CN108338977A - Eukaryotic cell membrane cladding bionic nano grain of anti-rotavirus infection and preparation method thereof - Google Patents
Eukaryotic cell membrane cladding bionic nano grain of anti-rotavirus infection and preparation method thereof Download PDFInfo
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Abstract
The invention discloses the eukaryotic cell membranes of anti-rotavirus infection to coat bionic nano grain and preparation method thereof.The kernel that the cellular membrane biomimetic nanoparticle of anti-rotavirus infection is coated by shell and the shell forms, and the shell is eukaryotic cell membrane, and the kernel is mesoporous silicon dioxide nano particle, and the eukaryotic cell membrane is 2 cell membranes of Caco or mdck cell film.The eukaryotic cell membrane cladding bionic nano grain of anti-rotavirus infection can effectively reduce rotavirus (RV) and be infected to normal small intestine cell.
Description
Technical field
The present invention relates to the eukaryotic cell membrane cladding bionic nano grain of viral infection resisting in biotechnology and its preparations
Method.
Background technology
Infantile diarrhea is the second largest reason for causing five years old global or less death of child, is only second to respiratory tract infection,
Middle rotavirus (Rotavirus, RV) infection is to cause infant's severe diarrhea and the first cause of dehydration.RV is felt at present
Diarrhea caused by dye there is no specific drug, clinical at this stage mainly to take symptomatic treatment means, mainly by timely fluid infusion, moisturizing and
Reduced using probiotics (such as probiotics), broad-spectrum antiviral medicament, immunomodulator, alimentary canal mucous membrane protective agent or
Mitigate the symptoms such as diarrhea, the vomiting of patient.
Vaccine inoculation becomes the means of diarrhea caused by effective and unique prevention and control RV infection.China is used by orchid
It develops and in the Oral Rotavirus univalent vaccine rood prestige of listing in 2001, in addition also Ge Lansu in state institute of biological products
The RotaTeq of the Rotarix and Merck ＆ Co., Inc.'s research and development of the production of SmithKline company.These vaccines are oral attenuated live vaccine, in people
Still there is the possibility for restoring virulence because retrograde mutation occurs in vivo, there are certain risks, and in world health group in 2013
Also confirm that there are entembole risks for Rotavirus Vaccine in the position paper knitted.
RV viruses are a kind of diplornavirus, belong to Reoviridae, and RV strain diversifications, according to important
Structural proteins VP6 types, divide RV viruses to 7 groups of A-G.Wherein A, B, C can infect the mankind, but be applied to face at present
The RV vaccines of bed are to be developed for A groups.VP7 is another important albumen of RV, can distinguish viral G serotypes according to VP7, so far
It is identified to have 14 serotypes.Because the serotype of different epidemic strains is different, and vaccine strain is single, so usually connecing
The case where infant is still infected by RV after kind vaccine.Therefore, the active drug or delivery system of prevention and treatment RV infection become
Urgent clinical needs carry out research and development important in inhibiting to it.In addition in the research of the infection mechanism of RV, RV mainly feels in vivo
Intestinal epithelial cell is contaminated, is also only capable of infection enterocyte and renal epithelial cell in vitro, but there may be a variety of for cell membrane surface
RV receptors, research find that sialic acid, nerve modulation fat, heat shock protein, a variety of integrins etc. all play in RV courses of infection
Important function, but it is complicated to interact, it is difficult to it finds suitable receptor blocking drug and effectively to control RV infection.
Modern medicines transmission system optionally delivers the medicament to lesions position and plays curative effect, and what is used at present is big
Mostly synthetic vectors, though can not have the complexity of human endogenous's property substance according to using purpose to carry out various structural modifications
With some functionality, and exogenous poisonous substance is considered as by body sometimes and excretes and can not play a role.In recent years, cell membrane
Bionic nano grain (cell membrane-camouflaged nanoparticles) causes researchers and greatly pays close attention to, it
The nanoparticle of synthesis is combined together with natural biologic material, by the mutual supplement with each other's advantages of the two, forms a kind of Novel imitation crude drug
Object delivery system.Cellular membrane biomimetic nanoparticle is mainly in the nanoparticle by using natural fine after birth as shell to contain synthesis
Core and realize.By this strategy, structure and the function of cell membrane are retained；And synthesize nanoparticle as core, not only
It plays a supportive role, medicine can also be carried or carries out structural modification.In recent years the document report about cellular membrane biomimetic nanoparticle both at home and abroad
In, the cell membrane of use has erythrocyte membrane, leucocyte film, tumor cell membrane, bacterial cell membrane etc..
Invention content
The technical problem to be solved by the present invention is to how anti-rotavirus infects.
In order to solve the above technical problems, the present invention provides the cellular membrane biomimetic nanoparticles of anti-rotavirus infection.
The cellular membrane biomimetic nanoparticle of anti-rotavirus provided by the present invention infection, the cellular membrane biomimetic nanoparticle by
The kernel composition of shell and shell cladding, the shell are eukaryotic cell membrane, and the kernel is mesoporous silicon dioxide nano
Grain, the eukaryotic cell membrane are Caco-2 cell membranes or mdck cell film.
In above-mentioned cellular membrane biomimetic nanoparticle, Caco-2 cells are human colon carcinoma epithelial cell, and mdck cell is that dog kidney is remote
Distal convoluted tubule epithelial cell (Madin-darby canine kidney).
In above-mentioned cellular membrane biomimetic nanoparticle, the grain size (diameter) of the cellular membrane biomimetic nanoparticle can be 80-120nm
(such as 90-96nm or 93-95nm)；Its polydispersity coefficient can be≤0.3 (such as≤0.25 or≤0.2).
In above-mentioned cellular membrane biomimetic nanoparticle, the Zeta potential of the cellular membrane biomimetic nanoparticle can be 0 to -20mV (such as 0
To -12mV or -10mV to -11mV).
The present invention also provides the methods for the cellular membrane biomimetic nanoparticle for preparing above-mentioned anti-rotavirus infection.
The method of the cellular membrane biomimetic nanoparticle provided by the present invention for preparing the infection of above-mentioned anti-rotavirus, including with true
Nucleus prepares eukaryocyte membranosomas, with the coating mesoporous silica dioxide nano particle of eukaryocyte membranosomas, obtains cell
Film bionic nano grain；The eukaryocyte is Caco-2 cells or mdck cell.
In the above method, the eukaryocyte membranosomas for preparing may include：1) it is cracked at 4 DEG C with cell pyrolysis liquid described true
Nucleus 2.5 hours, then carries out broken cell homogenate, then precipitation is collected by centrifugation, and obtains the eukaryotic cell membrane；It 2) will be described
The ultrasonication that eukaryotic cell membrane progress power is 50W and frequency is 20-25KHz 1 minute, hole is used with liposome extruder
Diameter is the filter membrane of 100-400nm, 200-400nm or 200nm, is squeezed out under the extrusion pressure of 0.3Mpa, it is thin to obtain the eukaryon
After birth corpusculum.
In the above method, the ultrasonication can carry out in ice bath.
In the above method, the precipitation that is collected by centrifugation may include：Supernatant is first collected with 500g centrifugations 5min, by the supernatant
Liquid is named as supernatant 1；Supernatant is collected using 10000g centrifugations 5min to the supernatant 1, which is named as
Clear liquid 2；100000g is carried out to the supernatant 2 and centrifuges 1h, collects precipitation, it is described to be precipitated as the eukaryotic cell membrane.
In the above method, the solute of the cell pyrolysis liquid can be Tris, MgCl2Inhibit with the protease of EDTA-free
Agent, solvent are water, Tris and MgCl2Concentration in the cell pyrolysis liquid is 10mM, and the pH of the cell pyrolysis liquid can
It is 7.4.
In the above method, it may include the eukaryon with the coating mesoporous silica dioxide nano particle of eukaryocyte membranosomas
The ultrasonication that cell membranosomas is 80W with mesoporous silicon dioxide nano particle progress power and frequency is 20-25KHz 2 minutes,
Use aperture for the filter membrane of 100-400nm, 200-400nm or 200nm with liposome extruder, under the extrusion pressure of 0.3Mpa
It squeezes out, obtains cellular membrane biomimetic nanoparticle.
It is described to use in the coating mesoporous silica dioxide nano particle of eukaryocyte membranosomas in the above method, at the ultrasonic wave
Reason can carry out in 20-25 DEG C of water-bath.
In the above method, the diameter of the eukaryocyte membranosomas can be 80-140nm, such as 100-120nm or 107-
110nm；Its polydispersity coefficient can be≤0.3 (such as≤0.25 or≤0.2).
In the above method, the Zeta potential of the eukaryocyte membranosomas can be -10mv to -25mV, such as -15mV to -
22mV or -18mV to -20mV.
In the above method, the diameter of the mesoporous silicon dioxide nano particle can be 70-110nm, such as 80-100nm or 80-
83nm；Its polydispersity coefficient can be≤0.2, such as≤0.1.
In the above method, the Zeta potential of the mesoporous silicon dioxide nano particle can be -20mv to -35mV, such as -26mV
To -28mV or -27mV.
Application of the above-mentioned cellular membrane biomimetic nanoparticle in preparing anti-rotavirus infection product (drug or vaccine) also belongs to
In protection scope of the present invention.
Above, the anti-rotavirus infection can be anti-rotavirus infection animal.The animal can be mammal.
The mammal can be true beast infraclass animal.The true beast infraclass animal can be primate.The primate can
For people or monkey.
Above, the anti-rotavirus infection can be anti-rotavirus infection cell.The cell can be mammal
Cell.The mammal can be true beast infraclass animal.The true beast infraclass animal can be primate.The Primates are dynamic
Object can be people or monkey.
(dog distal convoluted renal tubule epithelium is thin using Caco-2 cells (human colon carcinoma epithelial cell) and mdck cell by the present invention
Born of the same parents) two kinds of cells shell of the cell membrane as bionic nano grain, cladding has that physical stability is high, specific surface area and porosity
Greatly, in the inorganic mesoporous silica dioxide nano particle (MSN) for the features such as strong adsorption force, drug carrying ability are excellent, biocompatibility is preferable
Core.The MSN bionic nano grains for Caco-2 cell membranes or mdck cell the film cladding that the present invention is built, infect for RV, can be effective
RV is reduced to infect normal small intestine cell.The invention is expected to make for the treatment that RV infects and benefit our pursuits, and provides a kind of possibility
New way, for work out low toxicity, efficiently, the anti infantile RV with applications well foreground infect drug delivery system provide it is good
Theoretical foundation and practical advice enter clinic and provide referential experience for related drug delivery system and technology.
Description of the drawings
Fig. 1 is transmission electron microscope (TEM) collection of illustrative plates of MSN (A), MC-CMs (B) and MC-CM-MSN (C).In Fig. 1, arrow shows carefully
After birth, the bar in A are 200nm, and the bar in B and C is 100nm.
Fig. 2 is transmission electron microscope (TEM) collection of illustrative plates of CC-CM-MSN.In Fig. 2, bar 50nm.
Fig. 3 is the cell survival rate of each processing group.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation for the cellular membrane biomimetic nanoparticle that embodiment 1, anti-rotavirus infect and its viral infection resisting effect
Present embodiments provide the cellular membrane biomimetic nanoparticle of two kinds of anti-rotavirus infection, respectively Caco-2 cell membranes
Bionic nano grain and mdck cell film bionic nano grain.Caco-2 cellular membrane biomimetics nanoparticle and mdck cell film bionic nano grain
The kernel coated by shell and the shell forms, and kernel is mesoporous silicon dioxide nano particle, Caco-2 cellular membrane biomimetics
The shell of nanoparticle is Caco-2 cell membranes, and the shell of mdck cell film bionic nano grain is mdck cell film.
The preparation method of Caco-2 cellular membrane biomimetic nanoparticles includes preparing Caco-2 cell membranosomas with Caco-2 cells,
With the coating mesoporous silica dioxide nano particle of Caco-2 cell membranosomas, Caco-2 cellular membrane biomimetic nanoparticles are obtained.
The preparation method of mdck cell film bionic nano grain includes preparing mdck cell membranosomas with mdck cell, uses MDCK
The coating mesoporous silica dioxide nano particle of cell membranosomas obtains mdck cell film bionic nano grain.Specific preparation method is as follows：
1. the preparation of Caco-2 cellular membrane biomimetic nanoparticles
The preparation of 1.1 Caco-2 cell membranosomas
1.1.1 Caco-2 cell culture
Caco-2 cells, which use, contains 10% fetal calf serum, 1% glutamine, 1% nonessential amino acid, 1% glutamic acid
Sodium, the dual anti-MEM culture mediums of 1% Pen .- Strep, is positioned over 37 DEG C, 5%CO2Incubator in cultivate.Training is replaced every other day
Base is supported, uses 0.25% pancreatin containing EDTA as digestive juice, according to 1：3 ratios pass on.
1.1.2 the extraction of Caco-2 cell membranes
10% fetal calf serum, 1% glutamine, 1% nonessential amino acid, 1% sodium glutamate and 1% mould will be contained
The dual anti-MEM culture mediums of element-streptomysin are placed in 175cm2In culture bottle, at 37 DEG C, 5%CO2Incubator in culture Caco-2 it is thin
Born of the same parents wait for that cell fusion degree reaches 90%, 0.25% pancreatin of the 1ml containing EDTA are added, digest about 5min, 5ml fresh cultureds are added
Base is gently blown and beaten, and cell suspension is transferred in centrifuge tube, and 850rpm centrifuges 3min, discards upper layer culture medium, and PBS is added
(0.01M, pH 7.2-7.4), blows and beats mixing, and 850rpm centrifuges 3min, discards supernatant liquid, and be repeated twice washing step.Most
After discard supernatant liquid, be added 10ml cold cell pyrolysis liquids prepared in advance (pH 7.4, wherein solute are 10mM Tris,
10mM MgCl2The protease inhibitors of 1 EDTA-free is added with every 10ml cell pyrolysis liquids, solvent is water), it is placed in 4 DEG C
Cracking 2.5 hours after, be transferred in Dounce homogenizers and carry out broken cell homogenate, by broken cell suspension be transferred to from
In heart pipe, following substep differential centrifugation is carried out：Supernatant is first collected with 500g centrifugations 5min, which is named as supernatant
1；Supernatant is collected using 10000g centrifugations 5min to supernatant 1, which is named as supernatant 2；Supernatant 2 is carried out
100000g centrifuges 1h, collects precipitation, which is Caco-2 cell membranes.Caco-2 cell membrane freeze-dried powders are obtained after freeze-drying, are claimed
Big ration, it is spare.
1.1.3 the preparation of Caco-2 cells membranosomas (abbreviation CC-CMs)
The Caco-2 cell membrane freeze-dried powder 2mg for taking 1.1.2 are dispersed in PBS (0.01M, pH 7.2-7.4) solution, dilute
It releases to 6ml；After carrying out the ice-bath ultrasonic 1min that power is 50W and frequency is 20-25KHz with ultrasonic cell disintegration instrument；Use lipid
Body extruder, using the WHATMAN makrolon Nuclepore trace-etching-films in the apertures 200nm as filter membrane, 0.3Mpa's
It is squeezed out under extrusion pressure, obtains Caco-2 cell membranosomas (abbreviation CC-CMs), it is spare.
The preparation of 1.2 mesoporous silicon dioxide nano particles (MSN)
0.437g cetyl trimethylammonium bromides (CTAB) and 0.472g Brij58s (Brij58) are weighed (as template
Agent), it sets in 250ml beakers, the PB solution that the pH7.0 of 120ml room temperatures is added (weighs KH2PO43.43g and NaOH 0.58g, set
In 500ml water, mixing is placed at 4 DEG C, spare), magnetic agitation heats at 60 DEG C, makes to be completely dissolved.Wait for temperature of reaction system
After stabilization, under 60 DEG C of strong stirrings, 2.14ml tetraethyl orthosilicates (TEOS) are added dropwise, the reaction was continued to 8h.Reaction terminates
Afterwards, cooling, sample is transferred in centrifuge tube, 15000rpm centrifuges 15min, abandons supernatant.Isometric distilled water cleaning 2 is added
Secondary, 15000rpm centrifuges 15min；Washes of absolute alcohol 2 times, 15000rpm centrifuge 15min, abandon supernatant.Precipitation is taken, with 60ml
Acidic ethanol (takes in 2ml hydrochloric acid to 150ml absolute ethyl alcohols, mixing is to obtain the final product) dispersion, and 78 DEG C are heated to reflux, each 8h, totally 3 times,
Remove template.Absolute ethyl alcohol washs 3 times, and demineralizing acid is to get mesoporous silicon dioxide nano particle (MSN).It is scattered in ethyl alcohol, 4
It is preserved for a long time in DEG C refrigerator.The above-mentioned MSN being scattered in ethyl alcohol is taken when use, is set in centrifuge tube, 15000rpm centrifuges 15min,
Ethyl alcohol is discarded supernatant, distillation water washing is added 2 times, is dispersed in water with suitable concentration, surveys grain size and Zeta potential.
BET nitrogen adsorption detachment assays show the BET specific surface area (BET of the mesoporous silicon dioxide nano particle (MSN)
Surface area) it is 565.50 ± 4.21m2/ g, pore volume (Pore volume) is 0.73cm3Aperture is desorbed in/g, BJH
(BJH desorption diameter) is 2.13nm.The MSN grain sizes (diameter) are 81.88 ± 1.25nm, the polydispersion of grain size
Coefficient (PDI) is 0.068 ± 0.036, and Zeta potential is -27.10 ± 0.97mV.Illustrate mesoporous silicon oxide prepared by this method
Nanoparticle (MSN) grain size is smaller, in 100nm hereinafter, size distribution is more uniform；Particle surface bear electricity.
The preparation of 1.3 Caco-2 cellular membrane biomimetic nanoparticles (abbreviation CC-CM-MSN)
Caco-2 cell membranosomas (abbreviation CC-CMs) 2mg (freeze-dried powder dry weight) of 1.1.3 is taken, 1.2 MSN prepared are added
1mg, vortex mixed 1min, carry out that power is 80W and frequency is that 20-25KHz, 20-25 DEG C of water-baths are super with ultrasonic cell disintegration instrument
Sound 2min is used as filter with liposome extruder using the WHATMAN makrolon Nuclepore trace-etching-films in the apertures 200nm
Film squeezes out under the extrusion pressure of 0.3Mpa, obtains Caco-2 cellular membrane biomimetic nanoparticles (abbreviation CC-CM-MSN).
2. the preparation of mdck cell film bionic nano grain
The preparation of 2.1 mdck cell membranosomas
2.1.1 mdck cell culture
Mdck cell, which uses, contains 10% fetal calf serum, and the dual anti-DMEM culture mediums of 1% Pen .- Strep are positioned over
37 DEG C, 5%CO2Incubator in cultivate.Use 0.25% pancreatin containing EDTA as digestive juice, according to 1：5~1:10 ratios pass
Generation.
2.1.2 the extraction of mdck cell film
10% fetal calf serum will be contained, the dual anti-DMEM culture mediums of 1% Pen .- Strep are placed in 175cm2Culture bottle
In, at 37 DEG C, 5%CO2Incubator in cultivate mdck cell, wait for that cell fusion degree reaches 90%, 1ml be added containing EDTA's
0.25% pancreatin digests about 5min, and 5ml fresh cultures are added and gently blow and beat, and cell suspension is transferred in centrifuge tube,
850rpm centrifuges 3min, discards upper layer culture medium, and PBS (0.01M, pH 7.2-7.4) is added, and blows and beats mixing, 850rpm centrifugations
3min discards supernatant liquid, and is repeated twice washing step.Liquid is finally discarded supernatant, 10ml prepared cold cells in advance are added
(pH=7.4, wherein solute are 10mM Tris, 10mM MgCl to lysate2The protease of 1 EDTA-free is added with every 10ml
Inhibitor, solvent are water), be placed in 4 DEG C cracking 2.5 hours after, be transferred in Dounce homogenizers and carry out broken cell homogenate, will
Broken cell suspension is transferred in centrifuge tube, carries out following substep differential centrifugation：Supernatant is first collected with 500g centrifugations 5min
The supernatant is named as supernatant 1 by liquid；Supernatant is collected using 10000g centrifugations 5min to supernatant 1, which is ordered
Entitled supernatant 2；100000g is carried out to supernatant 2 and centrifuges 1h, collects precipitation, which is mdck cell film.After freeze-drying
To mdck cell film freeze-dried powder, weigh quantitative, it is spare.
2.1.3 the preparation of mdck cell membranosomas (abbreviation MC-CMs)
The mdck cell film freeze-dried powder 2mg for taking 2.1.2 is dispersed in PBS (0.01M, pH 7.2-7.4) solution, dilution
To 6ml；After carrying out the ice-bath ultrasonic 1min that power is 50W and frequency is 20-25KHz with ultrasonic cell disintegration instrument；Use liposome
Extruder, using the WHATMAN makrolon Nuclepore trace-etching-films in the apertures 200nm as filter membrane, in squeezing for 0.3Mpa
Go out under pressure and squeeze out, obtains mdck cell membranosomas (abbreviation MC-CMs), it is spare.
The preparation of 2.2 mdck cell film bionic nano grains (abbreviation MC-CM-MSN)
Mdck cell membranosomas (abbreviation MC-CMs) 2mg (freeze-dried powder dry weight) for taking 2.1.3, is added 1.2 MSN1mg, whirlpool
Rotation mixing 1min, carries out that power is 80W and frequency is 20-25KHz, 20-25 DEG C of water bath sonicator 2min with ultrasonic cell disintegration instrument,
With liposome extruder, using the WHATMAN makrolon Nuclepore trace-etching-films in the apertures 200nm as filter membrane,
It is squeezed out under the extrusion pressure of 0.3Mpa, obtains mdck cell film bionic nano grain (abbreviation MC-CM-MSN).
3, the characterization of cellular membrane biomimetic nanoparticle, cell membranosomas and mesoporous silicon dioxide nano particle
3.1 particle diameter distributions and Zeta potential
Take 1.3 Caco-2 cellular membrane biomimetic nanoparticles (abbreviation CC-CM-MSN), 2.2 mdck cell film bionic nano
The mdck cell membranosomas of grain (abbreviation MC-CM-MSN), the Caco-2 cell membranosomas (abbreviation CC-CMs) of 1.1.3,2.1.3
(abbreviation MC-CMs) and 1.2 mesoporous silicon dioxide nano particle (MSN), be dispersed in water with suitable concentration, with Nano-ZS 90
Type laser particle size and Zeta potential analyzer measure its grain size and its polydispersity coefficient (PDI) and Zeta potential respectively.It takes above-mentioned
Each sample drops on transmission electron microscope copper mesh, is put into draught cupboard and waits for that liquid volatilizes, and a drop 2% Salkowski's solution dyeing is added dropwise
2min sucks surplus liquid with filter paper, transmission electron microscope observation each sample form is used after dry.
The result shows that Caco-2 cellular membrane biomimetic nanoparticles (abbreviation CC-CM-MSN) and mdck cell film bionic nano grain
The grain size (diameter) of (abbreviation MC-CM-MSN) is respectively 94.60 ± 1.26nm and 95.37 ± 1.45nm, the polydisperse system of grain size
Number (PDI) is respectively 0.122 ± 0.042,0.131 ± 0.033.And MSN grain sizes (diameter) are 81.88 ± 1.25nm, grain size
Polydispersity coefficient (PDI) is 0.068 ± 0.036.Caco-2 cell membranosomas (abbreviation CC-CMs) and (letter of mdck cell membranosomas
Claim MC-CMs) grain size (diameter) be respectively 108.8 ± 1.26nm and 110.1 ± 1.41nm (Fig. 1 and Fig. 2), more points of grain size
Scattered coefficient is respectively 0.198 ± 0.031,0.187 ± 0.056.Exist outside MSN kernels in CC-CM-MSN and MC-CM-MSN
One tunic is dizzy, and thickness and cell membrane consistency of thickness, further proves, cell membrane is successfully coated in MSN nanoparticles
On core, cellular membrane biomimetic nanoparticle is built successfully.Caco-2 cellular membrane biomimetic nanoparticles (abbreviation CC-CM-MSN) and mdck cell
The Zeta potential of film bionic nano grain is respectively -10.03mV ± 1.64mV, -11.32mV ± 1.33mV, and Caco-2 cell membranes are small
The Zeta potential of body (abbreviation CC-CMs) and mdck cell membranosomas (abbreviation MC-CMs) respectively -18.58mV ± 0.45mV, -
The Zeta potential of 20.01mV ± 0.32mV, mesoporous silicon dioxide nano particle (MSN) are -27.10 ± 0.97mV.
4, the vitro Cytotoxicity Evaluation of cellular membrane biomimetic nanoparticle
4.1 MTT solution are prepared
MTT powder is dissolved with 1 × PBS (0.01M, pH 7.2-7.4) and is configured to the solution of 5mg/ml, filtration sterilization
After be dispensed into EP pipes, preserved under the conditions of being protected from light in -20 DEG C.It is diluted with culture medium when using every time, ensures, per 100 μ g of hole, to lead to
It is often 10 times of dilutions between continuous two pipe, 200 μ L is added per hole.
4.2 cell inoculation
Mdck cell is inoculated into 96 orifice plates according to the density of 4000 cells in every hole, in 37 DEG C, 5%CO2Culture
Being incubated in case for 24 hours keeps cell adherent.
4.3 dosings are incubated
Discard culture solution.Setting blank group (acellular, blank cultures, not dosing), control group (have cell, blank training
Support base, not dosing), MSN groups (having cell, MSN culture mediums), CC-CM-MSN groups (having cell, CC-CM-MSN culture mediums), MC-
CM-MSN groups (have cell, MC-CM-MSN culture mediums), in 37 DEG C, 5%CO2Incubator in be incubated for 24 hours.
Blank cultures are to contain 10% fetal calf serum, the dual anti-DMEM culture mediums of 1% Pen .- Strep.MSN is cultivated
Base be MSN described under 1.2 is added into blank cultures, and make finally obtained MSN contents be respectively 500,300,
200, the culture medium of 100,50,20,10 μ g/ml, CC-CM-MSN culture mediums are described under addition 1.3 into blank cultures
CC-CM-MSN, and finally obtained CC-CM-MSN contents is made to be respectively the culture medium of 200,100,50,20 μ g/ml, MC-
CM-MSN culture mediums are MC-CM-MSN described under 2.2 to be added into blank cultures, and make finally obtained MC-CM-
MSN contents are respectively the culture medium of 200,100,50,20 μ g/ml.
4.4 are added MTT
After dosing is incubated for 24 hours, culture solution is discarded, the MTT solution after serum free medium dilution is added, per 200 μ L of hole, after
It is continuous to be incubated 4h.Liquid is discarded supernatant, 150 μ L DMSO are then added per hole, being shaken on air bath oscillator makes to be completely dissolved.
4.5 OD values measure
It is measured every hole absorption values at 490nm with microplate reader.
4.6 data processing
Cell survival rate=(ODExperimental group-ODBlank group)/(ODControl group-ODBlank group) * 100%.
The results are shown in Figure 3, and when low concentration (concentration be less than 50 μ g/ml), CC-CM-MSN and MC-CM-MSN are to cell
Toxicity is less than MSN.Show in the concentration range of 10-50 μ g/ml, the cladding of cell membrane (CMs) reduces simple mesoporous dioxy
The toxicity of SiClx nanoparticle (MSN).
5. the external anti-rotavirus evaluation of effect of cellular membrane biomimetic nanoparticle
5.1 Virus culture
5.1.1 MA104 cell culture
MA104 cells (rhesus monkey Foetus renal cells) are cultivated using the dual anti-MEM containing 10% serum, 1% Pen .- Strep
Base, in 37 DEG C, 5%CO2Incubator in cultivate.
5.1.2 rotavirus proliferation is extracted with virus liquid
The MA104 cells for having grown up to fine and close single layer are taken, culture solution therein is discarded, 3 are washed with the MEM culture mediums without serum
Secondary, exhaust remaining maintaining liquid, and 0.5ml rotavirus liquid (human rotavirus Wa comes from Lanzhou Institute of Biological Products) is added,
37 DEG C of absorption 90min, rock once per 30min.Unadsorbed virus liquid is removed, the dimension that 10ml contains 0.5 μ g/ml pancreatin is added
Liquid is held, is put back in incubator, when lesion (CPE reaches +++) occurs in 75% cell, cell is alternately frozen in -20 DEG C~37 DEG C
Melt 3000rpm at 3 times, 4 DEG C and centrifuge 10min, it is rotavirus liquid to take supernatant, is preserved at -80 DEG C.
The external anti-rotavirus evaluation of effect of 5.2 two kinds of cellular membrane biomimetic nanoparticles
Experiment is set to be repeated three times, is repeated MA104 cells being equably layered in 96 orifice plates every time, is sucked culture medium, per hole
50 μ L, 100 TCID are added50Rotavirus liquid, be put into incubator and adsorb 2h, be divided into virus control group and 5 sample sets
(each sample sets are divided into as 6 dosage groups), 4 multiple holes of each dosage group are specific to be respectively：Virus control group, MSN groups (10 μ
G/ml groups, 20 μ g/ml groups, 50 μ g/ml groups, 100 μ g/ml groups, 200 μ g/ml groups, 300 μ g/ml groups), CC-CM-MSN groups (10 μ
G/ml groups, 20 μ g/ml groups, 50 μ g/ml groups, 100 μ g/ml groups, 200 μ g/ml groups, 300 μ g/ml groups), MC-CM-MSN groups (10 μ
G/ml groups, 20 μ g/ml groups, 50 μ g/ml groups, 100 μ g/ml groups, 200 μ g/ml groups, 300 μ g/ml groups), MC-CMs groups (10 μ g/ml
Group, 20 μ g/ml groups, 50 μ g/ml groups, 100 μ g/ml groups, 200 μ g/ml groups, 300 μ g/ml groups), CC-CMs groups (10 μ g/ml groups,
20 μ g/ml groups, 50 μ g/ml groups, 100 μ g/ml groups, 200 μ g/ml groups, 300 μ g/ml groups).Above-mentioned each group is carried out respectively simultaneously
Following processing：The MEM culture mediums that 100 μ L are free of serum are added per hole for virus control group, and MSN groups are added 100 μ L per hole and are trained containing MSN
Support base (the MSN amounts that various dose group is only added are different), 100 μ L culture mediums containing CC-CM-MSN are added per hole for CC-CM-MSN groups
100 μ L culture mediums containing MC-CM-MSN are added per hole for (the CC-CM-MSN amounts that various dose group is only added are different), MC-CM-MSN groups
100 μ L culture mediums containing MC-CMs (difference is added per hole for (the MC-CM-MSN amounts that various dose group is only added are different), MC-CMs groups
The MC-CMs amounts that dosage group is only added are different), (various dose group is only for 100 μ L culture mediums containing CC-CMs of addition per hole for CC-CMs groups
The CC-CMs amounts of addition are different).Set normal cell controls group simultaneously：MA104 cells are equably layered in 96 orifice plates, training is sucked
Base is supported, maintaining liquids of the 50 μ L of addition without virus, is put into incubator and adsorbs 2h per hole, and 100 μ L are added per hole and are free of serum
MEM culture mediums.
Wherein, culture medium containing MSN is that MSN described under 1.2 is added in the MEM culture mediums without serum above-mentioned to obtain
Culture medium, the MSN contents in the various dose group of culture medium containing MSN are respectively 10,20,50,100,200,300 μ g/ml.Contain
CC-CM-MSN culture mediums are that CC-CM-MSN described under 1.3 is added in the MEM culture mediums without serum above-mentioned to obtain
Culture medium, the CC-CM-MSN contents in the various dose group of culture medium containing CC-CM-MSN are respectively 10,20,50,100,200,300
μg/ml.Culture medium containing MC-CM-MSN is that MC-CM- described under 2.2 is added in the MEM culture mediums without serum above-mentioned
The culture medium that MSN is obtained, the MC-CM-MSN contents in the various dose group of culture medium containing MC-CM-MSN are respectively 10,20,50,
100、200、300μg/ml.Culture medium containing MC-CMs is described under addition 2.1.3 in the MEM culture mediums without serum above-mentioned
The obtained culture mediums of MC-CMs, the MC-CMs contents in the various dose group of culture medium containing MC-CMs are respectively 10,20,50,
100、200、300μg/ml.Culture medium containing CC-CMs is described under addition 1.1.3 in the MEM culture mediums without serum above-mentioned
The obtained culture mediums of CC-CMs, the CC-CMs contents in the various dose group of culture medium containing CC-CMs are respectively 10,20,50,
100、200、300μg/ml。
Above-mentioned each group is put back in incubator and is cultivated 3 days, observes cellular change under inverted microscope daily, records cell
Lesion effect (CPE) degree.Virus control group CPE is waited for up to 80% or more, and when cell controls group is normal, it is thin to work with MTT
Born of the same parents dye, and read A570Inhibiting rate of each sample group to virus is calculated according to the following formula in value：
Viral suppression (%)=(sample treatment group A570Virus control group A570)/(normal cell controls group A570Virus
Control group A570) × 100%, and calculate each sample group medium effective concentration IC50.It the results are shown in Table 1：
Inhibiting rate (%) of the 1. various dose cellular membrane biomimetic nanoparticle of table to RV viruses
By result it is found that cellular membrane biomimetic nanoparticle administration handles (CC-CM-MSN groups and MC-CM-MSN groups) to RV viruses
Well, inhibiting rate increases with dosage and is increased for the therapeutic effect (to the inhibiting effect of virus multiplication) of infection, and dose-effect is presented and closes
System, CC-CM-MSN groups and MC-CM-MSN group medium effective concentrations are respectively 52.8 μ g/ml and 66.5 μ g/ml；Wherein Caco-2
Cellular membrane biomimetic nanoparticle handles (CC-CM-MSN groups) and is slightly better than mdck cell film bionic nano grain processing (MC-CM-MSN groups).
The cell that individual mesoporous silicon dioxide nano particle processing (MSN groups) infects RV viruses is substantially without therapeutic effect (with virus
Control group is compared to no significant difference)；Individual Caco-2 cell membranosomas CMs processing (CC-CMs groups) and mdck cell membranosomas
CMs processing (MC-CMs groups) has certain inhibiting effect for RV virus multiplications, but is markedly less than CC-CM-MSN groups and MC-CM-
MSN groups.
It should be noted that in recent years both at home and abroad about in the document report of cellular membrane biomimetic nanoparticle, the cell membrane of use
There are erythrocyte membrane, leucocyte film, tumor cell membrane, bacterial cell membrane etc., there is not yet thin using Caco-2 cell membranes or MDCK
The report of after birth；By Caco-2 cell membranes or mdck cell film bionic nano grain for being even more to have no to appoint in terms of anti-RV viruses infection
What is reported.Moreover, natural Caco-2 cells or mdck cell by clasmatosis, cell membrane extraction and with nanoparticle in
Core co-extrusion pressure is formed in the complex process of bionic nano grain, and whether cell membrane surface structure and function can be maintained, if
The effect that absorption RV viruses can effectively be played like that as desired, can not predict；It needs by professional by a large amount of
Creative work obtains bionic nano plastochondria system and carries out ability susceptible of proof after antiviral relevant active testing.Though in addition, having
The cellular membrane biomimetic nanoparticle relevant report of other types, but the cell membrane extraction process and design parameter of variety classes cell are simultaneously
It differs, Caco-2 cells are compared with the cell (such as tumour cell, red blood cell, bacterial cell) of other existing researchs, training
It is longer to support the period, and extracting cell membrane and once at least needing to collect several ten million supreme hundred million cells can just carry out, therefore cell membrane
Far more than other cell types the time required to cell passage and collection before extraction, therefore there is abnormal wind in microbiological contamination or cell
Danger can not simply use for reference the experience of other cell membrane extractions also above other cell types.Furthermore Caco-2 or mdck cell film
After extraction, after MSN nanoparticle co-extrusion pressures, the preparation condition of bionic nano grain in document is shone, stabilization can not be formed
Bionic nano grain, the preparation method of the cellular membrane biomimetic nanoparticle for preparing anti-rotavirus infection in above-described embodiment 1 is
The suitable condition that inventor gropes to obtain by a large amount of process conditions, these experiments include：
1 cell membrane extraction conditions are investigated
The investigation of cell membrane extraction conditions is carried out using mdck cell.
The influence that 1.1 cell cracking times extracted cell membrane
To investigate the influence of cell cracking time, two experimental groups are designed：First group of pyrolysis time is 1h, second group of cracking
Time is 2.5h.The specific method is as follows：
10% fetal calf serum will be contained, the dual anti-DMEM culture mediums of 1% Pen .- Strep are placed in 175cm2Culture bottle
In, at 37 DEG C, 5%CO2Mdck cell is cultivated in incubator, waits for that cell fusion degree reaches 90%, and 1ml is added containing EDTA's
0.25% pancreatin digests about 5min, and 5ml fresh cultures are added and gently blow and beat, and cell suspension is transferred in centrifuge tube,
850rpm centrifuges 3min, discards upper layer culture medium, and PBS is added, and blows and beats mixing, and 850rpm centrifuges 3min, discards supernatant liquid, lay equal stress on
Multiple washing step twice.Liquid is finally discarded supernatant, 10ml prepared cold cell pyrolysis liquid (pH=7.4, wherein molten in advance are added
Matter is 10mM Tris, 10mM MgCl2With the protease inhibitors that 1 EDTA-free is added in every 10ml, solvent is water), it sets
After 4 DEG C crack 1 hour or 2.5 hours, it is transferred in Dounce homogenizers and carries out broken cell homogenate, by broken cell
Suspension is transferred in centrifuge tube, carries out following substep differential centrifugation：Supernatant is first collected with 500g centrifugations 5min, by the supernatant
It is named as supernatant 1；Supernatant is collected using 10000g centrifugations 5min to supernatant 1, which is named as supernatant 2；
100000g is carried out to supernatant 2 and centrifuges 1h, collects precipitation.The result shows that cracking 1 hour, it is not collected into membrane pellet,
Cracking has collected membrane pellet in 2.5 hours.
Influence of 1.2 centrifugal methods to cell membrane extraction efficiency
To investigate influence of the different centrifugal methods to cell membrane extraction efficiency, two experimental groups are designed.First group of centrifugation side
Method is：Supernatant is first collected with 500g centrifugations 5min, which is named as supernatant 1；To supernatant 1 using 10000g from
Heart 5min collects supernatant, which is named as supernatant 2；100000g is carried out to supernatant 2 and centrifuges 1h, collects precipitation.
Second group of centrifugal method be:Supernatant is first collected with 2000g centrifugations 12min, which is named as supernatant
1 ', 1h is centrifuged using 100000g to supernatant 1 ', collects precipitation.
Specific test method is as follows：Except 1.1 pyrolysis time is replaced with 2.5 hours, centrifugation is replaced with above-mentioned
Outside one group of centrifugation or second group of centrifugation, other operations are identical as 1.1.
The result shows that second group of centrifugation is not collected into membrane pellet, first group has been collected by centrifugation membrane pellet.
2, cell membranosomas (abbreviation CMs) preparation condition optimal screening
The Caco-2 cell membrane freeze-dried powder 2mg of 1.1.2 in Example 1, are dispersed in PBS solution, are diluted to 6ml,
It surveys grain size, which is named as CMs (after dispersion)；Carry out that power is 50W and frequency is 20- with ultrasonic cell disintegration instrument
It after the ice-bath ultrasonic 1min of 25KHz, surveys grain size again, which is named as CMs (after ultrasound)；With liposome extruder, divide
The WHATMAN makrolon Nuclepore trace-etching-films that aperture is 800nm, 400nm, 200nm, 100nm are not used to be used as filter
Film squeezes out 10 times repeatedly under the extrusion pressure of 0.3Mpa, the particle of acquisition is respectively designated as 800nm filter membranes, 400nm is filtered
Film, 200nm filter membranes and 100nm filter membranes.Above-mentioned is measured respectively with 90 type laser particle sizes of Nano-ZS and Zeta potential analyzer
The grain size and polydispersity coefficient (PDI) of grain.The results are shown in Table 2.Illustrate that ultrasound is very necessary to the formation of cell membranosomas；It is logical
It crosses and the selection result of different pore size filter membrane is known, through the apertures 200nm filter membrane cell membranosomas after extrusion, particle size is suitable,
And PDI values are minimum (dispersion homogeneity is best).Accordingly, it is determined that selecting the preparation of the apertures 200nm polyester film progress cell membranosomas.
The grain size and polydispersity coefficient of 2. each particle of table
| Particle | Grain size (nm) | PDI |
| CMs (after dispersion) | 1306 | 1.0 |
| CMs (after ultrasound) | 381.5 | 0.528 |
| 800nm filter membranes | 119.9 | 0.255 |
| 400nm filter membranes | 117.0 | 0.231 |
| 200nm filter membranes | 108.8 | 0.198 |
| 100nm filter membranes | 107.8 | 0.278 |
3, the preparation condition optimal screening of cellular membrane biomimetic nanoparticle (CM-MSN)
Caco-2 cell membranosomas (abbreviation CC-CMs) 2mg (freeze-dried powder dry weight) of 1.1.3 in Example 1 is added real
The MSN 1mg under 1.2, vortex mixed 1min in example 1 are applied, carries out that power is 80W and frequency is 20- with ultrasonic cell disintegration instrument
The aperture 400nm, 200nm and 100nm is respectively adopted with liposome extruder in 25KHz, 20-25 DEG C of water bath sonicator 2min
WHATMAN makrolon Nuclepore trace-etching-films squeeze out 10 times repeatedly as filter membrane under the extrusion pressure of 0.3Mpa,
Measure cellular membrane biomimetic nanoparticle (the abbreviation CM- of gained respectively with 90 type laser particle sizes of Nano-ZS and Zeta potential analyzer
MSN grain size) and polydispersity coefficient (PDI).The results are shown in Table 3, shows to select the filter membrane in the apertures 200nm to carry out nanoparticle
Cladding is squeezed out, the CM-MSN grain sizes obtained are close with desired value, and PDI values are lower, illustrate that size distribution is more uniform.
The particle size results of CM-MSN after 3. different pore size membrane filtration of table
| Filter sizes size (nm) | Grain size (nm) | PDI |
| 400 | 88.86 | 0.216 |
| 200 | 94.60 | 0.122 |
| 100 | 90.88 | 0.210 |
Claims (10)
1. the cellular membrane biomimetic nanoparticle of anti-rotavirus infection, the cellular membrane biomimetic nanoparticle is by shell and the shell packet
The kernel composition covered, the shell are eukaryotic cell membrane, and the kernel is mesoporous silicon dioxide nano particle, the eukaryotic cell membrane
For Caco-2 cell membranes or mdck cell film.
2. cellular membrane biomimetic nanoparticle according to claim 1, it is characterised in that：The grain of the cellular membrane biomimetic nanoparticle
Diameter is 80-120nm, 90-96nm or 93-95nm.
3. cellular membrane biomimetic nanoparticle according to claim 1 or 2, it is characterised in that：The cellular membrane biomimetic nanoparticle
Grain size polydispersity coefficient be≤0.3 ,≤0.25 or≤0.2.
4. according to any cellular membrane biomimetic nanoparticle in claim 1-3, it is characterised in that：The cellular membrane biomimetic is received
The Zeta potential of the grain of rice is 0 to -20mV, 0 to -12mV or -10 to -11mV.
5. cellular membrane biomimetic nanoparticle according to any one of claims 1-4, it is characterised in that：The cellular membrane biomimetic is received
The grain of rice is prepared according to method any in claim 6-10.
6. the method for preparing any cellular membrane biomimetic nanoparticle in claim 1-4, including prepared very with eukaryocyte
Nucleus membranosomas obtains cellular membrane biomimetic nanoparticle with the coating mesoporous silica dioxide nano particle of eukaryocyte membranosomas；
The eukaryocyte is Caco-2 cells or mdck cell.
7. according to the method described in claim 6, it is characterized in that：The eukaryocyte membranosomas for preparing includes：1) cell is used
Lysate cracks the eukaryocyte at 4 DEG C 2.5 hours, then carries out broken cell homogenate, then precipitation is collected by centrifugation, obtains institute
State eukaryotic cell membrane；2) ultrasonication 1 that eukaryotic cell membrane progress power is 50W and frequency is 20-25KHz is divided
Clock uses aperture for the filter membrane of 100-400nm, 200-400nm or 200nm with liposome extruder, in the extrusion pressure of 0.3Mpa
It is squeezed out under power, obtains the eukaryocyte membranosomas.
8. the method described according to claim 6 or 7, it is characterised in that：The precipitation that is collected by centrifugation includes：First centrifuged with 500g
5min collects supernatant, which is named as supernatant 1；To the supernatant 1 using in 10000g centrifugation 5min collections
The supernatant is named as supernatant 2 by clear liquid；100000g is carried out to the supernatant 2 and centrifuges 1h, collects precipitation, the precipitation
For the eukaryotic cell membrane.
9. according to any method in claim 6-8, it is characterised in that：It is coating mesoporous with the eukaryocyte membranosomas
Silica dioxide nano particle includes that the eukaryocyte membranosomas and mesoporous silicon dioxide nano particle are carried out power as 80W and frequency
For the ultrasonication 2 minutes of 20-25KHz, use aperture for 100-400nm, 200-400nm with liposome extruder or
The filter membrane of 200nm squeezes out under the extrusion pressure of 0.3Mpa, obtains cellular membrane biomimetic nanoparticle.
10. any cellular membrane biomimetic nanoparticle answering in preparing anti-rotavirus infection product in claim 1-4
With.
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| CN110269847A (en) * | 2019-06-21 | 2019-09-24 | 温州医科大学 | A kind of bionic nano material and the preparation method and application thereof for neutralizing bacteriotoxin |
| CN114128723A (en) * | 2021-11-09 | 2022-03-04 | 苏州大学 | Novel antiviral nano material and application thereof |
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| FARIDEH TAFAZOLI等: "NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells", 《JOURNAL OF VIROLOGY》 * |
| LANG RAO等: "Microfluidic Electroporation-Facilitated Synthesis of Erythrocyte Membrane-Coated Magnetic Nanoparticles for Enhanced Imaging-Guided Cancer Therapy", 《ACS NANO》 * |
| 魏潇萌等: "细胞膜仿生纳米粒的研究与应用", 《中国医药生物技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110269847A (en) * | 2019-06-21 | 2019-09-24 | 温州医科大学 | A kind of bionic nano material and the preparation method and application thereof for neutralizing bacteriotoxin |
| CN114128723A (en) * | 2021-11-09 | 2022-03-04 | 苏州大学 | Novel antiviral nano material and application thereof |
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