CN105582549A - Low-toxicity quantum dot preparation and preparation method and application thereof - Google Patents
Low-toxicity quantum dot preparation and preparation method and application thereof Download PDFInfo
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- CN105582549A CN105582549A CN201410557662.9A CN201410557662A CN105582549A CN 105582549 A CN105582549 A CN 105582549A CN 201410557662 A CN201410557662 A CN 201410557662A CN 105582549 A CN105582549 A CN 105582549A
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Abstract
The invention belongs to the field of biotechnology and nano-biomaterials and relates to preparation of a low-toxicity quantum dot preparation and its application. The prepared low-toxicity quantum dot preparation is a low-toxicity quantum dot preparation or composition which is composed of a quantum dot and one or more autophagy/reinforcing agents. The novel quantum dot preparation can guarantee luminous intensity of quantum dots. The preparation has advantages of simple preparation technology and extensive applicability, and can be used in large-scale production of low-toxicity quantum dots. The preparation can be used in treatment, diagnosis and detection of various diseases, including tracing of tumors, detection of inflammation and the like, and also can be used in imaging of internal organs or intracellular imaging and localization.
Description
Technical field
The invention belongs to biotechnology and nano meter biomaterial field, relate to a kind of low toxicity quantum dot preparation and its production and use, be specifically related to novel quantum dot preparation or composition that one or more cell autophagy inhibitor are made, this novel quantum dot preparation or composition can reduce the toxicity of traditional quantum dot preparation.
Background technology
Prior art discloses quantum dot (quantumdot, QD) and can be described as again semiconductor nanocrystals body (semiconductornanocrystal), and studying at present more is mainly CdX (X=S, Se, Te). Described quantum dot is due to its particle diameter little (approximately 1～100nm), and electronics and hole, by quantum confinement, can be with and become the discrete energy levels structure with molecular characterization continuously, therefore its optics behavior and some large molecules, for example, the aromatic hydrocarbon of many rings is very similar, can emitting fluorescence. Studies show that the volume size of quantum dot strictly controlling its light absorption and emission characteristic, crystal grain is less, specific area is larger, be distributed in surperficial atom just more, and the light activated positive electron on surface or negatron are subject to the constraint effect of passivated surface just larger, its dissimulated electricity can be just higher, the luminous energy absorbing is also higher, have quantum size effect (quantumsizeeffect), thereby make its absorption band blue shift, fluorescent emission peak position is corresponding blue shift also. Research also shows that independent quantum dot particle is easily subject to the impact of impurity and lattice defect, fluorescence quantum yield is very low, but work as taking it as core, coated with another kind of semi-conducting material, form after core-shell (core-shell) structure, quantum yield can be brought up to approximately 50%, even higher, and on extinction coefficient, have the increase of several times, thereby there is very strong fluorescent emission. The nano particle of synthetic multiple nucleocapsid structure has CdS/Ag at present2S、CdS/Cd(OH)2, CdS/ZnS, ZnS/CdSe, ZnSe/CdSe, CdS/HgS, CdS/PbS etc. and sandwich construction CdS/HgS/CdS. Described quantum dot is more stable than organic fluorescence molecule, and it can stand repeated multiple times exciting, and does not resemble easy generation fluorescent bleach organic fluorescence molecule.
In view of its unique Photophysics of quantum dot, it is widely used in cell in vitro mark. Than traditional organic fluorescent dye, the main advantage of quantum dot is that it has stronger bleach-resistant performance (from a few minutes by several hours), higher extinction coefficient (0.5～5 × 106L/(mol·cm－1)) and quantum yield. Have and studies show that by the quantum dot-labeled image obtaining to there is better contrast and definition, therefore can in most of fields, substitute organic dyestuff. In recent years, quantum dot has been obtained larger progress for imaging in cell, and the component in a lot of living cells and fixed cell and albumen are by quantum dot-labeled, imaging, as nucleus, mitochondria, microtubule, microfilament protein, Cytokeratin and endocytosis body etc.; Meanwhile, quantum dot is also used for studying epicyte protein and acceptor, comprises PSMA (PSMA), HER kinases, Glycine Receptors, thrombocytin transport protein, p-glycoprotein, Band III albumen and sodium-potassium ATPase etc. Quantum dot best one side in immunofluorescence application is that micro-quantum dot just can produce detection signal. Some researchs have reported that quantum dot has flickering phenomenon in cell sample, is called as " flicker "; This specific character of quantum dot can be brought up to single molecules level immunofluorescence detection sensitivity.
Quantum dot makes them become the in vivo preferential selection of fluorescence imaging and mark because having the advantage of the uniquenesses such as Near-infrared luminescence, long-time stability, high brightness fluorescent, anti-light bleaching. Akerman group, the quantum dot that sulfation targeting peptides is connected are expelled in canceration rat body, in histotomy, find that a large amount of polypeptide quantum dot probes are enriched in (AkermanME in cancerous issue, ChanW, AkkonmenLP.Proc.Natl.Acad.Sci., 2002.99 (20): 12617-21). Quantum dot is injected into respectively the subcutaneous of mouse and pig by Kim group, and their directed movements, to post lymph node, excite (5 × 10-3W/cm by low-yield near-infrared2) obtain the fluorescence imaging (KimS of pig lymph node 1cm depth, LimY, SolteseE.Nature.Biotechnology.200422:93-97), the above results is that the in-vivo imaging of living animal is laid a good foundation, also for clinical medicine imaging, operating accuracy provide foundation.
Along with updating of quantum dot synthetic technology, quantum dot is expected to one of fluorescent marker becoming tool potentiality, especially in the application of active somatic cell marker field. But being not yet generalized to so far in clinical and real life, is mainly because quantum dot itself all has certain toxicity in vivo and in vitro; One of trace it to its cause: in the mechanism of quantum dot and cell, their surface chemistry group causes minimum micropore to cell membrane, has changed the large molecular concentration of the inside and outside ion concentration of cell poor and cell interior Cell protection integrality and normal operation function; It two is, the interaction of quantum dot and cell has directly caused Apoptosis and cell autophagy, thus killer cell. Traditional quantum dot preparation because of its toxicity larger, and method of attenuating be physics and chemistry method, use other materials to be coated with. Not only affect the original characteristic of quantum dot, also affect its luminous and imaging.
Cell autophagy (autophagy) is again II type programmed death (type II programmedcelldeath), it is common " self-digestion " phenomenon (cellulardegradation) in most eukaryotes, can decompose organelle impaired or unnecessary in cell and albumen and produce nucleotides, the small-molecule substances such as amino acid synthesize new protein for cell, and can maintain the stable of the interior microenvironment of cell. In recent years along with the development of molecular biology and gene technology and the deep understanding to cell autophagy, find that the development of itself and various diseases, especially tumour is in close relations. Be transported to the difference of mode in lyase body cavity according to substrate in cell, mammalian cell autophagy can be divided into three kinds of modes: large autophagy (macroautophagy, hereinafter to be referred as autophagy, itself and tumor development and treatment relation are the closest), the autophagy (chaperone-mediatedautophagy, CMA) of little autophagy (microautophagy) and molecular chaperones mediation. Autophagy is the biological process of endochylema macromolecular substances and organelle a large amount of degradeds in duplicature packing bubble, it comprises: 1. under the stimulation of some factor such as hunger, anoxic, interfering effects of drug, the double membrane structure of autophagic vacuole starts to form gradually and be enclosed in to be degraded thing around; 2. the material that the complete moulding of autophagic vacuole also will be degraded is isolated from cytoplasm completely; 3. autophagosome and lysosome merge and form autophagy lysosome; 4. autophagy lysosome is finally dissolved by the hydrolase in lysosome, and catabolite can recycling in cell. Studies show that, autophagy can produce stress reaction to outside environment change and various stimulation to cell. Can there is the autophagy of reduced levels in cell, also claim basic autophagy under growth conditions. But, once be subject to extraneous stimulation, to deprive etc. as hunger, anoxic, high temperature, high-cell density or growth factor, the level of cell autophagy will raise rapidly. As in the situation that nutriment lacks, the interior non-viable non-apoptotic cell device generation of cell autophagy energy decomposer amino acid etc., for the synthetic new protein of cell, maintain the survival of cell, but, as the one of apoptosis, cell autophagy can directly or indirectly cause cell death by number of ways. This killing and wounding may be to occur by mechanism such as cell death inducing, necrosis, agings, may be also to make cell that autophagy death directly occur.
So far, still see that cells involved autophagy inhibitor and quantum dot make to obtain the report of new formulation.
Summary of the invention
The object of the invention is the defect for overcoming prior art, a kind of low toxicity quantum dot preparation is provided. Be specifically related to a kind of low toxicity quantum dot preparation and its production and use, especially the quantum dot preparation or the composition that are made up of low toxicity a kind of quantum dot and one or more cell autophagy/reinforcing agents, this novel quantum dot preparation or composition can reduce the toxicity of traditional quantum dot preparation.
Low toxicity quantum dot preparation of the present invention, is made up of a kind of quantum dot and one or more cell autophagy/reinforcing agents, and it ensures the luminous intensity of quantum dot in the toxicity that reduces traditional quantum dot preparation; The preparation technology of said preparation is simple, and applicability is wide, can be used for large-scale production low toxicity quantum dot. Said preparation mainly can be used for treatment, the diagnosis of various diseases and detects, and comprises the spike of tumour, the detection of inflammation etc. Also can be used for organ imaging or the interior imaging of cell and location in body simultaneously.
Quantum dot of the present invention includes but not limited to cadmium telluride quantum dot (CdTe), ZnS quantum dots (ZnS) and CdSe quantum dots (CdSe), preferred cadmium telluride quantum dot (CdTe) in embodiments of the invention.
In the present invention, cell autophagy inhibitor comprises but is not limited to: chloroquine (CQ), 3-MA, fertile graceful penicillin, Bava Lip river mycin A-1, ammonium chloride and LY294002; In embodiments of the invention, preferred cell autophagy inhibitor is chloroquine, 3-MA and ammonium chloride.
Quantum dot of the present invention can be made composition with cell autophagy inhibitor, and quantum dot wherein and the sequential use of one or more cell autophagies can reduce the toxicity of quantum dot.
The present invention also provides the application in the treatment in disease of described quantum dot, diagnosis, detection; And the application of described quantum dot preparation in the treatment of disease, diagnosis, detection.
Low toxicity quantum dot preparation of the present invention, can be absorbed and send fluorescence by cell and internal organs; There is lower toxicity than traditional quantum dot preparation.
Low toxicity quantum dot preparation of the present invention is made up of quantum dot preparation or the composition of low toxicity a kind of quantum dot and one or more cell autophagy/reinforcing agents, this novel quantum dot preparation can ensure the luminous intensity of quantum dot simultaneously. The preparation technology of said preparation is simple, and applicability is wide, can be used for large-scale production low toxicity quantum dot. Said preparation can be used for for the preparation of the treatment of various diseases, diagnosis and detects preparation, comprises the spike of tumour, the detection of inflammation etc. Simultaneously also can be used for preparing the preparation of imaging and location in organ imaging in body or cell.
Brief description of the drawings
Fig. 1 is the qualification result of the quantum dot preparation of preparation, wherein,
A is the schematic diagram of CdTe quantum dot in quantum dot solution;
B, C, D, E has shown that respectively the hydration particle diameter of described quantum dot is 12nm, and Zeta potential is-32.8mV, and maximum excitation wavelength is 257nm, and maximum emission wavelength is 655nm.
Fig. 2 is in-vivo imaging and the distribution situation of the quantum dot preparation of preparation.
Fig. 3 is the quantum dot preparation of preparation and the cytotoxicity comparative result of simple quantum dot.
Fig. 4 is the comparative result of the quantum dot preparation of preparation and the toxicity in vivo of common quantum dot, wherein,
Fig. 4 A has shown the liver of common quantum dot after 30 days, and more serious cell autophagy has all appearred in kidney and spleen,
Fig. 4 B has shown that each group has all been caused liver to a certain degree, kidney and spleen toxicity,
Fig. 4 C has shown that liver function index is abnormal, and the damage that the quantum dot preparation of preparation causes liver kidney spleen is significantly less than common amount.
Detailed description of the invention
Embodiment 1
1) prepare quantum dot preparation:
(1) preparation of quantum dot: by appropriate CdCl2, NaHTe and MPA insert in reactor, with the NaBH of pH=9.04After dissolving under the protection of nitrogen in 95 DEG C of successive reactions 12 hours, then in reactor, add a certain amount of Na2S, in 60 DEG C of successive reactions 1.5 hours, the Cd:Te:S:MPA ratio in reactor should be 1:0.4:0.012:4.16. Be stored in 2 DEG C;
(2) preparation of quantum dot preparation: chloroquine is dissolved in 0.01MPBS (pH=7.4) and makes the chloroquine solution of 10 μ M, after the quantum dot preparing is dissolved in this solution, make heavy metal concentration Cd2+The qualification of=1 μ M gained quantum dot preparation as shown in Figure 1, wherein scheming A is the schematic diagram of CdTe quantum dot in quantum dot solution, the hydration particle diameter that has shown the quantum dot of preparation in described accompanying drawing is 12nm, Zeta potential is-32.8mV, maximum excitation wavelength is 257nm, and maximum emission wavelength is 655nm; There is the due character of general quantum dot; 2) in-vivo imaging of quantum dot preparation and distribution
Above-mentioned quantum dot preparation is entered in Mice Body by tail vein injection with the dosage of every mouse of 100 μ L, after 24h, observe imaging and distribution (as shown in Figure 2) in intracorporeal organ, experimental result shows, this quantum dot preparation has stronger body inner glow effect, and is distributed widely in lung, liver, spleen, in kidney; And in experimentation, mouse shows no obvious abnormalities, prove the relative safety of said preparation;
3) cytotoxicity of novel quantum dot preparation and quantum dot contrast
It is to carry out respectively cell experiment after 20nM that quantum dot and novel quantum dot preparation are diluted to heavy metal concentration, jointly hatches after 0-72h with cell, and result shows, each experimental group is to HeG2Cell all has obvious growth inhibition effect, but show in the contrast of 48h and two groups of experiments of 72h, novel quantum dot preparation is compared simple quantum dot, there is lower toxicity, its CDCC is only 40%-60%, > 80% of common quantum dot toxicity, has shown the in vitro toxicity that it is lower;
4) toxicity in vivo of novel quantum dot preparation and common quantum dot contrast
The quantum dot of 0.1nmol-0.3nmol and novel quantum dot preparation are entered in Mice Body by tail vein injection respectively, observe respectively 30 days, in 30 days, respectively organize all without dead mouse, after being put to death, mouse gets haemanalysis its internal organs of dissection and analysis, result as shown in Figure 4, the liver of common quantum dot after 30 days, kidney and spleen have all occurred as shown in more serious cell autophagy (as Fig. 4 A), each group has all been caused liver to a certain degree, kidney and spleen toxicity (as shown in Figure 4 B) and liver function index abnormal (as shown in Figure 4 C), show through comparing result, the damage that novel quantum dot preparation causes liver kidney spleen is significantly less than the damage that common quantum dot causes, prove novel quantum dot preparation relative hypotoxicity in vivo, and make new formulation with cell autophagy inhibitor and can alleviate quantum dot toxicity in vivo.
Claims (9)
1. a low toxicity quantum dot preparation, is characterized in that, by a kind of quantum dot and one or more cell autophagies/The quantum dot preparation of reinforcing agent composition low toxicity, said preparation can be absorbed and send fluorescence by cell and internal organs; Its toxicityLow.
2. by low toxicity quantum dot preparation claimed in claim 1, it is characterized in that, described quantum dot is selected from telluriumCadmium quantum dot (CdTe), ZnS quantum dots (ZnS) is or/and CdSe quantum dots (CdSe).
3. by low toxicity quantum dot preparation claimed in claim 1, it is characterized in that, described quantum dot is selected from telluriumCadmium quantum dot (CdTe).
4. by low toxicity quantum dot preparation claimed in claim 1, it is characterized in that described cell autophagy inhibitorBe selected from chloroquine (CQ), 3-MA, fertile graceful penicillin, Bava Lip river mycin A-1, ammonium chloride or LY294002.
5. by low toxicity quantum dot preparation claimed in claim 1, it is characterized in that described cell autophagy inhibitorBe selected from chloroquine, 3-MA or ammonium chloride.
6. the composition being made up of a kind of quantum dot and one or more cell autophagy/reinforcing agents, is characterized in that instituteThe quantum dot of stating is selected from cadmium telluride quantum dot (CdTe), and ZnS quantum dots (ZnS) is or/and cadmium selenide quantumPoint (CdSe); Described cell autophagy inhibitor is selected from chloroquine (CQ), 3-MA, fertile graceful penicillin, Bava Lip riverMycin A-1, ammonium chloride or LY294002.
7. by composition claimed in claim 6, it is characterized in that described quantum dot and cell autophagy inhibitorSequential use.
Quantum dot preparation claimed in claim 1 for the preparation for the treatment of, diagnosis, detect in the preparation of diseaseApplication.
9. by the purposes of claim 8, it is characterized in that, described quantum dot for the preparation for the treatment of, diagnosis,Application in the preparation of detection disease.
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| CN104511017A (en) * | 2013-09-29 | 2015-04-15 | 复旦大学 | Drug composition reducing in-vivo and in-vitro toxicity of nano drug delivery material and preparation method thereof |
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| 于春艳等: "自噬溶酶体抑制剂氯化铵促进维生素K3诱导人宫颈癌HeLa 细胞凋亡的作用", 《中国病理生理杂志》 * |
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