WO2020098754A1 - 疏水性纳米生物探针 - Google Patents

疏水性纳米生物探针 Download PDF

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WO2020098754A1
WO2020098754A1 PCT/CN2019/118571 CN2019118571W WO2020098754A1 WO 2020098754 A1 WO2020098754 A1 WO 2020098754A1 CN 2019118571 W CN2019118571 W CN 2019118571W WO 2020098754 A1 WO2020098754 A1 WO 2020098754A1
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tat
nano
cells
cell
bqds
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阮刚
王军
谢金兵
梅玲
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南京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to the field of biomaterials and biomedicine, and in particular, to a nanobiological probe and its application for targeted delivery of bioactive molecules in cells, especially in living cells.
  • fluorescent probes play a key role in live cell imaging.
  • quantum dots QD, nano-scale crystals of semiconductor materials
  • optical properties Including extraordinary fluorescence intensity and light stability, size and composition adjustable with a narrow peak width of the fluorescence emission peak can be excited by a single light source multiple imaging, and can be used as an imaging probe for related optical and electron microscopes.
  • QD's current application in live cell imaging of specific subcellular structures or subcellular molecules is usually limited to the imaging of entities on cell membranes (eg, cell surface receptors).
  • a typical water-soluble QD is composed of bare hydrophobic QD used to generate fluorescence and a polymer coating used to dissolve and maintain fluorescence in water, with a diameter greater than 15 nm. This size is much larger than most intracellular proteins (a few nanometers in diameter), and is thought to cause great movement disorders in the crowded cytoplasm of gel-like structures with a pore size of about 80 nm.
  • the size of the exposed hydrophobic QD is close to the size of the protein, and intracellular vesicle capture is still an insurmountable obstacle: it is estimated that ⁇ 1% of endocytic proteins (And other types of large molecules) can escape from intracellular vesicle capture and reach the cytoplasm.
  • bypassing endocytosis by methods of physically destroying cell membranes usually requires additional equipment. The use of the equipment requires long technical exercises and laborious operations, resulting in significant cell death and incapacitation. Duplicate results.
  • the object of the present invention is to provide a nano-bioprobe capable of high-specific localization in tissues, organs and cells of an organism, with low toxicity and easy operation.
  • a nano-bioprobe is provided, and the structure of the nano-bioprobe from the inside to the outside is as shown in Formula I,
  • A-S is a nanoparticle, where S is the hydrophobic surface of the nanoparticle;
  • B is a biomolecule
  • L is an optional connection part that couples B to the surface of the quantum dot
  • n is the number of biomolecules coupled to the surface of the quantum dot, which is a positive integer greater than or equal to 1.
  • the nano-bioprobe is treated with a mixed solvent.
  • the solubility of the mixed-solvent-treated nanoprobe in an aqueous environment is ⁇ 1nM, preferably ⁇ 5nM, and more preferably ⁇ 50nM.
  • the mixed solvent includes at least one organic solvent and at least one non-organic solvent.
  • the organic solvent is selected from the group consisting of dimethylformamide (DMF), acetone, ethanol, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and ethyl acetate , Acetonitrile, formic acid, butanol, dimethyl methanol, propanol, acetic acid, or a combination thereof.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • ethyl acetate Acetonitrile, formic acid, butanol, dimethyl methanol, propanol, acetic acid, or a combination thereof.
  • the organic solvent is dimethylformamide (DMF).
  • the concentration of the organic solvent is 0.01v / v% to 50v / v%, preferably 0.1v / v% to 20v / v%, more preferably 0.5v / v% to 5v / v%.
  • the non-organic solvent is selected from water, phosphate buffer, cell culture fluid, blood, etc., or a combination thereof.
  • the A is selected from the group consisting of magnetic nanoparticles, metal nanoparticles, semiconductor nanoparticles (quantum dots), carbon nanoparticles, polymer nanoparticles, lipid nanoparticles, or a combination thereof.
  • the quantum dots include: cadmium selenide quantum dots, cadmium sulfide quantum dots, zinc sulfide quantum dots, zinc selenide quantum dots, silver sulfide quantum dots, silver selenide quantum dots, cadmium telluride quantum dots Dots, zinc telluride quantum dots, perovskite quantum dots, or a combination thereof.
  • the S is the hydrophobic surface of the nanoparticle itself or the surface of the hydrophobic ligand layer surrounding the nanoparticle.
  • the ligand is selected from the group consisting of tri-n-octylphosphine oxide, oleic acid, stearic acid, oleylamine, or a combination thereof.
  • said B includes small molecules or biological macromolecules.
  • the biomacromolecule includes proteins, nucleic acids, polysaccharides, polypeptides, or a combination thereof.
  • said B is a positively and / or negatively charged polar molecule.
  • the molecular weight of B is 1 to 500 kD, preferably 1 to 200 kD, and more preferably 1 to 80 kD.
  • n is a positive integer from 1 to 1000, preferably a positive integer from 1 to 10, and more preferably a positive integer from 1 to 3.
  • the B is a Tat peptide.
  • sequence of the Tat peptide is SEQ ID NO: 1.
  • the Tat peptide has the biological functions of cell penetration and nuclear targeting.
  • the structure of the nanobioprobe may not include L in Formula I.
  • the form of coupling includes: peptide bond, hydrogen bond, charge interaction, avidin-biotin binding, nucleic acid pairing interaction, or a combination thereof.
  • the coupling is achieved through ligand exchange and bioconjugation.
  • the coupling is accomplished by using a coupling agent.
  • the coupling agent includes: 1-hemiethylamine-3- (3-dimethylaminopropyl) carbodiimide (EDC), avidin (avidin) -biotin (biotin), nucleic acid pairing, or a combination thereof.
  • the B can be covalently coupled and / or non-covalently coupled to the A-S surface.
  • the covalent coupling includes: peptide bond generation reaction, biological orthogonal reaction, or a combination thereof.
  • the non-covalent coupling is selected from the group consisting of charge interaction, hydrogen bonding, van der Waals force, hydrophobic interaction, antibody antigen interaction, avidin-biotin binding, nucleic acid pairing interaction, or Its combination.
  • the diameter of the nanoprobe is 1 to 200 nm, preferably 1 to 20 nm, and more preferably 1 to 10 nm.
  • the nanoprobe does not include a fully hydrophilic polymer coating.
  • the quantum dot nanoprobe can be stable in a closed container at 4 ° C for ⁇ 3 days, preferably ⁇ 5 days, more preferably ⁇ 7 days.
  • the excitation light wavelength of the quantum dots is 100 to 1500 nm, preferably 200 to 1000 nm, and more preferably 250 to 600 nm.
  • the emission wavelength of the quantum dots is 400 to 2500 nm, preferably 400 to 2000 nm, and more preferably 450 to 1500 nm.
  • a nano-bioprobe product including:
  • first container and the second container may be the same or different containers.
  • a method for manufacturing a nano-bioprobe according to the first aspect of the present invention or a nano-bioprobe product according to the second aspect of the present invention comprising the steps of:
  • step (iii) Treat the hydrophobic nanoparticle-biomolecule conjugate obtained in step (ii) with a mixed solvent to obtain the nanobioprobe.
  • a method for intracellular delivery of biologically active molecules comprising coupling the biologically active molecules to the nanobioprobe described in the first aspect of the present invention, and culturing with cells ⁇ ⁇ Liquid mixing.
  • the method is performed in vitro.
  • the method is non-diagnostic and non-therapeutic.
  • the cells include: non-cancer cells or cancer cells.
  • the cancer cells include: cervical cancer cells, breast cancer cells, liver cancer cells, lung cancer cells, gastric cancer cells, prostate cancer, or a combination thereof.
  • the biologically active molecule includes: a drug that binds to DNA, a drug that binds to RNA, a drug that binds to intracellular proteins, a drug that binds to intracellular cytoskeleton, a drug that functions in mitochondria Drugs, drugs combined with intracellular sugar molecules, drugs combined with intracellular lipid molecules, or a combination thereof.
  • the biologically active molecules include: small molecules and biological macromolecules.
  • the biologically active molecule is an anticancer drug.
  • the anticancer drug includes doxorubicin, paclitaxel, docetaxel, cisplatin, or a combination thereof.
  • the biomacromolecule includes proteins, nucleic acids, polysaccharides, polypeptides, or a combination thereof.
  • the intracellular delivery is live cell delivery.
  • the delivery is targeted delivery.
  • the delivery is targeted delivery of the nucleus.
  • the percentage of drugs that achieve targeted delivery is ⁇ 20%, preferably ⁇ 70%, and more preferably ⁇ 90%.
  • a use of the nanoprobe according to the first aspect of the present invention for preparing a preparation, the preparation being used to deliver biologically active molecules into a living body.
  • the organism includes biological tissues, organs or cells.
  • the organism includes cancerous tissues, cancerous organs or cancer cells.
  • the tissue is skin tissue.
  • the positioning is performed in vitro or in vitro.
  • the preparation or pharmaceutical composition has a function of crossing a biological transmission barrier in a living body.
  • the biological transmission barrier includes a tissue barrier, an organ barrier, a cell barrier, and an organelle barrier.
  • Figure 1 shows the design of cS-bQD-BM ('SDot') for targeted intracellular QD delivery.
  • Figure 2 shows the preparation and physicochemical characterization of cS-bQD-Tat.
  • Figure 3 shows the intracellular targeting of cS-bQDs-Tat.
  • Optical microscope images at different delivery time points show that the targeted intracellular delivery of cS-bQDs-Tat is rapid.
  • the images of 15 minutes, 1 hour, 4 hours, and 8 hours are composite images of bright-field (displaying cells) and fluorescence (displaying cS-bQD-Tat) microscope images.
  • the 12-hour image is a fluorescent image showing the co-localization of the nucleus (blue) and cS-bQDs-Tat (green).
  • the co-localization of the nucleus and cS-bQDs-Tat leads to a complex color light blue. Scale bar, 20 ⁇ m.
  • Figure 4 shows a mechanism study of cS-bQDs-Tat cell transport (a-c) confirming that cS-bQD-Tat's excellent intracellular targeting is caused by three design parameters.
  • Figure 5 shows the single particle tracking (SPT) and the correlation function (pCF) analysis of cS-bQDs-Tat cell transport.
  • SPT single particle tracking
  • pCF correlation function
  • Figure 6 shows the results of using cS-bQDs-Tat (SDots-Tat) to enhance targeted intracellular delivery of drugs and macromolecules.
  • Doxorubicin is used as a drug model to kill cancer cells (HeLa cells).
  • Doxorubicin is used as a drug model to kill cancer cells (HeLa cells).
  • formulation QDs-DOX soluble QD
  • free DOX free DOX
  • conventional water-soluble QDs-Tat formulation QDs-Tat-DOX
  • SDots-Tat-DOX significantly enhances targeting The efficiency of intracellular delivery of doxorubicin and killing cancer cells.
  • ovalbumin (ova) as the intracellular macromolecule delivery model.
  • Tat peptide formulation Tat-ova-dye, dye is a small molecule dye TAMRA for fluorescence imaging
  • SDots-Tat formulation Tat-ova-SDots
  • the formula Tat-dye no ova, its size is much smaller than ova was used as a positive control.
  • Figure 7 shows the penetration and delivery effects of SDot-Tat-DOX in nude mouse skin and tumor models. Among them, the distribution of samples in tumor tissues at different locations is shown: (a) close to the skin, (b) middle location, (c) bottom location. Ruler 20 ⁇ m
  • Figure 8 shows the effect of QD-Tat-DOX penetration and delivery in nude mouse skin and tumor models. Among them, the distribution of samples in tumor tissues at different locations is shown: (a) close to the skin, (b) middle location, (c) bottom location. Ruler 20 ⁇ m
  • Figure 9 shows the change in tumor volume after administration.
  • the inventor After extensive and in-depth research, the inventor has developed a new type of quantum dot nanoprobe through extensive screening. Specifically, the inventors replaced a small part of the hydrophobic surface of the hydrophobic quantum dots with a bifunctional small molecule ligand cysteamine, and passed 1-hemiethylamine-3- (3-dimethylaminopropyl ) Carbodiimide (EDC) couples the biomolecule Tat peptide to the amino group on the surface of the quantum dot, and then applies a small amount of organic co-solvent DMF to process the resulting quantum dot nanoprobe.
  • EDC 1-hemiethylamine-3- (3-dimethylaminopropyl ) Carbodiimide
  • the quantum dot nanoprobe can overcome the obstacles of cell transport and achieve highly specific, efficient, stable, scalable, safe and convenient intracellular targeting in living cells, and can achieve high efficiency and precision Intracellular drug delivery.
  • the quantum dot nanoprobe can also achieve a biological transmission barrier across tissues and organs in living animals in living animals.
  • experimental results of transdermal administration to nude mice show that the quantum dot nanoprobe of the present invention can deliver drugs deeper into subcutaneous tumors, and has a significantly better ability to inhibit tumor growth than the control group.
  • nanobioprobe As used herein, “nanobioprobe”, “nanoprobe”, “bQDs-BM”, “QD nanoprobe”, “QD probe”, etc. are used interchangeably, and refer to the internal Nanoprobe with the structure shown in Formula I,
  • A-S is nanoparticle
  • S is the surface of the nanoparticle, where S is the hydrophobic surface of the nanoparticle;
  • B is a biomolecule
  • L is a connection portion that optionally couples B to the surface of the quantum dot
  • n is the number of biomolecules coupled to the surface of the nanoparticle, which is a positive integer greater than or equal to 1.
  • the A is selected from the group consisting of magnetic nanoparticles, metal nanoparticles, semiconductor nanoparticles (quantum dots), carbon nanoparticles, polymer nanoparticles, lipid nanoparticles, etc., or a combination thereof .
  • the quantum dots include: cadmium selenide quantum dots, cadmium sulfide quantum dots, zinc sulfide quantum dots, zinc selenide quantum dots, silver sulfide quantum dots, silver selenide quantum dots, cadmium telluride Quantum dots, zinc telluride quantum dots, perovskite quantum dots, or a combination thereof.
  • the S is the hydrophobic surface of the nanoparticle itself or the surface of the hydrophobic ligand layer surrounding the nanoparticle.
  • the choice of ligands is very wide, as long as one end of the molecule binds to the nanoparticles with a certain attraction, and the other end is hydrophobic.
  • the ligand is selected from the group consisting of tri-n-octylphosphine oxide, oleic acid, stearic acid, oleylamine, etc., or a combination thereof.
  • the biomolecule B may include proteins, nucleic acids, polysaccharides, polypeptides, etc., and may also include small molecules.
  • the B is a positively and / or negatively charged polar molecule.
  • the B includes a Tat peptide, and the sequence of the Tat peptide is YGRKKRRQRRR (SEQ ID NO: 1), which has the biological functions of cell penetration and nuclear targeting.
  • the nanobiological probe described in the present invention overcomes cell transport obstacles [such as cell membrane, intracellular vesicle capture (if the cell uptake mechanism is endocytosis), crowded cytoplasm, non-specific protein binding, and organelle membrane (if the target is located in the organelle Intra) and obstacles in organelles], and achieve highly specific, efficient, stable, scalable, safe and convenient intracellular QD targeting in living cells.
  • cell transport obstacles such as cell membrane, intracellular vesicle capture (if the cell uptake mechanism is endocytosis), crowded cytoplasm, non-specific protein binding, and organelle membrane (if the target is located in the organelle Intra) and obstacles in organelles
  • the design of the present invention involves changing a small portion of the exposed hydrophobic QD (eg, 50%, preferably 40%) with biomolecules (through ligand exchange and then bioconjugation) , Preferably 30%, more preferably 20%, more preferably 10%), and use a small amount of organic co-solvent to promote its dispersion in an aqueous environment (Figure 1).
  • this new QD probe integrates a variety of features that help overcome obstacles to cell transport, including: (1) large hydrophobic surface assists in crossing biofilms; (2) organic assists Solvents promote biological crossing membranes and reduce non-specific protein binding; (3) due to the elimination of the use of polymer coatings, the formation of ultra-small QD probes to promote their movement in crowded biological environments, and (4) natural biomolecules Biological functions can be utilized (Figure 1).
  • the nucleus located deep inside the cell and used as the cell command center is used as the intracellular target model.
  • the experimental results of this embodiment prove that the living cell nucleus targeting using SDots has near-perfect specificity and excellent The efficiency and reproducibility, high-throughput capability, minimized cytotoxicity, easy operation and other advantages.
  • the Tat peptide has the biological function of targeting the nucleus and penetrating the cell, and is used as a biomolecule (BM) in the design of SDot.
  • sDot can be used for combined imaging, tracking and correlation function analysis in living cells.
  • sDot's unique ability to overcome barriers to cell transport can be used to enhance the delivery of small drug molecules and biological macromolecules.
  • mixtureed solvent As used herein, “mixed solvent”, “organic solvent of the present invention”, “organic solvent” and “organic co-solvent”, etc. are used interchangeably, meaning that they can be used to perform the nanobiological probe described in the first aspect of the present invention
  • hydrophobic nanoparticles are limited by the poor targeted transmission effect and poor biocompatibility in living cells in biomedical applications, they need to be physically (such as hydrophilic polymer coating) or in biomedical applications.
  • Chemical methods (such as the surface covered with hydrophilic functional groups) modify the hydrophobic surface of the nanoparticles to a hydrophilic surface to improve the targeted transmission effect inside the living cells of the nanoparticles.
  • hydrophobic nanoparticles that have not been hydrophilized cannot be directly used for targeted delivery within living cells.
  • the present invention breaks this traditional cognition and realizes the direct application of hydrophobic nanoparticles in the biomedical field, especially in live cells, through the treatment of mixed solvents.
  • the kind is limited, so the present invention is applicable to various nanoparticles, including magnetic nanoparticles, metal nanoparticles, semiconductor nanoparticles (quantum dots), carbon nanomaterials, polymer nanoparticles, lipid nanoparticles, etc., or a combination thereof.
  • the mixed solvent includes at least one organic solvent and at least one non-organic solvent, wherein the organic solvent includes: dimethylformamide (DMF), acetone, ethanol, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), ethyl acetate, acetonitrile, formic acid, butanol, dimethyl methanol, propanol, acetic acid, or a combination thereof.
  • the organic solvent includes: dimethylformamide (DMF), acetone, ethanol, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), ethyl acetate, acetonitrile, formic acid, butanol, dimethyl methanol, propanol, acetic acid, or a combination thereof.
  • the concentration of the organic solvent in the mixed solvent should be controlled within a certain range to minimize the cytotoxicity.
  • the concentration of the organic solvent component is 0.01v / v% to 50v / v%, preferably 0.1v / v% to 20v / v%, more preferably 0.5v / v % To 5v / v%.
  • the nano bioprobe provided by the present invention can be used for intracellular delivery of bioactive molecules.
  • the biologically active molecule can be coupled to the nano-bioprobe, and the nano-bioprobe is delivered to a specific location in the cell with the precise positioning of the nano-bioprobe in the cell.
  • the biological transmission barrier includes a tissue barrier, an organ barrier, a cell barrier, and an organelle barrier.
  • Cadmium oxide (CdO, 99.99%), zinc nitrate hexahydrate (Zn (NO3) 2.6H2O, 98%), sulfur powder (S, 99.98%), selenium powder (Se, 100 mesh, 99.5%), 1-ten Octene (ODE, 90%), stearic acid (SA, 95%), trioctylphosphine (TOP, 90%), cysteamine (98%), 1-ethyl-3 (3-dimethyl Aminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS)) and chloroquine were purchased from Aldrich.
  • N, N-dimethylformamide (DMF, 99.5%), acetone (99.5%), ethanol (99.7%), dimethyl sulfoxide (DMSO, 99%) and chloroform (99%) were purchased from Sinopharm Chemical Reagent .
  • Tat peptide (sequence Ac-YGRKKRRQRRR) (SEQ ID NO: 1)
  • Tat-TAMRA that is, the TAMRA dye is inserted between the fifth and sixth residues of SEQ ID NO: 1
  • Tat-ovalbumin that is, Insert Ovalbumin between the 5th and 6th residues of the sequence SEQ ID NO: 1
  • Tat-Ovalbumin-TAMRA that is, insert TAMRA between the 5th and 6th residues of the sequence SEQ ID NO: 1 Dye, with ovalbumin inserted between the 6th and 7th residues
  • Hoechst 33342 was purchased from ThermoFisher Scientific.
  • Cytochalasin D was purchased from Shanghai Baili Biotechnology Co., Ltd.
  • Phospholipid-PEG was purchased from Avanti Lipids.
  • Doxorubicin hydrochloride (DOX, 98%) was purchased from Aladdin.
  • HeLa, MCF-7, NIH 3T3 and Hep G2 cells were purchased from KeyGEN BioTECH.
  • the morphology of the nanoparticles was revealed by transmission electron microscopy (TEM, JEM-200CX, JEOL).
  • the chemical surface properties of the nanoparticles were analyzed by Fourier transform infrared spectroscopy (FTIR, Nicolet Nexus 470, Thermo) and X-ray photoelectron spectroscopy (XPS, ESCALAB 250, Thermo).
  • FTIR Fourier transform infrared spectroscopy
  • XPS X-ray photoelectron spectroscopy
  • ESCALAB 250, Thermo The Malvern Zetasizer Nano ZS360 instrument was used to measure the dynamic light scattering (DLS) diameter of the nanoparticles (ie, the hydrodynamic diameter if the solvent is water) and the surface charge (zeta potential).
  • the fluorescence spectrum was obtained using a fluorescence spectrophotometer (HITACHI F-4600).
  • HeLa cells were used in most cell experiments and were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 ° C and 5% CO 2 according to the manufacturer's recommendations.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • the other cell lines used (MCF-7, NIH 3T3 and Hep G2) were also cultured under the conditions recommended by the manufacturer.
  • Live cell imaging studies were performed using a live cell rotating disk confocal imaging system consisting of a cell incubation chamber (IX3W, Tokai Hit), an epifluorescence microscope (IX-83, Olympus), a rotating disk confocal system, and electron multiplying charge coupled devices (EMCCD) camera (Evolve 512, Photometrics).
  • the cells were first seeded on a glass bottom cell culture dish at a confluence of ⁇ 35% (glass bottom thickness 0.17 mm) (Nest, China). After incubating for 18 hours at 37 ° C and 5% CO 2 , the cell culture medium was replaced with the dispersion of cS-bQDs-Tat.
  • the typical conditions for using cS-bQDs-Tat dispersion are a total solvent volume of 1nM nanoparticles, 1% DMF as an organic co-solvent in aqueous cell culture medium, 90% hydrophobic surface coverage on the nanoparticles, 1Tat peptide per nanoparticle, and fluorescence emission peak at 559nm.
  • cS-bQDs-Tat for example, 15 minutes, 1 hour, 4 hours, 8 hours, 12 hours, and 24 hours
  • the cells are washed three times with fresh medium to remove outside or outside surfaces Combine the cS-bQD and image the cells by a confocal microscopy system with a rotating disk of living cells.
  • the fluorescent dye Hoechst 33342 blue fluorescent color, 5 ⁇ M in cell culture medium was incubated with live cells for 20 minutes. Use MetaMorph and Image J software for image processing and analysis.
  • the targeted nucleus in QD cells is measured as the ratio of the amount of QD delivered to the nucleus to the total amount of QD in the cell. Measurements were performed by two different methods, namely confocal imaging and nuclear separation, which produced consistent results.
  • confocal imaging method a live cell rotating disk confocal imaging system is used for imaging.
  • the amount of QD in the nucleus or cell is quantified by measuring the fluorescence intensity generated by QD in the corresponding area.
  • the cell nucleus separation method the cells are washed with PBS, and the cell nucleus is separated from the remaining cells using a cell nucleus separation kit according to the manufacturer's instructions (KeyGEN).
  • the amount of QD in the nucleus (or the rest of the cell) is determined by measuring the intensity of QD fluorescence in the nucleus (or the rest of the cell) and the total amount of QD using a fluorescence spectrometer.
  • the QD in the cells was determined by summing the amount of QD in the nuclear part and the remaining cell parts.
  • the amount of QD measured using the cell separation method is slightly lower than the amount of QD measured by the confocal imaging method due to the loss of QD during the separation of cell nuclei from cell lysates.
  • HeLa cells were seeded on 96-well plates (Corning Costar, China) at a density of 6000 cells / well. After incubation for 24 hours, the cell culture medium was replaced with the preparation for testing (200 ⁇ L / well in complete Dulbecco ’s Modified Eagle ’s medium). After culturing the cells for a specific duration (eg 12 hours or 24 hours), 20 ⁇ l 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide (MTT) The solution (5 mg / ml) was added to each well with 180 ⁇ l of DMEM and incubated at 37 ° C. for 4 hours.
  • MTT 4,5-dimethylthiazol-2-yl
  • the insoluble crystals were dissolved in dimethyl sulfoxide (DMSO, 150 ⁇ l / well) and measured spectrophotometrically at a wavelength of 570 nm in an ELISA reader (RT-6000, Rayto, China).
  • the relative cell survival rate (%) compared to the control well containing only the cell culture medium (except cells) was calculated by dividing the optical density of the test well by the optical density of the control well. All samples were run in triplicate.
  • HeLa cells were incubated in the cell culture medium at 4 ° C for 1 hour, and then the medium was replaced with the nanoparticle preparation (in the cell culture medium).
  • HeLa cells were treated with a cell culture medium (2 ⁇ M) containing cytochalasin D for 30 minutes, and then the culture medium was replaced with a nanoparticle preparation (in the cell culture medium). After the nanoparticles were delivered for 1 hour, the cells were washed five times with phosphate buffer solution, and then imaged using a live cell rotating disk confocal microscope system.
  • HeLa cells were seeded on 96-well plates (Corning Costar, China) at a density of 6000 cells / well. After culturing the cells for 24 hours, the preparation was used instead of the cell culture medium (200 ⁇ L / well, complete Dulbecco ’s Modified Eagle ’s Medium). After incubating the cells for 12 hours and 24 hours, respectively, 120 ⁇ l / well of sample supernatant was collected, and the released lactate dehydrogenase (LDH) was analyzed by a commercial kit (C0017, Beyotime, China).
  • LDH lactate dehydrogenase
  • the LDH release value of the cells treated with the LDH releasing agent was set to 100% LDH release (ie, the total amount of LDH in intact cells), and the LDH release value of untreated cells was set to the negative control.
  • the optical density was measured by ELISA reader (RT-6000, Rayto, China) at wavelengths of 490 nm (test) and 630 nm (reference).
  • the relative LDH release is defined by the ratio of total LDH released by LDH in intact cells. Cell samples with less than 10% LDH release are considered as cells with intact cell membranes, following well-established standards. All samples were run in triplicate.
  • Intracellular vesicles were labeled with lipophilic fluorescent dye DiR (5 ⁇ M, incubated with live cells for 30 minutes). At a given time point, the cells were washed three times with PBS, and then imaged using a confocal imaging system with a live cell rotating disk. Co-localization with fluorescent nanoparticles is quantified by using Pearson correlation coefficients.
  • cS-bQDs-Tat 20nM
  • BSA bovine serum albumin
  • DMF organic cosolvent
  • the mixture was dialyzed against water (with respective organic solvent concentrations) with a molecular weight cutoff of 200k Daltons to remove free BSA (BSA not bound to the nanoparticle surface).
  • the amount of free BSA was determined by a UV-Vis absorption spectrometer and measured at 280 nm against a calibration curve (after the value became stable after 96 hours of dialysis).
  • the amount of BSA bound to the nanoparticle surface was calculated by subtracting the amount of free BSA from the BSA initially added to the mixture.
  • a live-cell rotating disk confocal microscope was performed to collect images (movies) for single particle tracking data analysis (on the same cell sample as the data analysis of the correlation function, without moving the sample).
  • the algorithm links the positions of particles between successive time frames, and then links the resulting track segments into complete trajectories.
  • the algorithm gives an optimized trajectory to minimize the effects caused by high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting.
  • the mean square displacement (MSD) of different durations ( ⁇ t) is calculated. Finding the best fit of the MSD- ⁇ t relationship in the following equation results in the motion pattern and corresponding characteristic constant:
  • a live cell rotating disk confocal microscope was performed to collect images (movies) for pCF data analysis (on the same cell sample as single particle tracking data analysis without moving the sample).
  • the program of the present invention contains data from a rotating disk scan.
  • the fluorescence intensity at different positions and at different time points is presented, where the x coordinate corresponds to the position point along the line (pixel) drawn on the image view, and the y coordinate corresponds to time.
  • the pCF of two points at a distance ⁇ r as a function of the propagation time ⁇ is calculated by the following formula
  • the maximum value of the derived pCF profile is determined as the average time it takes for the particles to travel a given distance.
  • a line is drawn in this direction and the pCF profile is calculated. Of the two positions on the line, one is inside the vesicle and the other is outside the vesicle.
  • DOX small molecule drug doxorubicin
  • a dispersion of nanoparticles eg cS-bQDs-Tat
  • DOX dissolved in an organic solvent
  • Dialysis molecular weight cut-off 100k Dalton
  • the free drug removed was measured by UV-Vis absorption spectroscopy against a calibration curve (absorption at 480 nm).
  • the drug load is then quantified based on the amount of free drug removed. It was found that the drug loading efficiency (percentage of successfully loaded drug relative to the total amount of drug added) of all nanoparticle formulations was> 70%.
  • the amount of drug molecules loaded per particle was estimated to be 70.
  • Tat-ova-SDot is prepared by conjugating Tat-ova with SDot using EDC chemistry.
  • Three different preparations ie Tat dye, Tat-ova-SDot and Tat-ova dye
  • Tat-ova-SDot were incubated with HeLa cells, and the distribution of fluorescent probes was imaged by a rotating disk confocal microscope at different time points after incubation .
  • Nuclear targeting is quantified by measuring the fluorescence intensity in the nuclear region relative to the fluorescence intensity in the entire cell region (100-200 cells per preparation).
  • cS-bQDs-Tat To prepare cS-bQDs-Tat, first replace a small portion (for example 10%) of the exposed hydrophobic QD (bQDs; surface ligand trioctylphosphine, or abbreviation) with a bifunctional small molecule ligand cysteamine in an organic solvent Is TOP) surface ( Figure 2a).
  • the detected organic solvents include dimethylformamide (DMF), acetone, ethanol and dimethyl sulfoxide (DMSO). DMF has the best overall performance.
  • cysteamine and Tat peptide With the addition of cysteamine and Tat peptide, the surface charge positive potential becomes higher and higher, which is related to TOP (the number of cationic functional groups in each molecule is zero), cysteamine (one cationic functional group in each molecule) It is consistent with the molecular structure of Tat peptide (with multiple cationic functional groups in each molecule). There are multiple cationic functional groups in each molecule) ( Figure 2b).
  • the FTIR and XPS results of cS-bQDs-Tat showed the characteristic peak of successful conjugation (amide bond formation).
  • the addition of cysteamine only increased the diameter of the nanoparticles measured by dynamic light scattering (DLS) by ⁇ 0.05 nm ( Figure 2c).
  • cS-bQDs-Tat largely maintains the fluorescent properties of the exposed hydrophobic QD (approximately 20% decrease in fluorescence quantum efficiency, Figure 2d). Overall, reducing the QD surface in the SDot system replaced by hydrophilic molecules or functional groups increases the fluorescence quantum efficiency. If no organic solvent co-solvent is added to compensate for natural evaporation loss, the cS-bQD-Tat probe can maintain the colloidal stability in a closed container at 4 ° C for about 5 days.
  • SDot overcomes some of the key limitations of the current two main methods of transferring hydrophobic QD to an aqueous environment. These two methods respectively use polymers or small molecules to cover the entire QD surface.
  • the use of polymers as coatings usually adds 7-30 nm to the diameter of the nanoparticles, even if the optimized polymer structure is used, the diameter will increase by at least 1.5-2 nm, thus limiting QD in crowded spaces, such as intracellular spaces Biotransport.
  • replacing the original ligand on the entire bQD surface with bifunctional small molecules often leads to poor fluorescence quantum efficiency and colloidal stability.
  • SDot provides minimized particle size (by eliminating the use of polymer coatings) and good fluorescence quantum efficiency and colloidal stability (by keeping most of the original surface ligands of bQD intact and using organic co-solvents to increase Water soluble).
  • all original surface ligands on bQDs were replaced by cysteamine, and 2) a small portion (10%) of surface ligands on bQD were replaced by cysteamine without adding any organic Co-solvent, the two sets of control experiments showed poor fluorescence and colloidal stability, so it was confirmed that a small part of surface ligand replacement and co-existence of organic co-solvent were needed to obtain the best physicochemical properties of SDot.
  • SDot also has a hydrophobic nanoparticle surface and an organic co-solvent, which can greatly enhance the transmission across the cell barrier.
  • Example 2 Intracellular targeting of cS-bQDs-Tat in living cells
  • cS-bQDs-Tat shows surprising intracellular targeting in living cells.
  • ⁇ 30 independent experiments by 3 independent researchers, ⁇ 200 cells were studied by fluorescence imaging in each experiment, among ⁇ 95% of the cells studied (about 5% of the studied cells were not Internalized QD), cS-bQDs-Tat reliably produces about 95% of nuclear targeting specificity (defined as the percentage of QD in the nucleus relative to the total QD in the cell) (Figure 3a includes more targeting Confocal image). This result is consistent with the result obtained by separating the cell nucleus from the cell suspension and then measuring the fluorescence spectrum.
  • cS-bQDs-Tat In the nucleus, cS-bQDs-Tat is often found to accumulate in the nucleolus, which is a specific intranuclear region that undergoes ribosomal biogenesis. This result indicates that cS-bQD-Tat can also overcome dyskinesia in the nucleus and achieve intranuclear targeting ( Figure 3b, including more confocal images of nucleolar targeting).
  • cS-bQDs-cysteamine ie, SDot without biomolecule Tat peptide
  • the nuclear function of cS-bQDs-Tat requires the biological function of the Tat peptide.
  • traditional Tat peptide coupled water-soluble QDs did not enter the nucleus, probably because they were trapped in intracellular vesicles (due to the large size of QDs compared to small molecules), this discovery supports new nanoparticle The importance of design for intracellular targeting.
  • the water-soluble QDs-Tat used is prepared by dissolving bQD in phospholipid-PEG micelles; in QDs-Tat, the number of Tat peptides conjugated to each QD and the number of peptides conjugated to each cS-bQDs-Tat the same.
  • Third, applying the commonly used vesicle-damaging drug chloroquine (50 ⁇ M, or 16 ⁇ g / mL) did not significantly improve the nuclear targeting of QDs-Tat, but it produced considerable cytotoxicity.
  • cS-bQD-Tat The nuclear targeting ability of cS-bQD-Tat was confirmed in all four cell types tested [HeLa (cervical cancer), MCF-7 (breast cancer), NIH 3T3 (fibroblast) and Hep G2 (liver cancer)] (Fig. 3d). All four low-concentration organic co-solvents used in cS-bQD-Tat (DMF, acetone, ethanol and DMSO; the concentration of organic co-solvent used was 1%; the concentration of nanoparticles used was 10%) caused little cytotoxicity (Figure 3e). SDot targeted delivery technology is convenient and scalable because the operation mainly involves simple mixing and culturing, and targeted delivery can be performed on millions of cells simultaneously.
  • Calcein is a water-soluble small molecule fluorescent dye that self-quenches at a concentration above a certain threshold and is commonly used as an indicator of the integrity of lipid vesicles.
  • nanoparticles can effectively and reliably escape intracellular vesicles without damaging the vesicle membrane (ie, efficient and non-invasive escape process), while small molecules (calcein, hydrophilic) , which is two orders of magnitude smaller than nanoparticles, unless the vesicle ruptures, it cannot effectively pass through the intracellular vesicle membrane.
  • Example 4 Single particle tracking and correlation function analysis of cS-bQDs-Tat cell transport dynamics
  • This example uses cS-bQDs-Tat cell transport as a model system, which demonstrates the comprehensive use of single particle tracking, correlation function analysis and rotating disc confocal microscope (iSPT-pCF-SDCM) to study the dynamic transport process of organisms.
  • Single particle tracking and pCF analysis were performed on the same sample at the same observation stage of the rotating disk confocal microscope. This combined analysis method provides supplementary information about the dynamic transmission process.
  • ⁇ 2,500 QD trajectories were obtained in ⁇ 30 cells using single particle tracking (Figure 5a shows a representative trajectory).
  • the trajectory of the fluorescent object is formed by connecting its positions at different time points, which are determined by using a commercial rotating disk confocal microscope. For each trajectory, calculate the quantitative relationship between the mean square displacement (MSD) and duration ( ⁇ t), classify the movement based on the MSD- ⁇ t relationship (including directional movement, normal diffusion, abnormal diffusion, and angular diffusion), and determine the corresponding movement The characteristic constant of the model.
  • MSD mean square displacement
  • ⁇ t duration
  • the study of directional motion in this example reveals an interesting distribution of velocity values.
  • the velocity distribution exhibits multiple Gaussian peaks, with peak multiples approximately (220nm / s, ⁇ 1x; 620nm / s, ⁇ 3x; 820nm / s, ⁇ 4x; 980nm / s, ⁇ 5x; 1,300nm / s, ⁇ 6 ⁇ , Figure 5b). This indicates that multiple molecular motors coordinate to promote directional motion.
  • dynein may be the main motor protein involved.
  • the peak value of the lowest speed is 220 nm / s, which is very consistent with the in vitro speed value of the disclosed dynein motor ( Figure 5b).
  • many directional motion trajectories were also found in the nucleus area, and the velocity distribution showed multiple peak-cut peaks approaching (the peak of the lowest velocity was 270 nm / s, Figure 5c).
  • Double correlation function (pCF) analysis was used to study the movement of vesicle escape in QD cells.
  • a cross-correlation function of the fluorescence intensity (time function) of a pair of location points, one inside the vesicle and the other outside the vesicle, showing the maximum peak at the time value corresponding to the transit time of the vesicle escape Direction of the line at each location point) ( Figure 5d).
  • a commercial rotating disc confocal microscope was used to measure the fluorescence intensity at different time points in the cells, and then the pCF was calculated using a written computer program.
  • Example 5 Enhancement of drug and macromolecule delivery with cS-bQDs-Tat
  • DOS-loaded cS-bQDs-Tat (formulation name SDots-Tat-DOX) only needs to act at a DOX dose of 0.1 ⁇ g / mL for 4 hours.
  • the same dose of free DOX preparation needs> 12 hours, or a higher dose (5 ⁇ g / mL) is used at the same time; the same dose of conventional TAT peptide conjugate loaded with DOX
  • the combined water-soluble QD (formulation name QDs-Tat-DOX) requires> 12 hours, or a higher dose (5 ⁇ g / mL) is used within the same time; the same DOX dose is the same as the conventional water-soluble QD loaded with DOX (without Tat peptide) (Formulation name QDs-DOX) needs >> 12h, or use a higher dose (>> 10 ⁇ g / mL) at the same time.
  • cS-bQDs-Tat can promote intracellular targeted delivery of biomacromolecules. Disorders of transport captured by intracellular vesicles are considered to be the main challenges of macromolecular-based therapies. It is well known that macromolecules [such as proteins and small interfering RNA (siRNA) delivered by Tat peptides] are almost completely (estimated> 99%) trapped in intracellular vesicles and therefore cannot enter the cytoplasm and organelles outside the vesicles (including the nucleus) ). The cS-bQDs-Tat was conjugated to the model protein ovalbumin (ova, MW45kDa), and cell delivery and imaging experiments were performed.
  • siRNA small interfering RNA
  • ovalbumin conjugated to cS-bQDs-Tat showed greatly enhanced delivery to the nucleus compared to the ovalbumin conjugated with Tat peptide (labeled with a fluorescent dye for imaging) ( ⁇ 25%, the control sample was almost For zero-core delivery, Figure 6b). Since the combination with SDot should increase the overall size of the transported cargo (twice or more), this result shows that the combination of hydrophobic nanoparticle surface and mixed solvent has such a significant effect on the biotransport enhancement that even the size of the cargo is sharp When increased, it can also greatly improve the target delivery of goods.
  • Example 6 Transdermal administration of cS-bQDs-Tat and its inhibitory effect on the growth of subcutaneous tumors
  • the SDot formulation used was the same as in Example 5.
  • the anti-cancer drug is doxorubicin (DOxorubicin, DOX for short), which kills cells by interacting with DNA in the nucleus.
  • the 100 nmol / L sample was directly applied to the skin outside the tumor by means of a syringe (this process takes about 2-5 minutes). After 24 hours, the nude mice were euthanized, and the tumor tissue was dissected out. Using the fluorescence emitted by quantum dots and DOX, observe the tumor tissue sections with a fluorescence microscope to investigate the transmission ability of penetrating the tumor.
  • step one When the tumor grows to 125 mm 3 , start the drug administration experiment. Apply the sample of 100 nmol / L directly to the skin outside the tumor by means of a syringe (this process takes about 2-5 minutes). The drug is administered once, and the drug is administered 5 times in total. The size of the tumor tissue is measured before each administration, and statistics are made.
  • SDot-Tat-DOX can enter the cell well and Efficiently targeted into the nucleus.
  • a KB tumor-bearing model was constructed to study the delivery of SDots-Tat-DOX penetrating nude mouse skin in the KB tumor model. Traditional water-dispersed quantum dots were loaded with DOX as a comparison sample.
  • Figure 1 shows the biological transmission results of SDot-Tat-DOX.
  • the tumor is divided into three sections from the position close to the skin as the tip, and sliced. Then use a laser confocal microscope to stain the tissues obtained in the three sections and observe.
  • Figure 1a is a picture taken by laser confocal of tumor tissue close to the skin. From the figure, it can be seen that there are many SDot-Tat-DOX entering the tumor tissue through the skin.
  • SDot-Tat fluorescence and DOX fluorescence are highly co-located (SDot-Tat fluorescence is green, DOX fluorescence is red, and the colocalized part will be yellow after merging), and many are distributed inside the nucleus.
  • Figure 1b is the data obtained from the tissue slice in the middle region of the tumor, showing that there are many SDot-Tat-DOX, and its distribution position is basically outside the cell nucleus.
  • Figure 1c is a picture of the tumor tissue farthest from the skin, and less SDot-Tat-DOX can be found.
  • QD-Tat-DOX Lipid-PEG (QD-DOX) -Tat
  • QD-DOX Lipid-PEG
  • FIG. 2 The results of the biotransmission are shown in FIG. 2.
  • QD-Tat-DOX can also enter the tumor tissue through the skin, but the penetration ability in the tumor is much lower than that in Figure 1 SDot-Tat-DOX.
  • Figures 2a, 2b, and 2c give the results of the tumor close to the skin part, the middle tumor part, and the tumor away from the skin part, respectively.
  • Figure 3 shows the results of comparative experiments on the ability to inhibit the growth of subcutaneous tumors. The medicine is still applied on the skin. According to the statistical results of tumor volume after administration, it can be found that SDot-Tat-DOX can greatly improve the ability to suppress tumor growth compared to free DOX and Lipid-PEG (QD-DOX) -Tat.

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Abstract

一种新型纳米生物探针及其在细胞内靶向递送生物活性分子的应用。具体地，涉及一种混合溶剂-裸露的疏水性QD-生物分子(cS-bQD-BM，或'SDot')。SDots以细胞核为模型靶标显示出非凡的细胞内靶向性能，包括近乎完美的特异性、优异的效率和重现性、高通量能力、最小化的毒性、易操作性，以及卓越的光学性质和胶体稳定性。

Description

疏水性纳米生物探针 技术领域
本发明涉及生物材料和生物医药领域，具体地，涉及一种纳米生物探针及其在细胞内，特别是活细胞内靶向递送生物活性分子的应用。
背景技术
荧光探针作为可视化的信号源，在活细胞成像中扮演着关键角色。与其他两类主要的荧光探针，即小分子荧光染料和遗传编码的荧光蛋白相比，量子点(QD，半导体材料的纳米级晶体)具有几种明显优越或独特的性质，尤其是光学性质，包括非凡的荧光强度和光稳定性、尺寸和组成可调的具有窄峰宽的荧光发射峰可通过单个光源激发多重成像，并且能够用作相关光学和电子显微镜的成像探针。然而，与荧光染料和荧光蛋白不同，QD目前在特定亚细胞结构或亚细胞分子的活细胞成像中的应用通常仅限于细胞膜上实体的成像(例如细胞表面受体)。所以到目前为止，将QD靶向活细胞内部的特定实体仅取得了有限的成功。将QD以高度特异性(理想接近100％)、高效、稳健、可扩展、安全和方便的方式实现细胞内靶向递送是被长期追求，但目前仍然难以实现的目标。
实现这一长期追求的目标需要以有效且微创的方式克服以下所有细胞转运障碍：(1)细胞膜，(2)细胞内囊泡捕获(如果细胞摄取机制是内吞作用)，(3)拥挤的细胞质，(4)非特异性结合，(5)细胞器膜(如果目标位于细胞器内)，和(6)细胞器内的障碍(如果目标是细胞器内的特定位置，图1)。克服所有这些细胞转运障碍对于各种生物学上重要的材料(例如小分子药物，大分子和纳米颗粒)，尤其是QDs的靶向递送是具有挑战性的。典型的水溶性QD是由用于产生荧光的裸露疏水QD和用于水溶解和维持荧光的聚合物涂层组成，直径大于15nm。这个尺寸比大多数细胞内蛋白质(直径几纳米)大得多，并且被认为在拥挤的具有约80nm孔径的凝胶状结构的细胞质中引起很大的运动障碍。此外，即使没有增加聚合物涂层的直径，裸露的疏水性QD的大小接近于蛋白质的大小，并且细胞内囊泡捕获仍然是一个难以克服的障碍：据估计，通常<1％的内吞蛋白质(和其他类型的大分子)可以逃避细胞内囊泡捕获并到达细胞质。此外，通过物理破坏细胞膜的方法(例如显微注射和电穿孔)绕过内吞作用通常需要额外的仪器，使用仪器需要长时间的技术练习和费力的操作，导致显著的细胞死亡，并产生不可重复的结果。
因此，本领域迫切需要开发一种能够在生物体的组织、器官以及细胞内进行高特异性定位、毒性小、易操作的纳米生物探针。
发明内容
本发明的目的就是提供一种能够在生物体的组织、器官以及细胞内进行高特异性定位、毒性小、易操作的纳米生物探针。
在本发明的第一方面，提供了一种纳米生物探针，所述纳米生物探针的从内向外的结构如式I所示，
A-S-L-B n    (式I)
式中，
A-S为纳米粒子，其中，S为纳米粒子的疏水性表面；
B为生物分子；
L为可选的将B偶联至量子点表面的连接部分；
n为偶联至量子点表面的生物分子的个数，为大于等于1的正整数。
在另一优选例中，所述纳米生物探针经过混合溶剂处理。
在另一优选例中，所述经过混合溶剂处理的纳米探针在水性环境中的溶解度为≥1nM，较佳地为≥5nM，更佳地为≥50nM。
在另一优选例中，所述混合溶剂包括至少一种有机溶剂和至少一种非有机溶剂。
在另一优选例中，所述混合溶剂中，有机溶剂选自下组：二甲基甲酰胺(DMF)、丙酮、乙醇、二甲基亚砜(DMSO)、四氢呋喃(THF)、乙酸乙酯、乙腈、甲酸、丁醇、二甲基甲醇、丙醇、乙酸，或其组合。
在另一优选例中，所述混合溶剂中，有机溶剂为二甲基甲酰胺(DMF)。
在另一优选例中，所述混合溶剂中，有机溶剂的浓度为0.01v/v％至50v/v％，较佳地为0.1v/v％至20v/v％，更佳地为0.5v/v％至5v/v％。
在另一优选例中，所述混合溶剂中，非有机溶剂选自水、磷酸盐缓冲液、细胞培养液、血液等，或其组合。
在另一优选例中，所述A选自下组：磁性纳米粒子、金属纳米粒子、半导体纳米粒子(量子点)、碳纳米粒子、聚合物纳米粒子、脂类纳米粒子中，或其组合。
在另一优选例中，所述量子点包括：硒化镉量子点、硫化镉量子点、硫化锌量子点、硒化锌量子点、硫化银量子点、硒化银量子点、碲化镉量子点、碲化锌量子点、钙钛矿量子点，或其组合。
在另一优选例中，所述S为纳米粒子本身的疏水表面或包裹纳米粒子的疏水性配位体层表面。
在另一优选例中，所述配位体选自下组：三正辛基氧膦、油酸、硬脂酸、油胺，或其组合。
在另一优选例中，所述B包括小分子或生物大分子。
在另一优选例中，所述生物大分子包括：蛋白质、核酸、多糖、多肽，或其组合。
在另一优选例中，所述B为带正电荷和/或负电荷的极性分子。
在另一优选例中，所述B的分子量为1至500kD，较佳地为1至200kD，更佳地为1至80kD。
在另一优选例中，所述n为1至1000的正整数，较佳地为1至10的正整数，更佳地为1至3的正整数。
在另一优选例中，所述B为Tat肽。
在另一优选例中，所述Tat肽的序列为SEQ ID NO:1。
在另一优选例中，所述Tat肽具有细胞穿透和细胞核靶向的生物学功能。
在另一优选例中，所述纳米生物探针的结构中可不包括式I中的L。
在另一优选例中，所述偶联的形式包括：肽键、氢键、电荷作用、抗生物素蛋白-生物素结合、核酸配对作用或其组合。
在另一优选例中，所述偶联通过配体交换和生物共轭实现。
在另一优选例中，所述偶联通过使用偶联剂完成。
在另一优选例中，所述偶联剂包括：1-半乙胺-3-(3-二甲基氨基丙基)碳二亚胺(EDC)、抗生物素蛋白(avidin)-生物素(biotin)、核酸配对或其组合。
在另一优选例中，所述B可以通过共价偶联和/或非共价偶联至所述A-S表面。
在另一优选例中，所述共价偶联包括：肽键生成反应、生物正交反应，或其组合。
在另一优选例中，所述非共价偶联选自下组：电荷作用、氢键、范德华力、 疏水性作用、抗体抗原作用、抗生物素蛋白-生物素结合、核酸配对作用，或其组合。
在另一优选例中，所述纳米探针的直径为1至200nm，较佳地为1至20nm，更佳地1至10nm。
在另一优选例中，所述纳米探针不包括全亲水性聚合物涂层。
在另一优选例中，所述量子点纳米探针在密闭容器中，4℃下可稳定存在≥3天，较佳地≥5天，更佳地≥7天。
在另一优选例中，所述量子点的激发光波长为100至1500nm，较佳地为200至1000nm，更佳地为250至600nm。
在另一优选例中，所述量子点的发射光波长为400至2500nm，较佳地为400至2000nm，更佳地为450至1500nm。
在本发明的第二方面，提供了一种纳米生物探针产品，包括：
(a)第一容器，以及位于第一容器中的如本发明第一方面所述的纳米生物探针；
(b)第二容器以及位于第二容器中的混合溶剂。
在另一优选例中，所述第一容器和第二容器可以是相同的或不同的容器。
在本发明的第三方面，提供了一种如本发明第一方面所述的纳米生物探针或本发明第二方面所述的纳米生物探针产品的制法，包括步骤：
(i)合成纳米粒子，所述纳米粒子表面为疏水性表面或包裹疏水性配位体层；
(ii)将生物分子偶联至纳米粒子表面，获得疏水性纳米粒子-生物分子偶联物；
(iii)使用混合溶剂对步骤(ii)所获得的疏水性纳米粒子-生物分子偶联物进行处理，获得所述纳米生物探针。
在本发明的第四方面，提供了一种生物活性分子的细胞内递送方法，包括将所述生物活性分子偶联至本发明第一方面所述的纳米生物探针，并与含有细胞的培养液混合。
在另一优选例中，所述方法是在体外进行的。
在另一优选例中，所述方法是非诊断性和非治疗性的。
在另一优选例中，所述细胞包括：非癌细胞或癌细胞。
在另一优选例中，所述癌细胞包括：宫颈癌细胞、乳腺癌细胞、肝癌细胞、肺癌细胞、胃癌细胞、前列腺癌，或其组合。
在另一优选例中，所述生物活性分子包括：与DNA结合的药物、与RNA结合的药物、与细胞内蛋白质结合的药物，与细胞内细胞骨架结合的药物、在细胞内线粒体起作用的药物、与细胞内糖分子结合的药物、与细胞内脂类分子结合的药物、或其组合。
在另一优选例中，所述生物活性分子包括：小分子和生物大分子。
在另一优选例中，所述生物活性分子为抗癌药物。
在另一优选例中，所述抗癌药物包括多柔比星(doxorubicin)、紫杉醇(paclitaxel)、多西他赛(docetaxel)、顺铂(cisplatin)，或其组合。
在另一优选例中，所述生物大分子包括：蛋白质、核酸、多糖、多肽，或其组合。
在另一优选例中，所述细胞内递送为活细胞内递送。
在另一优选例中，所述递送为靶向递送。
在另一优选例中，所述递送为细胞核靶向递送。
在另一优选例中，所述递送方法中，实现靶向递送的药物的百分比为≥20％，较佳地为≥70％，更佳地为≥90％。
在本发明的第五方面，提供了一种如本发明第一方面所述纳米探针的用途，用于制备一制剂，所述制剂用于向生物体内递送生物活性分子。
在另一优选例中，所述的生物体包括生物的组织、器官或细胞。
在另一优选例中，所述的生物体包括患癌组织、患癌器官或癌细胞。
在另一优选例中，所述组织为皮肤组织。
在本发明的第六方面，提供了一种本发明第一方面所述纳米生物探针的用途，用于制备一制剂或药物组合物，所述制剂或药物组合物用于生物体内靶向定位。
在另一优选例中，所述定位是体外或离体进行的。
在另一优选例中，所述制剂或药物组合物具有穿越生物体内的生物传输屏 障的功能。
在另一优选例中，所述生物传输屏障包括组织屏障、器官屏障、细胞屏障、细胞器屏障。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了用于靶向细胞内递送QD的cS-bQD-BM(‘SDot’)的设计。
图2显示了cS-bQD-Tat的制备和物理化学表征。
(a)cS-bQD-Tat的制备示意图。
(b)通过添加半胱胺和Tat肽的表面电荷(zeta电位)变化证实成功的表面修饰。
(c)通过动态光散射(DLS)测量的直径仅在添加半胱胺和Tat肽时分别增加～0.05nm和～0.5nm。
(d)荧光光谱表征显示cS-bQDs-Tat具有良好的荧光性质(与bQD相比荧光强度仅降低约20％)和良好的胶体稳定性(5天后水性分散体的荧光几乎没有变化)。
图3显示了cS-bQDs-Tat的细胞内靶向。
(a)具有cS-bQDs-Tat的HeLa细胞的鸟瞰图光学显微镜图像显示cS-bQD-Tat成功靶向细胞核，具有接近完美的靶向特异性。使用的光学显微镜物镜的放大倍数是20倍。该图像是显示细胞的明视场显微镜图像和显示细胞核(由细胞核染料Hoechst 33342，红色染色)和cS-bQDs-Tat(绿色)的荧光显微镜图像的复合物。细胞核和cS-bQDs-Tat的共定位导致复合色黄色。比例尺，60μm。
(b)具有cS-bQDs-Tat的HeLa细胞的特写镜头光学显微镜图像显示cS-bQD-Tat成功靶向细胞核，显示在细胞核内几乎所有cS-bQDs-Tat与特定结构核仁共定位。使用的光学显微镜物镜的放大倍数为60倍。比例尺，20μm。
(c)在不同递送时间点的光学显微镜图像显示cS-bQDs-Tat的靶向细胞内递送是快速的。15分钟、1小时、4小时和8小时的图像是明视场(显示细胞)和荧光(显示cS-bQD-Tat)显微镜图像的合成图像。12小时的图像是荧光图像，显示细胞核(蓝色)和cS-bQDs-Tat(绿色)的共定位。细胞核和cS-bQDs-Tat的共定位导致复合颜色浅蓝色。比例尺，20μm。
(d)cS-bQD-Tat的细胞内靶向在所有测试的四种不同细胞系中都是成功的。误差棒，平均值±s.e.m，n＝200个细胞。
(e)cS-bQD-Tat(SDot-Tat)的细胞毒性很小。误差线，平均值±s.e.m，n＝5。
图4显示了cS-bQDs-Tat细胞转运的机制研究(a-c)证实cS-bQD-Tat优良的细胞内靶向是由三个设计参数引起的。
(a)较大的纳米颗粒疏水表面覆盖率导致更好的靶向效应。
(b)更高的有机溶剂浓度导致更好的靶向效果。
(c)较小的纳米粒子尺寸导致更好的定位效果。
(d)细胞摄取cS-bQDs-Tat的胞吞作用抑制研究。使用的两种胞吞作用抑制剂，即低温和细胞松弛素D均未完全阻断cS-bQDs-Tat的细胞摄取。相反，对于常规的水溶性QDs-Tat(表面覆盖有亲水性聚乙二醇的QDs-Tat)，细胞摄取被所使用的两种内吞作用抑制剂中的每一种完全阻断。比例尺，20μm。
(e)cS-bQDs-Tat的细胞膜渗漏研究。对于测试的所有四种不同cS-bQD-Tat制剂，来自HeLa细胞的乳酸脱氢酶释放<10％，并且与仅具有HeLa细胞的对照样品的水平相似，表明在cS-bQD-Tat的递送过程期间细胞膜是完整的。误差棒，平均值±s.e.m，n＝5.
(f)使用亲脂性囊泡染料DiR的cS-bQDs-Tat的细胞内囊泡共定位研究。DiR呈绿色；cS-bQD-Tat为红色。左一至左四图显示了在递送过程的各个时间点的光学显微镜图像。右图显示了共定位(通过Pearson相关系数测量)随时间变化的量化结果。比例尺，20μm。
(g)使用荧光示踪剂钙黄绿素的细胞内囊泡膜完整性研究。在囊泡内部，钙黄绿素的荧光被淬灭，荧光弱且呈点状(左上图)；当囊泡破裂时，钙黄绿素被泄漏到细胞质中，其荧光弥漫而强烈(右上图)。在cS-bQDs-Tat(红色)的递送开始时，钙黄绿素荧光(绿色)是点状和弱的，并且在cS-bQDs-Tat和钙黄绿素之间存在部分共定位(左下图)；在递送结束时，钙黄绿素荧光保持点状和弱，并且cS-bQDs-Tat和钙黄绿素之间没有共定位(右下图)。比例尺，10μm。
(h)混合溶剂对与cS-bQDs-Tat结合的非特异性蛋白质的影响的体外研究。误差棒，平均值±s.e.m，n＝3.。***P<0.001。*P<0.05(学生t检验)。
图5显示了cS-bQDs-Tat的细胞转运的单粒子追踪(SPT)和对相关函数(pCF)分析。(a-c)是SPT结果。(d-f)是pCF结果。
(a)cS-bQDs-Tat的代表性轨迹。轨迹以红色显示；细胞核以蓝色显示；绘制线以显示细胞和细胞核的外周。比例尺，10μm。
(b)细胞质中定向运动速度的分布。插图显示了这种运动的代表性轨迹(红色)。
(c)核区定向运动速度的分布。插图显示了这种运动的代表性轨迹(红色)。
(d)用于分析逃逸细胞内囊泡的粒子的通过时间的代表性pCF曲线。插图显示了纳米颗粒的囊泡逃逸的示意图。
(e)在1％有机溶剂(DMF)存在下，SDot-Tat(cS-bQD-Tat)的囊泡逃逸通过时间明显短于QD-Tat(常规水溶性QD-Tat)。常规的水溶性QD-Tat(在没有有机溶剂的情况下)显示几乎没有囊泡逸出。***P<0.001(学生t检验)。
(f)cS-bQD-Tat的囊泡逃逸传播时间是各向同性的。对于研究的每个囊泡，测量在两个不同(垂直)方向上的囊泡逃逸传播时间的差异。
图6显示了用cS-bQDs-Tat(SDots-Tat)增强靶向细胞内递送药物和大分子的结果。
(a)以多柔比星(DOX)为药物模型杀死癌细胞(HeLa细胞)。使用SDots-Tat(配方SDots-Tat-DOX)与常规的可溶性QD(制剂QDs-DOX)，游离的DOX以及常规水溶性QDs-Tat(制剂QDs-Tat-DOX)相比，明显增强了靶向细胞内递送多柔比星及杀死癌细胞的效率。误差棒，平均值±s.e.m，n＝3。
(b)以卵清蛋白(ova)为细胞内大分子递送模型。与使用Tat肽(配方Tat-ova-dye，dye为小分子染料TAMRA用于荧光成像)相比，使用SDots-Tat(配方Tat-ova-SDots)增强了靶向细胞内递送卵清蛋白。虽然使用SDots-Tat增加了整体尺寸，但疏水性纳米级表面和混合溶剂的组合仍然增强了卵清蛋白向细胞核的靶向递送。配方Tat-dye(无ova，其尺寸比ova小得多)用作阳性对照。在Tat-ova-SDots和Tat-ova-dye的递送中使用了相同量的卵清蛋白。误差棒，平均值±s.e.m，n＝100-200个细胞。
图7显示了SDot-Tat-DOX在裸鼠皮肤和肿瘤模型中的渗透和递送效果。其中，显示了样品在不同位置的肿瘤组织中的分布:(a)靠近皮肤，(b)中间位置，(c)底部位置。标尺20μm
图8显示了QD-Tat-DOX在裸鼠皮肤和肿瘤模型中的渗透和递送效果。其中，显示了样品在不同位置的肿瘤组织中的分布:(a)靠近皮肤，(b)中间位置，(c)底部位置。标尺20μm
图9显示了给药后肿瘤体积的变化。
具体实施方式
本发明人经过广泛而深入的研究，通过大量的筛选，开发出一种新型的量子点纳米探针。具体地，本发明人用双功能小分子配体半胱胺取代一小部分裸露的疏水性量子点的疏水性表面，并通过1-半乙胺-3-(3-二甲基氨基丙基)碳二亚胺(EDC)将生物分子Tat肽与量子点表面氨基进行偶联，然后施用少量有机助溶剂DMF对所得量子点纳米探针进行处理。
实验表明，所述的量子点纳米探针能够克服细胞转运障碍，并在活细胞中实现了高度特异性、高效、稳定、可扩展、安全和方便的细胞内靶向，并且能够实现高效和精准的细胞内药物递送。
并且，所述的量子点纳米探针还能在活体动物中实现穿越活体动物中组织器官层次的生物传输屏障。例如，对裸鼠进行透皮给药的实验结果显示，本发明的量子点纳米探针能够将药物递送到皮下肿瘤更深处，并且具有显著的优于对照组的抑制肿瘤生长的能力。
在此基础上完成了本发明。
术语
纳米生物探针
如本文所用，“纳米生物探针”、“纳米探针”、“bQDs-BM”、“QD纳米探针”、“QD探针”等可互换使用，是指本发明所述的从内向外具有式I所示的结构的纳米探针，
A-S-L-B n    (式I)
式中，
A-S为纳米粒子
S为纳米粒子表面，其中，S为纳米粒子的疏水性表面；
B为生物分子；
L为可选地将B偶联至量子点表面的连接部分；
n为偶联至纳米粒子表面的生物分子的个数，为大于等于1的正整数。
在一个优选的实施方式中，所述A选自下组：磁性纳米粒子、金属纳米粒子、半导体纳米粒子(量子点)、碳纳米粒子、聚合物纳米粒子、脂类纳米粒子等，或其组合。在一个优选的实施方式中，所述量子点包括：硒化镉量子点、硫化镉量子 点、硫化锌量子点、硒化锌量子点、硫化银量子点、硒化银量子点、碲化镉量子点、碲化锌量子点、钙钛矿量子点，或其组合。
在一个优选的实施方式中，所述S为纳米粒子本身的疏水表面或包裹纳米粒子的疏水性配位体层表面。在本发明中，配位体的选择很广，只要该分子一端与纳米粒子用某种吸引力结合，另一端是疏水性的就可以。在一个优选的实施方式中，所述配位体选自下组：三正辛基氧膦、油酸、硬脂酸、油胺等，或其组合。
在本发明中，所述的生物分子B可包括：蛋白质、核酸、多糖、多肽等，也可包括小分子。优选地，所述B为带正电荷和/或负电荷的极性分子。
在一个优选的实施方式中，所述B包括为Tat肽，所述的Tat肽的序列为YGRKKRRQRRR(SEQ ID NO:1)，其具有细胞穿透和细胞核靶向的生物学功能。
本发明所述的纳米生物探针克服了细胞转运障碍[如细胞膜、细胞内囊泡捕获(如果细胞摄取机制是内吞作用)、拥挤的细胞质、非特异性蛋白质结合、细胞器膜(如果目标位于细胞器内)和细胞器内的障碍]，并在活细胞中实现了高度特异性，高效，稳定，可扩展，安全和方便的细胞内QD靶向。
本发明的设计(或称SDot-BM，或SDot)涉及用生物分子(通过配体交换，然后进行生物共轭)改变裸露疏水性QD的一小部分包面(例如50％，优选的40％，优选的30％，更优选的20％，更优选的10％)的性质，并使用少量有机助溶剂促进其在水性环境中的分散(图1)。
本发明设计的灵感来自于本发明人最近的发现，即裸露的疏水性QD经过少量有机助溶剂的处理后可以直接渗透生物膜而不会破坏膜的完整性。如本发明实施例部分的实验结果所示，这一新型QD探针集成了多种有助于克服细胞运输障碍的特征，包括：(1)大疏水表面协助穿越生物膜；(2)有机助溶剂促进生物穿越膜，并减少非特异性蛋白质结合；(3)由于消除使用聚合物涂层，形成超小QD探针，促进其在拥挤的生物环境中的运动，和(4)生物分子的天然生物功能可以被利用(图1)。
在一个优选的实施方式中，使用位于细胞内部深处，作为细胞指挥中心的细胞核作为细胞内靶标模型，该实施方式的实验结果证明了使用SDots的活细胞核靶向具有接近完美的特异性、优异的效率和重现性、高通量能力、最小化细 胞毒性、操作方便等优点。Tat肽具有靶向细胞核和穿透细胞的生物学功能，在SDot的设计中用作生物分子(BM)。
在另一个优选的实施方式中，展示了sDot可以用于活细胞中的组合成像，跟踪和对相关函数分析。在另一个优选的实施方式中，展示了sDot克服细胞转运障碍的独特能力可用于增强药物小分子和生物大分子的递送。
混合溶剂
如本文所用，“混合溶剂”、“本发明有机溶剂”、“有机溶剂”和“有机助溶剂”等可互换使用，是指能够使用其对本发明第一方面所述的纳米生物探针进行处理，从而增强本发明的纳米生物探针在水性溶液中的分散性，在生物膜中的运动能力和降低纳米生物探针与蛋白分子之间的非特异性结合的作用的溶剂。
由于各种疏水性纳米粒子在生物医学应用中均受到活细胞内部靶向传输效果差及生物相容性差的限制，因而在生物医学应用中均需通过物理(如亲水性聚合物包裹)或化学方法(如表面覆盖亲水性官能团)将纳米粒子的疏水表面改性为亲水表面以提高纳米粒子的活细胞内部靶向传输效果。目前生物医学领域内普遍认为未经亲水化处理的疏水性纳米粒子无法直接应用于活细胞内部靶向传输。
而本发明打破这一传统认知，通过混合溶剂处理的方式，实现了疏水性纳米粒子在生物医学领域特别是活细胞内靶向传输的直接应用，而本发明的原理并不受纳米粒子材料种类的限制，因此本发明适用于各种纳米粒子，包括磁性纳米粒子、金属纳米粒子、半导体纳米粒子(量子点)、碳纳米材料、聚合物纳米粒子、脂类纳米粒子等，或其组合。
在本发明中，混合溶剂包括至少一种有机溶剂和至少一种非有机溶剂，其中有机溶剂，包括：二甲基甲酰胺(DMF)，丙酮，乙醇、二甲基亚砜(DMSO)，四氢呋喃(THF)、乙酸乙酯、乙腈、甲酸、丁醇、二甲基甲醇、丙醇、乙酸，或其组合。
由于有机溶剂对细胞有一定的毒性，因此所述有机溶剂在混合溶剂中的浓度应控制在一定范围内，以使细胞毒性最小化。在一个优选的实施方式中，所述有机溶剂成分的浓度为0.01v/v％至50v/v％，较佳地为0.1v/v％至20v/v％，更佳地为0.5v/v％至5v/v％。
细胞内递送方法
本发明提供的纳米生物探针可用于生物活性分子的细胞内递送。具体地，所述生物活性分子可被偶联至纳米生物探针上，随着纳米生物探针在细胞内的精准定位，而被递送到细胞内的具体位置。
本发明的主要优点包括：
(1)明显改进纳米材料，赋予其高效、低创地穿越各种生物传输屏障(包括生物膜、拥挤复杂环境、非特异性吸附等)的能力。其中，所述的生物传输屏障包括组织屏障、器官屏障、细胞屏障、细胞器屏障。
(2)明显改进纳米材料在活细胞内部靶向输送的性能，包括特异性、效率、重复性、高通量、安全性、便易性等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，实施例所用的材料和试剂均为市售产品。
材料与方法
1.材料
氧化镉(CdO，99.99％)、六水合硝酸锌(Zn(NO3)2·6H2O，98％)、硫粉(S，99.98％)、硒粉(Se，100目，99.5％)、1-十八烯(ODE，90％)、硬脂酸(SA，95％)、三辛基膦(TOP，90％)、半胱胺(98％)、1-乙基-3(3-二甲基氨基丙基)碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS))和氯喹购自Aldrich。
N，N-二甲基甲酰胺(DMF，99.5％)、丙酮(99.5％)、乙醇(99.7％)、二甲基亚砜(DMSO，99％)和氯仿(99％)购自Sinopharm Chemical Reagent。
Tat肽(序列Ac-YGRKKRRQRRR)(SEQ ID NO:1)、Tat-TAMRA(即在SEQ  ID NO:1的第5、6个残基之间插入TAMRA染料)、Tat-卵清蛋白(即在序列SEQ ID NO:1的第5、6个残基之间插入卵清蛋白)和Tat-卵清蛋白-TAMRA(即在序列SEQ ID NO:1的第5、6个残基之间插入TAMRA染料，在第6、7个残基之间插入卵清蛋白)购自ChinaPeptides。
3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)、DiR、钙黄绿素、牛血清白蛋白(BSA)和细胞核分离试剂盒购自KeyGEN BioTECH。
Hoechst 33342购自ThermoFisher Scientific。
细胞松弛素D购自上海百利生物技术有限公司。
磷脂-PEG购自Avanti Lipids。
盐酸多柔比星(DOX，98％)购自Aladdin。
细胞系(HeLa，MCF-7，NIH 3T3和Hep G2细胞)及其培养基购自KeyGEN BioTECH。
2.cS-bQDs-Tat的制备
使用由Wenjin Zhang等人在文章《Scalable single-step noninjection synthesis of high-quality core/shell quantum dots with emission tunable from violet to near infrared》中披露的基于高温结晶的单步非注射生产方法合成表面覆盖有疏水性配位体三正辛基氧膦的疏水性QD[2012年发表于ACS Nano，6(12),pp11066-11073]。将疏水性QD(在DMF中)与半胱胺一起孵育0.5小时以交换QD的一小部分表面配体，使得所得QD在QD表面的一小部分上具有-NH 2(估计剩余的疏水表面覆盖率通常为90％)。使用EDC将Tat肽与QD表面上的-NH 2基团缀合，形成bQDs-Tat。将溶液(DMF，或其他有机溶剂，如丙酮，乙醇和DMSO)分散在水(或其他水性环境，如细胞培养基，有机溶剂与水的典型体积比为1:99)中，形成cS-bQDs-TAT。
3.cS-bQDs-Tat的物理化学表征
通过透射电子显微镜(TEM，JEM-200CX，JEOL)显现纳米颗粒的形态。通过傅立叶变换红外光谱(FTIR，Nicolet Nexus 470，Thermo)和X射线光电子能谱(XPS，ESCALAB 250，Thermo)分析纳米颗粒的化学表面性质。使用Malvern  Zetasizer Nano ZS360仪器测量纳米颗粒的动态光散射(DLS)直径(即，如果溶剂是水，则为流体动力学直径)和表面电荷(ζ电位)。使用荧光分光光度计(HITACHI F-4600)获得荧光光谱。
4.活细胞旋转盘共聚焦显微镜
HeLa细胞用于大多数细胞实验，并按照制造商的推荐，在37℃、5％CO 2下维持在补充有10％胎牛血清(FBS)的Dulbecco改良的鹰培养基(DMEM)中。使用的其他细胞系(MCF-7，NIH 3T3和Hep G2)也在制造商推荐的条件下培养。
使用活细胞旋转盘共聚焦成像系统进行活细胞成像研究，该系统由细胞孵育室(IX3W，Tokai Hit)，落射荧光显微镜(IX-83，Olympus)，旋转盘共聚焦系统和电子倍增电荷耦合器件(EMCCD)相机(Evolve 512，Photometrics)组成。首先将细胞以～35％汇合接种在玻璃底细胞培养皿上(玻璃底部厚度为0.17mm)(Nest，China)。在37℃和5％CO 2的条件下孵育18小时后，用cS-bQDs-Tat的分散体替换细胞培养基。除非另有说明，否则使用的cS-bQDs-Tat分散体的典型条件是总溶剂体积为1nM纳米颗粒，1％DMF作为水性细胞培养基中的有机助溶剂，纳米颗粒上90％疏水表面覆盖，每纳米颗粒1Tat肽，和荧光发射峰值559nm。与cS-bQDs-Tat孵育特定的持续时间(例如15分钟、1小时、4小时、8小时、12小时和24小时)后，用新鲜培养基洗涤细胞三次以除去在细胞外部或与细胞外表面结合的cS-bQD，并通过活细胞旋转盘共聚焦显微镜系统对细胞成像。为了对细胞核进行反染色，在成像之前(在细胞转运的特定时间点)，将荧光染料Hoechst 33342(蓝色荧光颜色，细胞培养基中5μM)与活细胞一起温育20分钟。使用MetaMorph和Image J软件进行图像处理和分析。
5.细胞内靶向的测量
将QD细胞内靶向细胞核测量为递送至细胞核的QD量与细胞中QD总量的比率。通过两种不同的方法进行测量，即共聚焦成像和细胞核分离，这产生了一致的结果。在共焦成像方法中，使用活细胞旋转盘共聚焦成像系统进行成像。通过测量由相应区域中的QD产生的荧光强度来量化细胞核或细胞中的QD的量。在细胞核分离方法中，用PBS洗涤细胞，并按照制造商的说明书(KeyGEN) 使用细胞核分离试剂盒将细胞核与其余细胞分离。通过使用荧光光谱仪测量细胞核部分(或细胞部分的其余部分)中的QD荧光强度以及QD的总量来确定细胞核(或细胞的其余部分)中的QD的量。通过总计核部分和其余细胞部分中QD的量来确定细胞中的QD。使用细胞核分离方法测量的QD量略低于通过共聚焦成像方法测量的QD量，这是由于在从细胞裂解物中分离细胞核的过程中QD的损失。
6.细胞活力分析(MTT测定)
为了评估特定制剂的细胞毒性，将HeLa细胞接种在96孔板(Corning Costar，China)上，密度为6000个细胞/孔。温育24小时后，将细胞培养基替换为用于测试的制剂(200μL/孔，在完全Dulbecco’s Modified Eagle’s培养基中)。在将细胞培养特定的持续时间(例如12小时或24小时)后，20μl 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)溶液(5mg/ml)向每个孔中加入180μlDMEM，并在37℃下孵育4小时。除去培养基后，将不溶性晶体溶于二甲基亚砜(DMSO,150μl/孔)中，并在ELISA读数器(RT-6000，Rayto，China)中以570nm的波长用分光光度法测量。通过测试孔的光密度除以对照孔的光密度计算与仅含有细胞培养基(除细胞外)的对照孔相比的相对细胞存活率(％)。所有样品一式五份运行。
7.内吞作用抑制研究
在物理抑制研究中，将HeLa细胞在4℃下在细胞培养基中孵育1小时，然后用纳米颗粒制剂(在细胞培养基中)替换培养基。在化学抑制研究中，用含有细胞松弛素D的细胞培养基(2μM)处理HeLa细胞30分钟，然后用纳米颗粒制剂(在细胞培养基中)替换培养基。在纳米颗粒递送1小时后，将细胞用磷酸盐缓冲溶液洗涤五次，然后使用活细胞旋转盘共聚焦显微镜系统进行成像。
8.细胞膜渗漏研究(LDH测定)
为了评估由特定制剂引起的细胞膜损伤，将HeLa细胞接种在96孔板(Corning Costar，China)上，密度为6000个细胞/孔。将细胞培养24小时后，用制剂代替细胞培养基进行测试(200μL/孔，完整的Dulbecco’s Modified Eagle’s  Medium)。将细胞分别温育12小时和24小时后，收集120μl/孔的样品上清液，并通过商业试剂盒(C0017，Beyotime，China)分析释放的乳酸脱氢酶(LDH)。将用LDH释放剂处理的细胞的LDH释放值设定为100％LDH释放(即，完整细胞中的总LDH量)，而将未处理细胞的LDH释放值设定为阴性对照。通过ELISA读数器(RT-6000，Rayto，China)在490nm(测试)和630nm(参考)的波长下测量光密度。相对LDH释放由完整细胞中LDH释放的总LDH比率定义。具有小于10％LDH释放的细胞样品被认为是具有完整细胞膜的细胞，遵循完善的标准。所有样品一式五份运行。
9.囊泡共定位研究
用亲脂性荧光染料DiR(5μM，与活细胞孵育30分钟)标记细胞内囊泡。在给定的时间点，用PBS洗涤细胞三次，然后用活细胞旋转盘共聚焦成像系统成像。通过使用Pearson相关系数来量化与荧光纳米颗粒的共定位。
10.囊泡膜完整性研究
通过使用荧光染料钙黄绿素研究细胞内囊泡的完整性。钙黄绿素分子(水溶性，250μM)通过胞吞作用(与细胞孵育30分钟)内化到活细胞的细胞内囊泡中。在该浓度下(具有完整的囊泡)，显示钙黄绿素的荧光自猝灭、点状和弱荧光。如果囊泡破裂，钙黄绿素分子释放到细胞质中，并显示出弥散和强烈的荧光。
11.体外蛋白结合研究
在室温下，在不同的有机助溶剂(DMF)浓度(包括0.5％，2％和5％，与磷酸盐缓冲液混合)下，将cS-bQDs-Tat(20nM)与牛血清白蛋白(BSA，15mg/mL)在水中混合。每个有机溶剂浓度使用三份样品。将混合物对水(具有各自的有机溶剂浓度)透析，截留分子量为200k道尔顿，以除去游离的BSA(未与纳米颗粒表面结合的BSA)。游离BSA的量通过UV-Vis吸收光谱仪测定，在280nm处相对于校准曲线测量(在透析96小时后值变得稳定后)。通过从最初添加到混合物中的BSA中减去游离BSA的量来计算与纳米颗粒表面结合的BSA的量。
12.单粒子跟踪
进行活细胞旋转盘共聚焦显微镜以收集图像(电影)用于单粒子追踪的数据分析(在与对相关函数的数据分析相同的细胞样品上，无需移动样品)。使用MATLAB计算机程序进行数据分析。简而言之，该算法在连续时间帧之间链接粒子的位置，然后将得到的轨道段链接成完整的轨迹。该算法给出了优化的轨迹，以最小化由高粒子密度，粒子运动异质性，暂时粒子消失以及粒子合并和分裂引起的影响。对于给定的轨迹，计算不同持续时间(Δt)的均方位移(MSD)。找到以下等式中MSD-Δt关系的最佳拟合导致运动模式和相应的特征常数：
方向运动：其中V是速度；
正常扩散：其中D是扩散系数；
异常扩散：其中α<1和D是扩散系数；
褶皱扩散：其中L2是围栅尺寸，D是扩散系数。
13.对相关函数(pCF)分析
进行活细胞旋转盘共聚焦显微镜以收集用于pCF数据分析的图像(电影)(在与单粒子跟踪的数据分析相同的细胞样品上，无需移动样品)。使用编写的MATLAB计算机程序进行数据分析。本发明的程序包含来自旋转磁盘扫描的数据。简言之，呈现不同位置和不同时间点的荧光强度，其中x坐标对应于沿图像视图上绘制的线(像素)的位置点，y坐标对应于时间。作为传播时间τ的函数的距离δr处的两个点的pCF由下式计算
以下等式：
Figure PCTCN2019118571-appb-000001
导出的pCF轮廓的最大值被确定为粒子行进给定距离所用的平均时间。为了研究在给定方向上的囊泡逃逸传播时间，在该方向上绘制线并计算pCF轮廓。对于线上的两个位置点，其中一个位于囊泡内，另一个位于囊泡外。
14.递送小分子药物多柔比星(DOX)
为了将DOX加载到纳米颗粒表面上，将纳米颗粒分散体(例如cS-bQDs-Tat)与DOX(溶解在有机溶剂中)在4℃下混合12小时。进行透析(截留分子量100k道 尔顿)以纯化载药纳米颗粒。通过UV-Vis吸收光谱法相对于校准曲线(480nm处的吸收)测量去除的游离药物。然后基于去除的游离药物的量来定量药物负载。发现所有纳米颗粒制剂的药物负载效率(成功负载的药物量相对于所添加药物的总量的百分比)>70％。估计每个颗粒加载的药物分子的量为70.研究每种不同制剂的一式三份样品。将载有药物的纳米颗粒分散体(在细胞培养基中)与HeLa细胞一起温育。在孵育开始后的不同时间点，通过MTT测定法测量癌细胞活力，并通过旋转盘共聚焦显微镜对纳米颗粒和DOX的分布进行成像。
15.蛋白质卵白蛋白(ova)的递送
通过使用EDC化学将Tat-ova与SDot缀合来制备Tat-ova-SDot。将三种不同的制剂(即Tat染料，Tat-ova-SDot和Tat-ova染料)与HeLa细胞一起孵育，并在孵育后的不同时间点通过旋转盘共聚焦显微镜对荧光探针的分布进行成像。开始。通过测量核区域中的荧光强度相对于整个细胞区域中的荧光强度(每种制剂100-200个细胞)来定量细胞核靶向。
实施例1：cS-bQDs-Tat的制备和物理化学表征
要制备cS-bQDs-Tat，首先在有机溶剂中用双功能小分子配体半胱胺取代一小部分(例如10％)裸露的疏水性QD(bQDs；表面配体三辛基膦，或简称为TOP)的表面(图2a)。检测的有机溶剂包括二甲基甲酰胺(DMF)、丙酮、乙醇和二甲基亚砜(DMSO)。DMF具有最佳的整体性能。然后通过1-半乙胺-3-(3-二甲基氨基丙基)碳二亚胺(EDC)化学方法与半胱胺缀合将Tat肽连接到QD表面，形成bQDs-Tat(图2a)。随后，将储存在有机溶剂中的bQDs-Tat在水中稀释，形成cS-bQDs-Tat(图2a)。通过zeta电位，傅里叶变换红外光谱(FTIR)和X射线光电子能谱(XPS)分析确认表面改性。随着半胱胺和Tat肽的加入，表面电荷正电位变得越来越高，这与TOP(每个分子中的阳离子官能团数量为零)，半胱胺(每个分子中一个阳离子官能团)和Tat肽(每个分子中有多个阳离子官能团)的分子结构一致。每个分子中有多个阳离子官能团)(图2b)。cS-bQDs-Tat的FTIR和XPS结果显示成功缀合的特征峰(酰胺键形成)。与bQD相比，添加半胱胺仅使通过动态光散射(DLS)测量的纳米颗粒直径增加～0.05nm(图2c)。与Tat的缀合在DLS 直径上增加另外约0.5nm(图2c)。通过透射电子显微镜(TEM)测量的纳米颗粒直径比DLS直径小约3nm。cS-bQDs-Tat在很大程度上维持裸露的疏水性QD的荧光特性(约20％荧光量子效率降低，图2d)。总体来说，减少由亲水分子或官能团取代的SDot系统中QD的表面增加了荧光量子效率。如不加入有机溶助溶剂补偿自然蒸发损耗，cS-bQD-Tat探针在密闭容器中在4℃下可维持胶体性稳定约5天，通过定期有机助溶剂再填充补偿有机助溶剂蒸发，其胶体稳定性可维持一个月以上(图2d)。SDots在水性环境中的溶解度可以轻易地达到50nM或更高。增加有机助溶剂的百分比会提高SDot水溶性。在实践中，SDot系统中使用的有机助溶剂的百分比低于3％(通常为1％)以使细胞毒性最小化。
SDot克服了当前两种主要的将疏水性QD转移到水性环境的方法的一些关键限制，这两种方法分别为使用聚合物或小分子覆盖整个QD表面。一方面，使用聚合物作为涂层通常在纳米颗粒直径上添加7-30nm，即使使用优化的聚合物结构直径也会增加至少1.5-2nm，因此会限制QD在拥挤空间中，如细胞内空间的生物转运。另一方面，用双功能小分子替换整个bQD表面上的原始配体经常导致欠佳的荧光量子效率和胶体稳定性。相比之下，SDot提供最小化的粒径(通过消除聚合物涂层的使用)和良好的荧光量子效率和胶体稳定性(通过保持bQD的大部分原始表面配体完整并使用有机助溶剂增加水溶性)。在两个对照样品上进行的实验，即1)bQDs上所有原始表面配体被半胱胺取代，和2)bQD一小部分(10％)表面配体被半胱胺取代并没有添加任何有机助溶剂，两组对照实验均显示出欠佳的荧光和胶体稳定性，因此确认需要小部分表面配体置换和有机助溶剂的共同存在以获得SDot的最佳物理化学性质。除了超小粒径外，SDot还具有疏水纳米粒子表面和有机助溶剂，可大大增强跨细胞屏障的传输。
实施例2：活细胞中cS-bQDs-Tat的细胞内靶向
cS-bQDs-Tat在活细胞中表现出惊人的细胞内靶向作用。在～30个独立实验中(由3个独立的研究人员进行)，每个实验中都通过荧光成像研究了～200个细胞，在研究的～95％的细胞中(约5％的研究细胞没有内化的QD)，cS-bQDs-Tat可靠地产生约95％的细胞核靶向特异性(定义为细胞核内QD量相对于细胞中QD总量的百分比)(图3a包括了更多的靶向共聚焦图像)。这个结果与通过从细胞 悬浮液中分离细胞核然后进行荧光光谱测量而获得的结果一致。在细胞核内，发现cS-bQDs-Tat经常在核仁中积累，核仁是进行核糖体生物发生的特定核内区域。该结果表明cS-bQD-Tat还可以克服细胞核内的运动障碍并实现细胞核内靶向(图3b，包括了更多核仁靶向的共聚焦图像)。
三项对照实验更多地阐明了cS-bQDs-Tat的细胞内靶向作用。首先，cS-bQDs-半胱胺(即没有生物分子Tat肽的SDot)不进入细胞核。这支持了cS-bQDs-Tat的细胞核靶向需要Tat肽的生物学功能的发现。其次，传统的Tat肽偶联水溶性QDs(QDs-Tat)没有进入细胞核，可能是因为它们被困在细胞内囊泡中(由于QDs与小分子相比体积庞大)，这个发现支持新纳米粒子设计对细胞内靶向的重要性。所用水溶性QDs-Tat是通过用磷脂-PEG胶束水溶化bQD制备而成；在QDs-Tat中，每个QD缀合的Tat肽数量与每个cS-bQDs-Tat上缀合的肽数量相同。第三，应用常用的囊泡破坏药物氯喹(50μM，或16μg/mL)没有显着改善QDs-Tat的细胞核靶向，却产生相当大的细胞毒性。这进一步支持了囊泡捕获是常规QD探针的细胞内靶向的主要障碍，并且表明本发明的QD探针设计在效力和非侵入性方面在客服囊泡捕获的能力显著优于氯喹。
cS-bQDs-Tat向细胞核的靶向递送过程快速且有效，表明其穿过细胞运输屏障的强大能力(图3c)。如在活HeLa细胞中的荧光共聚焦显微镜所示，在5-15分钟内，所有细胞中的几乎所有cS-bQDs-Tat都已经穿过细胞膜进入细胞(少数cS-bQDs-Tat仍然与细胞表面结合)；一些cS-bQDs-Tat已进入了细胞核(图3c)。到45-60分钟时，在一半以上的细胞中，一些cS-bQDs-Tat已进入细胞核(图3c)。到4-6小时，大约一半的cS-bQDs-Tat已进入细胞核(图3c)。到8-12小时，几乎所有cS-bQDs-Tat都进入了细胞核，并且通常在细胞核仁中积累(在时间依赖性研究中检查了100-200个细胞，图3c)。cS-bQDs-Tat的靶向核递送过程的速率与已有技术中的HeLa细胞的快速腺相关病毒感染(靶向细胞核)速率相当。cS-bQD-Tat的细胞核靶向能力在所有测试的四种细胞类型中得到证实[HeLa(宫颈癌)，MCF-7(乳腺癌)，NIH 3T3(成纤维细胞)和Hep G2(肝癌)](图.3d)。在cS-bQD-Tat中使用的所有四种低浓度有机助溶剂(DMF，丙酮，乙醇和DMSO；所用有机助溶剂浓度为1％；所用纳米颗粒浓度为10％)造成细胞毒性很小(图3e)。SDot靶向递送技术方便且可扩展，因为操作主要涉及简单的混合和培养，并且可以同时 在数百万个细胞上进行靶向递送。
实施例3：cS-bQDs-Tat的细胞运输机制研究
本实施例通过机理研究以获得对SDots的生物转运过程的了解。通过研究单独和系统地调整每个参数，证实了四个关键纳米探针参数(疏水纳米级表面，混合溶剂，小尺寸和生物分子)对SDot的细胞内靶向的重要性。如图4a，b，c所示，增加QD的疏水表面覆盖率(从0％到50％，到75％，到90％)，或增加水中助溶剂百分比(DMF从0.5％到1％))或减小的粒径(TEM直径从6.9nm到4.5nm，到3.4nm，到2.6nm)增强了cS-bQDs-Tat的核靶向性。将DMF百分比从1％进一步增加至1.5％并未显着改善靶向效应，表明混合溶剂给出的递送增强效果是可饱和的。另外，如前所述，来自纳米探针的Tat肽的缺失消除了细胞核靶向作用。
通过低温(物理抑制剂)或细胞松弛素D(化学抑制剂)阻断内吞作用仅部分阻止cS-bQDs-Tat进入细胞，而即便使用了细胞内吞作用抑制剂，常规Tat肽缀合的水溶性QD(QDs-Tat)在使用时也未表现出细胞内化(图4d)。这些结果表明，与QDs-Tat不同，cS-bQDs-Tat进入细胞部分通过独立于内吞作用的机制(直接穿透)。另外，这些结果对Tat肽介导的递送中的细胞摄取机制具有有趣的影响，即，改变货物的性质可导致Tat肽介导的递送中的细胞摄取机制的改变。通过乳酸脱氢酶(LDH)测定的细胞膜渗漏研究，对于所有测试的cS-bQD-Tat组成配方都给出<10％LDH释放结果(意味着没有显着的膜渗漏，类似于对照样品结果)，表明cS-bQDs-Tat进入细胞不会对细胞造成细胞膜损伤(使用的助溶剂浓度为1％；使用的纳米颗粒浓度为10nM)(图4e)。因此，部分cS-bQDs-Tat可以直接穿透细胞膜而不破坏膜的完整性。
通过细胞内囊泡染料的共定位研究所显示，胞吞作用在cS-bQDs-Tat的细胞内化中也发挥重要作用。使用囊泡染料DiR(亲脂性小分子标记脂质膜)的共定位研究表明，在cS-bQDs-Tat的细胞内转运过程的早期阶段，DiR和cS-bQDs-Tat之间存在显着的共定位，表明该部分cS-bQDs-Tat通过胞吞作用被细胞内化(除上述不依赖内吞作用的细胞内化)(图4f)。细胞内囊泡共定位程度随着时间的推移而降低，在cS-bQDs-Tat的细胞内转运过程的后期接近零。这表明最初捕获在囊泡中的纳米颗粒可以完全从囊泡陷阱中逸出(图4f)。
此外，另一种荧光染料，即钙黄绿素，用于探索cS-bQDs-Tat的囊泡逃逸机制。钙黄绿素是一种水溶性小分子荧光染料，在高于某一阈值的浓度下自猝灭，通常用作脂质囊泡完整性的指示剂。当囊泡完整时，钙黄绿素分子(基于其自猝灭性质选择的高浓度)被捕获在囊泡内部并显示点状和弱荧光(图4g，左上)；当囊泡破裂时，钙黄绿素分子释放到细胞质中并发出弥散的强荧光[图4g，右上方，细胞内囊泡被常用的囊泡破坏药物氯喹破坏50μM(16μg/mL)；随着这种药物的加入，许多细胞由于囊泡破坏而不健康]。结果发现，在整个cS-bQDs-Tat的细胞内转运过程中(在纳米粒子递送的早期阶段与钙黄绿素显著共定位，在纳米粒子递送的晚期递送阶段没有与钙黄绿素共定位)，钙黄绿素保持点状和弱荧光，表示囊泡膜的完整性(图4g，左下和右下)。因此，内吞的cS-bQDs-Tat可完全逃脱细胞内囊泡而不损害囊泡膜的完整性。通过直接观察可以观察到纳米颗粒(SDot)可以有效且可靠地逃避细胞内囊泡而不破坏囊泡膜(即，高效和非侵入性逃逸过程)，而小分子(钙黄绿素，亲水性)，比纳米粒子小两个数量级，除非囊泡破裂，否则不能有效地穿过细胞内囊泡膜。
与模型蛋白质牛血清白蛋白(BSA)结合的体外研究表明，SDot设计中的混合溶剂可以帮助减少纳米颗粒的非特异性蛋白质结合。将cS-bQDs-Tat(20nM)与BSA(15mg/mL)在不同浓度(0.5％，2％和5％)的有机助溶剂DMF存在下培养相同的时间。然后使用UV-Vis吸收光谱法测量与纳米颗粒表面结合的BSA。发现增加的有机溶剂浓度导致BSA与纳米颗粒表面结合的量显着减少(每种制剂的一式三份样品)，表明混合溶剂对减少纳米颗粒的蛋白质结垢的影响(图4h)。应该提到的是，有机溶剂浓度水平0％不包括在实验中，因为没有任何有机溶剂SDot不能具有良好的物理化学性质，如前所述。
实施例4：单粒子跟踪和对相关函数分析cS-bQDs-Tat的细胞运输动力学
本实施例使用cS-bQDs-Tat的细胞转运作为模型系统，展示了综合运用单粒子跟踪，对相关函数分析和旋转盘共聚焦显微镜(iSPT-pCF-SDCM)研究生物的动力学运输过程。在旋转盘共聚焦显微镜的相同观察阶段对相同样品进行单粒子跟踪和pCF分析。这种组合分析方法提供了有关动态传输过程的补充信息。
使用单粒子跟踪在～30个细胞中获得了～2,500个QD运动轨迹(图5a示出 了代表性轨迹)。荧光物体的轨迹通过连接其在不同时间点的位置而形成，所述不同时间点的位置通过使用商业旋转盘共聚焦显微镜确定。对于每个轨迹，计算均方位移(MSD)与持续时间(Δt)的定量关系，基于MSD-Δt关系对运动进行分类(包括定向运动，正常扩散，异常扩散和角度扩散)，并确定相应运动模式的特征常数。
本实施例对定向运动的研究揭示了速度值的有趣分布。在细胞质中，速度分布呈现多重高斯峰值，峰值近似倍数(220nm/s，～1×；620nm/s，～3×；820nm/s，～4×；980nm/s，～5×；1,300nm/s，～6×，图5b)。这表明多个分子马达协调作用以推动定向运动。考虑到所有cS-bQDs-Tat最终进入细胞核，且动力蛋白负责货物向细胞中细胞核的向内定向运动，所以动力蛋白可能是所涉及的主要运动蛋白。最低速度的峰值为220nm/s，这与已经披露的动力蛋白马达的体外速度值非常一致(图5b)。此外，在细胞核区域也发现了许多定向运动轨迹，并且速度分布显示出具有多个峰值切峰值接近(最低速度的峰值为270nm/s，图5c)。先前已报道核区域中的定向运动用于腺相关病毒的细胞转运，并且认为病毒在核区域中的定向运动是通过沿着核膜的内陷通道中的微管运动而给出的(而不是核质中的定向运动)。因此，本发明人推测cS-bQDs-Tat在核区域的定向运动是由沿着由核膜形成的这些通道中的微管的动力蛋白驱动运动引起的。考虑到通道的小直径(约50nm)的限制，在内陷通道中移动的cS-bQDs-Tat可能在细胞内囊泡外。这种运动是否(以及，如果是，如何)与cS-bQDs-Tat的核进入相关是一个待研究的问题。
双关联函数(pCF)分析用于研究QD细胞内囊泡逃逸的运动。一对位置点的荧光强度(时间函数)的互相关函数，一个位于囊泡内，另一个位于囊泡外，在对应于囊泡逃逸的通过时间的时间值处显示最大峰值(在穿过两个位置点的线的方向)(图5d)。使用商业旋转盘共聚焦显微镜测量细胞中不同位置点处在不同时间点的荧光强度，然后使用编写的计算机程序计算pCF。发现cS-bQDs-Tat的囊泡逃逸的通过时间为3.6ms(标准偏差1.7ms，对穿过3个细胞中的156个囊泡的膜绘制的312个线进行测量)(图5e)。这明显快于常规水溶性QDs-Tat在1％DMF存在情况下的囊泡逃逸传输时间(平均值15.4ms，标准偏差3.1ms；对穿过2个细胞中的120个囊泡的膜绘制的240个线进行测量)(图5e)。此外，在没有DMF的情 况下，常规的水溶性QDs-Tat几乎没有表现出囊泡逃逸(图5e)。使用1％DMF可以减少常规水溶性QD-Tat的囊泡逃逸传输时间的发现表明，混合溶剂也可以作为促进囊米材料囊泡逃逸的方便且独立的策略。此外，本发明人发现cS-bQDs-Tat的囊泡逃逸过程是各向同性的，因为它们在从相同囊泡逃逸的不同方向上表现出几乎相同的通过时间(图5f)。这个结论来自于对穿过3个细胞中的156个囊泡的膜绘制的312条线进行测量；为同一囊泡绘制的两条线具有垂直方向。这与在cS-bQDs-Tat的囊泡逃逸过程中囊泡保持完整的发现一致，因为破碎的囊泡膜将使纳米粒子的更容易从某一些方向逃逸(图4g)。
实施例5：用cS-bQDs-Tat增强药物和大分子的递送
本实施例研究了cS-bQDs-Tat作为小分子药物和生物大分子的一类生物传递载体被使用的潜力。通过吸附作用(～70DOX/颗粒)将DNA结合抗癌药物多柔比星(DOX)加载到cS-bQDs-Tat的颗粒表面上。在细胞培养实验中，以HeLa细胞(宫颈癌细胞系)作为模型癌细胞，本发明人发现装载DOX的cS-bQDs-Tat可以以更快的速度提供相同的癌细胞杀伤作用，并且药物剂量与对照DOX的配方相比更低(图6a)。例如，为了杀死30％的癌细胞，装载DOX的cS-bQDs-Tat(制剂名称SDots-Tat-DOX)仅需要在0.1μg/mL的DOX剂量下作用4小时。相比之下，为了达到相同的癌细胞杀伤效果，相同剂量的游离DOX制剂需要>12小时，或在相同时间内使用更高剂量(5μg/mL)；相同剂量的装载DOX的常规Tat肽缀合的水溶性QD(制剂名称QDs-Tat-DOX)需要>12小时，或在相同时间内使用更高剂量(5μg/mL)；DOX剂量相同装载DOX的常规水溶性QD(不含Tat肽)(配方名称QDs-DOX)需要>>12h，或在相同时间内使用更高剂量(>>10μg/mL)。负载DOX的cS-bQDs-Tat配方能节省时间(减少3倍)或减少剂量(减少50倍)的原因在于cS-bQDs-Tat具有前所未有的高效，特异递送药物进入药物起作用的细胞器(细胞核)中的能力和高效、特异，直接进入药物起作用的细胞器(细胞核)中的能力。双色荧光显微镜结果显示，在细胞转运过程中，DOX荧光与负载DOX的cS-bQDs-Tat的荧光之间存在高度共定位，表明在纳米颗粒表面解聚的DOX分子很少。值得指出的是，与游离DOX制剂相比，装载DOX的常规水溶性QDs-Tat(制剂名称QDs-Tat-DOX)未显著改善癌细胞杀伤效果(图6a)。这可能是 因为尽管QDs-Tat可以促进DOX进入位于细胞核外的微管组织中心(MTOC)的过程，但是装载DOX的水溶性QDs-Tat被困在细胞内囊泡中，无法克服囊泡捕获屏障。该结果再次强调了SDot技术的新纳米材料设计在克服细胞运输障碍方面的重要性。
本实施例的另一个发现是cS-bQDs-Tat可以促进生物大分子的细胞内靶向递送。细胞内囊泡捕获的转运障碍被认为是基于大分子的治疗的主要挑战。众所周知，大分子[例如由Tat肽递送的蛋白质和小干扰RNA(siRNA)]几乎完全(估计>99％)被捕获在细胞内囊泡中，因此无法进入囊泡外的细胞质和细胞器(包括细胞核)。将cS-bQDs-Tat与模型蛋白卵清蛋白(ova，MW45kDa)缀合，并进行细胞递送和成像实验。结果发现，与Tat肽偶联的卵白蛋白(用荧光染料标记用于成像)相比，与cS-bQDs-Tat结合的卵白蛋白显示出大大增强的向细胞核的递送(～25％，对照样品几乎为零核递送，图6b)。由于与SDot结合应该增加输送货物的总体尺寸(两倍或更多)，这一结果表明疏水纳米颗粒表面和混合溶剂的组合对生物转运增强效果是如此显着，以至于即使在货物的大小急剧增加时，它也可以极大地改善货物的目标输送。
实施例6：用cS-bQDs-Tat进行透皮给药及其抑制皮下肿瘤生长的效果研究
在本实施例中，所使用的SDot配方与实施例5中相同。其中，抗癌药物采用的是阿霉素/多柔比星(Doxorubicin,简称DOX)，该药物分子通过与细胞核中的DNA作用杀死细胞。
1.人口腔上皮癌裸鼠荷瘤模型的建立
取SPF级BALB/C雌性裸鼠(4周龄，20g左右)，将KB-3-1细胞(人口腔上皮癌细胞)于75cm培养瓶中培养，待生长至90％左右时，使用0.25％胰酶消化，培养基终止消化，常温离心，弃去上清液，而后使用PBS重复离心过程，最终加入PBS重悬。裸鼠左侧腹部消毒2-3次，将107/只KB细胞皮下注射至裸鼠的左侧腹部。裸鼠右耳标记编号，每隔两天称重记录体重，使用游标卡尺测量肿瘤体积大小，肿瘤体积计算公式V＝1/2*a*b2(a为长，b为宽)，待肿瘤体积生长至125mm 3，开始进行实验。
2.人口腔上皮癌裸鼠荷瘤模型透皮给药后生物传输能力的考察。
将100nmol/L的样品借助注射器直接涂抹在肿瘤外的皮肤上(此过程大概需要2-5min)，待24h后，对裸鼠进行安乐死，解剖取出肿瘤组织。利用量子点和DOX所发的荧光，用荧光显微镜对肿瘤组织切片进行观察，考察穿透肿瘤的传输能力。
3.人口腔上皮癌裸鼠荷瘤模型透皮给药对肿瘤生长抑制效果研究
重复步骤一中的实验，待肿瘤生长至125mm 3，开始进行给药实验，将100nmol/L的样品借助注射器直接涂抹在肿瘤外的皮肤上(此过程大概需要2-5min)，每隔48h给药一次，共给药5次，每次给药前测量肿瘤组织大小，并进行统计。
4.结果与分析
为了评估基于SDot-Tat体系的无创局部递送能力，通过将HeLa细胞与SDot-Tat-DOX共培养不同时间后通过激光共聚焦显微镜观察细胞内荧光，SDot-Tat-DOX能够很好的进入细胞且高效的靶向至细胞核内。为进一步考察SDot-Tat-DOX通过透皮递送的生物传输效果，构建KB荷瘤模型，研究SDots-Tat-DOX穿透裸鼠皮肤在KB肿瘤模型中的递送情况。传统水分散的量子点负载DOX后作为对比样品。
实验结果发现：虽然两组样品均能透过皮肤抵达肿瘤组织内部，但是在进入肿瘤后穿透肿瘤的能力显示了很大的不同。图1展示SDot-Tat-DOX的生物传输结果。如图1中所示，在对每块肿瘤组织切片时，将肿瘤从靠近皮肤的位置作为顶端，往下分为3段区域，进行切片。然后使用激光共聚焦显微镜对三段区域所得到的组织染色后观察。其中图1a中为靠近皮肤的肿瘤组织通过激光共聚焦拍摄得到的图片，从图中可以看出，通过皮肤进入肿瘤组织的SDot-Tat-DOX非常多，SDot-Tat荧光及DOX荧光高度共定位(SDot-Tat荧光为绿色，DOX荧光为红色，共定位部分会在合并后显示为黄色)，且很多是分布在细胞核内部的。图1b为肿瘤中间区域的组织片所获得的数据，显示具有很多SDot-Tat-DOX，其分布位置基本在细胞核外。图1c为而距离皮肤最远的肿瘤组织所获得图片，能发现较少的SDot-Tat-DOX。
同时作为对照，以QD-Tat-DOX(Lipid-PEG(QD-DOX)-Tat)作为对比样品，重复相同的实验，生物传输结果见图2。从图2中可以看出，QD-Tat-DOX 也可通过皮肤进入肿瘤组织，但在肿瘤中穿透能力远低于图1中SDot-Tat-DOX。在图2中，图2a,2b,2c分别给出肿瘤靠近皮肤部分，肿瘤中间部分，肿瘤远离皮肤部分的结果。图2a中显示，仅有小部分DOX进入细胞核内部；图2b显示，中间区域的组织片仅有很少的QD-Tat-DOX，其基本的分布位置均处在细胞核外；图2c显示，距离皮肤最远的肿瘤组织所获得图片基本未发现QD-Tat-DOX。
图3显示了对皮下肿瘤生长抑制能力的比较实验结果。仍然采用在皮肤上敷药。根据给药后肿瘤体积大小统计结果，可以发现，SDot-Tat-DOX相对于游离的DOX以及Lipid-PEG(QD-DOX)-Tat，能够大大提高遏制肿瘤的生长的能力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种纳米生物探针，其特征在于，所述纳米生物探针的从内向外的结构如式I所示，
    A-S-L-B n (式I)
    式中，
    A-S为纳米粒子，其中，S为纳米粒子的疏水性表面；
    B为生物分子；
    L为可选的将B偶联至量子点表面的连接部分；
    n为偶联至量子点表面的生物分子的个数，为大于等于1的正整数。
  2. 如权利要求1所述的纳米生物探针，其特征在于，所述纳米生物探针经过混合溶剂处理。
  3. 如权利要求2所述的纳米生物探针，其特征在于，所述混合溶剂包括至少一种有机溶剂和至少一种非有机溶剂。
  4. 如权利要求3所述的纳米生物探针，其特征在于，所述混合溶剂中，有机溶剂为二甲基甲酰胺(DMF)。
  5. 如权利要求1所述的纳米生物探针，其特征在于，所述B为Tat肽，所述Tat肽的序列为SEQ ID NO:1。
  6. 一种纳米生物探针产品，其特征在于，包括：
    (a)第一容器，以及位于第一容器中的如权利要求1至5中任一项所述的纳米生物探针；
    (b)第二容器以及位于第二容器中的混合溶剂。
  7. 一种如权利要求2至5中任一项所述的纳米生物探针或权利要求6所述的纳米生物探针产品的制法，其特征在于，包括步骤：
    (i)合成纳米粒子，所述纳米粒子表面为疏水性表面或包裹疏水性配位体层；
    (ii)将生物分子偶联至纳米粒子表面，获得疏水性纳米粒子-生物分子偶联物；
    (iii)使用混合溶剂对步骤(ii)所获得的疏水性纳米粒子-生物分子偶联物进行处理，获得所述纳米生物探针。
  8. 一种生物活性分子的细胞内递送方法，其特征在于，包括将所述生物活性 分子偶联至权利要求1至5中任一项所述的纳米生物探针，并与含有细胞的培养液混合。
  9. 一种如权利要求1至5中任一项所述纳米探针的用途，其特征在于，用于制备一制剂，所述制剂用于向生物体内递送生物活性分子。
  10. 如权利要求9所述的用途，其特征在于，所述的生物体包括生物的组织、器官或细胞。
  11. 如权利要求10所述的用途，其特征在于，所述组织为皮肤组织。
  12. 如权利要求9所述的用途，其特征在于，所述的生物体包括患癌组织、患癌器官或癌细胞。
  13. 一种如权利要求1至5中任一项所述纳米生物探针的用途，其特征在于，用于制备一制剂或药物组合物，所述制剂或药物组合物用于生物体靶向定位。
  14. 如权利要求13所述的用途，其特征在于，所述制剂或药物组合物具有穿越生物体内的生物传输屏障的功能。
  15. 如权利要求14所述的用途，其特征在于，所述生物传输屏障包括组织屏障、器官屏障、细胞屏障、细胞器屏障。
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