CN114432441A - 一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用 - Google Patents
一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用 Download PDFInfo
- Publication number
- CN114432441A CN114432441A CN202210027859.6A CN202210027859A CN114432441A CN 114432441 A CN114432441 A CN 114432441A CN 202210027859 A CN202210027859 A CN 202210027859A CN 114432441 A CN114432441 A CN 114432441A
- Authority
- CN
- China
- Prior art keywords
- nps
- solution
- photothermal
- nano
- cancer
- 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.)
- Granted
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 64
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 38
- 201000011510 cancer Diseases 0.000 title claims abstract description 35
- 239000002131 composite material Substances 0.000 title claims abstract description 14
- IRPLSAGFWHCJIQ-UHFFFAOYSA-N selanylidenecopper Chemical compound [Se]=[Cu] IRPLSAGFWHCJIQ-UHFFFAOYSA-N 0.000 title claims abstract description 10
- 238000002626 targeted therapy Methods 0.000 title description 6
- 108010074708 B7-H1 Antigen Proteins 0.000 claims abstract description 28
- 102000008096 B7-H1 Antigen Human genes 0.000 claims abstract description 28
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 claims abstract description 18
- 208000022679 triple-negative breast carcinoma Diseases 0.000 claims abstract description 17
- 230000000694 effects Effects 0.000 claims abstract description 11
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 3
- 239000010949 copper Substances 0.000 claims description 160
- 239000010931 gold Substances 0.000 claims description 35
- 238000011065 in-situ storage Methods 0.000 claims description 12
- 229910052737 gold Inorganic materials 0.000 claims description 11
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 10
- 238000001727 in vivo Methods 0.000 claims description 9
- 206010006187 Breast cancer Diseases 0.000 claims description 8
- 208000026310 Breast neoplasm Diseases 0.000 claims description 8
- 206010027476 Metastases Diseases 0.000 claims description 8
- 230000009401 metastasis Effects 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 8
- 239000006185 dispersion Substances 0.000 claims description 7
- 230000003993 interaction Effects 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- 230000006907 apoptotic process Effects 0.000 claims description 5
- 150000002343 gold Chemical class 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 229930182817 methionine Natural products 0.000 claims description 3
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 238000001802 infusion Methods 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 230000008685 targeting Effects 0.000 abstract description 18
- 210000004027 cell Anatomy 0.000 description 54
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 29
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 29
- 238000011282 treatment Methods 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 23
- 239000000243 solution Substances 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 16
- 239000002114 nanocomposite Substances 0.000 description 14
- 238000012986 modification Methods 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 239000000463 material Substances 0.000 description 12
- 230000004048 modification Effects 0.000 description 12
- 238000007626 photothermal therapy Methods 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 9
- 238000003384 imaging method Methods 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 230000001988 toxicity Effects 0.000 description 7
- 231100000419 toxicity Toxicity 0.000 description 7
- 230000008901 benefit Effects 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 5
- 238000002296 dynamic light scattering Methods 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 239000002086 nanomaterial Substances 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 4
- 238000002173 high-resolution transmission electron microscopy Methods 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 239000012221 photothermal agent Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000011729 BALB/c nude mouse Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 229910004042 HAuCl4 Inorganic materials 0.000 description 3
- 210000000577 adipose tissue Anatomy 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000001931 thermography Methods 0.000 description 3
- 239000012099 Alexa Fluor family Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- OSYYVEXNRXSIOA-UHFFFAOYSA-N [Cu].[Se].[Au] Chemical compound [Cu].[Se].[Au] OSYYVEXNRXSIOA-UHFFFAOYSA-N 0.000 description 2
- 238000002679 ablation Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- BWFPGXWASODCHM-UHFFFAOYSA-N copper monosulfide Chemical compound [Cu]=S BWFPGXWASODCHM-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 239000008204 material by function Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000011269 treatment regimen Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 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 1
- 238000012406 Annexin V-FITC/PI double staining Methods 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 206010010957 Copper deficiency Diseases 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 101000693007 Mus musculus Pancreatic alpha-amylase Proteins 0.000 description 1
- 241000935974 Paralichthys dentatus Species 0.000 description 1
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 1
- 229910018162 SeO2 Inorganic materials 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000779 annular dark-field scanning transmission electron microscopy Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005672 electromagnetic field Effects 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 239000002159 nanocrystal Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 229960002796 polystyrene sulfonate Drugs 0.000 description 1
- 239000011970 polystyrene sulfonate Substances 0.000 description 1
- 102000003998 progesterone receptors Human genes 0.000 description 1
- 108090000468 progesterone receptors Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000004054 semiconductor nanocrystal Substances 0.000 description 1
- 229940006186 sodium polystyrene sulfonate Drugs 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
- 238000000584 ultraviolet--visible--near infrared spectrum Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Images
Classifications
-
- 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
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
-
- 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
- A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/225—Microparticles, microcapsules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Nanotechnology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Radiology & Medical Imaging (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Medical Informatics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Materials Engineering (AREA)
- Manufacturing & Machinery (AREA)
- Composite Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明提供了一种PD‑1修饰的金复合硒化铜纳米粒子，具有更优的光热性能，以及对PD‑L1过表达的癌细胞具有特异性的靶向能力，对癌细胞具有显著的抑制和消融作用，尤其适用于治疗三阴性乳腺癌。
Description
技术领域
本发明属于生物功能材料技术领域，涉及一种PD-1修饰的金复合硒化铜纳米粒子，可以用于PD-1介导光热靶向治疗癌症，尤其是三阴性乳腺癌。
背景技术
光热治疗(PTT)是一种具有高特异性和很有前景的新型肿瘤治疗手段，具有时间短、毒副作用小、疗效显著等优点，近年来成为科学研究的热点。PTT是通过将光热试剂注入肿瘤组织中，在可见或近红外(NIR)光激发下，光热剂能够吸收近红外光，并将其转换为热能产生局部高温，导致癌细胞发生坏死或凋亡，从而达到癌症治疗的目的。光热转换效率(PCE)是光热试剂的一个关键因素，它直接决定光热治疗过程中所需要的激发光强度，高强度的激发光很容易对皮肤和组织造成损伤。因此，提高光热试剂的光热转化效率，降低激光使用强度，使用安全光强度的激光用于光热治疗，是光热治疗研究领域的关键点和难点。
作为世界各国的主要死因，如何预防和治疗癌症已成为世界范围内最重要的问题。在所有癌症中，女性乳腺癌已超过肺癌，成为2020年最常见的癌症。根据美国癌症协会的建议，乳腺癌的常规治疗包括手术、放疗、化疗和靶向治疗。然而，传统治疗方法存在一定的问题，例如手术过程中标记不充分，或放化疗的毒性。因此，迫切需要开发有效的诊断和治疗方法来治疗乳腺癌。由于纳米粒子介导的光声图像(PAI)和光热治疗(PTT)具有低侵袭性和高选择性，近年来备受关注。通过使用光热转换剂(PTAs)，将光能以非热辐射形式转化为声能或热能，分别用于诊断和治疗癌症。
在现有的PTAs中，铜硫属纳米材料表现出独特的光学性质，在近红外区具有优越的吸收能力和更好的光热转换效率，是一种较为理想的光声成像和光热治疗的材料。非化学计量的硒化铜纳米晶体(Cu2-xSe)表现出优异的光热转换性能，作为一种p型半导体纳米晶体，由于缺铜而产生的本征阳离子空穴(或空穴，h+)可以看作是自由载流子，通过局域表面等离子体共振与光的振荡电磁场相互作用，将光能转化为热能。Cu2-xSe在乳腺癌的PAI和PTT治疗中显示出良好的能力，但对于缺乏雌激素受体、孕激素受体和人表皮生长因子受体的三阴性乳腺癌(TNBCs)，如何在保持光热转换效率的同时有效靶向癌细胞是将Cu2-xSe引入TNBCs的前提。
除了铜硫属化合物作为单一光热试剂用于癌症治疗，科研工作者还将它们和其它功能材料联用，制备成复合纳米材料来增加其生物应用的功能。Lin和研究团队等报道了一种中空结构Pt-CuS的光热材料用于光声成像和近红外热成像。这项工作为多功能治疗策略开发了一个基于铜硫属复合物的纳米平台，并通过合理设计其结构拓宽了其生物学应用。这些复合材料相比于单一材料具有靶向、多模式协同治疗的优点。
发明内容
有鉴于此，本发明的目的在于提供一种PD-1修饰的金复合硒化铜纳米粒子，以期具有更优的光热性能，以及对PD-L1过表达的癌细胞具有特异性的靶向能力，从而对癌细胞产生显著的抑制和消融作用。
本发明提供了一种PD-1修饰的金复合硒化铜纳米粒子Cu2-xSe@Au-PD-1，于Cu2- xSe@Au纳米粒子的金颗粒上连接有PD-1。PD-1的修饰使得金复合硒化铜纳米粒子拥有了靶向PD-L1的基础能力。
其中，所述PD-1通过N端与金颗粒连接；进一步，所述PD-1通过N端的甲硫氨酸与金颗粒的金-硫醇相互作用与金颗粒连接。
其中，所述Cu2-x Se@Au纳米粒子，以Cu2-xSe为核，Au原位生长于Cu2-xSe外周，具有纳米核-卫星结构；所述Au通过(200)和(111)面与Cu2-xSe的(111)面晶格匹配；所述Cu2-xSe@Au纳米粒子中，Cu2-xSe核的平均尺寸为42.8±4.6nm，Au的平均尺寸为10.7±3.4nm。
本发明的Cu2-xSe@Au-PD-1纳米粒子采用下述步骤获得：
分别将含有Cu2-x Se@Au纳米粒子和PD-1的溶液混合均匀，0-8℃静置过夜，对经离心后的重液悬浮，得到Cu2-xSe@Au-PD-1；其中，含有Cu2-x Se@Au纳米粒子的溶液为Cu2-xSe@Au-PD-1在Tween-20溶液中的分散液，含有PD-1的溶液为PD-1在PBS溶液中的分散液。
具体方法为，在剧烈搅拌下，将2μL 1％Tween-20添加到上述制备的100μL 1mgmL-1Cu2-xSe@Au NPs溶液中；然后在2x PBS溶液中加入200μL的250μgμL-1PD-1；将两种溶液混合均匀后4℃静置过夜；6000rpm 4℃离心10min后弃上清，再用1x PBS悬浮，最终得到Cu2-xSe@Au-PD-1。
本发明提供的Cu2-xSe@Au-PD-1纳米粒子能够用于靶向PD-L1过表达的癌细胞并产生光热效应，从而可以在制备用于治疗癌症的药物方面得到广泛应用。通过对PD-L1过表达的癌细胞的特异性靶向能力，可以使Cu2-xSe@Au-PD-1纳米粒子在癌细胞周围聚集，然后通过激光照射发挥光热作用，使得癌细胞组织区域温度升高，从而达到抑制或杀伤癌细胞的目的。
其中，所述Cu2-xSe@Au-PD-1纳米粒子能够促进癌细胞的凋亡和抑制癌细胞的转移。
其中，所述癌症为三阴乳腺癌，所述癌细胞为乳腺癌细胞。
其中，所述Cu2-xSe@Au-PD-1在产生光热效应时，采用650～900nm的激光照射，功率优选为(0.75±0.25)W cm-2。合适的激光能量区域和照射强度最小化了对其他生物组织成分(如水)的附加影响，更利于生物组织环境的稳定。
其中，所述Cu2-xSe@Au-PD-1的用量为(10±7.5)mg kg-1,体内输入浓度优选为(1±0.5)mg/mL。合适的用量和浓度有利于足够剂量的纳米颗粒快速在癌细胞附近聚集，保证光热效率和经济效益。
本发明的优势在于，通过在Cu2-xSeNPs表面原位还原Au3+，合成了新型的Cu2-xSe@AuNPs。在Cu2-xSe上的Au NPs的数量可以进行精确地调控，以达到最佳的光热转换能力。原位生长的AuNPs不仅抑制了Cu2-xSe的毒性，而且还提供了修饰PD-1蛋白的位点，形成了Cu2- xSe@Au-PD-1纳米复合物。该纳米复合物对PD-L1过表达的4T1小鼠乳腺癌细胞(即三阴性乳腺癌(TNBC))表现出特异性靶向能力。因此，我们将其应用于PD-L1过表达的4T1裸鼠，光声成像和光热靶向治疗结果显示肿瘤被显着抑制和消融，治疗30天后未观察到肿瘤转移。因此，本发明所制备的新型的Cu2-xSe@Au NPs光热材料，在光热治疗领域是具有发展前途的复合材料，为TNBC的光热靶向治疗提供了一个有吸引力的机会，将该Cu2-xSe@Au NPs能够作为载体成功制备的Cu2-xSe@Au-PD-1纳米复合物，也被证实能够作为治疗TNBC的靶向光热试剂。
附图说明
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作优选的详细描述，其中：
图1：制备的Cu2-xSe@Au NPs的表征：(a)Cu2-xSe NPs和(b)Cu2-xSe@Au NPs的透射电镜图像；(c)Cu2-xSe NPs和(d)Cu2-xSe@Au NPs的高分辨透射电镜图像。(e)Cu2-xSe NPs和(f)Cu2-xSe@Au NPs的对应EDS元素分析。
图2：不同Au NPs浓度原位生长在Cu2-xSe NPs表面合成的Cu2-xSe@Au NPs的TEM图像：在Cu2-xSe NPs溶液中加入0.5mL,0.75mL,1mL,1.25mL,1.5mL和1.75mL的0.1％HAuCl4
图3：在功率密度为0.75W cm-2的808nm(a)和1064nm(b)激光连续照射10min下，纯水和Cu2-xSe NPs的光热加热曲线。
图4：在功率密度为0.75W cm-2的808nm激光连续照射10min(每个样品的浓度为100μg mL-1)下，水、Cu2-xSe NPs和不同AuNPs修饰Cu2-xSe@Au NPs的水溶液的(a)热图像和(b)相应的光热加热曲线。
图5：不同浓度的Cu2-xSe NPs和Cu2-xSe@Au NPs孵育4T1细胞48h后的细胞存活率。
图6：(a)在0.75W cm-2 808nm激光照射下，Cu2-xSe@Au NPs水溶液在不同浓度下的温度上升曲线；(b)不同功率密度(100μg mL-1)下Cu2-xSe@Au NPs水溶液在808nm激光照射下的温度上升曲线；(c)在0.75W cm-2 808nm激光照射下(浓度为100μg mL-1)，Cu2-xSe NPs和Cu2-xSe@Au NPs在三个辐照/冷却周期内的温度变化。
图7：在808nm激光辐照下Cu2-xSe(a)和Cu2-xSe@Au NPs(b)溶液的温度分布，激光关闭后自然冷却；(c-d)使用线性化的能量平衡来确定系统时间常数。
图8：Cu2-xSe NPs、Cu2-xSe@Au NPs和Cu2-xSe@Au-PD-1的(a)zeta电位分布和(b)动态光散射数据。(c)用Cu2-xSe@Au-PD-1纳米复合物孵育后PD-L1KO和PD-L1KO-PD-L1 4T1细胞中PD-1和PD-L1的蛋白印迹分析。
图9:PD-L1KO和PD-L1KO-PD-L1 4T1细胞与PBS和Cu2-xSe@Au-PD-1纳米复合物孵育后的免疫荧光。PD-L1用Alexa Fluor Plus 555标记并记录在AF555通道中；PD-1用AlexaFluor Plus 488标记并记录在AF488通道中；细胞核用DAPI标记。比例尺：10μm。右下角插入的图像被放大以展示共定位。比例尺：2μm。
图10:PD-L1KO和PD-L1KO-PD-L1 4T1细胞分别与PBS、Cu2-xSe@Au或Cu2-xSe@Au-PD-1纳米复合物孵育，并用808nm激光以0.75W/cm2功率照射10分钟。
图11：(a)体内PAI和PTTT治疗方案示意图。(b)4T1-PD-L1KO和4T1-PD-L1KO-PD-L1接种小鼠在注射Cu2-xSe@Au-PD-1后0、1、2、4、6、8、12和24h的光声图像。(c)4T1-PD-L1KO和4T1-PD-L1KO-PD-L1接种小鼠在注射Cu2-xSe@Au-PD-1后0、1、2、4、6、8、12和24h的热图像。(d)不同治疗14天后荷瘤小鼠和肿瘤的代表性图像。(e)各组肿瘤体积生长曲线，从第1天起每隔一天测量一次体积。(f)基于相同类型的癌细胞，G2、G3与G1组比较；G5、G6、T1与G4组比较肿瘤生长抑制率。采用非配对双尾t检验计算显着差异，n.s.：不显着，****：p<0.0001。数据以平均值±SD表示。
图12：(a)H&E染色、TUNEL和Ki67肝转移免疫组化的代表性图像。(b)不同处理后小鼠38天内的存活率，n＝7。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。需要说明的是，以下实施例中所提供的图示仅以示意方式说明本发明的基本构想，在不冲突的情况下，以下实施例及实施例中的特征可以相互组合。
本发明通过多步反应条件高效调控Cu2-xSe@Au NPs的形貌和尺寸，获得的Cu2-xSe@Au纳米颗粒能够作为有效载体，通过金-硫醇相互作用，在其表面上以修饰的方式负载PD-1，所制备的纳米复合物表现出优于裸Cu2-x Se的光热性能，对PD-L1过表达的癌细胞具有特异性靶向能力。
本发明的主要思路是：采用原位生长法在硒化铜纳米颗粒(Cu2-x Se NPs，其中0≤x≤1，为叙述方便，文中其余地方省略了关于x范围的表述)上原位生长金纳米颗粒(AuNPs)制备了一种核-卫星结构(Core Satellite Structure，CSS)的硒化铜-金复合光热纳米颗粒(Cu2-xSe@Au NPs)，对其表面进行修饰，使得制备的Cu2-x Se@Au NPs材料具有更好的靶向能力。Au NPs不仅能显著提高光热转换效率，还能降低裸Cu2-x Se的毒性，为后续光热靶向治疗(PTTT)提供一个有效的修饰位点。通过金-硫醇相互作用(N端的甲硫氨酸)将PD-1的细胞外结构域(NCBI参考序列：NP_032824.1)连接到Cu2-x Se表面的Au NPs上。所制备的纳米复合物表现出优于裸Cu2-x Se的光热性能，对PD-L1过表达的癌细胞具有特异性靶向能力。进一步将此复合物应用于TNBC荷瘤小鼠，光声和光热图像均表明肿瘤区域发生了富集，并观察到PTTT治疗后的显著抑制和消融。
一、实验和表征
材料合成：采用原位还原法制备硒化铜-金复合光热纳米颗粒(Cu2-x Se@Au NPs)：将1.6mL的聚苯乙烯磺酸钠(PSS)(10mg/mL，MW 70kDa)和5.5mL的去离子水加入圆底烧瓶中，随后加入0.1mL的0.2M SeO2和0.3mL 0.4M Vc的混合物。搅拌10min后，将0.1mL0.4M的CuSO4·5H2O和0.4mL 0.4M的Vc混合物加入烧瓶。30℃搅拌30min后，将温度升为45℃，持续搅拌8h左右，溶液变为墨绿色后，即Cu2-xSe纳米粒子制备完成，并通过10KDa的透析袋进行纯化，间隔1h换水1次，然后透析过夜，共透析24h。随后将纯化的Cu2-xSe纳米粒子离心重悬后置于4℃冰箱以备后续使用。
利用原位修饰等界面调控方法，在Cu2-xSe纳米材料上掺杂不同元素或复合不同的纳米材料对其进行界面修饰，将2ml的1mg mL-1Cu2-xSe NPs分散液加入到8mL含0.1％HAuCl4不同体积(Cu2-xSe@Au1 NPs、Cu2-xSe@Au2 NPs、Cu2-xSe@Au3 NPs分别为0.75mL，1.25mL和1.75mL)的去离子水中。在25℃下搅拌3h得到Cu2-xSe@Au NPs,10000rpm离心10min。最后，纯化的Cu2-xSe@Au NPs在4℃下重新分散在2mL去离子水(浓度：1mg mL-1)中备用。
在剧烈搅拌下，将2μL 1％Tween-20添加到上述制备的100μL 1mg mL-1Cu2-xSe@AuNPs溶液中。然后在2x PBS溶液中加入200μL的250μgμL-1PD-1。将两种溶液混合均匀后4℃静置过夜。6000rpm 4℃离心10min后弃上清，再用1x PBS悬浮，最终得到Cu2-xSe@Au-PD-1。
表征和仪器：采用Talos F200X透射电子显微镜(TEM)(Thermo)，在200kV的加速电压下对制备的样品的尺寸和形貌进行了表征。采用日立U-3600分光光度计获得了纳米粒子分散在水溶液中的紫外-可见-近红外光谱。使用Zetasizer Nano ZS(Malvern)进行动态光散射(DLS)和ζ电位表征。使用VLSM-808-B-4-SMA或VFLS-1064-B-MP-4W-1光纤激光器(Connet)获得高度稳定的激光源。使用FACSCantoTMⅡ流式细胞仪(BD)进行流式细胞分析。免疫荧光图像采用LSM 900激光扫描共聚焦显微镜(ZEISS)拍摄。光声图像和热图像分别采用VEVO LAZR-X小动物光声成像系统(VisualSonics)和Ti401 Pro红外热像仪(FLUKE)拍摄。采用XN 2000全自动血液分析仪(Sysmex)测定小鼠的常规血液指标。采用RM2016病理切片机(Leica)进行石蜡组织切片，H&E染色的石蜡切片采用ECLIPSE E100正置光学显微镜(Nikon)观察。
流式细胞术：4T1-PDL1KO和4T1-PDL1KO-PDL1细胞在含有10％FBS的高葡萄糖DMEM培养基中，在37℃ 10cm培养皿中培养。当细胞浓度达到1*107个细胞/培养皿时，用胰蛋白酶在37℃下消化3分钟，然后用培养基中和胰蛋白酶。将消化后的细胞转移至试管中，350g离心3分钟，弃上清，PBS洗涤细胞2次，重悬于含2％FBS的高糖DMEM培养基中。将细胞按1*106个细胞/组的浓度分成不同的组，每组加入PBS或Cu2-xSe@Au或Cu2-xSe@Au-PD-1。将细胞在37℃下轻轻摇动孵育2h。然后350g离心3分钟去除过量的Cu2-xSe@Au或Cu2-xSe@Au-PD-1，将细胞用PBS洗涤2次并重悬于含有2％FBS的高葡萄糖DMEM培养基中。然后用808nm激光以0.75W cm-2的功率密度对激光照射组照射10分钟。之后，所有组在37℃下再孵育6h，并按照操作手册将Annexin V-FITC/PI试剂盒应用于细胞。最后用流式细胞仪分析各组细胞的凋亡情况。
动物实验：
Cu2-x Se@Au的体内急性毒性：对BALB/c小鼠(6-8周，18-22g)进行编号，随机分为7组，每组小鼠尾静脉注射200μL不同浓度的Cu2-x Se@Au或相同体积的PBS。对小鼠进行连续14天的监测，每隔一天测量一次体重。14天后处死小鼠，并提取主要器官(心、肝、脾、肺、肾和脑)。所有器官均进行常规处理，按照标准程序进行石蜡组织切片，采用标准染色方案对石蜡切片进行H&E染色。所有小鼠自由进食和饮水，维持12小时光照/12小时黑暗的光循环，使用异氟醚麻醉。
体内光声光热成像：BALB/c裸鼠(雌性，6-8周，18-22g)编号，随机分为4组。培养4T1-PD-L1KO和4T1-PD-L1KO-PD-L1细胞，将105个细胞/小鼠注射至小鼠第4乳腺脂肪垫，将小鼠饲养7天以监测其致瘤性。当肿瘤体积达到～50mm3时，每组小鼠尾静脉注射相同体积的200μL 1mg/mL Cu2-x Se@Au或PBS。分别于注射后0、1、2、4、6、8、12、24h拍摄光声和光热图像。光热图像使用0.75w cm-2 808nm激光照射肿瘤10分钟，并使用红外热像仪记录光热图像。
体内光热治疗：BALB/c裸鼠(雌性，6-8周，18-22g)编号，随机分为7组(G1-G6和T1)，每组7只。培养4T1-PD-L1KO(G1-G3)和4T1-PD-L1KO-PD-L1(G4-G6和T1)细胞，并将105个细胞/小鼠注射到小鼠第4乳腺脂肪垫，小鼠饲养6天以监测其致瘤性。当肿瘤体积达到～30mm3时，每组小鼠尾静脉注射200μL PBS(G1、G4)、1mg/mL Cu2-x Se@Au(G2、G5)或1mg/mLCu2-x Se@Au-PD-1(G3、G6、T1)。每只小鼠(除G6外)在0.75w cm-2 808nm激光照射10分钟。注射后14天内监测肿瘤体积和体重，在第14天进行常规血液检查。
二、结果与分析
通过透射电镜(TEM)对材料的形貌和微观结构进行了表征。从图1a可以清楚地看出，Cu2-xSe构建为平均尺寸为42.8±4.6nm的球形颗粒。在Cu2-xSe NPs上原位还原生长AuNPs，其典型直径为10.7±3.4nm，形成Cu2-xSe@Au NPs作为纳米核卫星结构(CSS)(图1b)。为进一步确定纳米核心卫星的结构，采用高分辨率透射电子显微镜(HRTEM)和高角度环形暗场扫描透射电子显微镜(HAADF-STEM)分析。HRTEM显微照片显示，Cu2-xSeNPs的(111)面对应的分辨率为0.33nm的晶格间距(图1c)，0.24和0.20nm的晶格间距与Au的(200)和(111)面匹配良好(图1d)。HAADF-STEM图像(图1e和f)清晰地展示了Cu2-xSe@Au NPs的纳米核心卫星结构，较亮的黄色对比度显示出Cu2-xSe表面生长AuNPs。图1f所示的能谱(EDS)元素分析进一步证实了Cu、Se和Au元素的存在和分布。
通过改变0.1％HAuCl4的体积可以精确地控制Au NPs的数量，而过度修饰Au NPs可能会影响Cu2-xSe@Au NPs的结构和性能(图2)。
进一步采用两种不同波长的激光对光热转换进行了研究。图3显示了808nm(图3a)或1064nm(图3b)连续照射10min后，Cu2-xSe NPs与水的光热加热曲线。1064nm激光照射后，水的温度迅速升高，在55.3℃时达到最大值。而稳定状态下，在808nm下增加到34.5℃的速度要慢得多。因此，选择808nm激光来研究Cu2-xSe@Au NPs的光热特性，这主要是由于在650～900nm的近红外区域，水的吸收系数最低，在生物组织中渗透更深。
采用0.75W cm-2的808nm激光照射100μg mL-1Cu2-xSe NPs和Cu2-xSe@Au NPs 10分钟，记录热像和相应的温度曲线(图4a和4b)。Cu2-xSe@Au1和Cu2-xSe@Au2 NPs的稳定温度分别为61.2和63.2℃，高于Cu2-xSe NPs的58.4℃。然而，Au NPs的过度修饰可以在10分钟辐照后将稳态温度略微降低到54.8℃(Cu2-xSe@Au3 NPs)。
为了选择最合适的材料用于后续应用，我们进行了细胞活力试验来研究每个材料的生物相容性(图5)。Cu2-xSe在50μg mL-1浓度下也表现出较强的毒性。Au NPs修饰后，由于AuNPs具有较好的生物相容性，其毒性被显著抑制，但相对较少的Au NPs(Cu2-xSe@Au1 NPs)在高浓度下仍表现出毒性。为了平衡光热能力和毒性，Cu2-xSe@Au2 NPs被用于后续的实验。
图6a和b显示了Cu2-xSe@Au NPs光热转换的表观浓度和功率密度依赖关系。为了评价制备的Cu2-xSe NPs和Cu2-xSe@Au NPs的光热稳定性，记录了在0.75W cm-2 808nm激光辐照下三次辐照/冷却循环的温度分布。如图6c所示，经过三个周期的测试，Cu2-xSe NPs和Cu2- xSe@Au NPs都表现出稳定的光热转换能力。
将50μg/mL的Cu2-xSe NPs和Cu2-xSe@Au NPs溶液用808nm激光辐照500μL，关闭激光后自然冷却。监控的温度分布如图7所示。光热转换效率(η)由式1计算:
Tmax(K)为平衡温度，Tsurr(K)为环境的环境温度。Qdis为容器吸收光的热损失，I(W)为入射激光功率(0.75W cm-2)。A808是样品在808nm处的吸光度。式中h(W cm-2K-1)为传热系数，s(cm2)为容器表面积。HS的计算公式为：
其中τs为样品体系时间常数，mD和cD为溶剂的质量(g)和热容(4.2J/g)。根据计算光热转换效率(η)，Cu2-xSe@Au NPs的光热转换效率为54.53％，是Cu2-xSe NPs(35.84％)的1.52倍。证明所制备的材料的光热转换性能得到了大幅度提升。
采用动态光散射(DLS)和ζ-电位表征每个步骤的修饰(图8a和8b)。通过在Cu2-xSe上还原Au后，ζ-电位从-49.7mV增加到-15.8mV，并且由于PD-1的等电点(pKa)为5.27，在pH7.4 PBS缓冲液中带负电，经PD-1修饰后，ζ-电位变为-22.6mV。同时，每次修改后，流体动力直径也相应增大。上述结果均证明PD-1成功地结合在Cu2-xSe@Au上，形成了Cu2-xSe@Au-PD-1纳米复合物。然后我们将该复合物应用于4T1小鼠TNBC细胞中，建立了两种4T1细胞，PD-L1敲除(PD-L1KO)和PD-L1过表达(PD-L1KO-PD-L1)，以检测Cu2-xSe@Au-PD-1纳米复合物的靶向能力。蛋白印迹分析结果表明PD-1只能在PD-L1KO-PD-L1细胞中检测到，而在PD-L1KO细胞中未检测到PD-1或PD-L1(图8c)。
为了进一步证明PD-1靶向PD-L1，我们采用免疫荧光(IF)将PD-1和PD-L1共定位于4T1细胞表面，使用PBS缓冲液作为阴性对照(图9)。IF结果清楚地表明PD-1和PD-L1在PD-L1过表达的4T1细胞膜上共定位。然而，对于PD-L1敲除细胞，在细胞膜上几乎观察不到PD-1。考虑到PD-1被修饰在Cu2-xSe@Au-PD-1纳米复合物上，我们认为纳米复合物通过PD-1和PD-L1相互作用靶向癌细胞。
在确认了Cu2-xSe@Au-PD-1纳米复合物的靶向能力后，我们将其应用于癌细胞以验证光热效应的杀伤效率。将PD-L1KO和PD-L1KO-PD-L1细胞分别与PBS、Cu2-xSe@Au或Cu2-xSe@Au-PD-1纳米复合物一起孵育，并用808nm激光以0.75W/cm2照射10分钟。Annexin V-FITC/PI双染色用于流式细胞术检测细胞凋亡(图10)。对于在PBS缓冲液中孵育的两种细胞，808nm激光对细胞的伤害较小，这是因为水吸收的能量较少，温度没有上升太多。对于Cu2- xSe@Au来说，由于它不能特异性靶向癌细胞，其杀伤效率可以忽略不计。只有当Cu2-xSe@Au-PD-1纳米复合物应用于PD-L1过表达的4T1细胞时，才能观察到明显的凋亡，比例为54.2％。结合先前的研究结果，所制备的Cu2-xSe@Au-PD-1纳米复合物表现出优异的光热转换效率、低细胞毒性和对TNBC细胞的高靶向性，适合进一步应用于TNBC模型小鼠的光声成像和光热靶向治疗。
用Cu2-xSe@Au-PD-1纳米复合物对TNBC小鼠进行体内PAI和PTTT治疗。为了评估Cu2-xSe@Au-PD-1纳米复合物在肿瘤组织中的积累，在肿瘤达到～50mm3后24小时内监测PA和PT效应(图11a)。将PD-L1KO或PD-L1KO-PD-L1 4T1细胞接种于BALB/c裸鼠第4乳腺脂肪垫，尾静脉注射Cu2-xSe@Au-PD-1(10mg kg-1)后采集4T1荷瘤小鼠PA图像(图11b)。PD-L1KO-PD-L1组的PA信号在肿瘤部位清晰可见(以圆圈标出)，在注射后8小时左右达到峰值。与PD-L1KO组相比，PD-L1KO-PD-L1组的PA信号比PD-L1KO组高1.76倍，验证了Cu2-xSe@Au-PD-1在PD-L1KO-PDL1的肿瘤聚集是靶向作用的结果，并为肿瘤光热治疗的时间点提供了指导。在808nm激光照射下Cu2-xSe@Au-PD-1在肿瘤中的有效聚集能迅速升高肿瘤温度。根据体内热成像(图11c)，将Cu2-xSe@Au-PD-1溶液(10mg kg-1)注射到4T1荷瘤小鼠后，在1,2,4,6,8,12,24h时间点用808nm激光以0.75W/cm2功率照射10分钟，PD-L1KO-PD-L1组小鼠的肿瘤区域迅速变红变亮，证明了Cu2-xSe@Au-PD-1在体内的红外热成像能力。
如图11d所示，PBS(G1组)、Cu2-xSe@Au(G2组)、Cu2-xSe@Au-PD-1(G3组)接种4T1-PD-L1KO小鼠14天后，肿瘤体积无统计学差异，分别为930.05±177.11、935.59±160.33和911.75±78.43mm3。对于4T1-PD-L1KO-PD-L1接种小鼠，与PBS(G4组)或Cu2-xSe@Au(G5组)相比，用Cu2-xSe@Au-PD-1(T1组)处理后肿瘤显着抑制，分别为789.18±62.94、760.45±102.37和29.46±17.16mm3(图11e)。而即使没有照射，用Cu2-xSe@Au-PD-1(G6组)处理后的接种4T1-PD-L1KO-PD-L1小鼠也能观察到轻微的抑制，这可能是由靶向纳米复合物的内在抑制引起的。T1组的肿瘤抑制率高达97.43±2.59％，这也表明该PTTT治疗有效(图11f)。
在第30天分析各组的转移情况(图12a)。H&E染色表明G1—G6组小鼠肝脏出现严重的癌细胞定植，主要发生在血管周围，而T1组接受PTTT治疗后未发生明显肝转移。TUNEL的荧光免疫组织化学(IHC)显示未经适当处理的肝细胞高水平凋亡，Ki67蛋白染色显示G1—G6组肿瘤细胞高水平增殖和血管周围高水平生长。H&E染色和荧光IHC与之前的血液结果一致，表明我们的治疗可以抑制肝转移。此外，在喂养38天后，T1组小鼠全部存活，这也证明了该治疗方案的有效性(图12b)。
三、结论
本发明通过在Cu2-xSeNPs表面原位还原Au3+，合成了新型的Cu2-xSe@Au NPs。在Cu2- xSe上的Au NPs的数量可以进行精确地调控，以达到最佳的光热转换能力。原位生长的AuNPs不仅抑制了Cu2-xSe的毒性，而且还提供了修饰PD-1蛋白的位点，形成了Cu2-xSe@Au-PD-1纳米复合物。该纳米复合物对PD-L1过表达的4T1小鼠乳腺癌细胞(即三阴性乳腺癌(TNBC))表现出特异性靶向能力。因此，我们将其应用于PD-L1过表达的4T1裸鼠，光声成像和光热靶向治疗结果显示肿瘤被显着抑制和消融，治疗30天后未观察到肿瘤转移。因此，本发明所制备的新型的Cu2-xSe@Au NPs光热材料，在光热治疗领域是具有发展前途的复合材料，为TNBC的光热靶向治疗提供了一个有吸引力的机会，将该Cu2-xSe@Au NPs能够作为载体成功制备的Cu2-xSe@Au-PD-1纳米复合物，也被证实能够作为治疗TNBC的靶向光热试剂。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.一种PD-1修饰的金复合硒化铜纳米粒子Cu2-xSe@Au-PD-1，于Cu2-xSe@Au纳米粒子的金颗粒上连接有PD-1。
2.根据权利要求1所述的Cu2-xSe@Au-PD-1，其特征在于，所述PD-1通过N端与金颗粒连接；进一步，所述PD-1通过N端的甲硫氨酸与金颗粒的金-硫醇相互作用与金颗粒连接。
3.根据权利要求1所述的Cu2-xSe@Au-PD-1，其特征在于，所述Cu2-xSe@Au纳米粒子，以Cu2-xSe为核，Au原位生长于Cu2-xSe外周，具有纳米核-卫星结构；所述Au通过(200)和(111)面与Cu2-xSe的(111)面晶格匹配；所述Cu2-xSe@Au纳米粒子中，Cu2-xSe核的平均尺寸为42.8±4.6nm，Au的平均尺寸为10.7±3.4nm。
4.权利要求1-3任一项所述的Cu2-xSe@Au-PD-1纳米粒子的制备方法，包括下述步骤：
分别将含有Cu2-xSe@Au纳米粒子和PD-1的溶液混合均匀，0-8℃静置过夜，对经离心后的重液悬浮，得到Cu2-xSe@Au-PD-1；其中，含有Cu2-xSe@Au纳米粒子的溶液为Cu2-xSe@Au-PD-1在Tween-20溶液中的分散液，含有PD-1的溶液为PD-1在PBS溶液中的分散液。
5.根据权利要求4所述的Cu2-xSe@Au-PD-1纳米粒子的制备方法，其特征在于，在剧烈搅拌下，将2μL 1％Tween-20添加到上述制备的100μL 1mg mL-1Cu2-xSe@Au NPs溶液中；然后在2x PBS溶液中加入200μL的250μgμL-1PD-1；将两种溶液混合均匀后4℃静置过夜；6000rpm 4℃离心10min后弃上清，再用1x PBS悬浮，最终得到Cu2-xSe@Au-PD-1。
6.权利要求1-3任一项所述的Cu2-xSe@Au-PD-1纳米粒子制备用于治疗癌症的药物方面的应用。
7.根据权利要求6所述的应用，其特征在于，所述Cu2-xSe@Au-PD-1纳米粒子具体用于下述方面之一：
i.靶向PD-L1过表达的癌细胞并产生光热效应，
ii.促进癌细胞的凋亡和抑制癌细胞的转移。
8.根据权利要求6所述的应用，其特征在于，所述癌症为三阴乳腺癌，癌细胞为乳腺癌细胞。
9.根据权利要求6所述的应用，其特征在于，所述Cu2-xSe@Au-PD-1在产生光热效应时，采用650～900nm的激光照射，功率优选为(0.75±0.25)W cm-2。
10.根据权利要求6所述的应用，其特征在于，所述Cu2-xSe@Au-PD-1的用量为(10±7.5)mg kg-1,体内输入浓度优选为(1±0.5)mg/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210027859.6A CN114432441B (zh) | 2022-01-11 | 2022-01-11 | 一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210027859.6A CN114432441B (zh) | 2022-01-11 | 2022-01-11 | 一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114432441A true CN114432441A (zh) | 2022-05-06 |
| CN114432441B CN114432441B (zh) | 2023-04-25 |
Family
ID=81368112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210027859.6A Active CN114432441B (zh) | 2022-01-11 | 2022-01-11 | 一种pd-1修饰的金复合硒化铜纳米粒子及其在介导光热靶向治疗癌症方面的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114432441B (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203132A (zh) * | 2008-08-25 | 2011-09-28 | 安普利穆尼股份有限公司 | Pd-1拮抗剂的组合物和使用方法 |
| CN106075443A (zh) * | 2016-07-15 | 2016-11-09 | 上海工程技术大学 | 一种金包覆硒化铜纳米粒子及其制备方法与应用 |
| WO2018156649A1 (en) * | 2017-02-22 | 2018-08-30 | Flagship Pioneering, Inc. | Compositions of t cell modulator (tcm) molecules and uses thereof |
| US20190240186A1 (en) * | 2016-10-25 | 2019-08-08 | Council Of Scientific & Industrial Research | Gold nanoparticle based formulation for use in cancer therapy |
| US20210369862A1 (en) * | 2018-09-18 | 2021-12-02 | Universiteit Gent | Therapeutic nanoparticles and methods of use thereof |
-
2022
- 2022-01-11 CN CN202210027859.6A patent/CN114432441B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203132A (zh) * | 2008-08-25 | 2011-09-28 | 安普利穆尼股份有限公司 | Pd-1拮抗剂的组合物和使用方法 |
| CN106075443A (zh) * | 2016-07-15 | 2016-11-09 | 上海工程技术大学 | 一种金包覆硒化铜纳米粒子及其制备方法与应用 |
| US20190240186A1 (en) * | 2016-10-25 | 2019-08-08 | Council Of Scientific & Industrial Research | Gold nanoparticle based formulation for use in cancer therapy |
| WO2018156649A1 (en) * | 2017-02-22 | 2018-08-30 | Flagship Pioneering, Inc. | Compositions of t cell modulator (tcm) molecules and uses thereof |
| US20210369862A1 (en) * | 2018-09-18 | 2021-12-02 | Universiteit Gent | Therapeutic nanoparticles and methods of use thereof |
Non-Patent Citations (4)
| Title |
|---|
| J. BAO ET AL: "Gold-promoting-satellite to boost photothermal conversion efficiency of Cu2-xSe for triple-negative breast cancer targeting therapy" * |
| QIAN HUANG ET AL: "Boosting the Radiosensitizing and Photothermal Performance of Cu2–xSe Nanocrystals for Synergetic Radiophotothermal Therapy of Orthotopic Breast Cancer" * |
| YIFAN ZHANG ET AL: "Biomimetic Nanoemulsion for Synergistic Photodynamic-Immunotherapy Against Hypoxic Breast Tumor" * |
| 丁笠等: "纳米金用于肿瘤免疫治疗的研究进展" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114432441B (zh) | 2023-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dong et al. | Upconversion-mediated ZnFe 2 O 4 nanoplatform for NIR-enhanced chemodynamic and photodynamic therapy | |
| Geng et al. | Carbon dot/WS2 heterojunctions for NIR-II enhanced photothermal therapy of osteosarcoma and bone regeneration | |
| Ma et al. | Au nanoparticles with enzyme-mimicking activity-ornamented ZIF-8 for highly efficient photodynamic therapy | |
| Ning et al. | Cancer cell membrane-coated C-TiO2 hollow nanoshells for combined sonodynamic and hypoxia-activated chemotherapy | |
| Geng et al. | Multifunctional carbon dot/MXene heterojunctions for alleviation of tumor hypoxia and enhanced sonodynamic therapy | |
| Wang et al. | Prussian blue-coated lanthanide-doped core/shell/shell nanocrystals for NIR-II image-guided photothermal therapy | |
| US20230035080A1 (en) | Two-dimensional (2d) nanocomposite, preparation method, and use thereof | |
| Gao et al. | Ce6/Mn 2+-chelated polydopamine@ black-TiO 2 nanoprobes for enhanced synergistic phototherapy and magnetic resonance imaging in 4T1 breast cancer | |
| Zhang et al. | Peroxidase-like Fe 3 O 4 nanocomposite for activatable reactive oxygen species generation and cancer theranostics | |
| Cai et al. | Polypyrrole-coated UCNPs@ mSiO 2@ ZnO nanocomposite for combined photodynamic and photothermal therapy | |
| Lan et al. | Construction of a near-infrared responsive upconversion nanoplatform against hypoxic tumors via NO-enhanced photodynamic therapy | |
| Yang et al. | A Nd 3+ sensitized upconversion nanosystem with dual photosensitizers for improving photodynamic therapy efficacy | |
| Tang et al. | pH-Responsive Au@ Pd bimetallic core–shell nanorods for enhanced synergistic targeted photothermal-augmented nanocatalytic therapy in the second near-infrared window | |
| Chen et al. | Near-infrared persistent luminescence phosphors ZnGa2O4: Cr3+ as an accurately tracker to photothermal therapy in vivo for visual treatment | |
| Miao et al. | Recent advances in the biomedical applications of black phosphorus quantum dots | |
| CN110893237A (zh) | 铜钯合金纳米颗粒和自噬抑制剂在制备基于光热效应杀伤肿瘤的药物或试剂盒中的应用 | |
| Sun et al. | A Pd corolla–human serum albumin–indocyanine green nanocomposite for photothermal/photodynamic combination therapy of cancer | |
| Zhao et al. | Upconverting and persistent luminescent nanocarriers for accurately imaging-guided photothermal therapy | |
| Li et al. | A self-assembled nanoplatform based on Ag2S quantum dots and tellurium nanorods for combined chemo-photothermal therapy guided by H2O2-activated near-infrared-II fluorescence imaging | |
| Sun et al. | Metal–organic framework combined with CaO 2 nanoparticles for enhanced and targeted photodynamic therapy | |
| Chen et al. | Gold nanobipyramid@ copper sulfide nanotheranostics for image-guided NIR-II photo/chemodynamic cancer therapy with enhanced immune response | |
| Sheng et al. | Construction of PEGylated chlorin e6@ CuS-Pt theranostic nanoplatforms for nanozymes-enhanced photodynamic-photothermal therapy | |
| Yang et al. | Application of bismuth sulfide based nanomaterials in cancer diagnosis and treatment | |
| CN114652830A (zh) | 一种具有近红外二区吸收的纳米金壳颗粒的制备及其应用 | |
| Liu et al. | An all-in-one nanoplatform with near-infrared light promoted on-demand oxygen release and deep intratumoral penetration for synergistic photothermal/photodynamic therapy |
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 |