CN106729740B - A kind of surface combines the building of the stem cell cancer target system of drug-carrying nanometer particle - Google Patents
A kind of surface combines the building of the stem cell cancer target system of drug-carrying nanometer particle Download PDFInfo
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Abstract
The invention belongs to biological medicine technologies and nanosecond medical science technical field, and in particular to a kind of surface combines the building of the stem cell cancer target system of drug-carrying nanometer particle.Nanoparticulate carriers of the present invention using synthetic material biotin chitosan (Bio-Chi) as drug curcumin (Cur); again using biotinylated (B-) mescenchymal stem cell (MSCs) as cell carrier; the two is built bridge by Avidin and is connected, and stem cell-nanoparticle mixed carrier that surface combines drug-carrying nanometer particle is formed.The general formula of the cancer target delivery system of preparation is Cur/Bio-Chi-S-B-MSCs.Stem cell of the invention-nanoparticle mixed carrier, not only with the slow-releasing and controlled-releasing action of polymer nanoparticle, but also tumour taxis and low immunogenicity with stem cell carrier, a kind of new approach is provided as tumor-targeting drug delivery vector for stem cell.
Description
Technical field
The invention belongs to biological medicine technologies and nanosecond medical science technical field, and in particular to a kind of surface combines medicament-carried nano
The building of the stem cell cancer target system of grain.
Background technique
Although experienced development in 30 years, traditional anticancer drug still has non-specific target tumor position, dosage
The shortcomings that dependence toxic side effect.Slow controlled-release technology and targeted delivery technology, which are combined, helps to capture above-mentioned problem.Nanometer
The appearance of technology provides possibility for effective delivering of anti-tumor drug.The polymer nanoparticle of entrapped drug can reach long-term slow
Release or environmental response type release effect, maintain drug concentration in therapeutic domain for a long time, reduce toxic side effect.Medicine is carried to receive
The grain of rice also can be by EPR effect passive target tumor locus, or passes through surface linking ligand active targeting.But such target
Still have that body-internal-circulation is unstable easily quickly to be eliminated by reticuloendothelial system (RES), be difficult to across physiology screen to delivering mode
Hinder and can not effectively accumulate the problems such as reaching treatment concentration in tumor locus.
Erythrocyte surface is adsorbed in by pathogen and effectively escapes the inspiration that RES elimination acts on, and cell carrier starts to be answered
For anti-tumor drug targeted delivery.Mescenchymal stem cell (MSCs) because have essence tumour taxis and transfer ability,
Low immunogenicity and endogenous mutation rate, the cell for being easily isolated and becoming most application value the advantages that in vitro culture carry
Body.For focusing primarily upon delivering cell factor using MSCs as the research of carrier, (the relevant induction of tumor necrosis factor is withered at present
Die ligand/TRAIL, interleukins IL-12), enzyme and prodrug (cytosine deaminase CD and 5-flurocytosine 5-FC), oncolytic disease
Poison, for treating glioma.
Also there is research to attempt using MSCs as cell carrier in recent years, passes through cell endocytic or film surface knot with drug-carrying nanometer particle
The mode of conjunction constructs stem cell-nanoparticle mixed carrier with the delivery system of high-efficiency delivery anti-tumor drug.CN104771764A
Disclose a kind of stem cell cancer target system stemcells- (RGD-PPCD) for containing nano-prodrugn, with cell endocytic
Nano-prodrug is loaded on and prepares antineoplastic target system in stem cell by mode, combines targeting, sustained release and the acid of nano-prodrug
The characteristic of the long-range target tumor primary tumor and transfer stove of quick release characteristics and stem cell carrier.But in MSCs film surface knot
Closing drug-carrying nanometer particle building, stem cell-nanoparticle mixed carrier field is also rare is related to.The structure in such a way that film surface combines
The system built, anti-tumor drug will not directly act on stem cell carrier, can be reduced the influence to its activity, tumour taxis；
Nanoparticle drug release will not be influenced by stem cell carrier P glycoprotein outlet, film Passive diffusion, it is easier to which regulation release is bent
Line is effectively enriched with the purpose of simultaneously sustained release to realize drug in tumor locus.
Summary of the invention
The object of the present invention is to provide a kind of technologies of stem cell surface combination drug-carrying nanometer particle, control applied to cancer target
It treats.
Specifically, being the nanoparticulate carriers using synthetic material Bio-Chi as anti-tumor drug Cur, then with biotin
The MSCs of change receives grain of rice surface biological element and MSCs surface biological element, preparation as cell carrier, by Avidin bridge company
Stem cell-nanoparticle mixed carrier is delivered for cancer target, has following general formula: Cur/Bio-Chi-S-B-MSCs.
The MSCs is source of mouse fat mesenchymal stem cell.
The Bio-Chi is to be formed amido bond by the carboxyl of biotin and the amino of chitosan through condensation reaction and be made,
Structure are as follows:
Wherein the molecular weight of chitosan of Bio-Chi is 5-200k, and biotin degree of substitution is 1.91-45.82%.Preferred point
Son amount is 200k, degree of substitution 45.82%.
Synthetic method is as follows: chitosan being dissolved in 1% hydrochloric acid solution and is swollen overnight, with the 2- morpholine second sulphur of pH=5.5
Sour sodium buffer is diluted to required concentration.By biotin, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, N-
Hydroxysuccinimide is dissolved in the in the mixed solvent of DMSO and DMF, and 2h is stirred at room temperature.Stirring is added dropwise in the biotin of activation
In chitosan solution in, lasting stirring for 24 hours, is filtered to remove insoluble matter.It is placed in bag filter with deionized water dialysis 2 days, it is cold
It is lyophilized dry.
Drug-carrying nanometer particle Cur/Bio-Chi's the preparation method is as follows: by the ethanol solution containing 1mgCur, with volume
Than 1/4-3/4, it is added dropwise in 1% acetum containing 10mgBio-Chi that concentration is 2-10mg/mL.It is placed in 50 DEG C of water-baths
Persistently stir 30min.30min is handled with 50W Probe Ultrasonic Searching, dialyse 6h, crosses 0.8 μm of hydrophilic microporous filter membrane, is diluted to PBS
Required concentration.
In above-mentioned preparation process, the concentration that the volume ratio of ethanol solution and 1% acetum is 1/4, Bio-Chi is
10mg/mL.Cur/Bio-Chi obtained is placed in bag filter (MWCO=14,000), both ends tighten, immersion pH=7.4,
6.5, in 5.5 dissolution medium.Predetermined point of time collect dissolution medium, be added equivalent ethyl alcohol be acutely vortexed, according to λ=
Absorbance value at 431nm calculates burst size, draws release profiles.
The stem cell-nanoparticle mixed carrier, by Avidin build bridge connection Cur/Bio-Chi surface biological element and
B-MSCs surface biological element.Drug, polymer material, drug-carrying nanometer particle are detected first to the cytotoxicity of MSCs.2-200 will be contained
Cur solution, the Cur/Bio-Chi solution of μ g/mLCur, the Bio-Chi solution containing 0.05-5mg/mL are incubated for jointly with MSCs
For 24 hours, cells survival rate is detected with mtt assay.
The preparation method is as follows: by the PBS of MSCs and 0.5mL the concentration sulfo-NHS-LC-Biotin for being 0.1-1mg/mL
Solution is incubated for 20min.Meanwhile by 0.25mL concentration be 100-200 μ g/mL RBITC- Avidin PBS solution and 0.25mL
The PBS solution of the Cur/Bio-Chi of the g/mLCur of μ containing 25-100, which is protected from light, is incubated for 20min.By 0.5mLRBITC- Avidin and Cur/
The mixed solution and B-MSCs of Bio-Chi is incubated for 20min.
In above-mentioned preparation process, the concentration of sulfo-NHS-LC-Biotin is 0.5mg/mL, RBITC- Avidin it is dense
The concentration that degree is 150 μ g/mL, Cur/Bio-Chi is 100 μ g/mLCur.Cur/Bio- before being incubated for MSCs, after being incubated for
After Chi solution suitably dilutes, equivalent ethyl alcohol is added and is acutely vortexed, cell is quantitatively calculated according to the absorbance difference at λ=431nm
Drugloading rate.Cur/Bio-Chi-S-B-MSCs after the completion of incubation is observed with inverted fluorescence microscope, and is digested with pancreatin,
PBS is resuspended, and is detected with fluorescence of the flow cytometer to RBITC, Cur, qualitative detection MSCs adds Cur/Bio-Chi
It carries.
The surface that the present invention constructs combines the stem cell cancer target system of drug-carrying nanometer particle, will have the advantage that
1, it by anti-tumor drug specific delivery to tumor locus, and is enriched in tumor locus.
2, anti-tumor drug can continue slow release in tumor locus.
3, anti-tumor drug will not directly act on stem cell carrier, reduce the influence to its activity, tumour taxis.
4, the release of anti-tumor drug is only related with the interaction of polymer support, and release profiles are easily controllable.
Detailed description of the invention
The infared spectrum of Fig. 1 Chi and Bio-Chi；
Release profiles of Fig. 2 Bio-Chi in the medium of different pH；
Cytotoxicity of Fig. 3 Bio-Chi to MSCs；
The cytotoxicity of Fig. 4 Cur, Cur/Bio-Chi to MSCs；
The biotinylation of Fig. 5 MSCs is examined；
Fig. 6 Avidin concentration combines the influence of drug-carrying nanometer particle to the surface MSCs；
Fig. 7 Cur/Bio-Chi concentration combines the influence of drug-carrying nanometer particle to the surface MSCs.
Specific embodiment
Specific embodiment is to further explanation of the invention below, but following present invention that are merely to illustrate are not for limit
Determine the scope of the present invention.
The synthesis and characterization of embodiment 1:Bio-Chi
90mg chitosan is dissolved in 1% hydrochloric acid solution and is swollen overnight, with the 2-morpholine ethane sulfonic acid sodium buffer of pH=5.5
It is diluted to 20mL.120mg biotin, 84mgEDC, 56mgNHS are dissolved in the in the mixed solvent of 6mLDMSO and DMF, are stirred at room temperature
2h.The biotin of activation is added dropwise in the chitosan solution in stirring, lasting stirring for 24 hours, is filtered to remove insoluble matter.It is placed in
Deionized water dialysis 2 days in bag filter, freeze-drying.
Infrared spectrum analysis is carried out to the Bio-Chi of synthesis, as the result is shown in 1635.6cm-1There is the flexible of amido bond in place
There is the stretching vibration peak of the amido bond of biotin imidazolone ring at 1531.9cm-1 in vibration peak, shows that biotin and shell are poly-
Sugar has been chemically bonded (attached drawing 1).
The preparation and representation of embodiment 2:Cur/Bio-Chi
10mgBio-Chi is weighed, is dissolved in 1mL1% acetum, swelling is overnight.It is stirred at room temperature down, by 0.25mL4mg/
The Cur ethanol solution of mL is added dropwise in Bio-Chi solution.30min, the processing of 50W Probe Ultrasonic Searching are persistently stirred in 50 DEG C of water-baths
30min, dialysis 6h remove organic solvent, cross 0.8 μm of hydrophilic microporous filter membrane, be diluted to required concentration with PBS.
Partial size with laser particle size analyzer measurement Cur/Bio-Chi is 360.2 ± 2.6nm, and current potential is 9.6 ± 1.9mV.
Proper amount of nano grain solution is taken, equivalent ethyl alcohol is added and is acutely vortexed, the absorbance value at λ=431nm is measured, according to mark
Directrix curve calculates the Cur content in nanoparticle solution, and computational envelope rate according to the following formula: encapsulation rate %=WCur nanoparticle/WCur feeds intake×
100%.The encapsulation rate that Cur/Bio-Chi is calculated is 54.7 ± 2.4%.
The release profiles of embodiment 3:Cur/Bio-Chi
0.4mLCur/Bio-Chi is taken, is diluted to 2mL with 10mMPBS7.4, is placed in bag filter (MWCO=14,000),
Both ends tighten, and immerse in 10mMPBS (pH=7.4,6.5,5.5) of the 5mL containing 0.1% Tween 80.At 37 DEG C of constant temperature, revolving speed is
It is discharged under the conditions of 100rpm.In predetermined point of time 2,4,8,12,24,36,48,60,72,96,120,144,168h, collect
Dissolution medium simultaneously replaces 5mL fresh medium.1mL ethyl alcohol is added in the dissolution medium for taking 1mL to collect, and be acutely vortexed 5min, ultraviolet-
Visible spectrophotometer measures the absorbance value at λ=431nm, calculates burst size, draws release profiles.
The result shows that Cur/Bio-Chi is able to maintain lasting slow release Cur up to 7 days or more in pH=7.4 environment；
In acidic environment, there is a burst release in Cur/Bio-Chi, then sustained release again, release more completely, rate of release faster
(attached drawing 2).
Embodiment 4: the cytotoxicity of drug and carrier material
By the MSCs of logarithmic growth phase by 1 × 104, every hole cell inoculation in 96 orifice plates, 100 μ LDMEM high sugar trainings completely
Base/hole is supported, culture for 24 hours, makes cell confluency degree 70-80%.Culture medium is sucked, 200 μ L μ containing 2-200 g/mLCur is added in every hole
Cur solution, Cur/Bio-Chi solution, the Bio-Chi solution containing 0.05-5mg/mL.After for 24 hours, culture medium is sucked, PBS is clear
It washes, the sugared complete medium of DMEM high of 100 μ L solution containing 10%MTT (5mg/mL, pH=7.4PBS), 37 DEG C of continuation are added in every hole
It is incubated for 4h.Culture medium is gently absorbed, 150 μ LDMSO, shaken at room temperature 10min is added in every hole, with enzyme-linked immunosorbent assay instrument in 570nm
Measure absorbance.Cell to handle without cationic polymer calculates cells survival rate, cells survival rate (%) as control
=OD570 (experimental group)/OD570 (control group) × 100.
The result shows that the cytotoxicity of Bio-Chi is small, cell compatibility is good (attached drawing 3).Cur has MSCs certain thin
Cellular toxicity, but after Bio-Chi is encapsulated, cytotoxicity is substantially reduced, and is incubated for for 24 hours under the high concentration of 100 μ g/mLCur, carefully
Born of the same parents' survival rate remains to reach 80% or more, so being incubated for 20min and loading the cell toxicant that the Cur/Bio-Chi of low concentration is generated
Property negligible (attached drawing 4).
The cell surface biotinylation of embodiment 5:MSCs and inspection
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.The sulfo- for being 0.1-1mg/mL by MSCs and 0.5mL concentration
The PBS solution of NHS-LC-Biotin is incubated at room temperature 20min, using 0.5mLPBS solution as blank control.Medium is absorbed, is protected from light and adds
The PBS solution for entering the RBITC- Avidin that 0.5mL concentration is 50 μ g/mL, is incubated at room temperature 20min.Medium is absorbed, after pancreatin digestion
It is resuspended with PBS, is detected with fluorescence intensity of the flow cytometer to RBITC.
The result shows that hardly combining RBITC- Avidin without biotinylated MSCs (PBS group).Be 0.1 through concentration,
0.5, after the sulfo-NHS-LC-Biotin biotinylation of 1mg/mL, the RBITC fluorescence that B-MSCs is combined is gradually increased.When reaching
After to a certain concentration (>=0.5mg/mL), fluorescence labeled cell number and fluorescence intensity all tend towards stability (attached drawing 5).
The best Avidin concentration of embodiment 6:Cur/Bio-Chi-S-B-MSCs is investigated
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.The sulfo- for being 0.5mg/mL by MSCs and 0.5mL concentration
The PBS solution of NHS-LC-Biotin is incubated at room temperature 20min.Meanwhile the Avidin for by 0.25mL concentration being 100-200 μ g/mL
The PBS solution of the Cur/Bio-Chi of PBS solution and 0.25mL containing 50 μ g/mLCur, which is protected from light, is incubated for 20min.By 0.5mL Avidin
20min is incubated for the mixed solution and B-MSCs of Cur/Bio-Chi.Medium is absorbed, is resuspended after pancreatin digestion with PBS, uses streaming
Cell instrument detects the fluorescence intensity of Cur.
The result shows that increasing with Avidin concentration, cell surface drugloading rate can be made to increase in conjunction with more Cur/Bio-Chi
Add.But (150 μ g/mL of >) cell surface drugloading rate can reach saturation (attached drawing 6) after reaching a certain concentration.
The best Cur/Bio-Chi concentration of embodiment 7:Cur/Bio-Chi-S-B-MSCs is investigated
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.The sulfo- for being 0.5mg/mL by MSCs and 0.5mL concentration
The PBS solution of NHS-LC-Biotin is incubated at room temperature 20min.Meanwhile by 0.25mL concentration be 150 μ g/mL Avidin PBS
The PBS solution of the Cur/Bio-Chi of solution and 0.25mL μ containing 25-100 g/mLCur, which are protected from light, is incubated for 20min.By 0.5mL Avidin
20min is incubated for the mixed solution and B-MSCs of Cur/Bio-Chi.Medium is absorbed, is resuspended after pancreatin digestion with PBS, uses streaming
Cell instrument detects the fluorescence intensity of Cur.
The result shows that the concentration with Cur/Bio-Chi increases, cell surface drugloading rate is obviously increased.But consider cell
The concentration of the influence of toxicity, Cur/Bio-Chi should not be too large, so determining that concentration is 100 μ g/mL (attached drawing 7).
The drugloading rate of embodiment 8:Cur/Bio-Chi-S-B-MSCs detects
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.The sulfo- for being 0.5mg/mL by MSCs and 0.5mL concentration
The PBS solution of NHS-LC-Biotin is incubated at room temperature 20min.Meanwhile by 0.25mL concentration be 150 μ g/mL Avidin PBS
The PBS solution of the Cur/Bio-Chi of solution and 0.25mL μ containing 25-100 g/mLCur, which are protected from light, is incubated for 20min.By 0.5mL Avidin
20min is incubated for the mixed solution and B-MSCs of Cur/Bio-Chi.It is molten by forward and backward Cur/Bio-Chi is incubated for B-MSCs
Liquid is mixed with equivalent ethyl alcohol, vortex mixed respectively, measures the absorbance value difference at λ=431nm.It calculates cell surface and carries medicine
Amount.
The result shows that the surface MSCs drugloading rate is 54.26 ± 4.18pgCur/ cell.
Embodiment 1-8 the result shows that, stem cell-nanoparticle mixed carrier can pass through Avidin build bridge connection Cur/Bio-
The mode of Chi surface biological element and B-MSCs surface biological element constructs.A kind of surface that the present invention constructs combines drug-carrying nanometer particle
Stem cell cancer target system, the influence to stem cell Carriers Active can be reduced, be enriched in tumor locus and continue slowly to release
Drug is put to reach curative effect, provides a kind of new approach as tumor-targeting drug delivery vector for stem cell.
The above is only a specific embodiment of the invention, it is noted that is come for those of ordinary skill in the art
It says, without departing from the principle of the present invention, several improvement can also be made, these improvement also should be regarded as protection of the invention
Range.
Claims (5)
1. the stem cell cancer target system that a kind of surface combines drug-carrying nanometer particle, which is characterized in that using Bio-Chi as Cur
Nanoparticulate carriers, then using B-MSCs as cell carrier, the two is built bridge by Avidin and is connected, and has following general formula: Cur/
Bio-Chi-S-B-MSCs；The B-MSCs is biotinylated source of mouse fat mesenchymal stem cell；
The structure of the Bio-Chi are as follows:
The molecular weight of chitosan of the Bio-Chi is 5-200k, and biotin degree of substitution is 1.91-45.82%.
2. a kind of surface according to claim 1 combines the stem cell cancer target system of drug-carrying nanometer particle, feature exists
In, drug-carrying nanometer particle Cur/Bio-Chi's the preparation method is as follows: by the ethanol solution of the Cur containing 1mg, with volume ratio 1/
, it is added dropwise in 1% acetum for the Bio-Chi containing 10mg that concentration is 2-10mg/mL；
It is placed in 50 DEG C of water-baths and persistently stirs 30min, handle 30min with 50W Probe Ultrasonic Searching, dialyse 6h, and it is micro- to cross 0.8 μm of hydrophily
Hole filter membrane is diluted to required concentration with PBS.
3. a kind of surface according to claim 2 combines the stem cell cancer target system of drug-carrying nanometer particle, feature exists
In the concentration that the volume ratio of the ethanol solution and 1% acetum is 1/4, Bio-Chi is 10mg/mL.
4. a kind of surface described in claim 1 combines the preparation method of the stem cell cancer target system of drug-carrying nanometer particle,
It is characterized in that, is built bridge and connected by Avidin between the drug-carrying nanometer particle and MSCs, the preparation method is as follows:
The PBS solution of MSCs and 0.5mL the concentration biotinyl-N- hydroxy-succinimide for being 0.1-1mg/mL is incubated for
20min；
Meanwhile the PBS solution of the RBITC that 0.25mL concentration the is 100-200 μ g/mL Avidin marked and 0.25mL are contained into 25-
The PBS solution of the Cur/Bio-Chi of 100 μ g/mL Cur, which is protected from light, is incubated for 20min；
The mixed solution of 0.5mL RBITC- Avidin and Cur/Bio-Chi and B-MSCs are incubated for 20min.
5. the preparation side that a kind of surface according to claim 4 combines the stem cell cancer target system of drug-carrying nanometer particle
Method, which is characterized in that the concentration of biotinyl-N- hydroxy-succinimide is 0.5mg/mL, the Avidin of RBITC label
Concentration is that the concentration of 150 μ g/mL, Cur/Bio-Chi is 100 μ g/mL Cur.
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| CN114848844B (en) * | 2022-04-27 | 2023-06-20 | 中国药科大学 | Nano-engineering stem cell anti-tumor targeted drug delivery system and preparation method and application thereof |
| CN115998711A (en) * | 2022-12-26 | 2023-04-25 | 中国药科大学 | Targeted nano drug delivery system for reversing tumor drug resistance, and preparation method and application thereof |
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| CN105457037A (en) * | 2014-08-18 | 2016-04-06 | 复旦大学 | Stem cell tumor targeting system with internal nano-prodrug and preparation method thereof |
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| CN105457037A (en) * | 2014-08-18 | 2016-04-06 | 复旦大学 | Stem cell tumor targeting system with internal nano-prodrug and preparation method thereof |
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