CN114920869B - 两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用 - Google Patents
两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用 Download PDFInfo
- Publication number
- CN114920869B CN114920869B CN202210569438.6A CN202210569438A CN114920869B CN 114920869 B CN114920869 B CN 114920869B CN 202210569438 A CN202210569438 A CN 202210569438A CN 114920869 B CN114920869 B CN 114920869B
- Authority
- CN
- China
- Prior art keywords
- copolymer
- ciprofloxacin
- amphiphilic
- arginine
- block
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- MYSWGUAQZAJSOK-UHFFFAOYSA-N Ciprofloxacin Natural products C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 229920001577 copolymer Polymers 0.000 title claims abstract description 62
- 229960003405 ciprofloxacin Drugs 0.000 title claims abstract description 52
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 40
- 239000004475 Arginine Substances 0.000 title claims abstract description 29
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000001338 self-assembly Methods 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 30
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 26
- 238000003756 stirring Methods 0.000 claims description 21
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 20
- 230000015572 biosynthetic process Effects 0.000 claims description 19
- 238000003786 synthesis reaction Methods 0.000 claims description 18
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 15
- 150000005690 diesters Chemical class 0.000 claims description 15
- 239000008367 deionised water Substances 0.000 claims description 13
- 229910021641 deionized water Inorganic materials 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- LVZWSLJZHVFIQJ-UHFFFAOYSA-N Cyclopropane Chemical compound C1CC1 LVZWSLJZHVFIQJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 12
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 10
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 claims description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 239000012295 chemical reaction liquid Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
- 229940098779 methanesulfonic acid Drugs 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 6
- OMIGHNLMNHATMP-UHFFFAOYSA-N 2-hydroxyethyl prop-2-enoate Chemical compound OCCOC(=O)C=C OMIGHNLMNHATMP-UHFFFAOYSA-N 0.000 claims description 5
- KWTQSFXGGICVPE-WCCKRBBISA-N Arginine hydrochloride Chemical compound Cl.OC(=O)[C@@H](N)CCCN=C(N)N KWTQSFXGGICVPE-WCCKRBBISA-N 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 239000012065 filter cake Substances 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000003999 initiator Substances 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 5
- 239000000178 monomer Substances 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- CAEWJEXPFKNBQL-UHFFFAOYSA-N prop-2-enyl carbonochloridate Chemical compound ClC(=O)OCC=C CAEWJEXPFKNBQL-UHFFFAOYSA-N 0.000 claims description 5
- 150000003254 radicals Chemical class 0.000 claims description 5
- 150000003384 small molecules Chemical class 0.000 claims description 4
- 229940124350 antibacterial drug Drugs 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 51
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 21
- 239000003242 anti bacterial agent Substances 0.000 abstract description 16
- 206010059866 Drug resistance Diseases 0.000 abstract description 14
- 230000009471 action Effects 0.000 abstract description 9
- 239000012528 membrane Substances 0.000 abstract description 7
- 230000002209 hydrophobic effect Effects 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 2
- 229920001002 functional polymer Polymers 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 239000002861 polymer material Substances 0.000 abstract description 2
- 230000007480 spreading Effects 0.000 abstract description 2
- 238000003892 spreading Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 37
- 241000894006 Bacteria Species 0.000 description 19
- 241000588724 Escherichia coli Species 0.000 description 14
- 210000000170 cell membrane Anatomy 0.000 description 11
- 229940088710 antibiotic agent Drugs 0.000 description 9
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 125000003345 AMP group Chemical group 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 5
- 230000000845 anti-microbial effect Effects 0.000 description 5
- 229960004755 ceftriaxone Drugs 0.000 description 5
- VAAUVRVFOQPIGI-SPQHTLEESA-N ceftriaxone Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C(=O)NN1C VAAUVRVFOQPIGI-SPQHTLEESA-N 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 4
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 4
- 229930182566 Gentamicin Natural products 0.000 description 4
- 206010041925 Staphylococcal infections Diseases 0.000 description 4
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 125000004980 cyclopropylene group Chemical group 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 229960002518 gentamicin Drugs 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 4
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 3
- 239000012298 atmosphere Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 210000003292 kidney cell Anatomy 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 238000010526 radical polymerization reaction Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 206010034133 Pathogen resistance Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 229920006317 cationic polymer Polymers 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000009881 electrostatic interaction Effects 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- -1 H1N 1A in 2009 Species 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 229920002118 antimicrobial polymer Polymers 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000000733 zeta-potential measurement Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F218/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an acyloxy radical of a saturated carboxylic acid, of carbonic acid or of a haloformic acid
- C08F218/22—Esters containing nitrogen
-
- 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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- 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
- A61K47/58—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 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
-
- 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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6933—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nanotechnology (AREA)
- Polymers & Plastics (AREA)
- Immunology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及功能高分子材料技术领域,尤其涉及一种两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用。该共聚物自组装所得的抗菌纳米颗粒PAC@NPs可通过静电作用先靶向吸附于菌体细胞表面,随后具有刚性的疏水段环丙沙星似“纳米导弹”可刺穿菌膜进而充分发挥抗菌功效,致使细菌细胞不可逆转的“溶解”凋亡,菌体细胞根本没有任何机会发展耐药性。本发明为研发新型抗菌试剂和遏制细菌耐药性蔓延提供了新思路,较好的解决了现有广谱抗菌药环丙沙星使用中的缺陷和不足。
Description
技术领域
本发明涉及功能高分子材料技术领域,尤其涉及一种两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用。
背景技术
近年来,致病菌与病毒造成的感染性疾病越来越多,如2003年的SRSA、2009年的甲型H1N1、2013年的西非埃博拉病毒、以及2019年爆发的新型冠状病毒再到2021年的奥密克戎毒株,严重威胁着人类健康,对经济发展造成了不可估量的影响。纵观人类发展的历史,致病细菌、真菌及病毒一直是传播传染病的罪魁祸首。利用抗生素治疗感染性疾病是最为行之有效的办法,它的问世使死于细菌、病毒感染的人数大幅下降,也大大延长了人类的平均寿命,为人类的发展做出了巨大贡献。
如今,伴随着科学技术的进步,经过多年的蓬勃发展,抗生素种类日益丰富,目前已达上千种,成功进入临床的也不下几百种。然而,近年来,抗生素在临床、农畜牧业及日常生活中不受控制的过度使用导致细菌耐药性的产生,甚至出现了多重耐药的“超级细菌”。临床上的抗生素一般通过靶向干预细菌生命活动所必需的过程来发挥抗菌作用,如干扰核酸、蛋白质或细胞壁成分的合成以及叶酸代谢等。对于作用于单一特定靶标的传统小分子抗生素,若细菌细胞内的靶标由于基因突变而改变,该抗生素则不能继续发挥抗菌作用,使其丧失了原本功效,致使细菌产生耐药性。鉴于此,迫切需要开发一类能与细菌非特异性结合的安全低毒、高效且不会诱导细菌耐药性的新型抗菌剂。
阳离子特征聚合物是一类具有优异抗菌功效的抗菌试剂,这些聚合物发挥抗菌作用不需要特定靶标,其可通过静电作用吸附于细菌细胞表面进而使膜电位、膜通透性发生改变,甚至将细胞膜物理性地撕裂,最终导致细菌不可逆转的死亡。阳离子聚合物的非特异靶向的机械杀菌机制可以彻底将细菌细胞形态破坏,使其没有产生耐药性的机会。特别是,阳离子特征的抗菌肽(AMPs),作为生物体免疫***的重要防线,具有广谱高效、低毒、生物相容性良好和不易产生耐药性的特点。AMPs一般主要由亲水和疏水氨基酸残基组成,其亲水氨基酸通常是精氨酸或赖氨酸。在生理pH值7.4下,精氨酸或赖氨酸结构中的-NH2被质子化,赋予抗菌肽正电荷,通过静电相互作用可附着在菌体细胞壁,随后AMPs分子结构中的疏水残基可刺入细胞膜,造成菌体细胞不可逆转地物理性“溶菌”、凋亡,然而,AMPs的来源有限,直接合成也较困难,不能满足实际需要。因此,设计、合成仿生肽基聚合物十分必要,也具有重要价值。
与精氨酸结构片段相似,含胍基聚合物分子链中存在胍基基团,这可赋予含胍基聚合物与AMPs相似的抗菌功效和生物性质,含胍基的阳离子聚合物的抗菌性源于胍基的刚性平面氢键供体阵列和质子化,胍基抗菌聚合物通过静电作用和氢键配位机制能与所有细菌非特异性结合,这使其具有广谱高效、低毒和不易诱导耐药性的抗菌特性。同时,环丙沙星是一种具有广谱抗菌和抗病毒功效的传统药物,但其难溶性在一定程度上降低了在病灶部位的有效聚集浓度和应用范围。在临床上,医师为了治疗复杂的感染性疾病,经常通过加大剂量或联合用药来达到治疗目的。
发明内容
本发明为了弥补现有技术的不足,提供了一种两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用,解决了现有技术中存在的问题。
为实现上述发明目的,本发明采用的技术方案之一是:
一种两亲性精氨酸嵌段环丙沙星共聚物,结构式为:
其中,m=25-35,n=20-30。
进一步地,上述两亲性精氨酸嵌段环丙沙星共聚物结构式中,m=30,n=25。
本发明采用的技术方案之二是:
一种制备如上所述的两亲性精氨酸嵌段环丙沙星共聚物的方法,包括如下操作步骤:
(1)N-烯丙氧羰基-L-精氨酸(Alloc-L-Arginine,AA)的合成
将30.0g NaHCO3悬浮于120mL水中,加入20.0g精氨酸单盐酸盐,搅拌,20mL氯甲酸烯丙酯在30min内滴加到上述体系中,室温搅拌8h,4℃冷却过滤,用***和少量冷水洗涤、冷冻干燥得白色粉未26.4g,收率90.2%;
(2)丙烯酸环丙沙星乙二酯(ACE)的合成
将丙烯酸羟乙酯与环丙沙星按摩尔比为1.5:1的量,加入到装有甲磺酸和适量甲苯的三口烧瓶中,在N2保护下磁力搅拌,升温至125℃,5h后停止反应,将所得反应液经去离子水洗涤至中性,萃取并收集有机相;
(3)精氨酸嵌段环丙沙星共聚物(PAC)的合成
分别称取不同比例的步骤(1)的N-烯丙氧羰基-L-精氨酸(AA)和步骤(2)的丙烯酸环丙沙星乙二酯(ACE),置于恒压滴液漏斗中,再用适量无水乙醇和DMSO的混合溶剂溶解;然后,将占单体总质量1%的自由基引发剂AIBN充分溶解于THF中,将1/2体积的AIBN溶液分别加入上述恒压滴液漏斗和预先加有适量无水乙醇和DMSO的三口烧瓶内;在N2保护下,升温,将漏斗中的混合溶液缓慢滴入三口烧瓶中;滴加完毕后,继续回流反应24h,反应液经丙酮洗涤、乙醇透析,制得系列两亲性共聚物(PAC)。
进一步地,步骤(1)少量冷水用量为过滤后所得滤饼质量的2-4倍;步骤(2)中甲磺酸质量为环丙沙星质量的1%-4%,甲苯用量为环丙沙星物质的量的5-8倍;步骤(3)在N2保护下,升温至65-75℃;继续回流反应保持N2气氛、65-75℃条件。
本发明采用的技术方案之三是:
一种两亲性精氨酸嵌段环丙沙星共聚物纳米颗粒,由如上的制备方法制得的两亲性共聚物自组装制得;尺寸为200±50;多分散指数(PDI)为0.16。
进一步地,所述的两亲性共聚物自组装步骤包括如下操作:
称取两亲性共聚物(PAC)溶于四氢呋喃中,在匀速搅拌下缓慢滴加到去离子水中,匀速搅拌,所得混合液在去离子水中透析以除去小分子,每间隔一定时间更换一次透析液,得PAC共聚物的纳米颗粒PAC@PNs。
进一步地,操作中,两亲性共聚物与四氢呋喃采用2:1的重量体积比混合;去离子水与四氢呋喃用量相等;匀速搅拌时间为12h,透析时间72h,每间隔8h更换一次透析液。
本发明采用的技术方案之四是:
上述两亲性精氨酸嵌段环丙沙星共聚物纳米颗粒在抗菌药物中的应用。
本发明的有益效果:
本发明构建了一种以精氨酸为亲水段、环丙沙星为疏水段的两亲性共聚物(PAC),其在溶液中可通过自组装形成以精氨酸片段为壳,环丙沙星片段为核的带正电荷纳米颗粒(PAC@NPs)。这种新颖结构的抗菌纳米颗粒PAC@NPs可通过静电作用先靶向吸附于菌体细胞表面,随后具有刚性的疏水段环丙沙星似“纳米弹”可刺穿菌膜进而充分发挥抗菌功效,致使细菌细胞不可逆转的“溶解”凋亡,菌体细胞根本没有任何机会发展耐药性。PAC@NPs这种“致命”式、多模式的联合杀菌机制具有广谱、高效、快速杀菌和不易诱导细菌耐药性产生的优势,同时即使是多重耐药菌也难以抵抗这种杀菌历程。这为研发新型抗菌试剂和遏制细菌耐药性蔓延提供了新思路,较好的解决了现有广谱抗菌药环丙沙星使用中的缺陷和不足。
附图说明
图1为本发明实施例1制得的PAC@PNs在溶液中的形貌图;
图2为在不同PAC@NPs浓度下S.aureus(ATCC29219)的OD600随时间变化;
图3为S.aureus(ATCC29213)的菌落数随时间变化;
图4为VRE分别对头孢曲松和PAC@NPs的耐药性研究;
图5为E.coli(BL21)分别对庆大霉素和PAC@NPs的耐药性研究;
图6为E.coli(BL21)经PAC@NPs作用不同时间后的形貌;
图7为不同细菌细胞的荧光染色照片;
图8为PAC@NPs对肾细胞(Vero-E6)的毒性评价。
具体实施方式
为能清楚说明本方案的技术特点,下面通过具体实施方式,结合附图,对本发明进行详细阐述。
实施例1
一种两亲性精氨酸嵌段环丙沙星共聚物的制备方法,包括如下操作步骤:
(1)N-烯丙氧羰基-L-精氨酸(Alloc-L-Arginine,AA)的合成
将30.0g NaHCO3悬浮于120mL水中,加入20.0g精氨酸单盐酸盐,搅拌,20mL氯甲酸烯丙酯在30min内滴加到上述体系中,室温搅拌8h,4℃冷却过滤,用***和少量冷水洗涤、冷冻干燥得白色粉未26.4g,冷水用量为过滤后滤饼质量的3倍;收率90.2%;
(2)丙烯酸环丙沙星乙二酯(ACE)的合成
将丙烯酸羟乙酯与环丙沙星按摩尔比为1.5:1的量,加入到装有甲磺酸和适量甲苯的三口烧瓶中,在N2保护下磁力搅拌,升温至125℃,5h后停止反应,将所得反应液经去离子水洗涤至中性,萃取并收集有机相;甲磺酸用量为环丙沙星质量的3%,甲苯用量为环丙沙星物质的量的6倍;
(3)精氨酸嵌段环丙沙星共聚物(PAC)的合成
通过自由基聚合反应制备两亲性共聚物(PAC)。首先,分别称取6:5摩尔比例的N-烯丙氧羰基-L-精氨酸(AA)和丙烯酸环丙沙星乙二酯(ACE),将其置于恒压滴液漏斗中,再用适量无水乙醇和DMSO的混合溶剂溶解;随后,将占单体总质量1%的自由基引发剂AIBN充分溶解于THF中,将1/2体积的AIBN溶液分别加入上述漏斗和预先加有适量乙醇和DMSO的三口烧瓶里;在N2保护下,升温至70℃,将漏斗中的混合溶液缓慢滴入三口烧瓶中;滴加完毕后,在N2气氛,70℃下继续回流反应24h,反应液经丙酮洗涤,乙醇透析,最终制得两亲性共聚物(PAC)如下:
其中,m=30,n=25。
将上述得到的两亲性共聚物继续处理得到共聚物的纳米颗粒,处理步骤如下:
称取10mg共聚物(PAC)溶于5mL四氢呋喃中,在匀速搅拌下缓慢滴加到5mL去离子水中,匀速搅拌12h,所得混合液在去离子水中透析72h以除去小分子,每8h更换一次透析液,最终得到PAC共聚物的纳米颗粒PAC@PNs。
纳米颗粒的形成可以增加聚合物的正电荷密度和有效聚集浓度,从而增强杀菌效果。通过SEM观察纳米颗粒的形貌,结果如图1所示,其尺寸约为200±50nm;并进一步通过DLS研究测得PAC@PNs的粒径约为300nm,多分散指数(PDI)为0.16,由此说明,PAC@PNs为粒径较为均一的纳米颗粒。
实施例2
一种两亲性精氨酸嵌段环丙沙星共聚物的制备方法,包括如下操作步骤:
(1)N-烯丙氧羰基-L-精氨酸(Alloc-L-Arginine,AA)的合成
将30.0g NaHCO3悬浮于120mL水中,加入20.0g精氨酸单盐酸盐,搅拌,20mL氯甲酸烯丙酯在30min内滴加到上述体系中,室温搅拌8h,4℃冷却过滤,用***和少量冷水洗涤、冷冻干燥得白色粉未26.4g,冷水用量为过滤后滤饼质量的4倍;收率90%;
(2)丙烯酸环丙沙星乙二酯(ACE)的合成
将丙烯酸羟乙酯与环丙沙星按摩尔比为1.5:1的量,加入到装有甲磺酸和适量甲苯的三口烧瓶中,在N2保护下磁力搅拌,升温至125℃,5h后停止反应,将所得反应液经去离子水洗涤至中性,萃取并收集有机相;甲磺酸用量为环丙沙星质量的1%,甲苯用量为环丙沙星物质的量的5倍;
(3)精氨酸嵌段环丙沙星共聚物(PAC)的合成
通过自由基聚合反应制备两亲性共聚物(PAC)。首先,分别称取5:4摩尔比例的N-烯丙氧羰基-L-精氨酸(AA)和丙烯酸环丙沙星乙二酯(ACE),将其置于恒压滴液漏斗中,再用适量无水乙醇和DMSO的混合溶剂溶解;随后,将占单体总质量1%的自由基引发剂AIBN充分溶解于THF中,将1/2体积的AIBN溶液分别加入上述漏斗和预先加有适量乙醇和DMSO的三口烧瓶里;在N2保护下,升温至75℃,将漏斗中的混合溶液缓慢滴入三口烧瓶中;滴加完毕后,在N2气氛,75℃下继续回流反应24h,反应液经丙酮洗涤,乙醇透析,最终制得两亲性共聚物(PAC)如下:
其中,m=25,n=20。
将上述得到的两亲性共聚物继续处理得到共聚物的纳米颗粒,处理步骤同实施例1中两亲性共聚物继续处理得到共聚物的纳米颗粒的步骤。
实施例3
一种两亲性精氨酸嵌段环丙沙星共聚物的制备方法,包括如下操作步骤:
(1)N-烯丙氧羰基-L-精氨酸(Alloc-L-Arginine,AA)的合成
将30.0g NaHCO3悬浮于120mL水中,加入20.0g精氨酸单盐酸盐,搅拌,20mL氯甲酸烯丙酯在30min内滴加到上述体系中,室温搅拌8h,4℃冷却过滤,用***和少量冷水洗涤、冷冻干燥得白色粉未26.4g,冷水用量为过滤后滤饼质量的5倍;收率89.8%;
(2)丙烯酸环丙沙星乙二酯(ACE)的合成
将丙烯酸羟乙酯与环丙沙星按摩尔比为1.5:1的量,加入到装有甲磺酸和适量甲苯的三口烧瓶中,在N2保护下磁力搅拌,升温至125℃,5h后停止反应,将所得反应液经去离子水洗涤至中性,萃取并收集有机相;甲磺酸用量为环丙沙星质量的4%,甲苯用量为环丙沙星物质的量的8倍;
(3)精氨酸嵌段环丙沙星共聚物(PAC)的合成
通过自由基聚合反应制备两亲性共聚物(PAC)。首先,分别称取7:6摩尔比例的N-烯丙氧羰基-L-精氨酸(AA)和丙烯酸环丙沙星乙二酯(ACE),将其置于恒压滴液漏斗中,再用适量无水乙醇和DMSO的混合溶剂溶解;随后,将占单体总质量1%的自由基引发剂AIBN充分溶解于THF中,将1/2体积的AIBN溶液分别加入上述漏斗和预先加有适量乙醇和DMSO的三口烧瓶里;在N2保护下,升温至65℃,将漏斗中的混合溶液缓慢滴入三口烧瓶中;滴加完毕后,在N2气氛,65℃下继续回流反应24h,反应液经丙酮洗涤,乙醇透析,最终制得两亲性共聚物(PAC)如下:
其中,m=35,n=30。
将上述得到的两亲性共聚物继续处理得到共聚物的纳米颗粒,处理步骤同实施例1中两亲性共聚物继续处理得到共聚物的纳米颗粒的步骤。
一、抗菌活性评价
本发明制得的共聚物PAC具有两亲性结构,因此可以在溶液中自组装形成以精氨酸片段为壳,环丙沙星片段为核的纳米颗粒PAC@PNs,亲水精氨酸壳质子化后使纳米颗粒带有正电荷。而且,PAC结构中的亲水片段与胍基结构相似,这也赋予了PAC与天然抗菌肽相似的性质。通过MIC、抑菌率和MBC的测定评价实施例1制得的PAC@PNs在体外的抗菌活性。测试结果如表1,从表1中数据可以看出,带胍基性质的PAC@PNs的非特异性结合使其具有广谱、强效杀菌性能,包括对耐药细菌也表现出良好的抗菌效果,如MRSA(ATCC43300),VRE,MIC值在1-4μg/mL之间,MBC值在2-8μg/mL之间。其中,纳米颗粒对阳性菌S.aureus(BB2146)和阴性菌E.coli(BL21)的抗菌效果最好,MIC值均为1μg/mL,MBC值为2-3μg/mL。另外,在MIC条件下,测得PAC@PNs对所有菌株的抗菌率都达91%以上,即使对较难杀死的阴性菌也表现出优异的杀菌效果。
表1 PAC@PNs的抗菌性能测试
二、抗菌动力学评价
为测定PAC@NPs对革兰氏阳性菌S.aureus(ATCC29213)的杀菌动力学,实时监测在不同浓度PAC@NPs的作用下S.aureus(ATCC29213)的OD600的变化情况,以未经PAC@NPs作用的细菌生长作为对照。
从图2中可以看出,在对照组中,细菌的OD600值随着时间的延长而逐渐增加。当PAC@NPs浓度为0.5MIC(1μg/mL)时,S.aureus(ATCC29213)细菌细胞的生长速度滞后于对照组;当浓度为MIC(2μg/mL)值时,S.aureus(ATCC29213)的生长一直处于受抑制状态,说明制备的纳米颗粒具有高效的抗菌作用,且在连续孵育12h后,细菌悬浮液的OD600值没有明显变化,说明PAC@NPs的灭菌率较高且细菌细胞没有耐药性产生。当PAC@NPs浓度>MIC时,既使培养12h后,细菌细胞的生长也一直处于抑制状态,细菌浓度基本保持不变,且随着PAC@NPs浓度增加,其灭菌效果增强。同时,取不同时间点的S.aureus(ATCC29213)细菌细胞悬浮液,菌液经稀释后涂布于LB琼脂平板上,监测不同PAC@NPs浓度下细菌细胞的菌落数。结果如图3所示,从图3中可以清楚地看出,在PAC@NPs浓度为3μg/mL情况下,在2h内灭菌率就达90%以上,4h内使活菌数量降低了3个数量级,由此说明纳米颗粒PAC@NPs具有高效灭杀能力。
三、耐药性研究
细菌的耐药性,使人类健康、公共安全卫生受到巨大威胁。因此,研究细菌细胞是否容易对PAC@NPs产生耐药性是非常必要的。以VRE和E.coli(BL21)为测试菌株,将PAC@NPs浓度为1/2MIC孔中的测试菌株连续传代14次,通过分析PAC@NPs对每代细菌的MIC判断测试菌株是否发展了耐药性。
分别选择市售抗生素头孢曲松和庆大霉素作为对照。测试结果如图4和图5所示,对于VRE(图4),连续传代14次,头孢曲松对其MIC值在第14代后增加了近45倍;而制备的带正电荷的纳米颗粒PAC@NPs对其14代后的MIC值提高了近4倍,该变化幅度较小,也在误差允许的范围内,该结果说明,传统抗生素头孢曲松在对细菌重复给药刺激后,其优异的抗菌功效会逐渐下降,而PAC@NPs连续对细菌作用却不会诱导该细菌产生抗药性。从图5可以看出,庆大霉素对连续传代的E.coli(BL21)的MIC在第2代就出现了增长,表现出明显的抗药性,且随着传代次数的递增,在第14代后MIC提高了近50倍。然而,E.coli(BL21)连续传代14代后,PAC@NPs对该菌株的MIC值基本保持在误差允许的范围内浮动,没有发生明显增加。综上表明,测试菌株VRE和E.coli(BL21)对抗生素头孢曲松、庆大霉素容易产生明显抗性,而对PAC@NPs纳米颗粒不容易产生耐药性。这主要是因为PAC@NPs可以直接机械性地“溶菌”使细菌凋亡,因此赋予了PAC@NPs高效、广谱且不易诱导细菌耐药性产生的特点。
四、菌体细胞形貌监测
为探究纳米颗粒PAC@NPs的抗菌机制,通过SEM观察E.coli(BL21)经PAC@NPs作用前后的形貌,评价纳米颗粒对细菌形态的破坏。以未经PAC@NPs作用的细菌细胞作为空白对照(Con.)。从图6A中观察到,在没有经PAC@NPs作用的情况下,E.coli(BL21)细胞都表现出规律形态,具有完整光滑的表面。然而,当以致死剂量的PAC@NPs(2MIC)作用E.coli(BL21)细胞0.5h后,如图6B所示,细胞形貌发生了明显改变,细菌膜表面出现褶皱,变的不平滑,细胞间有相互融合的趋势。作用8h后,如图6C所示,细菌细胞结构已经完全坍塌、破裂,菌体细胞由于“溶菌”融合在了一起。由此说明纳米颗粒PAC@NPs可以将细菌细胞结构完全破坏,致使E.coli(BL21)没有任何机会产生耐药性。
五、荧光标记实验分析
为了证实PAC@NPs对细菌细胞膜的破坏,通过DAPI和PI荧光染色实验测定细胞膜通透性的变化。荧光染料DAPI能够穿透完整的细胞膜,与DNA结合发出蓝色荧光;DAPI不仅可以荧光标记有活性的细胞,失活的细菌细胞也可以。然而,PI只能对细胞膜受损的细胞标记并发出红色荧光。基于这一特性,可采用这两种荧光染色剂分别标记经PAC@NPs作用前后的细菌来判断细菌细胞膜是否完整。将PAC@NPs作用前后的MRSA(ATCC43300)或E.coli(BL21)用DAPI和PI染色,使用荧光倒置显微镜观察拍摄。结果如图7所示,在对照组中,经DAPI染色的MRSA和E.coli细胞发出蓝色荧光;而与PI孵育后都没有发出红色荧光,表明细菌细胞膜完好无损。然而,经PAC@NPs作用后的两种细菌与DAPI作用后依旧发出了蓝色荧光,用PI染色后发出了明显的红色荧光,表明细菌经PAC@NPs作用后,菌体细胞膜已受损,通透性发生了改变。这充分证实了PAC@NPs可以破坏细菌细胞膜结构。
六、菌体细胞Zeta电位分析
细菌细胞膜一般带负电,完好的膜电位是保证细胞正常生命活动的必要条件。为了探究PAC@NPs对细菌细胞膜电位的影响,监测细菌细胞Zeta电位经纳米颗粒作用前后的变化。将未经纳米颗粒作用的细菌悬浮液作为对照。测试结果如表2所示,测得MRSA、S.aureus、P.aeruginosa、E.coli细胞正常情况下的Zeta电位分别为-42.5、-40.6、-45.6、-47.8mV,经PAC@NPs作用8h后,细菌细胞的Zeta电位分别升至-7.6、-3.7、-12.3、-11.7mV,可以看出Zeta电位有了明显变化。该结果表明细菌细胞在带正电荷的纳米颗粒PAC@NPs(+28.6mV)作用下,切实可以扰乱细胞膜电位,并不破坏菌体细胞正常的生理活动。
表2细菌经PAC@NPs作用前后细胞的Zeta电位
七、PAC@NPs细胞毒性评价
采用MTT法评价纳米颗粒PAC@NPs对非洲绿猴的正常肾细胞(Vero-E6)的毒性。纳米颗粒的剂量为1-64μg/mL,与备好的正常肾细胞共同孵育,培养24h或48h后,经酶标仪分析计算细胞存活率。评价结果如图8所示,从图8中可以看出,当PAC@NPs的剂量低于16μg/mL时,Vero-E6的存活率均超过85%,表明纳米颗粒具有较低的毒性。这可能是由于哺乳动物真核细胞具有净中性电荷,对结构似胍基的精氨酸片段几乎没有亲和力,且PAC结构中的环丙沙星嵌段也具有较低的毒性;另外精氨酸片段属于肽成分,故具有良好生物相容性的特点,这些因素决定了PAC@NPs更低的细胞毒性。
本发明未详述之处,均为本技术领域技术人员的公知技术。
Claims (8)
2.根据权利要求1所述的一种两亲性精氨酸嵌段环丙沙星共聚物,其特征在于,所述m=30,n=25。
3.一种制备如权利要求1或2所述的两亲性精氨酸嵌段环丙沙星共聚物的方法,其特征在于,包括如下操作步骤:
(1)N-烯丙氧羰基-L-精氨酸的合成
取NaHCO3悬浮于一定量水中,加入精氨酸单盐酸盐,搅拌;将氯甲酸烯丙酯在一定时间内滴加到上述体系中,室温搅拌后,冷却过滤,用***和少量冷水洗涤、冷冻干燥得白色粉未;
(2)丙烯酸环丙沙星乙二酯的合成
将丙烯酸羟乙酯与环丙沙星按摩尔比为1.5:1的量,加入到甲磺酸和适量甲苯中,在N2保护下磁力搅拌,升温至125℃,5h后停止反应,将所得反应液经去离子水洗涤至中性,萃取并收集有机相;
(3)精氨酸嵌段环丙沙星共聚物的合成
分别称取不同比例的步骤(1)的N-烯丙氧羰基-L-精氨酸和步骤(2)的丙烯酸环丙沙星乙二酯,置于恒压滴液漏斗中,再用适量无水乙醇和DMSO的混合溶剂溶解;然后,将占单体总质量1%的自由基引发剂AIBN充分溶解于THF中,将1/2体积的AIBN溶液分别加入上述恒压滴液漏斗和预先加有适量无水乙醇和DMSO的三口烧瓶内;在N2保护下,升温,将漏斗中的混合溶液缓慢滴入三口烧瓶中;滴加完毕后,继续回流反应24h,反应液经丙酮洗涤、乙醇透析,制得系列两亲性共聚物。
4.根据权利要求3所述的一种制备两亲性精氨酸嵌段环丙沙星共聚物的方法,其特征在于,步骤(1)少量冷水用量为过滤后所得滤饼质量的2-4倍;步骤(2)中甲磺酸质量为环丙沙星质量的1%-4%,甲苯用量为环丙沙星物质的量的5-8倍;步骤(3)在N2保护下,升温至65-75℃;继续回流反应保持N2气氛、65-75℃条件。
5.一种两亲性精氨酸嵌段环丙沙星共聚物纳米颗粒,其特征在于,由如权利要求3或4的制备方法制得的两亲性共聚物自组装制得,尺寸为200±50nm。
6.根据权利要求5所述的两亲性精氨酸嵌段环丙沙星共聚物纳米颗粒,其特征在于,所述两亲性共聚物自组装步骤包括如下操作:
称取两亲性共聚物溶于四氢呋喃中,在匀速搅拌下缓慢滴加到去离子水中,匀速搅拌,所得混合液在去离子水中透析以除去小分子,每间隔一定时间更换一次透析液,得两亲性共聚物的纳米颗粒PAC@PNs。
7.根据权利要求6所述的两亲性精氨酸嵌段环丙沙星共聚物纳米颗粒,其特征在于,操作中,两亲性共聚物与四氢呋喃采用每1mL四氢呋喃加入2mg两亲性共聚物的重量体积比混合;去离子水与四氢呋喃用量相等;匀速搅拌时间为12h,透析时间72h,每间隔8h更换一次透析液。
8.如权利要求6或7所述的两亲性精氨酸嵌段环丙沙星共聚物纳米颗粒在抗菌药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210569438.6A CN114920869B (zh) | 2022-05-24 | 2022-05-24 | 两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210569438.6A CN114920869B (zh) | 2022-05-24 | 2022-05-24 | 两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114920869A CN114920869A (zh) | 2022-08-19 |
CN114920869B true CN114920869B (zh) | 2023-07-14 |
Family
ID=82811222
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210569438.6A Active CN114920869B (zh) | 2022-05-24 | 2022-05-24 | 两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114920869B (zh) |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0820597A (ja) * | 1994-07-07 | 1996-01-23 | Meiji Seika Kaisha Ltd | トロンビン阻害作用を有する複素環カルボニル化合物 |
CN1568996A (zh) * | 2004-05-10 | 2005-01-26 | 郭东宇 | 一种盐酸环丙沙星冻干制剂及制备方法 |
AU2006214655A1 (en) * | 2005-02-17 | 2006-08-24 | Medivas, Llc. | Polymer particle delivery compositions and methods of use |
EP1907444B1 (en) * | 2005-04-01 | 2009-08-19 | Intezyne Technologies Incorporated | Polymeric micelles for drug delivery |
US10709791B2 (en) * | 2014-11-12 | 2020-07-14 | University Of Washington | Stabilized polymeric carriers for therapeutic agent delivery |
CN105175625A (zh) * | 2015-09-23 | 2015-12-23 | 苏州大学 | 一种温敏性聚合物载体、制备方法及其应用 |
CN106750056B (zh) * | 2016-11-30 | 2019-04-16 | 华南师范大学 | 一种三氯生两亲性聚合物纳米粒子及其制法与抗菌应用 |
CN113024595B (zh) * | 2021-03-01 | 2022-05-31 | 桂林理工大学 | 1,3,5,7-四甲基环四硅氧烷基环丙沙星荧光探针及其在铁离子检测中的应用 |
CN113444211B (zh) * | 2021-06-11 | 2023-01-20 | 湖北大学 | 一种基于pisa的抗菌高分子纳米颗粒的制备方法和应用 |
CN113330931A (zh) * | 2021-06-29 | 2021-09-03 | 安徽德昌油茶专业合作社 | 一种油茶容器嫁接苗用环保栽培袋 |
-
2022
- 2022-05-24 CN CN202210569438.6A patent/CN114920869B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN114920869A (zh) | 2022-08-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Lam et al. | Antimicrobial polymeric nanoparticles | |
Liu et al. | Recent developments in polydopamine: an emerging soft matter for surface modification and biomedical applications | |
Liu et al. | Synthesis of graphene oxide-quaternary ammonium nanocomposite with synergistic antibacterial activity to promote infected wound healing | |
Zuo et al. | Enzyme-responsive Ag nanoparticle assemblies in targeting antibacterial against methicillin-resistant Staphylococcus aureus | |
Mukherjee et al. | Leucine-based polymer architecture-induced antimicrobial properties and bacterial cell morphology switching | |
Wang et al. | Antibacterial fluorescent nano-sized lanthanum-doped carbon quantum dot embedded polyvinyl alcohol for accelerated wound healing | |
CN107596368B (zh) | 一种细菌靶向纳米粒子的制备及其抑杀细菌的应用 | |
Wei et al. | Preparation of novel ferrocene-based shell cross-linked thermoresponsive hybrid micelles with antitumor efficacy | |
Mei et al. | Multivalent polymer–Au nanocomposites with cationic surfaces displaying enhanced antimicrobial activity | |
CN102973488A (zh) | 具有氧化还原/pH双重刺激响应性的纳米水凝胶及其制备方法和应用 | |
Chen et al. | Dual-functional antimicrobial coating based on the combination of zwitterionic and quaternary ammonium cation from rosin acid | |
CN104892949B (zh) | 一种谷胱甘肽/pH双重刺激响应离子交联型聚合物纳米水凝胶及其制备方法和应用 | |
Guo et al. | Acidity-activated charge-convertible silver nanocomposites for enhanced bacteria-specific aggregation and antibacterial activity | |
Xu et al. | Synergistic chemo-/photothermal therapy based on supercritical technology-assisted chitosan–indocyanine green/luteolin nanocomposites for wound healing | |
Li et al. | Selective capture, separation, and photothermal inactivation of methicillin-resistant Staphylococcus aureus (MRSA) using functional magnetic nanoparticles | |
CN114920869B (zh) | 两亲性精氨酸嵌段环丙沙星共聚物、纳米颗粒及其制备方法和应用 | |
Suga et al. | Surface Characteristics of Antibacterial Polystyrene Nanoparticles Synthesized Using Cationic Initiator and Comonomers | |
Ghaffarlou et al. | Poly (acrylic acid)-b-Poly (vinylamine) copolymer: decoration with silver nanoparticles, antibacterial properties, quorum sensing activity, and cytotoxicity on breast cancer and fibroblast cell lines | |
Luo et al. | Engineering Cationic Hyperbranched Polyureas for Combating Bacterial Biofilms | |
CN114807108B (zh) | 基于多巴胺聚合的活细胞表面功能化及应用 | |
CN115721725A (zh) | 一种靶向肝癌细胞的pH/GSH双响应型纳米药物载体及其制备方法 | |
Guo et al. | Facile core–shell nanoparticles with controllable antibacterial activity assembled by chemical and biological molecules | |
Zhen et al. | Ciprofloxacin peptide-based nanoparticles confer antimicrobial efficacy against multidrug-resistant bacteria | |
CN114052318B (zh) | 一种具有抗耐药菌作用的口罩及其制备方法和应用 | |
Xiao et al. | Lipase and pH-responsive diblock copolymers featuring fluorocarbon and carboxyl betaine for methicillin-resistant staphylococcus aureus infections |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |