CN106729740A - A kind of surface combines the structure of the stem cell cancer target system of drug-carrying nanometer particle - Google Patents
A kind of surface combines the structure 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 technology and nanosecond medical science technical field, and in particular to a kind of surface combines the structure of the stem cell cancer target system of drug-carrying nanometer particle.The present invention is using synthetic material biotin chitosan (Bio Chi) as the nanoparticulate carriers of medicine curcumin (Cur); again using biotinylated (B) mescenchymal stem cell (MSCs) as cell carrier; both are built bridge by Avidin and connected, and form the stem cell nanoparticle mixed carrier that surface combines drug-carrying nanometer particle.The formula of the cancer target delivery system of preparation is Cur/Bio Chi S B MSCs.Stem cell nanoparticle mixed carrier of the invention, the not only slow-releasing and controlled-releasing action with polymer nanoparticle, and tumour taxis and low immunogenicity with stem cell carrier, for stem cell provides a kind of new approach as tumor-targeting drug delivery vector.
Description
Technical field
The invention belongs to biological medicine technology and nanosecond medical science technical field, and in particular to a kind of surface combines medicament-carried nano
The structure of the stem cell cancer target system of grain.
Background technology
Although experienced the development of 30 years, traditional cancer therapy drug is still present non-specific target tumor position, dosage
The shortcoming of dependence toxic and side effect.Slow controlled-release technology and targeted delivery technology are combined to be helped to capture above-mentioned problem.Nanometer
The effective delivering for appearing as antineoplastic of technology is provided may.The polymer nanoparticle of entrapped drug can reach long-term slow
Release or environmental response type release effect, drug concentration is maintained in therapeutic domain for a long time, reduce toxic and side effect.Medicine is carried to receive
The grain of rice also can be by EPR effect passive target tumor locus, or by surface linking ligand active targeting.But such target
It is still present 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 cannot effectively accumulate the problems such as reaching treatment concentration in tumor locus.
Erythrocyte surface being adsorbed in by pathogen and effectively escaping RES elimination effects and inspired, cell carrier starts to be answered
For antineoplastic 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 turning into the advantages of in vitro culture most application value are carried
Body.(the related induction of TNF is withered to focus primarily upon delivering cell factor for the research with MSCs as carrier 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 with MSCs as cell carrier in recent years, tied by cell endocytic or film surface with drug-carrying nanometer particle
The mode of conjunction, builds stem cell-nanoparticle mixed carrier with the delivery system of high-efficiency delivery antineoplastic.CN 104771764
A discloses a kind of stem cell cancer target system stem cells- (RGD-PPCD) for containing nano-prodrugn, with cell endocytic
Mode nano-prodrug is loaded on prepared in stem cell antineoplastic target system, combine the targeting of nano-prodrug, sustained release with
The long-range target tumor primary tumor and the characteristic of MET of acid-sensitive release characteristics and stem cell carrier.But, on MSCs films surface
The field for building stem cell-nanoparticle mixed carrier with reference to drug-carrying nanometer particle also rare is related to.By way of film surface combines
The system of structure, antineoplastic will not directly act on stem cell carrier, can reduce the shadow to its activity, tumour taxis
Ring；Nanoparticle drug release will not also be influenceed by row, film Passive diffusion outside stem cell carrier P glycoprotein, it is easier to regulation and control release
Curve, to realize that medicine is effectively enriched with the purpose of simultaneously sustained release in tumor locus.
The content of the invention
It is an object of the invention to provide a kind of technology of stem cell surface combination drug-carrying nanometer particle, it is applied to cancer target and controls
Treat.
Specifically, it is nanoparticulate carriers using synthetic material Bio-Chi as antineoplastic Cur, then with biotin
The MSCs of change receives grain of rice surface biological element and MSCs surface biologicals element as cell carrier by Avidin bridge company, prepares
Stem cell-nanoparticle mixed carrier is delivered for cancer target, with below general formula：Cur/Bio-Chi-S-B-MSCs.
Described MSCs is mouse source fat mesenchymal stem cell.
Described Bio-Chi is to form amido link through condensation reaction with the amino of shitosan by the carboxyl of biotin to be obtained,
Structure is：
The chitosan molecule amount of wherein Bio-Chi is 5-200k, and biotin substitution value is 1.91-45.82%.Preferred point
Son amount is 200k, and substitution value is 45.82%.
Synthetic method is as follows：By shitosan be dissolved in 1% hydrochloric acid solution it is swelling overnight, with the 2- morpholine second sulphurs of pH=5.5
Sour sodium buffer solution is diluted to required concentration.By biotin, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, N-
Hydroxysuccinimide is dissolved in the mixed solvent of DMSO and DMF, and 2h is stirred at room temperature.The biotin of activation is added dropwise over stirring
In chitosan solution in, persistently stir 24h, be filtered to remove insoluble matter.It is placed in bag filter and is dialysed 2 days with deionized water, it is cold
It is lyophilized dry.
The preparation method of described drug-carrying nanometer particle Cur/Bio-Chi is as follows：By the ethanol solution containing 1mg Cur, with body
Product is added dropwise in 1% acetum containing 10mg Bio-Chi that concentration is 2-10mg/mL than 1/4-3/4.It is placed in 50 DEG C of water-baths
In persistently stir 30min.30min is processed with 50W Probe Ultrasonic Searchings, dialyse 6h, crosses 0.8 μm of hydrophilic microporous filter membrane, is diluted with PBS
To required concentration.
In above-mentioned preparation process, the volume ratio of ethanol solution and 1% acetum is for the concentration of 1/4, Bio-Chi
10mg/mL。
Obtained Cur/Bio-Chi is placed in bag filter (MWCO=14,000), two ends tighten, immersion pH=7.4,
6.5th, in 5.5 dissolution medium.Predetermined point of time collect dissolution medium, add equivalent ethanol be acutely vortexed, according to λ=
Absorbance at 431nm calculates burst size, draws release profiles.
Described stem cell-nanoparticle mixed carrier, by Avidin build bridge connection Cur/Bio-Chi surface biologicals element and
B-MSCs surface biologicals element.The cytotoxicity of medicine, polymeric material, drug-carrying nanometer particle to MSCs is detected first.2-200 will be contained
The Cur solution of μ g/mL Cur, Cur/Bio-Chi solution, the Bio-Chi solution containing 0.05-5mg/mL are incubated jointly with MSCs
24h, cells survival rate is detected with mtt assay.
Preparation method is as follows：It is the PBS of the sulfo-NHS-LC-Biotin of 0.1-1mg/mL by MSCs and 0.5mL concentration
Solution is incubated 20min.Meanwhile, it is the PBS solution and 0.25mL of the RBITC- Avidins of 100-200 μ g/mL by 0.25mL concentration
The PBS solution lucifuge of the Cur/Bio-Chi of the g/mL of μ containing 25-100 Cur is incubated 20min.By 0.5mL RBITC- Avidins and
The mixed solution of Cur/Bio-Chi is incubated 20min with B-MSCs.
In above-mentioned preparation process, the concentration of sulfo-NHS-LC-Biotin is 0.5mg/mL, RBITC- Avidins it is dense
Spend for the concentration of 150 μ g/mL, Cur/Bio-Chi is 100 μ g/mL Cur.
After Cur/Bio-Chi solution suitably dilution before being incubated with MSCs, after incubation, the violent whirlpool of equivalent ethanol is added
Rotation, cell drugloading rate is quantitatively calculated according to the absorbance difference at λ=431nm.By the Cur/Bio-Chi-S- after the completion of incubation
B-MSCs is observed with inverted fluorescence microscope, and is digested with pancreatin, and PBS is resuspended, with flow cytometer to the fluorescence of RBITC, Cur
Detected, loadings of the qualitative detection MSCs to Cur/Bio-Chi.
The surface that the present invention builds combines the stem cell cancer target system of drug-carrying nanometer particle, will be with following advantage：
1st, by antineoplastic specific delivery to tumor locus, and in tumor locus enrichment.
2nd, antineoplastic can continue slow release in tumor locus.
3rd, antineoplastic will not directly act on stem cell carrier, reduce the influence to its activity, tumour taxis.
4th, the release of antineoplastic is only relevant with the interaction of polymer support, and release profiles are easily controllable.
Brief description of the drawings
The infared spectrum of Fig. 1 Chi and Bio-Chi
Release profiles of Fig. 2 Bio-Chi in the medium of different pH
Cytotoxicities of Fig. 3 Bio-Chi to MSCs
The cytotoxicity of Fig. 4 Cur, Cur/Bio-Chi to MSCs
The biotinylation inspection of Fig. 5 MSCs
Fig. 6 Avidins concentration combines the influence of drug-carrying nanometer particle to MSCs surfaces
Fig. 7 Cur/Bio-Chi concentration combines the influence of drug-carrying nanometer particle to MSCs surfaces
Specific embodiment
Specific embodiment is further illustrated to of the invention below, but following present invention that are merely to illustrate are not for limit
Determine the scope of the present invention.
Embodiment 1：The synthesis of Bio-Chi and sign
By 90mg shitosans be dissolved in 1% hydrochloric acid solution it is swelling overnight, with the MES sodium buffer solution of pH=5.5
It is diluted to 20mL.120mg biotins, 84mg EDC, 56mg NHS are dissolved in the mixed solvent of 6mL DMSO and DMF, room temperature
Stirring 2h.The biotin of activation is added dropwise in the chitosan solution in stirring, 24h is persistently stirred, insoluble matter is filtered to remove.
It is placed in deionized water in bag filter to dialyse 2 days, freeze-drying.
Bio-Chi to synthesizing carries out infrared spectrum analysis, is as a result displayed in 1635.6cm-1There is the flexible of amido link in place
Vibration peak, the stretching vibration peak for occurring the amido link of biotin imidazolone ring at 1531.9cm-1 shows that biotin gathers with shell
Sugar has been chemically bonded (accompanying drawing 1).
Embodiment 2：The preparation of Cur/Bio-Chi and sign
Weigh 10mg Bio-Chi, be dissolved in the acetums of 1mL 1%, it is swelling overnight.It is stirred at room temperature down, by 0.25mL
The Cur ethanol solutions of 4mg/mL are added dropwise in Bio-Chi solution.30min is persistently stirred in 50 DEG C of water-baths, at 50W Probe Ultrasonic Searchings
Reason 30min, dialysis 6h removes organic solvent, crosses 0.8 μm of hydrophilic microporous filter membrane, and required concentration is diluted to PBS.
The particle diameter for determining Cur/Bio-Chi with laser particle size analyzer is 360.2 ± 2.6nm, and current potential is 9.6 ± 1.9mV.
Proper amount of nano grain solution is taken, adds equivalent ethanol to be acutely vortexed, the absorbance at λ=431nm is determined, according to standard curve meter
Calculate the Cur contents in nanoparticle solution, and computational envelope rate according to the following formula：Envelop rate %=WCur nanoparticles/WCur feeds intake× 100%.Meter
The envelop rate that calculation obtains Cur/Bio-Chi is 54.7 ± 2.4%.
Embodiment 3：The release profiles of Cur/Bio-Chi
0.4mL Cur/Bio-Chi are taken, 2mL is diluted to 10mM PBS 7.4, be placed in bag filter (MWCO=14,000)
In, two ends tighten, in 10mM PBS (pH=7.4,6.5,5.5) of the immersion 5mL containing 0.1% Tween 80.In 37 DEG C of constant temperature, rotating speed
To be discharged under the conditions of 100rpm.In predetermined point of time 2,4,8,12,24,36,48,60,72,96,120,144,168h, receive
Collection dissolution medium simultaneously changes 5mL fresh mediums.The dissolution medium of 1mL collections is taken, 1mL ethanol is added, be acutely vortexed 5min, it is purple
Outward-visible spectrophotometer determines the absorbance at λ=431nm, calculates burst size, draws release profiles.
Result shows, in pH=7.4 environment, Cur/Bio-Chi can keep continuing slowly to discharge Cur up to more than 7 days；
In sour environment, Cur/Bio-Chi occur in that one it is prominent release, then sustained release again, release more completely, rate of release faster
(accompanying drawing 2).
Embodiment 4：The cytotoxicity of medicine and carrier material
The MSCs of exponential phase is pressed and is inoculated in 96 orifice plates per 1 × 104, hole cell, 100 μ L DMEM sugar high are trained completely
Base/hole is supported, 24h is cultivated, makes cell confluency degree be 70-80%.Culture medium is sucked, 200 μ L μ containing 2-200 g/mL are added per hole
The Cur solution of Cur, Cur/Bio-Chi solution, the Bio-Chi solution containing 0.05-5mg/mL.After 24h, culture medium, PBS are sucked
Cleaning, per hole add 100 μ L solution containing 10%MTT (5mg/mL, pH=7.4PBS) DMEM sugared complete mediums high, 37 DEG C after
It is continuous to be incubated 4h.Gently absorb culture medium, per hole add 150 μ L DMSO, shaken at room temperature 10min, with enzyme-linked immunoassay instrument in
570nm mensuration absorbances.Using the cell that is processed without cationic polymer as control, cells survival rate, cells survival are calculated
Rate (%)=OD570 (experimental group)/OD570 (control group) × 100.
Result shows that the cytotoxicity of Bio-Chi is small, and cell compatibility is good (accompanying drawing 3).Cur has certain thin to MSCs
Cellular toxicity, but after being encapsulated through Bio-Chi, cytotoxicity is substantially reduced, and 24h is incubated under the high concentration of 100 μ g/mL Cur,
Cells survival rate remains to reach more than 80%, so being incubated the cell of the Cur/Bio-Chi generations of 20min and load low concentration
Toxicity is negligible (accompanying drawing 4).
Embodiment 5：The cell surface biotinylation of MSCs and inspection
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.It is the sulfo- of 0.1-1mg/mL by MSCs and 0.5mL concentration
The PBS solution incubation at room temperature 20min of NHS-LC-Biotin, with 0.5mL PBS solutions as blank.Medium is absorbed, lucifuge adds
Enter the PBS solution of the RBITC- Avidins that 0.5mL concentration is 50 μ g/mL, be incubated at room temperature 20min.Medium is absorbed, after pancreatin digestion
It is resuspended with PBS, the fluorescence intensity of RBITC is detected with flow cytometer.
Result shows, without biotinylated MSCs (PBS groups) hardly with reference to RBITC- Avidins.Be 0.1 through concentration,
0.5th, after the sulfo-NHS-LC-Biotin biotinylations of 1mg/mL, the RBITC fluorescence that B-MSCs is combined gradually strengthens.When reaching
After to finite concentration (>=0.5mg/mL), fluorescence labeled cell number and fluorescence intensity all tend towards stability (accompanying drawing 5).
Embodiment 6：The optimal Avidin concentration of Cur/Bio-Chi-S-B-MSCs is investigated
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.It is the sulfo- of 0.5mg/mL by MSCs and 0.5mL concentration
The PBS solution incubation at room temperature 20min of NHS-LC-Biotin.Meanwhile, it is the Avidin of 100-200 μ g/mL by 0.25mL concentration
PBS solution is incubated 20min with the PBS solution lucifuge of Cur/Bio-Chi of the 0.25mL containing 50 μ g/mL Cur.By 0.5mL Avidins
Mixed solution with Cur/Bio-Chi is incubated 20min with B-MSCs.Medium is absorbed, it is resuspended with PBS after pancreatin digestion, use streaming
Cell instrument is detected to the fluorescence intensity of Cur.
Result shows, with the increase of Avidin concentration, can increase cell surface drugloading rate with reference to more Cur/Bio-Chi
Plus.But (the μ g/mL of ＞ 150) cell surface drugloading rate can reach saturation (accompanying drawing 6) after reaching finite concentration.
Embodiment 7：The optimal Cur/Bio-Chi concentration of Cur/Bio-Chi-S-B-MSCs is investigated
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.It is the sulfo- of 0.5mg/mL by MSCs and 0.5mL concentration
The PBS solution incubation at room temperature 20min of NHS-LC-Biotin.Meanwhile, by the PBS of Avidin that 0.25mL concentration is 150 μ g/mL
Solution is incubated 20min with the PBS solution lucifuge of the Cur/Bio-Chi of 0.25mL μ containing 25-100 g/mL Cur.0.5mL is affine
The mixed solution of element and Cur/Bio-Chi is incubated 20min with B-MSCs.Medium is absorbed, it is resuspended with PBS after pancreatin digestion, with stream
Formula cell instrument is detected to the fluorescence intensity of Cur.
Result shows that the concentration with Cur/Bio-Chi increases, and cell surface drugloading rate substantially increases.But in view of cell
The influence of toxicity, the concentration of Cur/Bio-Chi is unsuitable excessive, so determining that concentration is 100 μ g/mL (accompanying drawing 7).
Embodiment 8：The drugloading rate detection of Cur/Bio-Chi-S-B-MSCs
MSCs is inoculated in 24 orifice plates, 37 DEG C of overnight incubations.It is the sulfo- of 0.5mg/mL by MSCs and 0.5mL concentration
The PBS solution incubation at room temperature 20min of NHS-LC-Biotin.Meanwhile, by the PBS of Avidin that 0.25mL concentration is 150 μ g/mL
Solution is incubated 20min with the PBS solution lucifuge of the Cur/Bio-Chi of 0.25mL μ containing 25-100 g/mL Cur.0.5mL is affine
The mixed solution of element and Cur/Bio-Chi is incubated 20min with B-MSCs.It is molten by forward and backward Cur/Bio-Chi is incubated with B-MSCs
Liquid, mixes, vortex mixed with equivalent ethanol respectively, determines the absorbance difference at λ=431nm.Calculate cell surface and carry medicine
Amount.
Result shows that MSCs surfaces drugloading rate is 54.26 ± 4.18pg Cur/ cells.
Embodiment 1-8 results show that stem cell-nanoparticle mixed carrier can be built bridge by Avidin and connect Cur/Bio-
The mode of Chi surface biologicals element and B-MSCs surface biologicals element builds.A kind of surface that the present invention builds combines drug-carrying nanometer particle
Stem cell cancer target system, the influence to stem cell Carriers Active can be reduced, be enriched with tumor locus and continue slowly to release
Medicine is put to reach curative effect, for stem cell provides a kind of new approach as tumor-targeting drug delivery vector.
The above is only specific embodiment of the invention, it is noted that come for one of ordinary skill in the art
Say, under the premise without departing from the principles of the invention, some improvement can also be made, these improvement also should be regarded as protection of the invention
Scope.
Claims (8)
1. a kind of surface combines the stem cell cancer target system of drug-carrying nanometer particle, it is characterised in that using Bio-Chi as Cur
Nanoparticulate carriers, then using B-MSCs as cell carrier, both are built bridge by Avidin and connected, with below general formula：Cur/
Bio-Chi-S-B-MSCs。
2. a kind of surface according to claim 1 combines the stem cell cancer target system of drug-carrying nanometer particle, and its feature exists
In described MSCs is mouse source fat mesenchymal stem cell.
3. a kind of surface according to claim 1 combines the stem cell cancer target system of drug-carrying nanometer particle, and its feature exists
In the structure of described Bio-Chi is：
4. a kind of Bio-Chi according to claim 3, it is characterised in that the chitosan molecule amount of described Bio-Chi is
5-200k, biotin substitution value is 1.91-45.82%.
5. a kind of surface according to claim 1 combines the stem cell cancer target system of drug-carrying nanometer particle, and its feature exists
In the preparation method of described drug-carrying nanometer particle Cur/Bio-Chi is as follows：By the ethanol solution containing 1mg Cur, with volume ratio 1/
4-3/4, is added dropwise in 1% acetum containing 10mg Bio-Chi that concentration is 2-10mg/mL.It is placed in 50 DEG C of water-baths and continues
Stirring 30min, 30min is processed with 50W Probe Ultrasonic Searchings, and dialyse 6h, crosses 0.8 μm of hydrophilic microporous filter membrane, needed for being diluted to PBS
Concentration.
6. the preparation method of a kind of drug-carrying nanometer particle Cur/Bio-Chi according to claim 5, it is characterised in that described
Ethanol solution and 1% acetum volume ratio for 1/4, Bio-Chi concentration be 10mg/mL.
7. a kind of surface according to claim 1 combines the structure of the stem cell cancer target system of drug-carrying nanometer particle, its
It is characterised by, being built bridge by Avidin between described drug-carrying nanometer particle and MSCs is connected, and preparation method is as follows：By MSCs with
0.5mL concentration is molten for the PBS of biotinyl-N- hydroxy-succinimides (sulfo-NHS-LC-Biotin) of 0.1-1mg/mL
Liquid is incubated 20min.Meanwhile, by 0.25mL concentration for 100-200 μ g/mL RBITC mark Avidin PBS solution with
The PBS solution lucifuge of the Cur/Bio-Chi of 0.25mL μ containing 25-100 g/mL Cur is incubated 20min.0.5mL RBITC- is affine
The mixed solution of element and Cur/Bio-Chi is incubated 20min with B-MSCs.
8. a kind of surface according to claim 7 combines the preparation side of the stem cell cancer target system of drug-carrying nanometer particle
Method, it is characterised in that the concentration of sulfo-NHS-LC-Biotin is 0.5mg/mL, and the concentration of RBITC- Avidins is 150 μ g/
The concentration of mL, Cur/Bio-Chi is 100 μ g/mL Cur.
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| CN114558125A (en) * | 2021-12-30 | 2022-05-31 | 厦门大学附属心血管病医院 | Urease-driven neutrophil drug delivery system and synthesis method thereof |
| CN114848844A (en) * | 2022-04-27 | 2022-08-05 | 中国药科大学 | Nano-engineered 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114558125A (en) * | 2021-12-30 | 2022-05-31 | 厦门大学附属心血管病医院 | Urease-driven neutrophil drug delivery system and synthesis method thereof |
| CN114558125B (en) * | 2021-12-30 | 2024-02-20 | 厦门大学附属心血管病医院 | Urease-driven neutrophil drug delivery system and synthesis method thereof |
| CN114848844A (en) * | 2022-04-27 | 2022-08-05 | 中国药科大学 | Nano-engineered stem cell anti-tumor targeted drug delivery system and preparation method and application thereof |
| 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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