CN105963717A - Composite nano-drug for integrated tumor diagnosis and treatment and preparation method thereof - Google Patents
Composite nano-drug for integrated tumor diagnosis and treatment and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a composite nano-drug for integrated tumor diagnosis and treatment and a preparation method thereof. A chemotherapeutic drug, a photodynamic drug and shRNA plasmids capable of reversing multidrug-resistant MDR1 genes of tumor cells are located through a nano-carrier, a magnetic nano-sphere with precious metal nano-particles arranged on the surface serves as a kernel of the nano-carrier, mesoporous silica with the surface modified with amino groups serves as a shell of the nano-carrier, and a polyelectrolyte thin film layer structure with pH response is formed on the surface of the kernel-shell structure through a layer-by-layer self-assembly technology. The composite nano-drug integrates targeted drug delivery, multimode imaging, chemotherapy, photodynamic therapy and gene therapy, can be applied to early diagnosis of tumors, image mediation and real-time monitoring treatment at the same time and overcomes the defect that tumor diagnosis and treatment are separate in the prior art; besides, the composite nano-drug is simple in preparation process, high in operability and suitable for industrial production.
Description
Technical field
The present invention relates to a kind of Multifunctional composite nanometer medicine for diagnosing tumor treatment integration, preparation method.
Background technology
Tumor is one of major disease threatening human health, especially malignant tumor at present.Malignant tumor is becoming the number one killer threatening human life, and annual tumor just steps up in mondial prevalence, and, the mortality rate of tumor is also constantly rising.The effectively treatment of tumor requires as early as possible, accurately finds, thus realizes treatment in time, improves therapeutic effect, but, a difficult problem of the diagnosis of current tumor and treatment always scientific circles.
In the early diagnosis of tumor, obtain the structure of tumor, metabolism and Biochemical Information with the imaging technique of different mode it is critical that.The diagnosing tumor technology commonly used clinically has X-ray Tomography (CT), positron emission tomography (PET), NMR (Nuclear Magnetic Resonance)-imaging (MRI) and fluorescence imaging (FOI), especially MRI and fluorescence imaging quite to be favored because of the non-intruding not damaged advantage of its uniqueness.
NMR (Nuclear Magnetic Resonance) imaging is clinical conventional imaging pattern, and it can provide high-quality organism structure information.Wherein, superparamagnetic nanoparticle can efficiently reduce the T2 (T2) of Hydrogen Proton, strengthens the NMR (Nuclear Magnetic Resonance) imaging contrast of T2 sequence.Therefore, the application in NMR (Nuclear Magnetic Resonance) imaging of the superparamagnetic nanoparticle has aroused great concern with widely studied, particularly the application in diagnosing tumor.
But, in actual case, single imaging pattern can not provide form and the function information of tumor completely, so development multi-modality imaging technology can give full play to the strong point of each imaging pattern, so can be greatly improved the precision of diagnosing tumor.Generally, injection of contrast agent is needed to carry out enhancing signal in imaging, if sequentially utilizing different imaging patterns to carry out detection may need patient is injected no contrast medium, the most time-consuming, and increase misery and the toxic and side effects of patient.So developing a kind of probe that can apply simultaneously to derived techniques can solve above problem, and the position that multimode probe is assembled can be as the reference point of each imaging modality images, and so fusion to different mode image has bigger help.
The method of clinical treatment malignant tumor mainly has excision, chemotherapy and radiotherapy etc. at present.Although excision is prefered method, but owing to infantile tumour site of pathological change compares concealment, and cancerous cell has the feature of high transfer, many cancer patients find that when starting to treat cancerous cell has occurred transfer phenomena, cause primary lesion position cannot to be effectively recognized and excise completely.In the case of can not taking operative treatment, it is considered as radiotherapy or chemotherapy according to organization type.Organization type is different, and first-selected method is the most different, and the squamous cell carcinoma such as lung is sensitive to lonizing radiation, and adenocarcinoma of lung is then more sensitive to chemotherapy.And the tumor of some privileged sites, even without metastasis, but operation is likely to result in serious complication, such as brain stem, hilus pulumonis, liver portal vein district, operation mishap once occurs, easily causes death.In this case, radiotherapy or chemotherapy are first-selected treatment means.Radiotherapy is to destroy cellular genetic material by high energy particle or light wave directly to be eliminated by tumor cell.But in implementation process, also can be by RADI to normal surrounding tissue cell, normal tissue cell is easily suddenlyd change by RADI, increases the risk forming new tumor.Chemotherapy, as a kind of systemic treatment means, utilizes chemicals to suppress the propagation of tumor, shift and infiltrate, can effectively kill tumor.Chemotherapy all has therapeutical effect to primary tumor, metastasis and subclinical metastasis, but mostly chemotherapy is to carry out non-selective killing for eugonic tissue, hemopoietic system and the immune system of human body are the most sensitive to chemicals simultaneously, and therefore chemotherapy also has major injury to hemopoietic system and immune system；Additionally, chemotherapeutics also has at tumor tissues the relatively low problem that is distributed, and tumor cell to chemotherapeutics generation toleration thus causes cells resistance, and then improves the difficulty of oncotherapy.
After the multidrug resistance (MDR) of tumor refers to a kind of antitumor drug of tumor cell contact and produces drug resistance, the antitumor drug that structurally and functionally mechanism is different had cross resistance simultaneously.MDR is the normal method of drug resistance of tumor cell, is also the failed main cause of chemotherapy of tumors.Therefore, MDR is currently used chemotherapy tumor problem demanding prompt solution.
Research tumor drug resistance mechanism is mainly started with from the product of MDR gene expression at present, and RNA perturbation technique can specificity and optionally silencing of target genes expression.For overcoming the multidrug resistance of tumor, avoid the biological cell toleration to chemotherapy by the expression of suppression MDR gene.The mechanism of RNA perturbation technique is exogenous double-stranded RNA, after entering cell such as siRNA, unwinding into positive-sense strand and antisense strand under the effect of the DBPA in kytoplasm, antisense strand combines the silencing complex (RISC) forming RNA induction again with more intracellular Cobra venom endonuclease or unwindase etc. subsequently.RISC is specific binding with the mRNA homology region of target gene, cuts mRNA at binding site enzymolysis, so that mRNA degraded causes corresponding silenced gene expression.SiRNA is the double stranded RNA sequence of about 22 base pairs length, in elecrtonegativity, soluble in water, has efficient gene silencing effect.
At present, traditional chemotherapy and gene silencing combined treatment tumor have obtained concern greatly, and obtain preferable therapeutic effect.But, this electronegative biomolecule delivery of siRNA is to the most highly difficult in tumor cell, therefore, by designing, transform, develop a kind of Multifunction pharmaceutical carrier, it is possible to effectively realizing RNA silence function is the ultimate challenge in the middle of siRNA target gene silence research.
Present stage clinical tumor Clinics and Practices separates, in time tumor can not be made due to its intrinsic limitation and accurately judge and effectively process, often it is delayed the treatment time that tumor patient is optimal, it is therefore desirable to the diagnosis and treatment medicine that research and development integrate functions as one, synchronously combine organic to both diagnosis and treatment, while realizing diagnosis, carry out the purpose treated.In the Clinics and Practices field of tumor, cause people's broad interest especially is exactly nano material and nanotechnology, Application of micron is embodied in its multifunctionality in the advantage of medicine conveying and imaging, many nano materials self have the characteristic promoting imaging capability, and the application of nanotechnology can also realize treatment and monitoring medicine action site in vivo and therapeutic effect simultaneously.Therefore, designing and build diagnosis and treatment integration composite Nano medicine to realize early diagnosis of tumor, Image-guided and the real-time treatment monitored simultaneously is the focus in the research of current Medical Technology and difficult point.
Summary of the invention
In view of mentioned above, the invention discloses described in the technical scheme literary composition specific as follows in terms of a kind of composite Nano medicine for tumor diagnosis and treatment integration, preparation method, the two:
First aspect disclosed by the invention is to provide a kind of composite Nano medicine for tumor diagnosis and treatment integration, and described composite Nano medicine is to combine chemotherapeutics, photo-dynamical medicine and the shRNA plasmid of energy reversing multiple medicine resistance of tumor cells MDR-1 gene by the way of nano-carrier is with physical absorption or chemical bonding；The nano-carrier of described combination drug has the magnetic nano-balls of noble metal nano grain as kernel with surface alignment, with surface modify amino mesoporous silicon oxide as shell, pass through layer-by-layer
(Layer-by-Layer, LbL) sequentially forms the thin layer that electronegative polyelectrolyte replaces with positively charged polyelectrolyte layer on described nucleocapsid structure surface；Described polyelectrolyte layer is the polymer with pH response.
For the composite Nano medicine of tumor diagnosis and treatment integration in the present invention, in the nano-carrier of its described combination drug, the material of magnetic nano-balls is Ni, Co, Fe, Fe3O4、γ-Fe2O3And MeFe2O4In any one, the material of the noble metal nano grain that point-like is arranged in magnetic nano-balls surface is Au or Ag；
Preferably, the nano-carrier kernel of described combination drug is the Fe that surface is modified with Au nanoparticle3O4The structure of nanosphere.
For the composite Nano medicine of tumor diagnosis and treatment integration in the present invention, its described chemotherapeutics is divided into alkylating agent, antimetabolite, antitumor antibiotic, plant, hormones and miscellany etc. according to common classification, hereinafter described medicine is Common Chemotherapy medicine, but not representing chemotherapeutics in the present invention and be confined to hereinafter described medicine, in actual case, type and patient profiles according to tumor select.Such as alkylating agent；nullRing phosphinylidyne (Cyclophosphamide)，Phosphinothioylidynetrisaziridine (Thiotepa)，Semustine (Semustine)，Mustine hydrochlcride (Chlormethine Hydrochloride)，Busulfan (Busulfan，Busulfan)，Chlorambucil (Chlorambucil)，Formylmerphalan (Formylmerphalan)，Carmustine (Carmustine)，Altretamine (Altretamine)，Lomustine (Lomustine)，Melphalan (DL-Phenylalanine Mustard)，Nitrocaphane (Nitrocaphane)，Ifosfamide (Ifosfamide)，Mitobronitol (Mitobronitol)；Antimetabolite；Cytosine arabinoside (Cytarabine), fluorouracil (Fluorouracil), methotrexate (Methotrexate), hydroxyurea (Hydroxycarbamide), ftorafur (Tegafur), Meisoindigotin (Meisoindigotin), mercaptopurine (Mercaptopurine)；Antitumor antibiotic；Actinomycin D (dactinomycin, Dactinomycin), mitomycin (Mitomycin), doxorubicin hydrochloride (Doxorubicin Hydrochloride), Bleomycin A5 hydrochloride. (Bleomycin A5Hydrochloride), epirubicin hydrochloride (Epirubicin Hydrochloride), NSC 654509 (Pirarubicin Hydrochloride), daunorubicin hydrochloride (Daunorubicin Hydrochloride)；Plant natural antitumor medicine；Homoharringtonine (Homoharringtonine) vincristine sulfate (leurocristine, Vincristine Sulfate), hydroxycamptothecin (Hydroxycamptothecin), etoposide (Etoposide), vindesine sulfate (Vindesine Sulfate), vinblastine sulfate (Vinblastine Sulfate), liquor epinephrinae bitartratis ophthalmicus vinorelbine (Vinorelbine Bitartrate), paclitaxel (Paclitaxel), catharanthine, vinorelbine alkali, Docetaxel, Oleum Curcumae, ginseng polysaccharide, colchicine, 9-aminocamptothecin, 7-ethyl-camptothecin, elemene；Hormone antineoplastic agent；Aminoglutethimide (Aminoglutethimide), tamoxifen (Tamoxifen), flutamide (Flutamide), gonadorelin (Gonadorelin), leuprorelin acetate (Leuprorelin Acetate), letrozole (Lelrozol)；Carboplatin (Carboplatin), procarbazine hydrochloride (methyl bar hydrazine, Procarbazine Hydrochloride), amsacrine (Amsacrine), citric acid dacarbazine (Dacarbazine Citrate), asparaginase (ASP, Asparaginase), cisplatin (Cisplatin), mitoxantrone hydrochloride (Mitoxantrone Hydrochloride).
For the composite Nano medicine of tumor diagnosis and treatment integration in the present invention, photo-dynamical medicine destroys cyto-architectural medicine for causing photodynamic reaction.Described photo-dynamical medicine, including, but not limited to hereinafter described medicine, should select according to actual case flexibly.Such as first generation photosensitizer；Hematoporphyrin derivative (hematoporphyrin deriva-tive, HPD), dihematoporphyrin ethers (dihaematoporphyrin ether, DHE) and Por-fimer sodium (Photofrin II)；Second filial generation photosensitizer；null5-ALA (5-ALA)，M-tetrahydroxy phenyl chlorin (meso-tetrahydroxyphenyl chlorin，m-TH-PC)，Etioporphyrin (ETIO) stannum (tin etiopurpurin，SnEtz)，Methylene blue (methylene blue) and methylene benzene are blue (toluidine blue)，Benzene porphyrin (benzoporphyrin) derivant and lutelium texaphyrins (Lu-Tex)，Benzoporphyrin derivative mono-acid (BPD-MA，vertoporfin)，Phthalein green grass or young crops class (Phthalocyanines)，Get Ke Sa porphyrin (Texaphyrins)，N-Radix Asparagi acyl group chlorin (N-aspartyl chlorin e6，Npe6)，Hypericin (hypercin)，Blood quinoline methyl ether (Her-mimether，HMME)；Deng.
Preferably, select chlorin e6 (Ce6) as photo-dynamical medicine, there is the longer triplet time in Ce6, effectively tumor cell can be carried out lethal effect, well absorption is had at region of ultra-red, fluorescence imaging can be carried out in live body, and Ce6 can quickly be metabolized out external.
In the present invention in the composite Nano medicine of tumor diagnosis and treatment integration, described electronegative polyelectrolyte is sodium alginate, polyacrylic acid or Polyethylene Glycol；Described positively charged polyelectrolyte is chitosan, polyvinylamine or polymine.
In the composite Nano medicine integrated for tumor diagnosis and treatment of the present invention, the concentration of described nano-carrier is 0.1～10mg/ml, the concentration of described chemotherapeutics is 0.1～10mg/ml, and the concentration of described photo-dynamical medicine is 0.1～10mg/ml, and the concentration of described plasmid is 10～500 μ g/ml.
Second aspect disclosed by the invention is to provide the preparation method of a kind of composite Nano medicine for tumor diagnosis and treatment integration, comprises the following steps:
Step A: previously prepared nano-carrier is dispersed in ultra-pure water, preparation concentration is 0.1～10mg/ml nano-carrier aqueous solution, described nano-carrier has the magnetic nano-balls of noble metal nano grain as kernel with surface alignment, the mesoporous silicon oxide of amino is modified as shell with surface, sequentially form, on described nucleocapsid structure surface, the thin layer that electronegative polyelectrolyte replaces with positively charged polyelectrolyte, until obtaining the target number of plies by layer-by-layer；Described polyelectrolyte layer is the polymer with pH response；
Step B: add chemotherapeutics in the nano-carrier aqueous solution that step A obtains and photo-dynamical medicine forms mixed solution, in mixed solution, the relationship between quality of solute is chemotherapeutics: photo-dynamical medicine: nano-carrier=1: 1: 1～5, and being placed in by described mixed solution and putting into pressure after shaking table loads 1～72 hour is 103Pa～105The vacuum tank of Pa is deposited 1～24 hour, the medicine not being carried on nano-carrier by centrifugal segregation, collect precipitate stand-by；
Step C: add the shRNA plasmid of energy reversing multiple medicine resistance of tumor cells MDR-1 gene in step B gained precipitate, the nano-carrier of described compound chemotherapeutics and photo-dynamical medicine and the mass ratio of plasmid are 10～100: 1, by the most static for described mixed solution 0.5～5 hour, prepare described composite Nano medicine.
In the present invention in the preparation method of the composite Nano medicine of tumor diagnosis and treatment integration in step A, it is preferable that nano-carrier has the magnetic nano-balls Fe being arranged with Au nanoparticle with superficial punctate3O4-Au is kernel, and it is shell that the mesoporous silicon oxide of amino is modified on surface, sequentially forms, in the modification of described nucleocapsid structure surface, the film layer structure that the polysaccharide of electronegative sodium alginate and positively charged shell replaces by layer-by-layer；
The preferably preparation method of nano-carrier is specific as follows:
A1: preparation Fe3O4-Au solution, add cetyl trimethylammonium bromide (CTAB) as template, stirring is to mix homogeneously, it is subsequently adding deionized water and ammonia, stir 1～4 hour under 30～50 DEG C of water bath condition, tetraethyl orthosilicate (TEOS) is added as silicon source in described solution, with ethyl acetate (EA) regulation pH to alkalescence, continue stirring reaction 4～8 hours under 30～50 DEG C of water bath condition, question response terminates, after being cooled to room temperature, product is centrifuged, taking precipitate dehydrated alcohol cleans 5～10 times repeatedly, product is dissolved in dehydrated alcohol and prepares the Fe containing template3O4-Au@mSiO2The solution of nanoparticle；
A2: by ammonium nitrate (NH4NO3) it is dissolved in heating in water bath after dehydrated alcohol, it is subsequently adding in step A1 gained solution, magnetic agitation makes solution mix homogeneously, condensing reflux 20～60 minutes, question response terminates, and naturally cools to room temperature, is centrifugally separating to obtain precipitate, with washes of absolute alcohol 5～10 times, freeze-dried process prepares Fe3O4-Au@mSiO2Nanoparticle, seals and preserves；
A3: the Fe that step A2 is obtained3O4-Au@mSiO2Nanoparticle dissolution is in dehydrated alcohol, magnetic agitation mix homogeneously under 60～100 DEG C of water bath condition, it is subsequently adding 3-aminopropyl triethyl silicane (APTES), by abundant for described mixed solution condensing reflux 4～8 hours, question response terminates, and is cooled to room temperature, will clean 5～10 times after product dehydrated alcohol, freeze-dried process, prepares Fe3O4-Au@mSiO2-NH2Nanoparticle, seals and preserves；
A4: preparation Fe3O4-Au@mSiO2-NH2Solution, and drip sodium alginate (ALG) solution wherein, described mixed solution at room temperature magnetic agitation to be reacted 1～4 hour, reaction terminates to process by centrifugation, and taking precipitate NaCl solution is washed 2～5 times, obtains Fe3O4-Au@mSiO2-ALG；
A5: the Fe that step A4 is obtained3O4-Au@mSiO2-ALG was dissolved in NaCl solution, and drips chitosan (CHI) solution wherein, by described mixed solution at room temperature magnetic agitation 1～4 hours, reaction terminates to process by centrifugation, taking precipitate is washed with deionized 2～5 times, by freeze-dried for gains process, obtains Fe3O4-Au@mSiO2-PEM composite nanoparticle, seals and preserves.
Step B and step C that are used for the preparation method of the composite Nano medicine of tumor diagnosis and treatment integration in the present invention carry out photo-dynamical medicine, the loading of chemotherapeutics and the loading of genomic medicine on step A gained nano-carrier respectively.Aberrant energy metabolism due to tumor cell and self regulation to specific protein, define the acid intracellular microenvironment different with normal cell, the composite Nano medicine that the present invention prepares has the characteristic of pH response, do not discharge under neutrality or weakly alkaline environment, stable release in sour environment, such that it is able to reduce the damage of Chemotherapeutic Drugs On Normal cell as far as possible；The composite Nano medicine that the present invention prepares has the characteristic of photoresponse, produces singlet oxygen or reactive oxygen free radical by preferred photo-dynamical medicine Ce6, thus kill tumor cell under excited state；The composite Nano medicine that the present invention prepares can realize drug targeting by externally-applied magnetic field and transport, and improves medication effect.
Compared to prior art, the method have the advantages that
1, the composite Nano medicine of the present invention integrates target administration, multi-modality imaging, chemotherapy, optical dynamic therapy, gene therapy, apply simultaneously to early diagnosis of tumor, Image-guided and the treatment of monitoring in real time, overcome the defect that prior art diagnosing tumor separates with treatment.
2, the composite Nano medicine of the present invention can be used as NMR (Nuclear Magnetic Resonance) imaging, fluorescence imaging and the bioprobe of CT these three imaging technique, different imaging technique based on same probe is used in combination the deficiency of the most complementary single imaging pattern, and can break the problems such as operating process use time-consuming, multiple contrast medium misery and the toxic and side effects that can increase patient with efficient solution diagnosis.
3, the magnetic target drug-supplying system that the composite Nano medicine of the present invention builds passes through gene silent technology, entrained gene is discharged under tumor intracellular acid condition, described gene is cut into siRNA intracellular, can the expression of effectively reticent drug resistant gene MDR-1, thus avoid the biological cell toleration to chemotherapy.
4, the composite Nano medicine of the present invention can pass through the targeting stimulating regulation and control composite Nano medicine and the drug release of magnetic, light and pH, thus realizes the Clinics and Practices to tumor.
5, the composite Nano pharmaceutical preparation of the present invention is simple, workable, be suitable to commercial production, can be used for multi-medicament Synergistic treatment tumor, and the Ce6 fluorescence imaging of tumor, MRI and CT imaging and the target administration of tumor and pH response are administered.
Accompanying drawing explanation
Fig. 1 is composite Nano medicine preparation flow schematic diagram；
Fig. 2 is nano-carrier (Fe3O4-Au@mSiO2-PEM) transmission electron microscope picture (TEM figure)；
Fig. 3 is the Drug-Release Behavior curve of composite Nano medicine；
Fig. 4 is nano-carrier (Fe3O4-Au@mSiO2-PEM) biocompatibility schematic diagram；
Fig. 5 is the laser scanning co-focusing microgram of MCF-7 cell endocytic composite Nano medicine；
Fig. 6 is the PI-Cam effect schematic diagram that MCF-7 is treated by composite Nano medicine；
Fig. 7 is the comparison diagram of mice right rear leg tumor size change under different dosing regimes；
Fig. 8 is CT/MR bimodal image, and wherein (a) is MR image, and (b) is CT imaging.
Detailed description of the invention
Based on the following embodiment and Figure of description, the present invention is further discussed below: as it is shown in figure 1, this schematic diagram is merely illustrative flow process prepared by composite Nano medicine of the present invention, the present invention is not limited to content shown in this accompanying drawing.
The present invention modifies formation surface alignment by surface the Fe of point-like Au grain (particle diameter is about 5～10nm)3O4Magnetic nano-balls, wraps up the Fe being previously formed with mesoporous silicon oxide (MSNs)3O4-Au nanoparticle forms nucleocapsid structure, the wherein selection of magnetic Nano ball material, and those skilled in the art combine common knowledge can be by Fe3O4Replace with Ni, Co, Fe, γ-Fe2O3Or MeFe2O4, then by prepared Fe3O4-Au@mSiO2Nanoparticle makes surface amination obtain Fe by amide reaction3O4-Au@mSiO2-NH2Nanoparticle, is modified sodium alginate (ALG) and chitosan (CHI) film layer structure alternately successively, thus prepares composite Nano carrier Fe by LBL self-assembly3O4-Au@mSiO2-PEM；Then composite Nano medicine Fe is prepared by loading photo-dynamical medicine, chemotherapeutics and genomic medicine3O4-Au@mSiO2(Dox/Ce6)-PEM/P-gp shRNA。
Embodiment 1:
Conveying medicine and the preparation method of the nano-carrier of gene, specifically comprise the following steps that altogether
A1: compound concentration is the Fe of 5mg/ml3O4-Au solution, add cetyl trimethylammonium bromide (CTAB) as template, stir 5 minutes to mix homogeneously, it is subsequently adding deionized water and ammonia, stir 2 hours under 40 DEG C of water bath condition, tetraethyl orthosilicate (TEOS) is added as silicon source in described solution, with ethyl acetate (EA) regulation pH to alkalescence, continue stirring reaction 6 hours under 40 DEG C of water bath condition, question response terminates, after being cooled to room temperature, product is centrifuged, taking precipitate dehydrated alcohol cleans 6 times repeatedly, product is dissolved in dehydrated alcohol and prepares the Fe containing template3O4-Au@mSiO2The solution of nanoparticle；
A2: by ammonium nitrate (NH4NO3) it is dissolved in heating in water bath after dehydrated alcohol, it is subsequently adding in step A1 gained solution, magnetic agitation makes solution mix homogeneously, condensing reflux 40 minutes, question response terminates, and naturally cools to room temperature, being centrifugally separating to obtain precipitate, with washes of absolute alcohol 6 times, freeze-dried process prepares Fe3O4-Au@mSiO2Nanoparticle, seals and preserves；
A3: the Fe that step A2 is obtained3O4-Au@mSiO2Nanoparticle dissolution is in dehydrated alcohol, magnetic agitation mix homogeneously under 80 DEG C of water bath condition, it is subsequently adding 3-aminopropyl triethyl silicane (APTES), by abundant for described mixed solution condensing reflux 6 hours, question response terminates, and is cooled to room temperature, will clean 6 times after product dehydrated alcohol, freeze-dried process, prepares Fe3O4-Au@mSiO2-NH2Nanoparticle, seals and preserves；
A4: preparation Fe3O4-Au@mSiO2-NH2Solution, and drip sodium alginate soln wherein, described mixed solution at room temperature magnetic agitation to be reacted 2 hours, reaction terminates to process by centrifugation, and taking precipitate NaCl solution is washed 3 times, obtains Fe3O4-Au@mSiO2-ALG；
A5: the Fe that step A4 is obtained3O4-Au@mSiO2-ALG is dissolved in NaCl solution, and drips chitosan solution wherein, and by described mixed solution at room temperature magnetic agitation 2 hours, reaction terminates to process by centrifugation, and taking precipitate is washed with deionized 3 times, by freeze-dried for gains process, obtains Fe3O4-Au@mSiO2-PEM composite nanoparticle, seals and preserves.
Nano-carrier (Fe is prepared according to above operation3O4-Au@mSiO2-PEM), its transmission electron microscope picture (TEM figure) is as shown in Figure 2, nano-carrier favorable dispersibility as can see from Figure 2, particle diameter is about 220nm, and polyelectrolyte (PEM) layer is successfully modified in the magnetic nano-balls Fe being arranged with Au nanoparticle with superficial punctate3O4-Au is kernel, and the nucleocapsid structure surface (Fe that mesoporous silicon oxide is shell of amino is modified on surface3O4-Au@mSiO2-NH2)。
Embodiment 2:
The preparation method of a kind of composite Nano medicine for tumor diagnosis and treatment integration, specifically comprises the following steps that
Step A: nano-carrier prepared by embodiment 1 is dispersed in ultra-pure water, preparation concentration is 1mg/ml nano-carrier aqueous solution, described nano-carrier has the magnetic nano-balls of noble metal nano particles as kernel with surface alignment, the mesoporous silicon oxide of amino is modified as shell with surface, sequentially form, on described nucleocapsid structure surface, the thin layer that electronegative polyelectrolyte replaces with positively charged polyelectrolyte, until obtaining the target number of plies by layer-by-layer；Described polyelectrolyte layer is the polymer with pH response；
Step B: add chemotherapeutics in the nano-carrier aqueous solution that step A obtains and photo-dynamical medicine forms mixed solution, in mixed solution, the relationship between quality of solute is chemotherapeutics: photo-dynamical medicine: nano-carrier=1: 1: 1～5, and being placed in by described mixed solution and putting into pressure after shaking table loads 1～72 hour is 103Pa～105The vacuum tank of Pa is deposited 1～24 hour, the medicine not being carried on nano-carrier by centrifugal segregation, collect precipitate stand-by；
Step C: add the shRNA plasmid of energy reversing multiple medicine resistance of tumor cells MDR-1 gene in step B gained precipitate, the nano-carrier of described compound chemotherapeutics and photo-dynamical medicine and the mass ratio of plasmid are 10～100: 1, by the most static for described mixed solution 0.5～5 hour, prepare described composite Nano medicine.
From fig. 3, it is found that chemotherapeutics discharges under sour environment, illustrate that composite Nano medicine of the present invention has good pH response.
Embodiment 3:
The blood compatibility of nano-carrier:
Step 1: take fresh Mouse Blood, 3000rpm/min is centrifuged 10 minutes removing supernatant and obtains erythrocyte, then cleaned 5 times with PBS continuously by product；
Step 2: erythrocyte is diluted 10 times with PBS, take the most respectively 0.1ml erythrocyte diluting fluid join 0.9ml concentration be 25 μ g/ml, number consecutively is 1～No. 5 pipe in the carrier solution of 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 400 μ g/ml, take 0.1ml erythrocyte diluting fluid and join numbered No. 6 pipes of 0.9ml PBS, as negative control, take 0.1ml erythrocyte diluting fluid and join numbered No. 7 pipes in 0.9ml distilled water, as positive control；
Step 3: put into after the solution of gained in step 2 1～No. 7 is slightly mixed and hatch in 37 DEG C of water-baths 3 hours, be then centrifuged 3 minutes under conditions of rotating speed is 3000rpm/min；
Step 4: measure in 1～No. 7 pipe of step 3 gained supernatant in the uv absorption that wave-length coverage is 450～750nm.
As shown in Figure 4, when nano-carrier concentration reaches 400 μ g/ml, the most do not produce haemolysis, by this nano-carrier of this description of test, there is good blood compatibility.
Embodiment 4:
The cell endocytic experiment of composite Nano medicine, specifically comprises the following steps that
Step 1: use 1640 culture medium culturing HBT's cells (MCF-7 cell), inoculate the same amount of cell being in exponential phase, incubated overnight 6 identical being provided with in 24 orifice plates of creep plate respectively；
Step 2: be resuspended in 1640 culture medium by the composite Nano medicine of the present invention, ultrasonic dissolution, in described composite Nano medicine, the concentration of chemotherapeutics is 2 μ g/ml, and the concentration of photo-dynamical medicine Ce6 is 4 μ g/ml；
Step 3: remove the culture medium in described 6 24 orifice plates, after all cleaning 3 times with PBS, add the composite Nano medicine of equivalent in 6 24 orifice plates, be placed in incubator hatching 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours respectively.
Step 4: removed the culture medium in 24 orifice plates hatched, 3 times are cleaned to remove not by the composite Nano medicine of cell endocytic with PBS, then drip the paraformaldehyde that mass fraction is 4% and fix cell 30 minutes, clean 3 times with PBS after having fixed, re-use DAPI to dye 10 minutes, clean 3 times with PBS；
Step 5: be placed under laser scanning co-focusing microscope by step 4 gained creep plate, observes the MCF-7 cell endocytosis situation to composite Nano medicine.
Fig. 5 is the confocal experiments design sketch of MCF-7 cell endocytic composite Nano medicine；Testing and contaminate nucleus by DAPI, excite as blueness, and Ce6 is excited into green under conditions of wavelength is 630nm under conditions of wavelength is 405nm, chemotherapeutics is excited into redness under conditions of wavelength is 488nm.Change according to intracellular each shade, after can be seen that nano-complex and MCF-7 cell hatch 0.5 hour altogether, nano-complex just can be by cell endocytic, and along with the increase of common incubation time, intracellular green and redness are the most gradually deepened, and reach maximum at 12 hours.Simultaneously it has also been discovered that, nano-complex is by after cell endocytic, polyelectrolyte (PEM) layer is decomposed, release chemotherapeutics and photo-dynamical medicine Ce6, chemotherapeutics diffuses into nucleus, and photo-dynamical medicine Ce6 is mainly distributed in Cytoplasm, this is conducive to the Synergistic treatment of tumor, improves antitumous effect.
In sum, along with the increase of composite Nano medicine and MCF-7 cell incubation time altogether, cell endocytic composite Nano medicine is gradually increased, and illustrates that composite Nano medicine can be good at by MCF-7 cell endocytic.
Embodiment 5:
Composite Nano vitro Drug synergistic antitumor effect is tested, and specifically includes following steps:
Step 1: use 1640 culture medium culturing MCF-7 cells, inoculates 1 × 10 in 96 orifice plates4Individual cell, incubated overnight；
Step 2: take 15 holes on 96 orifice plates of step 1 gained, and be divided into 5 groups, add 1640 culture medium of 100uL, the Fe of 100uL respectively in each group3O4-Au@mSiO2(Ce6) Fe of-PEM solution, 100uL3O4-Au@mSiO2(Dox) Fe of-PEM solution, 100uL3O4-Au@mSiO2(Dox/Ce6)-PEM solution and the Fe of 100uL3O4-Au@mSiO2(Dox/Ce6)-PEM/P-gp siRNA solution, wherein the concentration of chemotherapeutics (Dox) is 2ug/ml, and the concentration of photo-dynamical medicine Ce6 is 4ug/ml, is placed in incubator by above-mentioned 96 orifice plates and hatches 12 hours；
Step 3: the cell PBS solution after step 2 being hatched is washed 3 times, then using wavelength is 660nm, and power is 2W/cm2Laser instrument carry out 10 minutes laser and irradiate being loaded with Ce6 group, place into and carbon dioxide incubator hatched 60 hours, finally use CCK-8 method that therapeutic effect is detected.
Fig. 6 is the design sketch by the detection composite Nano medicine synergistic antitumor effect of PI-Calcein AM double-staining, calcium flavin-AM sloughs AM base by lipase effect in living cells can send strong green fluorescence, because calcium flavin-AM lipotropy is strong, propidium iodide (PI) can only pass dead cell, embeds the DNA in cell and produces red fluorescence.Therefore, in figure 6, living cells is colored as green, dead cell is colored as redness, it appeared that be loaded with in two groups of Ce6 and Dox and have part redness to occur from Fig. 6, there is certain therapeutic effect, but be loaded with Ce6 and Dox group simultaneously and be loaded with in Dox, Ce6 and P-gp sh RNA group almost the most red simultaneously, show that there is more preferable therapeutic effect, illustrate that composite Nano medicine has extraordinary synergistic therapeutic effect for killing tumor cell.
Embodiment 6:
The experiment of synergistic antitumor effect in composite Nano medicine body, specifically includes following steps:
Step 1: murine mammary tumor cells (EMT-6 cell) is inoculated in Balb/c female mice right rear leg, tumor to be seeded grows to experiment time a certain size；
Step 2: successful for inoculated tumour mice is randomly divided into 6 groups, is designated as 1～6 group respectively, and labelling 1 group is physiology saline control group, and 2 groups is Fe3O4-Au@mSiO2-PEM group, 3 groups is Fe3O4-Au@mSiO2(Ce6)-PEM group, 4 groups is Fe3O4-Au@mSiO2(Dox)-PEM group, 5 groups is Fe3O4-Au@mSiO2(Dox/Ce6)-PEM, 6 groups is Fe3O4-Au@mSiO2(Dox/Ce6)-PEM/P-gp siRNA group,
Step 3: dissolve respectively to be scattered in normal saline by the Nano medication of labelling in step 2 2～6 groups and make drug solution used by each group, wherein, 1 group be without medicine and with remaining group equivalent normal saline, using chemotherapeutics of the same race (Dox) and concentration to be 1mg/ml in 4 groups, 5 groups and 6 groups, in 3 groups, 5 groups and 6 groups, Ce6 concentration is 0.4mg/ml；By the drug administration of preparation in correspondence group mice, administering mode is administered in being preferably tumor, and dosage is 50 μ L；
Being administered latter 2 hours, use power is 2W/cm2, wavelength is the tumor locus 10 minutes of the laser illumination 3 groups of 660nm, 5 groups and 6 groups (being the group being loaded with Ce6) mices；
Each group of mice of this experiment is administered for Per-Hop behavior once, and altogether to 3 medicines, last observed and recorded respectively organizes tumor size, Mouse Weight situation of change.
Fig. 7 is the comparison diagram of mice right rear leg tumor size change after above-mentioned dosage regimen；It will be seen in fig. 7 that normal saline group and nano-carrier (Fe3O4-Au@mSiO2-PEM) organize the increase that all can not suppress mouse tumor, 3 groups that are loaded with photo-dynamical medicine Ce6 or 4 groups that are loaded with chemotherapeutics (Dox) have certain inhibitory action for tumor, and be loaded with photo-dynamical medicine Ce6 and 5 groups of chemotherapeutics (Dox) simultaneously and be loaded with 6 groups of photo-dynamical medicine Ce6, chemotherapeutics (Dox) and genomic medicine P-gp si RNA mouse tumor is had extraordinary inhibitory action, especially 6 groups have obvious therapeutic action.In sum, composite Nano medicine of the present invention has good synergistic therapeutic effect.
Embodiment 7:
Nano-carrier nuclear magnetic resonance, NMR (MRI) and CT (CT technology) bimodal imaging
Step 1: Fe prepared by embodiment 13O4-Au@mSiO2-PEM nano-carrier ultrasonic disperse is in deionized water, preparing concentration respectively is 0mg/ml, 0.8mg/ml, 1.6mg/ml, 2mg/ml, 4mg/ml solution, takes each group of solution 0.5ml respectively and joins mix homogeneously in the centrifuge tube containing 0.5mL agarose solution, solution to be mixed is cooled to room temperature, uses pico computer layer scanning technology (Micro-CT) to be scanned taking pictures；
Step 2: Fe prepared by embodiment 13O4-Au@mSiO2-PEM nano-carrier ultrasonic disperse is in deionized water, preparing concentration respectively is 0mg/ml, 0.4mg/ml, 0.8mg/ml, 1mg/ml, 2mg/ml solution, joins mix homogeneously in agarose solution, solution to be mixed is cooled to room temperature, and the imaging effect of nano-carrier is detected by the nuclear magnetic resonance imaging system using magnetic field size to be 3T.
As shown in Figure 8, due to nano-carrier (Fe3O4-Au@mSiO2-PEM) in be enclosed with Fe3O4Nanoparticle and Au nanoparticle, be therefore more satisfactory MRI and CT contrast agent.Wherein, can be seen that from figure (a), along with the increase of nano-carrier concentration, MR image is the most dimmed, and imaging effect gradually improves.Can be seen that from figure (b), along with the increase of nano-carrier concentration, CT image gradually brightens, and imaging effect gradually improves.In sum, the nano-carrier of the present invention can well carry out the bimodal imaging of MR Yu CT.
Claims (8)
1. for the composite Nano medicine of tumor diagnosis and treatment integration, it is characterised in that described composite Nano medicine is to be carried by nanometer
Body combines chemotherapeutics, photo-dynamical medicine and energy reversing multiple medicine resistance of tumor cells in the way of physical absorption or chemical bonding
The shRNA plasmid of MDR-1 gene；Described nano-carrier has the magnetic nano-balls of noble metal nano grain as kernel with surface alignment,
With surface modify amino mesoporous silicon oxide as shell, sequentially formed on described nucleocapsid structure surface by layer-by-layer
Electronegative polyelectrolyte and positively charged polyelectrolyte film layer structure alternately；Described electronegative polyelectrolyte and institute
Stating positively charged polyelectrolyte is the polymer with pH response.
Composite Nano medicine for tumor diagnosis and treatment integration the most according to claim 1, it is characterised in that described nanometer
In carrier, the material of magnetic nano-balls is Ni, Co, Fe, Fe3O4、γ-Fe2O3And MeFe2O4In any one.
Composite Nano medicine for tumor diagnosis and treatment integration the most according to claim 1, it is characterised in that described nanometer
In carrier, the material of noble metal nano grain is Au or Ag.
Composite Nano medicine for tumor diagnosis and treatment integration the most according to claim 1, it is characterised in that described band is born
The polyelectrolyte of electric charge is sodium alginate, polyacrylic acid or Polyethylene Glycol.
Composite Nano medicine for tumor diagnosis and treatment integration the most according to claim 1, it is characterised in that described band is just
The polyelectrolyte of electric charge is chitosan, polyvinylamine or polymine.
Composite Nano medicine for tumor diagnosis and treatment integration the most according to claim 1, it is characterised in that described nanometer
The concentration of carrier is 0.1～10mg/ml, and the concentration of described chemotherapeutics is 0.1～10mg/ml, the concentration of described photo-dynamical medicine
Being 0.1～10mg/ml, the concentration of described plasmid is 10～500ug/ml.
7. it is used for the preparation method of the composite Nano medicine of tumor diagnosis and treatment integration, it is characterised in that comprise the following steps:
Step A: previously prepared nano-carrier is dispersed in water, preparation concentration is 0.1～10mg/ml nano-carrier aqueous solution,
Described nano-carrier has the magnetic nano-balls of noble metal nano particles as kernel with surface alignment, modifies mesoporous the two of amino with surface
Silicon oxide is shell, sequentially forms electronegative polyelectrolyte and band by layer-by-layer on described nucleocapsid structure surface
Positive charge polyelectrolyte thin layer alternately, until obtaining the target number of plies；Described polyelectrolyte layer is the polymerization with pH response
Thing；
Step B: add chemotherapeutics in the nano-carrier aqueous solution that step A obtains and photo-dynamical medicine forms mixed solution,
In mixed solution, the relationship between quality of solute is chemotherapeutics: photo-dynamical medicine: nano-carrier=1: 1: 1～5, by described mixing
Solution is placed in and puts into pressure after shaking table loads 1～72 hour is 103Pa～105The vacuum tank of Pa is deposited 1～24 hour, pass through
Centrifugal segregation is not carried in the medicine on nano-carrier, collects precipitate stand-by；
Step C: add the shRNA matter of energy reversing multiple medicine resistance of tumor cells MDR-1 gene in step B gained precipitate
Grain, the nano-carrier of described compound chemotherapeutics and photo-dynamical medicine and the mass ratio of plasmid are 10～100: 1, by described mixing
The most static 0.5～5 hour of solution, prepares described composite Nano medicine.
The preparation method of the composite Nano medicine for tumor diagnosis and treatment integration the most according to claim 6, it is characterised in that
In described step A, nano-carrier has the magnetic nano-balls Fe being arranged with Au nanoparticle with superficial punctate3O4-Au is kernel, table
It is shell that the mesoporous silicon oxide of amino is modified in face, and by layer-by-layer, on described nucleocapsid structure surface, modification sequentially forms
Alginate films layer and chitosan film layer, the preparation method of described nano-carrier is specific as follows:
A1: preparation Fe3O4-Au solution, addition cetyl trimethylammonium bromide is as template, and stirring is to mix homogeneously, so
Rear addition deionized water and ammonia, stir 1～4 hour under 30～50 DEG C of water bath condition, add positive silicic acid in described solution
Ethyl ester, as silicon source, with ethyl acetate regulation pH to alkalescence, continues stirring reaction 4～8 under 30～50 DEG C of water bath condition little
Time, question response terminates, and after being cooled to room temperature, product is centrifuged, and taking precipitate dehydrated alcohol cleans 5～10 times repeatedly,
Product is dissolved in dehydrated alcohol and prepares the Fe containing template3O4-Au@mSiO2The solution of nanoparticle；
A2: ammonium nitrate is dissolved in heating in water bath after dehydrated alcohol, is subsequently adding in step A1 gained solution, and magnetic agitation makes
Solution mix homogeneously, condensing reflux 20～60 minutes, question response terminates, and naturally cools to room temperature, is centrifugally separating to obtain precipitate,
With washes of absolute alcohol 5～10 times, freeze-dried process prepares Fe3O4-Au@mSiO2Nanoparticle, seals and preserves；
A3: the Fe that step A2 is obtained3O4-Au@mSiO2Nanoparticle dissolution is in dehydrated alcohol, 60～100 DEG C of water-baths
Under the conditions of magnetic agitation mix homogeneously, be subsequently adding 3-aminopropyl triethyl silicane, by abundant for described mixed solution condensing reflux 4～
8 hours, question response terminated, and was cooled to room temperature, will clean 5～10 times, freeze-dried place after product dehydrated alcohol
Reason, prepares Fe3O4-Au@mSiO2-NH2Nanoparticle, seals and preserves；
A4: preparation Fe3O4-Au@mSiO2-NH2Solution, and drip sodium alginate soln wherein, described mixed solution is existed
Under room temperature, magnetic agitation is reacted 1～4 hour, and reaction terminates to process by centrifugation, and taking precipitate NaCl solution is washed 2～5 times,
Obtain Fe3O4-Au@mSiO2-ALG；
A5: the Fe that step A4 is obtained3O4-Au@mSiO2-ALG is dissolved in NaCl solution, and drips chitosan wherein
Solution, by described mixed solution at room temperature magnetic agitation 1～4 hours, reaction terminated to process by centrifugation, taking precipitate spend from
Sub-water washs 2～5 times, by freeze-dried for gains process, obtains Fe3O4-Au@mSiO2-PEM composite nanoparticle,
Seal and preserve.
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Application publication date: 20160928 |