CN109394733A - Tetrandrine PLGA nanoparticle of a kind of erythrocyte membrane encapsulating and preparation method thereof and its application - Google Patents
Tetrandrine PLGA nanoparticle of a kind of erythrocyte membrane encapsulating and preparation method thereof and its application Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology fields, and in particular to a kind of tetrandrine PLGA nanoparticle and the preparation method and application thereof of erythrocyte membrane encapsulating.It include erythrocyte membrane, PLGA and tetrandrine in the structure of the nanoparticle, the tetrandrine and the PLGA, which are combined, forms-PLGA nanometers of cores of tetrandrine, and the erythrocyte membrane is encapsulated in outside the tetrandrine-PLGA nanometers of core.The preparation method simple and effective.The nanometer formulation improves the biocompatibility of entire drug delivery system, realizes the long circulating and sustained release of tetrandrine in vivo, avoids the excessively high caused toxic side effect of blood peak concentration of drug of normal injection agent, has a good application prospect.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating and
Preparation method and application.
Background technique
Tetrandrine (Tetrandrine, TET), also known as hanfangchin A are Menispermaceae plant powder Radix Stephaniae Tetrandrae (Stephania
Tetrandra S.Moore) a kind of bisbenzylisoquinoline alkaloid for extracting in root tuber.Modern pharmacology research finds that powder is anti-
Own alkali in anti-inflammatory and antalgic, antiviral, pulmonary fibrosis resistant, anti-silicosis, antitumor, anti-hypertension, resist myocardial ischemia, anti-arrhythmia
Etc. have preferable effect, be clinically commonly used for treat rheumatalgia, arthralgia, silicosis, inflammatory lung disease, from
Body immunity disease and hypertension etc..
The preparation of the tetrandrine listed has " hanfangchin A piece " and " tetrandrine injection ".But there are still one
A little problems, mainly are as follows: tablet causes it to absorb not exclusively since its water-soluble is poor and there is the first pass effect of liver,
Bioavilability is low；And normal injection agent is metabolized quickly in vivo, blood concentration is held time short, and tetrandrine has in addition
Certain irritation, intravenous injection may cause local pain or phlebitis.
Summary of the invention
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide a kind of encapsulatings of erythrocyte membrane
Tetrandrine PLGA nanoparticle and the preparation method and application thereof.
PLGA refers to poly lactide-glycolide acid (poly (lactic-co-glycolic acid).Its mean point
Son amount range is 5000-100000.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating, the nanoparticle
Structure in include erythrocyte membrane, PLGA and tetrandrine, the tetrandrine and the PLGA are combined and are formed tetrandrine-
PLGA nanometers of cores, the erythrocyte membrane are encapsulated in outside the tetrandrine-PLGA nanometers of core.
Further, the partial size of the tetrandrine PLGA nanoparticle of the erythrocyte membrane encapsulating is 130nm-180nm.It is described
The partial size of the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating is also possible to 130nm-150nm.The erythrocyte membrane encapsulating
The partial size of tetrandrine PLGA nanoparticle is also possible to 150nm-170nm.The tetrandrine PLGA of the erythrocyte membrane encapsulating receives
The partial size of the grain of rice is also possible to 170nm-180nm.
In the present invention, the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating can be referred to RPTNs.
The second aspect of the present invention provides the preparation method of the tetrandrine PLGA nanoparticle of aforementioned erythrocyte membrane encapsulating,
Including at least following steps:
(1) tetrandrine and PLGA are dissolved in organic solvent and form organic phase；Emulsifier is dissolved in water and forms water phase；
(2) organic phase in step (1) is slowly added into water phase, removes the organic solvent, form nano-emulsion
(PTNs)；
(3) erythrocyte membrane is prepared；
(4) erythrocyte membrane prepared is squeezed out, obtains erythrocyte membrane vesica (RVs)；
(5) erythrocyte membrane vesica is added in nano-emulsion, squeezes out the tetrandrine contained to get the erythrocyte membrane
PLGA nanoparticle.
The step (3), (4) can also carry out before step (1) (2).
In one embodiment, in step (1), the emulsifier is in poloxamer, polyvinyl alcohol, polyethylene glycol
It is one or more.
In one embodiment, in step (1), the organic solvent is selected from acetone, methylene chloride, chloroform or second
One of acetoacetic ester is a variety of.
Further, in step (1), the tetrandrine, PLGA, emulsifier mass ratio are as follows: (0.5-5): (10-
50): (100-400).
In one embodiment, the tetrandrine, PLGA, emulsifier mass ratio can be with are as follows: (0.5-2): (10-
20): (100-200).In one embodiment, the tetrandrine, PLGA, emulsifier mass ratio can be with are as follows: (2-5):
(20-50): (200-400).In one embodiment, the tetrandrine, PLGA, emulsifier mass ratio can be with are as follows:
0.5:10:100.In one embodiment, the tetrandrine, PLGA, emulsifier mass ratio can be with are as follows: 5:50:400.
In one embodiment, the tetrandrine, PLGA, emulsifier mass ratio can be with are as follows: 2:20:200.In a kind of implementation
In mode, the tetrandrine, PLGA, emulsifier mass ratio can be with are as follows: 1:20:200.
Further, in step (2), the volume ratio of the organic phase and water phase are as follows: 1:20-2:20.
In one embodiment, in step (2), the method that removes acetone are as follows: slowly add the organic phase in step (1)
Magnetic agitation is carried out after entering into water phase.
Further, the magnetic agitation condition can be revolving speed 200rpm-500rpm, time 2h-6h.
In one embodiment, in step (3), erythrocyte membrane is prepared using hypotonic dialysis method.
Specifically, the following steps are included: animal takes blood, centrifugation removal blood plasma and leucocyte, hypotonic EDTA aqueous solution broken wall,
Up to the erythrocyte membrane after centrifugation, cleaning.
The animal is mammal, can be rodent, primate.Such as can be monkey, rabbit, goat,
Rat.
Taking blood mode can be abdominal aortic blood.
When centrifugation removal blood plasma and leucocyte, the centrifugal condition are as follows: 0-10 DEG C, 2000-5000rpm.
The mass concentration of the hypotonic EDTA aqueous solution are as follows: 0.1-1.0mM.
The broken wall number can be 3-10 times.
The dosage of the erythrocyte membrane and PLGA are as follows: the erythrocyte membrane that animal's whole blood described in 1mL extracts corresponds to 5mg-8mg
PLGA。
In one embodiment, in step (4), extrusion method is that ultrasound or mini-extruder extrusion instrument squeeze out.
In one embodiment, in step (5), extrusion method is that ultrasound or mini-extruder extrusion instrument squeeze out.
In step (4) or (5), the ultrasound condition is power 100w-200w, frequency 30-60KHz, time 0.5min-
10min。
In one embodiment, in step (4), the extrusion condition be by erythrocyte membrane pass sequentially through 800nm,
The polycarbonate membrane of 400nm and 200nm, each partial size get off 10-30 times out back to squeeze.
In one embodiment, in step (5), the extrusion condition is the poly- carbon that the mixed liquor is passed through to 200nm
Acid esters film squeezes out 10-30 times back and forth.
In step (5), the volume ratio of erythrocyte membrane vesica and nano-emulsion is 1:(5-10).In one embodiment, may be used
Think 1:5,1:8,1:10.
The third aspect of the present invention, the powder for providing a kind of erythrocyte membrane encapsulating that the preparation method by second aspect obtains are prevented
Own alkali PLGA nanoparticle.
The fourth aspect of the present invention provides the Fourstamen Stephania Root of the erythrocyte membrane encapsulating of aforementioned first aspect and third aspect offer
Alkali PLGA nanoparticle prepare anti-inflammation analgesis medicament, antiviral drugs, silicosis therapeutic agent, hypertension therapeutic drug, cardiac muscle lack
Purposes in blood therapeutic agent, treating irregular heart pulse drug or tumor therapeutic agent.
Compared with prior art, the invention has the following beneficial effects:
The tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating of the present invention solves the problems, such as drug poorly water-soluble,
Reduce its irritation, delays the release of drug；The biocompatibility for improving drug-loading system avoids it clear by immune system
It removes, realizes drug long circulating in vivo, extend blood halflife.The preparation method simple and effective.
Detailed description of the invention
Fig. 1 is partial size (A), PDI (B) and Zeta potential (C) lab diagram of PTNs, RPTNs and RVs；
Fig. 2 is that partial size (A), PDI (B) and Zeta potential (C) of the RPTNs in 15 days change lab diagram；
Fig. 3 is the aspect graph of PTNs (A), RPTNs (B) and RVs (C) under transmission electron microscope；
Fig. 4 is TET, PTNs and RPTNs In-vitro release curves；
Fig. 5 is the memebrane protein proof diagram of PTNs, RPTNs and RVs；
Fig. 6 is the Laser Scanning Confocal Microscope figure of PTNs and RPTNs；
Fig. 7 A is the fluidic cell figure that MCF-7/ADR absorbs PTNs and RPTNs；
Fig. 7 B is the fluidic cell figure of Raw 264.7 cellular uptake PTNs and RPTNs；
Fig. 8 is pharmacokinetic studies result figure in TET and RPTNs body.
Specific embodiment
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or according to condition proposed by each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.
The preparation of 1 nano-emulsion of embodiment (PTNs)
TET and PLGA (molecular weight 30000) are weighed respectively, and acetone, which is added, to be made to dissolve, so that the concentration of TET and PLGA point
Not Wei 1mg/mL, 20mg/ml, then 1mL organic phase is slowly added under conditions of magnetic agitation (revolving speed 300rpm)
In 20mL water phase, (emulsifier is poloxamer in water phase, and emulsifier quality is 0.2g), so that tetrandrine, PLGA, emulsifier
Mass ratio be three's mass ratio be 1:20:200, magnetic agitation 3h volatilization removal acetone, obtains a nanometer milk solution at normal temperature.
The preparation of 2. erythrocyte membrane of embodiment
Healthy rat is taken, is weighed, chloral hydrate anesthesia is injected intraperitoneally according to 100g/0.7mL, abdominal aortic blood will
In hematology aliquot to the EP pipe containing 0.05mL heparin sodium, supernatant leukocytic cream is removed in centrifugation (3800rpm, 5min, 4 DEG C),
PBS solution is added to mix, repeated washing three times, and is settled to 1mL with PBS.The every pipe of mixed liquid that above-mentioned constant volume obtains is dispensed into 4
It in a EP pipe containing 0.01mL heparin sodium, adds 0.9mLEDTA solution (0.2mM), mixes, make red blood cell in low permeability media
Middle cracking discharges hemoglobin, then adds 10 times of concentration PBS of 0.1mL and mixes, is centrifuged (13200r/min, 10min, 4
DEG C) cleaning, remove supernatant, retains bottom erythrocyte membrane, repeat this process three times or more, until supernatant is colourless.Remove supernatant
0.9mL EDTA solution is added afterwards, mixes, cleaning；Finally, two solencytes are combined into a pipe, and it is settled to EDTA solution
0.5mL is packed into valve bag, -80 DEG C of preservations.
The preparation of 3. erythrocyte membrane vesica (RVs) of embodiment
It takes and is managed according to the erythrocyte membrane two that embodiment 2 prepares, thawed in 37 DEG C of water-baths, first by erythrocyte membrane in power
Ultrasound 2min under 100W, frequency 40KHz, then again with film mini-extruder extrusion instrument by the obtained vesica of ultrasound pass sequentially through 800,400 and
The polycarbonate membrane of 200nm is squeezed out, and is squeezed out 10 times repeatedly under each partial size, is obtained RVs.
The preparation of the tetrandrine PLGA nanoparticle (RPTNs) of 4. erythrocyte membrane of embodiment encapsulating
The PTNs nano-solution for taking the RVs solution prepared according to embodiment 3 and being prepared according to embodiment 1, according to RVs
Solution is that 1:5 is mixed with the volume ratio of PTNs solution, is then carried out with film mini-extruder extrusion instrument by the polycarbonate membrane of 200nm
It squeezes out, squeezes out 10 times repeatedly, obtain RPTNs.
Parallel three groups of PTNs, RVs and RPTNs sample of preparation according to the method described above, measures it with Malvern laser particle analyzer
Partial size, PDI and Zeta potential.
As seen from Figure 1, the average grain diameter of three groups of PTNs and RPTNs is respectively 147.9nm and 164.1nm, partial size phase
Difference about 16nm, according to the literature, the average thickness of erythrocyte membrane is about 7-8nm, since nanoparticle is spherical structure, the grain measured
Diameter difference is consistent with two sides erythrocyte membrane thickness, it was demonstrated that the encapsulating of erythrocyte membrane；The PDI average value of PTNs and RPTNs is respectively
0.124 and 0.181, respectively less than 0.2, illustrate that nanoparticle size is uniform；The average potential of PTNs and RPTNs is respectively -6.34mV
It is far below PTNs current potential with the current potential of -17.1mV, RPTNs, also demonstrates the successful encapsulation of erythrocyte membrane.
The measurement of embodiment 5. encapsulation rate and drugloading rate
It takes PTNs solution appropriate, is added in processed bag filter, both ends are clamped, be placed in outside the dialysis of 10 times of volumes
In liquid (pH=7.4,1 × PBS), 37 DEG C, dialysis 4h takes dialyzate under magnetic agitation, 0.22 μm of miillpore filter, HPLC measurement are crossed
Its peak area.Encapsulation rate and drugloading rate are calculated by the following formula: quality/TET dosage of TET in encapsulation rate=nanoparticle
X100%；Quality/nanoparticle gross mass of TET in drugloading rate=nanoparticle.
The wherein determination condition of HPLC are as follows:
Chromatographic column: Agilent SB-C18 (50mm × 4.6mm, 5 μm)
Detector: Agilent Technologies UV detector
Mobile phase: methanol-acetonitrile -0.003mol/L KH2PO4Aqueous solution-triethylamine (50:30:20:0.05)
Flow velocity: 1mLmin-1
Column temperature: 30 DEG C
Detection wavelength: 280nm
Sample volume: 40 μ L
Detected by HPLC, the nanoparticle have good encapsulation rate and drugloading rate, respectively 83.97% and 4.03%.
The study on the stability of embodiment 6.RPTNs
The RPTNs that three batches are prepared according to the method for embodiment 4, sample is placed under room temperature, continuous measurement 15 days
Sample particle diameter, polydispersity coefficient and current potential investigate its stability.
By Fig. 2 result as it can be seen that RPTNs in 15 days partial size stablize 165 ± 3.1nm or so, PDI be maintained at 0.25 with
Under, current potential is first gradually decrease to -27.9mV or so, sample particle diameter and PDI from -18.1mV and has after the 10th day slightly increase
Trend, but it is basicly stable, it does not observe that phenomena such as precipitating or reuniting occur in nanoemulsions in experiment, illustrates RPTNs in 15 days
Character and distribution are basicly stable.
The morphologic observation of 7. nanoparticle of embodiment
It takes a piece of filter paper cut to be placed in culture dish, then supports net to there is film surface to be put in filter paper upwards in a piece of common carbon
On；Then the nanoparticle solution prepared is taken, 10 μ L nano fluids of drop are supported on the net, to air-dry, then drip 10 μ L ultrapure waters, wind in carbon
Dry, 10 μ L sodium phosphotungstate solution of drop dye 1-2min, suck extra dye liquor from edge with filter paper, toast under infrared baking lamp
30min is placed under transmission electron microscope and observes.
As Fig. 3 electron microscope it is found that PTNs made from this project has regular spherical structure, surface is smooth；RVs have compared with
The cavity structure of rounding, is evenly distributed；The visible clearly shell-core double-layer structure from the electron microscope of RPTNs indicates erythrocyte membrane
PLGA drug-carrying nanometer particle is successfully wrapped up.Three's partial size is about between 130-180nm, with Malvern particle instrument acquired results phase
Closely, the success of this preparation for also further demonstrating nanoparticle.
The extracorporeal releasing experiment of 8. nanoparticle of embodiment
Extracorporeal releasing experiment is carried out using dialysis.Take 5mL TET solution, PTNs solution and RPTNs solution in saturating respectively
It analyses in bag (MW 3500), binding sealing is placed in 1 × PBS of 50mL, and isothermal vibration carries out body under the conditions of 37 DEG C, 100rpm
Outer release takes out 1mL release liquid respectively at 0.5,1,2,4,8,12,24,36,48,72,96,120h and adds the fresh release of 1mL
External solution replaces whole release liquids when for 24 hours with the fresh 1 × PBS of 50mL.
By Fig. 4 result it is found that free TET discharges completely substantially in 4h；Within the 120h time, PTNs and RPTNs are aobvious
Slow releasing function is shown, and the slow release effect of PRTNs is substantially better than PTNs.In 0-4h, the phenomenon of burst release of PTNs and RPTNs are come
Derived from the free drug not wrapped up into nanoparticle, including some it is present between PTNs and erythrocyte membrane or is attached to nanoparticle table
The drug in face.When accumulative release time is 4h, the TET Cumulative release amount of PTNs is 37.37%, and the accumulative release of the TET of RPTNs
Amount is 32.65%；The equal slow release of PTNs and RPTNs after 4h, when accumulative release time is 120h, the TET of PTNs is accumulative to be released
It is high-volume 76.94%, and the TET Cumulative release amount of RPTNs is 73.21%.Aggregate analysis, TET of the RPTNs in various time points
Cumulative release amount averagely reduces 5.33% compared to PTNs, it was demonstrated that the encapsulating of erythrocyte membrane further enhances the sustained release effect of PTNs
Fruit.
The verifying of 9. memebrane protein of embodiment
Using the verifying of PAGE gel electrophoresis experiment, in the preparation process of RVs and RPTNs, whether to destroy neutral red thin
Memebrane protein on after birth.First using the protein concentration in BCA determination of protein concentration method calibration each sample, sample is adjusted by dilution
Product protein concentration makes identical, then takes erythrocyte membrane (RBCM), 100 μ L of RVs, RPTNs, PTN each sample respectively, is added on 25 μ L
Sample buffer, mixing is placed on 5min in 95 DEG C of water-baths, then takes the gel slab prepared successively each 15 μ L of loading, albumen Marker
Applied sample amount be 2 μ L, after electrophoresis, gel film is placed under the conditions of room temperature is protected from light and dyes 30min, be eventually adding destainer in
It is eluted overnight in decolorization swinging table, Protein Separation situation is photographed to record under gel imager and natural light.
As shown in Figure 5, after PAGE gel is separated by electrophoresis, albumen that RVs group, RPTNs group and RBCM group are presented
Pillar location is identical, and the no corresponding protein band of the PTNs group as negative control occurs, this illustrates that RVs and RPTNs are protected
Natural erythrocyte membrane protein type has been stayed, has further been also demonstrated in the preparation process of RPTNs, the processing such as ultrasound and extruding are not
Natural erythrocyte membrane protein can be made by apparent damage.It is therefore envisaged, the coating for remaining natural erythrocyte membrane protein is received
The grain of rice also has good biocompatibility, has and realizes macrocyclic potential in vivo.
10. laser co-focusing of embodiment observes intake of the MCF-7/ADR cell to nanoparticle
1. cell culture: MCF-7/ADR cell uses 1640 complete medium of RPMI, in 37 DEG C, 5%CO2Under the conditions of train
It supports.Cell, using the culture solution and frozen stock solution for being free of adriamycin, is greater than 50% and shape to cell density when passing on and freezing
State it is preferable when can in cultivating system plus adriamycin maintain drug resistance, adriamycin additional amount from 250ng/mL, 500ng/mL,
1000ng/mL is successively increased, if it is observed that cell state should improve serum dosing again when cell improves when poor.Before experiment
Reagent removal culture two weeks.
The preparation of the RVs of 2.DiO label: taking the 100 μ L of DiO stock solution of 2mg/mL, and 900 μ L PBS are added and are configured to concentration
It is spare for the working solution of 0.2mg/mL；The RVs solution 0.5mL prepared is separately taken, 0.5mL DiO working solution, room temperature condition is added
Under be protected from light incubation centrifugation (4000rpm, 30min, 4 DEG C) 8min, the precipitating finally obtained with 1 × PBS of 0.5mL dissolve it is spare.
3. carrying the preparation of the PTNs of Nile red: because without fluorescence, the TET in nanoparticle is replaced with Nile red by TET.
According to the preparation method of embodiment 1, the PLGA nanoparticle for carrying Nile red is obtained.
4. the preparation of the RPTNs of fluorescent marker: the PTNs of the RVs solution and load Nile red that take the fluorescent marker prepared is molten
Liquid obtains the RPTNs of fluorescent marker according to the coating method of embodiment 4.
5. cell administration: taking 12 porocyte culture plates of sterilizing, prepare 2 holes, the thin of one piece of φ=14mm respectively to be placed in hole
Born of the same parents' creep plate, being inoculated with 1mL cell density into hole is about 2*105The cell liquid of a/mL, in CO2It is cultivated for 24 hours in incubator.It is old to inhale abandoning
Cell culture fluid, with PBS cleaning twice of cell after, two holes is respectively added contain fluorescent marker PTNs and RPTNs (high molecular material is dense
Degree is 0.2mg/mL) serum deprivation culture solution 1mL, taken out after being incubated for 2h in incubator, subsequent experimental process, which should be noted that, keeps away
Light operation:
(1) culture medium is removed, PBS cleans 3 times (each 3min)；
(2) 4% paraformaldehyde 500ul is added, fixes cell 15min at room temperature；
(3) it inhales and abandons solution, PBS cleans 3 times (each 3min)；
(4) 500uL DAPI is added into each hole, contaminates core 8min；
(5) it inhales and abandons solution, PBS cleans 3 times (each 3min)；
(6) glass slide is taken, drop (about 50uL) anti-fluorescent quenching liquid is added dropwise to wave carrier piece center, takes creep plate to be buckled to tweezers
In (avoiding generation bubble as far as possible) on glass slide, with colourless nail sheet for oil seal after 10min；
(7) it is observed under laser confocal scanning microscope, using the exciting light of the excitation DAPI, 484nm of 405nm
Excite DiO, the excitation Nile red of 543nm.
As shown in fig. 6, blue-fluorescence represents the nucleus being colored, green fluorescence represents the RVs being colored, red fluorescence
Represent the PTNs being colored.After administration 2 hours, it can be observed that red and green fluorescence is both present in around nucleus, and
Both RPTNs groups position is overlapped, illustrates that PTNs and RPTNs in vitro can be by MCF-7/ADR cellular uptake and RPTNs
The integrality of its shell-core structure can be kept in the cell.
11. flow cytometer of embodiment observes the intake of Raw 264.7 and MCF-7/ADR cell to nanoparticle
When flow cytometer quantitative detection cell is to the intake of nanoparticle, for fluorescent marker PTNs solution prepare it is same
Embodiment 10, and the preparation of RPTNs is then made using the PTNs solution of RVs solution and fluorescent marker without fluorescent marker.So
After take sterilizing 12 porocyte culture plates, prepare 9 holes, be divided into PTNs group, RPTNs group and blank group, every group sets 3 multiple holes.To
It is about 2*10 that 1mL cell density is inoculated in hole5The cell liquid of a/mL, in CO2It is cultivated for 24 hours in incubator.It inhales and abandons old cell culture
Liquid, after twice of cell of PBS cleaning, experimental group is added that (macromolecular material concentration is containing fluorescent marker PTNs and RPTNs
Serum deprivation culture solution 1mL 0.2mg/mL), blank group are added equivalent serum deprivation culture solution, take out after being incubated for 2h in incubator.It moves
Except culture medium, PBS cleans 3 times (each 3min), is centrifuged after trypsin digestion cell, removes supernatant, and PBS is cleaned 3 times, finally weighed
It is suspended from 500uLPBS, with flow cytometer FL-2 Air conduct measurement cellular uptake situation.
Known by Fig. 7, after MCF-7/ADR cellular uptake PTNs and RPTNs, average fluorescent strength is respectively 1243 and 1183；
After Raw 264.7 cellular uptake PTNs and RPTNs, average fluorescent strength is respectively 302 and 204, significantly low to the intake of RPTNs
In PTNs (P < 0.01).This illustrates that erythrocyte membrane can reduce the intake of macrophage, reason in the cladding on nanoparticle surface
With Surface of Erythrocytes present on some identification Self substances albumen such as CD47, CD59 etc. it is related, they pass through combine it is huge
The signal adjusting protein on phagocyte surface to " hiding " macrophage phagocytosis.
Pharmacokinetic in 12. body of embodiment
Male rat 12 are taken, is randomly divided into 2 groups, 12h fasting, free water pass through tail vein respectively before rat is administered
Inject TET and RPTNs (dosage 250ug/kg), timing immediately after having injected.5min, 15min, 30min upon administration,
1h, 2h, 4h, 6h, 8h, 12h, for 24 hours, 36h, 48h, 72h eye socket take blood 0.5mL in the pretreated centrifuge tube of heparin sodium, shake up,
It is centrifuged (13000rpm, 10min), precision draws plasma sample 160uL, and it is 500ng/mL's that Dauricine (internal standard) concentration, which is added,
Working solution 20uL adds 820 μ L methanol extraction albumen, is vortexed and mixes 1min, and centrifugation takes supernatant to be measured with UPLC-MS.It adopts
Non- compartment model fitting is carried out to gained drug concentration-versus-time result with PKsolver pharmacokinetics software, calculates two groups
Pharmacokinetics relevant parameter.
The wherein determination condition of UPLC-MS are as follows:
Liquid-phase condition: chromatographic column Agilent Eclipse Plus-C18 RRHD (2.1mm × 50mm, 1.8 μm)；Flowing
0.1% aqueous formic acid of phase (A)-acetonitrile (B), 0~2.2min, 99%~60%A；2.2~2.3min, 60%~5%A；2.3
~3.3min, 5%A；3.3~3.4min, 5%~99%；3.4~4.5min, 99%；Flow velocity 0.4mL/min；Column temperature: 45 DEG C；
Sample volume: 2 μ L.
Mass Spectrometry Conditions: cation scanning；Detection mode: multiple-reaction monitoring (MRM)；Ion source: electric spray ion source；Collision
Gas (Collision Gas): medium；Ion source temperature (TEM): 550 DEG C；Ion spray voltage (Ion Spray
Voltage): 5000V；Gas curtain gas (Curtain Gas): 45psi；Atomization gas (GS1): 45psi；Assist gas (GS2): 45psi；
Remove cluster voltage (DP): 100v；Inject voltage (EP): 10v；Collision cell projects voltage (CXP): 15v；The collision voltage (CE) of TET
It is 54v, detects ion pair m/z 623.4~381.1, the collision voltage (CE) of Dauricine is 46v, detects ion pair 625.4
~206.1.
As shown in figure 8, free drug group is removed by the circulatory system quickly, and after administration for 24 hours, the TET blood of free drug group
Slurry concentration is 0.778ng/mL, and close to minimum quantitative limit concentration, and concentration remains to after 72h is administered in the TET blood plasma of RPTNs group
It is detected.Meanwhile pharmacokinetics relevant parameter is shown, the half-life period t of RPTNs group1/2With average residence time MRT (19.38h and
27.18h) it is longer than free drug group (6.56h and 2.73h), and two groups of the Cmax that reaches also has significant difference (P <
0.01).Therefore, after free TET being prepared into RPTNs, the slow release characteristic of drug can be significantly increased, extend drug in vivo
Circulation time.
Embodiment 13
The present invention also refers to embodiment 1-4 and is prepared for other kinds of RPTNs, and to nanoparticle partial size, PDI, encapsulation rate,
Drugloading rate and form are characterized.It is investigated by extracorporeal releasing experiment, memebrane protein activity, cell in vitro intake is tested and internal medicine
The dynamic different types of RPTNs of experiment investigation that learns is sustained long circulating action.
Type 1, the preparation of the method referring to described in embodiment 1-4, it is specific: (1) to weigh TET and PLGA respectively, be added three
Chloromethanes makes dissolution form organic phase, and organic phase is then slowly added into water under conditions of magnetic agitation (revolving speed 200rpm)
In phase (emulsifier is polyvinyl alcohol), so that the mass ratio of tetrandrine, PLGA, emulsifier is 0.5:10:100.At normal temperature
Magnetic agitation 6h volatilization removal chloroform, obtains a nanometer milk solution.(2) erythrocyte membrane two prepared according to embodiment 2 is taken
Pipe, thaw in 37 DEG C of water-baths, first by erythrocyte membrane at power 200W, frequency 60KHz ultrasound 0.5min, then use film again
Mini-extruder extrusion instrument squeezes out the polycarbonate membrane that the vesica that ultrasound obtains passes sequentially through 800,400 and 200nm, anti-under each partial size
It squeezes out 30 times again, obtains RVs.(3) the RVs solution prepared according to step (2) is taken to receive with the PTNs prepared according to step (1)
Rice solution is mixed for 1:10 with the volume ratio of PTNs solution according to RVs solution, then passes through 200nm with film mini-extruder extrusion instrument
Polycarbonate membrane squeezed out, repeatedly squeeze out 30 times, obtain RPTNs.
It is type 2, specific: (1) to weigh TET and PLGA respectively, chloroform, which is added, makes dissolution form organic phase, then exists
Organic phase is slowly added into water phase (emulsifier is polyvinyl alcohol) under conditions of magnetic agitation (revolving speed 200rpm), so that powder
Menispermine, PLGA, emulsifier mass ratio be 5:50:400.Magnetic agitation 2h volatilization removal methylene chloride, is received under room temperature
Rice milk solution.(2) it takes and is managed according to the erythrocyte membrane two that embodiment 2 prepares, thaw in 37 DEG C of water-baths, first erythrocyte membrane exists
Ultrasound 10min under power 150W, frequency 30KHz, the obtained vesica of ultrasound is then passed sequentially through 800 with film mini-extruder extrusion instrument again,
The polycarbonate membrane of 400 and 200nm is squeezed out, and is squeezed out 20 times repeatedly under each partial size, is obtained RVs.(3) it takes according to step
(2) the RVs solution prepared and the PTNs nano-solution prepared according to step (1), according to the body of RVs solution and PTNs solution
Product ratio is that 1:8 is mixed, and is then squeezed out with film mini-extruder extrusion instrument by the polycarbonate membrane of 200nm, is squeezed out 20 times repeatedly,
Obtain RPTNs.
Type 3, the preparation of the method referring to described in embodiment 1-4, it is specific: (1) to weigh TET and PLGA respectively, be added three
Chloromethanes makes dissolution form organic phase, and organic phase is then slowly added into water under conditions of magnetic agitation (revolving speed 200rpm)
In phase (emulsifier is polyvinyl alcohol), so that the mass ratio of tetrandrine, PLGA, emulsifier is 2:20:200.Magnetic at normal temperature
Power stirs 2h volatilization removal ethyl acetate, obtains a nanometer milk solution.(2) it takes and is managed according to the erythrocyte membrane two that embodiment 2 prepares,
Thaw in 37 DEG C of water-baths, first by erythrocyte membrane at power 100W, frequency 50KHz ultrasound 5min, then squeezed out again with film
Instrument squeezes out the polycarbonate membrane that the vesica that ultrasound obtains passes sequentially through 800,400 and 200nm, squeezes repeatedly under each partial size
25 times out, obtain RVs.(3) take the RVs solution prepared according to step (2) and the PTNs nanometer for preparing according to step (1) molten
Liquid is mixed for 1:5 with the volume ratio of PTNs solution according to RVs solution, then passes through the poly- carbon of 200nm with film mini-extruder extrusion instrument
Acid esters film is squeezed out, and is squeezed out 20 times repeatedly, is obtained RPTNs.
The RPTNs of type 1-3 is characterized referring to the method for embodiment 4-12, the results showed that
The encapsulation rate of RPTNs is 80.36% in type 1, and drugloading rate 5.08%, partial size 177.2nm, PDI are
0.179, current potential is -17.5mV, can keep stablizing in 15 days, and drug Cumulative release amount is 78.31% in 120h, and blood plasma partly declines
Phase is 16.97h.
RPTNs encapsulation rate is 78.60% in type 2, drugloading rate 3.78%, partial size 143.6nm, PDI 0.154,
Current potential is -16.7mV, can keep stablizing in 15 days, and drug Cumulative release amount is 82.30% in 120h, and plasma half-life is
16.52h。
RPTNs encapsulation rate is 82.38% in type 3, drugloading rate 3.96%, partial size 159.8nm, PDI 0.181,
Current potential is -18.0mV, can keep stablizing in 15 days, and drug Cumulative release amount is 74.62% in 120h, and plasma half-life is
18.82h。
The above 3 class RPTNs all has clearly shell-core double-layer structure, remains natural erythrocyte membrane protein type, can
Structural intergrity is kept when by MCF-7/ADR cellular uptake and significantly reduces the intake of macrophage.
To sum up, the present invention ties the good erythrocyte membrane of biocompatibility with high molecular degradable carrier material PLGA phase
Close, be made with slow releasing function drug-carrying nanometer particle RPTNs, and to nanoparticle partial size, PDI, encapsulation rate, drugloading rate and form into
Row characterization.Further, it is investigated by extracorporeal releasing experiment, memebrane protein activity, cell in vitro intake is tested and Internal pharmacokinetics are real
Test the sustained release long circulating action for having investigated the drug-loading system.The result shows that after free tetrandrine is prepared into RPTNs, energy
The slow release characteristic of drug is significantly increased, the circulation time of drug in vivo is extended.Nanoparticle prepared by the present invention this have clearly
Shell-core double-layer structure, partial size 164.1nm, PDI less than 0.2, current potential be -17.1mV, encapsulation rate 83.97%, drugloading rate
It is 4.03%, the basicly stable of its character and distribution can be kept in 15 days.It is natural due to being remained on RPTNs compared to PTNs
Erythrocyte membrane protein, so that macrophage Raw 264.7 reduces 32% to the phagocytosis of RPTNs.Add up release time in vitro
In 120h, the drug Cumulative release amount of RPTNs is 73.21%, and the slow release of drug, blood are also able to achieve after entering in vivo
Half-life period is 19.38h, is 2.95 times of free drug.The nanometer formulation improves the bio-compatible of entire drug delivery system
Property, realizes the long circulating and slow-release of tetrandrine in vivo, and the blood peak concentration of drug for avoiding normal injection agent excessively high draws
The toxic side effect risen, has a good application prospect.
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that under the premise of not departing from the method for the present invention, can also be made for those skilled in the art
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are equivalent embodiment of the invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical solution of the present invention
It is interior.
Claims (10)
1. a kind of tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating, include in the structure of the nanoparticle erythrocyte membrane,
PLGA and tetrandrine, the tetrandrine and the PLGA are combined and are formed-PLGA nanometers of cores of tetrandrine, the erythrocyte membrane
It is encapsulated in outside the tetrandrine-PLGA nanometers of core.
2. the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating according to claim 1, which is characterized in that described red thin
The partial size of the tetrandrine PLGA nanoparticle of after birth encapsulating is 130nm-180nm.
3. the preparation method of the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating according to claim 1, includes at least
Following steps:
(1) tetrandrine and PLGA are dissolved in organic solvent and form organic phase；Emulsifier is dissolved in water and forms water phase；
(2) organic phase in step (1) is slowly added into water phase, removes the organic solvent, form nano-emulsion；
(3) erythrocyte membrane is prepared；
(4) erythrocyte membrane prepared is squeezed out, obtains erythrocyte membrane vesica；
(5) erythrocyte membrane vesica is added in nano-emulsion, extrusion is received to get the tetrandrine PLGA that the erythrocyte membrane contains
The grain of rice.
4. the preparation method of the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating according to claim 3, feature exist
In further including one or more in following characteristics:
A. in step (1), the emulsifier is selected from one of poloxamer, polyvinyl alcohol, polyethylene glycol or a variety of；
B. in step (1), the tetrandrine, PLGA, emulsifier mass ratio are as follows: (0.5-5): (10-50): (100-400)；
C. in step (2), the volume ratio of the organic phase and water phase are as follows: 1:20-2:20；
D. in step (2), the method that removes organic solvent are as follows: be slowly added into the organic phase in step (1) laggard in water phase
Row magnetic agitation；
E. in step (3), erythrocyte membrane is prepared using hypotonic dialysis method；
F. in step (4), the extrusion condition is the polycarbonate that erythrocyte membrane is passed sequentially through to 800nm, 400nm and 200nm
Film, each partial size get off 10 times -30 times out back to squeeze；
G. in step (4) or (5), extrusion method is that ultrasound or mini-extruder extrusion instrument squeeze out；
H. in step (1), the organic solvent is selected from one of acetone, methylene chloride, chloroform or ethyl acetate or more
Kind.
5. the preparation method of the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating according to claim 4, feature exist
In in feature d, the magnetic agitation condition is revolving speed 200rpm-500rpm, time 2h-6h.
6. the preparation method of the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating according to claim 4, feature exist
In, in feature e, the hypotonic dialysis are as follows: animal takes blood, is centrifuged and removes blood plasma and leucocyte, hypotonic EDTA aqueous solution broken wall,
Up to the erythrocyte membrane after centrifugation, cleaning.
7. the preparation method of the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating according to claim 6, feature exist
In the dosage of the erythrocyte membrane and PLGA are as follows: the erythrocyte membrane that animal's whole blood described in 1mL extracts corresponds to 5mg-8mg PLGA.
8. the preparation method of the tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating according to claim 4, feature exist
In in feature g, the ultrasound condition is power 100w-200w, frequency 30-60KHz, time 0.5min-10min.
9. a kind of tetrandrine PLGA nanoparticle of erythrocyte membrane encapsulating, is made by any preparation method of claim 3-8.
10. the tetrandrine PLGA nanoparticle that the erythrocyte membrane as described in claims 1 or 2 or 9 is encapsulated is preparing anti-inflammatory and antalgic
Drug, antiviral drugs, silicosis therapeutic agent, hypertension therapeutic drug, curing myocardial ischemia drug, treating irregular heart pulse medicine
Purposes in object or tumor therapeutic agent.
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| CN110898029A (en) * | 2019-11-21 | 2020-03-24 | 东华大学 | Polydopamine coated drug-loaded PLGA material coated with erythrocyte membrane as well as preparation and application thereof |
| CN111184700A (en) * | 2019-04-10 | 2020-05-22 | 重庆大学 | Erythrocyte membrane encapsulated rapamycin nanoparticle, preparation method and application thereof |
| CN111494341A (en) * | 2020-05-26 | 2020-08-07 | 南开大学 | Nano cell membrane drug-loaded vesicle and preparation method and application thereof |
| CN113133988A (en) * | 2021-04-26 | 2021-07-20 | 西南医科大学附属医院 | Novel kidney-targeted nano drug delivery system subjected to biomimetic modification of erythrocyte membrane, preparation method and application |
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| CN113133988A (en) * | 2021-04-26 | 2021-07-20 | 西南医科大学附属医院 | Novel kidney-targeted nano drug delivery system subjected to biomimetic modification of erythrocyte membrane, preparation method and application |
| CN113133988B (en) * | 2021-04-26 | 2023-04-07 | 西南医科大学附属医院 | Erythrocyte membrane biomimetic-modified kidney-targeted nano drug-loading system, preparation method and application |
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Application publication date: 20190301 |