CN104174027A - Tumor vessel-tumor cell membrane-cell nucleus continuous targeted drug delivery system, as well as preparation method and application thereof - Google Patents
Tumor vessel-tumor cell membrane-cell nucleus continuous targeted drug delivery system, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a tumor vessel-tumor cell membrane-cell nucleus continuous targeted drug delivery system, as well as a preparation method and application thereof. The tumor vessel-tumor cell membrane-cell nucleus continuous targeted drug delivery system comprises mesoporous silica nano-particles, RGD polypeptide which is linked to the surfaces of the mesoporous silica nano-particles in a covalent manner and serves as a tumor vessel/tumor cell membrane targeting ligand, and a nuclear localization signal polypeptide sequence serving as a cell nucleus targeting ligand. Under the condition of intravenous injection, the drug-loaded system can be enriched in a tumor tissue by means of the targeting effect of the tumor vessel to reduce the uptake of normal tissues and reduce toxic and side effect, is capable of increasing the phagocytosis amount of tumor cells by means of the identification effect on the tumor cell membrane, and can be used for directly delivering anti-cancer drugs into a cell nucleus by means of the delivery performance of the cell nucleus to increase the concentration of effective drugs, so that an optimal treatment effect can be achieved.
Description
Technical field
The invention belongs to medical nano technical field of biological materials, relate to and a kind ofly there is good biocompatibility, high-specific surface area, homogeneous aperture and medicine and support with the medical targeted drug of efficient tumour-specific targeted therapy function and transport system and its preparation method and application.
Background technology
The shortcomings such as the medicine ubiquity of traditional treatment tumor is poor to tumor-selective, and toxic and side effects is large, how designing good drug delivery system becomes study hotspot in recent years.The drug delivery carrier that mesoporous silica nano-particle (MSNs) transports because its excellent medicine supports, controlled release properties is considered to have very much application prospect.Quantity research shows greatly, and the drug delivery system based on MSNs can improve utilization ratio of drug effectively, reduces Normocellular toxic and side effects, has significant cellular level anti-cancer properties.But its live body level is still faced huge challenge to the elimination effect of entity tumor.Its main cause is, body physiological circumstance complication, and especially under by intravenous injection mode administration condition, medicine-carried system will be faced its obstacle in tumor tissues enrichment of multiple obstruction, such as: blood vessel, cell membrane, nuclear membrane etc.Therefore, specifically drug delivery to tumor tissues has been become to the most critical strategy in anticancer nano medical research.Targeted drug transports can guarantee the effective enrichment of medicine at target lesions position, and the potential toxic and side effects of surrounding health tissue is down to minimum.Exploitation effectively can be identified tumor tissues by active targeting, and the drug delivery system of tumor cell and high responsive subcellular organelle is the key of targeted therapy.
Realize the targeted therapy in entity tumor, the problem that first will solve is how to realize medicine-carried system from blood vessel, to permeate specifically in tumor tissues in blood circulation process.At present, be mainly by passive target mechanism, i.e. so-called high infiltration and retention effect (EPR:enhanced permeability and retention).But EPR effect is to organize based on solid tumor that medium vessels is abundant, blood vessel wall gap is wider, poor structural integrity, lymphatic return disappearance, causes macromole class material and lipid granule to have selectivity high-permeability and anelasticity.It is subject to the impact of blood pressure, blood circulation time, the horizontal many factors of NO.So only depend on the passive target effect individual variation of EPR effect large, effect is unstable.Therefore, initiatively target tumor blood vessel is step primary in oncotherapy.Along with the proposition of " tumor growth dependence tumor vessel " theory, tumor vascular targeting treatment becomes a kind of approach novel, quite potential raising tumor efficiency.Angiogenesis is most important to tumor growth, and blood vessel directly contacts with blood, therefore realizes tumor vascular active targeting is probably improved to therapeutic effect.Research shows, integrin alpha
vβ
3there is high expressed on newborn tumor vascular endothelial cell surface, owe on the static endothelial cell membrane surface of normal blood vessels to express.This characteristic makes integrin alpha
vβ
3become the desirable target spot of tumor vascular targeting.And ring-type pentapeptide c (RGDyC) contain arginine-glycine-aspartic acid (Arg-Gly-Asp) can with integrin alpha
vβ
3specific binding, thus tumor growth and new vessels formation suppressed.Since from Pierschbacher reported first in 1984, it was cell recognition site, RGD peptide and derivant thereof just become the focus of various fields research.
Except the targeting on blood vessel level basis, should design the system that transports of the modularity restructuring of energy targeting identification surface of cell membrane receptor, because medicine-carried system enters after tumor tissues, cell membrane is that it is further absorbed maximum obstacle by tumor cell.In order to realize this goal, research worker has been done a large amount of research, and by using specific targeted molecular, as part, antibody etc., modification transports carrier, and exploitation has the efficient drug delivery system compared with high-affinity to tumor cell.It is worth mentioning that surface of cell membrane integrin alpha
vβ
3high expressed is also the speciality of Several Kinds of Malignancy (as breast carcinoma, carcinoma of prostate, ovarian cancer, glioblast cancer and malignant melanoma etc.), and therefore, the pharmaceutical carrier of rgd peptide grafting, as MSNs-RGD can strengthen drug effect greatly.
But, Cytoplasm is not medicine final destination conventionally, because most cancer therapy drugs, as amycin, be to reach to suppress endomitotic duplication of DNA the antitumaous effect that cell growth inhibiting is bred by oxidative damage and the inhibition topoisomerase II of induction activated dna, its action target spot is arranged in nucleus.Nanometer medicine-carried system discharges medicine to maintaining local drug concentration in correct position, realizes enhancing drug effect extremely important.Therefore, directly subcellular organelle, the especially nucleus to more fragile sensitivity by drug delivery of development, has very large meaning to significantly strengthening entity tumor curative effect.It is nucleus nuclear membrane that effective nucleus transports last obstacle, Cytoplasm and the exchange of nuclear all substances are all undertaken by the nucleopore being distributed on nuclear membrane, and external heterojunction structure must just likely enter in nucleus by nuclear localization signal (NLS) and nucleopore generation recognition reaction.Previous work shows, HIV activity factor TAT polypeptide is effective NLS, and suitable nano-particle enters in nucleus can efficiently to mediate particle diameter.
Summary of the invention
From discussing above, in order to ensure the effectively targeting specific treatment of intravenously administrable and tumor, develop multi-level targeting strategy and be very important.Therefore, the object of the present invention is to provide a kind of mesoporous silicon-dioxide-substrate drug delivery system with good biocompatibility, high-performance inhibition tumor growth and its preparation method and application, thereby under intravenous injection condition, realizing the omnidistance targeted drug of tumor vessel-tumor cell membrane-nucleus transports, overcome the toxic and side effects that cancer therapy drug is huge, the drug delivery system of performance better and safer is provided for realizing the efficient treatment of clinical specificity tumor.
On the one hand, the invention provides the continuous targeted drug of a kind of tumor vessel-tumor cell membrane-nucleus and transport system, comprise that mesoporous silica nano-particle and covalency are linked in the rgd peptide as tumor vessel/tumor cell membrane targeting part on described mesoporous silica nano-particle surface (being c (RGDyC): cyclo (Arg-Gly-Asp-D-Tyr-Cys)) and the nuclear localization signal peptide sequence as cell nucleus targeting part.
The present invention is at the surface of the mesoporous silica nano-particle as pharmaceutical carrier grafting tumor vessel/tumor cell membrane targeting part and cell nucleus targeting part simultaneously.First, utilize the neovasculature of tumor tissues, by RGD targeting identification tumor vessel, strengthen picked-up and the hold-up of tumor tissues.Secondly, the targeting combination by RGD to tumor cell membrane, promotes tumor cell endocytosis.Finally, to nuclear targeting, medicine cross-docking, to the nucleus of tumor Gao Min, is improved to active drug concentration in nucleus by nuclear localization signal peptide sequence.This continuous target administration strategy is expected to realize the omnidistance targeting under intravenously administrable, will farthest strengthen cancer cell death.
Preferably, described nuclear localization signal peptide sequence is TAT polypeptide, SV40T antigen, adenovirus etc.
Preferably, between described rgd peptide and described mesoporous silica nano-particle surface, go back chain and be connected to PEG.By first linking PEG on mesoporous silica nano-particle surface, link rgd peptide at the end of the chain of PEG again, thereby can improve the biocompatibility of nano-particle, reduce toxic and side effects and improve its blood circulation performance, promote the distribution of nano-particle in tumor tissues.
Preferably, the particle diameter of described mesoporous silica nano-particle is 20～50nm.By making mesoporous silica nano-particle there is suitable particle diameter, thereby can link enough tumor vessel/tumor cell membrane targeting part and cell nucleus targeting part, make this drug delivery system can pass through the nucleopore on nucleus nuclear membrane simultaneously, enter nucleus.In addition, the aperture of described mesoporous silica nano-particle can be 2～20nm.
On the other hand, the invention provides the continuous targeted drug of above-mentioned tumor vessel-tumor cell membrane-nucleus and transport the preparation method of system, comprise the following steps:
(1) mesoporous silica nano-particle and the silane coupler of end band amino are reacted to obtain the mesoporous silica nano-particle of surface band amino;
(2) mesoporous silica nano-particle of the surface band amino of gained is reacted to obtain surface graft with nuclear localization signal peptide sequence and NHS-PEG-MAL and have the mesoporous silica nano-particle of nuclear localization signal peptide sequence and PEG chain;
(3) surface graft of gained there is is the mesoporous silica nano-particle of nuclear localization signal peptide sequence and PEG chain react with rgd peptide with at PEG end of the chain grafting rgd peptide, thereby obtain the chain modified mesoporous silicon-dioxide-substrate medicine-carried system of dual peptide.
The present invention passes through multistep polypeptide modification technology at the surface of mesopore silicon dioxide nano material success grafting targeting part, tumor vessel/tumor cell membrane targeting part rgd peptide and cell nucleus targeting part covalency are linked to extra small mesoporous silica nano-particle (MSNs) surface of high degree of dispersion, synthesis technique is simple, without any pollution, output is high, efficiency is high, suitability for industrialized production very easily.Prepared nano material has extraordinary dispersibility and stability, in the diagnosis of disease and treatment, is with a wide range of applications.
Preferably, in step (1), the silane coupler of described end band amino is the amino silicane coupling agents such as 3-aminopropyl triethoxysilane (APTES), 3-aminopropyl trimethoxysilane, N-2-aminoethyl-3-aminopropyl methyl dimethoxysilane.
Preferably, in step (2), the mol ratio of described nuclear localization signal peptide sequence and described NHS-PEG-MAL is (1～100): 1.
On the one hand, the invention provides the continuous targeted drug of above-mentioned tumor vessel-tumor cell membrane-nucleus and transport system in the application of preparing in targeted anticancer medicine again.
Preferably, elecrtonegativity cancer therapy drug is supported in mesopore orbit by the electrostatic adsorption that transports the mesoporous silica particles inwall in system with described continuous targeted drug.
Preferably, the cancer therapy drug that action site is arranged in nucleus comprises amycin, cisplatin, camptothecine and some photosensitizer, as chlorin (Chorin e6), ZnPc, porphyrin etc.
The present invention by a kind of simple surperficial polypeptide modification technology tumor vessel/tumor cell membrane targeting part rgd peptide and the grafting of cell nucleus targeting part TAT polypeptide on MSNs surface, realize efficiently supporting of cancer therapy drug by means of the large specific surface area of MSNs, high pore volume and uniform pore size.In the present invention, mesoporous silica particles cancerous cell identification ability and the nucleus ride-through capability as pharmaceutical carrier given in the grafting of two peptide species (tumor vessel/tumor cell membrane targeting part and cell nucleus targeting part), thereby greatly improve targeting efficiency, strengthen chemotherapy effect, reduced toxic and side effects.This drug delivery system that shows In vitro cell experiment can effectively realize specificity cancerous cell cell nucleus targeting drug delivery, improves active drug concentration in nucleus, improves drug effect.Importantly, this continuous targeted drug transports system and has demonstrated high efficiency anti-tumor effect in live body level.Under intravenous injection condition, first this medicine-carried system realizes tumor vascular targeting effect, is enriched in tumor tissues, reduces the picked-up of normal structure, reduces toxic and side effects; By the recognition reaction to tumor cell membrane, increase the amount of engulfing of tumor cell and then; Finally transport performance by nucleus again, cancer therapy drug is directly transported in nucleus, improve active drug concentration, reach best therapeutic effect.And, synthesis technique of the present invention is simple, without any pollution (non-pollutant discharge), output is high, efficiency is high, excellent performance, the continuous targeted drug obtaining transports system and has excellent anti-cancer properties, and normal tissue is the obvious toxic and side effects of tool not, be the drug delivery carrier that has application prospect, therefore have broad application prospects.
Brief description of the drawings
Fig. 1 is the synthetic schematic diagram that continuous targeted drug of the present invention transports system;
Fig. 2 is the preparation flow figure that continuous targeted drug of the present invention transports system;
Fig. 3 is the TEM photo of the continuous every step product of targeting vector building-up process in example of the present invention, wherein, and b)～i) be respectively: b) MSNs, c) MSNs-NH
2, d) MSNs-PEG, e) and MSNs-RGD, f) MSNs-TAT, g) MSNs-RGD/TAT, h) low power MSNs, i) high power MSNs;
Fig. 4 is the uv-vis spectra of the MSNs-RGD/TAT of an example of the present invention;
Fig. 5 (a) is the burnt displaing micro picture of copolymerization of the cell targeted external qualitative sign of the MSNs-RGD/TAT of an example of the present invention;
Fig. 5 (b) is the distribution in Cytoplasm and nucleus of the Si element of cell targeted external quantitatively characterizing of the MSNs-RGD/TAT of an example of the present invention;
Fig. 6 (a) is that the MSNs-RGD/TAT of an example of the present invention distributes by tail vein injection to the body after in mice with tumor body 24 hours;
Fig. 6 (b) is that the MSNs-RGD/TAT of an example of the present invention distributes by tail vein injection to the body after in mice with tumor body 48 hours;
Fig. 6 (c) is the MSNs-RGD/TAT of an example of the present invention distribution in tumor tissue cell's core;
Fig. 7 (a) is by burnt the copolymerization in cell after the MSNs-RGD/TAT load anticancer drugs, doxorubicin (DOX) of an example of the present invention displaing micro picture, the burnt displaing micro picture of figure (i) copolymerization wherein, figure (ii) is the line sweep figure corresponding with it; A in figure
1-a
5be respectively: a
1) free DOX, a
2) DOXMSNs, a
3) DOXMSNs-RGD, a
4) DOXMSNs-TAT, a
5) DOXMSNs-RGD/TAT;
Fig. 7 (b) is by the quantitatively characterizing of the distribution in Cytoplasm and nucleus after the MSNs-RGD/TAT load anticancer drugs, doxorubicin (DOX) of an example of the present invention;
Fig. 8 is by the scattergram in tumor tissues after the MSNs-RGD/TAT load anticancer drugs, doxorubicin (DOX) of an example of the present invention;
Fig. 9 (a) be by the DOXMSNs-RGD/TAT of an example of the present invention under different pharmaceutical concentration with co-culture of cells cell survival rate after 24 hours;
Fig. 9 (b) be by the DOXMSNs-RGD/TAT of an example of the present invention under different pharmaceutical concentration with co-culture of cells cell survival rate after 48 hours;
Figure 10 (a) is the change curve of mice with tumor gross tumor volume after the DOXMSNs-RGD/TAT treatment of an example of the present invention;
Figure 10 (b) is the digital photograph that above-mentioned tumor changes, and wherein 1-6 represents respectively matched group, free DOX, DOXMSNs, DOXMSNs-RGD, DOXMSNs-TAT, DOXMSNs-RGD/TAT.
Detailed description of the invention
Further illustrate the present invention below in conjunction with accompanying drawing and following embodiment, should be understood that accompanying drawing and following embodiment are only for the present invention is described, and unrestricted the present invention.
One aspect of the present invention provides the continuous targeted drug of a kind of tumor vessel-tumor cell membrane-nucleus to transport system, this continuous targeted drug transports system taking mesoporous silica nano-particle as medicine carrying matrix, at its surface graft tumor vessel/tumor cell membrane targeting part and cell nucleus targeting part.This carrier cancerous cell identification ability and nucleus ride-through capability are given in the grafting of two peptide species, thereby have greatly improved targeting efficiency, have strengthened chemotherapy effect, have reduced toxic and side effects.Wherein, tumor vessel/tumor cell membrane targeting part can be can with the integrin alpha on tumor vascular endothelial cell surface and cancerous cell surface
vβ
3the polypeptide of specific binding, for example rgd peptide.Cell nucleus targeting part can be nuclear localization signal peptide sequence, includes but not limited to TAT polypeptide, SV40T antigen, adenovirus etc.
Fig. 1 illustrates that continuous targeted drug of the present invention transports the synthetic schematic diagram of system, as shown in the end product in Fig. 1, transport in system at continuous targeted drug of the present invention, cell nucleus targeting part can directly graft in the surface of mesoporous silica nano-particle, and tumor vessel/tumor cell membrane targeting part can graft in by PEG chain the surface of mesoporous silica nano-particle.By thering is PEG, can improve the biocompatibility of nano-particle, reduce toxic and side effects and improve its blood circulation performance, promote the distribution of nano-particle in tumor tissues.
Between each part and mesoporous silica nano-particle surface and PEG chain, can be connected by covalent bond.Particularly, can introduce amino on mesoporous silica nano-particle surface, cell nucleus targeting part and this amino form amido link and are linked in mesoporous silica nano-particle surface.PEG chain can be linked in mesoporous silica nano-particle surface with N-hydroxy-succinamide (NHS) with the amino covalent bond that reacts formed on mesoporous silica nano-particle surface by what be connected in its one end, and is linked in the end of PEG by being connected in covalent bond that the maleimide (MAL) of its other end reacts formed with sulfydryl on rgd peptide.
Particle diameter as the mesoporous silica nano-particle of pharmaceutical carrier can be 20～50nm.Like this, both can link enough tumor vessel/tumor cell membrane targeting part and cell nucleus targeting part, can pass through again the nucleopore on nuclear membrane, and make this drug delivery system can enter nucleus.
Again, the aperture of mesoporous silica nano-particle can be 2～20nm.So not only can support micromolecular cancer therapy drug, also can support large-sized gene and equal in mesopore orbit.
The present invention also provides the continuous targeted drug of above-mentioned tumor vessel-tumor cell membrane-nucleus to transport the preparation method of system.Fig. 1 illustrates that continuous targeted drug of the present invention transports the synthetic schematic diagram of system, and Fig. 2 illustrates that continuous targeted drug of the present invention transports the preparation flow figure of system.As shown in Figure 1, 2, preparation method of the present invention mainly comprises: surface amination, the grafting of core targeting part and the grafting of blood vessel/cell membrane targeting part of mesoporous silicon dioxide nano.Wherein, in the time of grafting core targeting part, can carry out the grafting of PEG chain, to save step, simple flow simultaneously.It should be understood that, can be also the grafting of first carrying out core targeting part, then carry out the grafting of PEG chain; Or first carry out the grafting of PEG chain, then carry out the grafting of core targeting part.In addition, also can first grafting blood vessel/cell membrane targeting part, remarry and connect core targeting part.Make continuous targeted drug and transport after system, can also support therein cancer therapy drug, obtain the chain modified mesoporous silicon-dioxide-substrate medicine-carried system of dual peptide.
Particularly, as shown in Figure 2, the preparation process of whole material can be subdivided into four steps, exemplifies as follows: first adopt surfactant guiding, alkali (or acid) catalysis and silicon source polycondensation-polymerization process to prepare the mesoporous SiO of particle diameter controlled (about 30nm), good dispersion
2nano-particle (step 1).Due to mesoporous SiO
2nano grain surface has the outstanding key of abundant silicon hydroxyl, after removing surfactant, select a kind of silane coupler (as amino silicane coupling agents such as 3-aminopropyl triethoxysilane-APTES, 3-aminopropyl trimethoxysilane, N-2-aminoethyl-3-aminopropyl methyl dimethoxysilanes) of end band amino, at a certain temperature with mesoporous SiO
2after nanoparticle reaction a period of time, centrifugal go out the mesoporous SiO of outer surface belt amino
2nano-particle (step 2).After fully washing with deionized water, by amidation process grafting TAT polypeptide amino and carboxyl, react grafting NHS-PEG by amino and N-hydroxy-succinamide (NHS)
1500-MAL (step 3).After further fully washing, react grafting rgd peptide (step 4) by maleimide with sulfydryl on rgd peptide.Realize medicine by the electrostatic interaction of mesopore orbit and cancer therapy drug again and efficiently support, thereby obtain the chain modified mesoporous silicon-dioxide-substrate medicine-carried system (step 5) of dual peptide.In the present invention, the cancer therapy drug that action site is arranged in nucleus includes but not limited to amycin, cisplatin, camptothecine and some photosensitizer, as chlorin (Chorin e6), ZnPc, porphyrin etc.
Above-mentioned having exemplified by surfactant and sol-gel process prepared mesoporous SiO
2nano-particle, but should be understood that the present invention does not limit mesoporous SiO
2the preparation method of nano-particle, as long as can obtain having the mesoporous SiO in appropriate particle size and aperture
2nano-particle.
The invention provides a kind of simple to operate, eco-friendly method synthesize there is good dispersion, good biocompatibility, uniform particle diameter is controlled, outer surface has nuclear localization signal peptide sequence (for example TAT) and rgd peptide mesoporous SiO
2nanoparticle.The diameter of nanoparticle is that 20～50nm is controlled, and the aperture on wall is that 2～20nm is controlled.Below, taking the chain modified mesoporous silicon-dioxide-substrate medicine-carried system (DOXMSNs-RGD/TAT) of dual peptide as example, an example of its preparation method is described.
(1) the synthetic mesoporous SiO of small particle diameter
2nanoparticle: this building-up process using deionized water as solvent, triethanolamine is as base catalyst, reacts the regular hour at a certain temperature, obtains controlled, the monodispersed mesoporous SiO of particle diameter
2nanoparticle.Roughly process is as follows: a certain amount of triethanolamine deionized water wiring solution-forming, add a certain amount of surfactant hexadecyltrimethylammonium chloride (CTAC) solution, at 95 DEG C, after vigorous stirring a period of time, dropwise add a certain amount of ethyl orthosilicate (TEOS).After magnetic stirring a period of time, can observe the solution shape (MSNs) that is creamy white.To after product collection, fully wash and remove unreacted material.
(2) mesoporous SiO
2nanoparticle surface is amino modified: this process is carried out chemical reaction using ethanol as solvent, reacts at a certain temperature after the regular hour, can be by the silane coupler grafting with amino to mesoporous SiO
2nanoparticle surface (obtains MSNs-NH
2).Roughly process is as follows: a certain amount of mesoporous SiO
2nanoparticle is dispersed in ethanol, stirs a period of time at a certain temperature.Then add a certain amount of APTES, centrifugal collection product after backflow certain hour, dehydrated alcohol obtains amidized mesoporous SiO after fully washing
2nanoparticle.
(3) the mesoporous SiO of amination
2nanoparticle surface TAT polypeptide modification: by amidized mesoporous SiO
2nanoparticle is dispersed in deionized water, the TAT polypeptide and the NHS-PEG that add a certain amount of EDC/NHS to activate
1500-MAL, reacts certain hour and obtains the mesoporous SiO of surface graft TAT polypeptide and PEG chain at a certain temperature
2nanoparticle.The TAT polypeptide and the NHS-PEG that wherein added
1500the mol ratio of-MAL can be (1～100): 1.Adopt EDC/NHS as condensing agent herein, but the invention is not restricted to this, other suitable condensing agent also can be suitable for.In addition, these two ends that are in PEG introduce NHS respectively and MAL carry out difunctionalization, but the invention is not restricted to this, also can introduce other functional group, as long as can make PEG form covalent bond in its one end with amino reaction, and react formation covalent bond with sulfydryl at its other end.Again, PEG is also not limited to PEG
1500, also can use PEG
2000, PEG
3500deng.
(4) mesoporous SiO
2nanoparticle surface rgd peptide modification: mesoporous SiO surface to TAT polypeptide and PEG chain
2nanoparticle is dispersed in deionized water, adds a certain amount of rgd peptide, reacts at a certain temperature certain hour, at PEG end of the chain grafting rgd peptide, obtains the mesoporous SiO of surface while grafting TAT polypeptide and rgd peptide
2nanoparticle (MSNs-RGD/TAT).
(5) model cancer therapy drug supports: selecting electronegative amycin (DOX:doxrubicin) is model drug, a certain amount of MSNs-RGD/TAT is disperseed in the PBS solution of a certain amount of DOX, after magnetic agitation certain hour, obtain at a certain temperature the chain modified mesoporous silicon-dioxide-substrate medicine-carried system (DOXMSNs-RGD/TAT) of dual peptide.
In above-mentioned preparation method, be first that structure directing agent, alkali are catalyst by surfactant, prepare particle diameter in about 30nm by sol-gel process, aperture is 2.7nm, have enrich pore passage structure mesoporous SiO
2nanoparticle (MSNs).Then by reacting between the outstanding key of the abundant silicon hydroxyl of nano grain surface and silane coupler, uniform amidized silane coupler grafting is arrived to nano grain surface.With the amidation process of TAT polypeptide and double-functional group PEG, TAT polypeptide and PEG success grafting are arrived to nano-particle outer surface by amino.Nano-particle outer surface is arrived in rgd peptide grafting by reaction by the sulfydryl of further rgd peptide and the maleimide of the PEG end of the chain, forms the dual polypeptide function nano carrier of grafting modification altogether.Finally, by the electrostatic adsorption of cancer therapy drug and mesopore orbit, realize efficiently supporting of medicine, obtain having the drug delivery system of continuous target function, this material is a kind of novel nanometer therapeutic agent.
The continuous targeting vector building-up process that Fig. 3 is illustrated in an example of the present invention (is the mesoporous SiO of small particle diameter MCM-41 type
2nanoparticle (MSNs) carries out surface amination through silane coupling A PTES, then progressively grafting cell nucleus targeting part TAT polypeptide and tumor vessel/tumor cell membrane targeting part rgd peptide, obtains having the pharmaceutical carrier MSNs-RGD/TAT of continuous target function) in the TEM photo of every step product.Therefrom can find out, the synthetic product of every step all has good dispersibility.
Fig. 4 illustrates the uv-vis spectra of MSNs-RGD/TAT.As can be seen from the figure, MSNs-RGD/TAT has TAT and rgd peptide simultaneously and lays respectively at the characteristic absorption peak of 495nm and 275nm, has proved the successful grafting of this two peptide species.
(biological property test)
MSNs-RGD/TAT of the present invention and the continuous targeting vector MSNs-RGD/TAT (DOXMSNs-RGD/TAT) that supports doxorubicin hydrochloride are carried out to biological property test, and analyze its result.
1, the Targeting Performance of the chain modified MSNs-RGD/TAT of dual peptide test:
1) external qualitative sign: first the good MSNs-RGD/TAT of FITC labelling is distributed to (100 μ g/mL) in cell culture medium.Seed cells into again in Laser Scanning Confocal Microscope special culture dish, in the time that cell density grows to 60%-70%, remove culture medium, add the cell culture medium that contains MSNs-RGD/TAT nanoparticle to cultivate altogether again 24h.Remove culture medium, wash not by cytophagic nanoparticle with PBS solution as far as possible.Finally use DAPI reagent transfect cell core, transfer under Laser Scanning Confocal Microscope and observe.
2) external quantitatively characterizing: will first the good MSNs-RGD/TAT of FITC labelling be distributed to (100 μ g/mL) in cell culture medium.Seed cells into again in the culture dish of 10cm, in the time that cell density grows to 60%-70%, remove culture medium, add the cell culture medium that contains MSNs-RGD/TAT nanoparticle to cultivate altogether again 24h.Remove culture medium, wash not by cytophagic nanoparticle with PBS solution as far as possible.Then use 0.25% trypsin digestion cell, centrifugal collecting cell.Then add 3mL cell pyrolysis liquid, cell lysis under ultrasound condition.Finally measure the content of Si in cell by ICP-AES.In order to measure Si content in nucleus, after incubation same as described above, trypsinization, centrifugal collecting cell.Then with 5 × 10
6the concentration of cells/mL adds nucleus separating medium (NaCl (5.845g), EDTA (0.292g), Triton X-100 (10g) are dissolved in Tris buffer (1L), mix and stir evenly).At 4 DEG C, leave standstill 10min, with the centrifugal 15min of 800rpm, obtain nucleus.Then add cell pyrolysis liquid cell lysis, then measure Si content in nucleus by ICP-AES.And in cell in total Si content and nucleus the difference of Si content be the content of Si in Cytoplasm.
3) in body, characterize: continuous targeting vector MSNs-RGD/TAT is distributed to (100 μ g/mL) in PBS, arrives (dosage: 20mg kg in mice with tumor body by tail vein injection
-1).After 24h, tumor tissues and main organs (conscience spleen lung kidney) are put to death and taken out to disconnected mice neck, after microwave ablation, use the Si content in IPC-AES quantitative tissue.
Fig. 5 (a) and Fig. 5 (b) illustrate the cell targeted vitro characterization of MSNs-RGD/TAT.Wherein Fig. 5 (a) is the burnt displaing micro picture of copolymerization of qualitative sign, and nucleus is dyeed by DAPI and presents blueness (point in Fig. 5 (a) little figure in the upper left corner).MSNs-RGD/TAT is presented green (point in the little figure of Fig. 5 (a) Zuo little Jiao) by FITC labelling.From picture, can find out that green fluorescence appears in Cytoplasm, prove that MSNs-RGD/TAT nanoparticle can successful targeted cells core.Fig. 5 (b) is the distribution in Cytoplasm and nucleus of the Si element of quantitatively characterizing.From figure, can further prove that MSNs-RGD/TAT can be at rgd peptide to HeLa surface of cell membrane integrin alpha
vβ
3specific recognition effect under realize efficient cell-targeting effect, the Si content in its whole cell improves greatly; And then under the effect of core targeting part TAT polypeptide, MSNs-RGD/TAT can pass through nucleus nuclear membrane, to realize in nuclear enrichment, in its nucleus, Si content, much larger than other groups, has shown the cancerous cell target function of MSNs-RGD/TAT excellence.
Fig. 6 (a)～Fig. 6 (c) illustrates interior sign of cell targeted body of the MSNs-RGD/TAT of an example of the present invention.Wherein Fig. 6 (a) and Fig. 6 (b) be respectively this MSNs-RGD/TAT by tail vein injection to the body distribution after in mice with tumor body 24 hours and 48 hours, as can be seen from the figure, MSNs-RGD/TAT nano-particle under continuous targeting efficiently concentrating in tumor tissues, effectively improve specificity, reduce the non-specific uptake of normal organ (conscience spleen lung kidney) to nano-particle, thereby be expected to reduce the toxic and side effects to health tissues.Fig. 6 (c) is the distribution of MSNs-RGD/TAT in tumor tissue cell's core, as can be seen here, MSNs-RGD/TAT can efficiently pass through nucleus nuclear membrane under TAT effect, is enriched in nucleus, its Si content, far above other groups, has been realized cell nucleus targeting effect in live body level.
2, the targeted drug of DOXMSNs-RGD/TAT transports performance test:
1) external qualitative sign: the MSNs-RGD/TAT (DOXMSNs-RGD/TAT) that has been coated doxorubicin hydrochloride (DOX) is distributed to (doxorubicin concentration: 1 μ g/mL) in cell culture fluid.Seed cells into again in the burnt culture dish of copolymerization, until cell density grows to 80-90%, remove cell culture fluid, add and disperseed the cell culture fluid of DOXMSNs-RGD/TAT to cultivate altogether 4h.Use again DAPI transfect cell core, finally transfer under Laser Scanning Confocal Microscope and observe.
2) external quantitatively characterizing: the MSNs-RGD/TAT (DOXMSNs-RGD/TAT) that has been coated doxorubicin hydrochloride (DOX) is distributed to (doxorubicin concentration: 5.6 μ g/mL) in cell culture fluid.Seed cells into again in the culture dish of 10cm, until cell density grows to 80-90%, remove cell culture fluid, add and disperseed the cell culture fluid of DOXMSNs-RGD/TAT to cultivate altogether 24h.Collecting cell, measures the amount of cancer therapy drug DOX in nucleus by standard curve method after separating nucleus.
3) characterize in body: the MSNs-RGD/TAT (DOXMSNs-RGD/TAT) that has been coated doxorubicin hydrochloride (DOX) is distributed in PBS solution, by tail vein injection to mice with tumor body.After 24h, tumor tissues is put to death and taken out to disconnected mice neck, carry out the distribution in tumor tissues at fluorescence microscopy Microscopic observation medicine after tumor vessel dyeing with FITC-CD31.
Fig. 7 (a) and Fig. 7 (b) illustrate the distribution in cell after MSNs-RGD/TAT load anticancer drugs, doxorubicin (DOX).In Fig. 7 (a), blueness is DAPI staining cell core, and redness is cancer therapy drug DOX.Wherein a
1-a
5be respectively: a
1) free DOX, a
2) DOXMSNs, a
3) DOXMSNs-RGD, a
4) DOXMSNs-TAT, a
5) DOXMSNs-RGD/TAT.From figure, can find out significantly a of Fig. 7 (a)
5in, the red fluorescence of medicine and nuclear blue-fluorescence are overlapping, illustrate that DOXMSNs-RGD/TAT can carry out cell nucleus targeting drug delivery effectively.The amount of DOX in further quantitative Cytoplasm and nucleus.Fig. 7 (b) shows, free DOX and HeLa co-culture of cells 24h still only have (54.6ng Dox/106cells) to be on a small quantity distributed in nucleus.Due to the active targeting of RGD to HeLa cell, HeLa cell and DOXMSNs-RGD cultivate medication amount (the 554ng DOX/10 in rear Cytoplasm altogether
6) have significant improvement with respect to free DOX, but nucleus Chinese medicine amount (86.5ng DOX/10
6cells) only has faint raising.Due to the active cell nucleus targeting effect of TAT polypeptide mediation, can effectively realize the nucleus transhipment of DOXMSNs-TAT, in nucleus, DOX drug level improves (506.7ng DOX/10 greatly
6cells).And under RGD and the combined effect of TAT polypeptide, nucleus Chinese medicine concentration is increased to 765.5ng DOX/10
6cells, is 14 times of free DOX, has shown the cell nucleus targeting drug delivery performance of continuous targeted drug transmission system excellence.
Fig. 8 illustrates the distribution in tumor tissues after MSNs-RGD/TAT load anticancer drugs, doxorubicin (DOX).The tumor vessel of FITC-CD31 labelling presents green (in figure brighter color), and cancer therapy drug DOX presents redness (in figure darker color).As can be seen from Figure, continuous target drug-carrying system can realize efficiently continuously targeted drug and transport in live body, drug distribution scope is wide, from blood vessel, be successfully penetrated in tumor tissues, embodied the continuous targeted drug of tumor vessel-tumor cell-neoplastic cell nuclei and transport the unique drug delivery performance of system.
3, the efficient anti-cancer properties of continuous target drug-carrying system (DOXMSNs-RGD/TAT)
1) vitro characterization: the MSNs-RGD/TAT (DOXMSNs-RGD/TAT) that has been coated doxorubicin hydrochloride (DOX) is distributed in cell culture fluid.Under different DOX concentration, cultivate respectively after 24h and 48h, detect cell survival rate.
2) in body, characterize: by DOXMSNs-RGD/TAT (dosage: 2mg kg
-1) enter in tumor-bearing mice body by intravenous injection, every other day measure gross tumor volume, weigh Mouse Weight.
Fig. 9 (a) and Fig. 9 (b) illustrate by DOXMSNs-RGD/TAT under different pharmaceutical concentration with co-culture of cells 24h and 48h after cell survival rate.By can obviously finding out in figure, DOXMSNs-RGD/TAT has the strongest cytotoxicity, and after itself and cytosis, cell survival rate is far below matched group.
Figure 10 (a) and Figure 10 (b) illustrate the chemotherapy effect evaluation of DOXMSNs-RGD/TAT in live body level, wherein Figure 10 (a) is the change curve of mice with tumor gross tumor volume after treatment, the digital photograph that Figure 10 (b) changes for tumor.Can find out, after continuous targeted drug transports system effect, tumor growth has obtained obvious inhibition, and its gross tumor volume has reduced 89% and reached extraordinary therapeutic effect.
The present invention adopts the surperficial polypeptide modification technology of multistep, tumor vessel/tumor cell membrane targeting part rgd peptide and cell nucleus targeting part TAT polypeptid covalence are linked to extra small mesoporous silica nano-particle (MSNs) surface of high degree of dispersion, realize efficiently supporting of cancer therapy drug by means of the large specific surface area of MSNs, high pore volume and the aperture of homogeneous.After this medicine-carried system tail vein injection is entered in mice with tumor body, medicine-carried system can initiatively be realized the enrichment at tumor tissues, and further by drug delivery to nucleus.By repeat administration, can effectively reduce tumor size, reach the object of high-efficient cleaning except malignant tumor.
Further exemplify embodiment below to describe the present invention in detail.Should understand equally; following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.The technological parameter that following example is concrete etc. is only also an example in OK range, and those skilled in the art can be done in suitable scope and be selected by explanation herein, and do not really want to be defined in the below concrete numerical value of example.
Embodiment 1
The synthetic mesoporous SiO of small particle diameter MCM-41 type
2nanoparticle (MSNs): 2g hexadecyltrimethylammonium chloride (CTAC) is dissolved in 20g deionized water.After solution becomes clarification, add 0.8g triethanolamine (TEB), vigorous stirring under 95 DEG C of conditions.After solution becomes clarification, dropwise add the ethyl orthosilicate (TEOS) of 1.5mL, and continuous stirring 1h.Product is cooled to after room temperature, adds dehydrated alcohol, makes product wadding heavy, and centrifugal collection product is also used absolute ethanol washing 3 times;
Utilize the alcoholic solution of hydrochloric acid to extract the surfactant in mesopore orbit: the mesoporous SiO that does not remove surfactant that takes 100mg
2nano-particle (MSNs), in the ultrasonic alcoholic solution (10%) that is dispersed in hydrochloric acid, 6h refluxes under 78 DEG C of conditions.Finally, centrifugal collection product, fully washs with dehydrated alcohol.Repeat this extraction process for several times, until CTAC is completely removed.
Embodiment 2
MSNs surface amino groups modification: take MSNs50mg in embodiment 1, ultrasonic being dispersed in 100mL dehydrated alcohol, 78 DEG C of constant temperature strong stirrings, then add 50 μ L 3-aminopropyl triethoxysilanes (APTES), centrifugal after backflow 4h, absolute ethanol washing, last centrifugal collection, obtains amino modified MSNs.
Embodiment 3
TAT polypeptide grafting: get 0.2mol (412.4mg) TAT polypeptide and 0.02mol NHS-PEG
1500-MAL (30mg), be dissolved in 20mL deionized water, then add respectively 0.2mol (38.34mg) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.5mol (57.54mg) N-hydroxy-succinamide (NHS), stirring at room temperature 15min.Then get amidized MSNs 20mg in embodiment 2, ultrasonic being scattered in above-mentioned TAT polypeptide solution.Centrifugal collection product MSNs-TAT/PEG after stirring at room temperature reaction 24h.
Embodiment 4
Rgd peptide grafting: take product 20mg in embodiment 3, ultrasonic being scattered in 20mL deionized water.Then add rgd peptide 0.2mol (118.9mg), centrifugal collection product after stirring at room temperature reaction 24h, fully washs and obtains product MSNs-RGD/TAT with deionized water.
In above-described embodiment 2-4 the TEM photo of the product of every step referring in Fig. 3 b)-i).Therefrom can find out, the synthetic product of every step all has good dispersibility.The uv-vis spectra of end product MSNs-RGD/TAT is referring to Fig. 4.As can be seen from the figure, MSNs-RGD/TAT has TAT and rgd peptide simultaneously and lays respectively at the characteristic absorption peak of 495nm and 275nm, has proved the successful grafting of this two peptide species.
Claims (10)
1. the continuous targeted drug of tumor vessel-tumor cell membrane-nucleus transports system, it is characterized in that, comprise mesoporous silica nano-particle and covalency be linked in described mesoporous silica nano-particle surface as the rgd peptide of tumor vessel/tumor cell membrane targeting part with as the nuclear localization signal peptide sequence of cell nucleus targeting part.
2. the continuous targeted drug of tumor vessel-tumor cell membrane-nucleus according to claim 1 transports system, it is characterized in that, described nuclear localization signal peptide sequence is TAT polypeptide, SV40T antigen and/or adenovirus.
3. the continuous targeted drug of tumor vessel-tumor cell membrane-nucleus according to claim 1 and 2 transports system, it is characterized in that, goes back chain and be connected to PEG between described rgd peptide and described mesoporous silica nano-particle surface.
4. transport system according to the continuous targeted drug of the tumor vessel-tumor cell membrane-nucleus described in any one in claims 1 to 3, it is characterized in that, the particle diameter of described mesoporous silica nano-particle is 20～50 nm, and aperture is 2～20nm.
5. in claim 1 to 4, the continuous targeted drug of the tumor vessel-tumor cell membrane-nucleus described in any one transports the preparation method of system, it is characterized in that, comprises the following steps:
(1) mesoporous silica nano-particle and the silane coupler of end band amino are reacted to obtain the mesoporous silica nano-particle of surface band amino;
(2) mesoporous silica nano-particle of the surface band amino of gained is reacted to obtain surface graft with nuclear localization signal peptide sequence and NHS-PEG-MAL and have the mesoporous silica nano-particle of nuclear localization signal peptide sequence and PEG chain;
(3) surface graft of gained there is is the mesoporous silica nano-particle of nuclear localization signal peptide sequence and PEG chain react with rgd peptide with at PEG end of the chain grafting rgd peptide, thereby obtain the chain modified mesoporous silicon-dioxide-substrate medicine-carried system of dual peptide.
6. preparation method according to claim 5, it is characterized in that, in step (1), the silane coupler of described end band amino is 3-aminopropyl triethoxysilane, 3-aminopropyl trimethoxysilane and/or N-2-aminoethyl-3-aminopropyl methyl dimethoxysilane.
7. according to the preparation method described in claim 5 or 6, it is characterized in that, in step (2), the mol ratio of described nuclear localization signal peptide sequence and described NHS-PEG-MAL is (1～100): 1.
8. in a claim 1 to 4, the continuous targeted drug of tumor vessel-tumor cell membrane-nucleus described in any one transports system in the application of preparing in targeted anticancer medicine.
9. application according to claim 8, is characterized in that, elecrtonegativity cancer therapy drug is supported in mesopore orbit by the electrostatic adsorption that transports the mesoporous silica particles inwall in system with described continuous targeted drug.
10. application according to claim 9, is characterized in that, the cancer therapy drug that action site is arranged in nucleus comprises amycin, cisplatin, camptothecine and photosensitizer, and described photosensitizer comprises chlorin (Chorin e6), ZnPc and/or porphyrin.
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