CN106924755A - The bionic nano particle and preparation method of a kind of Polymorphonuclear Leukocytes Membrane cladding of activation - Google Patents
The bionic nano particle and preparation method of a kind of Polymorphonuclear Leukocytes Membrane cladding of activation Download PDFInfo
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Abstract
The invention belongs to field of pharmaceutical preparations, it is related to bionic nano particle and preparation method that a kind of Polymorphonuclear Leukocytes Membrane of activation is coated.With polymer poly lactic-co-glycolic acid PLGA in the present invention, or copolymer of poly lactic acid PLA, or polycaprolactone co-polymer PCL is for material prepares nanoparticle as kernel and contains model drug, natural activation Polymorphonuclear Leukocytes Membrane is coated on nanoparticle surface as capsid, the biodegradable bionical particle nano-carrier system of the Polymorphonuclear Leukocytes Membrane cladding of activation is made.Show through inside and outside experiment, the bionical particle nano-carrier system can be directed to circulating tumor cell and micrometastasis stove bionical treatment system have targeting it is strong, it is efficient the characteristics of, it is that individuation and accurate treatment provide new thinking, with clinical practice meaning higher.
Description
Technical field
The invention belongs to technical field of medicine, it is related to the bionic nano preparation of antineoplastic, and in particular to one kind swashs
The bionic nano particle and preparation method of Polymorphonuclear Leukocytes Membrane cladding living, it is especially a kind of to contain having for fat-soluble medicine
Nano-carrier of imitative neutrophil leucocyte of polymer-cell membrane core shell structure and preparation method thereof.
Background technology
Data shows that malignant tumour is to endanger the primary killers of human health at present, is with invasion and transfer ability
Its most important physiological characteristic.In clinical practice, although the intervention means such as operation, chemotherapy can play one being in a short time set for
With, but prognosis is typically poor, and the recurrence and transfer of tumour are the ultimate challenges that treatment faces.Research display metastases medium vessels
It is main " highway " of each organ of tumour cell intrusion body, largely propagation comes off tumour epidermal cell, invades blood vessel
Form the circulating tumor cell (Circulating Tumor Cells, CTC) with invasiveness high and locomitivity, this tool
The CTC for having endothelial-like appearance reaches position of suitably living away from home at a distance, forms MET, as in the future fatal hidden danger,
It is the basic reason place of clinical cancer therapy failure.
Can be divided into various subgroups, including neutrophil leucocyte, lymphocyte and list prior art discloses human leukocytes
Nucleus etc., research finds that the neutrophil leucocyte for accounting for total white blood cells 70% or so has natural targeting to CTC and metastasis site
Effect, its molecular basis be inflammatory in the change of the adhesion molecule expression of both cell surfaces, i.e. tumor microenvironment because
Son activation neutrophil leucocyte, raises the adhesion molecule expression on its cell membrane, enhances the spy of neutrophil leucocyte and CTC expression high
Specific ligand affinity is so as to mediate the transfer of tumour.
Based on Basic of Biology of the neutrophil leucocyte to the efficient targetings of CTC is activated, present inventor intends providing a kind of
A kind of biodegradable bionical particle of the Polymorphonuclear Leukocytes Membrane cladding of activation, and in particular to tool that can contain fat-soluble medicine
Have a nano-carrier system of the imitative neutrophil leucocyte of polymer-cell membrane core shell structure, the system will in blood circulation height target
To and kill CTC, so as to suppress the transfer of tumour.
The content of the invention
It is an object of the invention to build a kind of the biodegradable bionical of Polymorphonuclear Leukocytes Membrane of Surface coating activation
Particle (neutrophil like nanopartiles, abbreviation NLP) and preparation method thereof.
The present invention is with polymer poly poly lactic coglycolic acid (poly (lactic-co-glycolic acid), letter
Claim PLGA), or copolymer of poly lactic acid (polylactic acid, abbreviation PLA), or polycaprolactone co-polymer
(Polycaprolactone, abbreviation PCL) for material prepares nanoparticle as kernel and contains model drug, in naturally activating
Property granulocyte film be coated on nanoparticle surface as capsid, be made activation Polymorphonuclear Leukocytes Membrane cladding it is biodegradable imitative
Raw particle nano-carrier system.In the nano-carrier system, the membrane protein of activation is on the one hand utilized, in blood long circulating
And height target killing CTC；On the other hand naturally raised using neutrophil leucocyte to the principle of inflammation part blood vessel, target inflammation
The early stage micrometastatic disease of state simultaneously discharges medicine, so as to suppress the transfer of tumour.
In the present invention, the model drug for using is Carfilzomib, bortezomib or taxanes based chemotherapy medicine, is FDA
The small molecule chemotherapeutic medicine of approval；Wherein, Carfilzomib, bortezomib or taxanes based chemotherapy medicine, can act on it is various with
The related path of transfer, resistance, inducing apoptosis of tumour cell suppresses metastases activity, by these chemotherapeutics with certain
Contain in above-mentioned nano-carrier, on the one hand can extend its half-life period in blood, induced circulation apoptosis of tumor cells；Separately
On the one hand the generation of matrix metalloproteinase and relevant inflammatory factors can be suppressed, early stage micrometastatic disease local inflammation ring is adjusted
Border, contains the field planting and invasion and attack of tumour cell.
In the present invention, polymer P LGA, PLA, the nanoparticle that PCL builds can contain hydrophobic drug, have as kernel
Mechanical stability higher, its molecular weight is 10000-30000Da.
In the present invention, the neutrophil leucocyte membrane vesicle is by thin from the isolated neutral grain of same species animal peripheral blood
Born of the same parents, are carried out the external evoked expression so as to raise related cell adhesion molecule, are extracted using differential centrifugation and obtained；
In the present invention, in the Polymorphonuclear Leukocytes Membrane surface related cell adhesion molecule of the activation, the neutrality of LPS lipopolysaccharides activation
Granulocyte film raises the expression of the memebrane proteins such as the Integrins of β 2, L-selectin, chemokine receptors, the integrins of β 1.
The present invention builds the bionical particle nano-carrier system of particle method by following methods：
1. from the same isolated neutrophil leucocyte of species animal peripheral blood, carried out external evoked related viscous so as to raise
The expression of attached molecule, differential centrifugation extracts the vesica obtained during cell membrane is scattered in the aqueous solution；
2. the nanoparticle of uniform particle diameter is obtained using emulsified vehicles volatility process as material with PLGA or PLA or PCL, while bag
Carry slightly solubility small molecule model medicine；
3. previously prepared neutrophil leucocyte membrane vesicle and PLGA or PLA or PCL nanoparticles are incubated and reciprocal film excessively altogether
Extruding.
In the present invention, the PBC source of species is mouse or rat.
In the present invention, the nanoparticle kernel and Cell membrane vesicles of the polymer construction are made by hydrophobic effect and electrostatic
With being combined, in polymer core pan coating film-proteolipid bilayer, the bionical grain of nanoscale of core shell structure is formed
Son.
In the present invention, the Cell membrane vesicles average grain diameter is 200nm or so, and polymer nanocomposite kernel average grain diameter is
80-100nm, ultimate size is in 100-120nm or so after coating Polymorphonuclear Leukocytes Membrane；Compared to the cell of micro-meter scale, than
Surface area is larger, can more efficiently play the effect of membrane structure.
It is early stage micrometastatic disease endothelial cell model that the present invention uses Human umbilical vein endothelial cells (HUVEC cells), with
It is circulating tumor cell mould that the vertebral plate viscosimeter of improvement produces certain shearing force to breast cancer lung high-transfer cell (4T1 cells)
Type, this two kinds of cells are art-recognized and know.
The present invention describes the preparation scheme that the medicine passs release system, and there is provided the result of the system inside and outside targeting
And pharmacodynamic evaluation.
The present invention simulates blood flow state by the cone and plate viscometer using improvement, evaluates in certain blood shearing force
Target functions of the lower NLP to the 4T1 tumour cells of flow regime is acted on, and Carfilzomib induced flow shape can be significantly improved
The ability of the apoptosis of the 4T1 cells of state.Lung metastases model nude mice live body near-infrared/bioluminescence double-mode imaging was it is demonstrated experimentally that should
Medicine can be specifically accumulated to Lung metastases focus.Internal pharmacodynamic evaluation monitoring display, compared to free drug and commonly
Nanometer formulation, the delivery system can significantly inhibit the formation of metastatic lesion, with obvious antitumous effect.
It is intravenous injection that novel targeted medicine obtained by the present invention passs its administering mode of release system.
The advantage of the invention is that：
Obtained bionical particle nano-carrier system is different from the active targeting nanoparticle of one or more molecular modifications
Son, the easy bionical particle of neutrophil leucocyte (Neutrophil-like particles, NLP) is preventing and treating metastases side
Face has its unique advantage：
1. the Polymorphonuclear Leukocytes Membrane surface related cell adhesion molecule for activating can highly target CTC；
2. PLGA or PLA or PCL kernels Drug loading capacity are strong, and tool slow release effect；
3. nanoparticle can escape the phagocytosis of autologous mononuclear macrophage using the camouflage of Polymorphonuclear Leukocytes Membrane, be different from
Traditional is polyethyleneglycol modified, for the long circulating for realizing preparation provides new approaches；
4. biocompatibility and safe：The surface coated Polymorphonuclear Leukocytes Membranes of NLP are in the absence of inhereditary material and carefully
Born of the same parents' device etc., immunogenicity is extremely low；The Biodegradable material that kernel is approved by FDA；Therefore there is security higher and doctor
Learn conversion prospect.
Brief description of the drawings：
Fig. 1 is the particle diameter and current potential phenogram of nanoscale medicine delivery system,
A in figure, B, C are respectively the particle diameter distribution of several nanometer formulations, average grain diameter and Zeta potential.
Fig. 2 is the transmission electron microscope picture of nanoscale medicine delivery system,
A in figure, B, C are respectively exposed PLGA nanoparticles, neutrophil leucocyte membrane vesicle and coating Polymorphonuclear Leukocytes Membrane
PLGA nanoparticle transmission electron microscope enlarged drawings, D is that the PLGA nanoparticles for being coated with Polymorphonuclear Leukocytes Membrane are scattered in the aqueous solution.
Fig. 3 is the qualitative detection result of nanoparticle surface adhesion and chemotactic protein,
A is western blot determination L-selectin in figure, and several albuminoids such as CXCR4, LFA-1, β 1intergrin are in sample
Expression in product；A () neutrophil leucocyte, (b) Polymorphonuclear Leukocytes Membrane, (c) is coated with the nanoparticle of Polymorphonuclear Leukocytes Membrane, (d)
By the post-stimulatory neutrophil leucocytes of LPS, (e) by the post-stimulatory Polymorphonuclear Leukocytes Membranes of LPS, (f) by LPS it is post-stimulatory in
The property coated nanoparticle of granulocyte film；B is that dying method with coomassie brilliant blue is determined by the post-stimulatory neutrophil leucocytes of LPS, neutral
Granulocyte film, the overall protein marking on nanoparticle surface.
Fig. 4 is the semi-quantitative results of western blot determination adhesion and chemotactic molecule expression,
In figure***p<0.001, refer to be dramatically increased compared with the neutrophil leucocyte stimulated without LPS.
Fig. 5 is that the intake in 4T1 breast cancer cells of the nanoscale medicine delivery system in static and flow regime is qualitative and quantitative
As a result,
A is the NP and NLP of the same concentrations for containing Coumarin-6 (fluorescence probe) in static and flowing 4T1 mammary gland in figure
Fluorescence microscope intake figure in cancer cell, B is average single 4T1 cellular uptakes various concentrations NP and NLP under static state
Intension scanning result high, C be shearing force be 188s-1Flow regime under average single 4T1 cellular uptakes various concentrations NP
With NLP intension scanning results high；**p<0.01,*p<0.05, refer to compared with the NP under 37 DEG C of inactive states, in 4T1 cells
Intake compared to dramatically increasing；###p<0.001,#p<0.05, refer to compared with the NP under 37 DEG C of flow regimes, in 4T1 cells
Intake compared to dramatically increasing.
Fig. 6 is that intake after nanoparticle is dispersed in fluid flow blood on HUVEC cells is qualitative and quantitative result, wherein,
A is the common focused view that the HUVEC cells stimulated without TNF-α are absorbed to NP, and B is stimulated without TNF-α
The common focused view that HUVEC cells are absorbed to NLP, C is the common focused view that the HUVEC cells stimulated by TNF-α are absorbed to NP, D
The common focused view absorbed to NLP of HUVEC cells stimulated by TNF-α, E be it is various in the case of intake quantitative result.
Fig. 7 is free CFZ, the 4T1 Apoptosis situations under NP-CFZ and NLP-CFZ nanoparticle induced flow states.
Fig. 8 is the Tissue distribution on mouse lung metastasis model using near infrared imaging detection NP and NLP nanoparticles.
Fig. 9 is the drug effect of nanometer formulation and free drug in the mouse lung metastasis model of tail vein injection luc-4T1 cells
As a result,
A is to give after the same amount of luc-4T1 cells of tail vein injection nude mice free CFZ, NP-CFZ and NLP-CFZ in figure
Bioluminescence living imaging detection after nanoparticle three times (the 1st day, the 7th day, the 14th day).B is each in the 16th day acquisition nude mice
Living imaging detection is carried out after individual main organs (heart, liver, spleen, lung, kidney).
Specific embodiment
The preparation of the Polymorphonuclear Leukocytes Membrane of the activation of embodiment 1 and sign
After the chloral hydrate anesthesia of mouse 5%, heart extracting blood is in the EP pipes for adding heparin.A 50ml centrifuge tube is taken, it is small
The heart adds 15ml mouse peripheral blood neutrophil leucocyte separating liquids.The liquid level of separating liquid is added on the careful draw blood sample of suction pipe
On, 500g centrifugations 25min.Two-layer ring-type milky cellular layer is will appear from after centrifugation, in centrifuge tube, upper cell is that single core is thin
Born of the same parents' layer, lower confluent monolayer cells are neutrophil leucocyte layer.The neutrophil leucocyte layer in separating liquid is carefully drawn with suction pipe, 3 times of cells are added
The erythrocyte cracked liquid of volume, gently piping and druming is mixed, and cracks 10min, 300g centrifugation 10min, abandons red supernatant.Repeat cracking step
Suddenly once, neutrophil leucocyte is obtained final product.To adding 10ml PBS to mix cell in gained cell, 250g centrifugation 10min abandon supernatant.
Repeated washing step, obtains pure neutrophil leucocyte.In order to stimulate the expression of neutrophil leucocyte upregulating adhesion molecule, 2 μ are added
g·mL-1LPS (a kind of lipopolysaccharides) be incubated 2h；
Polymorphonuclear Leukocytes Membrane is extracted using differential centrifugation, to addition 15mlIB-1 solution in above-mentioned gained neutrophil leucocyte
It is resuspended, it is transferred in glass homogenizer, it is homogenized 100 times on ice.By in resulting solution addition 50ml centrifuge tubes, 1000g is centrifuged
5min.Draw supernatant liquor, 10,000g centrifugation 20min.Draw supernatant liquor, add 6.5ml centrifuge tubes in, 100,000g from
Heart 1h.Supernatant liquor is discarded, precipitation is resuspended with tri-distilled water.After -80 DEG C of freezings of solution, vacuum freeze-drying obtains Polymorphonuclear Leukocytes Membrane, -
20 DEG C of preservations；
Neutrophil leucocyte and cell membrane, NLP lauryl sodium sulfate SDS are dissolved, is used after PAGE gel electrophoresises
Classical dying method with coomassie brilliant blue is characterized to the integrality of memebrane protein；
Using Western blot (Western blot) to LPS stimulate before and after neutrophil leucocyte, Polymorphonuclear Leukocytes Membrane and
A few class specific adhesion albumen of NLP are characterized；
Result shows：Found with the coomassie brilliant blue staining result of cell membrane by comparing NLP, on Polymorphonuclear Leukocytes Membrane
Protein band is basically identical with the protein band of NLP, while illustrating for cell membrane to be transferred to PLGA core surfaces, memebrane protein
It is successfully moved to PLGA nanoparticles surface.The small molecular weight protein of below electrophoresis leading portion 20kDa is located in complete neutrophil leucocyte
Small-molecule substance mostly in nuclear structures, cell membrane and this kind of material almost all of NLP protein bands are removed, and integrate
The molecular weight of albumen such as plain class, selectin and chemotactic protein focus mostly in 120kDa, and 38kDa, 300kDa or so are considerable on the diagram
Observe.Proved after 2 hours LPS are incubated and stimulate by immunoblot experiment, LFA-1 (Integrins of β 2), L-selectin,
CXCR4 (chemokine receptors), the integrins of β 1 etc. are expressed in each sample and significantly improved.
The coated Carfilzomib nanoscale medicine delivery system that carries of the Polymorphonuclear Leukocytes Membrane of the activation of embodiment 2 is prepared and characterized
Precision weighs the PLGA polymeric materials of 10mg, add Carfilzomib dichloromethane solution 1mL be dissolved as it is organic
Phase, adds the sodium taurocholate aqueous phase solution of 2mL1.0%, and 240w, 50s ultrasonic emulsification pour into a certain amount of 0.5% cholic acid immediately after
Sodium solution, magnetic agitation 5min, rotary evaporation takes out most organic solvent, and low-temperature and high-speed centrifugation (14500rpm, 4 DEG C, 60min) is obtained final product
Carry NP nanoparticles (abbreviation CFZ-NP) of Carfilzomib；
Precision weighs Polymorphonuclear Leukocytes Membrane, and using mini Avanti liposomes/nanometer extruder, aperture is crossed repeatedly is
The polycarbonate membrane of 200nm makes it assemble in water phase as small lipid vesicle, is 1 with mass ratio:1 PLGA materials are made
Standby nanoparticle uniformly mixes, ultrasonic 100Hz, 60s, obtains final product the NLP nanoparticles (CFZ-NLP) for carrying Carfilzomib；
Particle diameter, polydispersity (PDI) and the zeta current potentials of nanoparticle are determined using granularity/zeta potential measurements instrument.Using
Transmission electron microscope observing its form；
Result shows：Within 150nm, the coating of Polymorphonuclear Leukocytes Membrane makes nanoparticle to particle diameter before and after nanoparticle coated film
Particle diameter increases about 20nm or so, and Zeta potential is changed into -34mV or so from original -39mV or so, with neutrophil leucocyte membrane vesicle
Bubble is consistent.Two kinds of nanoparticle sizes are homogeneous, outward appearance rounding, and the PLGA kernels of CFZ-NLP can be clearly told by contrast
The dark neutrophil leucocyte membrane lipid layer of expoeridium.
The qualitative and quantitative study that embodiment 3NLP is absorbed on the 4T1 cells of static and flow regime
Qualitative analysis
A. the incubation process under inactive state：With every hole 2 × 104The density of individual 4T1 cells is inoculated in 24 well culture plates, in
Nutrient solution is sucked after incubation 24h is adherent in incubator, NLP and NP (the nanoparticle concentration for containing 0.1% Coumarin-6 is separately added into
400μg·mL-1), 4% paraformaldehyde is fixed after being incubated 2h, Hoechst dye nucleus；
B. the incubation process under flow regime：Certain shearing force, circulation are produced using Brookfield cone and plate viscometers
Water-bath ensures 37 DEG C of constant temperature, and 4T1 cell suspensions are according to 5 × 104The density of individual/mL adds 500 μ L in sample cell, is subsequently adding
NLP and NP (the μ gmL of nanoparticle concentration 800 for containing 0.1% Coumarin-6 of 500 μ L-1), in 188s-1Shear rate under
Supernatant is abandoned into cell centrifugation after incubation 2h, adds PBS to wash and use the resuspended counting of fresh medium, according to every hole 2 × 104's
Density is inoculated into 24 orifice plates, makes cell attachment 24h；
Intake differences of the fluorescence microscopy Microscopic observation NLP and NP in above-mentioned two situations on 4T1 cells, 4% poly
Formaldehyde is fixed, Hoechst dye nucleus；
Quantitative analysis
A.4T1 cell is with every hole 5 × 103Individual cell is inoculated in 96 well culture plates, after being incubated 24h, investigates under inactive state
The influence of nanoparticle concentration, incubation time and temperature to absorbing；
1. the influence of nanoparticle concentration：Respectively with NLP and the NP (nanometer for entering to contain 0.1% Coumarin-6 of various concentrations
Grain concentration 50-800 μ gmL-1), it is incubated 2h；
2. the influence of temperature：NLP the and NP solution for containing 0.1% Coumarin-6 will be entered respectively by various concentrations (nanoparticle
Concentration 50-800 μ gmL-1) 100 μ L are added per hole, it is respectively put into 37 DEG C and 4 DEG C incubation 2h；
After above-mentioned experiment terminates, nanoparticle solution is discarded, cell is fixed with 4% paraformaldehyde, Hoechst dye cores, finally
With the full-automatic high content screening instrument quantitative scannings of KineticScan；
B. certain shearing force is produced using Brookfield cone and plate viscometers, circulator bath ensures constant temperature simulation human body blood
Stream situation, by 4T1 suspensions according to 5 × 104The density of individual/mL adds 500 μ L in sample cell, and nanoparticle is investigated under flow regime
The influence of concentration, incubation time and temperature to absorbing；
1. the influence of nanoparticle concentration：Be separately added into 500 μ L various concentrations the NLP for entering to contain 0.1% Coumarin-6 and
NP (nanoparticle concentration 50-800 μ gmL-1) in sample cell, in 188s-1Shear rate under be incubated 2h；
2. the influence of temperature：NLP the and NP solution for containing 0.1% Coumarin-6 will be entered respectively by various concentrations (nanoparticle
Concentration 50-800 μ gmL-1) 500 μ L are added in sample cell, respectively in 37 DEG C and 4 DEG C, 188s-1Shear rate under be incubated
2h；
Supernatant is abandoned in the cell centrifugation processed according to conditions above, adds PBS to wash and use the resuspended counting of fresh medium, is pressed
According to every hole 5 × 103Density be inoculated into 96 orifice plates, make to discard nutrient solution after cell attachment 24h, cell is with 4% poly first
Aldehyde is fixed, Hoechst dye cores, finally with the full-automatic high content screening instrument quantitative scannings of KineticScan；
Result shows：Under static state, the 4T1 cellular uptakes of the culture orifice plate being directly incubated carry Coumarin-6 as glimmering
Green fluorescence intensity tools of the NLP of light probe compared to NP improves, but not substantially, and under flow regime
In the intake of 4T1 cells, NLP green fluorescences are significantly stronger than NP, and quantitative result is consistent with qualitative results, illustrate 4T1 cells pair
The intake of NLP is stronger compared to NP under blood shearing force, it may be possible to because surface of cell membrane various adhesion molecules are played
Concertedness effect, more firm adhesion has been mediated, so as to strengthen its intake；4T1 cells are 37 under static and flow regime
DEG C when intake be better than 4 DEG C and illustrate that this is a kind of endocytic processes of Energy Dependence.
Intake of the nanoparticle of embodiment 4 on HUVEC cells be qualitative and quantitative result
By people HUVEC cells according to every hole 2 × 104Individual density is inoculated in bottom and is covered with 24 orifice plates of cover glass, makes
Its adherent 24 hours on the cover slip, adding the solution of TNF-α stimulated 2 hours, is taken out cover glass with tweezers, and growth has cell
One face up, be fixed on Brookfield cone and plate viscometers specimen cup center with animal fat, addition contains Coumarin-6
NLP, NP nanoparticle (800 μ gmL-1PBS solution, in 188s-1Shear rate under be incubated 2 hours, circulator bath ensures permanent
37 DEG C of temperature, afterwards puts back in orifice plate cover glass, adds PBS to wash 3 times, and 4% paraformaldehyde is fixed, Hoechst dye nucleus,
Intakes of the fluorescence microscopy Microscopic observation NLP and NP in above-mentioned two situations on the HUVEC cells before and after by TNF-α activation
Difference.Quantitative determination is that the HUVEC processed according to conditions above is quantitative with the full-automatic high content screening instrument of KineticScan
Scanning；
Qualitative and quantitative result shows：Intake by the post-stimulatory HUVEC cells of TNF-α to two kinds of nanometer formulations has
Improved, and distribution in the cell is in tufted.The intake of NLP is significantly stronger than NP, but the HUVEC cells pair without stimulation oversaturation
There was no significant difference for the intake of NLP and NP, and the former is slightly strong.Endothelial cell of this result explanation under inflammatory stimulus is received to all
The intake of metric system agent is improved, but targets inflammation part endothelial cell for being coated with the nanometer formulation of Polymorphonuclear Leukocytes Membrane
Ability it is stronger, the bionic nano grain have similar to neutrophil leucocyte biological characteristics.
Embodiment 5 is dissociated CFZ, and the 4T1 Apoptosis under NP-CFZ and NLP-CFZ nanoparticle induced flow states is quantitatively tied
Really
By 4T1 cells according to 1 × 106The density of individual/mL is added in the sample cell of Brookfield cone and plate viscometers, is added
Free CFZ, three kinds of preparations of NP-CFZ and NLP-CFZ nanoparticles (Carfilzomib concentration 400ngmL-1), in 188s-1Shearing
Supernatant is abandoned into cell centrifugation after incubation 2h under speed.Washed with the PBS of precooling 3 times and counted with PBS re-suspended cells.One
Group with reference to Annexin V-FITC cell apoptosis detection kits, takes 0.5-1 × 10 immediately5Individual cell is resuspended in 200 μ L combinations
Liquid, adds 5 μ L Annexin V-FITC and 10 μ L PI dyeing liquors, room temperature avoid light place 15min to carry out flow cytometer immediately
Detection, observation of cell apoptosis situation.Another set is with 1 × 105The density in individual/hole is inoculated in 6 orifice plates, after being incubated 24 hours,
Digested, treatment is with first group；
Result shows：Streaming quantitative determination was carried out at once afterwards in 2 hours to flowing in vertebral plate viscosimeter, it was observed that control group
2 hours vigor of cell afterwards of flowing, apoptotic cell number seldom, illustrates mechanical force to cell not damaged.Flow 2 hours afterwards
There is different degrees of late apoptic in each administration group, and early apoptosis are little.Continue low concentration administration to be incubated 24 hours, monitor
Late apoptic number rises sharply after adding free CFZ, reaches 30.2% or so, and early apoptosis are 11.9% or so.Commonly receive
Grain of rice CFZ-NP group late apoptics are only 18.9% or so, and early apoptosis substantially increase to 20.2%, but CFZ-NLP group late periods wither
Die and dramatically increased with early apoptosis, respectively 25.3% and 39.9% or so.Speculate its reason, it is likely in flow regime
The increase of the membrane-mediated nanoparticle intake of lower neutrophil leucocyte, mediated cell a large amount of apoptosis.
Tissue distribution of the embodiment 6 using near infrared imaging detection NP and NLP nanoparticles on mouse lung metastasis model.
By 2 × 106Individual luc-4T1 cells are entered in 12 BALB/c mouse bodies of 18-22g by tail vein injection, treat the 14th
It when, carry out biodiversity resources detection, choose pulmonary metastases situation and the close nude mice of degree (8) be divided into 2 groups (n=4).
Tail vein injection contains the NLP of near-infrared probe DiR respectively, and NP nanoparticles carry out near infrared imaging and biological hair on the 24th hour
Light is detected, and (targeting is observed in the detection that (heart, liver, spleen, lung, kidney) is carried out under both of which to dissect each main organs；
Result shows：Tail vein injection luc-4T1 cell Lung metastases modelings success rate more than 90%, chooses identical transfer feelings
The nude mice of condition is imaged, and eliminates the interference of other factors, such as EPR effects etc..The nude mice entirety near-infrared fluorescent of NP groups
Intensity is less than NLP groups, and from dissecting from the lung tumors image for obtaining, two kinds of imaging patterns have no too many correlation, illustrate NP
It is relatively low to metastatic tumor specificity.And NLP groups signal good relationship under two kinds of imaging patterns, almost the tumour of metastasis site is all
With stronger near-infrared fluorescent signal.Illustrate to be coated with after Polymorphonuclear Leukocytes Membrane, metastasis site targeting is greatly improved.
Pharmacodynamic results of the preparation of embodiment 7 in the mouse lung metastasis model of tail vein injection luc-4T1 cells
The healthy nude mice 12 of 20 ± 2g of body weight is taken, 3 groups, tail vein injection equal amount (2 × 10 are randomly divided into6It is individual)
Luc-4T1 cells, the free CFZ of tail vein injection equivalent medicine, NP-CFZ and NLP-CFZ nanoparticles (5mg/ after half an hour
Kg), same amount of three kinds of preparations were given respectively at the 7th day and the 14th day, injection substrate daily was started in the 8th day carries out biology
Luminous living imaging detection and record.Being obtained at the 16th day after solution takes nude mice each main organs (heart, liver, spleen, lung, kidney) is carried out
Bioluminescence living imaging is detected；
Result shows：Bioluminescence fluorescence intensity is in good positive correlation with the size of internal knurl body, is as a result found, i.e.,
Make to be administered at once after tumor cell injection enters internal 30 minutes, the transfer of free CFZ to cancer cell has no obvious action,
Most nude mice lungs have formd larger metastatic lesion within 8th day.Treated according to the scheme of 3 times of administration in two weeks, finally
Pulmonary artery progress is not controlled.And the nude mice of CFZ-NP groups occurred in that the transfer of stronger degree at the 8th day, but rear
Gross tumor volume occurs in that diminution in continuous treatment, it is likely to lack the targeting of circulating tumor cell due to early stage, causes transfer
Success, but the later stage, due to EPR effects, nanometer formulation can be accumulated in tumor locus, so as to play curative effect.CFZ-NLP groups are treated
Effect significantly, most starts to be obviously reduced to the 16th day gross tumor volume is terminated for the 8th day, compared to other two kinds of preparations, illustrates this
The coated nano-delivery system drug effect of Polymorphonuclear Leukocytes Membrane is the most obvious.
Claims (10)
1. the bionic nano particle that a kind of Polymorphonuclear Leukocytes Membrane of activation is coated, it is characterised in that with polymer poly lactic acid-hydroxyl
Acetate multipolymer PLGA, or copolymer of poly lactic acid PLA, or polycaprolactone co-polymer PCL prepare nanoparticle as kernel for material
And model drug is contained, and natural activation Polymorphonuclear Leukocytes Membrane is coated on nanoparticle surface as capsid, it is made the neutrality of activation
The biodegradable bionical particle nano-carrier system of granulocyte film cladding.
2. the bionic nano particle that the Polymorphonuclear Leukocytes Membrane of the activation as described in claim 1 is coated, it is characterised in that used
Model drug is Carfilzomib, bortezomib or taxanes based chemotherapy medicine.
3. the bionic nano particle that the Polymorphonuclear Leukocytes Membrane of the activation as described in claim 1 is coated, it is characterised in that described
Bionic nano particle is the bionical particle of nanoscale of core shell structure, by polymer poly poly lactic coglycolic acid PLGA or poly-
Lactic acid copolymer PLA, or the nano inner core that polycaprolactone co-polymer PCL is built into, and neutrophil leucocyte membrane vesicle albumen
Lipid bilayer shell is combined by hydrophobic effect and electrostatic interaction.
4. the bionic nano particle that the Polymorphonuclear Leukocytes Membrane of the activation as described in claim 1 is coated, it is characterised in that described poly-
Compound PLGA, PLA, used as kernel, its molecular weight is 10000-30000Da to the nanoparticle that PCL builds, and can stablize and contain hydrophobicity
Medicine.
5. the bionic nano particle that the Polymorphonuclear Leukocytes Membrane of the activation as described in claim 1 or 4 is coated, it is characterised in that institute
Polymer nanocomposite kernel average grain diameter is stated for 80-100nm, ultimate size is 100-120nm after coating Polymorphonuclear Leukocytes Membrane.
6. the bionic nano particle that the Polymorphonuclear Leukocytes Membrane of the activation as described in claim 1 is coated, it is characterised in that in described
The vesica of property granulocyte film, by from the same isolated neutrophil leucocyte of species animal peripheral blood, carried out it is external evoked from
And the expression of related cell adhesion molecule is raised, extracted by differential centrifugation and obtained.
7. the bionic nano particle that the Polymorphonuclear Leukocytes Membrane of the activation as described in claim 6 is coated, it is characterised in that described to swash
In Polymorphonuclear Leukocytes Membrane surface related cell adhesion molecule living, Polymorphonuclear Leukocytes Membrane rise β 2 Integrins of LPS lipopolysaccharides activation,
The expression of L-selectin, chemokine receptors and the integrin memebrane proteins of β 1.
8. the bionic nano particle that the Polymorphonuclear Leukocytes Membrane of the activation as described in claim 6 is coated, it is characterised in that described
Neutrophil leucocyte membrane vesicle average grain diameter is 200nm.
9. the bionic nano particle of the Polymorphonuclear Leukocytes Membrane cladding that must be activated described in claim 1 is swollen for preparing targeting circulation
Purposes in oncocyte and early stage micrometastatic disease preparation.
10. the purposes as described in claim 9, it is characterised in that it is early wherein to use Human umbilical vein endothelial cells HUVEC cells
Phase micrometastatic disease endothelial cell model, is produced using the vertebral plate viscosimeter of improvement to breast cancer lung high-transfer cell 4T1 cells
Certain shearing force is circulating tumor cell model.
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