure 21-25.
  • tumor in vitro photos, tumor growth curve and tumor inhibition rate showed that the tumor volume of the control group increased rapidly.
  • the order of tumor inhibition rate of each preparation group was: polyion complex> doxorubicin solution> covalently coupled nanoparticles.
  • polyion complexes and covalently coupled nanoparticles can efficiently target A549 tumors, but according to the results of laser confocal microscopy, covalently coupled nanoparticles can not effectively release doxorubicin molecules, and can not inhibit tumors.
  • the polyion complex can quickly deliver the drug to the nucleus and exert its efficacy, so the polyion complex exhibits the best anti-tumor effect.
  • the results of tumor weight loss can reflect the health status of nude mice, and also indicate the non-specific toxicity of the preparation.
  • the weight of the control group and the covalently coupled nanoparticle group are almost the same, indicating that the toxicity of covalently coupled nanoparticles is almost negligible.
  • the solution group was the most non-specifically toxic; the polyion complex had some non-specific toxicity, but it was much smaller than the solution group and there was no significant difference from the control group.
  • the tumor inhibition index of each group we found that the polyion complex has both strong tumor inhibition and good targeting, so its tumor inhibition index is much larger than other groups.
  • the doxorubicin solution and the covalently coupled nanoparticles have a certain tumor inhibition rate, the tumor inhibition index is similar to that of the control group, indicating that the non-specific toxicity of the doxorubicin solution and the covalently coupled nanoparticles will cause the body to cause Certain damage, therefore, the tumor suppressor index comprehensively evaluates the results of the doxorubicin solution and the covalently coupled nanoparticle antitumor efficacy.
  • Control group tumor growth was active, showing obvious deep nuclear staining, nucleosis dysregulation, neovascularization (circle), and myocardial cells intact.
  • Doxorubicin solution group local necrosis of tumor cells, showing powdery staining; multiple myocardial ruptures, even cell lysis (arrow); local necrosis of the spleen (arrow).
  • Covalently coupled nanoparticle group tumor cells grow actively, showing large nuclear staining, but fewer new blood vessels; myocardial cells are intact.
  • Polyion complex group focal necrosis of tumor cells, even cell lysis (arrow), showing large area of pigmentation; myocardial cells intact.
  • polysialic acid and polyionic complexes formed by different kinds of cationic drugs In order to determine the efficacy of polysialic acid and polyionic complexes formed by different kinds of cationic drugs, we prepared polyionic complexes (polysialic acid with polysialic acid (average molecular weight of 30,000 Daltons) and different cationic antitumor drugs.
  • the mass ratio of the cationic drug is 10/1).
  • concentration of the cationic drug is 1 to 10 mg ⁇ mL -1
  • the mass ratio of polysialic acid to cationic drug is 5:1 to 50:1, but the PSA concentration cannot be greater than 50 mg. mL -1 , otherwise the viscosity of the formulation is too large to prepare a stable polyion complex.
  • mice were randomly divided into 13 groups (6 in each group), saline control group (10 mL ⁇ kg -1 ) and mitoxantrone hydrochloride solution (according to drug dosage 5 mg/kg).
  • Administration irinotecan solution (administered at a dose of 15 mg/kg), ibrutinib solution (administered at a dose of 10 mg/kg), oxaliplatin solution (administered at a dose of 10 mg/kg) , vinorelbine solution (administered at a dose of 10 mg/kg), bleomycin solution (administered at a dose of 10 mg/kg), polysialic acid-mitoxantrone polyion complex (according to a drug dose of 5 mg) /kg administration), polysialic acid-irinotecan polyion complex (administered at a dose of 15 mg/kg), polysialic acid-ilutinib polyion complex
  • Polysialic acid (average molecular weight 30,000 Daltons) and maleic acid cedar are prepared at a mass ratio of 4/1, 5/1, 10/1, 20/1 and 50/1, when maleic acid is used.
  • concentration of P. chinensis is 1 ⁇ 10mg ⁇ mL -1
  • mass ratio of polysialic acid to pilocarpine maleate is 5:1 ⁇ 50:1
  • PSA concentration cannot be greater than 50mg ⁇ mL -1 , otherwise the viscosity of the preparation Too large to produce a stable polyion complex.
  • mice were sacrificed on the 14th day, the tumor was exfoliated, weighed, and the tumor inhibition rate was calculated.
  • the results showed that the mice in the 4/1 polyion complex group showed a decrease in average body weight, and the mice in the 5/1, 10/1, 20/1, and 50/1 polyion complex groups showed no weight loss.
  • the tumor inhibition rate showed that the inhibition rate of the 4/1 polyion complex (51%) was significantly lower than 5/1 (83%), 10/1 (82%), 20/1 (85%) and 50/.
  • the 1 (80%) polyion complex group there was no significant difference between the 5/1, 10/1, 20/1 and 50/1 polyion complex groups.
  • the PSA-piscone group had an impurity of 0.15% and the commercial product had an impurity of 0.33%.
  • Toxicity test According to the routine toxicity test method, 20 healthy mice were randomly divided into 2 groups, one group was given cisplatin solution, and the other group was given PSA-cisplatin polyion complex (mass ratio 50/1), abdominal cavity. Injection (15 mg/kg), once every other day, a total of 3 doses, observed for 20 days. Two patients died on the 7th day in the cisplatin group and 5 died on the 20th day. In the PSA-cisplatin polyion complex group, there was no death. The results show that the polyion complex significantly reduces the toxicity of cisplatin.
  • Anti-tumor test 18 Kunming mice were randomly divided into 3 groups (6 in each group), saline control group (10 mL ⁇ kg -1 ) and cisplatin positive control group (4 mg ⁇ kg). -1 ) and PSA-cisplatin polyion complex group (mass ratio 50/1, 4 mg ⁇ kg -1 ). On the 4th day after inoculation (tumor volume of about 80-100 mm 3 ), it was administered intraperitoneally, once every 3 days, and administered a total of 3 times. The mice were sacrificed on the 17th day, the tumor was exfoliated, weighed, and the tumor inhibition rate was calculated.
  • Liposomes were prepared by mass ratio of HSPC/CH/PEG2000-DSPE of 3/1/1, and mitoxantrone hydrochloride was loaded by ammonium sulfate gradient method to obtain 1 mg ⁇ mL -1 mM of liposome.
  • a comparative study was conducted according to the conventional anti-S180 tumor experiment, intravenously (5 mg/kg), once every 3 days, for a total of 3 administrations. The mice were sacrificed on the 15th day, the tumor was exfoliated, weighed, and the tumor inhibition rate was calculated. The results showed that the inhibition rate of polyion complex (88%) was better than that of the liposome group (63%).
  • Example 21 Tumor inhibiting effect of different molecular weight polysialic acid and epirubicin polyion complex on S180
  • PSA-table soft ratio was prepared with reference to "Example 1"
  • the star polyion complex mass ratio of PSA to drug is 10:1
  • the final concentration of epirubicin is 1 mg ⁇ mL -1 .
  • bleomycin One of the undesirable effects of bleomycin is to cause pulmonary fibrosis. It has been reported in the literature that azithromycin has an intervention effect on bleomycin-induced pulmonary fibrosis in rats. Therefore, combine the two.
  • the bleomycin hydrochloride and the azithromycin citrate were respectively dissolved in water for injection to prepare a solution (10 mg/ml) to obtain a bleomycin solution and an azithromycin solution.
  • the PSA (having an average molecular weight of 30,000 to 50,000 Daltons) was dissolved in water for injection to prepare a 10 mg/ml PSA solution. Take 1 ml of bleomycin solution, 2 ml of azithromycin solution, mix with 20 ml of PSA solution, stir for 20 minutes, pass through a 0.2 ⁇ m microporous membrane, and freeze the resulting solution to obtain azithromycin-bleomycin-PSA polyion complex frozen. Dry goods.
  • the berberine hydrochloride and epirubicin hydrochloride were respectively dissolved in water for injection to prepare a solution (10 mg/ml) to obtain a berberine hydrochloride solution and a cobidobiine hydrochloride solution.
  • the PSA (average molecular weight of 30,000 Daltons) was dissolved in water for injection to prepare a 10 mg/ml PSA solution.
  • PSA concentration can not be greater than 50 mg ⁇ mL -1 , otherwise the viscosity of the preparation is too large to prepare a stable polyion complex
  • stirring After 60 minutes, a 0.2 ⁇ m microporous membrane was passed, and the resulting solution was freeze-dried to obtain a berberine-epiubicin-PSA polyion complex freeze-dried product.
  • the anti-S180 tumor experiment was carried out with reference to "Example 17 different ratios of polysialic acid and maleic acid sclerotium polyion complex for S180 tumor suppressive effect".
  • Three groups were established, saline group, berberine-epiubicin solution group, berberine-epiubicin-PSA group, administered at a dose of 5 mg/kg epirubicin hydrochloride, berberine-table
  • the tumor inhibition rate was 53% and 87% in the spirulina solution group and the berberine-epiubicin-PSA group, respectively, and 4 tumor detachment occurred in the berberine-epiubicin-PSA group.
  • clarithromycin disperse with water for injection, dilute hydrochloric acid at low temperature (4 ⁇ 10 ° C) and stirring, until all dissolved, to obtain a solution of clarithromycin (5 mg / ml).
  • a PSA (average molecular weight 10,000 Dalton) solution (100 mg/ml) was prepared with water for injection. 2 ml of clarithromycin solution was added dropwise to 1 ml of PSA solution, and the mixture was stirred and mixed to obtain a clarithromycin-PSA polyionic complex, and the pH was adjusted to 7 with a NaOH solution, followed by lyophilization.
  • mice The experimental mice were randomly divided into 3 groups (normal saline group/negative control group, clarithromycin solution group/positive control group, clarithromycin-PSA polyion complex group), 6 mice in each group, and the mice were subcutaneously subcutaneously.
  • the injection volume was 0.1 mL, and the first time and the total number of times each mouse in the doxorubicin scratched the administration site was recorded for 15 minutes.
  • the average time of the first grasping of the negative control group was 293 seconds, and the average number of scratches was 293 seconds.
  • the average time for the first control was 5.5 seconds, and the average number of scratches was 63.
  • the average time for the first scratching of PSA-clarithromycin was 213 seconds, and the average number of scratching was 7.3. The irritation is significantly reduced.
  • doxycycline hydrochloride An appropriate amount of doxycycline hydrochloride is taken, dispersed in water for injection, and dissolved by heating (55 to 60 ° C) to obtain a doxycycline solution (5 mg/ml).
  • a PSA (average molecular weight 30,000 Daltons) solution 100 mg/ml was prepared with water for injection. 1 ml of doxycycline solution was added dropwise to 1 ml of PSA solution, stirred and mixed to obtain a doxycycline-PSA polyion complex, which was adjusted to pH 6 with a NaOH solution, and then freeze-dried.
  • the experimental rats were randomly divided into 3 groups (normal saline group/negative control group, doxycycline solution group/positive control group, doxycycline-PSA polyion complex group), 6 rats in each group, and the right hind paw was injected into the rats.
  • the dose was 0.1 mL, and the first time and the total number of lameness of each rat's lameness were recorded within 15 min after injection.
  • the concentration of doxycycline solution and doxycycline-PSA polyion complex was 2.5 mg. /ml.
  • Table 4 The results are shown in Table 4.
  • the polyion complex greatly reduced the irritation of doxycycline.
  • Example 26 Inhibitory effect of doxorubicin- verapamil-PSA polyion complex on drug-resistant tumors
  • a solution of doxorubicin hydrochloride, verapamil hydrochloride and PSA (average molecular weight 30,000 Daltons) was prepared according to the ratio of mass ratio of doxorubicin hydrochloride to verapamil hydrochloride and PSA of 5/1/50. After mixing, stirring for 30 minutes, the microporous membrane was sterilized to obtain doxorubicin- verapamil-PSA polyion complex, and the final concentration of doxorubicin hydrochloride was 1 mg ⁇ mL -1 .
  • Anti-tumor study was carried out using HepG2, a liver cancer resistant cell (Gifts from China Medical University), divided into 4 groups, 6 in each group, saline-negative control group (10 mL ⁇ kg -1 ), doxorubicin positive control Group, doxorubicin- verapamil combination group, doxorubicin- verapamil-PSA polyion complex group.
  • intravenous administration 5 mg ⁇ kg -1 of doxorubicin
  • the mice were sacrificed on the second day of the last administration, the tumor was excised, weighed, and the tumor inhibition rate was calculated.
  • the tumor inhibition rate of doxorubicin was 22%, and the tumor inhibition rate of doxorubicin- verapamil combined group was The tumor inhibition rate of the 35% and polyion complex groups was 81%.
  • Metformin, clindaline and tetrandrine can also be combined in a similar manner, or even more than two combinations.

Landscapes

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

Abstract

本发明属于医药技术领域，涉及聚唾液酸(PSA)与阳离子化合物形成的聚离子复合物及其制备方法和应用。所述的聚离子复合物包含聚唾液酸PSA和阳离子化合物，PSA与阳离子化合物的质量比是2:1～50:1；所形成的聚离子复合物中包含一种或者一种以上的阳离子化合物。所述的聚离子复合物通过如下方法制备：(1)将阳离子化合物用纯水溶解，得溶液A；(2)将聚唾液酸(PSA)溶于纯水，得溶液B；(3)将溶液A与溶液B混合，得到聚离子复合物(PICs)液体。本发明的聚离子复合物的药效明显优于共价偶联纳米粒，不仅能够提高药物的疗效，还能降低其毒性。

Description

聚唾液酸与阳离子化合物形成的聚离子复合物及其制备方法和应用 技术领域
本发明属于医药技术领域，涉及聚唾液酸(PSA)与阳离子化合物形成的聚离子复合物及其制备方法和应用。
背景技术
1906年，德国著名的血液学家、免疫学说的奠基人Ehrlich提出了“魔弹”靶向给药的概念。在这一概念的启示下，世界各国医药研究人员孜孜不倦地探寻肿瘤靶向药物，并取得了相应的许多研究成果。如“粒径”影响着细胞的摄取机制与内吞途径(Patel B.,et al.Particle engineering to enhance or lessen particle uptake by alveolar macrophages and to influence the therapeutic outcome European Journal of Pharmaceutics and Biopharmaceutics 89(2015)163–174)，小粒径(小于100nm)的纳米载体可以直接进入淋巴结，大于200nm的纳米粒子则通过单核细胞摄取方式，间接进入淋巴结(Xiang SD,et al.Pathogen recognition and development of particulate vaccines:does size matter？[J].Methods,2006,40:1-9.)(Manolova V,Flace A,Bauer M,et al.Nanoparticles target distinct dendritic cell populations according to their size[J].Eur J Immunol,2008,38:1404-1413.)；小于500nm的纳米粒子可被DC高效摄取，大于500nm时,则更倾向于被巨噬细胞与皮肤中的郎格罕细胞摄取(Foged C,et al.Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model[J].Int J Pharm,2005,298:315-322.)；纳米粒子的“形态”同样会对细胞摄取水平与体内分布产生影响(Fifis T,Gamvrellis A,Crimeen-Irwin B,et al.Size-dependent immunogenicity:therapeutic and protective properties of nanovaccines against tumors[J].J Immunol,2004,173:3148-3154.)。非球形的纳米载体的血液循环时间长于球形粒子，被DC细胞摄取的水平也相对较低。但Gratton等的研究结果表明，杆状的纳米载体被细胞摄取的水平最高，随后依次为球形、圆柱体与立方体的纳米载体(Gratton SEA,et al.The effect of particle design on cellular internalization pathways[J].Proc Natl Acad Sci U S A,2008,105:11613-11618.)。“表面电荷”的影响，一般而言，由于细胞膜带有负电荷，与带负电或中性纳米载体相比，阳离子纳米载体能更快、更多地被吞噬细胞或树突状细胞(Dendritic cell，DC)摄取；进入细胞后，带负电或中性纳米载体定位在溶酶体，而带正电的纳米粒显示出溶酶体逃逸的能力，主要定位于细胞核周围区域，从而引起不同的免疫响应。
1986年，日本学者Maeda等发现，荷瘤小鼠在静脉注射伊文思蓝24小时后，伊文思蓝在肿瘤中的聚集量是正常组织与器官(如皮肤、心脏、肌肉、肾脏)的10～200倍，而且伊文思蓝在肿瘤中的清除更慢，由此正式提出实体瘤的“高渗透长滞留效应”(Enhanced permeability and retention effect，简称EPR效应)(Matsumura Y,Maeda H.A new concept for macromolecular therapeutics in cancer chemotherapy:mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs[J].Cancer research,1986,46(12 Part 1):6387-6392.)。肿瘤内的聚集量会随着分子质量及药时曲线下面积(Area under curve，AUC)的增加而增加，说明EPR效应具有分子质量与循环时间的依赖性(Maeda H.Macromolecular therapeutics in cancer treatment:The EPR effect and beyond[J].Journal of Controlled Release,2012,164(2):138-144.)。EPR效应的发现与特点，为构建肿瘤靶向药物递送系统提供了生物学基础，研究者们将其应用于肿瘤的靶向治疗，建立了PEG化纳米载体的技术，极大地延长了载体的循环时间，提高了靶向能力。然而，自从1990年首次报道PEG修饰脂质体以来，近30年的时间里，仅有一个利用高密度DSPE-PEG的PEG化技术，达到长循环目的，提高肿瘤组织药物浓度的多柔比星脂质体产品Doxil。虽然，Doxil降低了多柔比星心脏毒性，但又产生了新的不良反应，特别是“手足综合症”。另外，PEG化多柔比星脂质体(Doxil)在临床应用过程中发现，同一患者的不同时间，不同患者之间，均存在显著的差异。Doxil的清除与年龄、性别等有关，清除差异达到15倍(An open-label study to evaluate dose and cycle dependence of the pharmacokinetics of pegylated liposomal doxorubicin.Cancer Chemother Pharmacol 61(4):695–702.)。引起这些问题的一个原因是，枯否细胞吞噬处理Doxil而受损，此损伤可以持续28天(Yahuafai J,Asai T,Nakamura G,et al.Suppression in mice of immunosurveillance against PEGylated liposomes by encapsulated doxorubicin[J].Journal of Controlled Release,2014,192:167-173.)。
更为严重的是，由于PEG化制剂的循环时间得到极大延长，引起获得性免疫系统参与，产生抗PEG 抗体，导致抗PEG IgM快速识别而引发的PEG化制剂快速清除的ABC现象。值得关注与警惕的是，某些PEG修饰的大分子化合物在临床应用过程中，产生抗PEG抗体的发生率非常高，如2010年9月14日，美国FDA批准Krystexxa(pegloticase，聚乙二醇化重组尿酸酶)用于不能适应或不能忍受常规治疗痛苦的成人痛风患者，每两周静脉滴注一次(每次8mg)，即使pegloticase是采用两条分子量为10000道尔顿的mPEG修饰而得(酰胺键连接)，却导致了92％患者体内产生抗pegloticase抗体，无法继续使用，甚至面临被退市的风险。我们还必须面临极为糟糕的现实情况，1984年仅0.2％健康人体内存在抗PEG抗体，2009年这一数据则达到25％，而丙肝患者体内存在抗PEG抗体的比例更高，达到44％，导致目前临床使用的PEG化干扰素疗效下降，副作用增加。最为惨重的教训是，由麻省理工著名学者鲍伯兰格(罗伯·兰格，Robert Langer)教授主持开发的BIND-014，三期临床失败。BIND-014是基于“逃逸免疫反应的长循环与控制药物释放，并且能够主动靶向”的PEG化PLA/PLGA多西紫杉醇纳米粒，相比于常规的多西紫杉醇，在超过24小时后，BIND-014血浆药物浓度仍至少高100倍，瘤内药物浓度高10倍，在多个肿瘤模型中显示了极为优秀的肿瘤生长抑制作用。BIND-014的失败，其中一个可能的原因是人体内存在的抗PEG抗体，也与药物的释放速度、释放程度和免疫系统复杂反应有关。当然，肿瘤的复杂性、易变性与难治性，也会降低BIND-014的效果。最近两篇发表在Science杂志上的文章更加体现出来肿瘤治疗的艰难性与复杂性，其一是：肿瘤细胞能食“毒”而生，加速肿瘤细胞增殖。氨分子作为一种毒性代谢产物，正常情况下经由血液运输到肝脏，经肝脏代谢后进入泌尿系统排出体外。但研究人员发现，这些氨分子对肿瘤细胞没有毒性，反而加快了多种肿瘤细胞的生长，即“废物利用”(Spinelli J B,Yoon H,Ringel A E,et al.Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass[J].Science,2017,358(6365):941-946. http://www.sohu.com/a/213048801_208944)。其二是：肺癌细胞能够远程调控成骨细胞，即肺癌细胞与腿部骨骼中的成骨细胞能够远程互相促进。研究人员发现，肺癌模型的小鼠全身多处骨骼骨密度升高，新骨生成，成骨细胞增多，破骨细胞无显著变化，并且70％的肺癌患者中(包括未发生骨转移的患者)也存在这样的情况，从而体现出将癌症作为全身性疾病加以研究的重要性。通过小鼠肺癌模型证明，SiglecF高表达的中性粒细胞起了媒介作用(Engblom C,Pfirschke C,Zilionis R,et al.Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils[J].Science,2017,358(6367):eaal5081. http://www.sohu.com/a/213048801_208944； http://news.bioon.com/article/6713823.html)(备注：Siglec的全称是Sialic acid-binding immunoglobulin-type lectins，即唾液酸结合免疫球蛋白样凝集素，包括Siglec-1～Siglec-17，存在于中性粒细胞、单核细胞、巨噬细胞、髓系祖细胞、B细胞、DC、NK等细胞表面，SiglecF与SiglecE存在于小鼠与大鼠体内，人类比鼠类有更多的Siglec)。
在针对肿瘤的靶向药物递送系统(Targeted drug delivery system，TDDS)的研究过程中，人们逐渐意识到，良好的TDDS需要符合4个关键的条件：保留(retain)、逃避(evade)、靶向(target)与释放(release)。因此，对于通过静脉注射的纳米药物载体而言，这意味着要克服以下几个屏障：①血液屏障。例如血液循环中各种酶对纳米药物载体的降解，调理作用以及单核吞噬细胞系统(Mononuclear phagocyte system，MPS)的吞噬与清除，血浆蛋白的吸附造成的纳米药物载体聚集以及肾小球的滤过作用。②肿瘤组织屏障。肿瘤周围基质中致密的细胞外基质(Extracellular matrix，ECM)以及肿瘤间质高压(Interstitial fluid pressure，IFP)使得纳米药物载体难以穿过肿瘤间质而接触肿瘤细胞，大大削弱了针对肿瘤细胞而构建的TDDS的疗效。③肿瘤细胞屏障。纳米药物载体进入肿瘤细胞的过程存在着跨细胞膜屏障，其须具备适宜的表面特性及尺寸才能顺利跨过细胞膜进入细胞内。④肿瘤细胞内传递屏障。绝大部分纳米药物载体是以内吞途径进入肿瘤细胞，之后进入内涵体，继而进入溶酶体。内涵体与溶酶体是载体在细胞内传递的最主要的屏障，这是因为它们内部的酸性环境(pH 4.0～6.0)与酶易使载体及其包载的药物降解。
2013年，Chen等提出“肿瘤免疫循环”(Chen D S,Mellman I.Oncology meets immunology:the cancer-immunity cycle[J].Immunity,2013,39(1):1-10.)的概念，具体包括：①死亡癌细胞释放抗原；②肿瘤抗原提呈；③启动与激活；④T细胞运输至肿瘤部位；⑤T细胞浸润肿瘤；⑥T细胞识别癌细胞；⑦癌细胞被杀死。这些步骤分别发生在肿瘤、淋巴结与血液循环当中。
2016年，Wilhelm等的meta分析文章指出，进入肿瘤的纳米粒数量的平均值为0.7％(Wilhelm S, Tavares A J,Dai Q,et al.Analysis of nanoparticle delivery to tumours[J].Nature Reviews Materials,2016,1:16014.)，有学者采用氯膦酸脂质体耗竭枯否细胞(消除枯否细胞对纳米载体的清除作用)，肿瘤分布量提高20～100倍(最高可达150倍，肿瘤分布量为2％)，肝脏分布量下降(最高可达3倍)，但脾脏分布量大大提高(最高可达5倍)，即脾脏代偿性地替补枯否细胞功能(Wilhelm S,Tavares A J,Dai Q,et al.Analysis of nanoparticle delivery to tumours[J].Nature Reviews Materials,2016,1:16014.)，证明肝脏枯否细胞是影响纳米载体靶向肿瘤的重要因素之一。还有文指出，硬质纳米粒进入肝脏后，流动速度降低1000倍，与肝细胞接触增加7.5倍(Tsoi K M,MacParland S A,Ma X Z,et al.Mechanism of hard-nanomaterial clearance by the liver[J].Nature materials,2016.)。所有的这些事实均说明，免疫系统在处理纳米载体方面具有重要地位。
近年来，细胞治疗成为热点，美国FDA于2017年8月30日正式批准了诺华制药的CAR-T疗法Kymriah(定价47.5万美元)上市，用于治疗复发或难治性儿童B细胞急性淋巴细胞白血病，标志着史上首款CAR-T疗法的诞生，这必将掀起更大的热潮。然而，我们也必须意识到，细胞治疗和细胞作为药物载体同样存在免疫问题。例如，在体外将中性粒细胞与载体/药物孵育后再回输至体内，虽然能够提高治疗效果，但是回输的中性粒细胞大量聚集于肝脏、脾脏与肺部。一方面是由于供体与受体之间的配型问题，更重要的是中性粒细胞在分离纯化、体外孵育以及回输体内过程中，不可避免的受到了“损伤”，导致机体免疫系统迅速将“损伤”的中性粒细胞清除掉。
很有意思的是，“魔弹”靶向给药的概念是免疫学说奠基人Ehrlich于1906年提出的，而现有的靶向给药研究，特别是在MPS(单核吞噬细胞系统)和EPR(高渗透长滞留效应)指导下的PEG化策略，忽视了免疫系统的复杂性，没有将免疫系统放在首要位置，也没有用免疫学的思考方式来全面指导递药系统的设计。目前，纳米递药系统与细胞载药的研究文章很多，如果不全面考虑免疫系统的作用，我们的研究类似于“盲人摸象”，必然存在许许多多不能解决的问题。
综合之，应该将免疫系统放在首要位置。
既然，纳米粒进入体内后，无法避开免疫系统，产生复杂的免疫响应，就有必要用免疫学的思维方式来考虑药剂学的问题，从而提出免疫药剂学(Immunopharmaceutics或者Immunopharmacy)的概念，用免疫学的理论，完善药剂学相关理论的构建，指导处方设计、制备工艺、质量控制与合理应用。简而言之，免疫药剂学就是应用免疫学的基本理论、方法、技术和手段，研究药剂学中有关制剂产品设计的一门理论学科。由于免疫系统极为复杂，可谓是“牵一发而动全身”，各种因素都会影响免疫系统，包括药剂学的各种指标，如材料种类、药物种类、剂量、粒径大小、形状、荷电性、载药量等。免疫药剂学让我们意识到，在设计药物载体时，必须关注如下问题：①、所采用的材料是否为内源性物质，或者可以代谢且不引起(或者较少引起)免疫反应；②、与疾病关联的免疫细胞变化情况，如肿瘤和炎症的中性粒细胞膜上的L-选择素(慎重选择E选择素和P选择素，因为需要循环到病变部位才能够靶向结合)；③、不能增加免疫细胞的代谢负担(如药物与载体材料的连接需要避免酰胺键)；④、采用免疫系统正常的鼠模型进行纳米载体的评价，以及动物种属问题；⑤、要考虑细胞周期的问题，如阿霉素为细胞非周期特异性药物，PEG化脂质体、纳米粒等等，在较高剂量下，由于无选择性地伤害/杀灭免疫细胞，因此不产生抗PEG抗体，不能够发现ABC问题；但是，拓扑替康是细胞周期特异性药物，即使采用很高的剂量，也会产生抗PEG抗体，出现ABC现象；⑥、考虑药物杀灭或者伤害肿瘤细胞(包括正常细胞)所带来的免疫响应，产生不同的肿瘤免疫循环；⑦维护免疫细胞通路，确保药物载体进入病变部位；⑧进行细胞药代动力学研究等。很显然，一个成功的纳米粒，必须将免疫学与药剂学结合起来，必须正视免疫系统，关注免疫学及其相关领域的发展与新的研究成果。如果应用免疫药剂学的理论来分析纳米钻石、纳米硅、石墨烯(Gon lves G,Vila M,Portolés M T,et al.Nano-graphene oxide:a potential multifunctional platform for cancer therapy[J].Advanced Healthcare Materials,2013,2(8):1072-1090.)等不可代谢/或者难以代谢的材料，它们均不适合作为注射用靶向药物载体。另外，我们还必须重视，作为免疫应答的一种形式，在机体内的免疫记忆长达几十年，如果纳米载体选择不当，制剂处方组成等因素考虑不周，这种“非己”抗原必然引起机体的免疫反应，特别是在表观遗传学上，给人类带来几十年的影响，后果会很严重。
在现有的靶向递送药物体系设计中，虽然研究者意识到免疫系统对载体的免疫响应问题，采用了 PEG化技术，选择了“躲”的策略对待免疫系统的清除，但最终疗效不佳(因为药物载体在免疫系统面前“无处遁形”，不仅躲不掉，反而更加激发免疫响应，带来新问题，如ABC现象)。我们知道，在免疫学理论体系中，各种白细胞的百分比为：中性粒细胞50～70％；嗜酸性粒细胞1～4％；嗜碱性粒细胞0～1％；淋巴细胞20～40％；单核细胞为1～7％(3～8％)，其中具有吞噬能力的细胞为中性粒细胞与单核细胞。占比最多的中性粒细胞来源于骨髓的造血干细胞，在骨髓中分化发育后，进入血液或组织，其在骨髓、血液与结缔组织的分布数量比是28:1:25(大约52％、2％、46％，也有文献指出，骨髓里的中性粒细胞是血液里的中性粒细胞100倍)。中性粒细胞的血液循环时间(血液中存在时间)约为6-8小时，然后离开血液，穿过血管壁进入组织，进入组织后不再返回血液中来。最为重要的是，中性粒细胞是血液中的第一道防线，是冲锋陷阵的先头部队士兵，而单核吞噬细胞(巨噬细胞)处理伤亡士兵。当纳米载体进入体内时，最早响应的是中性粒细胞，我们由此提出“中性粒细胞吞噬系统”(Neutrophil phagocytic system，NPS)，完善了现有的MPS理论体系。因此，可以利用中性粒细胞靶向肿瘤与炎症部位，类似于“搭顺风车”。简而言之，可以基于“免疫药剂学”的思维方式，设计一种主动去“找”中性粒细胞的策略，而不是“躲”，达到高效靶向治疗的目的。
那么，如何去“找”？免疫学相关研究成果证明，在肿瘤与炎症等疾病体内的中性粒细胞表面上，存在着能够结合唾液酸的受体(Selectin、Siglec)，可以用唾液酸类物质修饰或者制备纳米载体，“找”到中性粒细胞，让中性粒细胞成为药物靶向载体，这也是一个智慧选择。另外，中性粒细胞存在“呼吸爆发”(中性粒细胞随着吞噬作用的开始，导致细胞膜紊乱而引起呼吸爆发，细胞耗氧量增加，产生大量的过氧化物及超氧化物，又称为“氧爆发”；再灌注组织重新获得氧供应的短时间内，也存在“氧爆发”)的情况，所设计的纳米载体不能在短时间内大量伤害中性粒细胞。因此，需要尽量选择内源性物质(紫杉醇白蛋白纳米粒Abraxane是一典型代表)，或者可以代谢而不引起免疫反应的物质。本发明选用唾液酸类物质进行相关研究，并意外地发现，聚唾液酸-药物聚离子复合物的药效远优于普通注射剂与共价偶联纳米粒(注：共价偶联物所形成的纳米粒，称为“共价偶联纳米粒”)。并且发现，中性粒细胞吞噬共价偶联纳米粒而形成许许多多吞噬小体，该小体不进入细胞核，导致抗肿瘤效果极大下降，这与文献报道的研究结果不一致(聚合物-药物偶联物纳米粒的药效优于未偶联的药物)(Feng Q,Tong R.Anticancer Nanoparticulate Polymer‐Drug Conjugate[J].Bioengineering&Translational Medicine,2016.)。
唾液酸(Sialic acid，SA)又称糖酸，是一类九碳单糖，它主要以短链残基的形式通过α-糖苷键连接于糖蛋白、糖脂与寡糖的末端，普遍存在于哺乳动物的细胞膜表面，其中红细胞及血管内皮细胞表面被高度唾液酸化。研究表明，红细胞经唾液酸酶处理后其寿命从原来的120天锐减到短短数小时。另外，许多病原体利用SA“装扮”自身，以掩蔽自身抗原表位，抑制补体的旁路激活途径，降低免疫原性进而成功逃脱宿主免疫系统的攻击。聚唾液酸(Polysialic acid，PSA)是多个SA单体以α-2,8与/或α-2,9连接的同聚物，其中以α-2,8连接的PSA无免疫原性且可生物降解。实验证明，PSA能够赋予所修饰分子较长的血液循环时间，目前的研究发现其在蛋白分子的修饰方面表现出较好的效果。有研究指出，PSA化门冬酰氨酶无免疫原性，且门冬酰氨酶的活性几乎不受影响，但使用PEG修饰后活性严重下降。另外，PSA在消除所修饰蛋白/多肽的免疫原性与抗原性的同时似乎并不影响它们与相应受体的结合。目前，PSA主要用于蛋白多肽类药物的修饰，将PSA进行氧化得到醛基，通过希夫碱反应与蛋白质多肽的氨基连接(N末端聚唾液酸化申请号：201310022631.9申请日：2007-07-25)。我们在国家自然科学基金支持下(资助项目号81373334)，通过在PSA分子上引入脂肪链，提高PSA在脂质体等载体中的锚定能力，发挥PSA的免疫伪装与靶向作用(一种聚唾液酸的脂质接枝衍生物及其应用申请号：201510603369.6申请日：2015-09-18)。同时，我们意识到PSA既可以发挥免疫伪装作用，又可以“搭顺风车”，与中性粒细胞等细胞表面上的Selectin、Siglec结合，利用中性粒细胞独特的变形穿过血管壁功能，在组织中释放药物，实现靶向递送药物目的，提高疗效。
发明内容：
本发明所解决的技术问题是克服现有技术的缺陷，基于“免疫药剂学”的理论，利用PSA分子中含有的大量负电基团，与阳离子化合物形成稳定的聚离子复合物(Polyion complexes,PICs)。所述聚离子复合物能够与中性粒细胞表面上的唾液酸受体(Selectin、Siglec)结合，“找”到中性粒细胞，以中性粒细胞作为药物载体，同时充分考虑免疫细胞的功能性特征，从而产生更优秀的药理活性。
本发明是通过如下技术方案实现的：
本发明所述的聚离子复合物包含聚唾液酸PSA和阳离子化合物，影响聚离子复合物性能与疗效的主要因素包括PSA与药物的质量比，以及PSA聚合度(平均分子量)。PSA与阳离子化合物的质量比是2:1～50:1，优选5:1～30:1，更优选5:1～20:1；所形成的聚离子复合物中包含一种或者一种以上的阳离子化合物。当阳离子化合物浓度是1～10mg·mL -1时，聚唾液酸与阳离子化合物的质量比是5:1～50:1，但是PSA浓度不能大于50mg·mL -1，否则制剂粘度过大，无法制备稳定的聚离子复合物。
本发明所述的PSA聚合度是2-600，优选为2-270；平均分子量为600-100000道尔顿(600Da～100kDa)；优选分子量为3000-80000道尔顿(3000Da～80kDa)，更优选5000-50000道尔顿(5000Da～50kDa)。
所述的阳离子化合物包括抗肿瘤、抗炎症、抗生素类化合物(氨基糖甙类，如链霉素、庆大霉素、卡那霉素、西索米星、妥布霉素、阿米卡星、奈替米星；大环内酯类，如红霉素、阿奇霉素、克拉霉素、罗红霉素、泰利霉素、地红霉素、氟红霉素；四环素类，如金霉素、土霉素、四环素、甲烯土霉素、强力霉素、二甲胺基四环素及其衍生物氨甲环素类化合物)。作为优选方案，所述的抗肿瘤药物选自下列药物中的一种或者多种：蒽环类药物，如多柔比星、表柔比星、吡柔比星与伊达比星；蒽醌类抗肿瘤药物，如马来酸匹杉琼、盐酸米托蒽醌；喜树碱类抗肿瘤药物，如拓扑替康、伊立替康、依喜替康(exatecan)、氨基喜树碱；替尼类抗肿瘤药，如吉非替尼、伊马替尼、尼罗替尼、舒尼替尼、拉帕替尼、托法替尼、克里唑替尼、马赛替尼、恩曲替尼(entrectinib)、依鲁替尼(ibrutinib)、阿法替尼(afatinib)、氟马替尼(flumatinib)、厄洛替尼(erlotinib)、来那替尼(neratinib)、艾乐替尼(alectinib)、阿帕替尼(Apatinib)、Talazoparib、lorlatinib(第三代ALK抑制剂)、TPX-0005(第四代ALK抑制剂)等；铂类抗肿瘤药物，如顺铂、卡铂、奈达铂、环铂、奥沙利铂、洛铂、Miriplatin Hydrate；其他阳离子化合物，包括博来霉素。除此之外，还包括生物碱，分为吡咯类生物碱、莨菪烷类生物碱、哌啶类生物碱、喹啉与喹诺酮类生物碱、吖啶酮类生物碱、喹唑啉类生物碱、咪唑类生物碱、异喹啉类生物碱、吲哚类生物碱、嘌呤及黄嘌呤类生物碱、大环类生物碱、萜类生物碱、甾类生物碱、有机胺类生物碱，如长春花碱、长春新碱、长春瑞滨、小檗碱、小檗胺等。
根据文献报道，二甲双胍(Metformin may prevent multidrug-resistant breast cancer  https://www.medicalnewstoday.com/articles/320278.php)、千金藤碱(韩立.盐酸千金藤碱逆转肿瘤多药耐药性的作用及其机制.2013.PhD Thesis.郑州大学.)、汉防己甲素(叶祖光；孙爱续.汉防己甲素对多柔比星或长春新碱耐药株人癌细胞的逆转抗药性作用.中国中药杂志,1996,21.6:369-371.)和维拉帕米(Clinical observation of verapamil combined with antitumor drugs in treating malignant pleural effusion.Biomedical Research 2017；28(19):8289-8294)(王坚成,et al.新型多柔比星抗耐药性隐形脂质体的体外细胞毒和体内毒性研究.药学学报,2005,40.5:475-480.)具有逆转多药耐药的作用，本发明也包括将它们与抗肿瘤药物联合应用。
本发明进一步提供了所述聚离子复合物(PICs)的制备方法包括：
(1)将阳离子化合物用纯水溶解，得溶液A(浓度0.1mg/ml～10mg/ml)；
(2)将聚唾液酸(PSA)溶于纯水，得溶液B(浓度1mg/ml～100mg/ml)；
(3)将溶液A与溶液B混合，得到聚离子复合物(PICs)液体(PSA与阳离子化合物的质量比是2/1～50/1)。
根据具体情况，可以采用葡萄糖、海藻糖、蔗糖、乳糖、甘露醇、山梨醇、木糖醇、甘油、氯化钠等物质，调节渗透压，达到符合注射要求。也可进行冷冻干燥或者喷雾干燥，获得固体产品。
处方中还可以加入其它辅料，如EDTA(二钠盐、钙钠盐)、泊洛沙姆、右旋糖酐、聚乙烯吡咯烷酮、多糖类物质(如人参多糖、黄芪多糖、香菇多糖)与pH调节剂等。
当所述的阳离子药物为抗肿瘤药物，尤其蒽环类药物，如多柔比星、表柔比星、吡柔比星或伊达比星时，PSA与阳离子药物之间的质量比为5/1～20/1，PSA的平均分子量为20000道尔顿(20kDa)～50000道尔顿(50kDa)，聚合度为：70～170。
本发明的复合物通过如下方法制备：
精密称取适量阳离子化合物，溶于适量的注射用水中，配制化合物溶液(浓度0.1mg/ml～10mg/ml，A)；精密称取PSA，溶于适量注射用水中，配制PSA溶液(浓度1mg/ml～100mg/ml，B)。将溶液A与 溶液B混合，持续搅拌一定时间，得到聚离子复合物(PICs)液体(PSA与阳离子化合物的质量比是2/1～50/1)。必要时，进行冷冻干燥或者喷雾干燥，得到固体制剂。
用于对比试验的PSA与阳离子药物的共价偶联物通过如下方法制备：
精密称取适量PSA，溶于甲酰胺中，加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl)与N-羟基丁二酰亚胺(NHS)，氮气保护下在冰水浴环境中活化20min。然后加入药物，在室温下反应12h。反应结束后，用重蒸水稀释反应液2倍，将其转移至截留分子量为1kDa的透析袋中，透析24小时，每4小时更换一次透析液。透析结束后，将透析袋中的混悬液通过0.8μm的微孔滤膜，即得聚唾液酸多柔比星共价偶联纳米粒(PSA-conjugated doxorubicin nanoparticles,PSANPs)。
本发明的优点：①尽管大量文献报道，材料与药物共价偶联物形成的前药的药效一般优于静电作用形成的复合物，但我们意外地发现，本发明的聚离子复合物的药效远优于共价偶联纳米粒；②发现中性粒细胞吞噬共价偶联纳米粒而形成许许多多吞噬小体，该吞噬小体不进入细胞核，从而导致抗肿瘤效果极大下降；③本发明具有操作简单、质量可控，成本低廉的特点，具有实际应用价值；④意外地发现，PSA-顺铂聚离子复合物的毒性大大降低，远低于顺铂溶液组；⑤意外地发现，PSA-米托蒽醌聚离子复合物的抑瘤率大于米托蒽醌脂质体。
附图说明
图1为聚离子复合物与共价偶联纳米粒的透射电子显微镜(TEM)成像图；
A：聚离子复合物B：共价偶联纳米粒；
图2为聚唾液酸-多柔比星共价偶联物的表征；
A：聚唾液酸红外图谱B：聚唾液酸核磁图谱C：聚唾液酸-多柔比星共价偶联物红外图谱D：聚唾液酸-多柔比星共价偶联物核磁图谱；
图3为聚离子复合物与共价偶联纳米粒的体外释放实验；
图4为密度梯度离心富集外周血中性粒细胞；
图5为流式细胞术检测中性粒细胞的纯度；
A和C：未标记的中性粒细胞D：荧光素抗体标记的中性粒细胞
图6为CCK8法检测多柔比星制剂对中性粒细胞活力的影响；
图7为中性粒细胞对多柔比星制剂的摄取量考察；
图8为中性粒细胞摄取多柔比星制剂后的释放行为考察；
图9为多柔比星制剂Wistar大鼠体内药动学行为；
图10为多柔比星制剂S180荷瘤昆明鼠体内荧光成像与组织分布；
图11为多柔比星制剂A549荷瘤裸鼠体内荧光成像与组织分布；
图12为流式细胞术分析A549肺癌细胞对多柔比星制剂体外摄取；
图13为激光共聚焦显微镜分析多柔比星制剂在A549肺癌细胞内的转运行为；
图14为S180荷瘤昆明鼠肿瘤生长曲线；
图15为多柔比星制剂对S180荷瘤昆明鼠肿瘤抑制率；
图16为S180荷瘤昆明鼠去瘤体重变化；
图17为S180荷瘤昆明鼠生存率；
图18为S180荷瘤昆明鼠肿瘤脱落现象；
图19为多柔比星制剂对S180荷瘤昆明鼠的抑瘤指数；
图20为S180荷瘤昆明鼠肿瘤与重要组织切片；
图21为A549荷瘤裸鼠肿瘤离体照片；
图22为A549荷瘤裸鼠肿瘤生长曲线；
图23为多柔比星制剂对A549荷瘤裸鼠肿瘤抑制率；
图24为A549荷瘤裸鼠去瘤体重变化；
图25为多柔比星制剂对A549荷瘤裸鼠的抑瘤指数；
图26为A549荷瘤裸鼠肿瘤与重要组织切片；
图27为不同药物聚离子复合物与相应药物溶液对S180肿瘤抑制率。
具体实施方式
下面结合实施例，更具体地说明本发明的内容。应当理解，本发明的实施并不局限于下面的实施例，对本发明所做的任何形式上的变通与/或改变都将落入本发明保护范围。
材料来源：
本发明中，所用PSA的聚合度分别为2、3、4、5、6、10、16、32、100、130～170、200～270，对应分子量分别为644.50，957.73，1270.97，1584.21，1897.45以及平均分子量为3000，5000，16000，30000，4～5万与6～8万道尔顿。聚合度2、3、4、5、6的PSA购买自美国Nacalai公司；聚合度为7至10的PSA购买自Nacalai tesque，日本；PSA(平均分子量11.0kDa，多分散系数(p.d.)1.17；平均分子量22.7kDa，多分散指数(p.d.)1.34；平均分子量39.0kDa，多分散系数(p.d.)1.40)，来自Camida，爱尔兰；平均分子量30kDa购自英国carbosynth公司(卡博森斯化学科技(苏州)有限公司)；其余分子量的PSA从江南大学获取或者自制。
实施例1聚唾液酸(平均分子量30000道尔顿，聚合度为100)多柔比星聚离子复合物的制备
精密称取10mg盐酸多柔比星(DOX)溶于5mL灭菌注射用水中，即得2mg·mL -1的盐酸多柔比星溶液(DOX-S)；精密称取50mg PSA溶于4mL灭菌注射用水中，即得10mg·mL -1的聚唾液酸溶液(PSA-S)。将PSA-S置于磁力搅拌器上，搅拌转速保持100rpm，然后DOX-S逐滴加入PSA-S中，孵育30min，即得聚唾液酸多柔比星聚离子复合物。最后，采用50％葡萄糖注射液调节至等渗，其中多柔比星终浓度为1mg·mL -1。处方筛选与优化结果见表1，电子显微镜图见附图1A。
表1聚离子复合物处方筛选与优化
Figure PCTCN2019074902-appb-000001
注：N.D.表示不能被检测到。
以粒径、载药量和4℃稳定性为指标，当聚唾液酸与多柔比星的质量比为5/1～10/1时，制剂稳定性大于6个月；当多柔比星浓度是1～10mg·mL -1时，聚唾液酸与多柔比星的质量比是5:1～50:1，优选为5:1～20:1，更优选为5:1～10:1。PSA浓度不能大于50mg·mL -1，否则制剂粘度过大，无法制备稳定的聚离子复合物。
当聚唾液酸/多柔比星(质量比)为5/1时，采用平均分子量为644.50，957.73，1270.97，1584.21，1897.45以及平均分子量为3000，5000，10000，20000，30000，4～5万与6～8万道尔顿的PSA制备多柔比星聚离子复合物，结果表明，当平均分子量小于3000时，制剂稳定性小于1个月(平均粒径大于230nm，难以采用过滤除菌)；当平均分子量为3000～10000时，制剂稳定性小于5个月(平均粒径大于200nm)；当平均分子量为10000～80000道尔顿时，其平均粒径为60-200nm，制剂稳定性大于6个月；当平均分子量为20000～50000道尔顿时，其平均粒径为80-160nm，制剂稳定性大于6个月。因此，PSA分子量为10000～80000道尔顿，优选为20000～50000道尔顿。
实施例2聚唾液酸多柔比星共价偶联纳米粒的制备
精密称取50mg PSA(平均分子量30000道尔顿)，溶于5mL甲酰胺中，加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl)63.9mg与N-羟基丁二酰亚胺(NHS)19.2mg，氮气保护下在冰水浴环境中活化20min。然后加入10mg DOX，在室温下反应12h。反应结束后，用重蒸水稀释反应液2倍，再将其转移至截留分子量为1kDa的透析袋中，1000倍体积的重蒸水中透析24小时，每4小时更换一次透析液。透析结束后，将透析袋中的混悬液通过0.8μm的微孔滤膜整粒，即得聚唾液酸多柔比星 共价偶联纳米粒(PSANPs)，其产率约为75％。处方筛选与优化结果见表2，红外图谱与核磁图谱见图2，电子显微镜图见附图1B。
表2共价偶联纳米粒处方筛选与优化
Figure PCTCN2019074902-appb-000002
注：N.D.表示不能被检测到。
以粒径和4℃稳定性为指标，当聚唾液酸与多柔比星的质量比为5/1～10/1时，所得到的偶联物纳米粒制剂稳定性大于6个月，但其平均粒径却大于聚唾液酸多柔比星聚离子复合物的粒径；当多柔比星浓度是1～10mg·mL -1时，聚唾液酸与多柔比星的质量比是5:1～50:1，PSA浓度不能大于50mg·mL -1，否则制剂粘度过大，无法制备稳定的聚离子复合物。
以下以实施例1和2中PSA平均分子量30000道尔顿，聚合度为100，聚唾液酸与多柔比星的质量比是5:1的组方制备的聚离子复合物作为实施例3-15的试验样品。
实施例3多柔比星制剂的体外释放考察
分别精密吸取1.0mL多柔比星溶液、聚离子复合物、共价偶联纳米粒，加入预处理过的透析袋中，置于100mL 10mM磷酸盐缓冲液中，避光，37℃±2℃恒温，50rpm搅拌，于第0.5、1、2、4、8、12、24小时分别吸取1.0mL透析液，并补加等量空白释放介质。将取出的1.0mL透析液采用酶标仪在激发470nm、发射580nm下测定并计算透析液中药物浓度，按照下列公式计算药物的累计释放量R n，结果见图3。
Figure PCTCN2019074902-appb-000003
其中，V 0为释放介质体积，C n为第n次取样时浓度，V为取样体积，M t为总的药物浓度。
多柔比星溶液组在中性与微酸性条件下均快速释放；共价偶联纳米粒在中性与微酸性条件下均基本不释放；聚离子复合物在两种pH条件下均存在缓释能力，但是在弱酸性条件下，其释放加快，具有pH敏感性。
实施例4密度梯度离心富集外周血中性粒细胞及纯度检测
中性粒细胞采用天津市灏洋生物制品科技有限责任公司的小鼠中性粒细胞分离液试剂盒(LZS1100)进行分离纯化，全过程样本、试剂及实验环境均在20±2℃的条件下进行。
1.制备抗凝血液，肝素加入量为10IU·mL -1全血。
2.取一支离心管，先加入分离液1：3ml，后加入80％浓度分离液1溶液(分离液1：样本稀释液＝4：1的混合液)：1.5ml，形成梯度界面。
3.制备血液样本：抗凝血液与红细胞沉降液按1：1比例混匀后，小心加于分离液梯度界面之上，800g离心20min。
4.离心后，血浆层下出现两层白色环状细胞层，取下层白色环状中性粒细胞层(会有少量红细胞混杂)，加3-5倍体积清洗液混匀，400g离心10min。
5.离心后弃上清，即得中性粒细胞。
随后，我们采用迪夫快速染色法(Diff-Quik Stain)对分离得到的中性粒细胞进行染色与光学显微镜下观察，可以看到细胞核呈现2-3瓣，中性粒细胞特征明显，纯度较高。
为了进一步定量确证中性粒细胞的纯度，我们采用Biolegend公司的FITC偶联单克隆抗小鼠 Ly-6G/Ly-6C(Gr-1)抗体特异性标记中性粒细胞。将分离纯化的小鼠中性粒细胞，用PBS洗一遍，加入含10％胎牛血清的RPMI-1640培养基轻轻吹打细胞，使之成为细胞悬液(1×10 6个/mL)，转移至细胞培养瓶中，置于37℃，5％CO 2培养箱中培养0.5h。取出培养瓶后，按250ng·mL -1加入FITC偶联单克隆抗小鼠Ly-6G/Ly-6C(Gr-1)抗体，于5％CO 2、37℃的培养箱中培养0.5h，收集细胞，5000rpm离心3min，去除上清液。加入PBS重悬细胞清洗，5000rpm离心3min，弃去上清，加入200μL PBS重新分散细胞，在流式细胞仪上检测样品的荧光强度，每个样品收集1×10 4个细胞，采用FITC通道检测细胞荧光强度，用FlowJo 7.6.1软件分析数据。其中A与C图为未标记抗体时的本底值，B与D图为抗体标记之后的检测值，中性粒细胞纯度为96.5％。(见图4，5)
实施例5 CCK8法检测多柔比星制剂对中性粒细胞活力的影响
为了考察多柔比星制剂对中性粒细胞活力的影响，又考虑到中性粒细胞的存活时间为6-8小时，无法使用MTT法(显色过程12小时)测定，因此，我们采用只需要4小时即可显色的CCK8法进行测定。
1.将中性粒细胞悬液100μl(约10 4个细胞)加入到96孔板(边缘孔用无菌水或PBS填充)。设置空白孔(有培养基，无细胞)与对照孔(培养基不加药，有细胞)，每组设定3复孔。
2.置37℃，5％CO2孵育0.5小时，倒置显微镜下观察。
3.每孔加入10μl待检测不同浓度多柔比星制剂，37℃孵育。
4.每孔加入10μl CCK-8溶液，37℃孵育4小时。
5.测定450nm各孔的吸光值。
6.结果分析：将各测试孔的OD值减去调零孔OD值或对照孔OD值。各重复孔的OD值取平均数。细胞活力％＝(加药细胞OD-空白OD)/(对照细胞OD-空白OD)×100％
CCK8实验结果显示，多柔比星浓度从1-100微克/毫升范围内，溶液组细胞活力均显著低于其他制剂组，聚离子复合物组细胞活力均小于共价偶联纳米粒组，但是10微克/毫升的多柔比星浓度下孵育细胞时，聚离子复合物与共价偶联纳米粒的中性粒细胞活力仍超过85％，因此，可以认为我们在进行后续试验时(多柔比星浓度为5微克/毫升)，中性粒细胞活力不受影响。结果见图6。
实施例6中性粒细胞对多柔比星制剂的摄取量考察
分离纯化的中性粒细胞加适量的不含胎牛血清的RPMI-1640培养基轻轻吹打细胞，使之成为细胞悬液，配制成浓度为5×10 5个/mL的细胞悬液，以1×10 6/孔的密度接种到6孔培养板中，每孔加2mL培养体系，置于37℃，5％CO 2培养箱中培养0.5h。取出培养板后，更换含有无菌多柔比星溶液、聚离子复合物与共价偶联纳米粒(多柔比星浓度均为5mg·mL -1)的培养液100μL，于5％CO 2、37℃的培养箱中培养0.5h，收集细胞，5000rpm离心3min，去除上清液。加入PBS重悬细胞清洗，5000rpm离心3min，弃去上清。其中一组中性粒细胞加入200μL PBS重新分散细胞，在流式细胞仪上检测样品的荧光强度，每个样品收集1×10 4个细胞，通过PE通道检测。用FlowJo 7.6.1软件分析数据。同时，另一组中性粒细胞采用加入500μL细胞裂解液处理后，5000rpm离心3分钟，取200μL上清液加入96孔板，采用酶标仪设置激发470nm、发射580nm下进行荧光检测，测定其中的多柔比星含量。结果见图7。
细胞摄取实验证明，中性粒细胞对聚离子复合物与共价偶联纳米粒的摄取能力远大于多柔比星溶液。由于多柔比星溶液以分子形式快速自由扩散进入细胞，一般情况下细胞对溶液的摄取速度远快于对纳米制剂的摄取速度。聚离子复合物与共价偶联纳米粒均采用聚唾液酸作为配体，能够与中性粒细胞表面的L-selectin受体特异性结合，提高摄取速度，增加摄取量。有一部分聚离子复合物可能发生解体现象，导致摄取量略低于共价偶联纳米粒。利用药物制剂的技术，结合细胞生物学方法，制备特殊的药物载体，进行细胞内的靶向与定位，从而将药剂学与细胞生物学有机地结合，体现出各自的优势，实现“1+1>2”的效果。因此，我们提出“细胞药剂学(Cellular pharmaceutics或者Cellular pharmacy)”的概念，有助于药物制剂技术与药物递送机制向细胞层面深入。细胞药剂学的另一任务是，为细胞实验提供质量可靠的制剂，确保细胞实验结果的可信与重现。
实施例7中性粒细胞摄取多柔比星制剂后的释放行为考察
为了考察中性粒细胞摄取多柔比星制剂后的保留能力，我们对中性粒细胞摄取制剂之后的释放行为进行考察。
中性粒细胞对多柔比星制剂摄取的操作同“实施例4”。将摄取完制剂的中性粒细胞用PBS重新分散成 细胞悬液(1×10 6个/mL)，将细胞悬液分装至无菌1.5mL离心管中，于5％CO 2、37℃的培养箱中培养，分别于1、2、4、8、12、24小时收集细胞，5000rpm离心3min，分离上清液与下层细胞沉淀。上层清液直接采用酶标仪设置激发470nm、发射580nm下进行荧光检测，测定细胞外的多柔比星含量。下层细胞沉淀采用加入500μL细胞裂解液处理后，5000rpm离心3分钟，取200μL上清液加入96孔板，采用酶标仪设置激发470nm、发射580nm下进行荧光检测，测定细胞内的多柔比星含量。结果见图8。
中性粒细胞的释放结果说明，多柔比星溶液组虽然也能够被中性粒细胞摄取一部分，但是中性粒细胞会被多柔比星溶液杀伤，并且快速释放其中的多柔比星；而聚离子复合物与共价偶联纳米粒均能够缓慢从中性粒细胞中释放出来。该结果表明，中性粒细胞可以将聚离子复合物与共价偶联纳米粒载于其内较长的时间，有机会将制剂递送至肿瘤、炎症等释放趋化因子(CXCL1)的部位。
实施例8多柔比星制剂Wistar大鼠体内药动学行为
将9只体重180-220g的Wistar大鼠随机分为3组，每组3只，即多柔比星溶液、共价偶联纳米粒与聚离子复合物。给药剂量以多柔比星计为5.0mg·kg -1，并于给药后0.083、0.25、0.5、1、2、4、8、12与24小时分别于眼眶静脉丛取血并置于肝素抗凝管中，5000rpm离心10min分离血浆，取100μL血浆，加入900μL甲醇/水(体积比50/50)，涡旋5分钟，10000rpm离心5分钟，取200μL上清液采用酶标仪在激发470nm、发射580nm下测定多柔比星浓度。结果见图9。
共价偶联纳米粒与聚离子复合物均体现出一定的长循环效果。其中共价偶联纳米粒稳定性较好，体内循环性最佳；聚离子复合物清除速度略快于共价偶联纳米粒，可能聚离子复合物在体内发生部分解体，被快速清除。
实施例9多柔比星制剂S180荷瘤昆明鼠体内荧光成像与组织分布
液氮中取出保种的S180细胞冻存管，迅速置于37℃水中复苏。将复苏的S180细胞悬液接种于昆明小鼠腹腔内(0.6mL/只)。6～7天后在无菌条件下抽取乳白色粘稠腹水，在倒置显微镜下计数，当肿瘤细胞活度大于95％时加生理盐水稀释成细胞混悬液，调整稀释倍数，使混悬液的瘤细胞数为1.8×10 7cells·mL -1。采用75％酒精消毒，将S180细胞混悬液接种于小鼠右前腋下的皮下组织，每只小鼠接种0.2mL，共接种12只。荷瘤后第3天，将小鼠随机分成4组，即对照组、多柔比星溶液、聚离子复合物与共价偶联纳米粒，每组3只。各组小鼠均于肿瘤体积到达1000mm 3后(约接种后第10～12天)开始进行活体成像。按多柔比星5mg·kg -1剂量尾静脉注射，分别于1,4,8,24小时进行活体荧光成像，在24小时之后脱颈处死小鼠，分离肿瘤与其他主要器官进行离体器官成像。结果见图10。
实验结果表明，多柔比星荧光强度较弱，在体成像不能够定量分析，我们通过离体器官成像进行ROI定量分析。通过定性与定量结果分析，我们发现肿瘤分布量顺序为：聚离子复合物>共价偶联纳米粒>多柔比星溶液。聚离子复合物与共价偶联纳米粒分别比多柔比星溶液在肿瘤中的分布量增加了795％与1005％；聚离子复合物与共价偶联纳米粒分别比多柔比星溶液在肝脏中的分布量增加了114％与163％；但是，聚离子复合物与共价偶联纳米粒分别比多柔比星溶液在肾脏中的分布量减少了74％与60％，在心脏中的分布量减少了78％与51％。聚离子复合物中，多柔比星带正电荷的氨基被周围大量聚唾液酸带负电荷的羧基所包裹；共价偶联纳米粒中的多柔比星的氨基通过酰胺键与PSA的羧基相连，因此，两种制剂中多柔比星的氨基糖结构无法与心肌细胞膜相结合，有效降低了游离多柔比星在心脏的累积量。总体而言，聚离子复合物与共价偶联纳米粒在体内利用NPS通路及EPR效应靶向肿瘤，但是中性粒细胞由于没有消化异物的能力，所以在吞噬异物后，极易被肝与肺中的巨噬细胞清除，导致肝肺中的分布量增加。
实施例10多柔比星制剂A549荷瘤裸鼠体内荧光成像与组织分布
液氮中取出保种的A549细胞冻存管，迅速置于37℃水中复苏。将复苏的A549细胞悬液置于含10％胎牛血清的RPMI-1640培养基中37℃，5％CO 2条件下培养。2～3次传代后，在倒置显微镜下计数，当肿瘤细胞活度大于95％时加生理盐水稀释成细胞混悬液，调整稀释倍数，使混悬液的瘤细胞数为1×10 7cells·mL -1。采用75％酒精消毒裸鼠的右后背皮下，将A549细胞混悬液接种于小鼠右后背的皮下组织，每只小鼠接种0.1mL，共接种12只。荷瘤后第18-20天，将小鼠随机分成4组，每组3只。按多柔比星5mg·kg -1剂量尾静脉注射，分别于1,4,8,24小时进行活体荧光成像，在24小时之后脱颈处死小鼠，分离肿瘤与其他主要器官进行离体器官成像。结果见图11。
聚离子复合物与共价偶联纳米粒分别比多柔比星溶液在肿瘤中的分布量增加了54％与87％；聚离子 复合物与共价偶联纳米粒分别比多柔比星溶液在肝脏中的分布量增加了53％与49％；聚离子复合物与共价偶联纳米粒在心脏中的分布量与多柔比星溶液相似；聚离子复合物与共价偶联纳米粒分别比多柔比星溶液在肾脏中的分布量增加了75％与73％；并且，聚离子复合物与共价偶联纳米粒分别比多柔比星溶液在A549荷瘤裸鼠肺中的分布量显著少于S180荷瘤昆明鼠肺中的分布量，说明S180荷瘤昆明鼠肺巨噬细胞对制剂的清除能力不同；但是两种动物模型肝脏巨噬细胞对制剂的清除能力类似。可能的原因是肝脏巨噬细胞对制剂清除不依赖外界因素，而肺中的巨噬细胞清除制剂可能依赖别的因素。由于昆明鼠体内拥有完整的免疫系统，S180肿瘤能够诱发昆明鼠产生较强的炎症反应，促使体内的中性粒细胞对制剂进行快速吞噬；而A549肿瘤对裸鼠诱导炎症反应较弱，有效激活中性粒细胞的能力下降，中性粒细胞摄取的制剂较少，所以中性粒细胞介导的制剂被巨噬细胞的摄取较少。肝脏中的枯否细胞可以直接高效的摄取制剂，因此肝脏对制剂的摄取情况类似。
实施例11 A549肺癌细胞对多柔比星制剂体外摄取与细胞内转运行为考察
收集处于对数生长期的A549细胞，用PBS洗一遍后加适量的新鲜培养基轻轻吹打细胞，使之成为细胞悬液，配制成浓度为2.5×10 5个/mL的细胞悬液，以5×10 5/孔的密度接种到6孔培养板中，每孔加2mL培养体系，置于37℃，5％CO 2培养箱中培养24h。取出培养板后，更换含有无菌多柔比星溶液、聚离子复合物与共价偶联纳米粒(多柔比星浓度均为5mg·mL -1)的培养液100μL，于5％CO 2、37℃的培养箱中培养4h，收集细胞，1500rpm离心5min，去除上清液。加入PBS重悬细胞清洗，1500rpm离心5min，弃去上清，加入200μL PBS重新分散细胞，在流式细胞仪上检测样品的荧光强度，每个样品收集1×10 4个细胞，通过PE通道检测。用FlowJo 7.6.1软件分析数据，得到平均荧光强度。
实验结果说明，多柔比星溶液以分子形式快速自由扩散进入A549细胞，因此摄取量最大；聚离子复合物与共价偶联纳米粒通过载体表面的唾液酸配体介导，被A549细胞主动摄取，摄取量显著大于对照组。
为了进一步研究载体在细胞内转运行为，取对数生长期的A549细胞，小心吸走培养皿中的培养基，PBS清洗一遍后加入新鲜的培养基，轻轻吹打细胞使其成为细胞悬液。取洁净细胞爬片在75％乙醇中浸泡5min，用无菌的PBS洗涤三遍，再用细胞培养液洗涤一遍。将盖玻片置于十二孔板内，接种细胞并置于5％CO 2，37℃的培养箱中培养过夜,使其约为50％～80％满。更换培养基为含多柔比星制剂的培养基，分别为含多柔比星溶液、聚离子复合物与共价偶联纳米粒的培养液(多柔比星浓度均为5mg/mL)，置于5％CO 2,37℃的培养箱中培养4h后。取出爬片细胞板，除去上清液，加入4％多聚甲醛固定细胞20min。固定完成后，弃去废液，使用PBS清洗三遍。加50μL DAPI工作液(细胞核染料)，室温避光孵育15min，染色结束后用冷的PBS清洗三次。轻轻甩干爬片，将抗荧光淬灭封片剂滴于载玻片上，从培养板中取出盖玻片，附着有细胞的一面向下扣在载玻片上，排除气泡，吸干溢出的封片剂，封边，最后爬片置于激光共聚焦显微镜下观察并拍照。结果见图12，13。
实验结果说明，多柔比星溶液以分子形式快速自由扩散进入A549细胞，并且进入细胞核，与DNA结合呈现红色，与细胞核蓝色染料叠加后呈现出明显的紫色细胞核；聚离子复合物被A549细胞摄取之后，进入细胞的内含体与溶酶体，随着pH的逐渐降低，聚离子复合物中的多柔比星加速释放，快速扩散，均匀分布于整个细胞中，但是细胞核区域多柔比星的分布量较溶液组少，与细胞核蓝色染料叠加后呈现出浅紫色；共价偶联纳米粒被A549细胞摄取后，在细胞质中呈现明显的点状分布，说明聚集在内含体与溶酶体中，细胞核区域几乎没有多柔比星分布，叠加后细胞核区域仅呈现细胞核染料的蓝色，说明共价偶联纳米粒通过胞吞胞饮途径进入A549细胞，由于多柔比星与聚唾液酸之间的酰胺键不易断裂，导致纳米粒中的多柔比星分子不能有效释放，未能进入细胞核发挥抑制作用。很显然，有必要采用“细胞药剂学”的理论对细胞靶向、细胞载体进行相关设计与研究，特别是针对不同处方组成和制剂理化特性等影响体外细胞实验结果的因素进行系统研究。
实施例12多柔比星制剂对S180荷瘤昆明鼠抗肿瘤研究
液氮中取出保种的S180细胞冻存管，迅速置于37℃水中复苏。将复苏的S180细胞悬液接种于昆明小鼠腹腔内(0.6mL/只)。6～7天后在无菌条件下抽取乳白色粘稠腹水，在倒置显微镜下计数，当肿瘤细胞活度大于95％时加生理盐水稀释成细胞混悬液，调整稀释倍数，使混悬液的瘤细胞数为1.8×10 7cells·mL -1。采用75％酒精消毒，将S180细胞混悬液接种于小鼠右前腋下的皮下组织，每只小鼠接种0.2mL，共接种24只。荷瘤后第3天，将小鼠随机分成4组，即对照组、多柔比星溶液、聚离子复合物 与共价偶联纳米粒，每组6只。各组小鼠均于肿瘤体积到达100mm 3后(接种后第3天)开始尾静脉注射给药，每3天1次，共给药5次(接种后第3、6、9、12与15天)，各组的单次给药剂量均为5mg多柔比星·kg -1，对照组5％葡萄糖注射液的给予量为10mL·kg -1。在整个药效学试验期间，记录肿瘤体积、体质量、死亡事件等数据。结果见图14-19。
抗肿瘤实验结果显示，抑瘤率依次为：聚离子复合物>多柔比星溶液>共价偶联纳米粒。多柔比星溶液肿瘤靶向性差，对正常组织的非特异性毒性较大，抑瘤率较低，体质量较小，中位生存时间较短；聚离子复合物与共价偶联纳米粒均能够高效靶向S180肿瘤，但是共价偶联纳米粒进入细胞后无法快速释放多柔比星分子，导致其抑瘤率小于聚离子复合物与多柔比星溶液。聚离子复合物进入细胞后，能够快速释放出多柔比星，抑制肿瘤生长，抑瘤效果最好。去瘤体重、生存分析与中位生存时间结果表明，三个制剂组的毒性强弱顺序为：多柔比星溶液>共价偶联纳米粒>聚离子复合物。由于多柔比星制剂的细胞毒性较大，对肿瘤的抑制作用较强，所以在这次试验中均体现出较好的抑瘤效果，同时也造成实验动物的非特异性损伤，这种损伤可以通过小鼠的体重直接反应出来。因此，为了更好地体现制剂的有效性与靶向性，兼顾制剂对肿瘤细胞的抑制和对机体非特异性的损伤，我们提出一个新的评价指标——“抑瘤指数(Tumor-inhibition index,TI index)”，可以分为“质量抑瘤指数”或者“体积抑瘤指数”两种计算形式，即“荷瘤动物质量/肿瘤质量”或者“荷瘤动物体积/肿瘤体积”，也可采用荷瘤动物质量/肿瘤体积、去瘤动物质量/肿瘤体积、去瘤动物质量/肿瘤质量，抑瘤指数越大，整体治疗效果越好。由于小鼠体质量较易获取，因此我们选择采用质量抑瘤指数形式进行计算分析。通过对各组抑瘤指数的比较，我们发现聚离子复合物远大于其他组，而多柔比星溶液与共价偶联纳米粒虽然具有一定的抑瘤率，但是其抑瘤指数与对照组相似，说明多柔比星溶液与共价偶联纳米粒的非特异性毒性对机体造成的损伤抵消了抗肿瘤效果。抑瘤指数将肿瘤生长情况与生存质量(Quality of life,QOL)有机地结合在一起，为客观评价抗肿瘤药物疗效提供更多的信息，获得更多的思考与启示，也充分体现了其在抗肿瘤实验过程中的重要价值。
在抗肿瘤实验5次给药之后，令人意外的是，发现聚离子复合物组的肿瘤出现脱落现象，这说明聚离子复合物不但能够高效靶向肿瘤细胞，还可以作用于肿瘤微环境，对肿瘤周围的细胞进行杀伤，造成肿瘤脱落。这种“脱落”与肿瘤部位的血管内皮细胞上表达的，且决定肿瘤转移与侵袭的“选择素”有关。唾液酸(SA)是选择素的配体，因此使用SA修饰载体在肿瘤靶向治疗中具有一定的价值，利用受体-配体介导的内吞作用，将SA与多柔比星以酯键相连接，大大增加药物转运效率，由此解决细胞摄取受阻问题。
然而，在免疫药剂学理论指导下的本次实验结果，与后续的研究结果均证明，中性粒细胞参与了聚离子复合物的摄取与递送过程，也有可能是一部分肿瘤相关中性粒细胞(Tumor associated neutrophils,TANs)直接摄取了利用EPR效应进入肿瘤部位的聚离子复合物而被杀伤。另一方面，昆明鼠是一种免疫健全的动物模型，当机体产生肿瘤时，机体会调动免疫系统，尤其是T细胞对肿瘤的杀伤作用，但是由于肿瘤微环境中存在着TAN与TAM等，抑制了T细胞的作用，SA/PSA制剂可以有效杀伤TAN(Faget J,Groeneveld S,Boivin G,et al.Neutrophils and Snail Orchestrate the Establishment of a Pro-tumor Microenvironment in Lung Cancer[J].Cell reports,2017,21(11):3190-3204.)，改善肿瘤微环境，T细胞发挥作用，导致肿瘤脱落。然而，胸腺免疫缺陷鼠体内，没有T细胞。尽管可以杀伤TAN，改善了肿瘤微环境，但没有肿瘤脱落现象。这也说明，对于肿瘤的治疗，最终途径还是得调动免疫系统，杀伤肿瘤。
细胞毒性实验显示，高浓度的制剂会对中性粒细胞产生细胞毒性。首先，中性粒细胞由于细胞因子的刺激，会产生较多分化，其中SiglecF高表达的中性粒细胞可以高效摄取制剂，并且这种中性粒细胞对肿瘤生长最有帮助，因此将其杀伤也是一种有效抑制肿瘤的策略；同时，摄取相对较少的中性粒细胞由于没有受到损伤，发挥着完整的生理学功能，可以在细胞因子的引导下递送至靶部位。
尽管有研究表明，PSA与表柔比星通过酰胺键偶联，体外抗肿瘤效果优于表柔比星，且具有较长的循环时间(Greco F,Arif I,Botting R,et al.Polysialic acid as a drug carrier:evaluation of a new polysialic acid–epirubicin conjugate and its comparison against established drug carriers[J].Polymer Chemistry,2013,4(5):1600-1609.)，但我们通过体内外实验意外发现，聚离子复合物的药效远优于共价偶联纳米粒。可能的原因：1.共价偶联纳米粒中的药物与PSA相互偶联，无法在细胞内释放，导致药物无法进入细胞核发挥药效；2.聚离子复合物可以高效快速靶向结合于中性粒细胞表面的L-selectin与SiglecF，在制剂解体 之前被中性粒细胞摄取，然后以中性粒细胞为载体在细胞因子的引导下递送至肿瘤靶部位。
实施例13 S180荷瘤昆明鼠肿瘤与重要组织切片
为了考察各多柔比星制剂对小鼠主要器官的毒性，在S180荷瘤昆明鼠抗肿瘤实验结束后，收集各组荷瘤小鼠的心、肝、脾、肺、肾、脑与肿瘤，用于HE病理切片。
石蜡切片制作
1组织固定
将新鲜剥离的心、肝、脾、肺、肾、肿瘤组织用PBS(pH 7.4)洗净后用4％多聚甲醛固定24h以上，固定结束后将组织从固定液取出，在通风橱内用手术刀将组织修平整。
2冲洗
将修整好的组织块放入包埋盒，将包埋盒置于蒸馏水下，用较低的流水速度冲洗组织，冲洗12～24h。
3脱水
将包埋盒放进吊篮里，置于脱水机内，用不同梯度的乙醇进行脱水，依次用75％乙醇作用4h，85％乙醇、90％乙醇各作用2h，95％乙醇作用1h，再用无水乙醇作用2次进行脱水。
4透明与浸蜡
将各组织转移至密闭容器内，二甲苯:乙醇(1:1，v/v)作用5～10min，二甲苯I作用5～10min，二甲苯II作用5～10min。透明结束后将各组织块浸没在完全融化的石蜡中(60℃恒温箱)1h，再将组织块转移到新的石蜡液中进行二次浸蜡1h。
5包埋
将浸好蜡的组织置于包埋机中进行包埋。先将融化的蜡放入包埋框，在蜡凝固之前，将组织从脱水盒内取出，按照包埋面的要求放入包埋框并贴上对应的标签。在-20℃冻台冷却，待蜡凝固后，将蜡块从包埋框中取出并对蜡块进行修整。
6切片
将蜡块置于石蜡切片机上连续切片，片厚5μm，将切片漂浮于摊片机上，40℃温水将组织展平，用防脱载玻片将组织捞起，并于60℃烘箱内烤片。待水烤干、蜡烤化后，将载玻片取出，常温保存备用。
7 HE染色
8石蜡切片脱蜡至水
将组织切片放入二甲苯I、二甲苯II溶液中各浸泡20min，100％乙醇、95％乙醇、85％乙醇、75％乙醇各浸泡5min，最后用蒸馏水冲洗10min。
9染核
切片入苏木素染液染8min，用蒸馏水冲洗去浮色，再用1％的盐酸酒精分化数20s，最后用蒸馏水冲洗返蓝。
10染质
切片入伊红染液中染色3min，再用蒸馏水冲洗20s。
11脱水封片
将切片依次用95％酒精I、95％酒精II各浸泡5min，再用无水乙醇Ⅰ、无水乙醇Ⅱ各浸泡5min，然后在二甲苯Ⅰ、二甲苯Ⅱ中各浸泡5min脱水透明，最后将切片从二甲苯中拿出晾干，用中性树胶封片。
12镜检拍照
待封片干燥完毕后，将载玻片置于倒置显微镜下观察。结果见图20。
肿瘤与重要组织切片显示，对照组：肿瘤生长活跃，呈现明显的核大深染，核浆失调，心肌细胞完整，脾脏和肾脏未见异常。多柔比星溶液组：肿瘤细胞局部坏死，呈现粉染；心肌多处断裂，甚至细胞溶解(箭头)；脾脏局部出现坏死(箭头)，肾小管出现萎缩(箭头)；共价偶联纳米粒组：肿瘤细胞出现局部坏死；心肌细胞完整；聚离子复合物组：肿瘤细胞可见多处灶性坏死，甚至细胞溶解(箭头)，呈现大面积粉染；心肌细胞完整。结果表明：由于多柔比星游离分子会快速被肾脏清除，因此溶液组的肾脏出现肾小管萎缩的现象。聚离子复合物与共价偶联纳米粒组肿瘤均出现坏死现象，但是聚离子复合 物组的肿瘤出现多处严重的、较大面积的坏死，这可能是由于聚离子复合物能够快速释放多柔比星分子，抑制肿瘤生长，而共价偶联纳米粒不易释放药物，导致抑制作用较弱。
实施例14多柔比星制剂对A549荷瘤裸鼠抗肿瘤研究
液氮中取出保种的A549细胞冻存管，迅速置于37℃水中复苏。将复苏的A549细胞悬液置于含10％胎牛血清的RPMI-1640培养基中37℃，5％CO 2条件下培养。2-3次传代后，在倒置显微镜下计数，当肿瘤细胞活度大于95％时加生理盐水稀释成细胞混悬液，调整稀释倍数，使混悬液的瘤细胞数为1×10 7cells·mL -1。采用75％酒精消毒，将A549细胞混悬液接种于小鼠右后背的皮下组织，每只小鼠接种0.1mL，共接种24只。荷瘤后第9天，将小鼠随机分成4组，即对照组、多柔比星溶液、聚离子复合物与共价偶联纳米粒，每组6只。各组裸鼠均于肿瘤体积到达100mm 3后(约接种后第9天)开始尾静脉注射给药，每3天1次，共给药5次(接种后第9、14、19与24天)，各组的单次给药剂量均为5mg多柔比星·kg -1，对照组5％Glu的给予量为10mL·kg -1。在整个试验期间，记录肿瘤体积、体质量、死亡事件等数据。结果见图21-25。
肿瘤离体照片、肿瘤生长曲线与抑瘤率结果显示，对照组肿瘤体积快速增长，各制剂组抑瘤率顺序为：聚离子复合物>多柔比星溶液>共价偶联纳米粒。活体成像实验表明，聚离子复合物与共价偶联纳米粒均能够高效靶向A549肿瘤，但是根据激光共聚焦显微镜观察结果，共价偶联纳米粒无法有效释放多柔比星分子，不能抑制肿瘤细胞生长。而聚离子复合物可以快速将药物递送至细胞核，发挥药效，因此聚离子复合物体现出最佳的抑瘤作用。
去瘤体重结果可以反映裸鼠的健康状态，同时也可说明制剂的非特异性毒性，对照组与共价偶联纳米粒组的去瘤体重几乎相同，说明共价偶联纳米粒的毒性几乎可以忽略；溶液组非特异性毒性最大；聚离子复合物有一定的非特异性毒性，但是远小于溶液组，并且与对照组不存在显著性差异。
通过对各组抑瘤指数的比较，我们发现聚离子复合物既有较强的肿瘤抑制作用，又有较好的靶向性，因此其抑瘤指数远大于其他组。而多柔比星溶液与共价偶联纳米粒虽然具有一定的抑瘤率，但是其抑瘤指数与对照组相似，说明多柔比星溶液与共价偶联纳米粒的非特异性毒性对机体会造成一定的损伤，因此，抑瘤指数综合评价多柔比星溶液与共价偶联纳米粒抗肿瘤药效的结果相似。
实施例15 A549荷瘤裸鼠肿瘤与重要组织切片
为了考察各多柔比星制剂对小鼠主要器官的毒性，在A549荷瘤裸鼠抗肿瘤实验结束后，收集各组荷瘤小鼠的心、肝、脾、肺、肾、脑与肿瘤，用于HE病理切片。
肿瘤与脏器的石蜡切片制作同“实施例13”。结果见图26。
对照组：肿瘤生长活跃，呈现明显的核大深染，核浆失调，新生血管明显(圈中)，心肌细胞完整。多柔比星溶液组：肿瘤细胞局部坏死，呈现粉染；心肌多处断裂，甚至细胞溶解(箭头)；脾脏局部出现坏死(箭头)。共价偶联纳米粒组：肿瘤细胞生长活跃，呈现核大深染，但是新生血管较少；心肌细胞完整。聚离子复合物组：肿瘤细胞可见灶性坏死，甚至细胞溶解(箭头)，呈现大面积粉染；心肌细胞完整。结果表明：聚离子复合物的抑瘤作用最佳，溶液组的抑瘤作用也比较明显，对照组与共价偶联纳米粒组的肿瘤细胞生长活跃，溶液组具有明显的心脏毒性与脾脏毒性，其他组的毒性并不明显。
实施例16多种聚离子复合物与其药物溶液抗S180肿瘤效果比较
为了确定聚唾液酸与不同种类的阳离子药物所形成聚离子复合物的疗效，我们制备了聚唾液酸(平均分子量30000道尔顿)与不同阳离子抗肿瘤药物的聚离子复合物(聚唾液酸与阳离子药物的质量比是10/1)，当阳离子药物浓度是1～10mg·mL -1时，聚唾液酸与阳离子药物的质量比是5:1～50:1，但是PSA浓度不能大于50mg·mL -1，否则制剂粘度过大，无法制备稳定的聚离子复合物。78只昆明小鼠接种鼠源S180肿瘤细胞后，随机分为13组(每组6只)，生理盐水对照组(10mL·kg -1)、盐酸米托蒽醌溶液(按药物剂量5mg/kg给药)、伊立替康溶液(按药物剂量15mg/kg给药)、依鲁替尼溶液(按药物剂量10mg/kg给药)、奥沙利铂溶液(按药物剂量10mg/kg给药)、长春瑞滨溶液(按药物剂量10mg/kg给药)、博来霉素溶液(按药物剂量10mg/kg给药)、聚唾液酸-盐酸米托蒽醌聚离子复合物(按药物剂量5mg/kg给药)、聚唾液酸-伊立替康聚离子复合物(按药物剂量15mg/kg给药)、聚唾液酸-依鲁替尼聚离子复合物(按药物剂量10mg/kg给药)、聚唾液酸-奥沙利铂聚离子复合物(按药物剂量10mg/kg给药)、聚唾液酸-长春瑞滨聚离子复合物(按药物剂量10mg/kg给药)、聚唾液酸-博来霉素聚离子复合物(按药物剂量10mg/kg给药)。 于接种后第4天(肿瘤体积约为80～100mm 3)腹腔注射给药，每3天给药一次，共给药3次。于第17天处死小鼠，剥离肿瘤，称重，计算抑瘤率。结果见图27。结果显示，聚离子复合物组的抑瘤率显著高于相应的溶液组，说明聚唾液酸与阳离子抗肿瘤药物所形成的聚离子复合物具有普遍适用性。
实施例17不同比例聚唾液酸与马来酸匹杉琼聚离子复合物对S180肿瘤抑制效果
聚唾液酸(平均分子量30000道尔顿)与马来酸匹杉琼按质量比4/1、5/1、10/1、20/1和50/1制备聚离子复合物，当马来酸匹杉琼浓度是1～10mg·mL -1时，聚唾液酸与马来酸匹杉琼的质量比是5:1～50:1，但是PSA浓度不能大于50mg·mL -1，否则制剂粘度过大，无法制备稳定的聚离子复合物。随后进行S180荷瘤昆明鼠抗肿瘤试验，将36只昆明小鼠接种鼠源S180肿瘤细胞，随机分为6组(每组6只)，生理盐水对照组(10mL·kg -1)、4/1聚离子复合物组、5/1聚离子复合物组、10/1聚离子复合物组、20/1聚离子复合物组与50/1聚离子复合物组。于接种后第4天(肿瘤体积约为80～100mm 3)腹腔注射给药，每3天给药一次，共给药3次。于第14天处死小鼠，剥离肿瘤，称重，计算抑瘤率。结果显示，4/1聚离子复合物组的小鼠出现平均体重下降，5/1、10/1、20/1与50/1聚离子复合物组的小鼠均未出现体重下降现象。抑瘤率结果显示，4/1聚离子复合物的抑瘤率(51％)显著低于5/1(83％)、10/1(82％)、20/1(85％)与50/1(80％)聚离子复合物组，5/1、10/1、20/1与50/1聚离子复合物组之间没有显著性差异。这充分说明聚离子复合物的处方比例直接影响治疗效果，而当聚离子复合物形成稳定的纳米颗粒时，处方比例对治疗效果的影响就会减弱甚至消除。
实施例18马来酸匹杉琼聚离子复合物与市售产品稳定性比较
精密称取20mg马来酸匹杉琼(Pix)溶于5mL灭菌注射用水中，即得4mg·mL -1的马来酸匹杉琼溶液(Pix-S)；精密称取100mg PSA溶于4mL灭菌注射用水中，并调整体积至5mL，即得25mg·mL -1的聚唾液酸溶液(PSA-S)。将PSA-S置于磁力搅拌器上，搅拌转速保持100rpm，然后Pix-S逐滴加入PSA-S中，孵育30min，即得PSA-匹杉琼聚离子复合物(PSA与匹杉琼的质量比是5/1)。最后，冷冻干燥，得到PSA-匹杉琼冻干品。进行稳定性加速试验(40℃，RH75％)，考察马来酸匹杉琼含量，同时与市售同类产品进行比较，结果见表3。
表3马来酸匹杉琼聚离子复合物与市售产品稳定性比较
Figure PCTCN2019074902-appb-000004
在十二月加速试验期间，PSA-匹杉琼组的杂质为0.15％，市售品的杂质为0.33％。
实施例19顺铂聚离子复合物的制备及其抗S180肿瘤药效学评价
取PSA分子量(60000～80000道尔顿)适量，精密称定，加入至100mL水中溶解。取顺铂适量，精密称定，加入到上述PSA溶液中，37℃水浴使顺铂充分溶解(PSA与顺铂的质量比50/1)，但是PSA浓度不能大于50mg·mL -1，否则制剂粘度过大，无法制备稳定的聚离子复合物，用NaOH调节其pH至7.0～7.5，室温下避光搅拌2h，之后冷冻干燥即得PSA-顺铂聚离子复合物。
毒性试验：按照常规毒性试验方法，取健康小鼠20只，随机分为2组，一组给顺铂溶液，另外一组给PSA-顺铂聚离子复合物(质量比50/1)，腹腔注射(15mg/kg)，隔天一次，共给3次药，观察20天。顺铂组中第7天死亡2只，第20天死亡5只；而PSA-顺铂聚离子复合物组，没有死亡。结果表明，聚离子复合物极显著地降低顺铂的毒性。
抗肿瘤试验：18只昆明小鼠接种鼠源S180肿瘤细胞后，随机分为3组(每组6只)，生理盐水对照组(10mL·kg -1)、顺铂阳性对照组(4mg·kg -1)与PSA-顺铂聚离子复合物组(质量比50/1，4mg·kg -1)。于接种后第4天(肿瘤体积约为80～100mm 3)腹腔注射给药，每3天给药一次，共给药3次。于第17天处死小鼠，剥离肿瘤，称重，计算抑瘤率。结果显示聚离子复合物抑瘤率(80％)显著优于顺铂溶液组(30％)，说明PSA-顺铂聚离子复合物能够有效提高顺铂抗肿瘤效果，同时，顺铂溶液组的小鼠均出现体重下降， 并有1只死亡现象，而聚离子复合物组没有小鼠死亡，体重也未下降。
实施例20米托蒽醌聚离子复合物与米托蒽醌脂质体抗S180比较
精密称取10mg盐酸米托蒽醌(Mit)溶于5mL灭菌注射用水中，即得2mg·mL -1的盐酸米托蒽醌溶液(Mit-S)；精密称取50mg PSA(平均分子量为30000道尔顿)溶于灭菌注射用水中，配制10mg·mL -1的溶液(PSA-S)。将PSA-S置于磁力搅拌器上，搅拌转速保持100rpm，然后将Mit-S逐滴加入PSA-S中，继续搅拌60min，即得PSA米托蒽醌聚离子复合物(质量比5/1)，米托蒽醌终浓度为1mg·mL -1。
采用HSPC/CH/PEG2000-DSPE质量比为3/1/1制备脂质体，以硫酸铵梯度法装载盐酸米托蒽醌，得到1mg·mL -1米托蒽醌脂质体。按照常规抗S180肿瘤实验进行比较研究，静脉注射(5mg/kg)，每3天给药1次，共给药3次。于第15天处死小鼠，剥离肿瘤，称重，计算抑瘤率。结果显示，聚离子复合物抑瘤率(88％)优于脂质体组的抑瘤率(63％)。
实施例21不同分子量聚唾液酸与表柔比星聚离子复合物对S180肿瘤抑制效果
不同分子量PSA(644.50，957.73，1270.97，1584.21，1897.45以及平均分子量为3.0kDa、11.0kDa、22.7kDa、39.0kDa、60kDa～80kDa、100kDa～200kDa)，参照“实施例1”制备PSA-表柔比星聚离子复合物(PSA与药物的质量比为10:1)，表柔比星终浓度为1mg·mL -1。
S180荷瘤昆明鼠抗肿瘤试验：78只昆明小鼠接种鼠源S180肿瘤细胞后，随机分为13组(每组6只)，生理盐水对照组(10mL·kg -1)、表柔比星阳性对照组(5mg·kg -1)与不同分子量的PSA-表柔比星聚离子复合物组。于接种后第4天(肿瘤体积约为80～100mm 3)腹腔注射给药(按表柔比星剂量5mg·kg -1)，每3天给药一次，共给药3次。于第17天处死小鼠，剥离肿瘤，称重，计算抑瘤率。结果显示不同分子量的聚离子复合物抑瘤率(37％，41％，40％，45％，43％，50％，52％，85％，90％，93％，92％)均优于表柔比星溶液组(30％)，说明PSA-表柔比星聚离子复合物能够有效提高表柔比星的抗肿瘤效果，同时，聚离子复合物可以有效降低药物的非特异性毒性。
实施例22阿奇霉素-博来霉素-PSA聚离子复合物的制备
博来霉素的一个不良作用是导致肺纤维化。有文献报道，阿奇霉素对博来霉素致大鼠肺纤维化具有干预作用。因此，将两者进行组合。
将盐酸博来霉素与枸橼酸阿奇霉素分别用注射用水溶解，配制成为溶液(10mg/ml)，得到博来霉素溶液和阿奇霉素溶液。再用注射用水溶解PSA(平均分子量为3万～5万道尔顿)，配制10mg/ml的PSA溶液。取博来霉素溶液1ml、阿奇霉素溶液2ml，与20mlPSA溶液混合，搅拌20分钟，过0.2μm的微孔滤膜，所得溶液进行冷冻干燥，得到阿奇霉素-博来霉素-PSA聚离子复合物冻干品。
实施例23小檗碱-表柔比星-PSA聚离子复合物
将盐酸小檗碱与盐酸表柔比星分别用注射用水溶解，配制成为溶液(10mg/ml)，得到盐酸小檗碱溶液和盐酸表柔比星溶液。再用注射用水溶解PSA(平均分子量为3万道尔顿)，配制10mg/ml的PSA溶液。取盐酸小檗碱溶液和盐酸表柔比星溶液各1ml，与10mlPSA溶液混合(注：PSA浓度不能大于50mg·mL -1，否则制剂粘度过大，无法制备稳定的聚离子复合物)，搅拌60分钟，过0.2μm的微孔滤膜，所得溶液进行冷冻干燥，得到小檗碱—表柔比星-PSA聚离子复合物冻干品。
参考“实施例17不同比例聚唾液酸与马来酸匹杉琼聚离子复合物对S180肿瘤抑制效果”进行抗S180肿瘤实验。设立3组，生理盐水组、小檗碱-表柔比星溶液组、小檗碱-表柔比星-PSA组，按照5mg/kg盐酸表柔比星的剂量给药，小檗碱-表柔比星溶液组和小檗碱-表柔比星-PSA组的抑瘤率分别为53％和87％，其中小檗碱-表柔比星-PSA组出现4只肿瘤脱落现象。
实施例24克拉霉素-PSA聚离子复合物降低刺激性
取适量克拉霉素，用注射用水分散，低温(4～10℃)和搅拌下，滴加稀盐酸，直至全部溶解，得到克拉霉素溶液(5mg/ml)。以注射用水配制PSA(平均分子量10000道尔顿)溶液(100mg/ml)。取2ml克拉霉素溶液逐滴加入1ml PSA溶液中，搅拌混合，得到克拉霉素-PSA聚离子复合物，用NaOH溶液调整pH至7，然后冷冻干燥即可。
小鼠抓挠实验
一般而言，大环内酯类抗生素存在极大的静脉刺激性，导致局部疼痛，严重者甚至导致静脉炎，因此采用“小鼠抓挠实验”研究相关的刺激性。
将实验小鼠随机分3组(生理盐水组/阴性对照组、克拉霉素溶液组/阳性对照组、克拉霉素-PSA聚离子复合物组)，每组6只小鼠，小鼠背部皮下注射给药，注射量均为0.1mL，记录15分钟多柔比星内每只小鼠抓挠给药部位的首次时间和总次数，结果阴性对照组的首次抓挠平均时间为293秒，平均抓挠次数为3.3；阳性对照组首次抓挠平均时间为55秒，平均抓挠次数为63；PSA-克拉霉素首次抓挠平均时间为213秒，平均抓挠次数为7.3。刺激性明显降低。
实施例25强力霉素-PSA聚离子复合物降低刺激性
取适量盐酸强力霉素，用注射用水分散，加热溶解(55～60℃)，得到强力霉素溶液(5mg/ml)。以注射用水配制PSA(平均分子量30000道尔顿)溶液(100mg/ml)。取1ml强力霉素溶液逐滴加入1ml PSA溶液中，搅拌混合，得到强力霉素-PSA聚离子复合物，用NaOH溶液调整pH至6，然后冷冻干燥即可。
大鼠舔足实验
将实验大鼠随机分3组(生理盐水组/阴性对照组、强力霉素溶液组/阳性对照组、强力霉素-PSA聚离子复合物组)，每组6只，大鼠右后足注射给药，注射量均为0.1mL，记录注射后15min内每只大鼠舔足的首次时间和总舔足次数，其中强力霉素溶液和强力霉素-PSA聚离子复合物浓度均为2.5mg/ml。结果见表4，聚离子复合物极大降低强力霉素的刺激性。
表4大鼠舔足实验结果
Figure PCTCN2019074902-appb-000005
实施例26多柔比星-维拉帕米-PSA聚离子复合物对耐药肿瘤的抑制效果
分别配制盐酸多柔比星、盐酸维拉帕米和PSA(平均分子量30000道尔顿)溶液，按照盐酸多柔比星与盐酸维拉帕米和PSA的质量比5/1/50的比例进行混合，搅拌30分钟，过微孔滤膜除菌，得到多柔比星-维拉帕米-PSA聚离子复合物，盐酸多柔比星终浓度为1mg·mL -1。
抗肿瘤实验：采用肝癌耐药细胞HepG2(中国医科大学惠赠)进行抗肿瘤研究，分为4组，每组6只，生理盐水阴性对照组(10mL·kg -1)、多柔比星阳性对照组、多柔比星-维拉帕米联合组、多柔比星-维拉帕米-PSA聚离子复合物组。于接种后第7天静脉注射给药(按多柔比星剂量5mg·kg -1)，每3天给药一次，共给药4次。于末次给药第二天处死小鼠，剥离肿瘤，称重，计算抑瘤率，多柔比星的抑瘤率为22％、多柔比星-维拉帕米联合组的抑瘤率为35％、聚离子复合物组的抑瘤率为81％。
二甲双胍、千金藤碱和汉防己甲素也可按照类似方法进行联合，甚至可以是两种以上的联合。

Claims (10)

  1. 聚唾液酸与阳离子化合物形成的聚离子复合物。
  2. 如权利要求1所述的聚离子复合物，其特征在于，聚唾液酸与阳离子化合物的质量比是2:1～50:1，优选5:1～30:1，更优选5:1～20:1。
  3. 如权利要求1或2所述的聚离子复合物，其特征在于，所述的聚唾液酸聚合度为2-600，优选为2-270。
  4. 如权利要求1-3任何一项所述的聚离子复合物，其特征在于，所述的聚唾液酸的平均分子量为600-100000道尔顿；优选为3000-80000道尔顿，更优选5000-50000道尔顿。
  5. 如权利要求1-4中任何一项所述的聚离子复合物，其特征在于，所述的阳离子化合物选自抗肿瘤药、抗炎症、抗生素、生物碱中的一种或几种；所述的抗肿瘤药物选自下列药物中的一种或者多种：蒽环类、蒽醌类、喜树碱类、替尼类、铂类，所述的蒽环类选自多柔比星、表柔比星、吡柔比星、伊达比星；所述的蒽醌类抗肿瘤药物选自马来酸匹杉琼、盐酸米托蒽醌；所述的喜树碱类抗肿瘤药物选自拓扑替康、伊立替康、依喜替康、氨基喜树碱；所述的替尼类抗肿瘤药物选自吉非替尼、伊马替尼、尼罗替尼、舒尼替尼、拉帕替尼、托法替尼、克里唑替尼、马赛替尼、恩曲替尼、依鲁替尼、阿法替尼、氟马替尼、厄洛替尼、来那替尼、艾乐替尼、阿帕替尼、Talazoparib、lorlatinib、TPX-0005；所述的铂类抗肿瘤药物选自顺铂、卡铂、奈达铂、环铂、奥沙利铂、洛铂、Miriplatin Hydrate；其他抗肿瘤药物为博来霉素；所述的生物碱包括吡咯类生物碱、莨菪烷类生物碱、哌啶类生物碱、喹啉与喹诺酮类生物碱、吖啶酮类生物碱、喹唑啉类生物碱、咪唑类生物碱、异喹啉类生物碱、吲哚类生物碱、嘌呤及黄嘌呤类生物碱、大环类生物碱、萜类生物碱、甾类生物碱、有机胺类生 物碱，如长春花碱、长春新碱、长春瑞滨、小檗碱、小檗胺；所述的抗生素包括氨基糖甙类、大环内酯类、四环素类，所述的氨基糖甙类选自链霉素、庆大霉素、卡那霉素、西索米星、妥布霉素、阿米卡星、奈替米星；所述的大环内酯类选自红霉素、阿奇霉素、克拉霉素、罗红霉素、泰利霉素、地红霉素、氟红霉素；所述的四环素类选自金霉素、土霉素、四环素、甲烯土霉素、强力霉素、二甲胺基四环素及其衍生物氨甲环素类化合物。
  6. 如权利要求5所述的聚离子复合物，其特征在于，当阳离子药物为蒽环类药物多柔比星、表柔比星、吡柔比星或伊达比星时，聚唾液酸与阳离子药物之间的质量比为5/1～20/1，聚唾液酸的平均分子量为20000道尔顿～50000道尔顿，聚唾液酸的聚合度为70-270。
  7. 如权利要求1-6任何一项所述的聚离子复合物，其特征在于，加入葡萄糖、海藻糖、蔗糖、乳糖、甘露醇、山梨醇、木糖醇、甘油、氯化钠，调节渗透压，冷冻干燥或者喷雾干燥，得固体粉末。
  8. 如权利要求1-7任何一项所述的聚离子复合物，其特征在于，所述的聚离子复合物中加入络合剂、pH调节剂、表面活性剂、多糖类物质，所述的络合剂选自EDTA二钠、EDTA钙钠，所述的表面活性剂选自泊洛沙姆，所述的多糖类物质选自人参多糖、黄芪多糖、香菇多糖。
  9. 如权利要求1所述的聚离子复合物的制备方法，其特征在于：
    (1)将阳离子化合物用纯水溶解，得溶液A，所述A的浓度为0.1mg/ml～10mg/ml；
    (2)将聚唾液酸溶于纯水，得溶液B，所述B的浓度为1mg/ml～100mg/ml；
    (3)将溶液A与溶液B混合，得到聚离子复合物液体。
  10. 权利要求1-8中任何一项所述的聚离子复合物在制备抗肿瘤药物、抗炎症药物中的应用。
PCT/CN2019/074902 2018-02-14 2019-02-13 聚唾液酸与阳离子化合物形成的聚离子复合物及其制备方法和应用 WO2019158060A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810151125.2 2018-02-14
CN201810151125.2A CN110152012B (zh) 2018-02-14 2018-02-14 聚唾液酸与阳离子化合物形成的聚离子复合物及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2019158060A1 true WO2019158060A1 (zh) 2019-08-22

Family

ID=67618890

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/074902 WO2019158060A1 (zh) 2018-02-14 2019-02-13 聚唾液酸与阳离子化合物形成的聚离子复合物及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN110152012B (zh)
WO (1) WO2019158060A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113827703A (zh) * 2020-06-08 2021-12-24 沈阳药科大学 一种蜂毒肽-聚唾液酸静电复合物的制备及其用途
CN112618728B (zh) * 2020-12-18 2022-07-29 绍兴文理学院 一种含有聚唾液酸基团的双重响应前药及其合成方法和其在药物制剂中的应用
CN114099469B (zh) * 2021-12-01 2024-03-22 齐齐哈尔医学院 一种复合纳米药物载体及其制备方法和应用
CN114469953B (zh) * 2022-02-14 2023-07-14 沈阳药科大学 一种具有协同作用的抗肿瘤药物组合物、纳米制剂及其制备方法和应用
CN115025241B (zh) * 2022-06-22 2023-08-01 西南民族大学 靶向cd22的纳米制剂及其制备方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080213349A1 (en) * 2006-09-11 2008-09-04 Deepak Ramesh Thakker Liposome Complexes Containing Pharmaceutical Agents and Methods
CN106554425B (zh) * 2015-09-18 2018-10-30 沈阳药科大学 一种聚唾液酸的脂质接枝衍生物及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FRANCESCA GRECO: "Polysialic Acid as A Drug Carrier: Evaluation of A New Polysialic Acid-epirubicin Conjugate and Its Comparison Against Established Drug Carriers", POLYM. CHEM., 30 November 2012 (2012-11-30), XP055633315 *
WU, JIANRONG ET AL.: "Advance in Applications of Polysialic, a Non-GAGs, Non-immunogenic Biomaterial", CHINA BIOTECHNOLOGY, vol. 37, no. 12, 31 December 2017 (2017-12-31), pages 96 - 102 *

Also Published As

Publication number Publication date
CN110152012B (zh) 2021-06-01
CN110152012A (zh) 2019-08-23

Similar Documents

Publication Publication Date Title
WO2019158060A1 (zh) 聚唾液酸与阳离子化合物形成的聚离子复合物及其制备方法和应用
Wang et al. Nanoparticle-based medicines in clinical cancer therapy
Kuerban et al. Doxorubicin-loaded bacterial outer-membrane vesicles exert enhanced anti-tumor efficacy in non-small-cell lung cancer
Xiong et al. A NIR light triggered disintegratable nanoplatform for enhanced penetration and chemotherapy in deep tumor tissues
Zhu et al. Chitosan-based nanoparticle co-delivery of docetaxel and curcumin ameliorates anti-tumor chemoimmunotherapy in lung cancer
Wang et al. Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicin-polymer conjugates
Fu et al. Tumor cell membrane-camouflaged responsive nanoparticles enable MRI-guided immuno-chemodynamic therapy of orthotopic osteosarcoma
CN110522919B (zh) 甘露糖受体靶向的组合物、药物及其制备方法和应用
Zhang et al. Redox-and light-responsive alginate nanoparticles as effective drug carriers for combinational anticancer therapy
Di-Wen et al. Improved antitumor activity of epirubicin-loaded CXCR4-targeted polymeric nanoparticles in liver cancers
Malhotra et al. Polymeric micelles coated with hybrid nanovesicles enhance the therapeutic potential of the reversible topoisomerase inhibitor camptothecin in a mouse model
Li et al. Improvement of pneumonia by curcumin-loaded bionanosystems based on platycodon grandiflorum polysaccharides via calming cytokine storm
KR20110056042A (ko) 종양세포 표적지향을 위한 나노 입자 및 이의 제조방법
CN105012956A (zh) 一种双功能肿瘤靶向脂质体给药系统及其制备和应用
Wang et al. Laser-triggered polymeric lipoproteins for precision tumor penetrating theranostics
CN110114072A (zh) 聚合物纳米颗粒
CN106344924A (zh) 一种联合代谢阻断的纳米剂型及其耐药逆转应用
Yang et al. Engineering prodrug nanomicelles as pyroptosis inducer for codelivery of PI3K/mTOR and CDK inhibitors to enhance antitumor immunity
Zhao et al. Construction of functional targeting daunorubicin liposomes used for eliminating brain glioma and glioma stem cells
Xu et al. Influence of lung cancer model characteristics on tumor targeting behavior of nanodrugs
Raza et al. Engineered tumor microvesicles modified by SP94 peptide for arsenic trioxide targeting drug delivery in liver cancer therapy
CN104784118A (zh) 具有内化作用的酸敏感脂质体给药系统及其制备方法和应用
Shaikh et al. Bleomycin loaded exosomes enhanced antitumor therapeutic efficacy and reduced toxicity
Qi et al. Exosomes separated based on the “STOP” criteria for tumor-targeted drug delivery
Wang et al. Uptake mechanism and endosomal fate of drug-phospholipid lipid nanoparticles in subcutaneous and in situ hepatoma

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19753994

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19753994

Country of ref document: EP

Kind code of ref document: A1