CN108543074A - The nano medicament carrying system and its preparation that a kind of excretion body for oncotherapy wraps up - Google Patents

The nano medicament carrying system and its preparation that a kind of excretion body for oncotherapy wraps up Download PDF

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CN108543074A
CN108543074A CN201810317543.4A CN201810317543A CN108543074A CN 108543074 A CN108543074 A CN 108543074A CN 201810317543 A CN201810317543 A CN 201810317543A CN 108543074 A CN108543074 A CN 108543074A
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cell
dox
nano
psinps
excretion body
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CN108543074B (en
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甘璐
雍土莹
杨祥良
张晓琼
别娜娜
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Huazhong University of Science and Technology
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Abstract

The invention discloses a kind of nano medicament carrying system that the excretion body for oncotherapy wraps up and its preparations, then the nano medicament carrying system of excretion body package is obtained after the extracellular row again using cell endocytic medicament-carried nano material, the medicament-carried nano material load has antitumor drug, including chemotherapeutics, the drug for immunization therapy, at least one of the drug for tumor microenvironment to be transformed.The present invention is improved by the composition of the outer component biomembrane to crucial package nano medicament carrying system and structure etc., provides a kind of new approach compared with prior art for the biological processing nanoparticle based on biomembrane.The present invention wraps up nano medicament carrying system using excretion body, can retain the composition and structure of excretion body significantly, and the nano medicament carrying system of obtained excretion body package is with good stability during blood circulation and tumor-targeting.

Description

The nano medicament carrying system and its preparation that a kind of excretion body for oncotherapy wraps up
Technical field
The invention belongs to nano materials and oncology, more particularly, to a kind of excretion body for oncotherapy The nano medicament carrying system of package and its preparation, especially a kind of tumour improving antitumor drug is accumulated and deep penetrates, tumour Preparation and purposes of the targeting of stem cell with the nano medicament carrying system of killing.
Background technology
The recurrence of tumour and transfer are the main factors for causing tumor patient survival rate not high.Studies have shown that tumour Recurrence has close contact with transfer and tumor stem cell (cancer stem cells, CSCs).The drug resistance of CSCs is to lead One of the main reason for causing oncotherapy failure includes mainly following several respects：(1) it is its table that CSCs high, which expresses abc transport body, The primary factor of existing drug resistance；(2) CSCs is in G0 phases stationary state for maintaining drug resistance particularly significant；(3) CSCs has More efficient DNA repair abilities；(4) CSCs is in the tumor tissues deep of anoxic, and antitumor drug is difficult to enter these cells. Currently, the treatment of CSCs includes the signal path of stationary state, low differentiation state and exception mainly for its Biological characteristics Deng.Although in vitro experiment, various inhibitors or accelerating agent can significantly inhibit the characteristic of CSCs, once enter body Interior, targeting and the specificity of therapeutic scheme are just particularly important, and small-molecule drug also can be by just when treating CSCs Normal histocyte intake, has potential toxic side effect.Therefore, the drug for CSCs treatments needs the tumor target for having certain To ability.
Nano medicament carrying system can be coupled targeted molecular, can be realized medicine due to its unique advantage, such as EPR effects, surface Object conveys altogether, highly beneficial to the targeted therapy of CSCs.Currently, a variety of nano medicament carrying systems pass through the resistance to medicine phases of total conveying CSCs The conditioning agent and specific killing drug of pass and surface modification CSCs targeting ligands etc., realize height of the drug in CSCs Accumulation.But this therapeutic strategy is still difficult to reach ideal CSCs therapeutic effects, essentially consists in：(1) CSCs is preferably targeted Nano medicament carrying system be required to height accumulate in tumor tissues, deep penetrate into tumor tissues deep and by CSCs it is a large amount of Intake, but the above method is difficult to meet these features simultaneously；(2) the general marker protein of CSCs neither ones, different CSCs Marker protein have differences to each other, and part marker protein in common stem cell also express by height, therefore passes through idol Join targeting ligand and improves CSCs targetings with potential threat；(3) often synthesis technology is complicated for above-mentioned nano medicament carrying system, and And there is potential toxicity and side effect due to its " non-self " feature.Therefore, development one kind is efficiently targeted and is killed CSCs and with extremely low toxic side effect therapeutic strategy it is very necessary.
Currently, the biological processing nanoparticle based on biomembrane is widely used in oncotherapy.Based on this method, Zhu Duote Different property is endowed nano medicament carrying system, for example, erythrocyte membrane package nanoparticle have longer blood long circulating ability, Tumor cell membrane package nanoparticle have efficiently homologous targeting ability, source of human stem cell nanoghosts with good Cancer target ability etc..This natural modification mode can avoid the potential toxic side effect such as PEG predicaments caused by chemical modification. But in this biological processing, the memebrane protein that the partial destruction of membrane structure causes part to play critical function is lost It loses, stability and tumor-targeting of the nano medicament carrying system of film package during blood circulation is caused to reduce.Excretion body It is extracellular vesica of the grain size of cell secretion between 30-100nm.Excretion body has good Biostatic in internal body Property and biocompatibility, low immunogenicity and hypotoxicity.The study found that since surface has special protein, excretion body surface Reveal fabulous cellular uptake and cancer target ability.Meanwhile some excretion body protein matter, such as CD81, CD9 etc. can drop Extracellular matrix is solved, and then raising tumour penetrates and CSCs targetings.Although these existing excretion bodies have good effect, But that there is yield is low, drugloading rate is few, preparation process the problem of time-consuming for existing excretion body technique, influences practical application.
Invention content
For the disadvantages described above or Improvement requirement of the prior art, the purpose of the present invention is to provide one kind being used for oncotherapy Excretion body package nano medicament carrying system and its preparation, wherein passing through the outer component to crucial package nano medicament carrying system The composition and structure of biomembrane, the overall flow technological design of corresponding preparation method and each step are (such as endocytosis, outer row Incubation step) parameter, condition etc. be improved, provided compared with prior art for the biological processing nanoparticle based on biomembrane A kind of new approach.The present invention wraps up nano medicament carrying system using excretion body, can retain the composition and knot of excretion body significantly The nano medicament carrying system of structure, obtained excretion body package is with good stability during blood circulation and tumor target Tropism.The present invention can especially solve the problems, such as that CSCs targetings and fragmentation effect are poor, which enters blood by intravenously administrable After liquid, it can efficiently accumulate and penetrate into tumour deep in tumor tissues, deep and significantly improve the intake behavior of CSCs； Significantly inhibit the growth of liver cancer and melanin pulmonary metastases, hence it is evident that reduce CSCs ratios.
To achieve the above object, according to one aspect of the present invention, a kind of excretion body packet for oncotherapy is provided The nano medicament carrying system wrapped up in, which is characterized in that the nano medicament carrying system of excretion body package is to utilize cell endocytic medicament-carried nano What then material obtained after the extracellular row again, the medicament-carried nano material load has antitumor drug, the antineoplastic Object includes chemotherapeutics, immunotherapy medicaments, at least one of the drug for reconstructing tumor microenvironment.
As present invention further optimization, the cell includes tumour cell, tumor stem cell, immunocyte, tumour At least one of associated fibroblast cell, mescenchymal stem cell, the inhibition cell of derived from bone marrow and regulatory T cells；Institute State tumour cell and the corresponding tumour of the tumor stem cell include acute leukemia, lymthoma, prostate cancer, thyroid cancer, Cancer of the esophagus, osteocarcinoma, gastric cancer, breast cancer, lung cancer, oophoroma, chorioepithelioma, cervix cancer, carcinoma of uterine body, liver cancer, bladder Cancer, cutaneum carcinoma, colon cancer or the carcinoma of the rectum；The immunocyte includes T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphs Cell, mast cell or mononuclear phagocyte system.
As present invention further optimization, the nano material in the medicament-carried nano material includes nano liposomes, carbon Based nano-material, Si-based nanometer material, metal nano material, semiconductor-quantum-point, up-conversion and polymer nanocomposite material At least one of material；The c-based nanomaterial preferably includes in graphene oxide, carbon nanotube, Nano diamond at least It is a kind of；The Si-based nanometer material preferably includes at least one of porous silicon, mesoporous silicon, silicon point；The metal nano material Preferably include at least one of Jenner's grain of rice, nano grain of silver, transition metal nanoparticle.
As present invention further optimization, the nano material in the medicament-carried nano material is nano-particle material, institute The grain diameter of nano-particle material is stated in 1-1000nm.
As present invention further optimization, the antitumor drug of the medicament-carried nano material load preferably includes treatment suddenly Property leukaemia, lymthoma, prostate cancer, thyroid cancer, cancer of the esophagus, osteocarcinoma, gastric cancer, breast cancer, lung cancer, oophoroma, chorion Epithelioma, cervix cancer, carcinoma of uterine body, liver cancer, carcinoma of urinary bladder, cutaneum carcinoma, the chemotherapeutics of colon and rectum carcinoma, for being immunized Medicine is one or more in the drug of tumor microenvironment for being transformed.
It is another aspect of this invention to provide that the present invention provides a kind of nanometer loads that the excretion body for oncotherapy wraps up The preparation method of medicine system, which is characterized in that include the following steps：
S1：Medicament-carried nano material is incubated altogether with cell；
S2：Centrifugation removes the medicament-carried nano material that the cell does not absorb；
S3：The fresh culture medium without medicament-carried nano material is added to continue to be incubated；
S4：The medicament-carried nano material extracellularly arranged through this is collected by centrifugation to wrap up to get to the excretion body for oncotherapy Nano medicament carrying system.
As present invention further optimization, in the step S1, the time being incubated altogether is 1-96h.
As present invention further optimization, the step S2 is specifically in a low temperature of 0-10 DEG C, with 100-1000g's Centrifugal force collects the cell, and the cell is cleaned using the phosphate buffer of precooling, until there is no free to receive in solution Rice drug-loading system；
The step S4 is specifically under 0-10 DEG C of low temperature, in the centrifugal force removal culture solution of 100-1000g Cell centrifuges the cell fragment in removal culture solution, with the centrifugal force 30- of 10000-20000g with 3000-5000g The nano medicament carrying system that 120min is extracellularly arranged；Using after the phosphate buffer cleaning of precooling to get to excretion body packet The nano medicament carrying system wrapped up in.
As present invention further optimization, in the step S3, the incubation is placed in cell incubator, 37 DEG C, 5%CO2Cell culture condition under be incubated, or make first be positioned over again after ultraviolet light 5-240min cell training It supports in case and is incubated 12-96h.
Another aspect according to the invention, the present invention provides the nanometer loads that the above-mentioned excretion body for oncotherapy wraps up Medicine system application in preparation of anti-tumor drugs.
Contemplated above technical scheme through the invention, compared with prior art, due to wrapping up nanometer using excretion body Drug-loading system forms the nano medicament carrying system wrapped up for the excretion body of oncotherapy, and advance load is specifically had antineoplastic The nano drug-carrying material of object is incubated altogether with cell, makes the cell endocytic medicament-carried nano material, and extracellular row is then recycled to obtain Outer row, the nano medicament carrying system that is wrapped up by excretion body.The nano drug-carrying wrapped up for the excretion body of oncotherapy in the present invention System is being obtained by way of the medicament-carried nano material for extracellularly arranging endocytosis, having following characteristics：The nano medicament carrying system It is made of the excretion body structure and internal medicament-carried nano material on surface；The nano medicament carrying system is in tumour cell and Tumor Stem Intake behavior and fragmentation effect in cell are substantially better than the medicament-carried nano material not wrapped up, and can more accumulate in Tumor tissues simultaneously penetrate into tumor tissues deep；The nano medicament carrying system can be with the antitumor drug of very low amount to a variety of swollen Tumor generates significant inhibition.
Present invention firstly provides using extracellular row's nano medicament carrying system, excretion body is wrapped in nano medicament carrying system table This preparation thinking of face.Existing nano medicament carrying system film technique for packing mainly by after broken biomembrane with nano medicament carrying system The method for being extruded through filter membrane jointly obtains, and this method is easy to cause the damage of membrane structure, and then leads to film surface work( The loss of energy albumen etc., this is unfavorable for stability, the Targeting Performance etc. of nano medicament carrying system.And the preparation in the present invention Method is milder, is capable of the complete structure of most complete reservation excretion body film, to play more preferably function.The present invention is for the first time The nano medicament carrying system that film package is built by extracellularly arranging nanoparticle, can be notable after stimulating cell using nano medicament carrying system 34 times of the yield or more of excretion body is improved, drugloading rate improves 2 times or more, and elapsed time can shorten half.Medicine is carried in the present invention The antitumor drug of nanomaterial loadings can be that direct chemotherapeutics, immunotherapy medicaments, the photo-thermal for inhibiting tumour growth is controlled Co-stimulators, the monoclonal for treating drug or small size nano particle or adjuvant therapy of tumors with photo-thermal effect are anti- At least one of body, or the drugs of tumor microenvironments such as tumor tissue cell's epimatrix, stromal cells can be influenced, example Acute leukemia, lymthoma, prostate cancer, thyroid cancer, cancer of the esophagus, osteocarcinoma, gastric cancer, breast cancer, lung cancer, ovum can such as be treated In nest cancer, chorioepithelioma, cervix cancer, carcinoma of uterine body, liver cancer, carcinoma of urinary bladder, cutaneum carcinoma, colon and rectum carcinoma chemotherapeutic It is one or more.
The incubation process that the present invention is further preferably arranged to endocytosis and outside controls, by the type and phase that control cell Close the Parameter Conditions (such as incubation time) of incubation process so that formed in medicament-carried nano material outer then outer through cell endocytic The excretion body formed is arranged, excretion body content is high, and it is good so that the nano medicament carrying system of excretion body package integrally has Stability and tumor-targeting.
Technical scheme of the present invention has the advantages that：
(1) excretion body structure is covered in medicament-carried nano material surface, improves the stability and drug of medicament-carried nano material Slow release effect；
(2) after medicament-carried nano material is with cell incubation, the yield of excretion body significantly improves；
(3) intake behavior of the nano medicament carrying system of excretion body package in tumour cell and CSCs significantly improves；
(4) nano medicament carrying system of excretion body package significantly increases the fragmentation effect of tumour cell and CSCs；
(5) nano medicament carrying system of excretion body package can highly be accumulated penetrates into tumor group in tumor tissues, deep Knit deep.
Description of the drawings
Figure 1A is the diameter characterization of E-PSiNPs of the present invention；
Figure 1B is the transmission electron microscope observation result of E-PSiNPs of the present invention, wherein left figure is control group, and right figure is Experimental group (scale in figure is 20nm)；
Fig. 2 is EDAX results of the E-PSiNPs of the present invention at FTEM；
Fig. 3 is that the common location of E-PSiNPs surface Cs D63 albumen of the present invention and inside PSiNPs observes result；
Fig. 4 is the Western blot results of the surfaces E-PSiNPs excretion body associated protein of the present invention；
Fig. 5 is intake behaviors of the DOX@E-PSiNPs of the present invention in H22 tumour cells；
Fig. 6 is intake behaviors of the DOX@E-PSiNPs of the present invention in H22CSCs；
Fig. 7 is intake behaviors of the DOX@E-PSiNPs of the present invention in B16CSCs；
Fig. 8 is cytotoxicities of the DOX@E-PSiNPs of the present invention to H22 tumour cells；
Fig. 9 A are shadows of the DOX@E-PSiNPs of the present invention to the H22 tumor colonies ball numbers cultivated in 3D soft fibre protein adhesives It rings；
Fig. 9 B are shadows of the DOX@E-PSiNPs of the present invention to the H22 tumor colonies ball sizes cultivated in 3D soft fibre protein adhesives It rings；
Figure 10 is cytotoxicities of the DOX@E-PSiNPs of the present invention to B16 tumour cells；
Figure 11 A are DOX@E-PSiNPs of the present invention to the B16 tumor colonies ball numbers cultivated in 3D soft fibre protein adhesives It influences；
Figure 11 B are DOX@E-PSiNPs of the present invention to the B16 tumor colonies ball sizes cultivated in 3D soft fibre protein adhesives It influences；
Figure 12 is accumulation behaviors of the DOX@E-PSiNPs of the present invention in the tumor tissues of tumor-bearing mice；
Figure 13 is the accumulation behavior in CSCs of the DOX@E-PSiNPs of the present invention in the tumor tissues of tumor-bearing mice；
Figure 14 penetrates behavior for the deep in DOX@E-PSiNPs of the present invention in vitro tumor colonies ball；
Figure 15 penetrates behavior for the deep in DOX@E-PSiNPs of the present invention in vivo tumor tissues；
After Figure 16 A enter liver cancer subcutaneous tumors model mice for DOX@E-PSiNPs of the present invention by intravenous injection, mouse Tumor growth curve；
Figure 16 B are after DOX@E-PSiNPs of the present invention enter liver cancer subcutaneous tumors model mice by intravenous injection, and mouse is swollen The weight of tumor tissue；
After Figure 16 C enter liver cancer subcutaneous tumors model mice for DOX@E-PSiNPs of the present invention by intravenous injection, mouse Life cycle experimental result；
Figure 17 A are after DOX@E-PSiNPs of the present invention enter liver cancer subcutaneous tumors model mice by intravenous injection, and mouse is swollen The ratio of side population cell in tumor tissue；
Figure 17 B are after DOX@E-PSiNPs of the present invention enter liver cancer subcutaneous tumors model mice by intravenous injection, and dispersion is small The tumor colonies ball number that the monodisperse cell that mouse tumor tissues obtain is formed in 3D soft fibre protein adhesives；
Figure 17 C are after DOX@E-PSiNPs of the present invention enter liver cancer subcutaneous tumors model mice by intravenous injection, and dispersion is small The tumor colonies ball size that the monodisperse cell that mouse tumor tissues obtain is formed in 3D soft fibre protein adhesives；
Figure 18 A are mouse lung after DOX@E-PSiNPs of the present invention enter melanin pulmonary metastases mouse by intravenous injection Portion's tumor nodule number；
Figure 18 B are lung tissue after DOX@E-PSiNPs of the present invention enter melanin pulmonary metastases mouse by intravenous injection H＆E coloration results；
After Figure 18 C enter melanin pulmonary metastases mouse for DOX@E-PSiNPs of the present invention by intravenous injection, mouse Life cycle experimental result；
Figure 19 A are after DOX@E-PSiNPs of the present invention enter melanin pulmonary metastases mouse by intravenous injection, and dispersion is small The tumor colonies ball number that the monodisperse cell that mouse lung tumors tubercle obtains is formed in 3D soft fibre protein adhesives；
Figure 19 B are after DOX@E-PSiNPs of the present invention enter melanin pulmonary metastases mouse by intravenous injection, and dispersion is small The tumor colonies ball size that the monodisperse cell that mouse lung tumors tubercle obtains is formed in 3D soft fibre protein adhesives.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below It does not constitute a conflict with each other and can be combined with each other.
Various tumour cells, drug and the experimental animal used in following embodiments：
H22 murine hepatocarcinoma cells, Bel7402 Bel7402 cells, mouse skin cancerous cell line B16, can be from U.S. ATCC companies of state or the CCTCC purchases of Chinese Typical Representative object collection.
BALB/C mice, C57 mouse are purchased from Wuhan University's medical experiment animal center, 18-20 grams of weight；
Adriamycin is purchased from Sigma companies.
Embodiment 1：The preparation of the nano medicament carrying system of excretion body package and characterization
1, experiment material and reagent
H22 murine hepatocarcinoma cells, adriamycin (band red fluorescence), boron-doping p<100>Silicon chip (resistance 0.00055-0.0015 Ω-cm) it is purchased from Virginia companies of the U.S.
2, experimental procedure
(1) silicon chip is placed in dense H2SO4With H2O2Mixed solution (volume ratio v:V=3:1) 15min in, ultra-pure water cleaning It is dried after 3 times spare.Silicon wafer polishing is installed in electrochemical corrosion device up, and it is 4 that volume ratio, which is added,:1 HF/ ethyl alcohol is molten Liquid, 165mA/cm2Current strength under persistently corrode silicon chip surface after 300s and kermesinus film occur, washes of absolute alcohol 3 times, Continuously add 3.3% HF/ ethanol solutions (mass ratio), 4.5mA/cm2Current strength under act on 90s, by porous silicon film It is removed from silicon matrix.HF solution is sucked, absolute ethyl alcohol gently cleans 3 times, collects porous silicon film, and transfer them to water-soluble Ultrasound 16h in liquid, 8000g centrifuge 20min, collect supernatant, are placed in 60 DEG C of water-baths and continue 3-6h to excite PSiNPs fluorescence (PSiNPs, i.e. porous silicon nanoparticle).
10mg PSiNPs are taken to be dissolved in 5mL ultra-pure waters, while it is lasting stirred that 1mL 10mg/mLDOX aqueous solutions are added Night, 10,000g centrifugation 10min abandon supernatant.Ultra-pure water cleaning precipitation, 10,000g centrifugation 10min, is repeated 3 times.Carry medicine PSiNPs (DOX@PSiNPs) is stored in ultra-pure water, and 4 DEG C preserve for use.
(2) 10 are taken7A H22 tumor cell cultures are in the sterile petri dish of a diameter of 10cm.It is discarded after being incubated overnight Culture medium after phosphate buffer (PBS) cleaning, is added serum-free RPMIs 1640 of the 6mL containing 200 μ g/mL PSiNPs and cultivates Base continues to be incubated.1-96h (such as 1h, 6h, 12h, for 24 hours, 48,96h) after, the cell in culture dish is collected by centrifugation；PBS cleanings are collected Cell after 6mL fresh cultures be added thereto continue to be incubated 12-96h (such as 12h, for 24 hours, 48h, 96h).Culture medium is taken, Supernatant is collected by centrifugation in 5000g, and 10000g or 20000g centrifugations 30min or 120min must be precipitated, the as outer porous silicon nanometer arranged Grain (E-PSiNPs).
(3) E-PSiNPs and PSiNPs are dissolved in PBS, using the nano particle size with 633nm He-Ne lasers and Zeta potentiometers detect its grain size and zeta Potential distributions.It specifically imposes a condition and is：Temperature is 25 DEG C, and equilibration time is 120s.Meanwhile thering is carbon film one side to be placed in upward on disposable PE gloves copper mesh, by certain density E-PSiNPs or PSiNPs It is added dropwise on copper mesh surface, balances 5min, after blotting copper mesh surface liquid with filter paper, copper mesh is placed on filter paper and is stood overnight, is made With tem observation sample topography；Energy-spectrum scanning, analysis sample element composition are carried out to sample using the scan pattern of FTEM.
3, experimental result
Experimental result finds, time of nano medicament carrying system and cell incubation (1h, 6h, 12h, for 24 hours, 48,96h), cell Continue the time (12h, for 24 hours, 48h, 96h) being incubated in fresh culture and last centrifugation obtains the porous of excretion body package The centrifugal force (10000g, 20000g) of silicon nanoparticle and time (30min, 120min) can obtain outer under these conditions Secrete the porous silicon nanoparticle of body package.It is in the case where ensureing cell survival rate that nano medicament carrying system and cell incubation is longer Time, the centrifugal force of bigger use shorter centrifugation time in the case where ensureing yield, are can guarantee in this way in higher yield The lower time for reducing consumption and the particular/special requirement to instrument and equipment.It is the preferred nano medicament carrying system of the present invention and cell incubation 12h, thin Born of the same parents be incubated in fresh culture for 24 hours, centrifugal force 20000g, centrifugation time be 30min (centrifugation 30min and 120min most Whole yield difference is little), higher yield is obtained in the shortest possible time.The final yield of excretion body of the present invention is 107 A cell can generate the excretion body of about 60 μ g, and conventional ultracentrifugation method collection can only obtain about 1.8 μ g excretion bodies, that is, make With 34 times of the yield or more that can significantly improve excretion body after nano medicament carrying system stimulation cell.
It is detected and is found by DLS, the average particle size distribution of E-PSiNPs and PSiNPs are 280nm and 120nm or so (figure 1A), zeta current potentials are -11mV.Transmission electron microscope picture shows that E-PSiNPs patterns are irregular and surface has one layer of membrane structure (Figure 1B).It is porous silicon nanoparticle (Fig. 2) that the presence of Si elements, which proves structure seen in figure really, in EDAX results.We make With same method (technological parameter size, used reagent type and concentration used in each step etc. remain unchanged) Jenner's grain of rice (Jenner's grain of rice that the porous silicon nanoparticle (DOX E-PSiNPs) of the load DOX of outer row has been prepared and has arranged outside Preparation process can refer to the prior art), obtained similar result.
Embodiment 2：The nano medicament carrying system surface film structure identification of excretion body package
1, experiment material and reagent
FITC-CD63 antibody, CD63 antibody, TSG101 antibody, Calnexin antibody
2, experimental procedure
(1) a certain amount of E-PSiNPs is taken, 500 μ L 5%BSA solution are added and are incubated 30min, continuously add 10 μ L FITC-CD63 antibody, 4 DEG C are rocked overnight incubation, and 20,000g centrifugation 30min abandon supernatant, and PBS is cleaned 3 times.Take 20 μ L FITC- The E-PSiNPs solution that CD63 is incubated is added dropwise on the burnt ware of copolymerization, and FV1000 confocal microscopies are used after fully spreading out The green fluorescence of FITC-CD63 antibody and the common location situation of PSiNPs red fluorescences, design parameter are：PSiNPs：Ex= 488nm, Em=680nm；FITC：Ex=488nm, Em=520nm.
(2) E-PSiNPs, excretion body and corresponding cell for taking same protein amount, using Western blot detect cell come Excretion body marker protein CD63 and TSG101 expression in the E-PSiNPs in source is made with the Calnexin albumen in endoplasmic reticulum source For negative control, β-action are used as internal reference.3, experimental result
Immunofluorescence dyeing result shows that the green fluorescence of FITC-CD63 antibody and PSiNPs red fluorescences are completely fixed altogether Position, illustrates the surfaces E-PSiNPs with the presence of CD63 albumen (Fig. 3).It is analyzed by westernblot, we further demonstrate that H22 There is a large amount of excretion body marker protein CD63 and TSG101 on the surfaces E-PSiNPs in source, and are made using Calnexin albumen For positive control, exclude E-PSiNPs from it is cytoplasmic may (Fig. 4, control group 1 are the product of cell lysis of H22 cells, Control group 2 is the excretion body for the H22 cells that conventional ultracentrifugation method is collected, experimental group E-PSiNPs).Likewise, The E-PSiNPs of Bel7402 cell origins is made using preparation method substantially the same manner as Example 1, also demonstrates Bel7402 There is also a large amount of excretion body marker protein CD63 and TSG101 on the surfaces E-PSiNPs of cell origin.
Embodiment 3：The intake behavior in tumour cell and its CSCs in vitro of the nano medicament carrying system of excretion body package
1, experiment material and reagent
H22 murine hepatocarcinoma cells, adriamycin, 3D soft fibres protein adhesive, 3D soft fibres protein adhesive (1) tumor cell in vitro are taken the photograph Take behavior
2×105A H22 suspends culture in tissue culture plate, removes culture medium afterwards for 24 hours, respectively into tissue culture plate The free DOX of a concentration of 0.5,1,2 μ g/mL of 1mL DOX, the free serum culture of DOX@PSiNPs or DOX@E-PSiNPs is added Base, 37 DEG C, 5%CO2It is incubated 4h under conditions of this normal cell culture, collects cell, PBS is cleaned 3 times, 250g centrifugations 10min.500 μ L precoolings PBS are added in cell precipitation, cell is resuspended, crosses 200 mesh screens, FC500 flow cytomery intracellulars DOX fluorescence.Specific detection parameters：Ex=488nm, transmitting light detection are FL2 fluorescence channels.
(2) external CSCs intakes
H22 cell inoculations are cultivated in 3D soft fibre protein adhesives and obtain H22CSCs.To 2 × 105Add in a H22CSCs Enter free DOX, DOX@PSiNPs and DOX the@E-PSiNPs, 37 DEG C, 5%CO of final concentration of 0.5,1, the 2 μ g/mL of DOX2Under the conditions of It is incubated 4h, collects cell, PBS is cleaned 3 times, and cell is resuspended in 500 μ L precoolings PBS, crosses 200 mesh screens, the inspection of FC500 flow cytometers Survey intracellular DOX fluorescence.Specific detection parameters：Ex=488nm, transmitting light detection are FL2 fluorescence channels.
3, experimental result
H22 cells are incubated altogether with the DOX@E-PSiNPs of H22 cell origins, flow cytometric analysis results show together DOX@PSiNPs are compared with free DOX, and DOX@E-PSiNPs processing group cell intracellular DOX fluorescence intensities improve 5 times or so (Fig. 5, control group 1 are free DOX；Control group 2 is DOX@PSiNPs, that is, loads the porous silicon nanometer of antitumor drug adriamycin Grain；Experimental group is DOX@E-PSiNPs).In H22CSCs, as DOX concentration increases, DOX@E-PSiNPs processing groups DOX is glimmering Luminous intensity gradually increases, and illustrates that concentration dependent is presented in CSCs endocytosis DOX@E-PSiNPs.In a concentration of 2 μ g/mL of DOX, 2.1 and 1.7 times for entering born of the same parents' amount and being free DOX and DOX@PSiNPs respectively of DOX@E-PSiNPs processing groups, show DOX@E- PSiNPs has better CSCs targetings ability, and (Fig. 6, control group 1 are free DOX, and control group 2 is DOX@PSiNPs, experimental group For DOX@E-PSiNPs).Likewise, our DOX@E-PSiNPs using Bel7402 cell origins, thin with Bel7402 by it Born of the same parents and its CSCs are incubated altogether, are also demonstrated DOX@E-PSiNPs and are shown significant tumour cell and CSCs intake behaviors.
Embodiment 4：The universality of intake behavior of the nano medicament carrying system of excretion body package in CSCs
In order to study whether DOX@E-PSiNPs targetings CSCs has universality, we pass through a certain number of B16 are thin Born of the same parents are seeded in 3D soft fibre protein adhesives, and culture collects after 5-7 days and obtains the tumor stem cell of a large amount of B16F10, further studies Intake behaviors of the B16CSCs to the DOX@E-PSiNPs of H22 cell origins.
1, experiment material and reagent
Mouse skin cancerous cell line B16, adriamycin, 3D soft fibre protein adhesives
2, experimental procedure
Mouse skin cancer cell B16 cell inoculations are cultivated in 3D soft fibre protein adhesives and obtain B16CSCs.To 2 × 105 It is added free DOX, the DOX@PSiNPs and DOX@E-PSiNPs of DOX final concentration of 0.5,1,2 μ g/mL in a B16CSCs, 37 DEG C, 5%CO2Under the conditions of be incubated 4h, collect cell, PBS is cleaned 3 times, and cells are resuspended in 500 μ L precoolings PBS, cross 200 mesh screens, FC500 flow cytomery intracellular DOX fluorescence.Specific detection parameters：Ex=488nm, transmitting light detection are logical for FL2 fluorescence Road.
3, experimental result
After B16CSCs is incubated altogether with the DOX@E-PSiNPs of H22 cell origins, flow cytometric analysis results show Under different pharmaceutical concentration, the B16CSCs intracellular DOX contents in DOX@E-PSiNPs processing groups be significantly better than free DOX and DOX@PSiNPs groups (Fig. 7, control group 1 are free DOX, and control group 2 is DOX@PSiNPs, and experimental group is DOX@E-PSiNPs), Illustrate that the DOX@E-PSiNPs in the sources H22 it is general can to show that DOX@E-PSiNPs targetings CSCs has by B16CSCs huge uptakes Adaptive.
Embodiment 5：The nano medicament carrying system of excretion body package is to the cytotoxicity in tumor cell in vitro and CSCs
1, experiment material and reagent
H22 murine hepatocarcinoma cells, Bel7402 Bel7402 cells, 3D soft fibre protein adhesives
2, experimental procedure
(1) tumor cell in vitro toxicity
H22 cells are with 8 × 103Cell density be seeded in 96 orifice plates, remove culture medium after being incubated overnight, 100 μ be added The free DOX of L a concentration of 0.25,0.5,1 μ g/mL containing DOX, the serum free medium of DOX@PSiNPs or DOX@E-PSiNPs, 37 DEG C, 5%CO2Under the conditions of be incubated for 24 hours after 10 μ L CCK-8 solution be added per hole be incubated 4h, use the detection of 318C microplate reader per hole The absorbance at 450nm.
(2) external CSCs toxicity
400/hole H22 cell inoculations are in 96 orifice plates for being covered with 1mg/mL 3D soft fibre protein adhesives, 37 DEG C, 5%CO2 Under the conditions of cultivate.After 5 days, discard culture medium, be added the 100 final concentration of 2 μ g/mL of μ L DOX free DOX, DOX@PSiNPs or DOX the@E-PSiNPs, 37 DEG C, 5%CO of H22 cell origins2Under the conditions of be incubated for 24 hours.It discards supernatant, being added 0.1% per hole expects Blue solution dyes 1min, discards dye liquor, and PBS is cleaned 5 times, and microscopically observation is simultaneously taken pictures, and tumour cell ball number living is counted With size.
3, experimental result
Tumor cell in vitro toxicity test the result shows that, compared to free DOX and DOX@PSiNPs, H22 cell origin DOX@E-PSiNPs show stronger fragmentation effect to H22 cells, and (Fig. 8, control group 1 are free DOX, and control group 2 is DOX@PSiNPs, experimental group are DOX@E-PSiNPs).In vitro in CSCs toxicity tests, DOX@E-PSiNPs and 3D soft fibres After the H22 tumor colonies ball grown in protein adhesive is incubated altogether, (Fig. 9 A, control group 1 are trip to the quantity of the tumor colonies ball of survival From DOX, control group 2 is DOX@PSiNPs, and experimental group is DOX@E-PSiNPs) and size (Fig. 9 B, control group 1 are free DOX, Control group 2 is DOX@PSiNPs, and experimental group is DOX@E-PSiNPs) significantly reduce, it is notable to show that DOX@E-PSiNPs have CSCs fragmentation effects.In conclusion DOX@E-PSiNPs have significant tumor cytotoxicity effect, while can efficiently kill Hinder CSCs.
Embodiment 6：Universality of the nano medicament carrying system of excretion body package to the cytotoxicity of tumour cell and CSCs
Whether there is universality to the lethal effect of CSCs for research DOX@E-PSiNPs, we examine H22 cells Fragmentation effects of the DOX@E-PSiNPs in source to the CSCs of mouse skin cancer cell B16.
1, experiment material and reagent
H22 murine hepatocarcinoma cells, mouse skin cancerous cell line B16,3D soft fibre protein adhesive
2, experimental procedure
(1) tumor cell in vitro toxicity test
B16 cells are with 8 × 103Cell density be seeded in 96 orifice plates, remove culture medium after being incubated overnight, 100 μ be added The free DOX of L a concentration of 0.25,0.5,1 μ g/mL containing DOX, the serum free medium of DOX@PSiNPs or DOX@E-PSiNPs, 37 DEG C, 5%CO2Under the conditions of be incubated for 24 hours after 10 μ L CCK-8 solution be added per hole be incubated 4h, use the detection of 318C microplate reader per hole The absorbance at 450nm.
(2) external CSCs toxicity tests
400/hole mouse skin cancer cell B16 is seeded in 96 orifice plates for being covered with 1mg/mL 3D soft fibre protein adhesives, and 37 DEG C, 5%CO2Under the conditions of cultivate.After 5 days, discard culture medium, be added the 100 final concentration of 2 μ g/mL of μ L DOX free DOX, DOX the@E-PSiNPs, 37 DEG C, 5%CO of DOX@PSiNPs or H22 cell origins2Under the conditions of be incubated for 24 hours.It discards supernatant, per hole 0.1% trypan blue solution is added, dyes 1min, discards dye liquor, PBS is cleaned 5 times, and microscopically observation is simultaneously taken pictures, and counts living Tumour cell ball number and size.
3, experimental result
Tumor cell in vitro toxicity test the result shows that, compared with free DOX, DOX@PSiNPs, the H22 of various concentration is thin The DOX@E-PSiNPs in born of the same parents source can significantly kill B16 cells, and it is general to show that DOX@E-PSiNPs killing tumor cells have Adaptive (Figure 10, control group 1 are free DOX, and control group 2 is DOX@PSiNPs, and experimental group is DOX@E-PSiNPs).External CSCs Toxicity test as a result, it has been found that, the DOX@E-PSiNPs in the sources H22 are incubated altogether with the B16 tumor colonies ball in 3D soft fibre protein adhesives Later, (Figure 11 A, control group 1 are free DOX to the quantity of the tumor colonies ball of survival, and control group 2 is DOX@PSiNPs, experimental group For DOX@E-PSiNPs) and size (Figure 11 B, control group 1 are free DOX, and control group 2 is DOX@PSiNPs, and experimental group is DOX@ E-PSiNPs it) significantly reduces, illustrates that DOX@E-PSiNPs killings CSCs has universality.
Embodiment 7：Accumulation behavior of the nano medicament carrying system of excretion body package in tumor tissues
1, experiment material and reagent
H22 murine hepatocarcinoma cells, BALB/c mouse
2, experimental procedure
(1) tumor tissue accumulation lab diagram；
2×106A H22 cancer cell subcutaneous is injected into outside BALB/c mouse (18-20g) thigh to build mouse H22 Subcutaneous tumors model.When tumor size reaches 250mm3When, tail vein injection 0.5mg/kg dissociates DOX, DOX@PSiNPs or DOX@ E-PSiNPs, rear cervical dislocation processing mouse, takes out mouse tumor and the heart, liver, spleen, lung, kidney for 24 hours, and PBS is cleaned 3 times, is inhaled Stem organization's surface moisture, each tissue weigh and 100 μ L RIPA lysates are added, and use tissue abrasion of tissue grinder.It has ground Cheng Hou draws lapping liquid, and 10,000g centrifugation 10min, taking-up supernatant solution is placed in 4 DEG C and is kept in dark place, to be measured.Various concentration DOX standard solution is prepared using the supernatant solution after each tissue blank control group grinding centrifugation.Existed using high performance liquid chromatography The fluorescence intensity of DOX in each tissue grinder supernatant solution is detected at 488nm.
3, experimental result
DOX E-PSiNPs enter tumor-bearing mice by tail vein injection, for 24 hours afterwards grind each tissue grinder to each tissue DOX contents carry out quantitative analysis in grinding fluid, as a result, it has been found that DOX@E-PSiNPs are free respectively in the DOX contents of tumor locus 3.9 times of DOX, 1.7 times of DOX@PSiNPs, illustrate DOX@E-PSiNPs have significant tumor tissues targeting ability (Figure 12, Control group 1 is free DOX, and control group 2 is DOX@PSiNPs, and experimental group is DOX@E-PSiNPs).
Embodiment 8：Intake behavior of the nano medicament carrying system of excretion body package in the CSCs of tumor tissues
1, experiment material and reagent
H22 murine hepatocarcinoma cells, BALB/c mouse
2, experimental procedure
2×106A H22 cancer cell subcutaneous is injected into outside BALB/c mouse (18-20g) thigh to build mouse H22 Subcutaneous tumors model.When gross tumor volume reaches 250mm3When, tail vein injection 0.5mg/kg dissociates DOX, DOX@PSiNPs, DOX@E- PSiNPs, rear cervical dislocation execution mouse, takes out tumor tissues, PBS is cleaned up for 24 hours.Tumor tissues are placed in stem cell It is cut into mung bean size in culture medium, removes culture medium, 1mg/mL Collagenase are added, 2h are digested at 37 DEG C, every 15min Rocking 1 time ensures fully to digest.Postdigestive tumor tissues are squeezed until not having block structure using coarse frosted glass plate, are drawn Postdigestive cell suspension, 1200g centrifuge 5min, abandon supernatant, after PBS cleanings cell precipitation 3 times by solution cross 200 mesh screens with Obtain single cell suspension.Single cell suspension is divided into two parts：A part is using FC500 flow cytometers in 488nm/FL2 conditions The fluorescing matter of DOX in lower all tumour cells of detection；Another part cell suspension is the same as 33342 dyestuff (final concentrations of Hoechst For 5 μ g/mL) mixing, it is protected from light at 37 DEG C and is incubated 90min, while by 50 μM of verapamil and 5 μ g/mL Hoechst 33342 As a control group, cell is placed on ice the cell being incubated altogether at once after the completion of incubation, and 250g centrifuges 5min at 4 DEG C, removes supernatant, in advance Cold PBS is cleaned 3 times, and cell is resuspended in precooling PBS, is preserved on ice.Use CytoFlex flow cytometry analysis side population cells Middle DOX fluorescing matters.Specific analytical method is as follows：
1) living cells is irised out in SSC-A and FSC-A scatter plots；
2) structure transverse and longitudinal coordinate is respectively Ex=405nm, Em=450nm and Ex=405nm, the scatterplot of Em=660nm Figure, comparison verapamil processing and untreated fish group, iris out a part of cell of disappearance, this part cell is side population cell；
3) excitation and transmission channel for using 488nm/585nm detect DOX fluorescence intensities in side population cell.
3, experimental result
After entering DOX E-PSiNPs in tumor-bearing mice body by tail vein injection according to the method for embodiment 7, by tumour Tissue is dispersed into individual cells after taking out, and is found by flow cytometry, compared to free DOX and DOX@PSiNPs processing Group, 64% and 71% has been respectively increased in side population cell intracellular DOX fluorescence intensities in DOX@E-PSiNPs processing group tumor tissues.With Upper result further proves that (Figure 13, control group 1 are DOX@E-PSiNPs with significant CSCs targetings ability in vivo experiment Free DOX, control group 2 are DOX@PSiNPs, and experimental group is DOX@E-PSiNPs).
Embodiment 9：The tumour deep of the nano medicament carrying system of excretion body package penetrates behavioral study
1, experiment material and reagent
H22 murine hepatocarcinoma cells, BALB/c mouse, 3D soft fibres protein adhesive, FITC-CD31 antibody
2, experimental procedure
(1) deep in vitro in 3D tumour cells ball penetrates behavior
4×102A/hole H22 cells are seeded according to method previously in 3D soft fibre protein adhesives.When tumour cell ball When growing into the 5th day, culture medium is discarded, PBS is cleaned 3 times, is separately added into free DOX, DOX@of the final concentration of 10 μ g/mL of DOX The serum free medium of PSiNPs or DOX@E-PSiNPs is incubated for 24 hours, removes culture medium, PBS is cleaned 3 times, by soft fibre in hole Protein adhesive is transferred in the burnt ware of copolymerization, uses FV1000 Laser Scanning Confocal Microscope Z axis scan pattern viewing distance tumour cell ball tables The DOX fluorescing matters of face different depth.Specific detection parameters setting is as follows：Ex=559nm, Em=590nm.
(2) the tumour deep in tumor-bearing mice body penetrates behavioral study
106A H22 liver cancer cells are subcutaneously injected into BALB/c mouse (18g or so) right lateral thigh root to build mouse H22 subcutaneous tumors models.When tumor volume growth to 250mm3When left and right, a concentration of 0.5mg/kg's of tail vein injection DOX is free DOX, DOX PSiNPs or DOX E-PSiNPs, for 24 hours after cervical dislocation put to death mouse, tumor tissues are taken out, at frozen section Reason.FITC-CD31 antibody is incubated 30min at 37 DEG C and is sliced blood vessel with marked tumor.Use FV1000 Laser Scanning Confocal Microscopes point The upper DOX red fluorescences of detection slice and FITC green fluorescences not at 559/580nm and 488/520nm.It is soft using Image J Part counts the DOX fluorescence distribution situations on from tumor vessel to the straight line of inside tumor.
3, experimental result
After DOX@E-PSiNPs are incubated altogether with tumor colonies ball, found by Laser Scanning Confocal Microscope Z axis scanned picture, The tumour cell ball DOX fluorescence of DOX@E-PSiNPs processing groups is significantly stronger than free DOX and DOX@under same penetration depth PSiNPs processing group (Figure 14).Further, DOX E-PSiNPs are entered by tail vein injection in tumor-bearing mice body, is taken out swollen Tumor tissue, slice, using FITC-CD31 antibodies on tumor blood vessel dye markers, confocal microscopy the result shows that, remote At 400 μm from blood vessel, DOX@E-PSiNPs groups still can detect apparent DOX fluorescence signals, and in DOX and DOX@ Farthest only apparent fluorescence signal is detected in 25 μm and 120 μm of depths, continue deeper into then without apparent in PSiNPs processing DOX fluorescence signals (Figure 15).Result above, which demonstrates DOX@E-PSiNPs, has apparent tumour deep penetration capacity.
Embodiment 10：The nano medicament carrying system of excretion body package is to the inhibition 1 of rat liver cancer subcutaneous tumors model, experiment Reagent and material
H22 murine hepatocarcinoma cells, BALB/c mouse
2, experimental procedure
2×106A H22 cancer cell subcutaneous is injected into subcutaneous to build mouse outside BALB/c mouse (18-20g) thigh Tumor model.When tumor size reaches 50mm3When, mouse is equally divided into 6 groups, every group 14, tail vein injection PBS, E- PSiNPs, DOX, DOX@PSiNPs, the high dose of DOX@E-PSiNPs (the final concentration of 0.5mg/kg of DOX) or 4mg/kg are free DOX was administered once every 2 days, 5 times in total, and the daily set time measures tumor size.The 17th day, every group after being administered for the first time 8 are randomly selected to simply continue to carry out life cycle experiment.Cervical dislocation put to death mouse, take out tumor tissues, clean, dry after weigh And it takes pictures.
(2) to tumor stem cell fragmentation effect
3, experimental result
Tumor growth curve is the result shows that DOX@E-PSiNPs significantly inhibit tumour growth, tumor inhibitory effect excellent Dissociating in high dose, (Figure 16 A, control group 1 are physiological saline group to DOX administration groups, and control group 2 is E-PSiNPs, and control group 3 is trip From DOX, control group 4 is DOX@PSiNPs, and control group 5 is the free DOX groups of high dose, and experimental group is DOX@E-PSiNPs).Administration After the completion, the weighing results of the tumor tissues of taking-up also indicate that, DOX@E-PSiNPs significantly suppress tumour growth (Figure 16 B, it is right It is physiological saline group according to group 1, control group 2 is E-PSiNPs, and control group 3 is free DOX, and control group 4 is DOX PSiNPs, control Group 5 is the free DOX groups of high dose, and experimental group is DOX@E-PSiNPs).Life cycle result is, it was also found that DOX@E-PSiNPs administrations Later, tumor-bearing mice life span extension 30 days, is significantly better than remaining experimental group, including high dose dissociate DOX groups (Figure 16 C, it is right It is physiological saline group according to group 1, control group 2 is E-PSiNPs, and control group 3 is free DOX, and control group 4 is DOX PSiNPs, control Group 5 is the free DOX groups of high dose, and experimental group is DOX@E-PSiNPs).
Embodiment 11：Fragmentation effect of the nano medicament carrying system of excretion body package to CSCs in rat liver cancer subcutaneous tumors model
1, experiment reagent and material
H22 murine hepatocarcinoma cells, BALB/c mouse
2, experimental procedure
Rat liver cancer subcutaneous tumors model administration is handled according to the method for case study on implementation 10, cervical dislocation after the completion of administration Mouse is put to death, tumor tissues are taken out, cleans and is separated into single tumor cell, carried out using CytoFlex flow cytometers Side group is analyzed, each tumor tissues side population cell ratio after detection different dosing processing.Monodispersed tumour cell is taken to count, with The cell density in 800/hole is seeded in 96 orifice plates of the soft fibre protein adhesives of 3D containing 1mg/mL, 37 DEG C, train under the conditions of 5%CO2 After supporting 5 days, the number and size of tumour cell ball are observed and counted.
3, experimental result
The above result shows that side group cell proportion significantly drops inside DOX E-PSiNPs administration treated tumor tissues It is low, illustrate DOX E-PSiNPs can significantly kill side population cell (Figure 17 A, control group 1 be physiological saline group, control group 2 be E- PSiNPs, control group 3 are free DOX, and control group 4 is DOX@PSiNPs, and control group 5 is the free DOX groups of high dose, and experimental group is DOX@E-PSiNPs).The individual cells of dispersion are seeded in 3D soft fibre protein adhesives and cultivate a period of time, as a result, it has been found that DOX@ The number for the tumor colonies ball that E-PSiNPs groups tumour cell is formed in 3D soft fibre protein adhesives (make a living by Figure 17 B, control group 1 Brine group is managed, control group 2 is E-PSiNPs, and control group 3 is free DOX, and control group 4 is DOX@PSiNPs, and control group 5 is high agent Amount is dissociated DOX groups, and experimental group is DOX E-PSiNPs) and size (Figure 17 C, control group 1 are physiological saline group, and control group 2 is E- PSiNPs, control group 3 are free DOX, and control group 4 is DOX@PSiNPs, and control group 5 is the free DOX groups of high dose, and experimental group is DOX@E-PSiNPs) it significantly reduces, illustrate that DOX@E-PSiNPs can greatly inhibit tumour cell dryness.
Embodiment 12：Inhibition of the nano medicament carrying system of excretion body package to mouse melanin pulmonary metastases
1, experiment reagent and material
Mouse skin cancerous cell line B16, C57 mouse
2, experimental procedure
5×105A B16F10 cell tail vein injections enter in C57BL/6 Mice Bodies.After 2 days, mouse is equally divided into 6 Group, every group 14, tail vein injection PBS, E-PSiNPs, DOX, DOX@PSiNPs, (DOX is final concentration of by DOX@E-PSiNPs 0.5mg/kg) or the high dose of 4mg/kg dissociate DOX, be administered once every 2 days, 5 times in total.The 17th day after being administered for the first time, 8 are randomly selected for every group to simply continue to carry out life cycle experiment.Cervical dislocation puts to death remaining 6 mouse, takes out lung tissue, observation is simultaneously Count lung's melanotic tumor tubercle number.Every group takes 3 lung tissues to be placed in 4%PFA, is handled at 4 DEG C for 24 hours, is sliced and H＆E is dyed Processing.
3, experimental result
DOX@E-PSiNPs are entered by tail vein injection in melanin pulmonary metastases Mice Body, we send out after the completion of administration The lung tumors tubercle number of existing DOX E-PSiNPs administration groups is substantially less than remaining control group, and (Figure 18 A, control group 1 are physiology salt Water group, control group 2 are E-PSiNPs, and control group 3 is free DOX, and control group 4 is DOX@PSiNPs, and control group 5 is swum for high dose From DOX groups, experimental group is DOX@E-PSiNPs).By lung tissue section and H＆E dyeing, we are swollen by micro- sem observation lung Tumor tubercle number has also obtained identical result, and (Figure 18 B, control group 1 are physiological saline group, and control group 2 is E-PSiNPs, control group 3 be free DOX, and control group 4 is DOX@PSiNPs, and control group 5 is the free DOX groups of high dose, and experimental group is DOX@E- PSiNPs).It is tested by survival time of mice, we further demonstrate that there is significant metastatic tumor to inhibit effect by DOX@E-PSiNPs Fruit, DOX E-PSiNPs administration group survival time of mice significantly improve (Figure 18 C, control group 1 be physiological saline group, control group 2 be E- PSiNPs, control group 3 are free DOX, and control group 4 is DOX@PSiNPs, and control group 5 is the free DOX groups of high dose, and experimental group is DOX@E-PSiNPs)。
Embodiment 13：Fragmentation effect of the nano medicament carrying system of excretion body package to CSCs in mouse melanin pulmonary metastases
1, experiment reagent and material
Mouse skin cancerous cell line B16, C57 mouse
2, experimental procedure
Mouse melanin pulmonary metastases are administered according to method in embodiment 12, after the completion of administration, in stem cell media Melanotic tumor tubercle in the middle lung tissue for taking out each administration processing group, is separated into single tumor cell and is counted, with 800 The cell density in a/hole is seeded in 96 orifice plates of the soft fibre protein adhesives of 3D containing 1mg/mL, 37 DEG C, CO2Under the conditions of cultivate 5 days Tumour cell ball size and number are observed afterwards.
3, experimental result
The tumor cell inoculation of dispersion is cultivated in 3D soft fibre protein adhesives, the tumour gram of formation is counted after a period of time The size and number of grand ball, as a result, it has been found that the number for the tumor colonies ball that the tumour cell of DOX@E-PSiNPs processing groups is formed (Figure 19 A, control group 1 are physiological saline group, and control group 2 is E-PSiNPs, and control group 3 is free DOX, and control group 4 is DOX PSiNPs, control group 5 are that high dose dissociates DOX groups, and experimental group is DOX@E-PSiNPs) and size (Figure 19 B, control group 1 are made a living Brine group is managed, control group 2 is E-PSiNPs, and control group 3 is free DOX, and control group 4 is DOX@PSiNPs, and control group 5 is high agent The free DOX groups of amount, experimental group are DOX@E-PSiNPs) it is substantially less than remaining administration group, show that DOX@E-PSiNPs administrations are handled CSCs dryness can be significantly inhibited.
Except employed in above-described embodiment specific cell category and antineoplastic species in addition to, be suitable for the present invention in lead to The medicament-carried nano Materials Cell for crossing outer row's endocytosis can also include tumour cell (acute leukemia, lymthoma, breast cancer, lung cancer, Oophoroma, chorioepithelioma, cervix cancer, liver cancer, carcinoma of urinary bladder, cutaneum carcinoma, colon cancer or the carcinoma of the rectum), tumor stem cell, exempt from Epidemic disease cell (includes T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphocytes, mast cell, mononuclear phagocyte system System), the suppression of the relevant fibrocyte of tumour, mescenchymal stem cell (Mesenchymal stem cells, MSCs), derived from bone marrow Cell (myeloid-derived suppressor cells, MDSCs) processed, regulatory T cells (Treg cells) etc., these are thin Born of the same parents are satisfied by the usual definition of this field；The antitumor drug that medicament-carried nano material loads in advance in the present invention may include that treatment is anxious Property leukaemia, lymthoma, breast cancer, lung cancer, oophoroma, chorioepithelioma, cervix cancer, liver cancer, carcinoma of urinary bladder, cutaneum carcinoma, It is one or more in the chemotherapeutic of colon and rectum carcinoma and various other entity tumors.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to The limitation present invention, all within the spirits and principles of the present invention made by all any modification, equivalent and improvement etc., should all include Within protection scope of the present invention.

Claims (10)

1. the nano medicament carrying system that a kind of excretion body for oncotherapy wraps up, which is characterized in that excretion body package is received Rice drug-loading system is obtained after the extracellular row again using cell endocytic medicament-carried nano material, and the medicament-carried nano material is negative It is loaded with antitumor drug, the antitumor drug includes in the drug of chemotherapeutics, immunotherapy medicaments, reconstruct tumor microenvironment At least one.
2. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that described thin Born of the same parents include tumour cell, tumor stem cell, immunocyte, tumour associated fibroblast cell, mescenchymal stem cell, derived from bone marrow Inhibit at least one of cell and regulatory T cells；The tumour cell and the corresponding tumour packet of the tumor stem cell Include acute leukemia, lymthoma, prostate cancer, thyroid cancer, cancer of the esophagus, osteocarcinoma, gastric cancer, breast cancer, lung cancer, oophoroma, suede Trichilemma epithelioma, cervix cancer, carcinoma of uterine body, liver cancer, carcinoma of urinary bladder, cutaneum carcinoma, colon cancer or the carcinoma of the rectum；The immunocyte packet Include T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphocytes, mast cell or mononuclear phagocyte system.
3. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that the load Nano material in medicine nano material include nano liposomes, c-based nanomaterial, Si-based nanometer material, metal nano material, At least one of semiconductor-quantum-point, up-conversion and polymer nano material；The c-based nanomaterial is preferably wrapped Include at least one of graphene oxide, carbon nanotube, Nano diamond；The Si-based nanometer material preferably include porous silicon, At least one of mesoporous silicon, silicon point；The metal nano material preferably includes Jenner's grain of rice, nano grain of silver, transition metal and receives At least one of grain of rice.
4. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that the load Nano material in medicine nano material is nano-particle material, and the grain size of the nano-particle material is in 1-1000nm.
5. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that the load The antitumor drug of medicine nanomaterial loadings preferably includes treatment acute leukemia, lymthoma, prostate cancer, thyroid cancer, food Road cancer, osteocarcinoma, gastric cancer, breast cancer, lung cancer, oophoroma, chorioepithelioma, cervix cancer, carcinoma of uterine body, liver cancer, carcinoma of urinary bladder, Cutaneum carcinoma, the chemotherapeutics of colon and rectum carcinoma, for immunotherapy medicaments or for being transformed in the drug of tumor microenvironment It is one or more.
6. a kind of preparation method for the nano medicament carrying system that excretion body for oncotherapy wraps up, which is characterized in that including with Lower step：
S1：Medicament-carried nano material is incubated altogether with cell；
S2：Centrifugation removes the medicament-carried nano material that the cell does not absorb；
S3：The fresh culture medium without medicament-carried nano material is added to continue to be incubated；
S4：The medicament-carried nano material extracellularly arranged through this is collected by centrifugation to get receiving to the excretion body package for oncotherapy Rice drug-loading system.
7. the preparation method of the nano medicament carrying system wrapped up as claimed in claim 6 for the excretion body of oncotherapy, feature It is, in the step S1, the time being incubated altogether is 1-96h.
8. the preparation method of the nano medicament carrying system wrapped up as claimed in claim 6 for the excretion body of oncotherapy, feature It is, the step S2 is specifically the cell to be collected with the centrifugal force of 100-1000g, using pre- in a low temperature of 0-10 DEG C Cold phosphate buffer cleans the cell, until there is no free nano medicament carrying systems in solution；
The step S4 is specifically to remove the cell in culture solution under 0-10 DEG C of low temperature with the centrifugal force of 100-1000g, The cell fragment in removal culture solution is centrifuged with 3000-5000g, is obtained with the centrifugal force 30-120min of 10000-20000g To the nano medicament carrying system extracellularly arranged；To get the nanometer wrapped up to excretion body after being cleaned using the phosphate buffer of precooling Drug-loading system.
9. the preparation method of the nano medicament carrying system wrapped up as claimed in claim 6 for the excretion body of oncotherapy, feature It is, in the step S3, the incubation is placed in cell incubator, in 37 DEG C, 5%CO2Cell culture condition under incubate It educates, or makes first to be incubated 12-96h with being positioned in cell incubator again after ultraviolet light 5-240min.
10. prepared by the nano medicament carrying system wrapped up for the excretion body of oncotherapy as described in claim 1-5 any one Application in antitumor drug.
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