CN104587474A - Gold-core-composite nano-carrier as well as preparation method and application thereof - Google Patents
Gold-core-composite nano-carrier as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a gold-core-composite nano-carrier as well as a preparation method and application thereof. According to the gold-core-composite nano-carrier, a silicon dioxide modified gold nanorod (Au@SiO2) is taken as an inner core and is coated with a layer of high polymer material sensitive to temperature and pH. The gold-core-composite nano-carrier can be used for coating various drug and probe molecules and transporting the drug and probe molecules to disease sites, and therefore, the targeted therapy is realized. After absorbing near-infrared laser, the gold-core-composite nano-carrier or a drug-loading nanoparticle can emit fluorescent light, and partial optical energy can be converted into heat to be applied to biological imaging, chemotherapy and thermal therapy; the gold-core-composite nano-carrier further has the pH-sensitive property and is capable of effectively releasing the drug molecules in a weakly acidic tumor microenvironment and cell lysosome; multiple tumor therapy functions of the gold-core-composite nano-carrier can be combined in use, so that the defects of a single tumor therapy method are overcome; the gold-core-composite nano-carrier is suitable for being applied to the complex tumor diagnosis and treatment.
Description
Technical field
The invention belongs to pharmaceutical carrier technical field, be specifically related to a kind of pharmaceutical carrier and its preparation method and application, particularly relate to a kind of pharmaceutical carrier based on golden core composite Nano, its preparation method and the application in tumor diagnosis and therapy thereof.
Background technology
In the past few decades, although the understanding of people to cancer has been made significant headway, cancer has been still one of principal disease affecting human health and life-span." Chinese tumor registration annual report in 2012 " display, the annual new cancer cases of China about 3,500,000, because of cancer mortality about 2,500,000.The high mortality of cancer is because traditional treatment of cancer weak effect, toxic and side effects cause greatly to a great extent.In treatment of cancer, thermotherapy and chemotherapy combined application have been proved to be able to the effect optimizing treatment of cancer.If thermotherapy itself thoroughly can not remove cancerous issue, temperature raises the increase of the tumor vessel permeability caused and vascular endothelial cell gap becomes large, also can increase the accumulation of chemotherapeutics in tumor, improves the curative effect of chemotherapy.But traditional thermotherapy and chemotherapy all lack the targeting for tumor tissues, can inevitably have side effects while bringing curative effect; And thermotherapy needs complicated operation just can reach the curative effect of expection.
Some nanoparticles that the energy of long-range conveying (such as magnetic field, laser, radio frequency, microwave, ultrasonic etc.) can be converted into heat are just studied by as novel thermotherapy carrier.Utilize these nanoparticles, the heat needed for thermotherapy can be controlled accurately by extraneous energy source, after outside energy is converted into heat, and the damage that can effectively prevent normal surrounding tissue to the heat of external diffusion by inside tumor.In addition, these nanoparticles can also as the carrier of chemotherapeutics effectively by medicament transport to tumor tissues.Visible, nanoparticle is applied to thermochemotherapy coupling can be transferred to tumor tissues by heat and chemotherapeutics simultaneously, avoids Conventional thermal to treat the side effect of coupling.
Nanoparticle is its precondition as the onset of Diagnosis and Treat carrier in effective enrichment of tumor locus.The nanoparticle be administered systemically can optionally be enriched in tumor tissues (being called " passive target ") by the infiltration of enhancing and delay (EPR) effect.In addition, the molecule with cell or tissue targeting often can be modified in the surface of nano-carrier, and the endocytosis mediated by surface receptor increases nanoparticle in tumor tissues or intracellular enrichment.But the performance be modified in zoopery of this active targeting is but usually barely satisfactory.Reason is that first nanoparticle needs just can reach tumor tissues by EPR effect on the one hand, and targeting modification effectively can not change the bio distribution of nanoparticle; But the blood circulation time of nanoparticle and the interaction with receptor thereof is easily reduced on the other hand.Outside energy stimulate, such as magnetic field and ultrasonic as new targeted approach prove living animal experiment in be effective.Also the temperature sensitive micelle of inducible system administration and the micromolecule of carrier band thereof tumor locus can be enriched in by external heat local organization.These stimulate the tactful prospect of carrying out neoplasm targeted therapy can the phase by outside energy, because the site of the stimulation in the external world, opportunity and intensity can control easily accurately.Up to the present, document and patent report near-infrared laser induced nano particle is rarely had to be applied to the transmission of tumor-targeting drug.
Summary of the invention
The object of the present invention is to provide a kind of pharmaceutical carrier and its preparation method and application, particularly a kind of pharmaceutical carrier based on golden core composite Nano, its preparation method and the application in tumor diagnosis and therapy thereof.
For reaching this goal of the invention, the present invention by the following technical solutions:
First aspect, the invention provides a kind of golden core composite Nano carrier, wherein, described carrier comprises with silicon dioxide modified gold nanorods kernel, and is coated on the macromolecular material of described gold nanorods core surface; Wherein, described macromolecular material is NIPA and acrylic acid copolymer.Second aspect, present invention also offers the preparation method of carrier as described in the first aspect of the invention, and wherein, the method comprises the following steps:
(1) in the gold nanorods aqueous solution containing cetyl trimethyl ammonium bromide, be that under the condition of 8.0-12.0, the alcoholic solution adding esters of silicon acis reacts at pH value, and carry out solid-liquid separation, obtain the gold nanorods that Surface coating has silicon dioxide layer;
(2) by monomer NIPA, acrylic acid, coupling agent, surfactant and initiator, under inert gas shielding, be heated to 60-80 DEG C of polyreaction 4-8 hour, prepare the macromolecular material of NIPA and acrylic acid copolymer;
(3) add 3-(methacryloxypropyl) propyl trimethoxy silicane in the gold nanorods obtained to step (1) and modify vinyl at silica surface, add the macromolecular material that step (2) obtains again, namely obtain described golden core composite Nano carrier.The third aspect, present invention also offers a kind of pharmaceutical composition, described pharmaceutical composition comprises carrier and the medicine of load on carrier, wherein, described carrier is the carrier described in first aspect present invention, or the carrier for being prepared by method described in second aspect present invention.
Fourth aspect, present invention also offers carrier as described in the first aspect of the invention and/or the application of the pharmaceutical composition described in third aspect present invention in the medicine for the preparation of diagnosis and/or treatment tumor.
The present invention is when utilizing golden core composite Nano carrier to carry out the active targeting treatment of induced with laser, its principle is under near-infrared laser irradiates, gold nanorods produces photo-thermal effect, make the temperature sensitive macromolecular material of outer bag quilt undergo phase transition size to reduce, local heating makes vascular space become large simultaneously, permeability increases, and the two synergism makes nanoparticle increase in sharp light-struck position enrichment, namely realizes the active targeting effect of induced with laser.
Novel medicine-carried system provided by the invention, have the several functions such as the active targeting of induced with laser, diagnosis, thermotherapy and chemotherapy, these functions can be united and applied in tumor diagnosis and therapy.
Compared with prior art, beneficial effect of the present invention is as follows:
(1) the golden core composite Nano carrier prepared of the present invention, the photothermal deformation effect of gold nanorods and the temperature-sensitive of macromolecular material are organically combined, irradiated by near-infrared laser and achieve the enrichment of carrier in tumor locus significant effective, i.e. photoinduced active targeting, the treatment curative effect for follow-up raising tumor has prepared necessary condition; And the conjunctive use of chemotherapy and thermotherapy two kinds of therapeutic modalities, can play the effect of Synergistic, also contribute to overcoming two kinds of therapeutic modalities shortcoming separately, obtain optimum therapeutic effect; Adopt this stimulation by outside energy to carry out the method for neoplasm targeted therapy, the site of environmental stimuli, opportunity and intensity can control easily accurately.
(2) the golden core composite Nano carrier that prepared by the present invention also has the function of multiple imaging: utilize the character that the optoacoustic of gold nanorods itself and x-ray tomography (CT) strengthen, can by the position of photoacoustic imaging and CT image-forming diagnose tumor and monitor therapy process; Utilize the characteristic of temperature sensitive macromolecule carrier band fluorescent molecular probe, golden core composite Nano carrier can also be used for near-infrared fluorescence imaging;
(3) the present invention is by the combination of materials of two or more different in kinds, prepare the complex of nanoscale, make its various composition Coordinated Play effect, opened up the application space of Nano medication in oncotherapy further, be particularly useful for the tumor diagnosis and therapy having complicated needs.
Accompanying drawing explanation
Fig. 1 is silicon dioxide coated gold nanorods (Au@SiO
2) transmission electron microscope picture;
Fig. 2 is that temperature sensing material bag is by Au@SiO
2form the transmission electron microscope picture of golden core composite Nano carrier; Wherein, Fig. 2 a represents that temperature sensitive layer thickness is the golden core composite Nano carrier of 110nm, Fig. 2 b represents that temperature sensitive layer thickness is the golden core composite Nano carrier of 20nm, Fig. 2 c represents that temperature sensitive layer thickness is the golden core composite Nano carrier of 80nm, and Fig. 2 d represents that temperature sensitive layer thickness is the golden core composite Nano carrier of 200nm;
Fig. 3 is the reversible change figure of the temperature variant particle diameter of golden core composite Nano carrier;
Fig. 4 is the golden particle diameter of core composite Nano carrier and the graph of a relation of pH;
Fig. 5 is the extinction spectra in time variation diagram of golden core composite Nano carrier in PBS;
Fig. 6 is the heating curve of golden core composite Nano carrier under same laser power is irradiated of variable concentrations;
Fig. 7 is the heating curve of golden core composite Nano carrier under different laser power is irradiated of same concentration;
Fig. 8 is gold nanorods (AuNRs), silicon dioxide gold-covered nano rod (Au@SiO
2), temperature sensing material bag is by the extinction spectra of silicon dioxide coated gold nanorods (Nanocom) and medicine-carried system (Nanocom-Dox) thereof;
Fig. 9 is that golden core composite Nano medicine-carried system (Nanocom-Dox) is at different pH and the drug release patterns under irradiating with or without laser;
Figure 10 is golden core composite Nano medicine-carried system (Nanocom-Dox) at 37 DEG C, drug release patterns in 100% serum;
Figure 11 is amycin (Dox), and golden core composite Nano medicine-carried system (Nanocom-Dox) and golden core composite Nano carrier (Nanocom) are on the impact of 4T1 cell viability;
Figure 12 is the absorption result of 4T1 cell to golden core composite Nano medicine-carried system (Nanocom-Dox);
Figure 13 is that golden core composite Nano medicine-carried system (Nanocom-Dox) and amycin (Dox) are total to positioning scenarios with 4T1 Cytolysosome;
Figure 14 is the circulation time figure of golden core composite Nano carrier (Nanocom) in mouse blood;
Figure 15 be carrier band near infrared fluorescent dye carrier (Nanocom-IR820) mice with tumor leg heating in water bath with do not heat time live body and in vitro fluorescence imaging;
Figure 16 be mice with after the leg heating in water bath of tumor, Nanocom-IR820 and IR820 is time dependent fluorescence imaging in Mice Body;
Figure 17 be after mouse vein administration tumor locus according to laser with not according to the photoacoustic imaging figure of laser;
Figure 18 is that intravenously administrable is according to the scattergram of golden core composite Nano carrier (Nanocom) after laser at Organs of Mice;
Figure 19 is the CT image after mouse vein administration;
Figure 20 is the heating curve that after mouse vein administration, tumor locus shines laser;
Figure 21 is the tumor suppression result of mouse vein administration;
Figure 22 is the tumor suppression condition optimizing of mouse vein administration;
Figure 23 is the pathological section of mice main organs;
Figure 24 is the tumor suppression result after mouse tumor inner injecting and administering.
Detailed description of the invention
Technical scheme of the present invention is further illustrated below by detailed description of the invention.Those skilled in the art should understand, described embodiment is only help to understand the present invention, should not be considered as concrete restriction of the present invention.
Golden core composite Nano carrier of the present invention comprises gold nanorods, the silicon dioxide layer on gold nanorods surface, and the macromolecular material of silica surface.
Macromolecular material in the present invention is a kind of macromolecular material to temperature and pH sensitivity, also can be described as temperature sensitive macromolecular material, it is obtained by NIPA and acrylic acid copolymer, this temperature sensitive macromolecular material is netted hydrogel structure, can be used for bag and carries various water miscible medicine and probe molecule.
In the present invention, the length of described gold nanorods is 5-100nm, such as, can be 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm.
According to golden core composite Nano carrier of the present invention, under preferable case, described gold nanorods length is 10-100nm; From being conducive to reaching tumor tissues to consider, more preferably described gold nanorods length is 15-80nm; Further preferred described gold nanorods length is 30-80nm.
In the present invention, the draw ratio of described gold nanorods is 1.5-20, such as, can be 1.5,2,2.5,3,4.5,5,6,8,12,15,18,20; Preferably, the draw ratio of described gold nanorods is 2-8; From being conducive to reaching tumor tissues and being suitable for controlling to consider by Submillineter Wave Technology, the draw ratio of further preferred described gold nanorods is 2.5-6, further preferred 3.0-4.5.
In the present invention, the thickness of described silicon dioxide layer is 3-50nm, such as, can be 3nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm; Described polymer material layer is netted hydrogel structure, its thickness is 5-300nm, such as, can be 5nm, 10nm, 15nm, 20nm, 30nm, 45nm, 50nm, 65nm, 80nm, 95nm, 100nm, 120nm, 150nm, 180nm, 200nm, 220nm, 250nm, 280nm, 300nm.
According to golden core composite Nano carrier of the present invention, described gold nanorods be coated with silicon dioxide.Under preferable case, the thickness of described silicon dioxide layer is 5-45nm; From being easy to preparation and being conducive to arriving tumor tissues consider, more preferably the thickness of described silicon dioxide layer is 10-30nm.
According to golden core composite Nano carrier of the present invention, described silicon dioxide modified gold nanorods pan coating has temperature sensitive macromolecule layer, and the thickness of described temperature sensitive macromolecule layer is 10-250nm; High and be conducive to arriving tumor tissues and consider from temperature sensitive thickness medicine carrying capacity, more preferably the thickness of described temperature sensitive macromolecule layer is 20-200nm.
Have the of the present invention golden core composite Nano carrier of said structure, drug loading is large, is enriched to tumor locus plays curative effect by laser irradiation and EPR effect.
The invention provides a kind of preparation method of golden core composite Nano carrier, according to golden core composite Nano support preparation method of the present invention, prepare described gold nanorods and the conventional method of this area can be adopted to carry out.Containing cetyl trimethyl ammonium bromide (CTAB) in described gold nanorods solution, under preferable case, the concentration of CTAB is 0.5-1.2mmol/L.When the concentration of CTAB in the gold nanorods solution prepared is higher than above-mentioned preferred concentration, the gold nanorods aqueous solution of required CTAB concentration can be obtained by centrifuge washing.
According to golden core composite Nano support preparation method of the present invention, under preferable case, in the gold nanorods concentration of gold element for 0.25-1.0mmol/L, the mol ratio of described gold nanorods GOLD FROM PLATING SOLUTION element and the element silicon of esters of silicon acis is 1:2-1:10.Solution ph can by this area the alkali compounds commonly used, as adjustments such as sodium hydroxide, potassium hydroxide, ammonia, under optimum condition, pH value is 8.5-10.5.Described esters of silicon acis is joined in described gold nanorods aqueous solution, can be disposable adding, also can add in batches, consider from the uniformity of the silicon dioxide layer obtained, preferably add in batches.Consider from operability, preferred 2-4 time, the interval time more preferably added for each time is identical.Reaction temperature and time, under preferable case, are 20-40 DEG C of reaction 12-48 hour.
According to golden core composite Nano support preparation method of the present invention, consider from the requirement of macromolecular polymerization reaction and solution dispersion, under preferable case, the consumption of 3-(methacryloxypropyl) propyl trimethoxy silicane is quality is gold nanorods (Au@SiO
2) 0.1-0.5 doubly, reaction 3-8h.High and be conducive to arriving tumor and consider from temperature sensitive thickness medicine carrying capacity, under preferable case, adding NIPA quality is Au@SiO
25-16 doubly; Adding acrylic acid quality is Au@SiO
20.4-1.2 doubly; Adding N,N methylene bis acrylamide quality is Au@SiO
20.7-2.2 doubly; Adding dodecyl sodium sulfate quality is Au@SiO
20.2-0.7 doubly; Logical nitrogen 30-60 minute, adding potassium peroxydisulfate quality is Au@SiO
20.2-0.7 doubly, 65-75 DEG C of polyreaction 4-6 hour.
The preferred golden core composite Nano support preparation method of the present invention, comprises the following steps:
(1) with cetyl trimethyl ammonium bromide modify gold nanorods solution for raw material, wherein, the length of gold nanorods is 5-100nm, and draw ratio is 1.5-10.0; In the gold nanorods concentration of gold element for 0.1-2mmol/L; The concentration of cetyl trimethyl ammonium bromide is 0.1-10mmol/L;
(2) in the solution of step (1), sodium hydroxide or ammonia spirit is added, pH value of solution is made to be in 8.0-12.0, then the methanol or the alcoholic solution that add silester for three times is divided, every minor tick 10-60min, react on 20-80 DEG C and carry out 1-4 days, wherein, respectively in gold element and element silicon, the gold nanorods in described gold nanorods aqueous solution and the mol ratio of described silester are 1:1-50;
(3) after above-mentioned steps (2) completes, add 3-(methacryloxypropyl) propyl trimethoxy silicane, quality is 0.02-2 times of gold nanorods, reaction 3-24h;
(4) by centrifugal for the reaction solution in step (3), make nanoparticle sedimentation wherein, remove unreacted 3-(methacryloxypropyl) propyl trimethoxy silicane in supernatant; Then add monomer NIPA, quality is 5-20 times of gold nanorods; Add acrylic acid, quality is 0.5-5 times of gold nanorods; Add N,N methylene bis acrylamide, quality is 0.5-5 times of gold nanorods; Add dodecyl sodium sulfate, quality is 0.1-1 times of gold nanorods, logical nitrogen 20-60min, add potassium peroxydisulfate, quality is 0.1-1 times of gold nanorods, 60-80 DEG C of polyreaction 4-8 hour, cool to room temperature after reaction, namely obtains phase transition temperature at the golden core composite Nano carrier of 34-42 DEG C.
Present invention also offers a kind of golden core composite Nano medicine-carried system, described medicine-carried system comprises above-mentioned carrier and the medicine of load on above-mentioned carrier.From being easy to, bag carries, the high and stability of drug loading is considered, under optimum condition, described medicine is one or both in doxorubicin hydrochloride, indocyanine green (ICG) and new indocyanine green (IR820).
According to golden core composite Nano medicine-carried system of the present invention, under optimum condition, the weight ratio of described pharmaceutical carrier and described drug solution Chinese medicine can be 2-25, and the carrying drug ratio of described golden core composite Nano medicine-carried system is 2.4-24.5%.
In the present invention, to the method for drug loading on above-mentioned carrier, there is no particular limitation, can adopt known various method.Such as, described can be contacted with described pharmaceutical carrier by drug solution by the method for drug loading on carrier.Contact Temperature is 20-40 DEG C, and time of contact is 1-3 days.Preferred described contact is carried out under magnetic stirring.
According to golden core composite Nano medicine-carried system of the present invention, there is multiple imaging function, may be used for the diagnosis of various disease, Real-Time Monitoring drug delivery and distribution situation, and be used for monitoring therapeuticing effect; In addition, this carrier can absorb near-infrared laser, and light energy conversion is become heat, on the one hand may be used for thermotherapy, can be used for drives medication release on the other hand, realizes treating the multi-mode of disease such as cancer.
The present invention also provides the application of above-mentioned golden core composite Nano medicine-carried system in diagnosing tumor and/or treatment.
Golden core composite Nano medicine-carried system provided by the invention has the multiple functions such as diagnosis, chemotherapy and thermotherapy, and these functions can use in conjunction.
The kind of tumor does not specially require, and only needs the medicine be applicable to according to the kinds of loads of tumor.Such as the tumor of applicable Diagnosis and Treat comprises: breast carcinoma, pulmonary carcinoma, melanoma, hepatocarcinoma, skin carcinoma, cervical cancer, bladder cancer, cancer of pancreas, gastric cancer etc.
The laser instrument used in the present invention is femtosecond titanium-doped sapphire pulse laser and 808 nanometer continuous-wave lasers.This two kinds of laser instrument are not limited in actual therapeutic process.As long as there is the wavelength of near infrared band and power enough makes material warms can to the laser instrument of more than 42 DEG C.
Below will be described the present invention by embodiment, but the present invention to be not limited in following embodiment, the reagent used by following examples or test instrunment, if no special instructions, to be commercially available can acquisition.
The preparation of embodiment 1 gold medal core composite Nano carrier
1) silicon dioxide coated gold nanorods (Au@SiO
2) preparation
In the beaker of 20mL, mixed with the aqueous solution (concentration is 0.01mol/L) of the gold chloride of 250 μ L by the aqueous solution (concentration is 0.1mol/L) of the cetyl trimethyl ammonium bromide of 7.5mL, adding water to cumulative volume is 9.4mL; Then the sodium borohydride aqueous solution (concentration is 0.01mol/L) of the cooled 0.6mL of frozen water is added; Vigorous stirring 3min, is placed in 27 DEG C of calorstat 3-5h, obtains gold nano grain seed solution.
In the beaker of 150mL, add the cetyl trimethyl ammonium bromide (concentration is 0.1mol/L) of 100mL respectively, the aqueous solution of chloraurate (concentration is 0.01mol/L) of 5mL, the silver nitrate aqueous solution (concentration is 0.01mol/L) of 0.8mL, the aqueous sulfuric acid (concentration is 0.5mol/L) of 2mL, and the aqueous ascorbic acid of 800 μ L (concentration is 0.1mol/L); Then add the gold nano grain seed solution obtained above of 240 μ L, be placed in 30 DEG C of calorstats and react 12h, obtain gold nanorods aqueous solution.The chances are 3.4 for the draw ratio of the gold nanorods that this step obtains.
The gold nanorods solution centrifugal of above-mentioned for 40mL preparation is washed twice, all makes for twice residual volume be 2.53mL.Add water to 40mL after first time completes again to disperse, add water to 20mL after second time completes and again disperse.In the gold nanorods aqueous solution finally obtained, cetyl trimethyl ammonium bromide concentration is 0.8mmol/L, and in the gold nanorods concentration of gold element for 0.5mmol/L.Then the sodium hydrate aqueous solution adjust ph under agitation adding 0.1mol/L is 10.0, then adds the ethyl orthosilicate methanol solution (concentration is 20 volume %) of 40 μ L every 30min, altogether adds three times.Reaction under agitation carries out 24 hours at 27 DEG C, obtains gold nanorods (the Au SiO that pan coating has meso-porous titanium dioxide silicon layer
2, as shown in Figure 1).The silicon dioxide coated gold nanorods that this step prepares, silicon dioxide layer thickness is 30nm, and the aperture of mesoporous silicon oxide is 3-4nm.
2) preparation of golden core composite Nano carrier (Nanocom)
In 50mL round-bottomed flask, add 20mL above-mentioned Au@SiO
2aqueous solution, then add 2.5 μ L 3-(methacryloxypropyl) propyl trimethoxy silicanes (being dissolved in 100 μ L methanol), room temperature reaction 5h.
By centrifugal for above-mentioned reaction solution, 5mL aqueous dispersion is used after removing supernatant, then 10mL NIPA aqueous solution (concentration is 100mmol/L) is added respectively, 1.1mL acrylic acid aqueous solution (concentration is 100mmol/L), 2.0mL N, N-methylene-bisacrylamide aqueous solution (concentration is 50mmol/L), 200 μ L sodium dodecyl sulfate aqueous solutions (concentration is 100mmol/L), magneton stirs, logical nitrogen 40 minutes, then 1.68mL persulfate aqueous solution (concentration is 10mmol/L) is added with syringe, under nitrogen atmosphere, 70 DEG C are reacted 4 hours.Obtain phase transition temperature the temperature sensitive high score attached bag of about 39 DEG C by silicon dioxide modified gold nanorods material (Fig. 2).The preparation of electron microscopic sample is by after centrifugal for material solution washing once, drip on the copper mesh containing common carbon film and dry, drip phosphotungstic acid aqueous solution (mass fraction is 2%) dyeing 7min again, then dry after siphoning away Salkowski's solution with filter paper, the golden core composite Nano carrier after dyeing can be obtained.
Fig. 2 represents that temperature sensing material bag is by Au@SiO
2the transmission electron microscope picture of the golden core composite Nano carrier formed, wherein, Fig. 2 a represents that diameter of nano particles is 300nm, and temperature sensitive macromolecule layer thickness is 110nm, finely disseminated golden core composite Nano carrier; Fig. 2 b represents that length is 140nm, width is 110nm, and temperature sensitive macromolecule layer thickness is the golden core composite Nano carrier of 20nm; Fig. 2 c represents that diameter is 250nm, and temperature sensitive macromolecule layer thickness is the golden core composite Nano carrier of 80nm; Fig. 2 d represents that diameter is 480nm, and temperature sensitive macromolecule layer thickness is the golden core composite Nano carrier of 200nm.
Embodiment 2
The present embodiment, for illustration of the temperature variant reversible process of particle diameter of golden core composite Nano carrier, can judge the phase transition temperature of temperature sensitive golden core composite Nano carrier by change of size speed.
By the golden core composite Nano carrier of preparation in embodiment 1, centrifugal washing once, then gets 2mL in cuvette, surveys its size at different temperatures with laser particle analyzer.Heat up gradually from 25 DEG C to 35 DEG C every 5 DEG C, 35 DEG C heat up to 43 DEG C gradually every 2 DEG C, and 43 DEG C heat up to 50 DEG C gradually every 3 or 4 DEG C, each temperature spot balance 5min, and measure the size of golden core composite Nano carrier at such a temperature.Then lower the temperature gradually from 50 DEG C, each temperature spot balance 5min, with identical temperature in temperature-rise period under measure the size of golden core composite Nano carrier, whether reversible to judge the change of size of golden core composite Nano carrier.As shown in Figure 3, the change of size of this golden core composite Nano carrier is maximum about 39 DEG C changes, and therefore its phase transition temperature is 39 DEG C, and change of size is reversible in intensification or temperature-fall period.
Embodiment 3
The present embodiment is for illustration of the golden particle diameter of core composite Nano carrier and the relation of pH.
Get the golden core composite Nano carrier of preparation in 7 parts of embodiments 1 in the little centrifuge tube of 1.5mL, every part of 1mL, centrifugal washing once, then respectively with the PBS aqueous dispersion that pH is 4.5,5.0,5.5,6.0,6.5,7.0,7.4, be placed in the current potential pond of laser particle analyzer successively, the hydration particle diameter of golden core composite Nano carrier under measuring this pH, and curve plotting.Fig. 4 display is from pH 7.4 to pH 6.0, and the change of size of golden core composite Nano carrier is little, and as pH<6.0, the particle diameter of golden core composite Nano carrier strengthens with acidity and obviously reduces, and shows the character of pH sensitivity.
Embodiment 4
The present embodiment is for illustration of golden core composite Nano carrier stability in physiological conditions.
The golden core composite Nano carrier solution of preparation in Example 1, centrifugal washing once, disperses with PBS.Respectively at 4 hours, 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days and 14 days, get 200 μ L microplate reader and detect the extinction spectra of solution under 400-1000nm wavelength, judged the stability of carrier by spectrum change.As shown in Figure 5, carrier is very stable in PBS, and spectrum change is little.
Embodiment 5
The present embodiment is for illustration of the ramp case of golden core composite Nano carrier under laser irradiates of variable concentrations.
The golden core composite Nano carrier solution of preparation in embodiment 1, centrifugal washing once, with the aqueous dispersion of same volume, wherein the concentration of Au is 70 μ g/mL, be diluted to respectively again 4 times (concentration of Au is 17.5 μ g/mL), 8 times (concentration of Au is 8.8 μ g/mL), 16 times (concentration of Au is 4.4 μ g/mL), 32 times (concentration of Au is 2.2 μ g/mL), each taking-up 400 μ L is placed in the little centrifuge tube femtosecond laser irradiation of 1.5mL, and (wavelength is 760nm, power is 500mW, spot diameter 2mm), with the maximum temperature in thermal imaging system record temperature-rise period.Fig. 6 is the curve that the maximum temperature of solution in temperature-rise period is done Au concentration in carrier, can see that carrier solution about the 5min of variable concentrations reaches maximum temperature, and then temperature keeps balance.
Embodiment 6
The present embodiment is for illustration of the ramp case of golden core composite Nano carrier under the laser of different capacity irradiates.
The golden core composite Nano carrier solution of preparation in embodiment 1, centrifugal washing once, then dilutes 4 times (Au concentration is 17.5 μ g/mL).Take out 400 μ L be placed in the little centrifuge tube femtosecond laser of 1.5mL irradiate (wavelength is 760nm, power is 100 respectively, 200,300,400mW, spot diameter 2mm), with the maximum temperature in thermal imaging system record temperature-rise period.Fig. 7 is the curve that the maximum temperature of solution in temperature-rise period is done Au concentration in carrier, and under can seeing different laser power, carrier solution about 5.5min reaches maximum temperature, then keeps balance.
The preparation of embodiment 7 gold medal core composite Nano medicine-carried system
Get the golden core composite Nano carrier solution (1.74mg/mL) of preparation in 1mL embodiment 1, centrifugal washing once, disperses by the PBS solution of 1.5mL 0.4mg/mL amycin, and room temperature lower magnetic force stirs 12h.Then centrifugal, washing twice, merges supernatant, measures the absorbance of supernatant at 480nm place by microplate reader, substitutes into the content that standard curve calculates amycin in supernatant.Carrying drug ratio and envelop rate are by formulae discovery below: carrying drug ratio (%)=carrier Chinese medicine quality/(carrier quality+carrier Chinese medicine quality) * 100%; Envelop rate (%)=carrier Chinese medicine quality/input medicine total amount * 100%.As can be seen from Table 1, the carrying drug ratio of amycin and envelop rate can reach 92% and 24.5% respectively.By reducing the input amount of amycin, carrying drug ratio and the envelop rate of amycin can be reduced.
Table 1
| Gold core composite Nano carrier (mg/mL) | Amycin (mg/mL) | Carrying drug ratio (%) | Envelop rate (%) |
| 1.16 | 0.4 | 24.5 | 92 |
| 1.16 | 0.3 | 17.8 | 90 |
| 1.16 | 0.2 | 7.8 | 89 |
| 1.16 | 0.05 | 2.4 | 75.1 |
Embodiment 8
The present embodiment is for illustration of the preparation of golden core composite Nano carrier carrier band medicine and molecular probe simultaneously.
To get in 1mL embodiment 1 the golden core composite Nano carrier solution (1.74mg/mL) of preparation, once, with the aqueous dispersion of 1.5mL 0.2mg/mL amycin and 0.2mg/mL indocyanine green or new indocyanine green, room temperature lower magnetic force stirs 12h in centrifugal washing.Then centrifugal, wash 1 time.Measure the extinction spectra of supernatant under 400-1000nm wavelength by microplate reader, find the absworption peak not having indocyanine green or new indocyanine green, namely indocyanine green or new indocyanine green are all loaded in carrier.
Embodiment 9
The present embodiment is for illustration of the change of the extinction spectra before and after golden core composite Nano carrier modification and medicine carrying.
Respectively get the medicine carrying material of preparation in the gold rod of preparation in 200 μ L embodiments 1, silicon dioxide coated gold rod, golden core composite Nano carrier and embodiment 7, centrifugal washing once, dilute 1 times, detect the extinction spectra of solution under 400-1000nm wavelength by microplate reader.As shown in Figure 8, the maximum absorption band of gold rod is at 740nm, after the excellent bag silicon of gold, maximum absorption wavelength is at 760nm, and the maximum absorption band of golden core composite Nano carrier is also at 760nm, and after carrying amycin, maximum absorption band is 730nm and occurs obvious amycin absworption peak at 480nm place.
Embodiment 10
The present embodiment for illustration of golden core composite Nano medicine-carried system at different pH with or without the drug release behavior under laser irradiation condition.
To get in 2 parts of 0.5mL embodiments 7 medicine carrying material of preparation, centrifugally remove supernatant, disperse with the PBS buffer of pH 5.0 and pH 7.4 respectively.Interval is carried out centrifugal at regular intervals, and take supernatant away and measure doxorubicin content in supernatant by microplate reader, the PBS buffer of residue same volume and pH disperses again, proceeds release reaction.Laser irradiates release experiment and adopts similar step to carry out, and lasing condition is respectively 780nm wavelength, 200 or 300mW power, 2mm spot diameter.Solution temperature is monitored by thermal imaging system, and 200mW laser irradiates solution final temperature and is raised to 37 DEG C, and 300mW laser irradiates solution temperature and is finally raised to 48 DEG C.As shown in Figure 9, amycin in acid condition (pH 5.0) than (pH 7.4) release under neutrallty condition faster and more, and laser irradiate cause intensification to promote drug release.
Embodiment 11
The present embodiment is for illustration of the drug release behavior of golden core composite Nano medicine-carried system in 37 DEG C of water-baths, 100% hyclone.
To get in 0.5mL embodiment 7 medicine carrying material of preparation, centrifugally remove supernatant, disperse with 100% hyclone.Interval is carried out centrifugal at regular intervals, take supernatant away and measure (the fluorescent method detection: excitation wavelength is 480nm of doxorubicin content in supernatant by microplate reader, emission wavelength is 595nm), 100% hyclone of residue same volume disperses again, proceeds release reaction.As shown in Figure 10, medicine-carried system release in serum (release 60% in 24 hours) is faster than release in PBS (Fig. 9,24h release 40%), may be cause containing more salt and albumen in serum.
Embodiment 12
The present embodiment for illustration of anticancer drugs, doxorubicin, golden core composite Nano medicine-carried system and golden core composite Nano carrier itself on the impact of mammary carcinoma 4T1 cell viability.
1) cell culture
By mammary carcinoma 4T1 cell culture in containing in the DMEM of 10% hyclone or 1640 fluid mediums, be placed in 37 DEG C, the incubator of 5% carbon dioxide cultivates.
2) cell viability measures
With 6000 cells/well density, cell is inoculated in 96 orifice plates.After adherent 24h, add containing concentration be 0.1,1.0,5.0,10 μm of ol/L amycin or containing the golden core composite Nano medicine carrying material (from embodiment 7) of same doxorubicin concentration or the DMEM of golden core composite Nano carrier (from embodiment 1, concentration is identical with the molar concentration of golden core composite Nano medicine carrying material) or 1640 fluid mediums (containing 10% hyclone).After process 12h, 24h, 48h, 72h, remove culture medium; Every hole adds 110 μ L, and containing the cell culture fluid of 10% (volume ratio) CCK-8, hatch 2h in incubator after, microplate reader measures 450nm place absorbance, and 600nm is as reference wavelength.After often organizing absorbance deduction blank solution absorbance, every hole respective value divided by the absorbance of matched group as cell viability.Often group arranges 6 parallel holes.As shown in figure 11, golden core composite Nano carrier itself of the present invention does not have toxicity to cell viability measurement result, and golden core composite Nano medicine carrying material can play the anticancer effect the same with anticancer drugs, doxorubicin.
Embodiment 13
The present embodiment is for illustration of the absorption of mammary carcinoma 4T1 cell to golden core composite Nano medicine-carried system of the present invention.
1) cell culture
With embodiment 12.
2) cellular uptake quantitative determination
Cultured cells in step 1 is inoculated in 6 orifice plates, 80,000 cells/well, adds 2mL DMEM or 1640 fluid mediums (containing 10% hyclone), be placed in 37 DEG C, 5% CO2 gas incubator cultivates 24h.Then in the DMEM containing 10% hyclone or 1640 culture medium of new preparation, add the amycin of 5 μm of ol/L and the golden core composite Nano medicine carrying material (from embodiment 7) of 5 μm of ol/L equivalence doxorubicin concentration respectively, respectively getting 2mL after mixing adds in 6 orifice plates, often kind of concentration arranges 4 parallel holes, 37 DEG C, cultivate in the incubator of 5% carbon dioxide.Different time points (3,6,12,24h) use trypsin digestion and cell respectively, 800g is centrifugal removes supernatant, with the resuspended preparation cell suspension of 0.5mL Hanks buffer, then uses flow cytomery intracellular Fluorescence value.Detected parameters: take the streaming laser voltage of fixing in experimentation, 488nm laser excitation, collects FL2 access detection signal.Gathering the Mean Fluorescence of 10,000 cells, by several Duplicate Samples calculating mean value, is the fluorescent quantitation value of this cellular uptake amycin (Dox) of this time point and golden core composite Nano medicine carrying material (Nanocom-Dox).Get fluorescent quantitation value, Origin software is mapped, and analysis of cells takes in the trend of amycin.As Figure 12 can find out, the effective carrier band amycin of golden core composite Nano carrier energy of the present invention, and make it to enter cell.
Embodiment 14
The present embodiment is the positioning scenarios in tumor cell for illustration of amycin and golden core composite Nano medicine carrying material.
1) cell culture
Mammary carcinoma 4T1 cell is inoculated in 35mm co-focusing imaging culture dish, with the DMEM containing 10% hyclone or 1640 cultivate based on 37 DEG C, cultivate 24h in the incubator of 5% carbon dioxide.Then wash twice by phosphate buffered solution, add the culture medium of the golden core composite Nano medicine carrying material containing 5 μm of ol/L equivalence amycin containing preparation in 5 μm of ol/L amycin or embodiment 7, process cell 4h.
2) mensuration of intracellular targeting
Abandon cell culture medium, with phosphate buffered solution washed cell three times, add serum-free without phenol red DMEM or 1640 culture medium, with the fluorescence observing amycin under laser confocal microscope.After Figure 13 shows and hatches 4h, with containing the celluar localization image after the serum-free DMEM of lysosome dyestuff (LysoTracker Green) of 100nmol/L or 1640 culture medium, 37 DEG C of incubated cell 45min, can find out that the amycin that part gold core composite Nano is carrier carried and lysosome are located altogether, and amycin itself is located altogether with lysosome is less, illustrate that golden core composite Nano medicine carrying material enters cell and is positioned lysosome, and the amycin of its carrier band does not discharge completely, show the effect of slow release.
Embodiment 15
The present embodiment is for illustration of golden core composite Nano carrier circulation time in blood.
1) gold nanorods of Bovine Serum Albumin Modified and silicon dioxide coated gold nanorods is prepared
By gold nanorods in embodiment 1 and silicon dioxide coated gold rod, once, be the bovine serum albumin ultrasonic disperse of 1% with mass fraction, reaction is spent the night in centrifugal washing.
2) administration and drawing materials
Get the gold nanorods of 200 μ L bovine serum albumin dispersions and silicon dioxide coated gold nanorods respectively, and the golden core composite Nano carrier of preparation in embodiment 1, centrifugal washing once, with 200 μ L PBS aqueous dispersion, then tail vein injection is in the male balb/c Mice Body in 6-8 week, get blood respectively at 3min, 10min, 1h, 2h and 24h eyeball, then respectively take out 100 μ L in the little centrifuge tube of 1.5mL, frozen in-20 DEG C.Often organize three mouse and do parallel control.Respectively get 3 part of 200 μ L material solution in addition, clear up survey gold content together with blood sample.
3) Specimen eliminating detect gold content with inductivity coupled plasma mass spectrometry
Sample in step 1 is added nitric acid (MOS level) to transfer in fine taper bottle or beaker, then add 4mL nitric acid (MOS level), reaction is spent the night.Then each sample adds 3mL 30% hydrogen peroxide (MOS level), be put into heating and decompose on electric hot plate, electric plate temperature heats up gradually from 50 DEG C to 150 DEG C, about 1mL is remained Deng solution, sample is taken off from electric hot plate, 2.5mL chloroazotic acid (nitric acid: hydrochloric acid volume ratio=1:3) is added after cooling, continue to clear up on electric hot plate (about 100 DEG C), about 0.5mL is remained Deng solution, take off sample from electric hot plate, cool rear mixed acid solution (containing 2% nitric acid and 1% hydrochloric acid) and be diluted to 3 grams.The standard solution (0.5,1,5,10,50,100ppb) of a series of gold is configured, for carrying out quantitatively Gold Samples content with nitration mixture.Finally use the gold content of inductivity coupled plasma mass spectrometry examination criteria solution, blood sample and material itself, calculate the percentage composition of material in blood with excel form.As shown in figure 14, the blood circulation time of the golden core composite Nano carrier of temperature sensitive macromolecule layer bag quilt is the longest.Therefore, the gold rod modified compared to serum albumin and silicon dioxide coated gold excellent, golden core composite Nano carrier more may play curative effect in a large amount of enrichment of tumor.
Embodiment 16
The present embodiment increases the enrichment of golden core composite Nano carrier at tumor locus for illustration of heating in water bath.
1) mouse inoculated tumour
With depilatory cream to the right rear leg depilation of 6-8 week male balb/c mice, then by 1*10
6individual mammary carcinoma 4T1 cell is inoculated in the right rear leg of mice, grows to 1000mm etc. tumor
3heating in water bath experiment is carried out in left and right.
2) heating in water bath increases the enrichment at tumor tissues of medicine and fluorescence molecule
After mouse anesthesia in step 1,5min in 42 DEG C of waters bath with thermostatic control is immersed by there being the right rear leg of tumor, in Mice Body in the golden core composite Nano carrier tail vein injection to step 1 of the carrier band near infrared fluorescent dye then prepared in tail vein injection 200 μ L embodiment 12, again mice right rear leg is continued to soak 30min in 42 DEG C of waters bath with thermostatic control, then the fluorescence (excitation wavelength is 690nm, and emission wavelength is 700-900nm) of Maestro 2 multispectral small animal imaging systems axiol-ogy nir dye is used.Figure 15 shows golden core composite Nano carrier having heating in water bath than under the condition not having heating in water bath, more in the enrichment at mouse tumor position; After sacrifice, take out tumor and other organs, carry out in vitro near-infrared fluorescence imaging and detect, show golden core composite Nano carrier after heating in water bath, increase in tumor tissues enrichment, reduce in the accumulation of spleen and kidney.
By the golden core composite Nano carrier (prepared by embodiment 8) of 200 μ L near infrared fluorescent dyes and carrier band same concentration near infrared fluorescent dye by the Mice Body in tail vein injection to step 1, again mice right rear leg is continued to soak 30min in 42 DEG C of waters bath with thermostatic control, then use the enrichment condition (excitation wavelength of fluoroscopic examination is 690nm, and emission wavelength is 700-900nm) of Maestro 2 multispectral small animal imaging system Real-Time Monitoring dyestuff simultaneously.As Figure 16, obviously than dyestuff, these are many in tumor enrichment for the carrier carried fluorescent dye of golden core composite Nano, and after 6h, the fluorescence of the fluorescent dye that golden core composite Nano is carrier carried does not obviously reduce, and the fluorescence of dyestuff itself changes decay clearly in time.
Embodiment 17
The present embodiment irradiates for illustration of laser can increase the enrichment of golden core composite Nano carrier at tumor locus.
1) observe laser by photoacoustic imaging and irradiate the enrichment of rear golden core composite Nano carrier in tumor
By 1*10
6individual mammary carcinoma 4T1 cell is inoculated in the back of 6-8 week balb/c nude mice, waits gross tumor volume to grow to 150mm
3shi Jinhang laser enrichment experiment.After mouse anesthesia, the golden core composite Nano carrier (prepared by embodiment 1) that intravenous injection 200 μ LPBS disperses, then at once with femtosecond laser (760nm, 500mW, 2mm spot diameter) irradiate 20min, then observe the enrichment of material in tumor by photoacoustic imaging.Do not have injection material and injection material not according to laser mouse in contrast.As shown in figure 17, laser irradiates obviously can increase the accumulation of carrier at tumor locus.
2) content of material in tumor is measured by inductivity coupled plasma mass spectrometry
With depilatory cream to the right rear leg depilation of 6-8 week male balb/c mice, then by 1*10
6individual mammary carcinoma 4T1 cell is inoculated in the right rear leg of mice, grows to 100mm etc. tumor
3laser enrichment experiment is carried out in left and right.After mouse anesthesia, the golden core composite Nano carrier (prepared by embodiment 1) that intravenous injection 200 μ L PBS disperses, then at once with femtosecond laser (760nm, 500mW, 2mm spot diameter) irradiate 20min, put to death mouse respectively at administration 30min and 24h and take out tumor and other organs.Tail intravenously administrable but not according to laser mouse in contrast.Often group arranges 3 Duplicate Samples.Tumor and internal organs are weighed, is then put in fine taper bottle or beaker, clears up sample according to the method in embodiment 15, and measure the gold content in each internal organs with inductivity coupled plasma mass spectrometry.By Excel Form Handle experimental result, result represents the percentage composition (%ID/g, Figure 18) of injected dose in every gram of tissue.Result shows that the Au content of the tumor locus of irradiating laser adds about 7.6 times than non-irradiated mice, and still exists after the 24h of this enrichment effect after laser has irradiated.Therefore, near-infrared laser irradiates the enrichment that can considerably increase tumor locus nano-carrier, for further oncotherapy lays the first stone.
Embodiment 18
The present embodiment may be used for X ray computer Tomography for illustration of golden core composite Nano carrier.
The all male balb/c mices of 6-8, after anesthesia, the golden core composite Nano carrier (prepared by embodiment 1) that vein or subcutaneous injection 200 μ L PBS disperse, then at once with the imaging of X ray computer tomoscanner.The living imaging figure of Figure 19 to be intravenously administrable gold content be 1mg/kg body weight, can find out that gold element signal is very strong, mainly be distributed in liver and spleen.Therefore, golden core composite Nano carrier can be used as a kind of CT contrast agent, is used for carrying out bio-imaging.
Embodiment 19
The present embodiment is for illustration of the ramp case of tumor locus after mouse vein administration.
1) mouse inoculated tumour
With depilatory cream to the right rear leg depilation of 6-8 week male balb/c mice, then by 1*10
6individual mammary carcinoma 4T1 cell is inoculated in the right rear leg of mice, and after 4 days, gross tumor volume reaches 100mm
3left and right, carries out laser intensification and Experiment on therapy.
2) tumor ramp case
Golden core composite Nano carrier solution (25mg/mL in the embodiment 1 of 200 μ L, gold content is 950 μ g/mL), (gold content is 950 μ g/mL to golden core composite Nano medicine carrying solution in embodiment 7, equivalence doxorubicin concentration is 1mg/mL), PBS and 1mg/mL Doxorubicin solution through tail vein injection to the mice in step 1, then at once with femtosecond laser (760nm, 500mW, 2mm spot diameter) irradiate 20min, with the maximum temperature in thermal imaging system record temperature-rise period.Figure 20 is the curve that the maximum temperature of mouse tumor in temperature-rise period is done irradiation time, can see that PBS and amycin group show the increasing extent of temperature of about 7 DEG C, and the temperature of carrier and medicine carrying group about improves 40 DEG C, has obvious photothermal deformation ability.
Embodiment 20
The present embodiment is for illustration of the tumor suppression result after tumor-bearing mice intravenously administrable.
1) mouse inoculated tumour
With embodiment 19.
2) inhibiting tumor assay
Golden core composite Nano carrier solution (25mg/mL in the embodiment 1 of 200 μ L, gold content is 950 μ g/mL), (gold content is 950 μ g/mL to golden core composite Nano medicine carrying solution in embodiment 7, equivalence doxorubicin concentration is 1mg/mL), PBS and 1mg/mL Doxorubicin solution to the mice in step 1, then uses femtosecond laser (760nm, 500mW through tail vein injection at once, 2mm spot diameter) irradiate 20min, monitor temperature-rise period with thermal imaging system.Tail intravenously administrable but not according to laser mouse in contrast.Laser irradiates the latter second day size by vernier caliper measurement tumor, uses the size of vernier caliper measurement tumor every three days backward, monitors 14 days always.When 14 days, put to death mouse, take out tumor and weigh, take pictures.Figure 21 is respectively by the tumor suppression result that gross tumor volume and weight represent, can find out that carrier and medicine carrying group are according to after laser, tumor growth obviously receives suppression.Thermotherapy is inherently enough to the growth of Tumor suppression; Medicine carrying group and amycin group tumour inhibiting rate almost, illustrate that medicine-carried system can arrive the effect that tumor plays chemotherapy.
Embodiment 21
The present embodiment is for illustration of the tumor suppression condition optimizing of tumor-bearing mice intravenously administrable.
1) mouse inoculated tumour
With embodiment 19.
2) tumor suppression condition optimizing
Golden core composite Nano carrier solution (25mg/mL in the embodiment 1 of 200 μ L, gold content is 950 μ g/mL), (gold content is 950 μ g/mL with the golden core composite Nano medicine carrying solution in embodiment 7, equivalence doxorubicin concentration is 1mg/mL), through tail vein injection to the mice in step 1, then irradiate 10min with 808nm continuous wave laser at once, be raised to 45 DEG C, 50 DEG C and about 60 DEG C respectively by regulating laser power control tumor final temperature.By the variations in temperature in thermal imaging system monitoring temperature-rise period.As Figure 22, when 45 DEG C, mainly chemotherapy works; The synergy of thermotherapy and chemotherapy is embodied when 50 DEG C; When 60 DEG C, major embodiment thermotherapy is to the lethal effect of tumor.Visible, control laser power and make tumor temperature be raised to about 50 DEG C to be most suitable treatment condition, can tumor growth to be restrained, turn avoid the too high side effect caused of temperature.
Embodiment 22
The present embodiment can not cause lesion tissue for illustration of golden core composite Nano carrier, and can Tumor suppression transfer.
Each group of mouse internal organs (heart, liver, spleen, lung, kidney) in embodiment 20 are taken out, does pathological section.Concrete steps are as follows: be put in by each internal organs in 10% formalin fixing, by tissue sampling, embed in wax stone, section, with h and E dyeing, then with the pathological change of observation by light microscope tissue.As Figure 23, compare with PBS group, carrier and medicine carrying group are according to after laser, and obvious pathology damage does not appear in each internal organs (heart, liver, spleen, lung and kidney); Due to the tumor cell that mammary carcinoma 4T1 cell is high malignancy, there is obvious neoplasm metastasis in the lung of PBS group, the lung of Dox group has a small amount of metastasis, and there is not any neoplasm metastasis in lung after carrier and medicine carrying group oncotherapy, illustrate that golden core composite Nano carrier can grow by Tumor suppression, again can Tumor suppression transfer.
Embodiment 23
The present embodiment is for illustration of the blood biochemistry result after golden core composite Nano carrier intravenously administrable treatment.
Before being put to death by each group of mouse in embodiment 20, eyeball gets blood, the blood taken out leaves standstill 3h, then 3000 rpms of centrifugal 15min, collect serum above, alanine aminotransferase (ALT), aspartate amino transferase (AST), glutamyl transpeptidase (γ-GT), total bilirubin (TBIL), total protein (TP), albumin (ALB), globulin (GLOB), alkali phosphatase (ALP) is measured by biochemistry analyzer, whether normal to judge each group of mouse liver function.Table 2 is the blood biochemistry results after mouse vein drug treatment.
Table 2
| Blood biochemistry result | PBS | Dox | Nanocom+ laser | Nanocom-Dox+ laser |
| ALT(U/L) | 28.50±9.19 | 25.67±4.04 | 35.00±2.82 | 42.61±7.28 |
| AST(U/L) | 173.50±9.19 | 146.50±28.99 | 137.50±6.36 * | 138.8±35.54 |
| AST/ALT | 6.37±1.73 | 5.47±2.16 | 3.95±0.50 * | 3.14±1.11 * |
| TBIL(μmol/L) | 3.10±0.85 | 1.60±0.17 * | 1.06±0.34 * | 1.38±0.38 * |
| TP(g/l) | 48.15±2.90 | 47.20±0.72 | 48.38±2.37 | 47.90±3.13 |
| ALB(g/l) | 29.15±1.63 | 30.60±0.72 | 29.91±1.63 | 30.23±1.89 |
| GLOB(g/l) | 19.00±1.27 | 16.60±1.40 | 18.47±1.22 | 17.67±1.32 |
| ALP(U/L) | 50.50±2.12 | 39.50±2.12 | 61.33±7.37 | 54.22±9.02 |
| ALB/GLOB | 1.50±0.00 | 1.87±0.21 | 1.63±1.67 | 1.73±0.09 |
As can be seen from Table 2, as compared to PBS with Dox group, gold core composite Nano carrier (Nanocom+ laser) and golden core composite Nano medicine carrying group (Nanocom-Dox+ laser) have lower [AST/ALT] value and total bilirubin (TBIL) content, effectively inhibit the growth of tumor after carrier and the treatment of medicine carrying group are described, thus reduce the impact of tumor on liver.In conjunction with the data result of all blood biochemistry, illustrate that carrier and medicine carrying group can not cause hepatic injury in tumor therapeutic procedure.
Embodiment 24
The present embodiment is for illustration of the tumor suppression result of injectable drug in tumor.
1) mouse inoculated tumour
With embodiment 19.
2) inhibiting tumor assay
Golden core composite Nano carrier solution (25mg/mL in the embodiment 1 of 50 μ L, gold content is 950 μ g/mL), (gold content is 950 μ g/mL to golden core composite Nano medicine carrying solution in embodiment 7, equivalence doxorubicin concentration is 1mg/mL), PBS and 1mg/mL Doxorubicin solution, in the tumor of direct injection mice in step 1, then at once with femtosecond laser (760nm, 500mW, 2mm spot diameter) irradiate tumor 20min, monitor temperature-rise period with thermal imaging system.Intratumor injection medicine but not according to laser mouse in contrast.Laser irradiates the latter second day size by vernier caliper measurement tumor, uses the size of vernier caliper measurement tumor every three days backward, monitors 14 days always.When 14 days, put to death mouse, take out tumor and weigh, take pictures.Figure 24 is respectively by the tumor suppression result that gross tumor volume and weight represent, can find out that carrier and medicine carrying group are according to after laser, tumor growth obviously receives suppression.Medicine carrying group is better according to the tumor killing effect of laser than vehicle group according to laser, embodies the synergy of thermotherapy and chemotherapy combined application.Compare with the thermotherapy of intravenously administrable and Results of Chemotherapy, although intratumor injection is higher than the gold content being enriched to tumor by intravenously administrable a lot of to the gold content of tumor locus, but the result that the therapeutic outcome of intratumor injection does not have intravenously administrable is good, reason may be that the feature that tumor tissues has an interstitial high pressure makes intratumor injection medicine uneven, so do not have vein treatment effective.Golden core composite Nano carrier provided by the invention, there is macrocyclic character, therefore be applicable to by intravenous mode administration, by the photothermal deformation of gold nanorods and temperature sensitive high molecular temperature-sensitive under near-infrared laser irradiates, can in the enrichment of tumor locus significant effective, and then realize the conjunctive use of heat-transformation treatment, for neoplasm targeted therapy provides a kind of New Policy.
Applicant states, the present invention illustrates process of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned processing step, does not namely mean that the present invention must rely on above-mentioned processing step and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, the concrete way choice etc. of raw material selected by the present invention, all drops within protection scope of the present invention and open scope.
Claims (10)
1. a golden core composite Nano carrier, is characterized in that, described carrier comprises with silicon dioxide modified gold nanorods kernel, and is coated on the macromolecular material of described gold nanorods core surface;
Wherein, described macromolecular material is NIPA and acrylic acid copolymer.
2. carrier according to claim 1, is characterized in that, the length of described gold nanorods is 5-100nm; The draw ratio of described gold nanorods is 1.5-20;
Preferably, the thickness of described silicon dioxide layer is 3-50nm; Described polymer material layer is netted hydrogel structure, and its thickness is 5-300nm.
3. a preparation method for golden core composite Nano carrier as claimed in claim 1 or 2, it is characterized in that, the method comprises the following steps:
(1) in the gold nanorods aqueous solution containing cetyl trimethyl ammonium bromide, be that under the condition of 8.0-12.0, the alcoholic solution adding esters of silicon acis reacts at pH value, and carry out solid-liquid separation, obtain the gold nanorods that Surface coating has silicon dioxide layer;
(2) by monomer NIPA, acrylic acid, coupling agent, surfactant and initiator, under inert gas shielding, be heated to 60-80 DEG C of polyreaction 4-8 hour, prepare the macromolecular material of NIPA and acrylic acid copolymer;
(3) add 3-(methacryloxypropyl) propyl trimethoxy silicane in the gold nanorods obtained to step (1) and modify vinyl at silica surface, add the macromolecular material that step (2) obtains again, namely obtain described golden core composite Nano carrier.
4. method according to claim 3, is characterized in that, in the gold nanorods aqueous solution described in step (1), in the gold nanorods concentration of gold element for 0.1-2mmol/L, the length of described gold nanorods is 5-100nm; The draw ratio of described gold nanorods is 1.5-20;
Preferably, respectively in gold element and element silicon, the gold nanorods in described gold nanorods aqueous solution and the mol ratio of described esters of silicon acis are 1:1-50; In the mol ratio of the described gold nanorods of gold element and cetyl trimethyl ammonium bromide for 1:1-20;
Preferably, the temperature of described reaction is 20-80 DEG C, and the time of reaction is 0.04-10 days;
Preferably, described esters of silicon acis is quanmethyl silicate, tetraethyl orthosilicate, one or more in silicic acid orthocarbonate or silicic acid four butyl ester;
Preferably, described alcohol is methanol, ethanol or other molecular weight be less than 200, be one or more of the alcohol apoplexy due to endogenous wind of liquid under normal temperature and pressure.
5. the method according to claim 3 or 4, is characterized in that, in step (2), described coupling agent is N,N methylene bis acrylamide, and described surfactant is dodecyl sodium sulfate, and described initiator is potassium sulfate.
6. the method according to any one of claim 3-5, is characterized in that, the method comprises the following steps:
(1) with cetyl trimethyl ammonium bromide modify gold nanorods solution for raw material, wherein, the length of gold nanorods is 5-100nm, and draw ratio is 1.5-10.0; In the gold nanorods concentration of gold element for 0.1-2mmol/L; The concentration of cetyl trimethyl ammonium bromide is 0.1-10mmol/L;
(2) in the solution of step (1), sodium hydroxide or ammonia spirit is added, pH value of solution is made to be in 8.0-12.0, then the methanol or the alcoholic solution that add silester for three times is divided, every minor tick 10-60min, react on 20-80 DEG C and carry out 1-4 days, wherein, respectively in gold element and element silicon, the gold nanorods in described gold nanorods aqueous solution and the mol ratio of described silester are 1:1-50;
(3) after above-mentioned steps (2) completes, add 3-(methacryloxypropyl) propyl trimethoxy silicane, quality is 0.02-2 times of gold nanorods, reaction 3-24h;
(4) by centrifugal for the reaction solution in step (3), make nanoparticle sedimentation wherein, remove unreacted 3-(methacryloxypropyl) propyl trimethoxy silicane in supernatant; Then add monomer NIPA, quality is 5-20 times of gold nanorods; Add acrylic acid, quality is 0.5-5 times of gold nanorods; Add N,N methylene bis acrylamide, quality is 0.5-5 times of gold nanorods; Add dodecyl sodium sulfate, quality is 0.1-1 times of gold nanorods, logical nitrogen 20-60min, add potassium peroxydisulfate, quality is 0.1-1 times of gold nanorods, 60-80 DEG C of polyreaction 4-8 hour, cool to room temperature after reaction, namely obtains phase transition temperature at the golden core composite Nano carrier of 34-42 DEG C.
7. a pharmaceutical composition, comprises carrier and the medicine of load on carrier, it is characterized in that, described carrier is the carrier described in claim 1 or 2, or the carrier for being prepared by method according to any one of claim 3-6.
8. pharmaceutical composition according to claim 7, is characterized in that, described medicine be in doxorubicin hydrochloride, cisplatin, metal fullerene, DNA, siRNA, protein drug, indocyanine green or rhodamine any one or multiple.
9. the pharmaceutical composition according to claim 7 or 8, is characterized in that, with the weight of described carrier for benchmark, the carrying drug ratio of described pharmaceutical composition is 1-40 % by weight.
10. the carrier described in claim 1 or 2 and/or the application of the pharmaceutical composition described in any one of claim 7-9 in the medicine for the preparation of diagnosis and/or treatment tumor.
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