AFP antibody modification PLGA loads DCN nanoparticle anti-tumor drugs targeting
Technical field
The invention belongs to field of biomedicine technology, is related to a kind of anti-tumor drugs targeting, is directed to more particularly to one kind
The target therapeutic agent of primary hepatoma.
Background technology
Primary hepatoma (hepatocellular carcinoma, HCC) is most common and death rate highest is disliked
One of property tumour.China's HCC incidences of disease occupy the world first, and still increase year by year, be the weight of the serious threat health of our people
Big disease.HCC seriously restricts its curative effect because of the features such as grade of malignancy is high, transfer is early, high recurrence rate, plus traditional therapy pair
The very big negative effect that body's immunity is brought, accelerate tumour progression, aggravation, poor prognosis.Pay close attention to simultaneously Efforts To Develop HCC
Therapy study, the particularly significant and real society of tool outstanding to China and medical significance.With molecular biology and it is immunologic not
Disconnected development, HCC generations, development mechanism understanding are deepened continuously, HCC gene target treatment has turned into clinical tumor research
Focus.
Traditional gene vector system is divided into viral vector and the class of non-virus carrier two, and existing viral vector has immunogene
The defects of property, oncogenicity;Then there is the problem of low transmission efficiency for non-virus carrier.Meanwhile the mode of these genes transfection is all
Lack it is cell targeted, and carrier carry target gene by transfection act on tumour cell after, often in the short time
Interior to be destroyed by intracellular enzymic digestion, action time is short, and bioavilability is low.These problems result in traditional gene therapy effect
It is bad, have become the bottleneck that repressor gene treatment further develops.
Nano-gene carrier is a kind of new gene vector system, and it is using nano particle as carrier, by therapeutic gene bag
It is rolled in nano particle or adsorbs on its surface, nano particle is entered cell by endocytosis and discharge treatment base
Cause, so as to play gene therapy efficiency.Polylactic acid-polyglycolic acid (PLGA) nano particle is that one kind has good biocompatibility
And the biomaterial of degradability, it has been approved by the FDA in the United States and has been used in human body.Also there is PLGA nano particles nucleotides protection to make
With by modifying the pendant carboxylic group of PLGA ends, the part or antibody of target cell being coupled, realize gene therapy
Active targeting.
Decorin (decorin, DCN) is a pleiotropism of proteoglycans (proteoglycan, PG) family
Molecule, cell propagation is adjusted by core protein cell cycle regulation and combination cell factor etc., differentiation is formed with matrix.In recent years
Great mass of data shows:1) DCN is a kind of effective growth of tumour cell and migration inhibitor, can suppress a variety of different tissues
The growth of tumour cell in source, and induce its apoptosis;2) find that DCN is Met (HGFR) antagonism part, is being prevented again recently
Played a role in tumor invasion, infiltration and transfer;3) DCN can also the reversing tumor cell resistance to the action of a drug.
DCN fully shows that it is controlled in tumour to the complicated binding ability of a variety of target spots and noticeable antitumor action
Medical value in treatment.The DCN gene therapy studies have shown thats DCN that foreign scholar is carried out using adenovirus vector has local and remote
Journey specificity antineoplastic acts on.There are indivedual reports, DCN contents are reduced can be as the index of prediction poor prognosis.Before this seminar
Phase research data shows no matter DCN is over-expressed in vivo, or is used as recombinant protein or gene transfecting cell, can suppress more
Growth of tumour cell, the inducing apoptosis of tumour cell of kind different tissue sources, and DCN can the reversing tumor cell resistance to the action of a drug.
The content of the invention
The purpose of the present invention is the problem of presence for traditional genophore, there is provided a kind of AFP antibody based on new support
Modify PLGA loads DCN nanoparticle anti-tumor drugs targeting.
AFP antibody modifications PLGA loads DCN of the present invention nanoparticle anti-tumor drugs targeting is based on DCN and suppresses swollen
The multiple antitumor actions such as tumor cell proliferation, transfer, Tumor Angiongesis and the reversing tumor cell resistance to the action of a drug, matter is recombinated with DCN
Grain is used as core medicine, selects high degree of specificity hepatoma targeting character AFP monoclonal antibody molecules, uses and given birth to biodegradable polymer
Drug-carrying nanometer particle PLA/glycolic compound (PLGA) load DCN antioncogenes plasmid of thing material preparation is simultaneously coupled liver cancer
Targeting AFP monoclonal antibody molecules, build the therapeutic nano-particles of AFP mAb-PLGA-rhDCN.
The preparation method of AFP antibody modifications PLGA loads DCN of the present invention nanoparticle anti-tumor drugs targeting be by
PLGA, which is scattered in dichloromethane solution, is used as oil phase, and pIRES2-EGFP-DCN plasmids, which are scattered in the 1wt% PVA aqueous solution, to be made
For aqueous phase, colostrum is made by the oil phase is mixed with water;The colostrum is instilled to stir in the 0.3wt% PVA aqueous solution and formed
Emulsion;The dichloromethane in emulsion is removed, nano-particle is collected by centrifugation, is suspended in pH7.4 PBS and nanoparticle is made
Dispersion;Into nanoparticle dispersion add 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC)/
N-hydroxysuccinimide (NHS), the nanoparticle activated is reacted, add AFP monoclonal antibodies, reaction obtains AFP mAb-PLGA-
The therapeutic nano-particles of rhDCN.
Wherein, 1- (3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and n-hydroxysuccinimide of addition
Mol ratio be 2: 1.
Further, in the above-mentioned preparation method of the present invention, the activation is that reaction 40min is stirred at room temperature;It is described anti-
Body coupling reaction time 6h.
The anti-tumor drugs targeting of the present invention utilizes the spy that secretion of hepatoma AFP and antibody are combined with antigentic specificity
Property so that AFP mAb-PLGA-rhDCN nano-particles can actively combine liver cancer cells and enter into the cell, slowly discharge DCN
Plasmid and the corresponding product for expressing DCN gene codes, play its GVT, realize special targets of the DCN to liver cancer cells
Inhibitory action.The present invention anti-tumor drugs targeting avoid be transferred to using transfection methods such as liposomes cell gene it is rapid
The problem of degraded, the functioning efficiency of rotaring redyeing gene is improved, while realize long-acting inhibitory action of the target gene to target cell.
Anti-tumor drugs targeting prepared by the present invention can be used for the targeted therapy of primary hepatoma, have great city
Field potentiality.
Brief description of the drawings
Fig. 1 is the mode of appearance figure of AFP mAb-PLGA-rhDCN nanoparticles prepared by the present invention.
Fig. 2 is the grain size distribution of AFP mAb-PLGA-rhDCN nanoparticles prepared by the present invention.
Fig. 3 is the Zeta potential figure of AFP mAb-PLGA-rhDCN nanoparticles prepared by the present invention.
Fig. 4 is the In-vitro release curves figure of AFP mAb-PLGA-rhDCN nanoparticles prepared by the present invention.
Fig. 5 is the situation that AFP mAb-PLGA-rhDCN nanoparticles prepared by the present invention enter HepG2 cells.
Fig. 6 is proliferation inhibition rate of the various concentrations AFP mAb-DCN-PLGA nanoparticles to HepG2 cells.
Fig. 7 is influence of the various concentrations AFP mAb-DCN-PLGA nanoparticles to HepG2 cell transfer abilities.
Fig. 8 is influence of the various concentrations AFP mAb-DCN-PLGA nanoparticles to HepG2 cell invasion abilities.
Embodiment
Embodiment 1
Take 20mg PLGA to add in 1ml dichloromethane, polymer is uniformly dispersed as oil phase (O);Take dissolved with 500 μ g
The 1wt% PVA aqueous solution 2ml of pIRES2-EGFP-DCN plasmids are as aqueous phase (W1).Oil phase (O) is mixed with aqueous phase (W1), ice
Water bath sonicator 5min (ultrasonic power 300W, super 1s stop 2s), obtains colostrum (W1/O).Then obtained colostrum is instilled into 10ml
In the 0.3wt%PVA aqueous solution (W2), magnetic agitation 5min forms emulsion (W1/O/W2).Obtained emulsion is rotated and removes dichloro
Methane, centrifugation obtain PLGA-rhDCN nanoparticles.
Obtained PLGA-rhDCN nanoparticles are suspended in 2mL PBSs dispersion is made, add 200 μ g
EDC and NHS mixture (EDC and NHS mol ratio are 20: 10), is stirred at room temperature reaction 40min, 10000r/min centrifugation
5min, the PLGA-rhDCN nanoparticles precipitation activated.Precipitation is collected, is washed twice with 2ml PBSs (pH 7.4),
Again it is scattered in PBS, adds 200 μ g AFP monoclonal antibodies, stirring reaction 6h, PBS is centrifuged repeatedly, washed, and obtain AFP
MAb-DCN-PLGA nanoparticles, freeze and preserve.
Embodiment 2
The μ g of nanoparticle 10 prepared by Example 1, which are scattered in 1ml PBSs, is made suspension, drips and is supported in plating carbon
On film (Electronic Speculum copper mesh), dry.The nanometer appearance feature shown in Fig. 1 is observed under a scanning electron microscope.In figure A for ×
4000 times, B is × 5000 times.Using the particle size distribution range and Zeta potential of laser particle analyzer measure nanoparticle, as a result such as Fig. 2
And Fig. 3, the particle size range of nanoparticle are mainly distributed between 100~300nm, Zeta potential is between -20~-13mV.
From Fig. 1, Fig. 2 and Fig. 3, prepared nanoparticle particle size belongs to the ingestible optimum range of cell.If
The nanoparticle particle diameter of preparation is excessive, particle can be caused to be difficult to enter cell, particle diameter is too small, then may destroy package-contained gene
Biological structure, while decline the validity of coupled antibody, lose the effect of its targeting anti-tumor.Nanoparticle prepared by the present invention exists
Ensure the target gene plasmid of effectively parcel institute load, on the premise of ensureing nanoparticle structure functional completeness, do as far as possible
To the particle diameter of nanoparticle is minimized, it is easy to cellular uptake.
Embodiment 3
The μ g of nanoparticle 20 prepared by Example 1, are resuspended in the centrifuge tube equipped with 3ml PBSs and seal, will
Centrifuge tube is placed in 37 DEG C of constant temperature oscillators, 100r/min centrifugations, respectively at 1h, 12h, 24h, 36h, 48h, and 72h and hereafter every
Centrifuged every 24h, take 2ml supernatants, detect the plasmid amount of release, while supplement the fresh PBSs of 2ml, continue constant temperature oscillation, directly
To 240h time point.Preparation is calculated, draws curve such as Fig. 4.
The accumulative release rate average value that first 24h is obtained through measurement is 35.01%, and slowly release, is released in 240h afterwards
The rate of putting reaches 79.3%.
In Fig. 4 results, unstable combination is formed because part load gene plasmid is adsorbed in nano material, so as to cause
There is release rate in the 24h of beginning and increase phenomenon suddenly.This phenomenon of burst release can be such that medicine is quickly formed in tumor tissues
One higher treatment concentration, its antitumor action of fast onset.Gradual degraded with PLGA in the cell afterwards, persistently delays
The gene of slow steady release package-contained, realizes long-term inhibitory action of the DCN to HepG2 cells, and avoid and utilize lipid
The transfection methods such as body enter the rapid of the gene of cell and are degraded, and improve the functioning efficiency of gene.
Embodiment 4
AFP monoclonal antibodies are replaced with IgG antibody, other are identical with the step of embodiment 1, and IgG-DCN-PLGA is prepared
Nanoparticle, as AFP mAb-DCN-PLGA nanoparticle check experiment materials.
Embodiment 5
Fluoresceincarboxylic acid (FAM) mark is first carried out to antibody respectively, the PLGA-rhDCN nanoparticles PLGA to carrying DCN plasmids
Nano-particle carries out rhodamine (Rhodamine) fluorescence labeling, and nucleus is marked with Hoechst33342.According still further to embodiment 1
With 4 steps, the nanoparticle of double fluorescence labeling is prepared respectively.
HepG2 cells in exponential phase are inoculated in six orifice plates, after cultivating 12h, take 10 μ g fluorescence labelings
AFP mAb-DCN-PLGA nanoparticles are incubated as experimental group jointly with cell, the IgG-DCN-PLGA nanometers of 10 μ g fluorescence labelings
Grain is incubated as negative control group, respectively in 4h, 12h, under laser confocal microscope × 630 times jointly with cell
(488nm excites FAM, 543nm to excite rhodamine, and 350nm excites Hoechst33342) observes the common location situation of two kinds of fluorescence.
As a result as shown in figure 5, wherein blue-fluorescence mark nucleus, green and red fluorescence are double to nano-particle progress glimmering
Signal, yellow fluorescence are green rear color overlapping with red fluorescence.There are a large amount of fluorescence to assemble around experimental group (a) nucleus,
Illustrate that AFP mAb-PLGA-rhDCN nanoparticles have biological targeting effect, HepG2 cells can actively absorb receiving for AFP monoclonal antibodies modification
The grain of rice, and action time is longer, intake is more;And there was only a small amount of fluorescence aggregation around negative control group (b) nucleus, explanation
IgG without targeting can not mediate nanoparticle largely to enter cell.
Embodiment 6
Inhibitory action of the AFP mAb-PLGA-rhDCN nanoparticles to HepG2 cells is evaluated by MTT experiment.Take the logarithm the phase
HepG2 cells be inoculated in 96 orifice plates, 180 μ L DMEM nutrient solution culture 12h, it is respectively 5 μ g/mL, 10 μ to take 20 μ L final concentrations
G/mL, 15 μ g/mL AFP mAb-PLGA-rhDCN nanoparticles are added in culture cell respectively as basic, normal, high dosage experiments
Group, the IgG-DCN-PLGA nanoparticles for taking 20 μ L to dilute add in culture cell and are used as negative control group, to be added without nanometer
The culture cell of grain after being incubated 24h, 48h, 72h, discards nutrient solution, added fresh without serum as blank control group
The μ L of DMEM nutrient solutions 100 and 20 μ L MTT solution, nutrient solution is discarded after being incubated 4h, add 150 μ L DMSO per hole, use ELIASA
Each hole OD values are determined under 490nm, every group sets three multiple holes.The relative inhibition of cell is calculated as follows nanoparticle:(blank
Hole OD values-experimental port OD values)/blank well OD value × 100%.
Experimental result is as shown in fig. 6, compared with IgG non-specific antibody groups, the AFP mAb-PLGA-rhDCN of experimental group
Nanoparticle substantially increases to the proliferation inhibition rate of HepG2 cells, and strengthens with the rise of activity, with prolonging for action time
Grow and strengthen.
Embodiment 6
Shadow using scratch damage measuring AFP mAb-PLGA-rhDCN nanoparticles to HepG2 cell transfer abilities
Ring.The cell of exponential phase is taken, is blown and beaten, single cell suspension is formed, by every hole 1 × 105Individual cell is inoculated in 6 orifice plates.It is secondary
After day cell attachment, using sterile pipette tips cut, relevant nanometer grain processing cell is absorbed by the experiment packet of embodiment 5.It is right after 24h
Cut position is taken pictures, as shown in fig. 7, carrying out graphical analysis.
Fig. 7 scratch experiment results show that experimental group cell acts on through various concentrations AFP mAb-PLGA-rhDCN nanoparticles
Scratch width value afterwards prompts migration energy of the AFP mAb-PLGA-rhDCN nanoparticles to HepG2 cells apparently higher than control group
Power has inhibitory action, and certain dose-dependant trend is presented.
Embodiment 7
AFP mAb-PLGA-rhDCN nanoparticles are determined to HepG2 cell invasions using Transwell cells Matrigel
The influence of ability.After the cell grown in logarithmic phase is digested with pancreatin, nutrient solution suspends, by every hole 1 × 105Individual cell connects
Kind, according to the experiment packet of embodiment 5, after carrying out nanoparticle stimulation 24h, carries out following operation in 6 hole Transwell plates:
Cell is washed twice with PBS, and 4% paraformaldehyde fixes 20min;PBS is washed three times, each 5min;0.1%Trition X-100 permeabilizations
Cell 15min;PBS is washed three times, each 5min;Haematoxylin dyeing 20min, washes 10min, and cell is penetrated into film after basket is returned takes
Under, it is put on slide, microscopy after mounting.It is random to take 3 high power field of view, cell Xia Shi faces cell number is counted, is as penetrated
Matrigel cell number, experimental result are as shown in Figure 8.
Fig. 8 results show that experimental group penetrates theca cell number and downward trend is presented, say compared with untreated fish group and blank group
Bright AFP mAb-PLGA-rhDCN nanoparticles have inhibitory action to the invasive ability of HepG2 cells.