CN105031649A - Doped self-assembled nano fiber structure and preparation method thereof - Google Patents
Doped self-assembled nano fiber structure and preparation method thereof Download PDFInfo
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- CN105031649A CN105031649A CN201510534572.2A CN201510534572A CN105031649A CN 105031649 A CN105031649 A CN 105031649A CN 201510534572 A CN201510534572 A CN 201510534572A CN 105031649 A CN105031649 A CN 105031649A
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Abstract
The invention discloses a doped self-assembled nano fiber structure and a preparation method thereof. Through synergistic interaction of molecules, nano fiber can obviously change near-infrared absorption of a cyanine dye, thereby obviously improving photo-acoustic performance and photo-thermal performance. Phosphatase response synchronous-assembly processes and diagnosis and treatment performance of assembly materials are verified respectively through cell and in vivo experiments. Particularly, through caudal vein injection, after 4 hours, tumors and peripheral normal tissues can be very clearly distinguished. According to the invention, the cyanine dye-doped nano fiber with in situ formation of tumor tissues can be used for fluorescence, photo-acoustic imaging and photo-thermal therapy.
Description
Technical field
The invention belongs to biological medicine material and nanosecond medical science field, be specifically related to one utilize the endogenous phosphatase of tumor to form indocyanine green (ICG) at tumor tissue specificity to adulterate self-assembled nanometer fibre structure, its preparation method and institute's metal nanometer material can be used for the diagnosis and treatment integration of tumor.
Background technology
Tumor thermotherapy has become a kind of important oncotherapy mode, and it is by exogenous irritant, such as, light, radio frequency, microwave, ultrasonic and magnetic field make cancerous tissue or cancerous cell heating dead, reach therapeutic purposes with this.Especially, photo-thermal therapy is present study hotspot, and because it has specific selectivity over time and space, and invasiveness is extremely low.When carrying out photo-thermal therapy, photothermal deformation agent absorbs and changes luminous energy, produces reach lethal temperature at tumor region.But pharmacokinetics and the chronobiological safety of most photothermal deformation agent of report are at present very poor, therefore all can not be used for clinical.
Summary of the invention
Primary and foremost purpose of the present invention is to provide one to utilize endogenous phosphatase to form ICG (1) doping self-assembled nanometer fibre structure (5) at tumor tissue specificity;
Another object of the present invention there are provided a kind of method that tumor tissues original position forms nanostructured;
Another object of the present invention is that the alkali phosphatase under phosphatase effect responds peptide (2) and the common self assembly of ICG (1) forms a kind of nanofiber;
Another object of the present invention is that in body, the nanofiber of self assembly has extraordinary tumour-specific;
The nanofiber that another object of the present invention is to provide self assembly in a kind of described body has the application of fluorescence imaging, photoacoustic imaging and photo-thermal therapy.
Object of the present invention can be achieved through the following technical solutions:
A kind of nanofiber, this nanofiber is obtained by assembling altogether by the product of the alkali phosphatase response peptide of phosphatase effect and cyanine dyes.
Wherein, cyanine dyes of the present invention is preferably indocyanine green, and except ICG, the analog of all cyanine dyes and derivant are all applicable to being doped in nanofiber.
The purposes of aforesaid nanofiber, it is preparing the application in tumour diagnostic reagent.
The purposes of aforesaid nanofiber, it treats the application in reagent in preparation tumor photo-thermal.
A preparation method for nanofiber, this nanofiber is obtained by assembling altogether by the product of the alkali phosphatase response peptide of phosphatase effect and ICG, and method comprises the steps:
ICG and NapFFKYp polypeptide is dissolved in the PBS of PH7-8 jointly, and both molar ratio range are 1:2-1:100, then adds the nanofiber that alkali phosphatase forms ICG doping, and the additional proportion scope of alkali phosphatase is 0.1-1U.
Beneficial effect of the present invention:
1. the invention provides a kind of tumor tissues original position and form nanostructured; Indocyanine green (ICG) is a kind of near-infrared tricarbocyanine, and ICG is made up of two lipotropy polycyclic moiety, is connected, and add a sulfo group in each multi-ring outside by polymethine chain, to strengthen its stability in water.Whole molecule is amphipathic, energy and plasma protein, especially albumin non-specific binding.After tail vein injection, ICG molecule is absorbed by hepatic parenchymal cells very soon, then with bile excretion.But its half-life only has 2-4 minute.In addition, ICG can not active targeting tumor, and its tumor accumulation efficiency is low especially.Therefore, a kind of effective mode is needed to extend circulation, improve tumor accumulation efficiency and holdup time.Through process of the present invention, contrast other nano-carrier, this supermolecule system of the present invention can avoid the picked-up of being rich in reticuloendothelial system organ effectively, thus has higher tumor accumulation efficiency and comparatively bearing tumor holdup time.
2. the nanofiber of the present invention's self assembly in vivo has extraordinary tumour-specific;
3. in the present invention, in body, the nanofiber of self assembly has the application of fluorescence imaging and photoacoustic imaging;
4. in the present invention, the nanofiber of self assembly has good photo-thermal therapy effect.
5. neoplasm in situ provided by the invention formed nanostructured may be used for tumor drug delivery, at body diagnosis and detection, treatment and curative effect evaluation etc.
Accompanying drawing explanation
Fig. 1 enzyme induction Supramolecular self assembly schematic diagram
Fig. 2 ICG (1, a) and alkali phosphatase response peptide NapFFKYp (2, structural formula b)
The transmission electron microscope photo of Fig. 3 ICG doping self-assembled nanometer fiber
The atomic force microscopy of Fig. 4 ICG doping self-assembled nanometer fiber
Fig. 5 fluorescence imaging monitoring vivo tumor original position forms self-assembled nanometer fiber 5.HeLa mice with tumor grouping tail vein injection 1+2 (1,10mg/kg; 2,100mg/kg) or 1 (1,10mg/kg) after, different time points near-infrared fluorescence imaging picture, and the fluorescence imaging picture of 24 h before harvest tumor tissue in vitro and main organs.
Fig. 6 photoacoustic imaging monitoring vivo tumor original position forms self-assembled nanometer fiber 5.HeLa mice with tumor grouping tail vein injection 1+2 (1,10mg/kg; 2,100mg/kg) or 1 (1,10mg/kg) after, representative two and three dimensions photoacoustic imaging picture (4 and 24 hours) of different time points tumor region.
Fig. 7 tumor tissues original position forms the photo-thermal therapy effect of ICG doping self-assembled nanometer fiber.Tumor growth curve after HeLa tumor-bearing mice photo-thermal therapy.
Detailed description of the invention
The present invention can be made to be illustrated more clearly in below by way of specific embodiment:
1. the preparation of nanofiber:
ICG (10 μ g/mL) and NapFFKYp polypeptide (500 μ g/mL) are dissolved in PBS (PH7-8,1mL) jointly.The alkali phosphatase adding 1 unit forms the nanofiber of ICG doping.Same method making does not have the nanofiber of ICG in contrast.
2. in the research of cell aspect
1) cultured cell: humanized's cervical cancer cell HeLa cell line and murine mammary cancerous cell 4T1 derive from AmericanTypeCultureCollection (ATCC), and at 37 DEG C of 5%CO
2incubator in cultivate.
2) forming nanofiber in cell: form nanofibrous structures to study in cell, HeLa cell 5000/hole being seeded in 96 orifice plates and cultivating 24 hours, then adding different sample incubation: No. 1 sample 10 μ g/mL; No. 2 sample 500 μ g/mL; No. 1 sample 10 μ g/mL+2 sample 500 μ g/mL; No. 1 sample 10 μ g/mL+2 sample 500 μ g/mL+5 μm of ol/LCinnGEL2Me.
3) cytotoxicity experiment: use the survival rate of cell counting CCK-8 kit measurement tumor cell to confirm the cytotoxicity of No. 5 samples.Matched group is left intact, and all experimental grouies all use matched group homogenization process to compare.HeLa cell 5000/hole is seeded in 96 orifice plates and cultivates 24 hours, then add different sample incubation 24 hours, wherein No. 1 sample 10 μ g/mL, No. 2 sample 10-500 μ g/mL.The CCk-8 experimental evaluation cytoactive of standard.Result gets the meansigma methods of 5 repeating holes.
4) near infrared light induction light hot-cast socket: all samples aqueous solution is all in plastic test tube, with 808 nanometer laser (1W/cm
2, 3min) irradiate.Adjustment spot size makes it cover whole liquid level.Real-time thermal imaging is carried out by FLIR thermal imaging photographic head.FLIR monitoring of software analyzes the temperature variations of sample.
5) the photo-thermal curative effect of external near-infrared induction: in vitro in photo-thermal therapy experiment, HeLa cell is seeded in 96 orifice plates with the density in 5000/hole and cultivates 24 hours, and adds No. 1 or No. 5 sample (1,10 μ g/mL; 2,500 μ g/mL) hatch 4 hours, then irradiate 3 minutes (power respectively: 0.05,0.1,0.25,0.5, and1W/cm with 808 nanometer lasers
2.After process, cell continues cultivation 24 hours, by the CCK-8 measuring cytoactive of standard.Without laser irradiation group in contrast, all results all get the meansigma methods of 5 repeating holes.
3. mouse tumor original position forms nanofibrous structures and performance study
1) animal model: the animal that all experimental implementation are all passed through according to clinical center animal health and the use committee of NIH uses and health care system.Female athymic nude mice (six weeks, 20-25g), at nude mice foreleg subcutaneous injection 2 × 10
6the PBS solution of HeLa or 4T1 tumor cell sets up mouse tumor model.When gross tumor volume reaches 60mm
3after, tumor-bearing mice is used for carrying out fluorescence, photoacoustic imaging and photo-thermal therapy.
2) living body fluorescent imaging
Self-assembled nanometer fiber is formed, when Hela tumor reaches 60mm in order to study vivo tumor original position
3during volume, by No. 1, mouse tail vein injection or No. 1+No. 2 (1,10mg/kg; 2,100mg/kg) sample.Then within 0.5,1,2,4,24,48, and168 hour after injectable drug, by MaestroII small animal living body imaging (CaliperLifeSciences, Hopkinton, MA), imaging is carried out to nude mice.The exciter filter used is 710nm to 760nm, and launching optical filter is the long flux optical filter of 800nm.Range of receiving is 780nm to 590nm (step level: 10nm).And after drug injection 24 hours, the internal organs taking out animal main carry out in vitro fluorescence imaging, analyze the bio distribution of ICG.
As Fig. 5, for having injected 1+2 group, because the existence of ICG monomer is observed in the fluorescence signal distribution of the stage whole body that starts most.Along with the development of time, in the enhancing that the fluorescence of tumor region slightly continues, this also makes tumor region and normal surrounding tissue within 1 hour, just can significantly come respectively in injection later.Contrary, only injecting the mice of 1 medicine, the medicine 1 of most is absorbed by liver in injection for 30 minutes later fast, and is removed fast in 4 hours by biliary system.24 h before harvest tumor tissue in vitro and main organs carry out fluorescence imaging, found that in the mice group defining 5 only have tumor region have very strong should signal, in other organs, there is no the residual of discovery 1.Contrary, 1 injection group mice does not almost have fluorescence signal at tumor region.
4) live body photoacoustic imaging
Photoacoustic imaging for in-vivo tumour is studied, HeLa tumor tumor-bearing mice tail vein injection 1 or 1+2 (1,10mg/kg; 2,100mg/kg), at different time points, utilize photoacoustic imaging system to carry out the collection of the ultrasonic and photoacoustic imaging signal of two and three dimensions cross section to tumor region.As shown in Figure 6, strong than 1 of the photoacoustic signal having injected each time point of 5.Found that of contrast fluorescence imaging, these optoacoustic results further demonstrate neoplasm in situ and form 5, are conducive to the tumor accumulation efficiency of raising 1.
5) live body photo-thermal therapy effect
Based on the result of fluorescence before the present invention and photoacoustic imaging, 24 and 48 hours two time points are selected to implement photo-thermal therapies respectively.When the HeLa gross tumor volume of tumor-bearing mice reaches 60mm
3time, by mouse tail vein injection 1+2 (1,5mg/kg; 2,100mg/kg) or 1 (1,5mg/kg).Use SC300 near infrared imaging instrument (FLIR, Arlington, VA) to carry out thermal imaging, analyzed by ExaminIRimagesoftware (FLIR) software.Light source is 808 nanotube diode light-pumped solid state laser systems.
HeLa tumor-bearing mice is divided into 7 groups at random, often organizes 5 mices: (1) blank group (contrast); (2) PBS group is penetrated in illumination is injected; (3) No. 5 Sample Injection groups; (4) No. 1 sample light group is injected; (5) No. 2 samples light group is injected; (6) No. 5 samples in 24 h light groups are injected; (7) No. 5 medicines in 48 h light groups are injected. all use identical laser to irradiate (808nm, 0.8W/cm in all photo-thermal experiments
2, 5min).Every other day use vernier caliper measurement gross tumor volume, and according to formula V=AB
2* 0.5 calculates gross tumor volume, wherein A is the major diameter of tumor, and B is the minor axis (mm) of tumor.Each measurement result is all by starting tumor volume normalization before treatment.Tumor growth curve as shown in Figure 7, inject No. 5 sample sets and no matter implement photo-thermal therapy 24 hours or 48 hours, tumor can be melted effectively, and does not all recur.By contrast, other experiment contrast groups are all growing with similar speed.
Claims (6)
1. a nanofiber, is characterized in that, this nanofiber responds the product of peptide and the analog of cyanine dyes or derivant by the alkali phosphatase of phosphatase effect to obtain by assembling altogether.
2. a kind of nanofiber as claimed in claim 1, is characterized in that: described cyanine dyes is ICG.
3. the purposes of nanofiber as claimed in claim 1, it is preparing the application in tumour diagnostic reagent.
4. the purposes of nanofiber as claimed in claim 1, it treats the application in reagent in preparation tumor photo-thermal.
5. the purposes of nanofiber as claimed in claim 1, it is for the drug delivery of tumor.
6. a preparation method for nanofiber, this nanofiber is obtained by assembling altogether by the product of the alkali phosphatase response peptide of phosphatase effect and ICG, and method comprises the steps:
ICG and NapFFKYp polypeptide is dissolved in the PBS of PH7-8 jointly, and both molar ratio range are 1:2-1:100, then adds the nanofiber that alkali phosphatase forms ICG doping, and the additional proportion scope of alkali phosphatase is 0.1-1 unit.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111335040A (en) * | 2020-04-14 | 2020-06-26 | 广西大学 | Biomass-based stepped dual-temperature/pH/near-infrared stimulus-responsive intelligent nanofiber and preparation method and application thereof |
| CN111909239A (en) * | 2020-07-29 | 2020-11-10 | 苏州大学 | Self-assembled polypeptide molecule with bacterial flocculation and antibacterial properties and application thereof |
| CN112691191A (en) * | 2020-12-25 | 2021-04-23 | 哈尔滨工业大学（威海） | Temperature photoacoustic imaging and accurate control method and system based on nano photothermal preparation |
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| CN104707143A (en) * | 2008-11-07 | 2015-06-17 | 克洛克斯科技公司 | Combination of an oxidant and a photoactivator for the healing of wounds |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104707143A (en) * | 2008-11-07 | 2015-06-17 | 克洛克斯科技公司 | Combination of an oxidant and a photoactivator for the healing of wounds |
Non-Patent Citations (1)
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| PENG HUANG等: ""Tumor-Specific Formation of Enzyme-Instructed Supramolecular Self-Assemblies as Cancer Theranostics"", 《ACS NANO》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111335040A (en) * | 2020-04-14 | 2020-06-26 | 广西大学 | Biomass-based stepped dual-temperature/pH/near-infrared stimulus-responsive intelligent nanofiber and preparation method and application thereof |
| CN111335040B (en) * | 2020-04-14 | 2021-02-19 | 广西大学 | Biomass-based stepped dual-temperature/pH/near-infrared stimulus-responsive intelligent nanofiber and preparation method and application thereof |
| CN111909239A (en) * | 2020-07-29 | 2020-11-10 | 苏州大学 | Self-assembled polypeptide molecule with bacterial flocculation and antibacterial properties and application thereof |
| CN112691191A (en) * | 2020-12-25 | 2021-04-23 | 哈尔滨工业大学（威海） | Temperature photoacoustic imaging and accurate control method and system based on nano photothermal preparation |
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