CN104726491A - Novel applications of prussian blue-chitosan composite nanoparticle as gene carrier - Google Patents
Novel applications of prussian blue-chitosan composite nanoparticle as gene carrier Download PDFInfo
- Publication number
- CN104726491A CN104726491A CN201310711145.8A CN201310711145A CN104726491A CN 104726491 A CN104726491 A CN 104726491A CN 201310711145 A CN201310711145 A CN 201310711145A CN 104726491 A CN104726491 A CN 104726491A
- Authority
- CN
- China
- Prior art keywords
- prussian blue
- composite nanoparticle
- chitosan composite
- chitosan
- nanoparticle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses novel applications of a prussian blue-chitosan composite nanoparticle as a gene carrier. The prussian blue-chitosan composite nanoparticle is taken as the gene carrier, and is capable of absorbing near infrared laser effectively, and generating heat; so that entering of the prussian blue-chitosan composite nanoparticle into cells is promoted, and gene transfection effect is improved substantially. And in addition, prussian blue and chitosan are both clinical drugs, and possess excellent in vivo biosecurity; a preparation method of the prussian blue-chitosan composite nanoparticle is simple, and green; cost is low; and the application prospect of the prussian blue-chitosan composite nanoparticle in gene transfection is promising.
Description
Technical field
The invention belongs to biomedical materials field, relate to the new opplication of Prussian blue chitosan composite nanoparticle as genophore.
Technical background
Transgenic technology is a kind of very important molecular biology method, and has a lot of advantages with the carrier that nanoparticle prepares gene transfer.A good gene transfer vector need possess following condition: its biological safety is good, can realize biological degradation in vivo, toxicity and immunogenicity less; The size to fit of genophore, efficiently can enter smoothly in cell paste and transmit foreign gene; The nucleic acid molecule with negative electricity can be combined efficiently, have and well encapsulate effect; Carrier is more stable in physiological conditions, not easily occurs to assemble and forms precipitation; Transfection is better, effectively can realize the stable gene transfection of various kinds of cell.
Chitosan is a kind of widely used pharmaceutical carrier and gene vector material, has good biocompatibility, and can carry out the features such as abundant metabolism in vivo, being widely used in the middle of scientific research and clinical practice, is a kind of ideal gene transfection material.
Prussian blue is the antidote applied clinically for a long time, and its biological safety is good, and Side effect is in vivo very low, is a kind of well pharmaceutical carrier.Nearest research finds that Prussian blue is a kind of well optothermal material, can produce heat kill tumour cell when irradiating with 808nm laser apparatus.
Adopt the Prussian blue optothermal material that waits of 808nm laser illumination can produce higher temperature, but when controlling irradiation time and power, thermogenetic for light temperature can be controlled within limits, when the survival rate that guarantee cell is higher, by the permeability promoting cell surface membrane that heats up, thus strengthen the transgenosis effect of this carrier.
The present invention has a lot of advantages and good application prospect: first, and Prussian blue and chitosan is all the biomaterial applied clinically, and its biological safety is guaranteed; Secondly, Prussian blue chitosan composite nanoparticle has very little particle diameter and higher positive potential, can efficient adsorption nucleic acid, has and well encapsulates effect; Again, when transgeneic procedure, adopt the laser apparatus of 808nm wavelength to carry out fixed point and irradiate, effectively can control the temperature of irradiation area, thus reach the function strengthening transgenosis effect in target area and privileged site; Finally, preparation method's mild condition of this composite nanoparticle, simple to operate, desired raw material is easy to get safely, cheap.
Summary of the invention
An object of the present invention is the new opplication of Prussian blue chitosan composite nanoparticle as genophore.
Two of object of the present invention is to provide above-mentioned Prussian blue chitosan composite nanoparticle strengthens transfection efficiency in cell experiment using method by photothermy, especially for the transfection method of reporter gene GFP gene for zooblast.
For reaching above object, technical scheme of the present invention is:
The preparation method of Prussian blue chitosan composite nanoparticle take chitosan as skeleton, thereon the Prussian blue crystal of growth in situ, centrifugally namely obtains Prussian blue chitosan composite nanoparticle through washing.Detailed process is as follows:
(1) chitosan is dissolved in dilute hydrochloric acid stirs, after being filtered by the filter-sterilizer of 0.22 μm of millipore filtration, add the Tripotassium iron hexacyanide and iron protochloride, add the acetone that triploid is long-pending after stirring, 10000g obtains composite nanoparticle with acetone cleaning-drying after centrifugal 30 minutes.The molecular weight ranges of wherein said chitosan is 2-30 ten thousand, dissolves completely in the dilute hydrochloric acid of 0.5mol/L, and concentration is 0.75mg/ml; Described DNA solution concentration is 1mg/m; Described Prussian blue preparation method successively adds 20ml, the potassium ferricyanide solution of 1mM and the solution of ferrous chloride of 20ml, 1mM in the chitosan solution of 80ml, and after fully stirring, color gradually becomes mazarine, proves Prussian blue crystal formation.
(2) the above-mentioned Prussian blue chitosan composite nanoparticle prepared can be applied to gene transfection.Detailed process is as follows:
Prussian blue chitosan composite nanoparticle is dissolved ultrasonic in water, it is mixed in the medium according to a certain percentage with nucleic acid, make nucleic acid in its surface adsorption, in the HeLa cell that carrier-DNA solution is entered to cultivate, with 808nm laser apparatus, it is irradiated after hatching 1 hour, make it better enter cell by photothermy, then gain fresh culture and continue cultivation 48 hours, in the gene transfection effect of this carrier of fluorescence microscopy Microscopic observation.
Compare with existing genophore, Prussian blue chitosan composite nanoparticle of the present invention is developed as novel Gene transfer vector, has following outstanding advantage:
1 the present invention prepares first and has prepared Prussian blue chitosan composite nanoparticle with Prussian blue and these two kinds of extraordinary biomaterials of biological safety of chitosan, and is applied to gene transfection, and the method is easy and simple to handle, and raw material is easy to get, cheap.
2 the present invention are irradiated Prussian blue chitosan composite nanoparticle with 808nm laser apparatus first, strengthen membrane passage facilitate transgenosis effect by photothermy.Method simple practical, can realize strengthening transfection efficiency to the cell of specific region, have good operability.
3 compared with other gene particles, and it is good that Prussian blue chitosan composite nanoparticle has biological safety, and particle diameter is less is easy to picked-up, and price is lower, can be carried out the transfection efficiency of controls local, transfection efficiency high by illumination.
Accompanying drawing explanation
Fig. 1 is transmission electron microscope figure and the dynamic light scattering figure of Prussian blue chitosan composite nanoparticle of the present invention; Fig. 2 is the uv-absorbing figure (a:0.015mg/ml of Prussian blue chitosan composite nanoparticle under different concns; B:0.031mg/ml; C:0.0625mg/ml; D:0.125mg/ml; E:0.25mg/ml; F:0.5mg/ml); Fig. 3 be Prussian blue chitosan composite nanoparticle in water, the photo in PBS buffered soln and substratum: (a) aqueous solution (b) PBS solution (c) cell culture fluid; Fig. 4 is the thetagram after Prussian blue chitosan composite nanoparticle uses 808nm laser illumination in aqueous; Fig. 5 be Prussian blue chitosan composite nanoparticle and nucleic acid by the picture a carrying out electrophoresis after different ratios mixing and absorption, 0; B, 0.1; C, 0.2; D0.4; E, 0.8; F, 1.6; G3.2; H, 6.4; Fig. 6 is after Prussian blue chitosan composite nanoparticle and nucleic acid absorption, with the MTT survivorship curve figure of HeLa cell after 808nm laser illumination; Fig. 7 is that Prussian blue chitosan composite nanoparticle and commercial transgene carrier polymine (PEI) are to the MTT graphic representation of the HeLa cell that the toxicity of HeLa cell contrasts; Fig. 8 is Prussian blue chitosan composite nanoparticle after irradiation 24 hours, and the shows fluorescent microscopy images of the cell transfecting after 48 hours and 72 hours, contrasts as commercial transgene carrier (PEI) and blank group.
Embodiment
To contribute to understanding the present invention by following embodiment, but not limit content of the present invention.
Embodiment 1
Low-molecular weight chitoglycan is dissolved in 0.5mol/L dilute hydrochloric acid and stirs, its final concentration is made to be 0.75mg/ml, after degerming by 0.22 μ filtering with microporous membrane, successively 20ml is added in the chitosan solution of 80ml, the potassium ferricyanide solution of 1mM and the solution of ferrous chloride of 20ml, 1mM, fully stir, color wherein adds the long-pending acetone of triploid after gradually becoming mazarine, 10000g obtains composite nanoparticle with acetone cleaning-drying after centrifugal 30 minutes.
Embodiment 2
Predict size of particles by transmission electron microscope and dynamic light scattering method uniform and stable, its size is about 4nm (Fig. 1). measure its ultraviolet absorption curve, find that it has obvious absorption peak (Fig. 2) at 700nm place.By water-soluble after Prussian blue chitosan composite nanoparticle, in PBS damping fluid and substratum, find dissolution homogeneity stable (Fig. 3). then investigate its temperature variation by after this carrier 808nm laser illumination, find that it has good temperature rise effect (Fig. 4)
Embodiment 3
This solution is mixed evenly according to different proportionings from nucleic acid in culture medium solution, after static 20 minutes, sample is added in the gel of 1% agarose, observe after 20 minutes with 110v electrophoresis, can find that Prussian blue chitosan composite nanoparticle well can be combined with nucleic acid (Fig. 5).Subsequent experimental is carried out according to the Prussian blue chitosan composite nanoparticle of complete adsorbs nucleic acid and the ratio of nucleic acid, this nucleic acid complexes and cytomixis are hatched rear 808nm laser radiation, investigate the relation (Fig. 6) of its MTT survival rate and irradiation time, and the cytotoxicity of itself and commercial transgene carrier polymine (PEI) is compared, therefrom can find out the cytotoxicity of Prussian blue chitosan composite nanoparticle very low (Fig. 7).
Embodiment 4
Prussian blue chitosan nano composite particles is mixed in the medium with green fluorescent protein GFP gene, static about 20 minutes, then put it in Tissue Culture Dish with cell incubation 1 hour, then with the power of 1W/cm2,1 is irradiated to it with the laser apparatus of 808nm, 2, 4, 8 minutes, then substratum is outwelled, change fresh perfect medium into and continue cultivation 24 hours, 48 hours and 72 hours, then observe with fluorescent microscope, find the prolongation with incubation time and irradiation time, the efficiency gene transfection of Prussian blue chitosan composite nanoparticle is also more and more higher.
Claims (6)
1. the new opplication of Prussian blue chitosan composite nanoparticle, is characterized in that: described Prussian blue chitosan composite nanoparticle is used for the carrier as gene transfection.
2. the new opplication of Prussian blue chitosan composite nanoparticle according to claim 1, is characterized in that: the near infrared region that described composite nanoparticle is 650-1300nm at wavelength has strong absorption.
3. the Prussian blue chitosan composite nanoparticle according to claim 1 and 2, is characterized in that: the molecular weight ranges of described chitosan is 2-30 ten thousand.
4., according to the Prussian blue chitosan composite nanoparticle described in claim 1,2 and 3, it is characterized in that: the particle size range of described nanoparticle is between 2-1000nm.
5. Prussian blue chitosan composite nanoparticle according to claim 1 is used for the new opplication of genophore, it is characterized in that: in gene transfection process, the wavelength region of laser used is between 650-1300nm.
6. the Prussian blue chitosan composite nanoparticle according to claim 1 and 2 is used for the new opplication of genophore, it is characterized in that: described nanoparticle can produce heat under near infrared light effect, increase cell to the picked-up of particle, thus improve the efficiency of gene transfection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310711145.8A CN104726491A (en) | 2013-12-20 | 2013-12-20 | Novel applications of prussian blue-chitosan composite nanoparticle as gene carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310711145.8A CN104726491A (en) | 2013-12-20 | 2013-12-20 | Novel applications of prussian blue-chitosan composite nanoparticle as gene carrier |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104726491A true CN104726491A (en) | 2015-06-24 |
Family
ID=53450904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310711145.8A Pending CN104726491A (en) | 2013-12-20 | 2013-12-20 | Novel applications of prussian blue-chitosan composite nanoparticle as gene carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104726491A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111821283A (en) * | 2020-07-23 | 2020-10-27 | 华侨大学 | Zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane and preparation method of zinc glutamate-coated Prussian blue nanoparticles |
| CN114950533A (en) * | 2022-03-21 | 2022-08-30 | 湖南农业大学 | Preparation method and application of prussian blue nanoflower and nanoflower structure adjusting method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986608A (en) * | 2006-12-18 | 2007-06-27 | 武汉大学 | Preparing process and application of nano chitosan particle |
-
2013
- 2013-12-20 CN CN201310711145.8A patent/CN104726491A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986608A (en) * | 2006-12-18 | 2007-06-27 | 武汉大学 | Preparing process and application of nano chitosan particle |
Non-Patent Citations (4)
| Title |
|---|
| GUANGLEI FU,ET AL: "Prussian blue nanoparticles operate as a new generation of photothermal ablation agents for cancer therapy", 《CHEM. COMMUN》 * |
| LIANGZHU FENG，ET AL: "Polyethylene Glycol and Polyethylenimine Dual-Functionalized Nano-Graphene Oxide for Photothermally Enhanced Gene Delivery", 《SMALL》 * |
| QIAN ZHANG，ET AL: "Fabrication and electrochemical study of monodisperse and size controlled Prussian blue nanoparticles protected by biocompatible polymer", 《ELECTROCHIMICA ACTA》 * |
| XIAO-DA LIA,ET AL: "Chitosan stabilized Prussian blue nanoparticles for photothermally enhanced photothermally enhanced gene delivery", 《COLLOIDS AND SURFACES B: BIOINTERFACES》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111821283A (en) * | 2020-07-23 | 2020-10-27 | 华侨大学 | Zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane and preparation method of zinc glutamate-coated Prussian blue nanoparticles |
| CN111821283B (en) * | 2020-07-23 | 2021-11-30 | 华侨大学 | Zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane and preparation method of zinc glutamate-coated Prussian blue nanoparticles |
| CN114950533A (en) * | 2022-03-21 | 2022-08-30 | 湖南农业大学 | Preparation method and application of prussian blue nanoflower and nanoflower structure adjusting method |
| CN114950533B (en) * | 2022-03-21 | 2023-10-20 | 湖南农业大学 | Prussian blue nanoflower preparation method and application thereof, and nanoflower structure adjusting method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Genchi et al. | Barium titanate nanoparticles: promising multitasking vectors in nanomedicine | |
| Lee et al. | Recent nanoparticle engineering advances in microalgal cultivation and harvesting processes of biodiesel production: a review | |
| Pylaev et al. | A novel cell transfection platform based on laser optoporation mediated by Au nanostar layers | |
| Wang et al. | Responsive graphene oxide hydrogel microcarriers for controllable cell capture and release | |
| Lyu et al. | A universal platform for macromolecular deliveryinto cells using gold nanoparticle layers via the photoporation effect | |
| AU773342B2 (en) | A method for introducing foreign matters into living cells | |
| CN104109534B (en) | A kind of preparation of nitrogen-doped graphene quantum dot two-photon fluorescence probe and application thereof | |
| Kang et al. | A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode | |
| JP2018504110A (en) | Modified Cell by Putting Material into the Cell without Vehicle [Detailed Description of the Invention] | |
| JP2007523096A (en) | Metal complex solution and its application | |
| CN106497563B (en) | A kind of carbon quantum dot nano material and its preparation method and application | |
| Qu et al. | Photothermal scaffolds/surfaces for regulation of cell behaviors | |
| Wang et al. | Using porous magnetic iron oxide nanomaterials as a facile photoporation nanoplatform for macromolecular delivery | |
| CN104379725B (en) | By the method that near infrared ray mode carries out selective cell adhesion/come off, cell patterning and cell harvesting | |
| Nguyen-Ngoc et al. | Sol–gel process for vegetal cell encapsulation | |
| Sardo et al. | Mini-review: potential of diatom-derived silica for biomedical applications | |
| CN104726491A (en) | Novel applications of prussian blue-chitosan composite nanoparticle as gene carrier | |
| KR100988945B1 (en) | Simulation Device of Cell Induction·Fixation in Target Part of Blood Vessels Using Microchannels and Method for Simulation Using the Same | |
| CN104136625B (en) | The method that green algae is quickly and efficiently harvested using cationic organic clay | |
| WO2021109132A1 (en) | Composite material and preparation method therefor and application thereof | |
| Ma et al. | Inorganic nanoparticles-based systems in biomedical applications of stem cells: opportunities and challenges | |
| Driscoll et al. | Fabrication and characterization of a biomaterial based on extracellular-vesicle functionalized graphene oxide | |
| Liu et al. | Nanozymatic magnetic nanomotors for enhancing photothermal therapy and targeting intracellular SERS sensing | |
| CN116064393B (en) | Method for stimulating macrophage to secrete exosome by near infrared light and exosome | |
| CN102206665A (en) | Nano calcium phosphate/polymer complex gene transfection reagent and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150624 |
|
| WD01 | Invention patent application deemed withdrawn after publication |