CN110743011A - Pharmaceutical system based on polyamino acids, method for the production thereof and use thereof - Google Patents
Pharmaceutical system based on polyamino acids, method for the production thereof and use thereof Download PDFInfo
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- CN110743011A CN110743011A CN201810723852.1A CN201810723852A CN110743011A CN 110743011 A CN110743011 A CN 110743011A CN 201810723852 A CN201810723852 A CN 201810723852A CN 110743011 A CN110743011 A CN 110743011A
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- drug
- polyamino acid
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- micelle
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- 229920001308 poly(aminoacid) Polymers 0.000 title claims description 6
- 238000004519 manufacturing process Methods 0.000 title description 2
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- 229940079593 drug Drugs 0.000 claims abstract description 122
- 239000000693 micelle Substances 0.000 claims abstract description 115
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- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 43
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 43
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical class C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 claims abstract description 41
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 24
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- 229940127024 acid based drug Drugs 0.000 claims abstract description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethyl sulfoxide Natural products CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 44
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- 239000006184 cosolvent Substances 0.000 claims description 19
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- SURLGNKAQXKNSP-DBLYXWCISA-N chlorin Chemical compound C\1=C/2\N/C(=C\C3=N/C(=C\C=4NC(/C=C\5/C=CC/1=N/5)=CC=4)/C=C3)/CC\2 SURLGNKAQXKNSP-DBLYXWCISA-N 0.000 claims description 16
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- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 18
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 12
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- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
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- APTMUIAOGSSQEM-UHFFFAOYSA-N 2-(2,2-dihydroxyethyldisulfanyl)ethane-1,1-diol Chemical compound OC(O)CSSCC(O)O APTMUIAOGSSQEM-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- FBDOJYYTMIHHDH-OZBJMMHXSA-N (19S)-19-ethyl-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-2,4,6,8,10,14,20-heptaen-18-one Chemical compound CC[C@@]1(O)C(=O)OCC2=CN3Cc4cc5ccccc5nc4C3C=C12 FBDOJYYTMIHHDH-OZBJMMHXSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- 238000003917 TEM image Methods 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
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- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 2
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 2
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
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- 238000012406 Annexin V-FITC/PI double staining Methods 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000003915 DNA Topoisomerases Human genes 0.000 description 1
- 108090000323 DNA Topoisomerases Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
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- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
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- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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- 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
- A61K47/62—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 a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Dispersion Chemistry (AREA)
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- Inorganic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
A drug system based on polyamino acid, a preparation method and application thereof, relating to the technical field of medicine. The drug system based on the polyamino acid is a linear drug-loaded micelle which is formed by adopting the polyamino acid as a drug carrier to encapsulate the camptothecin dimer and modifying the drug carrier by the dialdehyde polyethylene glycol. The application of the polyamino acid-based drug system of the embodiment of the invention is mainly used for preparing drugs for treating lung cancer.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a polyamino acid-based medicine system, and a preparation method and application thereof.
Background
Cancer is one of the major diseases that currently seriously affect human health and threaten human life. The conventional cancer treatment methods mainly include surgical treatment, chemotherapy and radiotherapy, wherein the method of surgical resection increases pain of patients and damages primordial qi, and is very expensive, and the method of chemotherapy or radiotherapy kills cancer cells in patients and damages red blood cells and white blood cells, so that the patients are not sufferer, and thus more effective cancer treatment methods are always sought. Camptothecin (CPT), an alkaloid extracted from the chinese plant Camptothecin, is a broad-spectrum anticancer drug that can induce apoptosis of cancer cells by binding to DNA topoisomerase I-DNA complex, inhibiting DNA replication and RNA synthesis. Two camptothecin derivatives, topotecan and irinotecan, are currently approved by the FDA for clinical use. However, camptothecin has the problems of poor water solubility, unstable lactone ring, non-specific absorption, high toxic and side effects, low bioavailability and the like, so that it is necessary to construct a multifunctional prodrug delivery system to help CPT exert a better anticancer effect.
The polymer micelle is a core-shell structure formed by self-assembling a hydrophilic section and a hydrophobic section of an amphiphilic polymer, and has the advantages of biodegradability, capability of improving the stability of a medicament, prolonging the circulation time of the medicament, reducing the toxic and side effects of the medicament and the like as a common medicament delivery system. In addition, the polymer micelle can also realize the responsive release of the drug at a tumor part through modification, or realize the combined treatment of the tumor by loading a plurality of drugs, genes, photosensitizers and the like together.
Although the polymer micelle can be used as a drug delivery system, at present, a linear multifunctional drug-loading system which is formed by encapsulating camptothecin dimer by using a polyamino acid compound as a drug carrier and can effectively improve the anticancer effect of camptothecin does not appear.
Disclosure of Invention
The invention aims to provide a drug system based on polyamino acid, which adopts the polyamino acid as a drug carrier to entrap camptothecin dimer, and can effectively improve the tumor treatment effect of the drug.
Another object of the present invention is to provide a process for the preparation of pharmaceutical systems based on polyamino acids which is simple.
The invention also aims to provide application of the polyamino acid-based medicine system, which is mainly used for preparing the medicine for treating lung cancer.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a drug system based on polyamino acid, which is a linear drug-loaded micelle formed by mainly adopting polyamino acid as a drug carrier to entrap camptothecin dimer and modifying the drug carrier by dialdehyde polyethylene glycol.
Further, in the preferred embodiment of the present invention, the polyamino acid may be grafted with a photosensitizer chlorin.
Further, in a preferred embodiment of the present invention, the polyamino acid is a diblock copolymer formed by ring-opening polymerization of lysine-N-carboxylic anhydride and phenylalanine-N-carboxylic anhydride, and the molecular structure of the polyamino acid is as follows:
Further, in the preferred embodiment of the present invention, the molecular weight of the dialdehyde polyethylene glycol is 5000-8000 Da.
The invention provides a preparation method of the drug system based on the polyamino acid, which comprises the following steps:
dissolving polyamino acid and camptothecin dimer in a cosolvent, then dropwise adding the mixture into water, then dialyzing the mixture with water to remove the cosolvent, and then adding dialdehyde polyethylene glycol for mixing to obtain the linear drug-loaded micelle.
Further, in a preferred embodiment of the present invention, the co-solvent is dimethyl sulfoxide.
Further, in a preferred embodiment of the present invention, the mass ratio of polyamino acid to camptothecin dimer is 1: 1-6.
Further, in a preferred embodiment of the present invention, the mass ratio of the polyamino acid to the dialdehyde polyethylene glycol is 1: 3-4.
Further, in the preferred embodiment of the present invention, the polyamino acid is grafted to the photosensitizer chlorin, and then dissolved in a co-solvent with the camptothecin dimer.
The invention provides application of the drug system based on the polyamino acid in preparing a drug for treating lung cancer.
The pharmaceutical system based on polyamino acid, the preparation method and the application of the pharmaceutical system have the beneficial effects that at least: the drug system based on the polyamino acid is a linear drug-loaded micelle which is formed by adopting the polyamino acid as a drug carrier to encapsulate the camptothecin dimer and modifying the drug carrier by the dialdehyde polyethylene glycol. The application of the polyamino acid-based drug system of the embodiment of the invention is mainly used for preparing drugs for treating lung cancer.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a nuclear magnetic spectrum of a polyamino acid in an example of the present invention;
FIG. 2 is a transmission electron micrograph of PEG-PKF/DCPT drug loaded micelle of example 1 of the present invention;
FIG. 3 is a transmission electron micrograph of PEG-PKF blank micelles of example 1 of the present invention;
FIG. 4 is a graph showing the drug release behavior of PEG-PKF/DCPT drug-loaded micelles of example 1;
FIG. 5 is a graph showing the results of critical micelle concentration of PEG-PKF blank micelles in example 1 of the present invention;
FIG. 6 is a graph showing the stability results of drug-loaded micelles of examples 1-3 of the present invention;
FIG. 7 is a graph showing the zeta potential results of the drug-loaded micelle of example 1 of the present invention;
FIG. 8 is a schematic diagram showing the results of cell uptake of the drug-loaded micelle of example 1;
FIG. 9 is a graph showing the cytotoxicity results of drug-loaded micelles of example 1 of the present invention;
FIG. 10 is a schematic diagram showing the apoptosis results of the drug-loaded micelle of example 1 of the present invention;
fig. 11 is a schematic diagram showing cytotoxicity results of the drug-loaded micelle of example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The polyamino acid-based pharmaceutical system of the present invention, and the preparation method and use thereof are specifically described below.
The embodiment of the invention provides a drug system based on polyamino acid, which can be a linear PEG-PKF/DCPT drug-loaded micelle formed by taking polyamino acid (PLys-PPhe, PKF) as a drug carrier to entrap camptothecin Dimer (DCPT) and modifying the drug carrier by dialdehyde polyethylene glycol (PEG). Aiming at the defects of poor selectivity, uncontrollable release, low drug-loading rate and the like of the antitumor drug camptothecin, the embodiment adopts polyamino acid as a drug carrier, the polyamino acid is used as a novel biodegradable material, the biocompatibility is good, the immunogenicity is avoided, no acidic degradation product is generated, the functional groups are multiple, the core-shell structure can be self-assembled in water, and the antitumor drug camptothecin dimer is encapsulated to form a nano drug-loading system, and the nano drug-loading system has the pH-responsive separable PEG shell and the reduction sensitive drug-release property, and is used for improving the tumor treatment effect of the drug. Compared with the common spherical drug-loaded micelle, the linear drug-loaded micelle can be more effectively taken in by cells, and has longer blood circulation time, thereby better exerting the treatment effect. The PEG-PKF/DCPT drug-loaded micelle is linear, can remove PEG in a tumor weakly acidic environment to improve the electropositivity of the micelle, so that the PEG can be effectively absorbed by cancer cells, and glutathione with high concentration in the cancer cells is utilized to break disulfide bonds in the DCPT, so that the anticancer drug camptothecin is reduced and responsively released, the proliferation of the cancer cells is remarkably inhibited, and the apoptosis of the cancer cells is induced.
Furthermore, a photosensitizer chlorin (Ce6) can be grafted on the polyamino acid (PKF), and the corresponding drug system based on the polyamino acid is a linear PEG-PKF-Ce6/DCPT drug-loaded micelle which is formed by adopting the polyamino acid grafted photosensitizer chlorin (PKF-Ce6) as a drug carrier to entrap camptothecin Dimer (DCPT) and modifying the drug carrier by dialdehyde polyethylene glycol (PEG). The photodynamic therapy is a micro/noninvasive cancer treatment method, which utilizes photosensitizer to generate singlet oxygen under the irradiation of light with specific wavelength to damage cancer cells, thereby realizing the accurate treatment of tumors. The PEG-PKF-Ce6/DCPT drug-loaded micelle realizes the combined treatment of chemotherapy and photodynamic, thereby playing a more effective anti-tumor role.
In this example, the polyamino acid (PLys-PPhe, PKF) is a diblock copolymer formed by ring-opening polymerization of lysine-N-carboxylic anhydride (lys (z) -NCA) and phenylalanine-N-carboxylic anhydride (Phe-NCA), and the molecular structure of the polyamino acid is as follows:
In the embodiment, the polyamino acid grafted photosensitizer chlorin (PKF-Ce6) is obtained by reacting photosensitizer chlorin (Ce6), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and then adding polyamino acid (PKF).
In this example, camptothecin Dimer (DCPT) was obtained by activating Camptothecin (CPT) with triphosgene and 4-dimethylaminopyridine, and then reacting with dihydroxyethyl disulfide.
In this example, bishydroxy polyethylene glycol (PEG) was reacted with p-carboxybenzaldehyde in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and dichloromethane. The molecular weight of the polyethylene glycol with the dialdehyde group is 5000-8000Da, and is preferably 6000 Da.
The embodiment of the invention also provides a preparation method of the drug system based on the polyamino acid, wherein the preparation method of the PEG-PKF/DCPT drug-loaded micelle comprises the following steps:
s1, dissolving polyamino acid and camptothecin dimer in a cosolvent, wherein the mass ratio of the polyamino acid to the camptothecin dimer is 1: 1-6, slowly adding dimethyl sulfoxide (DMSO) as a cosolvent into water while stirring, dialyzing with water to remove the cosolvent, and specifically transferring the dropwise added solution into a dialysis bag to dialyze the water to obtain the linear PKF/DCPT drug-loaded micelle.
Camptothecin Dimer (DCPT) is obtained by activating Camptothecin (CPT), triphosgene and 4-dimethylaminopyridine and then reacting with dihydroxyethyl disulfide, and the specific preparation method comprises the following steps: adding anhydrous dichloromethane into the CPT, performing water bath ultrasound, and stirring under the protection of nitrogen to enable the CPT to be in a highly suspended state; adding triphosgene and 4-dimethylaminopyridine, adding a mixed solution of tetrahydrofuran of dihydroxyethyl disulfide and dichloromethane after the solution is clarified, and reacting in a dark place; then dialyzing, freezing and drying to obtain a crude product, and purifying by a silica gel chromatographic column to obtain the product DCPT.
S2, adding dialdehyde polyethylene glycol into the PKF/DCPT drug-loaded micelle for mixing, wherein the mass ratio of the polyamino acid to the dialdehyde polyethylene glycol is generally 1: 3-4, obtaining the linear PEG-PKF/DCPT drug-loaded micelle.
The preparation method of the dialdehyde polyethylene glycol comprises the following steps: the bishydroxy polyethylene glycol and the p-carboxybenzaldehyde are mixed and react in the presence of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDC) and dichloromethane to obtain the bishydroxy polyethylene glycol.
Or the polyamino acid can be grafted with photosensitizer chlorin, and the preparation method of the corresponding PEG-PKF-Ce6/DCPT drug-loaded micelle comprises the following steps:
s1, grafting polyamino acid to photosensitizer chlorin, and then, according to the mass ratio of 1: 1-6, dissolving polyamino acid grafted photosensitizer chlorin and camptothecin dimer in cosolvent DMSO, slowly dropwise adding into water while stirring, transferring the dropwise added solution into a dialysis bag, and dialyzing water to obtain the linear PKF-Ce6/DCPT drug-loaded micelle.
The specific preparation method of the polyamino acid grafted photosensitizer chlorin (PKF-Ce6) is as follows: taking photosensitizer chlorin (Ce6), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to react in a cosolvent, then adding polyamino acid solution to react, and dialyzing in the cosolvent to obtain the PKF-Ce 6.
S2, according to the mass ratio of the polyamino acid to the polyethylene glycol of the dialdehyde in the PKF-Ce6/DCPT drug-loaded micelle being 1: 3-4, adding polyethylene glycol of dialdehyde into the PKF-Ce6/DCPT drug-carrying micelle for mixing to obtain the linear PEG-PKF-Ce6/DCPT drug-carrying micelle.
The embodiment of the invention also provides application of the polyamino acid-based medicine system in preparing a medicine for treating lung cancer. The drug system based on the polyamino acid has good stability, has the properties of pH responsiveness in polyethylene glycol removal and reduction-sensitive drug release, is good in selectivity, can be significantly internalized by lung cancer cells, has the effect of inducing apoptosis of the lung cancer cells, and has a significant inhibiting effect on proliferation of the lung cancer cells. Moreover, after the photosensitizer chlorin is grafted, the chemotherapy and photodynamic combined treatment is realized, and the lung cancer cells can be killed more efficiently.
The features and properties of the present invention are described in further detail below with reference to examples.
Preparing main raw materials:
preparation of camptothecin Dimer (DCPT): adding 2.187mmol of CPT into 20mL of anhydrous dichloromethane in a dry environment, performing water bath ultrasound for 3min, and stirring for 5min under the protection of nitrogen to enable the CPT to be in a highly suspended state; then 0.765mmol of triphosgene and 4.812mmol of 4-dimethylaminopyridine are added, after the solution is clarified, 0.723mmol of tetrahydrofuran and dichloromethane (1:9) mixed solution of dihydroxyethyl disulfide is added, and the mixture is reacted for 24 hours in a dark place; dialyzing in DMSO for 12h with a dialysis bag with a molecular weight of 500, dialyzing in pure water for 48h, freeze-drying to obtain crude product, and purifying with silica gel chromatographic column to obtain DCPT product.
Preparing polyethylene glycol with dialdehyde: the dihydroxy polyethylene glycol and p-carboxybenzaldehyde are mixed according to a molar ratio of 1: 6, and reacting in the presence of 12 times of mole number (compared with the dihydroxy polyethylene glycol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and dichloromethane to obtain the dialdehyde polyethylene glycol PEG.
Preparation of polyamino acid (PKF): firstly, N-hexylamine is taken as an initiator to initiate the ring-opening polymerization of lysine-N-carboxylic anhydride (Lys (Z) -NCA) protected by benzyloxycarbonyl to obtain PLys (Z); then PLys (Z) was mixed with previously synthesized phenylalanine-N-carboxyanhydride (Phe-NCA) in a ratio of 30: 20, 30: 40, 30: feeding materials according to a molar ratio of 60, taking N, N-Dimethylformamide (DMF) as a solvent, and reacting for 48 hours at room temperature; then pouring the solution into a dialysis bag with the molecular weight of 3500Da for dialysis for 48h for purification, and putting the product into a freeze dryer for freeze-drying to obtain white flocculent solid, namely PLys (Z) -PPhe; then carrying out deprotection reaction of benzyloxycarbonyl on the synthesized PLys (Z) -PPhe material, completely dissolving the PLys (Z) -PPhe material by trifluoroacetic acid, then adding a hydrobromic acid solution containing 30% acetic acid, slowly pouring the solution into anhydrous ether obtained by precooling in advance for settling after 1h, filtering a solid product, and dissolving by DMF; pouring the solution into a dialysis bag with molecular weight of 3500Da for dialysis for 48h for purification in the same way as the above operation; finally, the product is put into a freeze dryer for freeze drying, and white flocculent solid is obtained, namely the product PLys-PPhe (PKF).
Molecular structure of the prepared PKFIs constructed as
Wherein m is 30 and n is 40. The nuclear magnetic spectrum of PKF is shown in FIG. 1.
The preparation process of the polyamino acid grafted photosensitizer chlorin (PKF-Ce6) is as follows: and (2) reacting Ce 618 mg, 20mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 20mg of N-hydroxysuccinimide (NHS) in 3mL of DMSO for 30min, adding a 12mg/mL PKF solution, reacting for one day, transferring into a 3500Da dialysis bag, and dialyzing for one day in DMSO to obtain PKF-Ce 6.
Example 1
The embodiment provides a drug system based on polyamino acid, in particular to a PEG-PKF/DCPT drug-loaded micelle, which is prepared according to the following preparation method:
PKF and DCPT are mixed according to the ratio of 1: 2 in the weight ratio of the solvent, and then slowly dropwise adding the mixture into the aqueous solution while stirring. After dripping, transferring the solution into a 3500Da dialysis bag, dialyzing water, and removing DMSO to obtain the PKF/DCPT drug-loaded micelle.
Adding 3 times of dialdehyde polyethylene glycol (6000Da) according to the mass of PKF in the PKF/DCPT drug-loaded micelle, and mixing to obtain the PEG-PKF/DCPT drug-loaded micelle.
In addition, PKF of the same mass as PKF/DCPT drug-loaded micelles was dissolved in DMSO, and then slowly added dropwise to the aqueous solution, with stirring. After dropping, the solution was transferred into a 3500Da dialysis bag, dialyzed against water, and DMSO was removed to obtain a PKF blank micelle. And then, adding 3 times of mass of dialdehyde-modified PEG according to the mass of the PKF in the PKF blank micelle, and mixing to obtain the PEG-PKF blank micelle.
Diluting the PEG-PKF/DCPT drug-loaded micelle and the PEG-PKF blank micelle into proper concentrations, dripping the diluted PEG-PKF/DCPT drug-loaded micelle and PEG-PKF blank micelle onto a copper net for a transmission electron microscope, drying the liquid, and observing the appearance of the liquid, wherein the appearance is shown in figures 2 and 3, figure 2 is a transmission electron microscope image of the PEG-PKF/DCPT drug-loaded micelle, and figure 3 is a transmission electron microscope image of the PEG-PKF blank micelle. It can be seen from the figure that: the PEG-PKF/DCPT drug-loaded micelle is in a nano-scale linear shape, and the PEG-PKF blank micelle is in a nano-scale spherical shape.
Example 2
The embodiment provides a drug system based on polyamino acid, in particular to a PEG-PKF/DCPT drug-loaded micelle, which is prepared according to the following preparation method:
PKF and DCPT are mixed according to the ratio of 1: 4 in the cosolvent DMSO, then slowly dropwise adding the mixture into the aqueous solution, and dropwise adding the mixture while stirring. After dripping, transferring the solution into a 3500Da dialysis bag, dialyzing water, and removing DMSO to obtain the PKF/DCPT drug-loaded micelle.
Adding 4 times of dialdehyde polyethylene glycol (6000Da) according to the mass of PKF in the PKF/DCPT drug-loaded micelle, and mixing to obtain the PEG-PKF/DCPT drug-loaded micelle.
Example 3
The embodiment provides a drug system based on polyamino acid, in particular to a PEG-PKF/DCPT drug-loaded micelle, which is prepared according to the following preparation method:
PKF and DCPT are mixed according to the ratio of 1: 6 is dissolved in a cosolvent DMSO, and then is slowly dripped into the aqueous solution, and is dripped while stirring. After dripping, transferring the solution into a 3500Da dialysis bag, dialyzing water, and removing DMSO to obtain the PKF/DCPT drug-loaded micelle.
Adding 3 times of dialdehyde polyethylene glycol (6000Da) according to the mass of PKF in the PKF/DCPT drug-loaded micelle, and mixing to obtain the PEG-PKF/DCPT drug-loaded micelle.
Example 4
The embodiment provides a drug system based on polyamino acid, in particular to a PEG-PKF-Ce6/DCPT drug-loaded micelle, which is prepared according to the following preparation method:
mixing PKF-Ce6 and DCPT according to the mass ratio of 1: 2 is dissolved in a cosolvent DMSO, then slowly dropped into the aqueous solution, and dropped while stirring. After dripping, transferring the solution into a 3500Da dialysis bag, dialyzing water, and removing DMSO to obtain the PKF-Ce6/DCPT drug-loaded micelle.
Adding 3 times of dialdehyde polyethylene glycol (6000Da) by mass according to the mass of PKF in the PKF-Ce6/DCPT drug-loaded micelle, and mixing to obtain the PEG-PKF-Ce6/DCPT drug-loaded micelle.
Example 5
The embodiment provides a drug system based on polyamino acid, in particular to a PEG-PKF-Ce6/DCPT drug-loaded micelle, which is prepared according to the following preparation method:
mixing PKF-Ce6 and DCPT according to the mass ratio of 1: 5 is dissolved in a cosolvent DMSO, then slowly added dropwise to the aqueous solution, and added dropwise with stirring. After dripping, transferring the solution into a 3500Da dialysis bag, dialyzing water, and removing DMSO to obtain the PKF-Ce6/DCPT drug-loaded micelle.
Adding 4 times of dialdehyde polyethylene glycol (6000Da) by mass according to the mass of PKF in the PKF-Ce6/DCPT drug-loaded micelle, and mixing to obtain the PEG-PKF-Ce6/DCPT drug-loaded micelle.
Example 6
The embodiment provides a drug system based on polyamino acid, in particular to a PEG-PKF-Ce6/DCPT drug-loaded micelle, which is prepared according to the following preparation method:
mixing PKF-Ce6 and DCPT according to the mass ratio of 1: 6 is dissolved in a cosolvent DMSO, then slowly dropped into the aqueous solution, and dropped while stirring. After dripping, transferring the solution into a 3500Da dialysis bag, dialyzing water, and removing DMSO to obtain the PKF-Ce6/DCPT drug-loaded micelle.
Adding 3 times of dialdehyde polyethylene glycol (6000Da) by mass according to the mass of PKF in the PKF-Ce6/DCPT drug-loaded micelle, and mixing to obtain the PEG-PKF-Ce6/DCPT drug-loaded micelle.
The polyamino acid-based pharmaceutical systems of the present invention are assayed by the following tests.
Drug release property of drug-loaded micelle
The PEG-PKF/DCPT drug-loaded micelle solution of example 1 was taken and observed in PBS with pH values of 7.4 and 6.5, and the drug release behavior of the PEG-PKF/DCPT drug-loaded micelle is shown in FIG. 4 by comparing the drug release behavior in the reducing environment.
The result shows that in the environment without reducing agent DTT, the micelle is stable no matter at pH7.4 and pH6.5, and no CPT is released; in the presence of DTT, a large amount of CPT can be released in a short time, and the property of reducing sensitive drug release is shown.
Critical micelle concentration of blank micelle
Critical micelle concentration of PEG-PKF blank micelle in example 1 was determined using pyrene fluorescence probe method: equal volume of 6.0X 10-6Adding a solution of pyrene in mol/L in acetone into a series of centrifuge tubes of 4.0mL, and standing for 24 hours in a dark place to completely volatilize the acetone. PEG-PKF blank micelle solution with different concentrations is added into the mixture, and the concentration range is 2 x 10-6To 1mg/mL, to give a final pyrene concentration of 6.0X 10 per tube-7mol/L, and standing overnight at room temperature in dark. Measured by a fluorescence spectrometer, the ratio of fluorescence intensities at 373nm and 384nm was calculated. The logarithm of the concentration (Log C) is plotted as the abscissa, I373/I384The ratio is plotted on the ordinate, the concentration at the inflection point of the curve is 0.0121mg/mL, which is the critical micelle concentration of the PEG-PKF blank micelle, as shown in FIG. 5, and the result shows that the micelle can maintain the structural integrity at low concentration.
Stability of drug-loaded micelle
The PKF/DCPT drug-loaded micelles and PEG-PKF/DCPT drug-loaded micelles of examples 1 to 3 were taken to be 1mL in a 2mL centrifuge tube, 10% FBS was added respectively, after mixing uniformly, the particle sizes of the drug-loaded micelles at different time points (0, 0.5, 1, 2, 4, 8, 10, 12, 24h) were detected by DLS to examine the stability thereof, and the stability results of the PKF/DCPT drug-loaded micelles and PEG-PKF/DCPT drug-loaded micelles of examples 1 to 3 are shown in fig. 6.
The result shows that the PEG modified drug-loaded micelle (PEG-PKF/DCPT drug-loaded micelle) has better stability.
Zeta potential of drug-loaded micelle
The PKF/DCPT drug-loaded micelle and the PEG-PKF/DCPT drug-loaded micelle of example 1 are respectively taken and respectively placed in 1mL of a 2mL centrifuge tube, 1mL of PBS with pH7.4 and pH6.5 is respectively added, after uniform mixing, the zeta potential of different drug-loaded micelle groups is detected by using DLS, and the zeta potential result of the drug-loaded micelle is shown in figure 7.
The result shows that after the PKF/DCPT drug-loaded micelle is modified by dialdehyde PEG, the potential is reduced under the condition of pH7.4, but the potential is reversely increased under the weak acidic condition of pH6.5, which shows that the drug-loaded micelle has the function of improving the electropositivity of the micelle in a pH response manner.
Fifth, cell internalization of drug-loaded micelles
Taking 2-5 generation A549 and H460 lung cancer cells, and mixing according to 1 × 105The cells/well were seeded on 12-well plates. After overnight incubation to allow cells to adhere, 2ug/ml of rhodamine-labeled PEG blank micelles (spherical) and PKF/DCPT drug-loaded micelles (linear) of example 1 and different pH media (pH7.4 and pH6.5) were administered, respectively. After the cells were cultured for 6h, the cells were collected and resuspended in 400ul PBS, and fluorescence detection was performed on a flow cytometer, and the results of cell uptake of the drug-loaded micelle are shown in FIG. 8.
The result shows that the linear PKF/DCPT drug-loaded micelle has a remarkably enhanced cell internalization effect and is easier to be internalized by cells.
Sixthly, the inhibition effect of the PEG-PKF/DCPT drug-loaded micelle on the lung cancer cells
Taking 2-5 generation A549 and H460 lung cancer cells, and mixing according to 5 × 103The/well cells were seeded in 96-well plates. After overnight incubation to allow cells to adhere, different concentrations of CPT, DCPT, PKF/DCPT from example 1 and PEG-PKF/DCPT were administered separately, and control groups were set, each group having three parallel wells. After 24h or 48h of cell culture, the cells were incubated with MTT for 4h to form the formazan product, which, after solubilization in DMSO, was measured for OD values with a microplate reader at a wavelength of 570 nm. The experiment was performed 3 times in total, the average was calculated, the percentage of cell viability was calculated from the control group, and the toxicity results of the drug-loaded micelles to lung cancer cells are shown in fig. 9.
The result shows that the PEG-PKF/DCPT drug-loaded micelle can obviously inhibit the proliferation of lung cancer cells.
Seventhly, the drug-loaded micelle induces the apoptosis of the lung cancer cells
Annexin V-FITC/PI double staining test: 2-5 generations of A549 and H460 lung cancer cells are taken and inoculated on a 6-well plate according to 2 multiplied by 105/well cells. After overnight culture to allow cells to adhere, CPT, DCPT, PKF/DCPT of example 1 and PEG-PKF/DCPT were administered at a concentration of 10. mu.M, respectively, and a control group was set. After the cells are cultured for 24H, the cells are collected, and are dyed with Annexin V-FITC and PI dye for 20min in a dark place, then are resuspended with 400ul PBS, and are detected on a flow cytometer, and the apoptosis result of the lung cancer cells of the drug-loaded micelle is shown in figure 10, wherein A is H460 cells, and B is A549 cells.
The result shows that the PEG-PKF/DCPT drug-loaded micelle can effectively induce the apoptosis of the lung cancer cells.
Eighthly, the inhibiting effect of PEG-PKF-Ce6/DCPT drug-loaded micelle on lung cancer cells
Taking 2-5 generation A549 and PC-9 lung cancer cells, and mixing according to the ratio of 8 × 103The/well cells were seeded in 96-well plates. After overnight culture for cell adherence, different concentrations of CPT, DCPT, Ce6, physical mixture of free CPT and Ce6 (CPT + Ce6), physical mixture of free DCPT and Ce6 (DCPT + Ce6) and PEG-PKF-Ce6/DCPT drug-loaded micelle of example 5 were administered, and after 8h treatment, 660nm light source (100 mW/cm) was used for each group of Ce 6-containing groups2) Irradiation for 10min, four parallel wells per group. After 24h of cell culture, the cells were incubated with MTT for 4h to form the formazan product, which, after solubilization in DMSO, was measured for OD values with a microplate reader at a wavelength of 570 nm. The experiment was performed 3 times in total, the average value was calculated, the percentage of cell viability was calculated from the control group, and the toxicity results of the drug-loaded micelles to the lung cancer cells are shown in fig. 11.
The results show that PEG-PKF-Ce6/DCPT drug-loaded micelles exhibit stronger antitumor effects compared to chemotherapy alone (CPT) and photodynamic therapy alone (Ce 6).
In conclusion, the medicine system based on the polyamino acid provided by the embodiment of the invention adopts the polyamino acid as a medicine carrier to entrap the camptothecin dimer, so that the tumor treatment effect of the medicine can be effectively improved; the preparation method of the drug system based on the polyamino acid is simple; the application of the polyamino acid-based drug system of the embodiment of the invention is mainly used for preparing drugs for treating lung cancer.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A drug system based on polyamino acid is characterized in that the polyamino acid is mainly used as a drug carrier to entrap camptothecin dimer, and the linear drug-carrying micelle is modified by dialdehyde polyethylene glycol.
2. The polyamino acid based drug system according to claim 1, wherein the polyamino acid may further have grafted thereto a photosensitizer chlorin.
3. Polyaminoacid-based pharmaceutical system according to claim 1, wherein the polyaminoacid is a diblock copolymer formed by ring opening polymerization of lysine-N-carboxylic anhydride and phenylalanine-N-carboxylic anhydride, the polyaminoacid having the following molecular structure:
wherein, m is 20-60, n is 20-60.
4. The polyamino acid based drug system according to claim 1, wherein the molecular weight of said dialdehyde polyethylene glycol is 5000-8000 Da.
5. A process for the preparation of a polyamino acid based pharmaceutical system according to claim 1, comprising the steps of:
dissolving polyamino acid and camptothecin dimer in a cosolvent, then dropwise adding the mixture into water, then dialyzing the mixture with water to remove the cosolvent, and then adding dialdehyde polyethylene glycol for mixing to obtain the linear drug-loaded micelle.
6. The method of claim 5, wherein the co-solvent is dimethyl sulfoxide.
7. The method of claim 5, wherein the mass ratio of the polyamino acid to the camptothecin dimer is 1: 1-6.
8. The method for preparing a polyamino acid-based drug system according to claim 5, wherein the mass ratio of the polyamino acid to the dialdehyde polyethylene glycol is 1: 3-4.
9. The method of claim 5, wherein the polyamino acid is grafted to the photosensitizer chlorin, and then dissolved in the cosolvent together with the camptothecin dimer.
10. Use of a polyamino acid based pharmaceutical system according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of lung cancer.
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