CN108671236A - Special target breast cancer cell nanometer drug-loading system and preparation method thereof - Google Patents
Special target breast cancer cell nanometer drug-loading system and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of special target breast cancer cell nanometer drug-loading systems and preparation method thereof, including：After being chemically crosslinked the YSA small peptides of targeting EphA2 receptors and the specific D4 small peptides of targeting EGFR receptor in the mesoporous silicon dioxide nano particle sublist face of synthesis, it is made in loading treatment mammary cancer chemotherapy drug in the duct of mesoporous silicon oxide；It expresses the specific binding of EphA2, EGFR by YSA small peptides, D4 small peptides with breast cancer cell membrane height respectively, realizes the dual-target effect for carrying medicine MSN systems.The present invention has the characteristics that breast cancer specific target tropism, active targeting, drug delivery are reliable and stable, the receptor that drug-loading system high expression different from breast cancer cell membrane is effectively improved by dual-target combines, and the adverse reaction to normal cell is substantially reduced while enhancing anticancer effect.
Description
Technical field
The present invention relates to a kind of MSN drug-loading systems and preparation method thereof of special target breast cancer cell membrane height expression, belong to
In the research and development of cancer cell targeted drug and preparing technical field.
Background technology
Breast cancer is the most common cancer of women and the second largest reason of woman cancer associated death all over the world.In south
Some lesser developed countries in America, Africa and Asia, the incidence of breast cancer are increasing, increased the reason is that life style
Change and new screening sequence.In the U.S. in 2015, new case estimative figure was 234,190, and estimation death toll reaches
To 40 730 people./ 10 to ten/8ths women can obtain breast cancer, the flourishing state including North America and European Union
The breast cancer deaths case of family has reduced, and PYLL analysis is mainly since early detection and efficient system are treated.Chemotherapy
It is one of the standard strategy of breast cancer treatment.Adriamycin (DOX) is one of most common anti-breast cancer medicines, especially metastatic
Breast cancer medicines.But gastrointestinal reaction, myocardium toxicity and other non-specificity is led to due to cytotoxicity caused by drug pair
Effect limits the effect of clinical treatment.The chemotherapeutics of nano-carrier, which is used in combination, can control therapeutic agent in blood transportation mistake
Release in journey discharges again after reaching cancerous tissue.Therefore, adverse reaction is reduced while anticancer effect can be enhanced.
Due to the meso-hole structure that its high ratio/surface area and surface have, mesoporous silicon dioxide nano particle
(mesoporous silica nanoparticles, abbreviation MSN) is the nano-carrier that can effectively carry out drug delivery, is drawn
People have been played greatly to pay close attention to.Eph receptors are the largest receptor tyrosine kinase family, are divided into A types and Type B.Eph receptors are in embryo
Key effect is played in development and the human diseases including cancer.We have found that tyrosine kinase receptor (EphA2) and table
Skin growth factor receptor (EGFR) has a high expression on breast cancer cell membrane, thus using have respectively with EGFR and EphA2
Receptor-specific in conjunction with D4 and YSA small peptides come functionalization carry medicine MSN systems, target combination cell film EGFR and EphA2 high tables
The breast cancer cell reached effectively improves tumor cell surface height by dual-target and expresses EGFR and EphA2 breast cancer cells
Ingestion of medicines amount, realize efficient targeted therapy.
Invention content
Goal of the invention：In order to overcome the deficiencies in the prior art, it is thin that the present invention provides a kind of special target breast cancer
Born of the same parents' height expresses the load medicine MSN systems and preparation method thereof of EphA2 and EGFR, and there is specific target tropism, active targeting, drug to pass
The features such as reliable and stable is sent, the drug that surface height expresses EGFR and EphA2 breast cancer cells is effectively improved by dual-target
Intake realizes efficient targeted therapy.
Technical solution：To achieve the above object, the technical solution adopted by the present invention is：
A kind of special target breast cancer cell nanometer drug-loading system, passes through the mesoporous silicon dioxide nano in synthesis
After particle surface is chemically crosslinked targeting factor D4 polypeptides, in loading anti-breast cancer medicine in the duct of mesoporous silicon dioxide nano particle
Object (generally adriamycin) and be made；It passes through the specificity of D4 polypeptides and the high expressed receptor albumen EGFR in breast cancer cell surface
It is implemented in combination with the targeting for carrying medicine MSN systems；The D4 polypeptide sequences include following sequence：Leu-Ala-Arg-Leu-Leu-
Thr。
Further, the load medicine MSN system surfaces, which are also chemically crosslinked, targeting factor YSA small peptides, by YSA small peptides with
The specific binding of the high expressed receptor albumen EphA2 in breast cancer cell surface realizes that carrying medicine MSN systems further targets work
With；The YSA short peptide sequences include following sequence：
Tyr-Ser-Ala-Tyr-Pro-Asp-Ser-Val-Pro-Met-Met-Ser。
Further, the particle size range for carrying medicine MSN systems is 50-200nm, and dispersion index is in 0.1-0.2.
Further, the anti-breast cancer medicines are adriamycin.
The preparation side of the nanometer drug-loading system of the special target breast cancer cell height expression EphA2 and EGFR
Method includes the following steps：
(1) MSN is synthesized：Weigh 0.3198g CTAB, 2.815ml EtOAc, 545.0ml H2O、9.695ml NH4After OH
Mixing makes CTAB fully dissolve, and the mixed liquor of 0.72ml TEOS and 0.88ml APTES is added and blows and beats 20 times or more and mixes
It is even, it is reacted for 24 hours under the conditions of 22 DEG C；Then 13000rpm rotating speeds centrifuge 30 minutes, ultrasonic ethyl alcohol wash 2 times, obtained sample adds
Enter 240ml ethyl alcohol and the dense HCL of 480ul react 3h under the conditions of 60 DEG C；
(2) MSN surface modifications YSA and D4 polypeptide：The meso-porous titanium dioxide for taking a concentration of 18mg/ml steps (1) of 25 μ L to prepare
YSA the and D4 polypeptides of silicon and a concentration of 1mg/300 μ L of 150 μ L are protected from light stirring for 24 hours, are sufficiently mixed；
(3) chemotherapeutics-adriamycin is loaded：Since YSA and D4 segments contain carboxyl, it is condensed according to amino and carboxyl
The principle of reaction carries out mesopore silicon dioxide nano material surface modification, i.e., the mesoporous silicon oxide material prepared 2mg steps (2)
The adriamycin of material and 0.5mg are dissolved in the phosphate buffer that 1mL pH value is 7.2 (PBS), vibrate mixing overnight at room temperature,
12000rpm rotating speeds centrifuge 30 minutes, discard supernatant liquid.
Advantageous effect：The load medicine MSN systems of special target breast cancer cell height expression EphA2 and EGFR provided by the invention
And preparation method thereof, compared with the existing technology, has the following advantages：
1, preparation process is simple, and stability is good, effectively controls release of the medicine in breast cancer cell tissue, simultaneously
Promote drug release by low ph environment in tumour cell, substantially reduce the adverse reaction to normal cell, realizes to tumour
The efficient and targeting chemotherapeutics of cell transmits；
2, so that drug long-acting can be recycled in blood, drug-loading system and breast cancer cell are effectively improved by dual-target
The receptor of different high expression combines on film, to increase the ingestion of medicines amount of breast cancer cell, enhances anticancer effect.
Description of the drawings
Fig. 1 is the Electronic Speculum phenogram of 1 intermediary hole silica of the embodiment of the present invention；
Fig. 2 is the Zeta potential figures of MSN after surface modification in the embodiment of the present invention 1；
Fig. 3 is adriamycin release efficiency figure of the embodiment of the present invention 2 under condition of different pH；
Fig. 4 is that the embodiment of the present invention 4 inhibits tumor cell proliferation design sketch；
Fig. 5 is the embodiment of the present invention 4 to apoptosis of tumor cells impact effect figure；
Fig. 6 is that breast cancer animal model toxic effect figure is administered in the embodiment of the present invention 5.
Specific implementation mode
The present invention is further described with reference to the accompanying drawings and embodiments.
Embodiment 1：Special target carries preparation and the characterization of medicine MSN systems
Stirring makes CTAB after weighing 0.3198g CTAB+2.815ml EtOAc+545.0ml H2O+9.695ml NH4OH
(0.72mlTEOS+0.88ml APTES) mixed liquor (piping and druming 20 times or more mixing) is added in fully dissolving, and 22 DEG C of reactions are for 24 hours；And
13000rpm rotating speeds centrifugation 30min, ultrasonic ethyl alcohol are washed 2 times afterwards, gained sample+60 DEG C of (the dense HCL of 240ml ethyl alcohol+480ul) reactions
3h.Applied chemistry synthetic method changes the different experimental conditions synthesis size mesoporous silicon dioxide nano different with grain size
Grain, scanning electronic microscope examination nano particle, the prompt of Electronic Speculum inspection result, mesoporous silica particles uniformity, aperture
Size is identical, is evenly distributed consistent (as shown in Figure 1).
It is rich in amino according to meso-porous titanium dioxide silicon face, YSA and D4 small peptides contain carboxyl, and it is anti-that condensation occurs for amino and carboxyl
The principle answered carries out mesopore silicon dioxide nano material surface modification YSA and D4.A concentration of 18mg/ml of 25 μ L are taken to prepare mesoporous
The YSA and D4 of silica and a concentration of 1mg/300 μ L of 150 μ L are protected from light stirring for 24 hours, are sufficiently mixed.
In order to further confirm that whether successfully YSA and D4 have been gone up in modification to meso-porous titanium dioxide silicon face, and We conducted Zata
As a result potential measurement is shown in Fig. 2.Zeta potential testing result is shown, when mesoporous silicon oxide surface modification carboxyl is that its current potential is low
In the mesoporous silicon oxide being not decorated, after surface modification YSA and D4, due to free-NH2 so that mesoporous dioxy
The point of SiClx increases.As a result it prompts in mesoporous silicon oxide surface modification YSA and D4 success.
Embodiment 2：Special target height expresses the external slow release experiment that EGFR carries medicine MSN systems
Confirm that mesoporous silicon oxide (MSN) nano material of same weight under condition of different pH imitates the loading of adriamycin
Rate.Concrete operations are summarized as follows：The adriamycin of the Metaporous silicon dioxide material of 2mg and 0.5mg is dissolved in 1mL difference pH value
In phosphate buffer (PBS), mixing overnight is vibrated at room temperature, and 12000rpm rotating speeds centrifuge 30 minutes, and supernatant is taken to be divided
Photometric determination, mesoporous silicon oxide is in different pH value to the efficiency of loading of same concentrations adriamycin.In neutral meta-alkali
(pH7.4) under approximating anatomy's normal body fluid environment, mesoporous silicon oxide drugloading rate is maximum, and the supernatant medicament contg of centrifugation is most
It is low.When solution is acid (pH5.4) namely close to the environmental pH of inside tumor cells when, mesoporous silicon oxide drugloading rate
Minimum, the adriamycin for being conducive to load in this way is released from carrier.Experimental result in Fig. 3 prompts, under the conditions of pH7.4,
The adriamycin that MSN/DOX/YSA/D4 is released is minimum.
Embodiment 3：The external intake that breast cancer cell MCF7 carries special target medicine MSN systems is tested
Most common tumor chemotherapeutic drug Doxorubicin (Doxorubincin, DOX) also known as adriamycin, this chemotherapeutics
It can be used for acute leukemia, malignant lymphoma, breast cancer, lung cancer, oophoroma, bone and soft tissue sarcoma, the nephroblastoma, wing
Guang cancer, thyroid cancer, prostate cancer, G. cephalantha, carcinoma of testis, gastric cancer, liver cancer etc..This chemotherapeutics has preferable water
Dissolubility, it is most important that this medicine sends out red fluorescence under ultraviolet light irradiation, can be by fluorescence microscope fluorescence intensity
To judge the nano particle situation into cell.
When breast cancer MCF7 cells passage 24 is small, when the fusion rate between cell reaches 80% or so, we add respectively
Enter to load the mesoporous dioxy of the mesopore silicon dioxide nano material of adriamycin and the loading adriamycin of surface modification YSA and D4 small peptide
SiClx nano material after 37 DEG C are continued culture 24 hours, using DAPI dyes nucleus, is entered thin with fluorescence microscope
The mesopore silicon dioxide nano material of born of the same parents adriamycin amount (adriamycin sends out red fluorescence under ultraviolet light irradiation, fluorescence it is strong
What degree represented mesoporous silicon oxide takes the photograph dose).
DAPI is the blue dyes for contaminating nucleus, and adriamycin has the characteristics that autofluorescence, sent out under ultraviolet light
Red fluorescence.The breast cancer cell MCF7 of culture respectively with load drug mesoporous silicon oxide (MSN-DOX) and surface modification
The mesoporous silicon oxide of YSA, D4 small peptide together 37 DEG C culture 24 hours after, confocal microscopy enter intracellular Ah mould
Element amount (red fluorescence intensity).The experimental results showed that the amount that MSN-DOX/YSA/D4 enters cell is significantly more than MSN-DOX.
Embodiment 4：Special target carries the external inhibition Cells Proliferation of Human Breast Cancer of medicine MSN systems and promotes apoptosis effect
We are filled using free adriamycin (DOX), the mesoporous silicon oxide of MTT and cell apoptosis assay detection various concentration
The mesoporous silicon oxide for carrying adriamycin (MSN-DOX) and the surface modification solvable segments of EphA7 loads adriamycin (EphA7-MSN-
DOX after) acting on tumour cell (illustrating using intestinal cancer HCT116 as representative) 48 hours, the survival rate and apoptosis rate of cell are seen
Examine the therapeutic effect of mesoporous silicon oxide drug-loading system (see Fig. 4).
As a result, it has been found that free adriamycin is most strong to the toxic effect of cell, this is because free adriamycin directly into
Enter cell, disposable big metering acts on cell, causes cell death, this effect that can also cause poison is secondary to make to normal cell
With.After mesoporous silicon oxide drug-loading system loading adriamycin enters cell, slow release, under blood of human body neutral environment, no
Adriamycin is released, only enters under tumour cell acidic environment and just starts to discharge adriamycin.This result also confirms that surface is repaiied
Decorations YSA the mesoporous silicon oxide drug-loading systems of D4 segments the meso-porous titanium dioxide that do not modify is better than to the lethal effect of tumour cell
Silicon drug-loading system.
Apoptosis detection is carried out according to kit specification, is mainly included the following steps：
(1) 10 × Binding Buffer are diluted to 1 × Binding Buffer with deionized water；
(2) cell cultivated with the pancreatin digestion without EDTA, PBS are centrifuged 5~10 minutes after washing in room temperature 2000rpm,
Collect cell；
(3) primary with (4 DEG C) resuspension cells of 1 × PBS of precooling, 2000rpm is centrifuged 5 minutes, washs cell；
(4) 1 × Binding Buffer suspension cells of 300 μ L are added；
(5) it after the Annexin V-FITC mixings of 5 μ L being added, is protected from light, is incubated at room temperature 15 minutes；
(6) PI is marked：The PI dyeing for adding 5 μ L in 5 minutes before upper machine.
(7) on before machine, 1 × Binding Buffer of 200 μ L are added.
The results are shown in Figure 5 for apoptosis, and the apoptosis ratio of tumour cell is in mesoporous silicon oxide/DOX and mesoporous silicon oxide
YSA is modified, there are apparent otherness (P between D4 small peptides and blank control group<0.001) meso-porous titanium dioxide of small peptide, is modified
Toxicity of the silicon to breast cancer cell>Simple mesoporous silicon oxide loads adriamycin.
Embodiment 5：Special target carries the internal effect for inhibiting breast cancer cell of medicine MSN systems
Breast cancer cell line (MCF7) establishes animal model.The nude mice for choosing 6 week old is divided into experimental group (DOX/MSN/YSA/
D4) and control group (PBS, phosphate buffer), every group has nude mice 6.After tumor cell culture passes on 24 hours, pancreatin digestion
15mL plastic centrifuge tubes are sucked afterwards, and 900rpm is centrifuged 10 minutes, and PBS is added and cleans 3 times.1640 culture medium 0.5mL, which are added, to be made carefully
Born of the same parents uniformly mix.Cell concentration is counted with blood counting chamber, the inoculated tumour cell of every nude mice is adjusted to 1 × 107Cell.With card
Jie's seedling syringe is by 0.2mL tumor cell injections to nude mice back leg subcutaneous tissue.When tumor volume measurement reaches 20mm3 from naked
Caudal vein starts to inject experimental drug.Mesoporous silicon dioxide nano metering is 10mg/kg weight, and injection in every 5 days is primary.30 days
After put to death nude mice, cubing is carried out to tumor tissues, and carries out histopathologic examination (Fig. 6).The result shows that DOX/MSN/
YSA/D4 is substantially better than blank control group to the therapeutic effect of breast cancer.The result shows that DOX/MSN/YSA/D4 is to breast cancer
Therapeutic effect is substantially better than blank control group.
It is above-mentioned the experimental results showed that, special target height express EphA2 and EGFR carry medicine MSN systems have it is excellent external
Anti-breast cancer therapeutic effect and preferable biological safety, do not show internal organs apparent cytotoxicity.Further
, work(can also be carried out with the D4 polypeptides of high expression EGFR and EphA2 specific bindings and amino acid short peptide YSA respectively using having
Energyization carries medicine MSN systems, and the breast cancer cell of targeting combination cell surface EGFR and EphA2 high expression passes through dual-target
The ingestion of medicines amount for effectively improving the different high expression breast cancer cells in surface, realizes efficient targeted therapy.
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (5)
1. a kind of special target breast cancer cell nanometer drug-loading system, which is characterized in that by mesoporous the two of synthesis
After silicon oxide nanoparticle surface chemistry is crosslinked targeting factor D4 polypeptides, loaded in the duct of mesoporous silicon dioxide nano particle
Anti-breast cancer medicines and be made；The specific binding that EGFR is expressed by D4 polypeptides and breast cancer cell surface height realizes that MSN is carried
The targeting of medicine system；The D4 polypeptide sequences include following sequence：Leu-Ala-Arg-Leu-Leu-Thr.
2. a kind of special target breast cancer cell nanometer drug-loading system according to claim 1, which is characterized in that
The medicine MSN system surfaces that carry also are chemically crosslinked the YSA small peptides for having targeting EphA2 receptors, pass through YSA small peptides and breast cancer cell
The specific binding of surface height expression EphA2, realizes and carries the further targeting of medicine MSN systems；The YSA short peptide sequences packet
Include following sequence：Tyr-Ser-Ala-Tyr-Pro-Asp-Ser-Val-Pro-Met-Met-Ser.
3. special according to a kind of any special target breast cancer cell nanometer drug-loading systems of claim 1-2
Sign is that the particle size range for carrying medicine MSN systems is 50-200nm, and dispersion index is in 0.1-0.2.
4. special according to a kind of any special target breast cancer cell nanometer drug-loading systems of claim 1-2
Sign is that the anti-breast cancer medicines are adriamycin.
5. the preparation method of special target breast cancer cell nanometer drug-loading system according to claim 2, feature
It is, includes the following steps：
(1) MSN is synthesized：Weigh 0.3198g CTAB, 2.815ml EtOAc, 545.0ml H2O、9.695ml NH4It is mixed after OH
Stirring makes CTAB fully dissolve, and the mixed liquor of 0.72ml TEOS and 0.88ml APTES is added and blows and beats 20 times or more mixing,
It is reacted for 24 hours under the conditions of 22 DEG C；Then 13000rpm rotating speeds centrifuge 30 minutes, ultrasonic ethyl alcohol wash 2 times, obtained sample is added
240ml ethyl alcohol and the dense HCL of 480ul react 3h under the conditions of 60 DEG C；
(2) MSN surface modifications YSA and D4 polypeptide：Take mesoporous silicon oxide prepared by a concentration of 18mg/ml steps (1) of 25 μ L and
YSA the and D4 polypeptides of a concentration of 1mg/300 μ L of 150 μ L are protected from light stirring for 24 hours, are sufficiently mixed；
(3) chemotherapeutics-adriamycin is loaded：The adriamycin of Metaporous silicon dioxide material and 0.5mg prepared by 2mg steps (2)
It is dissolved in the phosphate buffer that 1mL pH value is 7.2, vibrates mixing overnight at room temperature, 12000rpm rotating speeds centrifuge 30 points
Clock discards supernatant liquid.
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Application publication date: 20181019 |