CN106620720A - Modification of carrier gas microbubbles having different charges respectively through step-by-step method and one-step method, and preparation of microspheres through one-step method - Google Patents
Modification of carrier gas microbubbles having different charges respectively through step-by-step method and one-step method, and preparation of microspheres through one-step method Download PDFInfo
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Abstract
The present invention discloses a method for modifying poly ethyl alpha-cyanoacrylate carrier gas microbubbles by using a step-by-step method and a one-step method so as to make the carrier gas microbubbles have positive charges on the surface, and discloses a method for preparing microspheres having different charges and having methylene blue supported on the surface by using a one-step method or a step-by-step method. According to the present invention, the preparation process is simple; the positively charged carrier gas microbubbles prepared through the preparation method have the uniform particle size, the surface charge is the positive charge and is 11-50 mV, the positively charged carrier gas microbubbles have the gene loading function, and the surface of the carrier gas microbubbles has the charge; and the methylene blue supported microspheres have the small particle size, have the positive charge or negative charge on the surface, can be adopted as the micro-nano contrast agent, and can further be used as the drug or gene vector for targeting drug and gene delivery.
Description
Technical field
The present invention relates to various preparation methods of a kind of carrier gas microvesicle with different electric charges and its carrier gas microvesicle, specifically related to surface prepares the preparation method of area load fluorescent material and positively or negatively charged microballoon with the positive charge carrier gas microvesicle method of fractional steps and one-step method surface modification method, and one-step method.
Background technology
Microbubble contrast agent mainly by shell and is wrapped in the gas core of inside and constitutes, and the shell of the microvesicle compound that can be birdsed of the same feather flock together by protein, lipid, polysaccharide or macromolecule is constituted.Medical microbubble contrast agent is widely used in myocardium acoustic contrast, acute focal inflammation, thrombus, the diagnosis of tumour.By strengthening the backscatter signals of tissue and the Doppler signal of blood, ultrasonic microbubble can realize noninvasive development.Carrier gas microbubble contrast agent can also carry gene or medicine reaches target tissue, and fixed point release medicine is beneficial to the treatment of disease.Additionally, by area load autofluorescence material, built-up fluorescence drug bearing microsphere, as pharmaceutical carrier and fluorescent tracing carrier, is widely used in medical field.
At present the application of contrast preparation is relatively broad, its particle size, surface an electrically charged key factor for being to limit its range of application.A diameter of 6-9um of capillary, if being relatively large in diameter for contrast preparation, cannot be introduced into capillary and developed.Additionally, the surface institute band positive and negative charge and the quantity of electric charge of ultrasonic microbubble, also determine the range of application of contrast preparation.In recent years, the load medicine or DNA loaded nanoparticle of difference in functionality and microballoon medical treatment aspect are widely studied.The particle diameter of function nano grain is generally less than 100nm, and the particle size of functional microsphere is generally hundreds of nanometer.Because its particle diameter is less, can be phagocytized by cells and enter target cell, and realize the purpose of medicament slow release, gene delivery or targeted therapy.
Carrier gas microvesicle or functional microsphere to prepare material varied, for example, according to different therapeutic purposes, people prepare nanoparticle, microballoon or the microvesicle of difference in functionality using multiple material biomaterials such as poly (lactic acid-glycolic acid), polycaprolactone, polyethyleneimine, polylysines.A-cyanoacrylate class loading glue has preferable biocompatibility, is widely used in clinical operation adhesive.Shitosan is unique alkaline polysaccharide present in nature.Due to the physicochemical properties with good biocompatibility, degradability and uniqueness, shitosan is all widely used in biomedical sectors such as drug loading, gene delivery vector, anti-biotic materials.
The present invention, as base material, is prepared for carrier gas microvesicle and microballoon using ECA.Using two methods of the method for fractional steps and one-step method, surface modification is carried out to carrier gas microvesicle using shitosan so as to which surface carries positive charge, so as to being that it has gene loading functional.Using one-step method, it is prepared for surface and carries different electric charges, and the ECA microballoon of area load methylene blue, this microballoon not only has the potentiality of micro-nano imaging, and can be also used for the targeting transmission of gene and medicine.
The content of the invention
First purpose of the present invention is to provide a kind of carrier gas microvesicle with difference lotus.
Second object of the present invention is to provide a kind of method of fractional steps modification carrier gas microvesicle and makes the method that it carries positive charge.
Third object of the present invention is to provide a kind of one-step method and prepares method of the surface with positive charge carrier gas microvesicle.
Fourth object of the present invention is to provide a kind of one-step method and prepares negatively charged and area load methylene blue microballoon method.
5th purpose of the present invention is to provide a kind of method of fractional steps and prepares positively charged and area load methylene blue microballoon method.In order to solve above-mentioned technical problem, the technical solution used in the present invention is：
It is double by poly- ECA, shitosan, polyethylene glycol, Triton X-100 or 3,7- with different electric charge carrier gas microvesicles（Dimethylamino）Phenthazine -5- father-in-law's chlorides, dextran-70 are constituted.
The first preparation method：For preparing the preparation method with different electric charge carrier gas microvesicles of claim 1, made with following methods of fractional steps：
Step one, 100-200mL ultra-pure waters are measured, add-the 0.5mL of Aqueous Solutions of Polyethylene Glycol 0.25 of 0.5g/mL, pH value of solution to be adjusted to 2-3, add 1.0-2.0mL the ECAs ,-1.5h of high-speed stirred 1 to obtain carrier gas microbubble suspensions；Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 2000-5000 to form；
The suspension of step 2, the gained of centrifugation step one, it is 1.8-2.8um on-load carrier gas microvesicles to obtain electric charge for-50-- 30mV, particle diameter；
Step 3, by step 2 gained microbubble solution be diluted to 35mL, add-the 5mL of chitosan solution 3 of 0.5g/mL, it is the positively charged carrier gas microvesicles of 2.2-3.2um fully to mix 1-2h, milli-Q water 3-4 times, and centrifugation and obtain electric charge for 30-50mV, particle diameter.
Second preparation method：For preparing the preparation method with different electric charge carrier gas microvesicles of claim 1, made with following one-step method：Measure 100-200mL ultra-pure waters, add-the 0.5mL of Aqueous Solutions of Polyethylene Glycol 0.25 of 0.5g/mL, add-the 5mL of chitosan solution 3 of 0.5g/mL, pH value of solution is adjusted to 2-3, adds 1.0-2.0mL the ECAs ,-1.5h of high-speed stirred 1, obtain carrier gas microbubble suspensions, milli-Q water 3-4 times, and it is 11-30mV that centrifugation obtains electric charge, particle diameter is the positively charged carrier gas microvesicles of 1.6-2.6um；Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 2000-5000 to form, and the carrier gas microvesicle of gained carries positive charge.
The third preparation method：For preparing the preparation method with different electric charge carrier gas microvesicles of claim 1, made with following one-step method：Take 100mL0.5% dextran-70 solution, pH to 2-3 is adjusted, 0.02mmol methylene blues are added, adds 1mL concentration to be 0.1g/mL polyethylene glycol, add 1mL ECAs, 4-17h of stirring, washing gained suspension 2-3 times is simultaneously centrifuged, and obtains electric charge for-70-- 50mV, particle diameter is the microballoon of 500-800nm, microballoon carries negative electrical charge and area load methylene blue, and the molecular weight of methylene blue is 7000, and molecular weight polyethylene glycol is 2000.
4th kind of preparation method：For preparing the preparation method with different electric charge carrier gas microvesicles of claim 1, made with following methods of fractional steps：
Step one, the dextran-70 solution 100mL for measuring 0.5%, adjust pH to 2-3, add 0.02mmol methylene blues, add 1mL ECAs, stir 4-5h, and it is 440-480nm to obtain particle diameter, and current potential is-37-- 45mV；
Step 2, the chitosan solution for adding 1mL0.2-0.8g/mL, continue to stir 4-17h, washing gained suspension 2-3 times is simultaneously centrifuged, electric charge is obtained for 20-30mV, particle diameter is the microballoon of 400-600nm, microballoon is with positive charge and loads methylene blue, and the molecular weight of methylene blue is 7000, and chitosan is low viscosity shitosan.
The present invention has the advantages that compared with prior art：The invention provides a kind of method of fractional steps with different electric charge carrier gas microvesicles and microballoon is modified and preparation method with one-step method, compared with the conventional method, the method that the present invention is provided has simple process, the electrically charged controllable, uniform particle sizes of institute, Stability Analysis of Structures and is easy to the characteristics of preserving, with the potentiality for realizing industrialization production.
Description of the drawings
Below in conjunction with the accompanying drawings the present invention will be further described in detail.
Fig. 1 is the zeta current potentials of negatively charged microvesicle.
Fig. 2 is the microscopic optical structure of carrier gas microvesicle.
Fig. 3 is the particle diameter distribution of the positively charged carrier gas microvesicle in surface.
Fig. 4 is with negative electrical charge and the microballoon zeta current potentials of area load methylene blue.
Fig. 5 is with negative electrical charge and the microspherulite diameter of area load methylene blue is distributed.
Fig. 6 is with positive charge and the microballoon zeta current potentials of load methylene blue.
Fig. 7 is with positive charge and the microspherulite diameter distribution of load methylene blue.
Fig. 8 is adsorption function of the variable concentrations carrier gas microvesicle to gene PET28A.
Specific embodiment
With reference to Fig. 1-8 and specific embodiment, the present invention is further illustrated, and embodiments of the invention are in order that those skilled in the art better understood when the present invention, but to the present invention not carry out any restriction.
Embodiment 1
One kind tool positive charge carrier gas microvesicle, is made with following methods of fractional steps：
Step one, 100mL ultra-pure waters are measured, add 0.5
The Aqueous Solutions of Polyethylene Glycol 0.25mL of g/mL, pH value of solution is adjusted to 2.1.0 mL ECAs, the h of high-speed stirred 1 are added to obtain carrier gas microbubble suspensions；The Aqueous Solutions of Polyethylene Glycol, is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 2000 to form.
Step 2, the gained suspension of centrifugation step one, it is 2.8 to obtain electric charge for -50mV, particle diameter
Um on-load carrier gas microvesicles（Fig. 1）.
Step 3, step 2 gained microbubble solution is diluted to into 35 mL, adds the chitosan solution 3mL of 0.5g/mL, fully mix 1 h.Milli-Q water 3 times, and it is 3.2 that centrifugation obtains electric charge for 30 mV, particle diameter
The positively charged carrier gas microvesicles of um.
One kind tool positive charge carrier gas microvesicle, is made with following one-step method：
100mL ultra-pure waters are measured, 0.5 is added
The Aqueous Solutions of Polyethylene Glycol 0.25mL of g/mL, adds the chitosan solution 3mL of 0.5g/mL, pH value of solution to be adjusted to 2.1.0mL ECAs, high-speed stirred 1h is added to obtain carrier gas microbubble suspensions；Milli-Q water 3 times, and it is the positively charged carrier gas microvesicles of 1.6 um that centrifugation obtains electric charge for 11mV, particle diameter.The Aqueous Solutions of Polyethylene Glycol, is, by the polyethylene glycol that number-average molecular weight is 2000, to be dissolved in ultra-pure water configuration and form.
One kind tool negative electrical charge and area load methylene blue microballoon, are made with following one-step method：
The dextran-70 solution of 100mL 0.5% is taken, pH to 2 is adjusted.0.02 mmol methylene blues are added, it is 0.1g/mL polyethylene glycol that 1mL concentration is added dropwise, and adds 1mL ECAs, stirs 4h.Continue to stir 4h, washing gained suspension 2 times is simultaneously centrifuged, obtain electric charge be -70 mV, particle diameter be microballoon of the 500 nm surfaces with negative electrical charge.The molecular weight of the methylene blue is 7000, and the molecular weight polyethylene glycol is 2000.
One kind tool positive charge and area load methylene blue microballoon, are made with following methods of fractional steps：
0.5% mL of dextran-70 solution 100 is measured, pH to 2 is adjusted.0.02 mmol methylene blues are added, 1mL ECAs are added, 4h is stirred.It is -37 mV that particle diameter is obtained for 440 nm, current potential.
Afterwards, the chitosan solution of 1mL 0.2g/mL is added, continues to stir 4h.Washing gained suspension 2 times is simultaneously centrifuged, and it is 20mV to obtain electric charge, and particle diameter is the positively charged microballoons of 400nm.The molecular weight of the methylene blue is 7000, and the chitosan is low viscosity shitosan.
Embodiment 2
One kind tool positive charge carrier gas microvesicle, is made with following methods of fractional steps：
Step one, 200 mL ultra-pure waters are measured, add the Aqueous Solutions of Polyethylene Glycol 0.5mL of 0.5 g/mL, pH value of solution to be adjusted to 3.2.0 mL ECAs, the h of high-speed stirred 1.5 are added to obtain carrier gas microbubble suspensions；The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 5000 to form.
Step 2, the gained suspension of centrifugation step one, obtain electric charge for -30mV（Fig. 1）, particle diameter is 1.8um on-load carrier gas microvesicles（Fig. 2）.
Step 3, step 2 gained microbubble solution is diluted to into 35 mL, adds the chitosan solution 5mL of 0.5g/mL, fully mix 2 h.Milli-Q water 4 times, and it is the positively charged carrier gas microvesicles of 2.2um that centrifugation obtains electric charge for 50 mV, particle diameter（Fig. 3）.
One kind tool positive charge carrier gas microvesicle, is made with following one-step method：
200 mL ultra-pure waters are measured, the Aqueous Solutions of Polyethylene Glycol 0.5mL of 0.5 g/mL is added, adds the chitosan solution 5mL of 0.5g/mL, pH value of solution to be adjusted to 3.2.0 mL ECAs, the h of high-speed stirred 1.5 are added to obtain carrier gas microbubble suspensions；Milli-Q water 4 times, and it is 2.6 that centrifugation obtains electric charge for 30 mV, particle diameter
The positively charged carrier gas microvesicles of um.The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 5000 to form.
One kind tool negative electrical charge and area load methylene blue microballoon, are made with following one-step method：
The dextran-70 solution of 100mL 0.5% is taken, pH to 3 is adjusted.0.02 mmol methylene blues are added, it is 0.1g/mL polyethylene glycol that 1mL concentration is added dropwise, and adds 1mL ECAs, stirs 5h；Continue to stir 17 h.Washing gained suspension 3 times is simultaneously centrifuged, and obtains electric charge for -50 mV（Fig. 4）, particle diameter is 800 nm（Fig. 5）Microballoon of the surface with negative electrical charge.The molecular weight of the methylene blue is 7000.The molecular weight polyethylene glycol is 2000.
One kind tool positive charge and area load methylene blue microballoon, are made with following methods of fractional steps：
0.5% mL of dextran-70 solution 100 is measured, pH to 3 is adjusted.0.02 mmol methylene blues are added, 1mL ECAs are added, 5h is stirred.It is 480 nm to obtain particle diameter, and current potential is -45 mV.
Afterwards, the chitosan solution of 1mL 0.8g/mL is added, continues to stir 17 h.Washing gained suspension 3 times is simultaneously centrifuged, and obtains electric charge for 30 mV（Fig. 6）, particle diameter is 600 nm（Fig. 7）Positively charged microballoon.The molecular weight of the methylene blue is 7000, and the chitosan is low viscosity shitosan.
Embodiment 3
One kind tool positive charge carrier gas microvesicle, is made with following methods of fractional steps：
Step one, 150 mL ultra-pure waters are measured, add the Aqueous Solutions of Polyethylene Glycol 0.4mL of 0.5 g/mL, pH value of solution to be adjusted to 2.1.5mL ECAs, the h of high-speed stirred 1.2 is added to obtain carrier gas microbubble suspensions；The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 4000 to form.
Step 2, the gained suspension of centrifugation step one, it is -40mV to obtain electric charge, and particle diameter is 2.2um on-load carrier gas microvesicles.
Step 3, step 2 gained microbubble solution is diluted to into 35 mL, adds the mL of chitosan solution 4 of 0.5g/mL, fully mix 1.5 h.Milli-Q water 3 times, and it is 40 mV that centrifugation obtains electric charge, particle diameter is the positively charged carrier gas microvesicles of 2.8um.The carrier gas microvesicle has gene loading functional, as shown in figure 8, the adsorption function for variable concentrations carrier gas microvesicle to gene PET28A.
One kind tool positive charge carrier gas microvesicle, is made with following one-step method：
150 mL ultra-pure waters are measured, the mL of Aqueous Solutions of Polyethylene Glycol 0.4 of 0.5 g/mL is added, adds the mL of chitosan solution 4 of 0.5g/mL, pH value of solution to be adjusted to 3.1.5 mL ECAs, the h of high-speed stirred 1.2 are added to obtain carrier gas microbubble suspensions；Milli-Q water 4 times, and it is the positively charged carrier gas microvesicles of 2.0um that centrifugation obtains electric charge for 25 mV, particle diameter.The Aqueous Solutions of Polyethylene Glycol, is, by the polyethylene glycol that number-average molecular weight is 4000, to be dissolved in ultra-pure water configuration and form.
One kind tool negative electrical charge and area load methylene blue microballoon, are made with following one-step method：
The dextran-70 solution of 100mL 0.5% is taken, pH to 3 is adjusted.0.02 mmol methylene blues are added, it is 0.1g/mL polyethylene glycol that 1mL concentration is added dropwise, and adds 1mL ECAs, stirs 4.5h；Continue to stir 13 h.Washing gained suspension 2 times is simultaneously centrifuged, obtain electric charge be -60 mV, particle diameter be microballoon of the 600 nm surfaces with negative electrical charge.The molecular weight of the methylene blue is 7000.The molecular weight polyethylene glycol is 2000.
One kind tool positive charge and area load methylene blue microballoon, are made with following methods of fractional steps：
0.5% mL of dextran-70 solution 100 is measured, pH to 3 is adjusted.0.02 mmol methylene blues are added, 1mL ECAs are added, 5h is stirred.It is -40 mV that particle diameter is obtained for 400 nm, current potential.
Afterwards, the chitosan solution of 1mL 0.4g/mL is added, continues to stir 12 h.Washing gained suspension 3 times is simultaneously centrifuged, and it is the positively charged microballoons of 500 nm to obtain electric charge for 25 mV, particle diameter.The molecular weight of the methylene blue is 7000, and the chitosan is low viscosity shitosan.
Embodiment 4
One kind tool positive charge carrier gas microvesicle, is made with following methods of fractional steps：
Step one, 150 mL ultra-pure waters are measured, add the Aqueous Solutions of Polyethylene Glycol 0.5mL of 0.5 g/mL, pH value of solution to be adjusted to 3.1.5 mL ECAs, the h of high-speed stirred 1 are added to obtain carrier gas microbubble suspensions；The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 4600 to form.
Step 2, the gained suspension of centrifugation step one, it is -35 mV to obtain electric charge, and particle diameter is 2.5um on-load carrier gas microvesicles.
Step 3, step 2 gained microbubble solution is diluted to into 35 mL, adds the chitosan solution 3.5mL of 0.5g/mL, fully mix 2 h.Milli-Q water 4 times, and it is the positively charged carrier gas microvesicles of 2.8um that centrifugation obtains electric charge for 45 mV, particle diameter.
One kind tool positive charge carrier gas microvesicle, is made with following one-step method：
150 mL ultra-pure waters are measured, the mL of Aqueous Solutions of Polyethylene Glycol 0.4 of 0.5 g/mL is added, adds the chitosan solution 3.5mL of 0.5g/mL, pH value of solution to be adjusted to 2.1.5 mL ECAs, high-speed stirred 1h are added to obtain carrier gas microbubble suspensions；Milli-Q water 4 times, and it is 25 mV that centrifugation obtains electric charge, particle diameter is the positively charged carrier gas microvesicles of 2.4um.The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 4600 to form.
One kind tool negative electrical charge and area load methylene blue microballoon, are made with following one-step method：
The dextran-70 solution of 100mL 0.5% is taken, pH to 3 is adjusted.0.02 mmol methylene blues are added, it is 0.1g/mL polyethylene glycol that 1mL concentration is added dropwise, and adds 1mL ECAs, stirs 4h；Continue to stir 17 h.Washing gained suspension 3 times is simultaneously centrifuged, obtain electric charge be -60 mV, particle diameter be microballoon of the 650 nm surfaces with negative electrical charge.The molecular weight of the methylene blue is 7000.The molecular weight polyethylene glycol is 2000.
One kind tool positive charge and area load methylene blue microballoon, are made with following methods of fractional steps：
0.5% mL of dextran-70 solution 100 is measured, pH to 3 is adjusted.0.02 mmol methylene blues are added, 1mL ECAs are added, 5h is stirred.It is -39 mV that particle diameter is obtained for 450 nm, current potential.
Afterwards, the chitosan solution of 1mL 0.6g/mL is added, continues to stir 15 h.Washing gained suspension 3 times is simultaneously centrifuged, and it is the positively charged microballoons of 550 nm to obtain electric charge for 28 mV, particle diameter.The molecular weight of the methylene blue is 7000, and the chitosan is low viscosity shitosan.
Embodiment 5
One kind tool positive charge carrier gas microvesicle, is made with following methods of fractional steps：
Step one, 200 mL ultra-pure waters are measured, add the Aqueous Solutions of Polyethylene Glycol 0.5mL of 0.5 g/mL, pH value of solution to be adjusted to 2.1.0 mL ECAs, the h of high-speed stirred 1.5 are added to obtain carrier gas microbubble suspensions；The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 2000 to form.
Step 2, the gained suspension of centrifugation step one, it is -40mV to obtain electric charge, and particle diameter is 2.0um on-load carrier gas microvesicles.
Step 3, step 2 gained microbubble solution is diluted to into 35 mL, adds the mL of chitosan solution 4 of 0.5g/mL, fully mix 1.5 h.Milli-Q water 4 times, and it is the positively charged carrier gas microvesicles of 3.0um that centrifugation obtains electric charge for 45 mV, particle diameter.
One kind tool positive charge carrier gas microvesicle, is made with following one-step method：
200 mL ultra-pure waters are measured, the Aqueous Solutions of Polyethylene Glycol 0.5mL of 0.5 g/mL is added, adds the mL of chitosan solution 4 of 0.5g/mL, pH value of solution to be adjusted to 3.2.0 mL ECAs, the h of high-speed stirred 1.2 are added to obtain carrier gas microbubble suspensions；Milli-Q water 3 times, and it is the positively charged carrier gas microvesicles of 1.8um that centrifugation obtains electric charge for 20 mV, particle diameter.The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 2000 to form.
One kind tool negative electrical charge and area load methylene blue microballoon, are made with following one-step method：
The dextran-70 solution of 100mL 0.5% is taken, pH to 2 is adjusted.0.02 mmol methylene blues are added, it is 0.1g/mL polyethylene glycol that 1mL concentration is added dropwise, and adds 1mL ECAs, stirs 5h；Continue to stir 11 h.Washing gained suspension 2 times is simultaneously centrifuged, obtain electric charge be -60 mV, particle diameter be microballoon of the 550 nm surfaces with negative electrical charge.The molecular weight of the methylene blue is 7000.The molecular weight polyethylene glycol is 2000.
One kind tool positive charge and area load methylene blue microballoon, are made with following methods of fractional steps：
0.5% mL of dextran-70 solution 100 is measured, pH to 3 is adjusted.0.02 mmol methylene blues are added, 1mL ECAs are added, 5h is stirred.It is -42 mV that particle diameter is obtained for 460 nm, current potential.
Afterwards, the chitosan solution of 1mL 0.8g/mL is added, continues to stir 15 h.Washing gained suspension 3 times is simultaneously centrifuged, and it is the positively charged microballoons of 480 nm to obtain electric charge for 30 mV, particle diameter.The molecular weight of the methylene blue is 7000, and the chitosan is low viscosity shitosan.
Embodiment 6
One kind tool positive charge carrier gas microvesicle, is made with following methods of fractional steps：
Step one, 120 mL ultra-pure waters are measured, add the mL of Aqueous Solutions of Polyethylene Glycol 0.3 of 0.5 g/mL, pH value of solution to be adjusted to 2.1.2 mL ECAs, high-speed stirred 1h are added to obtain carrier gas microbubble suspensions；The Aqueous Solutions of Polyethylene Glycol, is, by the polyethylene glycol that number-average molecular weight is 5000, to be dissolved in ultra-pure water configuration and form.
Step 2, the gained suspension of centrifugation step one, it is -40mV to obtain electric charge, and particle diameter is 2.6um on-load carrier gas microvesicles.
Step 3, step 2 gained microbubble solution is diluted to into 35 mL, adds the chitosan solution 4mL of 0.5g/mL, fully mix 1h.Milli-Q water 3 times, and it is the positively charged carrier gas microvesicles of 3.0 um that centrifugation obtains electric charge for 35 m, particle diameter.
One kind tool positive charge carrier gas microvesicle, is made with following one-step method：
120 mL ultra-pure waters are measured, the mL of Aqueous Solutions of Polyethylene Glycol 0.4 of 0.5 g/mL is added, adds the mL of chitosan solution 4 of 0.5g/mL, pH value of solution to be adjusted to 3.1.2 mL ECAs, the h of high-speed stirred 1 are added to obtain carrier gas microbubble suspensions；Milli-Q water 3 times, and it is 30 mV that centrifugation obtains electric charge, particle diameter is the positively charged carrier gas microvesicles of 2.4um.The Aqueous Solutions of Polyethylene Glycol, is, by the polyethylene glycol that number-average molecular weight is 5000, to be dissolved in ultra-pure water configuration and form.
One kind tool negative electrical charge and area load methylene blue microballoon, are made with following one-step method：
The dextran-70 solution of 100mL 0.5% is taken, pH to 2 is adjusted.0.02 mmol methylene blues are added, it is 0.1g/mL polyethylene glycol that 1mL concentration is added dropwise, and adds 1mL ECAs, stirs 4 h；Continue to stir 16h.Washing gained suspension 2 times is simultaneously centrifuged, obtain electric charge be -65 mV, particle diameter be microballoon of the 560 nm surfaces with negative electrical charge.The molecular weight of the methylene blue is 7000.The molecular weight polyethylene glycol is 2000；
One kind tool positive charge and area load methylene blue microballoon, are made with following methods of fractional steps：
0.5% mL of dextran-70 solution 100 is measured, pH to 3 is adjusted.0.02 mmol methylene blues are added, 1mL ECAs are added, 5h is stirred.It is -42 mV that particle diameter is obtained for 470 nm, current potential.
Afterwards, the chitosan solution of 1mL 0.8g/mL is added, continues to stir 17 h.Washing gained suspension 2 times is simultaneously centrifuged, and it is the positively charged microballoons of 450 nm to obtain electric charge for 25mV, particle diameter.The molecular weight of the methylene blue is 7000, and the chitosan is low viscosity shitosan.
The present invention can be summarized with others without prejudice to the concrete form of the spirit or essential characteristics of the present invention.Therefore, no matter from the point of view of that point, the embodiment above of the present invention can only all be considered the description of the invention and can not limit invention, claims indicate the scope of the present invention, and the scope of the present invention is not pointed out in above-mentioned explanation, therefore, any change in the implication and scope suitable with claims of the present invention, is all considered as being included within the scope of the claims.
Claims (5)
1. there are different electric charge carrier gas microvesicles, it is characterised in that be made up of poly- ECA, shitosan, polyethylene glycol, Triton X-100, methylene blue, dextran-70.
2. it is used to prepare the preparation method with different electric charge carrier gas microvesicles described in claim 1, it is characterised in that made with following methods of fractional steps：
Step one, 100-200mL ultra-pure waters are measured, add-the 0.5mL of Aqueous Solutions of Polyethylene Glycol 0.25 of 0.5g/mL, pH value of solution to be adjusted to 2-3, add 1.0-2.0mL the ECAs ,-1.5h of high-speed stirred 1 to obtain carrier gas microbubble suspensions；The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 2000-5000 to form；
The suspension of step 2, the gained of centrifugation step one, it is 1.8-2.8um on-load carrier gas microvesicles to obtain electric charge for-50-- 30mV, particle diameter；
Step 3, by step 2 gained microbubble solution be diluted to 35mL, add-the 5mL of chitosan solution 3 of 0.5g/mL, it is the positively charged carrier gas microvesicles of 2.2-3.2um fully to mix 1-2h, milli-Q water 3-4 times, and centrifugation and obtain electric charge for 30-50mV, particle diameter.
3. it is used to prepare the preparation method with different electric charge carrier gas microvesicles described in claim 1, it is characterised in that made with following one-step method：Measure 100-200mL ultra-pure waters, add-the 0.5mL of Aqueous Solutions of Polyethylene Glycol 0.25 of 0.5g/mL, add-the 5mL of chitosan solution 3 of 0.5g/mL, pH value of solution is adjusted to 2-3, adds 1.0-2.0mL the ECAs ,-1.5h of high-speed stirred 1, obtain carrier gas microbubble suspensions, milli-Q water 3-4 times, and it is 11-30mV that centrifugation obtains electric charge, particle diameter is the positively charged carrier gas microvesicles of 1.6-2.6um；The Aqueous Solutions of Polyethylene Glycol is to be dissolved in ultra-pure water configuration by the polyethylene glycol that number-average molecular weight is 2000-5000 to form, and the carrier gas microvesicle of gained carries positive charge.
4. it is used to prepare the preparation method with different electric charge carrier gas microvesicles described in claim 1, it is characterised in that made with following one-step method：Take 100mL0.5% dextran-70 solution, pH to 2-3 is adjusted, 0.02mmol methylene blues are added, adds 1mL concentration to be 0.1g/mL polyethylene glycol, add 1mL ECAs, 4-17h of stirring, washing gained suspension 2-3 times is simultaneously centrifuged, and obtains electric charge for-70-- 50mV, particle diameter is the microballoon of 500-800nm, microballoon carries negative electrical charge and area load methylene blue, and the molecular weight of the methylene blue is 7000, and the molecular weight polyethylene glycol is 2000.
5. it is used to prepare the preparation method with different electric charge carrier gas microvesicles described in claim 1, it is characterised in that made with following methods of fractional steps：
Step one, the dextran-70 solution 100mL for measuring 0.5%, adjust pH to 2-3, add 0.02mmol methylene blues, add 1mL ECAs, stir 4-5h, and it is 440-480nm to obtain particle diameter, and current potential is-37-- 45mV；
Step 2, the chitosan solution for adding 1mL0.2-0.8g/mL, continue to stir 4-17h, washing gained suspension 2-3 times is simultaneously centrifuged, electric charge is obtained for 20-30mV, particle diameter is the microballoon of 400-600nm, microballoon is with positive charge and loads methylene blue, and the molecular weight of the methylene blue is 7000, and the chitosan is low viscosity shitosan.
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| CN107537044A (en) * | 2017-08-29 | 2018-01-05 | 重庆医科大学 | A kind of chitosan nano microvesicle and preparation method thereof and purposes |
| CN112933248A (en) * | 2021-02-04 | 2021-06-11 | 北京大学第三医院（北京大学第三临床医学院） | Sentinel lymph node double-development tracer, preparation method and application |
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| CN107537044A (en) * | 2017-08-29 | 2018-01-05 | 重庆医科大学 | A kind of chitosan nano microvesicle and preparation method thereof and purposes |
| CN112933248A (en) * | 2021-02-04 | 2021-06-11 | 北京大学第三医院（北京大学第三临床医学院） | Sentinel lymph node double-development tracer, preparation method and application |
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