CN111184874B - Hydrophobic nano biological probe - Google Patents

Hydrophobic nano biological probe Download PDF

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CN111184874B
CN111184874B CN201811353772.8A CN201811353772A CN111184874B CN 111184874 B CN111184874 B CN 111184874B CN 201811353772 A CN201811353772 A CN 201811353772A CN 111184874 B CN111184874 B CN 111184874B
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tat
cells
hydrophobic
bqds
quantum dot
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CN111184874A (en
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阮刚
王军
谢金兵
梅玲
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Nanjing University
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Nanjing University
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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

Abstract

The invention provides a novel nano biological probe and application thereof in targeting delivery of bioactive molecules in cells, and particularly discloses a mixed solvent-naked hydrophobic QD-biological molecule (cS-bQD-BM, or 'SDot') for the first time. SDots exhibit extraordinary intracellular targeting properties with the nucleus as a model target, including near perfect specificity, excellent efficiency and reproducibility, high throughput capability, minimized toxicity, ease of handling, and excellent optical properties and colloidal stability.

Description

Hydrophobic nano biological probe
Technical Field
The invention relates to the field of biological materials and biomedicine, in particular to a nano biological probe and application thereof in targeting delivery of bioactive molecules in cells, particularly living cells.
Background
Fluorescent probes play a key role in live cell imaging as a visual signal source. Quantum dots (QDs, nano-scale crystals of semiconductor materials) have several significantly superior or unique properties, especially optical properties, including extraordinary fluorescence intensity and light stability, a fluorescence emission peak with narrow peak width that is adjustable in size and composition, can be excited for multiple imaging by a single light source, and can be used as imaging probes for related optical and electron microscopes, compared to the other two main classes of fluorescent probes, i.e., small molecule fluorescent dyes and genetically encoded fluorescent proteins. However, unlike fluorescent dyes and fluorescent proteins, the current use of QDs for live cell imaging of specific subcellular structures or molecules is generally limited to imaging of entities on the cell membrane (e.g., cell surface receptors). So to date, targeting QDs to specific entities inside living cells has met with only limited success. Achieving intracellular targeted delivery of QDs in a highly specific (ideally close to 100%), efficient, robust, scalable, safe and convenient manner is a long sought after, but currently still elusive goal.
Achieving this long-sought goal requires overcoming all of the following cell transport barriers in an effective and minimally invasive manner: (1) cell membranes, (2) intracellular vesicle capture (if the cellular uptake mechanism is endocytosis), (3) crowded cytoplasm, (4) nonspecific binding, (5) organelle membranes (if the target is located within an organelle), and (6) obstacles within organelles (if the target is a specific location within an organelle, figure 1). Overcoming all of these cellular transport barriers is challenging for targeted delivery of various biologically important materials (e.g., small molecule drugs, macromolecules, and nanoparticles), especially QDs. Typical water-soluble QDs consist of bare hydrophobic QDs for fluorescence generation and a polymer coating for water dissolution and fluorescence maintenance, with diameters greater than 15 nm. This size is much larger than most intracellular proteins (a few nanometers in diameter) and is thought to cause significant dyskinesias in crowded gel-like structures of cytoplasm with a pore size of about 80 nm. Furthermore, even without increasing the diameter of the polymer coating, the size of the bare hydrophobic QDs approaches that of proteins, and intracellular vesicle trapping remains a formidable obstacle: it is estimated that generally < 1% of endocytosed proteins (and other types of macromolecules) can escape intracellular vesicle capture and reach the cytoplasm. Furthermore, bypassing endocytosis by methods of physically disrupting cell membranes (e.g., microinjection and electroporation) often requires additional instrumentation, requires long technical exercises and laborious manipulations to use the instrumentation, results in significant cell death, and produces unrepeatable results.
Therefore, there is an urgent need in the art to develop a nanobiotrobe that can be positioned in a cell with high specificity, has low toxicity, and is easy to handle.
Disclosure of Invention
The invention aims to provide a nano biological probe which can perform high-specificity positioning in cells, has low toxicity and is easy to operate.
In a first aspect of the present invention, there is provided a nanobioprobe, which has a structure from inside to outside as shown in formula I,
A-S-L-Bn(formula I)
In the formula (I), the compound is shown in the specification,
A-S are nanoparticles, wherein S is the hydrophobic surface of the nanoparticle;
b is a biomolecule;
l is an optional linking moiety coupling B to the surface of the quantum dot;
n is the number of biomolecules coupled to the surface of the quantum dot and is a positive integer greater than or equal to 1.
In another preferred example, the nano biological probe is treated by a mixed solvent.
In another preferred embodiment, the solubility of the nanoprobe treated by the mixed solvent in the aqueous environment is more than or equal to 1nM, preferably more than or equal to 5nM, and more preferably more than or equal to 50 nM.
In another preferred embodiment, the mixed solvent includes at least one organic solvent and at least one non-organic solvent.
In another preferred embodiment, in the mixed solvent, the organic solvent is selected from the group consisting of: dimethylformamide (DMF), acetone, ethanol, dimethyl sulfoxide (DMSO), Tetrahydrofuran (THF), ethyl acetate, acetonitrile, formic acid, butanol, dimethyl methanol, propanol, acetic acid, or a combination thereof.
In another preferred embodiment, in the mixed solvent, the organic solvent is Dimethylformamide (DMF).
In another preferred embodiment, the concentration of the organic solvent in the mixed solvent is 0.01 to 50 v/v%, preferably 0.1 to 20 v/v%, and more preferably 0.5 to 5 v/v%.
In another preferred embodiment, the non-organic solvent in the mixed solvent is selected from water, phosphate buffer, cell culture fluid, blood, etc., or a combination thereof.
In another preferred embodiment, a is selected from the group consisting of: magnetic nanoparticles, metal nanoparticles, semiconductor nanoparticles (quantum dots), carbon nanoparticles, polymer nanoparticles, lipid nanoparticles, or combinations thereof.
In another preferred example, the quantum dot includes: cadmium selenide quantum dots, cadmium 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.
In another preferred embodiment, S is the hydrophobic surface of the nanoparticle itself or the surface of the hydrophobic ligand layer coating the nanoparticle.
In another preferred embodiment, the ligand is selected from the group consisting of: tri-n-octylphosphine oxide, oleic acid, stearic acid, oleylamine, or a combination thereof.
In another preferred embodiment, the B comprises a small molecule or a biological macromolecule.
In another preferred embodiment, the biomacromolecule comprises: a protein, a nucleic acid, a polysaccharide, a polypeptide, or a combination thereof.
In another preferred embodiment, said B is a positively and/or negatively charged polar molecule.
In another preferred embodiment, the molecular weight of B is 1 to 500kD, preferably 1 to 200kD, more preferably 1 to 80 kD.
In another preferred embodiment, 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.
In another preferred embodiment, said B is a Tat peptide.
In another preferred embodiment, the Tat peptide has the sequence SEQ ID NO 1.
In another preferred embodiment, the Tat peptide has biological functions of cell penetration and nuclear targeting.
In another preferred embodiment, the structure of the nanobody probe may not include L in formula I.
In another preferred embodiment, the coupled form comprises: peptide bonds, hydrogen bonds, charge interactions, avidin-biotin binding, nucleic acid pairing interactions, or combinations thereof.
In another preferred embodiment, the coupling is achieved by ligand exchange and bioconjugation.
In another preferred embodiment, the coupling is accomplished by using a coupling agent.
In another preferred embodiment, the coupling agent comprises: 1-hemi-ethylamine-3- (3-dimethylaminopropyl) carbodiimide (EDC), avidin (avidin) -biotin (biotin), nucleic acid pairing, or combinations thereof.
In another preferred embodiment, said B may be coupled to said a-S surface by covalent coupling and/or non-covalent coupling.
In another preferred embodiment, the covalent coupling comprises: a peptide bond formation reaction, a bio-orthogonal reaction, or a combination thereof.
In another preferred embodiment, the non-covalent coupling is selected from the group consisting of: charge interactions, hydrogen bonding, van der waals forces, hydrophobic interactions, antibody-antigen interactions, avidin-biotin binding, nucleic acid pairing interactions, or combinations thereof.
In another preferred embodiment, the diameter of the nanoprobe is 1 to 200nm, preferably 1 to 20nm, more preferably 1 to 10 nm.
In another preferred embodiment, the nanoprobe does not comprise a fully hydrophilic polymer coating.
In another preferred example, the quantum dot nanoprobe can stably exist in a closed container for more than or equal to 3 days, preferably more than or equal to 5 days, and more preferably more than or equal to 7 days at 4 ℃.
In another preferred embodiment, the excitation light wavelength of the quantum dot is 100 to 1500nm, preferably 200 to 1000nm, and more preferably 250 to 600 nm.
In another preferred embodiment, the emission wavelength of the quantum dots is 400 to 2500nm, preferably 400 to 2000nm, and more preferably 450 to 1500 nm.
In a second aspect of the present invention, there is provided a nanobioprobe product comprising:
(a) a first container, and a nanobody probe according to the first aspect of the present invention in the first container;
(b) a second container and a mixed solvent in the second container.
In another preferred embodiment, the first and second containers may be the same or different containers.
In a third aspect of the present invention, there is provided a method for preparing the nanobioprobe according to the first aspect of the present invention or the nanobioprobe product according to the second aspect of the present invention, comprising the steps of:
(i) synthesizing nano particles, wherein the surface of each nano particle is a hydrophobic surface or is wrapped with a hydrophobic ligand layer;
(ii) coupling biological molecules to the surfaces of the nanoparticles to obtain hydrophobic nanoparticle-biological molecule conjugates;
(iii) (iii) treating the hydrophobic nanoparticle-biomolecule conjugate obtained in step (ii) with a mixed solvent to obtain the nano-bioprobe.
In a fourth aspect of the invention, there is provided a method for intracellular delivery of a biologically active molecule comprising coupling said biologically active molecule to a nanobody probe according to the first aspect of the invention and mixing with a culture medium containing cells.
In another preferred embodiment, the method is performed in vitro.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In another preferred embodiment, the cell comprises: non-cancerous or cancerous cells.
In another preferred embodiment, the cancer cell comprises: cervical cancer cells, breast cancer cells, liver cancer cells, lung cancer cells, stomach cancer cells, prostate cancer cells, or a combination thereof.
In another preferred embodiment, the bioactive molecule comprises: a drug that binds to DNA, a drug that binds to RNA, a drug that binds to an intracellular protein, a drug that binds to an intracellular cytoskeleton, a drug that acts on an intracellular mitochondrion, a drug that binds to an intracellular sugar molecule, a drug that binds to an intracellular lipid molecule, or a combination thereof.
In another preferred embodiment, the bioactive molecule comprises: small molecules and biological macromolecules.
In another preferred embodiment, the bioactive molecule is an anti-cancer drug.
In another preferred embodiment, the anticancer drug comprises doxorubicin (doxorubicin), paclitaxel (paclitaxel), docetaxel (docetaxel), cisplatin (cispain), or a combination thereof.
In another preferred embodiment, the biomacromolecule comprises: a protein, a nucleic acid, a polysaccharide, a polypeptide, or a combination thereof.
In another preferred embodiment, the intracellular delivery is live intracellular delivery.
In another preferred embodiment, the delivery is targeted delivery.
In another preferred embodiment, the delivery is nuclear targeted delivery.
In another preferred embodiment, the delivery method is such that the percentage of drug that achieves targeted delivery is 20% or more, preferably 70% or more, and more preferably 90% or more.
In a fifth aspect of the invention, there is provided a use of a nanoprobe according to the first aspect of the invention for the preparation of a formulation for delivering a biologically active molecule into a cell.
In a sixth aspect of the invention, there is provided a use of the nanobody probe of the first aspect of the invention for targeting in living cells.
In another preferred embodiment, said localization is performed in vitro or ex vivo.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows the design of cS-bQD-BM ('SDot') for targeted intracellular delivery of QDs.
FIG. 2 shows the preparation and physicochemical characterization of cS-bQD-Tat.
(a) Schematic diagram of the preparation of cS-bQD-Tat.
(b) Successful surface modification was confirmed by the addition of cysteamine and changes in the surface charge (zeta potential) of the Tat peptide.
(c) The diameter measured by Dynamic Light Scattering (DLS) increased by-0.05 nm and-0.5 nm, respectively, only upon addition of cysteamine and Tat peptide.
(d) The characterization of the fluorescence spectrum shows that cS-bQDs-Tat has good fluorescence properties (fluorescence intensity is only reduced by about 20% compared with bQD) and good colloidal stability (there is almost no change in fluorescence of the aqueous dispersion after 5 days).
FIG. 3 shows intracellular targeting of cS-bQDs-Tat.
(a) Bird's eye view optical microscope images of HeLa cells with cS-bQDs-Tat show that cS-bQD-Tat successfully targets the nucleus with near-perfect targeting specificity. The magnification of the optical microscope objective used is 20 times. The image is a composite of bright field microscope images showing cells and fluorescence microscope images showing nuclei (stained by the nuclear dye Hoechst 33342, red) and cS-bQDs-Tat (green). The co-localization of the nucleus and cS-bQDs-Tat results in a complex color yellow. Scale bar, 60 μm.
(b) Close-up optical microscope images of HeLa cells with cS-bQDs-Tat show that cS-bQD-Tat successfully targets the nucleus, showing that nearly all cS-bQDs-Tat is co-localized with a specific structural nucleolus within the nucleus. The magnification of the optical microscope objective used was 60 times. Scale bar, 20 μm.
(c) Light microscopy images at different delivery time points show that targeted intracellular delivery of cS-bQDs-Tat is rapid. The images at 15 minutes, 1 hour, 4 hours and 8 hours are composite images of bright field (showing cells) and fluorescence (showing cS-bQD-Tat) microscope images. The 12 hour image is a fluorescence image showing co-localization of nuclei (blue) and cS-bQDs-Tat (green). The co-localization of the nucleus and cS-bQDs-Tat results in a bluish complex color. Scale bar, 20 μm.
(d) Intracellular targeting of cS-bQD-Tat was successful in all four different cell lines tested. Error bars, mean ± s.e.m, n is 200 cells.
(e) cS-bQD-Tat (SDot-Tat) is less cytotoxic. Error bar, mean ± s.e.m, n ═ 5.
FIG. 4 shows the mechanistic studies of cellular transport of cS-bQDs-Tat (a-c) confirm that the excellent intracellular targeting of cS-bQD-Tat is caused by three design parameters.
(a) Larger nanoparticle hydrophobic surface coverage results in better targeting effect.
(b) Higher organic solvent concentrations lead to better targeting effects.
(c) Smaller nanoparticle sizes lead to better localization effects.
(d) Endocytosis inhibition of cellular uptake of cS-bQDs-Tat was studied. Neither of the two endocytosis inhibitors used, i.e., hypothermia and cytochalasin D, completely blocked cellular uptake of cS-bQDs-Tat. In contrast, for conventional water-soluble QDs-Tat (QDs-Tat with hydrophilic polyethylene glycol coated surface), cellular uptake was completely blocked by each of the two endocytosis inhibitors used. Scale bar, 20 μm.
(e) cell membrane leakage study of cS-bQDs-Tat. For all four different cS-bQD-Tat formulations tested, lactate dehydrogenase release from HeLa cells was < 10%, and levels similar to control samples with only HeLa cells, indicating that the cell membrane was intact during the delivery process of cS-bQD-Tat. Error bar, mean ± s.e.m, n ═ 5.
(f) Intracellular vesicle co-localization studies of cS-bQDs-Tat using the lipophilic vesicle dye DiR. DiR is green; cS-bQD-Tat is red. The left one to four figures show optical microscope images at various time points of the delivery process. The right panel shows the quantification of co-localization (measured by Pearson correlation coefficients) over time. Scale bar, 20 μm.
(g) Intracellular vesicle membrane integrity studies using the fluorescent tracer calcein. Inside the vesicle, the fluorescence of calcein is quenched, weak and punctate (upper left panel); when the vesicles burst, calcein is leaked into the cytoplasm, and its fluorescence diffuses and becomes intense (upper right panel). At the beginning of the delivery of cS-bQDs-Tat (red), calcein fluorescence (green) is punctate and weak, and there is partial co-localization between cS-bQDs-Tat and calcein (bottom left panel); at the end of delivery, calcein fluorescence remained punctate and weak, and there was no co-localization between cS-bQDs-Tat and calcein (lower right panel). Scale bar, 10 μm.
(h) In vitro study of the Effect of mixed solvents on nonspecific proteins binding to cS-bQDs-Tat. Error bar, mean ± s.e.m, n ═ 3. P < 0.001. P <0.05 (student's t-test).
FIG. 5 shows Single Particle Tracking (SPT) and correlation function (pCF) analysis of cellular transport of cS-bQDs-Tat. (a-c) is the SPT result. (d-f) is the pCF result.
(a) Representative trace of cS-bQDs-Tat. The track is displayed in red; cell nuclei are shown in blue; lines are drawn to show the periphery of the cell and nucleus. Scale bar, 10 μm.
(b) Distribution of directional movement velocities in the cytoplasm. The inset shows a representative trajectory of this motion (red).
(c) Distribution of the directional movement speed of the nucleus region. The inset shows a representative trajectory of this motion (red).
(d) Representative pCF curves for analysis of transit times of particles escaping intracellular vesicles. The inset shows a schematic of the vesicular escape of the nanoparticles.
(e) The vesicle escape time of SDot-Tat (cS-bQD-Tat) in the presence of 1% organic solvent (DMF) was significantly shorter than QD-Tat (conventional water-soluble QD-Tat). Conventional water-soluble QD-Tat (in the absence of organic solvents) shows little vesicle escape. P <0.001 (student's t-test).
(f) The vesicle escape propagation time of cS-bQD-Tat is isotropic. For each vesicle studied, the difference in vesicle escape propagation times in two different (perpendicular) directions was measured.
FIG. 6 shows the results of enhancing targeted intracellular delivery of drugs and macromolecules with cS-bQDs-Tat (SDots-Tat).
(a) Cancer cells (HeLa cells) were killed using Doxorubicin (DOX) as a drug model. The use of SDots-Tat (formulation SDots-Tat-DOX) significantly enhances the efficiency of targeted intracellular delivery of doxorubicin and killing of cancer cells compared to conventional soluble QDs (formulation QDs-DOX), free DOX and conventional water-soluble QDs-Tat (formulation QDs-Tat-DOX). Error bar, mean ± s.e.m, n ═ 3.
(b) Ovalbumin (ova) is taken as an intracellular macromolecular delivery model. The use of SDots-Tat (formulation Tat-ova-SDots) enhances targeted intracellular delivery of ovalbumin compared to the use of Tat peptides (formulation Tat-ova-dye, dye is a small molecule dye TAMRA for fluorescence imaging). Although the use of SDots-Tat increases overall size, the combination of hydrophobic nanoscale surfaces and mixed solvents still enhances targeted delivery of ovalbumin to the nucleus. The formulation Tat-dye (without ova, with a much smaller size than ova) was used as a positive control. The same amount of ovalbumin was used in the delivery of Tat-ova-SDots and Tat-ova-dye. Error bars, mean ± s.e.m, n ═ 100-.
Detailed Description
The present inventors have conducted extensive and intensive studies and, through a large number of screenings, have developed a novel quantum dot nanoprobe. Specifically, the inventor replaces a small part of exposed hydrophobic quantum dot hydrophobic surface with bifunctional small molecule ligand cysteamine, couples biomolecule Tat peptide with quantum dot surface amino through 1-half ethylamine-3- (3-dimethyl aminopropyl) carbodiimide (EDC), and then applies a small amount of organic cosolvent DMF to process the obtained quantum dot nanoprobe.
Experiments show that the quantum dot nanoprobe can overcome cell transport obstacles, realize high-specificity, high-efficiency, stable, expandable, safe and convenient intracellular targeting in living cells, and realize high-efficiency and accurate intracellular drug delivery.
The present invention has been completed based on this finding.
Term(s) for
Nano biological probe
As used herein, "nanoprobe", "bQDs-BM", "QD nanoprobe", "QD probe", and the like, which may be used interchangeably, refer to a nanoprobe according to the present invention having a structure represented by formula I from the inside to the outside,
A-S-L-Bn(formula I)
In the formula (I), the compound is shown in the specification,
A-S are nanoparticles
S is the surface of the nanoparticle, wherein S is the hydrophobic surface of the nanoparticle;
b is a biomolecule;
l is a linking moiety that optionally couples B to the surface of the quantum dot;
n is the number of biomolecules coupled to the surface of the nanoparticle and is a positive integer greater than or equal to 1.
In a preferred embodiment, a is selected from the group consisting of: magnetic nanoparticles, metal nanoparticles, semiconductor nanoparticles (quantum dots), carbon nanoparticles, polymer nanoparticles, lipid nanoparticles, or the like, or combinations thereof. In a preferred embodiment, the quantum dot comprises: cadmium selenide quantum dots, cadmium 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.
In a preferred embodiment, S is the hydrophobic surface of the nanoparticle itself or the surface of the hydrophobic ligand layer encapsulating the nanoparticle. In the present invention, the ligand is selected widely, as long as one end of the molecule is bound to the nanoparticle with a certain attractive force and the other end is hydrophobic. In a preferred embodiment, the ligand is selected from the group consisting of: tri-n-octylphosphine oxide, oleic acid, stearic acid, oleylamine, and the like, or combinations thereof.
In the present invention, the biomolecule B may include: proteins, nucleic acids, polysaccharides, polypeptides, etc., and may also include small molecules. Preferably, B is a positively and/or negatively charged polar molecule.
In a preferred embodiment, the B comprises a Tat peptide having the sequence YGRKKRRQRRR (SEQ ID NO:1) which has the biological functions of cell penetration and cell nucleus targeting.
The nano biological probe overcomes the cell transport obstacles such as cell membranes, intracellular vesicle capture (if the cellular uptake mechanism is endocytosis), crowded cytoplasm, nonspecific protein combination, organelle membranes (if the target is positioned in an organelle) and obstacles in the organelle, and realizes high specificity, high efficiency, stability, expandability, safety and convenience in intracellular QD targeting in living cells.
The design of the present invention (or SDot-BM, or SDot) involves modifying the properties of a small fraction (e.g. 50%, preferably 40%, preferably 30%, more preferably 20%, more preferably 10%) of the surface coverage of bare hydrophobic QDs with biomolecules (via ligand exchange followed by bioconjugation) and using small amounts of organic co-solvents to facilitate their dispersion in an aqueous environment (figure 1).
The inspiration designed by the present invention comes from the recent discovery by the present inventors that bare hydrophobic QDs, after treatment with a small amount of organic co-solvent, can directly penetrate a biological membrane without destroying the integrity of the membrane. As shown by the experimental results in the examples section of the present invention, this novel QD probe integrates a variety of features that help overcome cell trafficking obstacles, including: (1) the large hydrophobic surface assists in crossing the biofilm; (2) the organic cosolvent promotes the biological crossing of the membrane and reduces the nonspecific protein combination; (3) due to the elimination of the use of polymer coatings, ultra-small QD probes are formed, facilitating their movement in crowded biological environments, and (4) the natural biological functions of biomolecules can be exploited (fig. 1).
In a preferred embodiment, the nucleus located deep inside the cell, which serves as a cell command center, is used as an intracellular targeting model, and the experimental results of this embodiment demonstrate that the targeting of viable nuclei using SDots has the advantages of near perfect specificity, excellent efficiency and reproducibility, high throughput capability, minimized cytotoxicity, convenient operation, etc. Tat peptides have biological functions of targeting the nucleus and penetrating cells and are used as Biomolecules (BM) in the design of SDot.
In another preferred embodiment, it is demonstrated that sDot can be used for combined imaging, tracking and analysis of correlation functions in living cells. In another preferred embodiment, the unique ability of sDot to overcome cellular transport barriers is demonstrated to be useful for enhancing the delivery of small drug molecules and biomacromolecules.
Mixed solvent
As used herein, "mixed solvent", "organic solvent of the present invention", "organic solvent", and "organic co-solvent", and the like, which are used interchangeably, refer to a solvent with which the nanobody probe according to the first aspect of the present invention can be treated, thereby enhancing the dispersibility of the nanobody probe of the present invention in an aqueous solution, the motility in a biological membrane, and the effect of reducing the nonspecific binding between the nanobody probe and a protein molecule.
Since various hydrophobic nanoparticles are limited by poor targeting transmission effect and poor biocompatibility inside living cells in biomedical applications, the hydrophobic surface of the nanoparticles needs to be modified into a hydrophilic surface by physical (such as hydrophilic polymer coating) or chemical methods (such as surface covering with hydrophilic functional groups) in order to improve the targeting transmission effect inside the living cells of the nanoparticles. At present, it is generally considered in the biomedical field that hydrophobic nanoparticles which are not subjected to hydrophilization treatment cannot be directly applied to targeting transmission inside living cells.
The invention breaks the traditional cognition, realizes the direct application of the hydrophobic nanoparticles in the biomedical field, particularly the targeted transmission in living cells by a mixed solvent treatment mode, and the principle of the invention is not limited by the types of the nanoparticle materials, so the invention is suitable for various nanoparticles, including magnetic nanoparticles, metal nanoparticles, semiconductor nanoparticles (quantum dots), carbon nanomaterials, polymer nanoparticles, lipid nanoparticles and the like, or the combination of the magnetic nanoparticles, the metal nanoparticles, the semiconductor nanoparticles, the carbon nanomaterials, the polymer nanoparticles and the lipid nanoparticles.
In the present invention, 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.
Since the organic solvent has a certain toxicity to cells, the concentration of the organic solvent in the mixed solvent should be controlled within a certain range to minimize the cytotoxicity. In a preferred embodiment, the concentration of the organic solvent component is from 0.01 to 50 v/v%, preferably from 0.1 to 20 v/v%, more preferably from 0.5 to 5 v/v%.
Intracellular delivery method
The nano biological probe provided by the invention can be used for intracellular delivery of bioactive molecules. In particular, the bioactive molecule can be coupled to a nanobiotrobe and delivered to a specific location within the cell following the precise localization of the nanobiotrobe within the cell.
The main advantages of the invention include:
(1) the nanomaterials are significantly improved, giving them the ability to efficiently and minimally invasively traverse a variety of biological transport barriers, including biofilms, crowded complex environments, non-specific adsorption, etc.
(2) The performance of targeted delivery of the nano material in living cells is obviously improved, and the performance comprises specificity, efficiency, repeatability, high throughput, safety, convenience and the like.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The materials and reagents used in the examples were all commercially available products unless otherwise specified.
Materials and methods
1. Material
Cadmium oxide (CdO, 99.99%), zinc nitrate hexahydrate (Zn (NO3) 2.6H 2O, 98%), sulfur powder (S, 99.98%), selenium powder (Se, 100 mesh, 99.5%), 1-octadecene (ODE, 90%), stearic acid (SA, 95%), trioctylphosphine (TOP, 90%), cysteamine (98%), 1-ethyl-3 (3-dimethylaminopropyl) 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 peptides (sequence Ac-YGRKKRRQRRR) (SEQ ID NO:1), Tat-TAMRA (i.e.inserting TAMRA dye between residues 5 and 6 of SEQ ID NO:1), Tat-ovalbumin (i.e.inserting ovalbumin between residues 5 and 6 of sequence SEQ ID NO:1) and Tat-ovalbumin-TAMRA (i.e.inserting TAMRA dye between residues 5 and 6 of sequence SEQ ID NO:1 and ovalbumin between residues 6 and 7) were purchased from Chinapeptides.
3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT), DiR, calcein, Bovine Serum Albumin (BSA), and Nuclear isolation kit were purchased from KeygEN BioTECH.
Hoechst 33342 was purchased from ThermoFisher Scientific.
Cytochalasin D was purchased from bailey biotechnology limited, shanghai.
phospholipid-PEG was purchased from Avanti Lipids.
Doxorubicin hcl (DOX, 98%) was purchased from aladin.
Cell lines (HeLa, MCF-7, NIH 3T3 and Hep G2 cells) and their media were purchased from KeygEN BioTECH.
Preparation of cS-bQDs-Tat
Hydrophobic QDs surface-coated with the hydrophobic ligand tri-n-octyloxyphosphine were synthesized using a high temperature crystallization-based single-step non-injection production process disclosed in the article "Scalable single-step synthesis of high-quality core/shell quality with injection of structured from vitamin to near information" by Wenjin Zhang et al [ published in ACS Nano, 6(12) in 2012 ], pp11066-11073]. Incubating hydrophobic QDs (in DMF) with cysteamine for 0.5 hours to exchange a small portion of the surface ligands of the QDs, such that the resulting QDs have-NH on a small portion of the QD surface2(the remaining hydrophobic surface coverage is estimated to be typically 90%). Contacting Tat peptide with-NH on QD surface using EDC2The groups are conjugated to form bQDs-Tat. The solution (DMF, or other organic solvent such as acetone, ethanol and DMSO) is dispersed in water (or other aqueous environment such as cell culture medium, typical volume ratio of organic solvent to water is 1:99) to form cS-bQDs-TAT.
Physicochemical characterization of cS-bQDs-Tat
The morphology of the nanoparticles was visualized 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). The Dynamic Light Scattering (DLS) diameter (i.e., hydrodynamic diameter if the solvent is water) and surface charge (zeta potential) of the nanoparticles were measured using a Malvern Zetasizer Nano ZS360 instrument. A fluorescence spectrum was obtained using a fluorescence spectrophotometer (HITACHI F-4600).
4. Living cell rotating disc confocal microscope
HeLa cells were used for most of the cell experiments and were recommended by the manufacturer at 37 ℃ with 5% CO2The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). The other cell lines used (MCF-7, NIH 3T3 and Hep G2) were also cultured under the conditions recommended by the manufacturer.
Living cell imaging studies using a living cell rotating disk confocal imaging system consisting of a cell incubation chamber (IX 3W)Tokai Hit), epifluorescence microscope (IX-83, Olympus), rotating disk confocal system and Electron Multiplying Charge Coupled Device (EMCCD) camera (Evolve 512, Photometrics). Cells were first seeded at-35% confluence onto glass-bottom cell culture dishes (glass bottom thickness 0.17mm) (Nest, China). At 37 ℃ and 5% CO2After 18 hours of incubation under conditions of (1), the cell culture medium was replaced with a dispersion of cS-bQDs-Tat. Unless otherwise indicated, typical conditions for the cS-bQDs-Tat dispersion used are a total solvent volume of 1nM nanoparticles, 1% DMF as an organic cosolvent in aqueous cell culture medium, 90% hydrophobic surface coverage on the nanoparticles, 1Tat peptide per nanoparticle, and a fluorescence emission peak of 559 nM. After incubation with cS-bQDs-Tat for a specified duration (e.g., 15 minutes, 1 hour, 4 hours, 8 hours, 12 hours, and 24 hours), the cells are washed three times with fresh medium to remove cS-bQD bound to the outside or outer surface of the cells, and the cells are imaged by a live cell spinning disc confocal microscope system. To counterstain the nuclei, the fluorescent dye Hoechst 33342 (blue fluorescent color, 5 μ M in cell culture medium) was incubated with live cells for 20 minutes prior to imaging (at a specific time point of cell transport). Image processing and analysis were performed using MetaMorph and Image J software.
5. Measurement of intracellular targeting
Intracellular targeting of QDs to the nucleus was measured as the ratio of the amount of QDs delivered to the nucleus to the total amount of QDs in the cell. Measurements were performed by two different methods, confocal imaging and nuclear separation, which yielded consistent results. In the confocal imaging method, imaging is performed using a living cell rotating disk confocal imaging system. The amount of QDs in the nucleus or cell is quantified by measuring the intensity of fluorescence generated by the QDs in the corresponding region. In the method of nuclear isolation, cells were washed with PBS and nuclei were separated from the remaining cells using the nuclear isolation kit according to the manufacturer's instructions (KeyGEN). The amount of QDs in the nucleus (or the rest of the cell) is determined by measuring the QD fluorescence intensity in the nucleus fraction (or the rest of the cell fraction) and the total amount of QDs using a fluorescence spectrometer. QD in cells was determined by summing the amount of QD in the core fraction and the remaining cell fraction. The amount of QDs measured using the cell nucleus isolation method is slightly lower than the amount of QDs measured by the confocal imaging method due to the loss of QDs during the process of isolating the cell nucleus from the cell lysate.
6. Cell viability assay (MTT assay)
To assess the cytotoxicity of a particular formulation, HeLa cells were seeded in 96-well plates (Corning Costar, China) at a density of 6000 cells/well. After 24 hours incubation, the cell culture medium was replaced with the formulation for testing (200. mu.L/well in complete Dulbecco's Modified Eagle's medium). After culturing the cells for a specified duration (e.g., 12 hours or 24 hours), 20. mu.l of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) solution (5mg/ml) was added to each well 180. mu.l DMEM and incubated at 37 ℃ for 4 hours. After removal of the medium, the insoluble crystals were dissolved in dimethyl sulfoxide (DMSO, 150. mu.l/well) and measured spectrophotometrically at a wavelength of 570nm in an ELISA reader (RT-6000, Rayto, China). Relative cell viability (%) compared to control wells containing cell culture medium alone (except for cells) was calculated by dividing the optical density of the test wells by the optical density of the control wells. All samples were run in five replicates.
7. Endocytosis inhibition study
In physical inhibition studies, HeLa cells were incubated in cell culture medium for 1 hour at 4 ℃ and then the medium was replaced with the nanoparticle formulation (in cell culture medium). In the chemical inhibition study, HeLa cells were treated with cytochalasin D containing cell culture medium (2 μ M) for 30 minutes, and then the medium was replaced with the nanoparticle preparation (in cell culture medium). After 1 hour of nanoparticle delivery, cells were washed five times with phosphate buffer solution and then imaged using a live cell spinning disc confocal microscope system.
8. Cell membrane leakage study (LDH assay)
To assess cell membrane damage caused by a particular formulation, HeLa cells were seeded in 96-well plates (Corning Costar, China) at a density of 6000 cells/well. After culturing the cells for 24 hours, the test was performed with the preparation instead of the cell culture Medium (200. mu.L/well, intact Dulbecco's Modified Eagle's Medium). After incubating the cells for 12 hours and 24 hours, respectively, 120. mu.l/well of the sample supernatant was collected and analyzed for released Lactate Dehydrogenase (LDH) by a commercial kit (C0017, Beyotime, China). LDH release values for cells treated with LDH-releasing agent were set to 100% LDH release (i.e. total LDH amount in intact cells), while LDH release values for untreated cells were set to negative controls. The optical density was measured by an ELISA reader (RT-6000, Rayto, China) at a wavelength of 490nm (test) and 630nm (reference). Relative LDH release is defined by the total LDH ratio released by LDH in intact cells. Cell samples with less than 10% LDH release were considered to be cells with intact cell membranes, following well established standards. All samples were run in five replicates.
9. Vesicle co-localization study
Intracellular vesicles were labeled with the lipophilic fluorescent dye DiR (5 μ M, incubated with live cells for 30 min). At a given time point, cells were washed three times with PBS and then imaged with a live cell spinning disk confocal imaging system. Co-localization with fluorescent nanoparticles was quantified by using Pearson correlation coefficients.
10. Vesicle membrane integrity study
The integrity of intracellular vesicles was studied by using the fluorescent dye calcein. Calcein molecules (water soluble, 250 μ M) are internalized into intracellular vesicles of living cells by endocytosis (incubation with cells for 30 minutes). At this concentration (with intact vesicles), the fluorescence of calcein is self-quenched, punctate, and weakly fluorescent. If the vesicles break, the calcein molecules are released into the cytoplasm and show diffuse and intense fluorescence.
11. In vitro protein binding studies
The cS-bQDs-Tat (20nM) was mixed with bovine serum albumin (BSA, 15mg/mL) in water at room temperature under different concentrations of organic cosolvent (DMF), including 0.5%, 2% and 5%, mixed with phosphate buffer. Three samples were used for each organic solvent concentration. The mixture was dialyzed against water (with respective organic solvent concentrations) to remove free BSA (BSA not bound to the nanoparticle surface) with a molecular weight cut-off of 200 kdaltons. The amount of free BSA was determined by UV-Vis absorption spectroscopy and measured at 280nm against a calibration curve (after the value had stabilized 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.
12. Single particle tracking
Live cell spinning disk confocal microscopy was performed to collect images (movies) for data analysis for single particle tracking (without moving the sample on the same cell sample as the data analysis for the correlation function). Data analysis was performed using a MATLAB computer program. Briefly, the algorithm links the positions of the particles between successive time frames and then links the resulting track segments into a complete trajectory. The algorithm gives an optimized trajectory to minimize the effects caused by high particle density, particle motion heterogeneity, temporal particle disappearance, and particle merging and fragmentation. For a given trajectory, the Mean Squared Displacement (MSD) is calculated for different durations (Δ t). Finding the best fit to the MSD- Δ t relationship in the following equation results in a motion pattern and corresponding characteristic constants:
directional movement: wherein V is the velocity;
normal diffusion: wherein D is the diffusion coefficient;
abnormal diffusion: wherein α <1 and D are diffusion coefficients;
and (3) wrinkle diffusion: where L2 is the wrap gate dimension and D is the diffusion coefficient.
13. Analysis of the correlation function (pCF)
Live cell spinning disk confocal microscopy was performed to collect images (movies) for pCF data analysis (on the same cell sample as single particle tracked data analysis, without moving the sample). Data analysis was performed using a MATLAB computer program written. The program of the present invention contains data from a rotating disk scan. Briefly, the fluorescence intensity is presented at different locations and at different points in time, where the x-coordinate corresponds to the location point along a line (pixel) drawn on the image view and the y-coordinate corresponds to time. The pCF of two points at a distance δ r as a function of the propagation time τ is calculated by the following equation
The following equation:
Figure BDA0001865548930000171
the maximum value of the derived pCF profile is determined as the average time taken for the particle to travel a given distance. To investigate the vesicle escape propagation time in a given direction, a line was drawn in that direction and the pCF profile was calculated. For two position points on the line, one is located inside the vesicle and the other is located outside the vesicle.
14. Delivery of the Small molecule drug Doxorubicin (DOX)
To load DOX onto the nanoparticle surface, the nanoparticle dispersion (e.g., cS-bQDs-Tat) is mixed with DOX (dissolved in an organic solvent) at 4 ℃ for 12 hours. Dialysis (molecular weight cut-off 100 kdalton) was performed to purify the drug-loaded nanoparticles. The removed free drug was measured by UV-Vis absorption spectroscopy against a calibration curve (absorption at 480 nm). Drug loading is then quantified based on the amount of free drug removed. The drug loading efficiency (percentage of the amount of drug successfully loaded relative to the total amount of drug added) was found to be > 70% for all nanoparticle formulations. The amount of drug molecules loaded per particle was estimated to be 70. triplicate samples of each different formulation were studied. Drug-loaded nanoparticle dispersions (in cell culture media) were incubated with HeLa cells. At different time points after the start of incubation, cancer cell viability was measured by MTT assay and nanoparticle and DOX distribution was imaged by rotating disc confocal microscopy.
15. Delivery of protein ovalbumin (ova)
Tat-ova-SDot was prepared by conjugating Tat-ova to SDot using EDC chemistry. Three different formulations (i.e., Tat dye, Tat-ova-SDot and Tat-ova dye) were incubated with HeLa cells and the distribution of the fluorescent probes was imaged by a rotating disk confocal microscope at different time points after incubation. And starting. Nuclear targeting was quantified by measuring the fluorescence intensity in the nuclear region relative to the fluorescence intensity in the entire cellular region (100-200 cells per formulation).
Example 1: preparation and physicochemical characterization of cS-bQDs-Tat
To prepare cS-bQDs-Tat, the surface of a small fraction (e.g., 10%) of the bare hydrophobic QDs (bQDs; the surface ligand trioctylphosphine, or shortly TOP) is first replaced with cysteamine, a bifunctional small molecule ligand, in an organic solvent (fig. 2 a). Organic solvents tested included Dimethylformamide (DMF), acetone, ethanol, and dimethyl sulfoxide (DMSO). DMF has the best overall performance. The Tat peptide was then attached to the QD surface by conjugation with cysteamine via 1-cysteine-3- (3-dimethylaminopropyl) carbodiimide (EDC) chemistry to form bQDs-Tat (figure 2 a). Subsequently, bQDs-Tat stored in an organic solvent is diluted in water to form cS-bQDs-Tat (FIG. 2 a). Surface modification was confirmed by zeta potential, fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. With the addition of cysteamine and Tat peptide, the positive potential of the surface charge becomes higher and higher, consistent with the molecular structure of TOP (zero number of cationic functional groups per molecule), cysteamine (one cationic functional group per molecule) and Tat peptide (multiple cationic functional groups per molecule). Multiple cationic functional groups per molecule) (fig. 2 b). FTIR and XPS results of cS-bQDs-Tat show characteristic peaks (amide bond formation) for successful conjugation. The addition of cysteamine only increased the nanoparticle diameter measured by Dynamic Light Scattering (DLS) by-0.05 nm compared to bQD (fig. 2 c). Conjugation to Tat increased by about another 0.5nm in diameter of the DLS (fig. 2 c). The nanoparticle diameter as measured by Transmission Electron Microscopy (TEM) was about 3nm smaller than the DLS diameter. The fluorescence properties of the bare hydrophobic QDs were largely maintained by cS-bQDs-Tat (about 20% reduction in fluorescence quantum efficiency, FIG. 2 d). Overall, reducing the surface of QDs in SDot systems substituted with hydrophilic molecules or functional groups increases the fluorescence quantum efficiency. If the natural evaporation loss is compensated without adding the organic cosolvent, the cS-bQD-Tat probe can maintain colloidal stability for about 5 days at 4 ℃ in a closed container, and the colloidal stability can be maintained for more than one month by regularly refilling the organic cosolvent to compensate the evaporation of the organic cosolvent (FIG. 2 d). Solubility of SDots in aqueous environments can easily reach 50nM or higher. Increasing the percentage of organic co-solvent increases the SDot water solubility. In practice, the percentage of organic co-solvent used in the SDot system is below 3% (typically 1%) to minimize cytotoxicity.
SDot overcomes some of the key limitations of the current two major approaches to transferring hydrophobic QDs to an aqueous environment, which are to cover the entire QD surface with polymers or small molecules, respectively. On the one hand, the use of polymers as coatings typically add 7-30nm in nanoparticle diameter, which increases by at least 1.5-2nm even with optimized polymer structure diameter, thus limiting the biological transport of QDs in crowded spaces, such as intracellular spaces. On the other hand, replacing the original ligands on the entire bQD surface with bifunctional small molecules often results in poor fluorescence quantum efficiency and colloidal stability. In contrast, SDot provides minimized particle size (by eliminating the use of a polymer coating) and good fluorescence quantum efficiency and colloidal stability (by keeping most of the original surface ligands of bQD intact and using organic cosolvents to increase water solubility). Experiments performed on two control samples, i.e., 1) all of the original surface ligands on the bQDs were replaced with cysteamine, and 2) bQD a small portion (10%) of the surface ligands were replaced with cysteamine without the addition of any organic co-solvent, both control experiments showed poor fluorescence and colloidal stability, thus confirming that the co-presence of a small portion of surface ligand substitutions and organic co-solvent was required to obtain the best physicochemical properties of SDot. In addition to the ultra-small particle size, SDot has a hydrophobic nanoparticle surface and an organic cosolvent, which can greatly enhance transport across cellular barriers.
Example 2: intracellular targeting of cS-bQDs-Tat in living cells
cS-bQDs-Tat shows surprising intracellular targeting in living cells. In 30 independent experiments (performed by 3 independent investigators), 200 cells were studied in each experiment by fluorescence imaging, and in 95% of the studied cells (approximately 5% of the studied cells did not have internalized QDs), cS-bQDs-Tat reliably produced a nuclear targeting specificity (defined as the percentage of the amount of QDs in the nucleus relative to the total amount of QDs in the cell) of approximately 95% (fig. 3a includes more targeted confocal images). This result is consistent with that obtained by isolating nuclei from a cell suspension and then performing fluorescence spectroscopy. Within the nucleus, cS-bQDs-Tat is found to accumulate frequently in the nucleolus, a specific intranuclear region where ribosome biogenesis occurs. This result indicates that cS-bQD-Tat can also overcome dyskinesia in the nucleus and achieve nuclear targeting (fig. 3b, included a more nucleolar targeted confocal image).
Three control experiments more elucidated the intracellular targeting role of cS-bQDs-Tat. First, cS-bQDs-cysteamine (i.e., SDot without the biomolecule Tat peptide) does not enter the nucleus. This supports the discovery that nuclear targeting of cS-bQDs-Tat requires the biological function of the Tat peptide. Second, the finding that traditional Tat peptide-coupled water-soluble QDs (QDs-Tat) do not enter the nucleus, probably because they are trapped in intracellular vesicles (due to the bulkiness of QDs compared to small molecules), supports the importance of new nanoparticle design for intracellular targeting. The water-soluble QDs-Tat is prepared by dissolving bQD in water with phospholipid-PEG micelle; in QDs-Tat, the number of Tat peptides conjugated per QD is the same as the number of peptides conjugated per cS-bQDs-Tat. Third, the application of the commonly used vesicle-disrupting drug chloroquine (50. mu.M, or 16. mu.g/mL) did not significantly improve the nuclear targeting of QDs-Tat, but produced considerable cytotoxicity. This further supports vesicle capture as a major obstacle to intracellular targeting of conventional QD probes and suggests that the QD probe design of the present invention is significantly superior to chloroquine in its ability to overcome vesicle capture in terms of potency and non-invasiveness.
The targeted delivery process of cS-bQDs-Tat to the nucleus was fast and efficient, indicating its powerful ability to cross the cell trafficking barrier (fig. 3 c). As shown by fluorescence confocal microscopy in live HeLa cells, within 5-15 minutes almost all of the cS-bQDs-Tat in all cells has crossed the cell membrane into the cell (a few cS-bQDs-Tat remain bound to the cell surface); some of the cS-bQDs-Tat had entered the nucleus (FIG. 3 c). By 45-60 minutes, some of the cS-bQDs-Tat had entered the nucleus in more than half of the cells (FIG. 3 c). By 4-6 hours, about half of the cS-bQDs-Tat had entered the nucleus (FIG. 3 c). By 8-12 hours, almost all of the cS-bQDs-Tat entered the nucleus and was normally accumulated in the nucleolus (100-200 cells were examined in a time-dependent study, FIG. 3 c). The rate of targeted nuclear delivery process of cS-bQDs-Tat is comparable to the rate of rapid adeno-associated virus infection (nuclear targeting) of HeLa cells in the prior art. The nuclear targeting ability of cS-bQD-Tat was demonstrated in all four cell types tested [ HeLa (cervical carcinoma), MCF-7 (breast carcinoma), NIH 3T3 (fibroblast) and Hep G2 (liver carcinoma) ] (FIG. 3 d). All four low concentrations of organic co-solvents (DMF, acetone, ethanol and DMSO; 1% concentration of organic co-solvent used; 10% concentration of nanoparticles used) used in cS-bQD-Tat caused little cytotoxicity (FIG. 3 e). SDot targeted delivery technology is convenient and scalable because the manipulations involve mainly simple mixing and culturing, and targeted delivery can be performed on millions of cells simultaneously.
Example 3: study of cell trafficking mechanism of cS-bQDs-Tat
This example was conducted by a mechanistic study to gain insight into the biological transport process of SDots. The importance of four key nanoprobe parameters (hydrophobic nanoscale surface, mixed solvents, small size and biomolecules) for intracellular targeting of SDot was confirmed by studying each parameter individually and systematically. As shown in fig. 4a, b, c, increasing the hydrophobic surface coverage of QDs (from 0% to 50%, to 75%, to 90%), or increasing the percent of co-solvent in water (DMF from 0.5% to 1%) or decreasing the particle size (TEM diameter from 6.9nm to 4.5nm, to 3.4nm, to 2.6nm) enhances the nuclear targeting of cS-bQDs-Tat. Further increasing the DMF percentage from 1% to 1.5% did not significantly improve the targeting effect, indicating that the delivery enhancing effect given by the mixed solvents was saturable. In addition, as previously described, the deletion of Tat peptide from nanoprobes eliminates the nuclear targeting effect.
Blockade of endocytosis by low temperature (physical inhibitor) or cytochalasin D (chemical inhibitor) only partially prevented cS-bQDs-Tat from entering cells, whereas conventional Tat peptide-conjugated water-soluble QDs (QDs-Tat) did not exhibit cellular internalization when used, even with an inhibitor of endocytosis (fig. 4D). These results indicate that, unlike QDs-Tat, cS-bQDs-Tat enters the cellular moiety through a mechanism independent of endocytosis (direct penetration). In addition, these results have an interesting effect on the cellular uptake mechanism in Tat peptide-mediated delivery, i.e. altering the properties of the cargo may lead to an alteration of the cellular uptake mechanism in Tat peptide-mediated delivery. Cell membrane leakage studies by Lactate Dehydrogenase (LDH) assay gave < 10% LDH release results (meaning no significant membrane leakage, similar to control sample results) for all of the cS-bQD-Tat composition formulations tested, indicating that entry of cS-bQDs-Tat into cells did not cause cell membrane damage to the cells (1% co-solvent concentration used; 10nM nanoparticles concentration used) (FIG. 4 e). Thus, the moiety cS-bQDs-Tat can penetrate directly into the cell membrane without destroying the integrity of the membrane.
Endocytosis also plays an important role in cellular internalization of cS-bQDs-Tat as shown by co-localization studies of intracellular vesicle dyes. Co-localization studies using the vesicular dye DiR (lipophilic small molecule-labeled lipid membrane) showed that there was significant co-localization between DiR and cS-bQDs-Tat during the early stages of its intracellular transport process, indicating that this fraction of cS-bQDs-Tat was internalized by cells via endocytosis (in addition to the endocytosis-independent cells described above) (FIG. 4 f). The degree of intracellular vesicle co-localization decreases with time, approaching zero late in the intracellular transport process of cS-bQDs-Tat. This indicates that the nanoparticles initially trapped in the vesicle can escape completely from the vesicle trap (fig. 4 f).
In addition, another fluorescent dye, calcein, was used to explore the vesicle escape mechanism of cS-bQDs-Tat. Calcein is a water-soluble small molecule fluorescent dye that self-quenches at concentrations above a certain threshold, and is commonly used as an indicator of lipid vesicle integrity. When the vesicles were intact, calcein molecules (high concentration selected based on their self-quenching properties) were trapped inside the vesicles and showed punctate and weak fluorescence (fig. 4g, top left); when the vesicles burst, the calcein molecules are released into the cytoplasm and diffuse strong fluorescence is emitted [ fig. 4g, top right, intracellular vesicles are destroyed by the commonly used vesicle-destroying drug chloroquine to 50 μ M (16 μ g/mL); with the addition of this drug, many cells are unhealthy due to vesicle destruction ]. It was found that calcein remained punctate and weakly fluorescent throughout the intracellular transport of cS-bQDs-Tat (significantly co-localized with calcein during the early phase of nanoparticle delivery, and not co-localized with calcein during the late phase of nanoparticle delivery), indicating the integrity of the vesicular membrane (fig. 4g, bottom left and bottom right). Therefore, endocytosed cS-bQDs-Tat can escape the intracellular vesicles completely without compromising the integrity of the vesicle membrane. It can be observed by direct observation that nanoparticles (SDot) can efficiently and reliably escape intracellular vesicles without disrupting the vesicle membrane (i.e., a highly efficient and non-invasive escape process), while small molecules (calcein, hydrophilic) are two orders of magnitude smaller than nanoparticles and cannot efficiently cross intracellular vesicle membranes unless the vesicles break.
In vitro studies of binding to the model protein Bovine Serum Albumin (BSA) indicate that mixed solvents in the SDot design can help reduce non-specific protein binding of nanoparticles. The cS-bQDs-Tat (20nM) was incubated with BSA (15mg/mL) in the presence of different concentrations (0.5%, 2% and 5%) of the organic cosolvent DMF for the same time. BSA bound to the nanoparticle surface was then measured using UV-Vis absorption spectroscopy. It was found that increased organic solvent concentration resulted in a significant reduction in the amount of BSA bound to the nanoparticle surface (triplicate samples of each formulation), indicating the effect of mixed solvents on reducing the protein fouling of the nanoparticles (FIG. 4 h). It should be mentioned that the organic solvent concentration level of 0% is not included in the experiment, since SDot cannot have good physicochemical properties without any organic solvent, as previously described.
Example 4: single particle tracking and analysis of cellular trafficking kinetics of cS-bQDs-Tat for correlation function
This example uses the cellular transport of cS-bQDs-Tat as a model system to demonstrate the study of the kinetic transport process of organisms using single particle tracking in combination with rotating disk confocal microscopy (iSPT-pCF-SDCM). Single particle tracking and pCF analysis were performed on the same samples at the same observation stage of a rotating disk confocal microscope. This combined analysis method provides supplementary information about the dynamic transmission process.
Using single particle tracking, 2,500 QD motion trajectories were obtained in-30 cells (representative trajectories are shown in figure 5 a). The trajectory of the fluorescent object is formed by connecting its positions at different points in time, which are determined by using a commercial rotating disk confocal microscope. For each trajectory, a quantitative relationship of Mean Squared Displacement (MSD) to duration (Δ t) is calculated, the motion is classified based on MSD- Δ t relationship (including directional motion, normal diffusion, abnormal diffusion and angular diffusion), and the characteristic constants of the corresponding motion pattern are determined.
The present example of the study of directional motion reveals an interesting distribution of velocity values. In the cytoplasm, the velocity distribution exhibited multiple Gaussian peaks with approximate multiples of the peak (220nm/s, -1 ×; 620nm/s, -3 ×; 820nm/s, -4 ×; 980nm/s, -5 ×; 1,300nm/s, -6 ×, FIG. 5 b). This indicates that multiple molecular motors act in concert to drive directional motion. Given that all cS-bQDs-Tat eventually enters the nucleus and dynein is responsible for the inward directed movement of cargo into the nucleus in the cell, dynein may be the major motor protein involved. The peak value of the lowest velocity was 220nm/s, which is in good agreement with the in vitro velocity values of the dynein motor already disclosed (FIG. 5 b). In addition, many directional motion trajectories were also found in the nucleus region, and the velocity profile showed multiple peaks close together (peak at lowest velocity 270nm/s, FIG. 5 c). It has previously been reported that the directed movement in the nuclear region is used for cellular transport of adeno-associated virus, and it is believed that the directed movement of the virus in the nuclear region is given by the movement of microtubules in invaginated channels along the nuclear membrane (rather than in the nucleoplasm). Thus, the present inventors speculate that the directed movement of cS-bQDs-Tat in the nuclear region is caused by motor protein-driven movement along microtubules in these channels formed by the nuclear membrane. Given the small diameter (about 50nm) limitations of the channel, the cS-bQDs-Tat that moves within the invaginated channel may be outside the intracellular vesicle. Whether (and, if so, how) such movement is associated with nuclear entry of cS-bQDs-Tat is a question to be investigated.
A dual correlation function (pCF) analysis was used to study the movement of vesicle escape within QD cells. The cross-correlation function of the fluorescence intensity (function of time) for a pair of position points, one located inside the vesicle and the other outside the vesicle, shows a maximum peak (in the direction of the line crossing the two position points) at the time value corresponding to the transit time for the vesicle to escape (fig. 5 d). Fluorescence intensities at different time points at different location points in the cells were measured using a commercial rotating disk confocal microscope and then pCF was calculated using a written computer program. The passage time for vesicle escape of cS-bQDs-Tat was found to be 3.6ms (standard deviation 1.7ms, measured on 312 lines drawn across the membrane of 156 vesicles out of 3 cells) (fig. 5 e). This is significantly faster than the vesicle escape transit time of conventional water-soluble QDs-Tat in the presence of 1% DMF (mean 15.4ms, standard deviation 3.1 ms; measured on 240 lines drawn across the membrane of 120 vesicles out of 2 cells) (fig. 5 e). Furthermore, conventional water-soluble QDs-Tat shows little vesicle escape in the absence of DMF (fig. 5 e). The finding that the vesicle escape transit time of conventional water-soluble QD-Tat can be reduced using 1% DMF suggests that mixed solvents can also serve as a convenient and independent strategy for promoting vesicle escape of the bulk material. Furthermore, the inventors found that the vesicle escape process of cS-bQDs-Tat is isotropic, as they show almost the same transit time in different directions from the same vesicle escape (fig. 5 f). This conclusion is from measurements taken on 312 lines drawn across the membrane of 156 vesicles out of 3 cells; the two lines drawn for the same vesicle have perpendicular directions. This is consistent with the finding that vesicles remain intact during vesicle escape of cS-bQDs-Tat, as the fragmented vesicle membrane will make it easier for the nanoparticles to escape from some direction (fig. 4 g).
Example 5: enhanced delivery of drugs and macromolecules with cS-bQDs-Tat
This example investigated the potential of cS-bQDs-Tat to be used as a class of biological delivery vehicles for small molecule drugs and biological macromolecules. DNA-bound anticancer drug Doxorubicin (DOX) was loaded onto the particle surface of cS-bQDs-Tat by adsorption (. about.70 DOX/particle). In cell culture experiments with HeLa cells (cervical cancer cell line) as model cancer cells, the inventors found that DOX-loaded cS-bQDs-Tat can provide the same killing of cancer cells at a faster rate and at a lower drug dose compared to the formulation of control DOX (fig. 6 a). For example, to kill 30% of cancer cells, DOX-loaded cS-bQDs-Tat (formulation name SDots-Tat-DOX) need only be applied at a dose of DOX of 0.1. mu.g/mL for 4 hours. In contrast, to achieve the same cancer cell killing effect, the same dose of free DOX formulation required >12 hours, or a higher dose (5 μ g/mL) used over the same time period; the same dose of conventional Tat peptide conjugated water soluble QDs loaded with DOX (formulation name QDs-Tat-DOX) required >12 hours, or a higher dose (5 μ g/mL) was used over the same time; DOX dose conventional water soluble QDs loaded with DOX (without Tat peptide) (formulation name QDs-DOX) required > >12h, or higher doses (> 10. mu.g/mL) were used over the same time period. The reason why the DOX-loaded cS-bQDs-Tat formulation can save time (3-fold reduction) or reduce dose (50-fold reduction) is that cS-bQDs-Tat has unprecedented high efficiency, the ability to specifically deliver drugs into the drug-acting organelles (nuclei) and the ability to efficiently, specifically, directly into the drug-acting organelles (nuclei). Results of the double-color fluorescence microscope show that high co-localization exists between DOX fluorescence and fluorescence of the cS-bQDs-Tat loaded with DOX in the cell transport process, and the DOX molecules disaggregated on the surface of the nanoparticle are few. It is worth noting that conventional water-soluble QDs-Tat (formulation name QDs-Tat-DOX) loaded with DOX did not significantly improve the cancer cell killing effect compared to the free DOX formulation (fig. 6 a). This is probably because, although QDs-Tat can promote the process of DOX entering the microtubule tissue center (MTOC) located outside the nucleus, water-soluble QDs-Tat loaded with DOX is trapped in intracellular vesicles and fails to overcome the vesicle trapping barrier. This result again underscores the importance of the new nanomaterial design of SDot technology in overcoming cell trafficking obstacles.
Another finding of this example is that cS-bQDs-Tat can facilitate intracellular targeted delivery of biological macromolecules. Transport barriers to intracellular vesicle capture are considered to be a major challenge for macromolecule-based therapies. It is well known that macromolecules [ e.g. proteins delivered by Tat peptides and small interfering rnas (sirnas) ] are almost completely (estimated > 99%) trapped in intracellular vesicles and therefore cannot enter the cytoplasm and organelles (including the nucleus) outside the vesicles. The cS-bQDs-Tat was conjugated to a model protein, ovalbumin (ova, MW45kDa), and cell delivery and imaging experiments were performed. As a result, it was found that ovalbumin bound to cS-bQDs-Tat showed greatly enhanced delivery to the nucleus (. about.25%, almost zero nuclear delivery in the control sample, FIG. 6b), compared to Tat peptide-conjugated ovalbumin (labeled with fluorescent dye for imaging). Since the overall size of the cargo to be transported should be increased (two times or more) in combination with SDot, this result indicates that the combination of the hydrophobic nanoparticle surface and the mixed solvent is so significant in the biological transport enhancing effect that it can greatly improve the targeted transport of the cargo even when the size of the cargo is sharply increased.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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<120> hydrophobic Nanobiotic Probe
<130> P2018-1973
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10

Claims (14)

1. A nano biological probe is characterized in that the structure of the nano biological probe from inside to outside is shown as a formula I,
A-S-L-Bn(formula I)
In the formula (I), the compound is shown in the specification,
A-S are quantum dots, wherein S is the hydrophobic surface of the quantum dot;
b is a biomolecule Tat peptide;
l is an optional linking moiety coupling B to the surface of the quantum dot;
n is the number of biomolecules coupled to the surface of the quantum dot and is a positive integer greater than or equal to 1;
coupling a biomolecule Tat peptide and amino on the surface of a quantum dot by 1-half ethylamine-3- (3-dimethyl aminopropyl) carbodiimide;
the nano biological probe is dispersed in a mixed solvent;
the mixed solvent comprises an organic solvent and at least one non-organic solvent;
in the mixed solvent, an organic solvent is dimethylformamide DMF;
in the mixed solvent, the concentration of the organic solvent is 0.5-1 v/v%;
the diameter of the nano biological probe is 1 to 10 nm;
the hydrophobic surface coverage is 50%, 60%, 70%, 75%, 80% or 90%.
2. The nanobioprobe of claim 1, wherein the solubility of the nanobioprobe in aqueous environment is 1nM or more.
3. The nanobioprobe of claim 1, wherein S is the hydrophobic surface of the quantum dot itself or the surface of the hydrophobic ligand layer coating the quantum dot.
4. The nanobioprobe of claim 1, wherein the Tat peptide has the sequence of SEQ ID No. 1.
5. The nanobioprobe according to claim 1, which is stable in a closed container at 4 ℃ for 3 days or more.
6. The nanobioprobe of claim 1, wherein the excitation wavelength of the quantum dot is 100 to 1500 nm.
7. The nanobioprobe of claim 1, wherein the quantum dot emits light at a wavelength of 400 to 2500 nm.
8. A nano-bio probe product, comprising:
(a) a first container, and the nanobiotrobe of claim 1 located in the first container;
(b) a second container and a mixed solvent in the second container.
9. A method for preparing the nanobiotrobe of claim 1 or the nanobiotrobe product of claim 8, comprising the steps of:
(i) synthesizing quantum dots, wherein the surfaces of the quantum dots are hydrophobic surfaces or wrapped with hydrophobic ligand layers;
(ii) coupling biological molecules to the surfaces of the quantum dots to obtain hydrophobic quantum dot-biological molecule conjugates;
(iii) (iii) treating the hydrophobic quantum dot-biomolecule conjugate obtained in the step (ii) by using a mixed solvent to obtain the nano-biological probe.
10. A method for intracellular delivery of a biologically active molecule comprising coupling the biologically active molecule to the nanobiotrobe of claim 1 and mixing with a culture medium containing cells;
the method is performed in vitro, and the method is non-diagnostic and non-therapeutic.
11. Use of the nanobody probe of claim 1 for the preparation of a formulation for the intracellular delivery of a biologically active molecule.
12. The use of claim 11, wherein the cell comprises: non-cancerous or cancerous cells.
13. The use of claim 12, wherein the cancer cells comprise: cervical cancer cells, breast cancer cells, liver cancer cells, lung cancer cells, stomach cancer cells, prostate cancer cells, or a combination thereof.
14. Use of the nanobody probe of claim 1 for targeting inside living cells;
the localization is performed in vitro;
and the localization is non-diagnostic and non-therapeutic.
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