CN104492396B

CN104492396B - Preparation method of target liposomes based on protein molecular trace and products thereof

Info

Publication number: CN104492396B
Application number: CN201410755690.1A
Authority: CN
Inventors: 李翀; 龙莹莹; 张焱; 陈章宝
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2014-12-10
Filing date: 2014-12-10
Publication date: 2016-08-17
Anticipated expiration: 2034-12-10
Also published as: CN104492396A

Abstract

The invention discloses preparation method of target liposomes based on protein molecular trace and products thereof, concrete grammar is acrylamides and N N methylene-bisacrylamide to be dissolved in PBS solution or water, ultrasonic, obtains mixed liquor A；Olefinic recycle key modifies silicon dioxide liposome, is then scattered in by protein molecular in the silicon dioxide liposome solutions that ethylene linkage is modified and stirs, and vacuum outgas must mix liquid B；Mixed solution A being added dropwise in mixed solution B under nitrogen protection, nitrogen blowing, stir prepolymerization, then under nitrogen protection, dropping Ammonium persulfate. and tetramethylethylenediamine cause polymerization, continue to stir, dialysis, must be based on the target liposomes of protein molecular trace；The target liposomes based on protein molecular trace prepared, as core, acrylamides and N, N ' methylene-bisacrylamide can be used for mediating active targeting pass medicine as shell, the target protein of energy initiative recognition lesions position by silicon dioxide modified liposome.

Description

Preparation method of target liposomes based on protein molecular trace and products thereof

Technical field

The invention belongs to chemical field, be specifically related to the preparation method of target liposomes based on protein molecular trace, further relate to by The product that the method prepares.

Background technology

Molecular imprinting (molecular imprinting technique, MIT), also known as molecular imprinting, molecular brand skill Art or molecular templating techniques, be a kind of emerging molecular recognition technology, its objective is that preparation has specific selectivity to template molecule The polymer of identification ability, i.e. molecularly imprinted polymer (MIPs).Molecular imprinting is developed so far, and prints compared to little molecule Reaching its maturity of mark technology, protein and other engram technology also relatively lags behind.Molecular engram material and template protein In conjunction with being similar to the specific binding of " antigen-antibody ", imprinted material is at high temperature, high pressure, extreme pH condition and organic reagent In can keep stable, it is simple to store, thus research and development can in physiological conditions identification of protein thus simulate natural process point Sub-trace " man-made antibody " has the biggest potential using value.

Compared with traditional blotting techniques, surface molecule print is in material surface because of the recognition site preparing resulting polymers, can have Effect is improved the template molecule that traditional method causes and is excessively embedded, is difficult to the problems such as removing, and this has for macromolecule blots such as protein Significance.For making gained imprinted polymer particle, homogenization (microsphere, nanoparticle etc.), researchers often use core -shell structure material realizes surface imprinted target, it may be assumed that with microsphere well prepared in advance or nanoparticle as support matrix (" core "), It is " shell " that the imprinted polymer formed on it covers surface.The trace strategy of this nucleocapsid structure function needed for usual trace The material that a class is new, i.e. support matrix material is introduced again outside monomer.Earth silicon material has surface and is prone to modify, stablize Property strong, chemical/biological inertia, particle diameter and the feature such as dispersibility is controlled and its be applied to biomedicine field already, therefore, two Silicon oxide is the support matrix material that a class is ideal, relies on silicon dioxide granule to carry out surface imprinted being expected to and realizes template egg White effectively identifies and has preferable potential using value.

Mediate by modifying the part for specified disease tissue target molecule (mostly being protein) on pharmaceutical carrier surface and improve Drug-loading system is to realize active targeting to pass the important channel of medicine to the identification of target area.Due to biological species molecule (as polypeptide, antibody, Aptamer etc.) often face the problem of internal stability, and surface molecule print has " artificial antibody "/" artificial ligand " Potentiality and imprinted sites stability strong, therefore, carry out for disease relevant target protein and surface imprinted likely pass for active targeting Medicine provides new means.

Summary of the invention

In view of this, an object of the present invention is to provide the preparation method of target liposomes based on protein molecular trace, should Preparation method is simple, quick, low cost；The two of the purpose of the present invention be provide by said method prepare based on protein molecular The target liposomes of trace.

For achieving the above object, the present invention provides following technical scheme:

The preparation method of target liposomes based on protein molecular trace, comprises the steps: to take acrylamides and N-N Methylene-bisacrylamide be in mass ratio 1～5:1 be dissolved to pH6.8～7.4, concentration be 0.02～0.1M PBS solution or water in, Ultrasonic, obtain mixed liquor A；Olefinic recycle key modifies silicon dioxide liposome, and then protein molecular is scattered in the dioxy that ethylene linkage is modified Stirring in SiClx liposome solutions, vacuum outgas obtains mixed liquid B；It is 1:1～1:10 by volume by mixed liquor A under nitrogen protection Being added dropwise in mixed liquid B, nitrogen blowing, stir prepolymerization, then the lower dropping Ammonium persulfate. of nitrogen protection and tetramethylethylenediamine draw Send out polymerization, continue stirring, dialyse, must be based on the target liposomes of protein molecular trace.

In the present invention, acrylamides is preferably NIPA or acrylamide, acrylamides and N-N methylene-bisacrylamide mass ratio is preferably 2:1；PBS solution preferably pH 7.4, concentration are that the PBS of 0.02M is molten Liquid.

Preferably, the preparation method of described silicon dioxide liposome is: first use thin film dispersion ultrasonic method to prepare liposome, then Add through the scattered tetraethyl orthosilicate of ethanol solution in liposome, under stirring, regulate pH to 8～10.4 with sodium hydroxide solution, It is stirred overnight, dialysis, obtains silicon dioxide liposome.

It is furthermore preferred that the addition of described tetraethyl orthosilicate presses phospholipid: tetraethyl orthosilicate: H₂The mol ratio of O is 1:8:344.

It is furthermore preferred that the volume fraction of described ethanol solution is 5%～25%.

Preferably, phospholipid and cholesterol prepare the method for liposome to be dissolved in volume ratio by described thin film dispersion ultrasonic method is 1:1 Chloroform is with the mixed solution of methanol, and decompression rotary evaporation removes organic solvent, forms transparent lipid membrane, adds water hydratable, super Sound, obtains liposome.

It is furthermore preferred that the mass ratio of described phospholipid and cholesterol is 7:3, the liposome formed under this ratio is more conducive to follow-up modification.

In the present invention, phospholipid is preferably soybean lecithin or Ovum Gallus domesticus Flavus lecithin.

Preferably, described ultrasonic condition is under the conditions of 800W ultrasonic for 50 times, each ultrasonic 10s, continues after the 10s of interval Ultrasonic.

Preferably, the condition of described aquation is aquation 1 hour under the conditions of 37 DEG C.

It is furthermore preferred that described protein molecular is staphylococcus aureus protein A or matrix metalloprotease MMPs.

2, the target liposomes based on protein molecular trace prepared by described preparation method.

The beneficial effects of the present invention is: the invention discloses the preparation method of target liposomes based on protein molecular trace, system Preparation Method is simple, low cost, and fast, the protein molecular imprinted polymer prepared, using liposome as central support, utilizes fat The original medicine carrying of plastid with pass medicine advantage, and particle diameter is easy to control；Owing to having modified layer of silicon dioxide thin film in the periphery of liposome, The stability of medicine can be improved, reduce the impact of the unfavorable factors such as acidity, temperature and enzyme and there is medicament slow release ability, in addition On silicon materials surface by introducing ethylene linkage, albumen and monomer material crosslinking copolymerization can be made in shell surface, make the monomer material amount of input Low, moreover it is possible to the gelation that after avoiding polymerization, dispersion easily occurs, and make non-aggregated trace granule more easily collecting, with the most real Existing surface imprinted, the liposome after trace is used for medicine carrying, it is possible to increase the targeting of liposome.

Accompanying drawing explanation

In order to make the purpose of the present invention, technical scheme and beneficial effect clearer, the present invention provides drawings described below:

Fig. 1 is the silicon dioxide liposome after the silicon dioxide modified liposome (S-LIP) of infrared spectrum measurement, ethylene linkage are modified (MS-LIP) and staphylococcus aureus trace target liposomes (SPA-MIPs) architectural feature peak (a is SPA-MIPs, B be MS-LIP, c be S-LIP).

Fig. 2 is the target liposomes (SPA-MIPs) of staphylococcus aureus trace and non-imprinted polymer (NIPs) is dynamically inhaled Attached experimental result picture.

Fig. 3 is target liposomes (SPA-MIPs) and the suction of non-imprinted polymer (NIPs) isothermal of staphylococcus aureus trace Attached experimental result picture.

Fig. 4 is the non-specific adsorption experimental result picture of the target liposomes (SPA-MIPs) of staphylococcus aureus trace.

Fig. 5 is that staphylococcus aureus carries the SPA imprinted polymer (FAM-SPA-MIPs) of 6-Aminofluorescein to bag, bag carries The non-imprinted polymer of 6-Aminofluorescein (FAM-NIPs) and the picked-up experimental result picture of 6-Aminofluorescein (6-FAM).

Fig. 6 is that staphylococcus aureus carries the SPA imprinted polymer (FAM-SPA-MIPs) of 6-Aminofluorescein to bag, bag carries Staphylococcus aureus is pressed down by the non-imprinted polymer of 6-Aminofluorescein (FAM-NIPs) and 6-Aminofluorescein (6-FAM) Bacterium experimental result picture.

Fig. 7 is target liposomes (MMP-2-MIPs) and the non-imprinted polymer (NIPs) of matrix metalloproteinase trace Dynamic adsorption experimental result picture.

Fig. 8 is target liposomes (MMP-2-MIPs) and the non-imprinted polymer (NIPs) of matrix metalloproteinase trace Adsorption isotherm experiment result figure.

Fig. 9 is target liposomes (MMP-2-MIPs) and the non-imprinted polymer (NIPs) of matrix metalloproteinase trace Specific adsorption experimental result picture.

Figure 10 is that U937 cell carries the matrix metalloproteinase MMP-2 imprinted polymer of amycin to bag (DOX-MMP-2-MIPs) the picked-up experimental fluorescence microscope of the non-imprinted polymer (DOX-NIPs) of amycin is carried with bag Result figure.

Detailed description of the invention

Below in conjunction with accompanying drawing, the preferred embodiment of the present invention is described in detail.The reality of unreceipted actual conditions in embodiment Proved recipe method, generally according to normal condition or according to the condition proposed by manufacturer.

Embodiment 1, the preparation of target liposomes of staphylococcus aureus protein A (SPA) trace and evaluation thereof

The preparation method of the target liposomes of SPA trace, comprises the steps:

(1) thin film dispersion ultrasonic method is used to prepare liposome, method particularly includes: weigh soybean lecithin 10.5mg, cholesterol 4.5mg, dissolves with methanol mixed solution 1mL with the chloroform that volume ratio is 1:1, and then under the conditions of 37 DEG C, decompression rotates steaming Send out and remove organic solvent, form one layer of uniform transparent lipid membrane, add pure water 1mL in 37 DEG C of aquations 1h, utilize ultrasonic carefully Born of the same parents' crusher ultrasonic 50 times at 800W, is spaced 10s and continues ultrasonic, obtain liposome (LIP) after each ultrasonic 10s；

(2) use sol method to prepare surface and modify the liposome of silicon dioxide, method particularly includes: by phospholipid: tetraethyl orthosilicate (TEOS): H₂O mol ratio be 1:8:344 (mol/mol) be slowly added in liposome through volume fraction be 10% ethanol Scattered TEOS, the most under agitation by the sodium hydroxide solution regulation pH to 8～10.4 of 0.1mol/L, room temperature (18-25 DEG C) is stirred overnight, the silica precursor not deposited in removing system of being dialysed by mixture, obtains surface and modifies dioxy The liposome of SiClx, named silicon dioxide liposome (S-LIP)；

(3) ethylene linkage modifies silicon dioxide liposome, method particularly includes: by 3-sulfydryl-1-propane sulfonic acid sodium (MPS) of 50 μ L It is slowly dropped in the ethanol of 250 μ L after disperseing half an hour, then takes 10 μ L dispersant liquid drops and be added to the lipid of 2ml silicon dioxide In body, room temperature (18-25 DEG C) is stirred overnight, and obtains the silicon dioxide liposome (MS-LIP) that ethylene linkage is modified；

(4) preparation of the target liposomes of SPA trace, method particularly includes: take acrylamide (AAM) 4mg and N-N sub- Bisacrylamide (MAA) 2mg be dissolved into 20ml pH be 7.4, concentration be 0.02M PBS in, ultrasonic dissolution, Obtain mixed solution A, then 2mg SPA be dispersed in the silicon dioxide liposome solutions that 1mL ethylene linkage is modified and be positioned over room temperature Stirring 0.5h, then mixture is carried out vacuum outgas 10min, obtain mixed solution B, take 200 μ L mixing under nitrogen protection molten Liquid A is added dropwise in mixed solution B, and nitrogen blowing 10min, and the lower prepolymerization 1h of room temperature (18-25 DEG C) stirring, nitrogen is protected Lower dropping 7 μ L Ammonium persulfate. (APS, 10%, W/W) and 4.5 μ L tetramethylethylenediamine (TEMED, 5%, W/V) In mixture, cause polymerization, be positioned over room temperature (18-25 DEG C) stirring 24h the most again, dialyse one week with pure water, remove template Molecule and unreacted reagent, lyophilizing to constant weight i.e. S. aureus L-forms surface protein SPA imprinted polymer (referred to as SPA-MIPs).

In the present embodiment, acrylamide can directly replace with NIPA, and NIPA and N-N The mass ratio of methylene-bisacrylamide is 1～5:1, and makes NIPA and N-N di-2-ethylhexylphosphine oxide in mixed solution A The total concentration of acrylamide is 1～5mg/ml；During dispersion TEOS, the volume fraction of ethanol is 5%～25%；PBS solution is PH6.8～7.4, concentration are all to realize goal of the invention in the range of 0.02～0.1M.

The synthesis of non-imprinted polymer (NIPs) is in addition to being added without template protein molecule, and other preparation methoies are ibid.

Targeting preparation constructed by the present invention is carried out preliminary characterization: weigh S-LIP and SPA-MIPs prepared at 25 DEG C Under, use Malvern laser particle analyzer to measure its size and distribution situation, and be repeated three times.Result shows, S-LIP Mean diameter be 147.9 ± 40.5nm, PI value be 0.214 ± 0.015, its dispersibility is relatively good, through trace be polymerized after SPA-MIPs Mean diameter be 200.5 ± 60.5nm, PI value be 0.22 ± 0.018, show that SPA-MIPs preparation particle diameter is less, be evenly distributed. It addition, use infrared spectrum measurement S-LIP, MS-LIP, SPA-MIPs architectural feature peak, particularly as follows: first by S-LIP, MS-LIP, SPA-MIPs freeze-dried process respectively, then presses with S-LIP, MS-LIP, SPA-MIPs respectively by KBr Mass ratio is that 100:1 is placed in mix homogeneously in agate mortar, finally rolls, and uses Fourier infrared spectrograph to survey after tabletting Fixed, measurement result is as shown in Figure 1.Result shows, S-LIP (c in Fig. 1) is at 1087cm^-1Place occurs in that Si-O-Si key Characteristic absorption peak；MS-LIP (b in Fig. 1) is at 1712cm^-1Place occurs in that it is Si-C=C characteristic absorption peak, shows dioxy The surface of SiClx has successfully been grafted ethylene linkage.SPA-MIPs (a in Fig. 1) through trace be polymerized after, 1712cm^-1The absworption peak at place Disappear, show that trace type liposome SPA-MIPs is successfully prepared.

The adsorption experiment of the target liposomes of SPA trace: for investigating the SPA-MIPs the prepared specific adsorption to SPA Ability, uses following methods to verify it.

Dynamic adsorption is tested: first SPA is carried out FITC fluorescent labeling, specifically takes 4mg SPA and be dissolved in 0.2M Na₂PO₄Molten In liquid, separately take 0.16mg FITC and be dissolved in 0.2M Na₂PO₄In solution, the mol ratio making FITC Yu SPA is 1.2:1, magnetic Under power stirring, the solution containing SPA is added dropwise in the solution containing FITC, lucifuge reaction 1.5h；After reaction terminates, at 8000r/min Under the conditions of centrifugal 10min to remove precipitation SPA, take supernatant Sephadex G-50 gel column and carry out isolated and purified, lyophilizing, Obtain FITC-SPA.Then by 5mg SPA-MIPs and NIPs respectively with the 0.2mg/ml FITC-SPA solution of 5ml in 37 DEG C Oscillation incubation, takes out 1mL respectively at 0.25h, 0.5h, 1h, 2h and 4h, is centrifuged 10min with 4500r/min, in mensuration The fluorescence intensity of clear liquid, result is as shown in Figure 2.Result shows, SPA-MIPs and NIPs to the absorption of FITC-SPA along with The increase of time and increase, reach dynamic equilibrium at 2h.Further, during dynamic adsorption balance, SPA-MIPs is to FITC-SPA About adsorbance is NIPs 5 times, illustrate that albumen is had well by the molecular engram target polymer carrier that the present invention builds Weight absorbability.

Adsorption isotherm experiment: accurately weigh 1mg SPA-MIPs and NIPs and be respectively placed in centrifuge tube, respectively with 1ml's The FITC-SPA solution of 0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml vibrates under the conditions of 37 DEG C Hatching 2h, be centrifuged 10min with 4500r/min after hatching, Aspirate supernatant measures fluorescence intensity, and result is as shown in Figure 3.Knot Fruit shows, the ratio of adsorption of template molecule is directly proportional by polymer to FITC-SPA concentration, when the concentration of FITC-SPA reaches During 0.4mg/ml, ratio of adsorption reaches balance；Compared with NIPs, SPA-MIPs has higher adsorption capacity to FITC-SPA With preferable selectivity.

Non-specific adsorption test: choose matrix metalloproteinase MMP-2, lysozyme (LYZ), bovine serum albumin (BSA), Streptavidin (SA) for competition albumen, carry out FITC fluorescent labeling according to the method described above, respectively FITC-MMP-2, FITC-LYZ, FITC-BSA, FITC-SA, then weigh 1mg SPA-MIPs and NIPs and be respectively placed in centrifuge tube, add Enter 1ml 0.2mg/ml FITC-MMP-2, FITC-LYZ, FITC-BSA, FITC-SA and FITC-SPA solution in After 37 DEG C are placed on agitator vibration absorption 2h, measuring the fluorescence intensity in supernatant respectively, result is as shown in Figure 4.Result shows Showing, SPA-MIPs only has higher absorption property to SPA, and relatively low to the absorption property of other four kinds competition albumen, Suitable to the absorption property of SPA with NIPs.Imprinted polymer SPA-MIPs constructed by the present invention is to template molecule in this explanation SPA has higher specific selectivity.

On the basis of investigation on the target liposomes molecular level of SPA trace, also investigate in its bacteria levels.Due to The SPA albumen of trace is a kind of protein separated from aureus cell wall, is the main component of cell wall antigen. Therefore, first the molecular engram targeted system constructed by the present invention is carried out bag and carries 6-Aminofluorescein (6-FAM), pass through streaming Cell measures the antibacterial picked-up to different several preparations to evaluate their targeting.Bag carries the trace of 6-Aminofluorescein The method of polymer (FAM-SPA-MIPs) and non-imprinted polymer (FAM-NIPs) is with reference to the target liposomes of SPA trace (SPA-MIPs) with the preparation method of non-imprinted polymer (NIPs), when difference is to prepare liposome, contain with 1ml The PBS solution of 1mg 6-FAM replaces hydrating fluid, and remaining step is identical with the preparation method of SPA-MIPs and NIPs. Then FAM-SPA-MIPs, FAM-NIPs is the dilutest with normal saline with 6-FAM (the 6-FAM concentration of three is identical) Release 5 times, staphylococcus aureus bacterium solution (1 × 10⁸CFU/mL) dilution 100 times；Again by FAM-SPA-MIPs, FAM-NIPs Or 6-FAM mixes for 1:1 by volume with staphylococcus aureus bacterium solution respectively, it is placed in 37 DEG C of thermostatic water tanks, simultaneously with phase With concentration antibacterial as a control group, hatch after 6h centrifugal 10min under the conditions of 7000r/min, remove supernatant, it is heavy to retain Form sediment, in precipitation, add the fresh culture 5mL mixing of sterilizing, survey fluorescence intensity level with flow cytometer, result such as Fig. 5 institute Show.Result shows, after staphylococcus aureus hatches 6h with FAM-SPA-MIPs, FAM-NIPs and 6-FAM respectively, takes the photograph Taking bag, to carry the fluorescence intensity of fluorescein preparation different, antibacterial combine the amount of FAM-SPA-MIPs be significantly higher than FAM-NIPs and 6-FAM, fluorescence intensity is 2.2 times and 2.8 times of FAM-NIPs and 6-FAM respectively.Show that FAM-SPA-MIPs is to gold Staphylococcus aureus has higher external binding ability.

Secondly, the molecular engram targeted system constructed by the present invention is carried out erythromycin medicine carrying, by investigating it to golden yellow Fructus Vitis viniferae The inhibitory action of coccus verifies its targeting further.Bag carries the imprinted polymer (EM-SPA-MIPs) of erythromycin and non-print The target liposomes (SPA-MIPs) of the preparation method reference SPA trace of mark polymer (EM-NIPs) and non-imprinted polymer (NIPs) manufacture method, difference is when preparing liposome, is that 1:15 weighs by the mass ratio of erythromycin with liposome Erythromycin 1mg is dissolved in 200 μ l ethanol, weighs soybean lecithin 10.5mg, cholesterol 4.5mg is dissolved in chloroform: methanol (1: 1) in mixed solution 800 μ l, three is all added in eggplant-shape bottle, remaining steps and the target liposomes of SPA trace (SPA-MIPs) consistent with the preparation method of non-imprinted polymer (NIPs).The concentration of erythromycin measures and uses high-efficient liquid phase color Spectrometry (HPLC), testing conditions is as follows: chromatographic column: C18 post；Column temperature: 25 DEG C；Detection wavelength: 210nm；Flowing phase: 0.1mol/L ammonium dihydrogen phosphate buffer (pH 6.5 adjusted by triethylamine): acetonitrile volume ratio is 70:30；Flow velocity: 1.0mL/min； Sample size 50 μ L.Testing result shows, is 100～500 μ g mL at erythromycin concentration^-1In the range of erythromycin concentration (X) with Good linear relationship is had between HPLC chromatogram peak area (Y).Standard curve equation is: Y=30054X+56254 (R²=0.9991), Basic, normal, high content assaying precision RSD is respectively 3.46%, 2.18% and 1.52%, and the response rate is respectively 96.54 ± 2.05%, 98.73 ± 2.59%, 100.26 ± 3.02%.

Finally, measure erythromycin liposome encapsulation: take erythromycin liposome and be divided into two parts, a through Sephadex G-50 Gel column purified pool, another part is directly diluted to same volume, and both of which adds methanol breakdown of emulsion, measures respectively in two parts of samples Erythromycin concentration C and C₀.Computational envelope rate (EE): EE=C/C₀× 100%.After testing, erythromycin liposome EM-LIP Envelop rate average (n=3) be 91.25 ± 1.48%, the envelop rate average (n=3) of erythromycin imprinted polymer (EM-SPA-MIPs) It is 86.25 ± 2.53%.

When mensuration EM-SPA-MIPs is to the fungistatic effect of staphylococcus aureus, according in staphylococcus aureus picked-up experiment The incubation method used, first uses normal saline by EM-SPA-MIPs, EM-NIPs, EM (the EM concentration of three is identical) Dilution 5 times, staphylococcus aureus bacterium solution (1 × 10 respectively⁸CFU/mL) dilution 100 times；Again by EM-SPA-MIPs, EM-NIPs, EM mix for 1:1 by volume with staphylococcus aureus bacterium solution respectively, are placed in 37 DEG C of thermostatic water tanks, identical The antibacterial of concentration as a control group, is centrifuged 10min under the conditions of 7000r/min, removes supernatant, retain precipitation after hatching 6h, The fresh culture 5mL mixing of sterilizing is added in precipitation.By sample suspension in 96 orifice plate point samples, it is placed in constant incubator Middle cultivation, and survey its OD value respectively at 0h, 1h, 2h, 4h, 6h, 8h, experimental result is as shown in Figure 6.Result shows, The OD value of EM-SPA-MIPs group and EM-NIPs group is below the OD value of non-administration group (Control group), with NIL group Comparing, the OD of EM-SPA-MIPs group is significantly lower than EM-NIPs group, and the EM-SPA-MIPs inhibitory action to antibacterial is described Being better than EM-NIPs, the Targeting Effect further illustrating molecular engram targeting preparation constructed by the present invention is obvious.

Embodiment 2, the preparation of target liposomes of matrix metalloproteinase MMP-2 trace and evaluation thereof

The preparation of the target liposomes of matrix metalloproteinase MMP-2 trace, specifically comprises the following steps that

(1) thin film dispersion ultrasonic method is used to prepare liposome, method particularly includes: weigh soybean lecithin 10.5mg, cholesterol 4.5mg, dissolves with chloroform and the solution 1ml that methanol volume ratio is 1:1, and the rotary evaporation that then reduces pressure under the conditions of 37 DEG C removes Organic solvent, forms one layer of uniform transparent lipid membrane, adds pure water 1mL in 37 DEG C of aquations 1h, utilizes ultrasonic cell disrupte The ultrasonic 5min of instrument (800W 50 times, is spaced 10s after each ultrasonic 10s), obtains liposome (LIP)；

(2) use sol method to prepare surface and modify the liposome of silicon dioxide, method particularly includes: by phospholipid: tetraethyl orthosilicate (TEOS): H₂O mol ratio be 1:8:344 be slowly added in liposome through volume fraction be the 10% scattered TEOS of ethanol, The most under agitation with the sodium hydroxide solution regulation pH to 8.50 of 0.1mol/L, room temperature (18-25 DEG C) is stirred overnight, The silica precursor not deposited in removing system of being dialysed by mixture again, obtains surface and modifies the liposome of silicon dioxide, name For silicon dioxide liposome (S-LIP)；

(3) ethylene linkage modifies silicon dioxide liposome, method particularly includes: by 3-sulfydryl-1-propane sulfonic acid sodium (MPS) of 50 μ L It is slowly dropped in the ethanol of 250 μ L after disperseing half an hour, then takes its 10 μ L dispersant liquid drop and be added to the fat of 2ml silicon dioxide In plastid, room temperature (18-25 DEG C) is stirred overnight, and obtains the silicon dioxide liposome (MS-LIP) that ethylene linkage is modified；

(4) preparation of matrix metalloproteinase MMP-2 imprinted polymer, method particularly includes: take acrylamide (AAM) 4mg With N-N methylene-bisacrylamide (MAA) 2mg be dissolved into pH be 7.4, concentration be 0.02M PBS in, ultrasonic molten Solve, obtain mixed solution A, then 2mg matrix metalloproteinase MMP-2 is dispersed in the titanium dioxide silicone grease that 1mL ethylene linkage is modified In plastid solution and be positioned over 0.5h is stirred at room temperature, then mixture is carried out vacuum outgas 10min, obtain mixed solution B, at nitrogen Take 200 μ L mixed solution A under gas shielded to be added dropwise in mixed solution B, and nitrogen blowing 10min, room temperature (18-25 DEG C) is stirred Mix lower prepolymerization 1h, the lower dropping 7 μ L Ammonium persulfate .s (APS, 10%, W/W) of nitrogen protection and 4.5 μ L tetramethylethylenediamines (TEMED, 5%, W/V) causes polymerization in mixture, is positioned over and 24h is stirred at room temperature, and dialyses one week with pure water, Removing template molecule and unreacted reagent, lyophilizing to constant weight i.e. matrix metalloproteinase MMP-2 imprinted polymer is named MMP-2-MIPs.Dialyse one week with pure water, remove template molecule and unreacted reagent.Lyophilizing i.e. obtains matrix metal to constant weight The target liposomes (MMP-2-MIPs) of protease MMP-2 trace.

The adsorption experiment of the target liposomes of matrix metalloproteinase MMP-2 trace: the matrix metalloprotease prepared for investigation The target liposomes of the enzyme MMP-2 trace specific adsorption ability to matrix metalloproteinase MMP-2, uses following methods pair It is verified.

First, matrix metalloproteinase MMP-2 has been carried out FITC fluorescent labeling: take 4mg matrix metalloproteinase MMP-2 It is dissolved in 0.2M Na₂PO₄In solution, separately take 0.16mgFITC and be dissolved in 0.2M Na₂PO₄In solution, make FITC and matrix metal The mol ratio of protease MMP-2 is 1.2:1.Under magnetic agitation, the solution containing matrix metalloproteinase MMP-2 is added In solution containing FITC, lucifuge reaction 1.5h.After reaction terminates, centrifugal 10min under the conditions of 8000r/min, to remove precipitation Matrix metalloproteinase MMP-2.Take supernatant Sephadex G-50 gel column and carry out isolated and purified, lyophilizing, obtain FITC The matrix metalloproteinase MMP-2 of labelling.

Dynamic adsorption is tested: by 5mg MMP-2-MIPs and NIPs respectively with the 0.2mg/ml FITC-MMP-2 solution of 5ml In 37 DEG C of oscillation incubations, take out 1ml respectively at 0.25h, 0.5h, 1h, 2h, 4h, be centrifuged 10min with 4500r/min, Measuring the fluorescence intensity of supernatant, result is as shown in Figure 7.Result shows, MMP-2-MIPs and NIPs is to FITC-MMP-2 Absorption increase over time and increase, 2h when, i.e. reach dynamic equilibrium.Further, during dynamic adsorption balance, To FITC-MMP-2 about adsorbance is NIPs 5 times of MMP-2-MIPs, illustrate the molecular engram targeting that the present invention builds Polymer support has good absorbability to albumen.

Adsorption isotherm experiment: accurately weigh 1mg MMP-2-MIPs and NIPs and be respectively placed in centrifuge tube, respectively with 1ml's The FITC-MMP-2 solution of 0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml shakes under the conditions of 37 DEG C Swinging and hatch, take out after 2h, be centrifuged 10min with 4500r/min, Aspirate supernatant measures fluorescence intensity, and result is as such as Fig. 8 institute Show.Adsorption isotherm experiment shows, template molecule is adsorbed the change along with FITC-MMP-2 concentration and changes by polymer, when When the concentration of FITC-MMP-2 is less than 0.2mg/ml, ratio of adsorption rapid increase, when the concentration of FITC-MMP-2 reaches During 0.2mg/ml, ratio of adsorption reaches balance, and compared with NIPs, MMP-2-MIPs has higher suction to FITC-MMP-2 Attached capacity and preferable selectivity.

Specific adsorption is tested: specifically have chosen staphylococcus aureus surface Protein S PA, lysozyme (LYZ), bovine serum albumin In vain (BSA), Streptavidin (SA) is competition albumen, and the FITC labelling of competition albumen is with reference to the preparation of FITC-MMP-2 Method, obtains FITC-SPA, FITC-LYZ, FITC-BSA, FITC-SA respectively.The concrete operations of specific adsorption test are: Weigh a series of 1mg MMP-2-MIPs and NIPs to be respectively placed in centrifuge tube, add the 0.2mg/ml's of 1ml FITC-SPA, FITC-LYZ, FITC-BSA, FITC-SA and FITC-MMP-2 solution is placed on agitator in 37 DEG C and shakes After swinging absorption 2h, measuring the fluorescence intensity in supernatant respectively, result is as shown in Figure 9.Result shows, MMP-2-MIPs is only MMP-2 is had higher absorption property, and relatively low, with NIPs to MMP-2 to the absorption property of other competition albumen Absorption property similar.Template molecule MMP-2 is had by this explanation imprinted polymer MMP-2-MIPs constructed by the present invention There is higher specific selectivity.

On this basis, the target liposomes of matrix metalloproteinase MMP-2 trace is carried out the inspection on cellular level, Owing to MMP-2 type matrix metalloproteinase enzyme is the one of tumor cell outer matrix metalloproteases enzyme, therefore, by testing The targeting of tumor cell is evaluated its trace ability by card MMP-2 imprinted polymer.In this experiment, select tumor thin Born of the same parents U937, fluorescent probe is amycin.

Carry amycin matrix metalloproteinase MMP-2 trace target liposomes (DOX-MMP-2-MIPs) with bag carry Ah The preparation method of the non-imprinted polymer (DOX-NIPs) of mycin is with the target liposomes of matrix metalloproteinase MMP-2 trace (MMP-2-MIPs), with non-imprinted polymer (NIPs), when difference is to do liposome, ammonium sulphate gradient is used to be prepared into Liposome to bag amycin: weigh soybean lecithin 10.5mg, cholesterol 4.5mg, is 1:1 by chloroform and methanol volume ratio Solution 1ml dissolve, then under the conditions of 37 DEG C reduce pressure rotary evaporation remove organic solvent, form one layer of uniform transparent lipid Thin film, adds pure water 1mL in 37 DEG C of aquations 1h, and (800W 50 times, surpasses every time to utilize the ultrasonic 5min of ultrasonic cell disrupte instrument 10s it is spaced after sound 10s), obtain liposome (LIP)；Dialyse to water 4h by gained LIP.With 0.9% normal saline 10mg/ml Doxorubicin solution, then the LIP after dialysis and the Doxorubicin solution of 150 μ l are hatched 20min 60 DEG C of conditions, hatch Remove free amycin through Sephadex G-50 gel column after completing, the liposome (DOX-LIP) carrying amycin must be wrapped.

Then measure U937 cell the picked-up of DOX-MMP-2-MIPs Yu DOX-NIPs is tested, evaluate institute of the present invention structure The targeting of the target liposomes of the trace built.Concrete operations are: by 10^⁴U937 cell overnight incubation, respectively with DOX-SPA-MIPs, DOX-NIPs (the DOX concentration of the two is identical) hatch 2h, then wash off with PBS and are not taken the photograph by cell The medicinal liquid taken, then fixes 30min with paraformaldehyde, and the cell after fixing dyes through DAPI, at fluorescence microscopy Microscopic observation The picked-up result of cell, result is as shown in Figure 10.Result shows, can around the nucleus hatched with DOX-MMP-2-MIPs Observe stronger red fluorescence, and the red fluorescence that the cell observation hatched with DOX-NIPs arrives is more weak, thus, can be abundant Illustrating, DOX-MMP-2-MIPs has more preferable external binding ability to U937 cell.

Finally illustrating, preferred embodiment above is only in order to illustrate technical scheme and unrestricted, although by above-mentioned The present invention is described in detail by preferred embodiment, it is to be understood by those skilled in the art that can in form and In details, it is made various change, without departing from claims of the present invention limited range.

Claims

1. the preparation method of target liposomes based on protein molecular trace, it is characterized in that, comprise the steps: to take acrylamides and N-N methylene-bisacrylamide is that 1 ~ 5:1 is dissolved to pH6.8 ~ 7.4, concentration is the PBS solution of 0.02 ~ 0.1M in mass ratio or in water, ultrasonic, obtaining mixed liquor A, in gained mixed liquor A, the total concentration of acrylamides and N-N methylene-bisacrylamide is 1 ~ 5mg/ml；Olefinic recycle key modifies silicon dioxide liposome, is then scattered in by protein molecular in the silicon dioxide liposome solutions that ethylene linkage is modified and stirs, and vacuum outgas obtains mixed liquid B；Under nitrogen protection, mixed liquor A is added dropwise in mixed liquid B for 1:1 ~ 1:10 by volume, nitrogen blowing, stirs prepolymerization; then the lower dropping Ammonium persulfate. of nitrogen protection and tetramethylethylenediamine cause polymerization; continue stirring, dialyse, must be based on the target liposomes of protein molecular trace；

Described silicon dioxide liposome is prepared by following methods: first use thin film dispersion ultrasonic method to prepare liposome, then add through the scattered tetraethyl orthosilicate of ethanol solution in liposome, regulate pH to 8 ~ 10.4 with sodium hydroxide solution under stirring, be stirred overnight, dialysis, obtains silicon dioxide liposome.

Preparation method the most according to claim 1, it is characterised in that: the addition of described tetraethyl orthosilicate presses phospholipid: tetraethyl orthosilicate: H₂The mol ratio of O is 1:8:344.

Preparation method the most according to claim 1, it is characterised in that: the volume fraction of described ethanol solution is 5% ~ 25%.

Preparation method the most according to claim 1, it is characterized in that: described thin film dispersion ultrasonic method prepare the method for liposome be phospholipid and cholesterol are dissolved in chloroform that volume ratio is 1:1 with in the mixed solution of methanol, decompression rotary evaporation removes organic solvent, form transparent lipid membrane, add water hydratable, ultrasonic, obtain liposome.

Preparation method the most according to claim 4, it is characterised in that: the mol ratio of described phospholipid and cholesterol is 2 ~ 3:1.

Preparation method the most according to claim 4, it is characterised in that: described ultrasonic condition is under the conditions of 800W ultrasonic for 50 times, each ultrasonic 10s, continues ultrasonic after the 10s of interval.

Preparation method the most according to claim 4, it is characterised in that: the condition of described aquation is aquation 1 hour under the conditions of 37 DEG C.

8. according to the preparation method described in any one of claim 1 ~ 7, it is characterised in that: described protein molecular is staphylococcus aureus protein A or matrix metalloprotease MMPs.

9. the target liposomes based on protein molecular trace prepared by the preparation method described in any one of claim 1 ~ 8.