CN103539096A - Preparation method of hydroxyl zinc phosphate nano hollow spheres for drug carrier - Google Patents
Preparation method of hydroxyl zinc phosphate nano hollow spheres for drug carrier Download PDFInfo
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Abstract
The invention relates to a preparation method of hydroxyl zinc phosphate nano hollow spheres for a drug carrier. In the method, zinc nitrate and diammonium hydrogen phosphate are used as reactants, the reaction temperature is controlled below 0-25 DEG C, and the hydroxyl zinc phosphate nano hollow spheres are prepared by a chemical coprecipitation method. Compared with the prior art, the method provided by the invention has the advantages of simple process, low cost, low toxicity, large drug loading capacity, quick cell entering, slow release control on drug and the like.
Description
Technical field
The present invention relates to the synthetic field of inorganic hollow nano material, more particularly, relate to a kind of inorganic materials of the novel nano level hollow structure as pharmaceutical carrier.
Background technology
Hollow Nano particle is obtaining great concern nearly ten years because it has good pattern, lower density, larger specific surface area and wide application prospect.Compare with full particle, hollow structure has how outstanding chemistry and physical property.In numerous hollow beads, the characteristic of the aspects such as that inorganic hollow nano particle has is optical, electrical, magnetic, electrochemistry and catalysis, this surface inorganic hollow nano particle be a kind of more common, more diversified with organic hollow Nano Particle Phase ratio, the changeable class material of horn of plenty more.Therefore within the most several years, for the inorganic hollow particle with large cavity, have extremely many research, proved that it is in catalysis, the aspects such as drug conveying and medical imaging are widely used.
In synthesizing inorganic nano-hollow material method, template synthesis method is the most common.Template synthesis method is by modifying template, making it have induction inorganic precursor (salt or alkoxide) and accumulate in surperficial ability; Then take template after karyomorphism becomes inorganic shell, template must profit be removed by some way, finally obtains the particle of hollow structure.Template can be divided into two kinds of hard template method and soft template methods.Hard template method refers to utilizes stiff molecule as the synthetic method of template.The feature of hard template method is that shape and the size of final product all depends on template substantially.Chemical compound lot is used as polymkeric substance, inorganic non-metallic, metallic particles all can be used as hard template.In preparing hollow bead process, having three steps is must be obligato: 1. pair template is carried out finishing/functionalization to obtain needed surface property; 2. utilize different methods and reactive force inorganic shell or inorganic precursor on template surface covers; 3. optionally remove template and finally obtain hollow ball (by selective dissolution or roasting).The advantage of hard template method is to guarantee the pattern of product and the homogeneity of size, also can apply to synthetic various inorganic hollow materials.Yet its deficiency is also fairly obvious: its building-up process is consuming time and complicated, energy consumption is also very large and also have the risk that hollow structure is collapsed to exist while removing template while removing template.Advantage maximum for hard template method is easily being removed with its template.That can use as soft template is not limited only to emulsion droplet liquid, and tensio-active agent, or other supramolecule particulates, also comprise polyphosphazene polymer collective and bubble.Compare with hard template method, soft template method not only can remove template by gentle condition and method, and the drop using as template etc. can will have the cavity inside that therapeutic action or bioactive material are introduced product simply again effectively.Although yet having had larger progress aspect control product size, homogeneity and microstructure, when in the face of the problems referred to above, utilize the synthetic hollow product of soft template method to be still difficult to overcome completely.This also will be owing to the characteristic of soft template.For bio-medical material, template complex process, material property controllability difficulty is large, and manufacturing cost is high, is unfavorable for enterprise scale production; On the other hand, mould is pulled agent meeting and is brought potential biological safety.Therefore, simple, the mild condition of technique, green synthetic non-template legal system are favourably welcome for inorganic nano hollow ball.
Another impressive progress prepared by hollow inorganic spheres is to utilize the strategy of non-template to carry out synthesizing inorganic nanometer hollow granule, it is Oswald that moral maturing process (Ostwald ripening process), this method not only combines the advantage of soft/hard template method, also avoided soft, hard template method to remove difficulty (for hard template) as template, pattern is the deficiency such as controlled (for soft template) well simultaneously.You moral slaking mechanism (inside-out OsMald ripening process) have following ultimate principle most basic Oswald from inside to outside; Larger crystal is all grown compared with small-crystalline preferably by absorbing solvability.In a colloidal state aggregate, the crystallite less, crystallinity is poorer, more loose can dissolve in liquid phase gradually, and become larger, crystallinity better, " nutrition " of more fine and close microcrystalline growth originate.In the process of crystal growth, because the size difference of nanocrystal, the concentration of crystal growth unit is different in mother liquor.Be subject to ordering about of surperficial energy minimization, metastable nano particle produces gathering because of the minimizing of solution degree of supersaturation.Once the particle aggregation of different size is together, larger particle will absorb less particle further growth gradually, and at large aggregate intermediate formation cavity, and the shell of cavity is thick also because the solute of middle cavity constantly increases to external diffusion.With Oswald your moral aging machine, being made as basic non-template method can well solve in hard template method, needed to remove the problem of template and the problem that soft template method product appearance and size differs simultaneously.Yet this kind of special effect only appears in some special compound, do not have enough abundant basis credential can further support this mechanism.
The most attractive place of inorganic hollow particle is embodied in it and applies widely.The specific surface area, low density of utilizing its good pattern, super large with and in the characteristic of the aspects such as optical, electrical, magnetic, inorganic hollow particle has important application in fields such as catalysis, lithium cell, biological medicine and gas detection.While being used as for biological medical material, the purposes of the inorganic hollow particle maximum that composition is simple, performance is controlled, biological safety is good is loaded and delivering medicament as drug carrier exactly, and controls the release of medicine in cell and body.
Summary of the invention
Object of the present invention is exactly that a kind of preparation method of the hydroxyl zinc phosphate nano-hollow ball for pharmaceutical carrier is provided in order to overcome the defect of above-mentioned prior art existence.
Object of the present invention can be achieved through the following technical solutions: a kind of preparation method of the hydroxyl zinc phosphate nano-hollow ball for pharmaceutical carrier, it is characterized in that, the method is that to take zinc nitrate and Secondary ammonium phosphate be reactant, control temperature of reaction at 0-25 ℃, by chemical coprecipitation, make hydroxyl zinc phosphate nano-hollow ball.
Described chemical coprecipitation is specially: the solution that zinc nitrate is mixed with to 33.4mM, the trimethylolethane that adds 1.Owt%, adjusting temperature of reaction is 0-25 ℃, after trimethylolethane dissolve complete dissolves, slowly add and the isopyknic ammonium dibasic phosphate solution of zinc nitrate, adopt ammoniacal liquor to regulate the pH value of reaction solution to maintain 8-9, reaction 24h, to guarantee crystal growth completely, ageing 12h is above to improve homogeneity and the crystallinity of precipitation again, final product is removed unnecessary trimethylolethane to neutrality by centrifuge washing, carry out lyophilize to carry out next stage sign.
The reaction mol ratio of described zinc nitrate and Secondary ammonium phosphate is 1.67:1 by Zn:P.
In described chemical coprecipitation reaction process, by ammoniacal liquor, regulate reacting liquid pH value to maintain 8-9.
The drug loading > 16wt% of described hydroxyl zinc phosphate nano-hollow ball during as the carrier of anticancer drugs, doxorubicin.
Compared with prior art, positively effect of the present invention is: 1. aspect synthetic preparation, the nano-hydroxy phosphoric acid zinc that adopts non-template method, low temperature to prepare cavity structure possesses synthesis technique and aftertreatment is simple, cost is low and feature that can large-scale production; 2. aspect biomedical applications, possess that toxicity is low, drug loading large, enter cell rapidly, control the feature of medicament slow release, there is potential using value in nano drug-carrying field.
Accompanying drawing explanation
Fig. 1. the XRD diffracting spectrum of (0,20,35,50,65,80 and 95 ℃) synthetic product under differing temps;
Fig. 2. under differing temps, the FTIR of (0,20,35 and 50 ℃) synthetic product absorbs collection of illustrative plates;
Fig. 3. the SEM photo (a) 0 of synthetic product under differing temps; (b) 20; (c) 35; (d) 50 ℃;
Fig. 4. the TGM photo (a) 0 of synthetic product under differing temps; (b) 20; (c) 35; (d) 50 ℃;
Fig. 5. the nitrogen Adsorption and desorption isotherms of synthetic product at 0 and 20 ℃;
Fig. 6. the different differential pore volumes that calculate according to the nitrogen desorption isotherm of synthetic product at 0 and 20 ℃ and the graph of a relation of pore size distribution;
Fig. 7. the synthetic sample of differing temps with different concns (10,50 μ g/mL) with NIH/3T3 co-cultivation the relative cytoactive data plot after 24,48 hours; Using and do not add the NIH/3T3 cell of any sample cultivation as blank;
Fig. 8. the protein adsorption curve of synthetic hollow ball sample (0 and 20 ℃) under differing temps; The protein of studying in experiment is bovine serum albumin (BSA) and antalzyme protein (LSZ);
Fig. 9. the Zorubicin drug loading of (0,20 and 50 ℃) synthetic gained nano particle under differing temps;
Figure 10. (a) load Zorubicin sample (synthesis temperature is 20 ℃) TEM photo afterwards; (b) the energy dispersion X ray spectrum of region 1 and region 2 correspondences in a figure;
Fig. 1 l.Hela cell and sample (synthesis temperature is 0 ℃) the co-cultivation laser confocal microscope photo after a series of time at 37 ℃ that loads Zorubicin; Nucleus is used Hoechst33342 staining agent to process;
Figure 12 .Hela cell and sample (synthesis temperature is 20 ℃) the co-cultivation laser confocal microscope photo after a series of time at 37 ℃ that loads Zorubicin; Nucleus is used Hoechst33342 staining agent to process;
Figure 13 .Hela cell and Zorubicin be the laser confocal microscope photo of co-cultivation after a series of time at 37 ℃; Nucleus is used Hoechst33342 to dye core agent and processes.
Embodiment
Below in conjunction with accompanying drawing, the present invention is elaborated, but the effect of example is only to explain and non-limiting the present invention.
One, experiment material and equipment.
1, experiment material:
Zinc nitrate hexahydrate (Zn (NO
3)
26H
2o, AR) and Secondary ammonium phosphate ((NH
4)
2hPO
4, AR) buy in Chemical Reagent Co., Ltd., Sinopharm Group.(content is (with MH for strong aqua
3meter) 25%-28%, AR) buy in Shanghai joint-trial company limited.1,1,1-trimethylolethane (1,1, l-Tris (hydroxymethyl) ethane (TME), 97%) is bought the Aesar in Alfa.95% ethanol (CH
3cH
2oH, AR) buy the Yang Yuan Chemical Co., Ltd. in Changshu City.(Doxorubicin hydrochloride (DOXHCl) AR) buys in Beijing Hua Feng associating scientific & technical corporation Lipodox.Hydrolith (CaH
2, AR), buy the Solution on Chemical Reagents in Shanghai company in Chinese Medicine group.Methyl-sulphoxide (C
2h
6sO, Dimethyl sulfoxide (DMSO), AR) buys in Chemical Reagent Co., Ltd., Sinopharm Group, with hydrolith (CaH
2) be dried also and use after underpressure distillation.Ultrapure water (ρ > 18M Ω), the U.S.'s Milli-Q of Pall company water purification machine makes.3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent), DMEM dehydrated medium (high sugar), non-essential amino acid, green grass or young crops-Streptomycin sulphate, 0.25% trypsinase, 0.02%EDTA Digestive system, foetal calf serum (FBS), phosphate buffered saline buffer (PBS) is bought the company in Sigma.
2, major equipment:
X-ray diffractometer adopts the D8advance diffractometer of German Bruker company, and source of radiation is Cr target.Use the EQUINOX55 type Fourier transformation infrared spectrometer of German Bruker company to measure FTIR spectrum, Sample Scan scope 4000 to 400cm
-1, scan pattern is transmission spectrum.Electronic Speculum morphology analysis adopts FEI Siron200 (20kV, 1.0nm) field emission scanning electron microscope (U.S. FEI Co.), and JEM-2010HT (200kV) transmission electron microscope (Jeol Ltd.).Use ASAP2010 M+C (U.S. Micromeritics company), with Brunauer-Emmett-Teller (BET) model analysis sample specific surface area, by desorption isotherm, with Barrett-Joyner-Halenda (BJH) equation, draw pore structure data.Inductive coupling plasma emission spectrograph adopts the Scientific iCAP6300 ICP Spectrometer of U.S. Thermo company, for the element zinc (Zn) of sample and the quantitative analysis of phosphorus (P) content.
Two, experimental technique
1. the preparation of hydroxyl zinc phosphate nano-hollow ball
Utilize simple chemical precipitation method synthesis of hydroxy zinc phosphate nano-hollow ball, in whole experimentation, solvent has all adopted ultrapure water.Select Zn(NO
3)
26H
2o and (NH
4)
2hPO
4for reaction raw materials is respectively as Zn
2+and PO
4 3-source of supply.Concrete reactions steps is as follows: at 33.4mM Zn (NO
3)
26H
2in O solution, add after 1.0wt% trimethylolethane (TME), Heating temperature is set to respectively 0 ℃, and 20 ℃, 35 ℃, 50 ℃, 65 ℃, 80 ℃, 95 ℃.After TME dissolves completely, slowly splash into (the NO with Zn
3)
26H
2isopyknic 20.0mM (the NH of O solution
4)
2hPO
4solution, has so just guaranteed that the molar ratio of Zn/P is 1.67.The pH value of reaction is used strong aqua (containing MH
3amount 25-28%) regulate and control.Whole reaction process continues 24h to guarantee crystal growth completely, then ageing 12h is above to improve homogeneity and the crystallinity of precipitation.Final product is removed unnecessary TME to neutrality by centrifuge washing, carries out lyophilize to carry out next stage sign.
2, cell cultures
3T3cells is cultivated in having the DMEM nutrient solution of 10% foetal calf serum (containing 200u/mL penicillin, Streptomycin sulphate).The cell of recovery is cultivated routinely operation and is placed in 37 ℃, 5% CO
2in (relative humidity 95%) incubator, cultivate, within approximately 2 to 3 days, change a nutrient solution.
3, material in vitro toxicity detects
Experiment grouping: be divided into experimental group and blank group.Experimental group is for being added in the cell of synthetic sample at 0,20,35 and 50 ℃, and blank group is without adding material cultured cells.The concentration of nano particle in nutrient solution is chosen for respectively: 10 and 50 μ g/mL.After at the bottom of the l cell strain NIH/3T3 Growth of Cells confluent culture bottle going down to posterity 48 hours, use 0.25% tryptic digestion, make cell suspension, adjustment cell density is 4X10
4individual/mL.With 8000, every hole cell/200 μ L DMEM culture medium inoculated in 96 well culture plates.37 ℃, 5% CO
2in incubator, cultivate the adherent and moderate rear use of density of observation of cell 24 hours.At 0,20,35 and 50 ℃, synthetic sample carries out after sterilising treatment with uv light irradiation, take phosphate buffered saline buffer (phosphate buffered saline/PBS) as 0 ℃ of solvent configuration, 20 ℃, synthetic sample suspension liquid at 35 ℃ and 50 ℃, concentration is respectively 50 and 250 μ g/mL.After ultrasonic being uniformly dispersed, with the amount of every hole 50 μ L, add in 96 orifice plates, each concentration of each sample arranges 5 multiple holes, so just obtained being respectively 10 with the cultivation final concentration of the different samples of 50 μ g/mL.At 37 ℃, 5% CO
2in incubator, cultivate respectively after 24,48 hours, substratum and sample solution in sucking-off 96 orifice plates, clean after twice with PBS is careful, adds the 200 every hole of μ L DNEM nutrient solutions, every hole adds the MTT liquid 20 μ L (preparing with PBS) of 5mg/mL again, continues to cultivate 4 hours.Cultivation completes after sucking-off MTT solution, adds the methyl-sulphoxide (DMSO) in the 200 every holes of μ L to shake 10 minutes to dissolve the bluish voilet crystallization producing.Select 490nm wavelength, in microplate reader, detect the optical density value (Optical density, OD) in each hole, take that what do not add that cell only adds nutrient solution is blank, record OD value.OD value is higher, illustrates that the propagation of NIH/3T3 cell is stronger.Each experiment repeats 3 times, averages.According to following formula, calculate cell survival rate:
Cell survival rate=experimental group OD value/blank group OD value * 100%
4, the protein adsorption characteristic of material
According to the measurement result of specific surface area, (total specific surface area is 1.0m to a certain amount of hydroxyl zinc phosphate
2) with the protein solution of different concns (from 0 to 1mg/cm
3) at room temperature stir 24 hours after blend after, by suspension under 10,000rpm rotating speed centrifugal 10 minutes, and collect the supernatant liquor after centrifugal.The absorbance that in supernatant liquor, the concentration of protein is recorded under 280nm by ultraviolet spectrophotometer, and corresponding protein standard curve calculation draws.By experiment, the variation of strength of solution can draw the protein adsorption quantity of hydroxyl zinc phosphate.
5, the medicine carrying characteristic of material
1mg DOXHCl is dissolved in 5mL ultrapure water, again 5mg sample (is respectively to 0,20 and 50 ℃ of samples) add this solution, under room temperature, stir after 24h, by precipitation centrifuge washing and collect the supernatant liquor after centrifugal for the first time, after removing free Zorubicin lyophilize completely, obtain loading the sample of Zorubicin.Adopted uv-absorbing to carry out the mensuration of drug loading.The DOXHCl solution that configuration concentration is respectively 5,10,25,50,75,100,150,200mg/mL carries out the test of Zorubicin uv-absorbing, obtains the uv-absorbing typical curve of Zorubicin.According to typical curve, calculate the concentration C of residual ionization Zorubicin in solution
1, known initial doxorubicin concentration is C
0, liquor capacity is V, and medicine carrying sample quality is m, and the drug loading of sample can calculate by following formula:
Sample drug loading (wt%)=((C
0-C
1) V/m) * 100%
It is sample drug loading (wt%)=(drug loading quality/medicine carrying material quality) * 100%
6, medicine-carried system cell in vitro administration
Adopt comparatively conventional laser co-focusing (confocal laser scanning microscopy (CLSM)) observing samples in vitro by the behavior of Cell uptake.Because contained medicine is anticancer drugs, doxorubicin, therefore choose cervical cancer cell Hela, as experimental cell, be.Experiment grouping: be divided into medicine carrying material group and pure medicine control group.Experimental group is the cell that is added in 0 and 20 ℃ of medicine carrying sample, and pure medicine control group is and adds pure Zorubicin cultured cells.Incubation time is respectively 15,30,60 and 120min.After at the bottom of the Hela Growth of Cells confluent culture bottle going down to posterity 48 hours, use 0.25% tryptic digestion, make cell suspension, adjustment cell density is 5x10
5individual/mL.With every hole 1 * 10
5individual cell/200 μ L DMEM culture medium inoculated is in having placed in advance 12 well culture plates of sterility cover slide.37 ℃, 5% CO
2in incubator, cultivate the adherent and moderate rear use of density of observation of cell 24 hours.Use pure DMEM, with 10 μ g/mL Zorubicin equivalent concentration configuration medicine carrying sample and pure Zorubicin solution.Remove substratum in hole, every hole adds the solution that configures in 1mL D, and drug solution in sucking-off hole after incubation time and Hela co-cultivation according to target adds that PBS is careful to be cleaned three times.Every hole adds 400 μ L paraformaldehydes that cell fixedly after 30min, is washed away with PBS; Following every hole adds 400 μ L to dye core agent Hoechst33342 to dye core and process 10min, with PBS, wash away and dye core agent again, finally by mountant, there is the cover glass of cell to seal up for safekeeping on slide glass growth, with laser co-focusing (confocal laser scanning microscope (CLSM)) instrument, observe.
Three, experimental result and discussion.
This example is used X-ray diffractometer (XRD) and infrared spectra (FTIR) to characterize the product component of synthesized, uses constituent content and composition in inductive coupling plasma emission spectrograph (ICP) assay products.Meanwhile, use the appearance structure of scanning electron microscope (SEM) and transmission electron microscope (TEM) observation product.In cytotoxicity experiment, adopt mtt assay and detect in conjunction with microplate reader.In medicine carrying experiment, adopt transmission electron microscope to judge in conjunction with energy spectrometer (EDX) whether medicine enters nano material cavity inside.Finally adopt comparatively laser confocal microscope (CLSM) observing samples in vitro by the behavior of Cell uptake.
When Fig. 1 is pH=8.5,0,20,35,50,65,80, the XRD figure of synthetic different samples spectrum at 95 ℃.80 ℃ of samples with the sample of 65 ℃ and 95 ℃ are the same Zn
3(PO
4)
2(H
2o)
4diffraction peak, only compares with 95 ℃ of samples, and the sample diffraction peak of 80 and 65 ℃ obviously weakens, and crystallinity is poorer.Three kinds of products under high temperature are not target products, thus after phenetic analysis all no longer carry out.At lower temperature, (0～50 ℃) synthetic gained sample has diverse peak crystallization, and three strongest ones peak position is respectively 2 θ=11.44 °, 19.94 ° and 28.36 °.In PDF card spectrum library, do not find the card of answering in contrast.This show pH=8.5, temperature lower than the condition of 50 ℃ under, can synthesize the good type material of a kind of crystallinity.With zinc, entirely replacing the synthetic hydroxyl zinc phosphate of calcium is a kind of brand-new material, ought to have own special XRD diffraction spectrogram.
FTIR spectrum is the important method of identification function group, and therefore, we carry out the composition of analytic sample surface group with FTIR.Fig. 2 has shown functional group's infrared absorption peak of synthetic product under differing temps.At 2800-3650cm
-1broad peak and 1635cm
-1the little absorption peak of left and right is all O-H key chattering absorption peak hydroxyl in product itself or planar water.1430cm
-1the small peak at place is the absorption peak of C=O, has shown may contain carbonate (CO in sample
3 2-).And zine ion appears in the surface of phosphatic rock and really can absorb the carbonic acid gas (CO in atmosphere
2).927-1160cm
-1unimodal phosphate radical (the PO that belongs at place
4 3-) middle P-O ν 3 stretching vibrations.432cm
-1place weak absorption peak and 497-608cm
-1the bimodal of place is respectively due to phosphate radical (PO
4 3-) ν 2 and ν 4 flexural vibration cause.Infrared result shown 0,20, contains hydroxyl and phosphate radical (PO at 35,50 ℃ in synthetic sample
4 3-), and may contain carbonate (CO
3 2-).
The molar ratio that has guaranteed Zn, P element in when reaction is Zn/P=1.67, and in order to check Zn, the P element mol ratio in product, by 0,20, synthetic sample has carried out ICP test at 35,50 ℃.
The Zn of synthetic sample and P constituent content under table 1 differing temps
By upper table, can be learnt, the Zn/P mol ratio of all products is all slightly larger than 1.67 these feed ratio to some extent, and this is also that product absorbs the CO in atmosphere because in reaction process
2, make a part of phosphate radical (PO in product
4 3-) by carbonate (CO
3 2-) institute replaces, and finally cause Zn/P mol ratio to rise.This conclusion is also consistent with the conclusion of infared spectrum, C=O absorption peak wherein really due to the carbonate (CO containing in product
3 2-) and produce.
The microscopic appearance of product is an important indicator of nano material all the time, has adopted SEM and TEM to 0 ℃ in example, and 20 ℃, 35 ℃, synthetic sample microscopic appearance characterizes at 50 ℃.In Fig. 3, SEM has proved at 0 ℃ dry straightly, 20 ℃, and 35 ℃, at 50 ℃, the size of synthetic sample is all at Nano grade, and particle diameter is in 30～50nm left and right, although nanoparticle agglomerates is very serious, but still the homogeneity that can see product is better, the particle of near-spherical is also fairly obvious.Due to reasons such as particle are too little, electroconductibility is poor, oneself is surperficial through seeing through completely for SEM electron beam, causes the surface topography of sample to observe by photo.In TEM picture in Fig. 4, first can see it being equally also because reuniting effect product particle has all been gathered in together, but still can observe comparatively homogeneous of nanoparticle size, all in 30-50nm left and right.At 0,20 ℃, synthetic sample has obvious hollow structure, through measuring and calculating, draws: its wall thickness is in 7nm left and right, and aperture is in 28nm left and right.And synthetic sample is still full particle at 35 and 50 ℃.This result shows by synthetic method used herein and condition, can synthesize the nano-hydroxy phosphoric acid zinc with hollow structure below 20 degree.
Adopt the further microvoid structure character of 0 ℃ and 20 ℃ synthetic sample of research of nitrogen adsorption desorption.According to 0 ℃, the nitrogen Adsorption and desorption isotherms figure (Fig. 5) of 20 ℃ of synthetic samples is known, and both hollow structure feature thermoisopleths are very similar, and hysteresis loop is to be all first parallel to X-axis (relative barometric pressure) then with almost vertical angle, to rise suddenly.Two samples all have the Adsorption and desorption isotherms of IV type, and in relative barometric pressure 0.99 to 0.75 scope, have hysteresis loop to occur.Pore structure in this result surface sample is all mesoporous.The data such as the specific surface area calculating from thermoisopleth, pore volume and mean pore size have all been enumerated out in table 2.The sample of 0 ℃ and 20 ℃ has respectively 78.13 and 75.31m
2the specific surface area of/g, and the mean pore size of 29.8nm and 25.3nm, this result is not only consistent with graph of pore diameter distribution (Fig. 6), has also confirmed observable result in TEM before.Data results in this example is calculated mean pore size by Barrett-Joyner-Halenda (BJH) analytical method by desorption opisometer by software respectively, and specific surface area and pore volume are to use Barrett-Emmett-Teller (BET) method to calculate.
The specific surface area of two kinds of samples of table 2, the concrete numerical value of pore volume and mean pore size
Vitro cytotoxicity data are summed up as shown in Figure 7: along with the increase of incubation time and sample concentration, sample also progressively increases the toxicity of NIH/3T3 cell, but the concentration of toxicity maximum is 50 μ g/mL 50 ℃ of groups samples and the relative cytoactive still having after cell cultures 48h more than 70%.And there are 0 ℃ of hollow structure and 20 ℃ of samples, all there is more than 75% relative cytoactive.This result shown, all synthetic samples all have lower vitro cytotoxicity for NIH/3T3 normal cell.
In example, adopt these two kinds of typical protein of bovine serum albumin (BSA) and antalzyme protein (LSZ) to carry out the protein adsorption characteristic of study sample.There is the protein adsorption experiment of hollow structure sample (0 ℃ and 20 ℃) as shown in Figure 8: wherein, two kinds of samples have all been shown the adsorption curve of stronger intimate linearity to bovine serum albumin.And for antalzyme protein, in the adsorption curve of two kinds of samples, all shown saturated adsorption value.In experiment, two kinds of samples are obviously greater than the adsorptive capacity to antalzyme protein to the adsorptive capacity of bovine serum albumin.For bovine serum albumin, high adsorption capacity can reach 8.66 ± 0.23mgm
-2(0 ℃) and 9.54 ± 0.37mgm
-2(20 ℃); For antalzyme protein, corresponding saturated extent of adsorption is 2.60 ± 0.14mgm
-2(0 ℃) and 2.53 ± 0.18mgm
-2(20 ℃).
Fig. 9 has shown the drug loading at 0,20 and 50 ℃ of synthetic sample.The sample wherein with hollow structure has respectively the high drug load of 16.28 ± 0.44wt% (0 ℃) and 16.73 ± 0.13wt% (20 ℃), and solid sample (50 ℃) only has the Zorubicin content of 7.45 ± 0.07wt%.Such result means, no matter hollow specimen is on surface or can loads Zorubicin at cavity inside, and solid sample only can utilize surface adsorption Zorubicin, and hollow structure can improve the drug loading of sample really greatly.
In order further to prove above-mentioned viewpoint, in example, supplemented the TEM picture of sample after medicine carrying, and analyzed in conjunction with corresponding EDX spectrogram.Figure 10 shown 20 ℃ of medicine carrying samples TEM picture (Figure 10 a) and the EDX in corresponding SP1 and SP2 region can spectrogram (Figure 10 b).SP1 and the SP2 sample area after corresponding medicine carrying and the white space on sample side respectively wherein.In sample table (copper mesh) due to transmission electron microscope, contain C and Cu element, so can not be only prove with the existence that (sample area after medicine carrying) detects C element in SP1 region can drug loading in sample cavity.Therefore, this example utilizes adjacent SP2 region (white space on sample side) in contrast, usings the Cu element (8keV place) of same amount as interior mark in can spectrogram, come more corresponding C constituent content number.Result shows, the C constituent content that SP1 region (sample area after medicine carrying) detects is greater than adjacent SP2 region (white space on sample side) significantly, be the provable existence at SP1 region organism (Zorubicin), also proved that the cavity inside of sample has loaded Zorubicin simultaneously.
Laser co-focusing picture presentation different medicine carryings sample 0 ℃ (Figure 11) and 20 ℃ (Figure 12) two kinds of samples to take pure Zorubicin (Figure 13) be reference, with Hela cell co-culture medicine distribution situation in cell paste after 15,30,60 and 120 minutes.Use pure DMEM for solvent, with 10 μ g/mL Zorubicin equivalent concentration, configured medicine carrying sample and pure Zorubicin solution.Nucleus is used Hoechst33342 to dye core agent processing and in picture, demonstrates blue-fluorescence (secondary series in Figure 11-13).From Figure 13, can find, no matter the length of pure Zorubicin incubation time, the red fluorescence of its feature all only appears in nucleus, has shown that Zorubicin has completely all entered in nucleus.And 0 ℃ be mainly all gathered in tenuigenin with 20 ℃ of two kinds of medicine carrying samples (seeing Figure 11 and Figure 12) in initial 30min, increase along with incubation time, in the photo of 60min group, can see that red fluorescence has also appeared in nucleus, composite photograph has also clearly demonstrated the purple fluorescence of overlapping generation, illustrates that oneself warp of Zorubicin now loading discharges and entered nucleus in cell.This result also shown, it is by endocytosis that medicine carrying sample enters cell, and pure Zorubicin can permeate through cell membranes diffuse into cell interior.The result demonstration of laser confocal microscope, medicine carrying sample can be conveyed into Zorubicin Hela cell interior effectively.
Four, conclusion.
This example utilizes the process of non-template, low temperature synthesis of hydroxy zinc phosphate nano-hollow ball, and technique is very simple, reaction conditions is gentle and it is green synthetic pollution-free to belong to.Vitro cytotoxicity experiment shows that this nano material cell toxicity is lower.Utilize this nano-hollow ball to have good protein adsorption ability and Drug loading capacity, its carrier as anticancer drugs, doxorubicin has higher drug loading (> 16wt%).By confocal laser scanning microscope, show that this drug-loading system can promptly enter cell by drug conveying.These results have all shown that hollow Nano hydroxyl zinc phosphate has potential using value in medicine carrying field.Its cavity characteristics is more given its potentiality that develop to some extent in inorganic hollow particle Application Areas, such as can be applicable to catalysis, load genes/proteins matter or as the aspects such as other nano material of template synthetic kernel shell structure.
Claims (4)
1. the preparation method for the hydroxyl zinc phosphate nano-hollow ball of pharmaceutical carrier, it is characterized in that, the method is that to take zinc nitrate and Secondary ammonium phosphate be reactant, controls temperature of reaction at 0-25 ℃, by chemical coprecipitation, makes hydroxyl zinc phosphate nano-hollow ball.
2. the preparation method of a kind of hydroxyl zinc phosphate nano-hollow ball for pharmaceutical carrier according to claim 1, is characterized in that, the reaction mol ratio that feeds intake of described zinc nitrate and Secondary ammonium phosphate is 1.67:1 by Zn:P.
3. the preparation method of a kind of hydroxyl zinc phosphate nano-hollow ball for pharmaceutical carrier according to claim 1, is characterized in that, in described chemical coprecipitation reaction process, by ammoniacal liquor, regulates reacting liquid pH value to maintain 8-9.
4. the preparation method of a kind of hydroxyl zinc phosphate nano-hollow ball for pharmaceutical carrier according to claim 1, it is characterized in that the drug loading > 16wt% of described hydroxyl zinc phosphate nano-hollow ball during as the carrier of anticancer drugs, doxorubicin.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107032315A (en) * | 2016-11-26 | 2017-08-11 | 华东理工大学 | A kind of preparation method of trbasic zinc phosphate nanocages |
| CN108569683A (en) * | 2018-04-23 | 2018-09-25 | 南京信息工程大学 | A kind of trbasic zinc phosphate containing the crystallization water and preparation method thereof and purposes with photocatalyst |
| CN111265493A (en) * | 2018-12-05 | 2020-06-12 | 沈阳药科大学 | Preparation method and application of phospholipid-coated polyacrylic acid/zinc phosphate nanoparticles |
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2013
- 2013-10-08 CN CN201310465267.3A patent/CN103539096A/en active Pending
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| 马晓飞: "纳米空心羟基磷酸锌的制备及其载药特性研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107032315A (en) * | 2016-11-26 | 2017-08-11 | 华东理工大学 | A kind of preparation method of trbasic zinc phosphate nanocages |
| CN108569683A (en) * | 2018-04-23 | 2018-09-25 | 南京信息工程大学 | A kind of trbasic zinc phosphate containing the crystallization water and preparation method thereof and purposes with photocatalyst |
| CN108569683B (en) * | 2018-04-23 | 2022-01-04 | 南京信息工程大学 | Zinc phosphate containing crystal water, preparation method thereof and application of zinc phosphate as photocatalyst |
| CN111265493A (en) * | 2018-12-05 | 2020-06-12 | 沈阳药科大学 | Preparation method and application of phospholipid-coated polyacrylic acid/zinc phosphate nanoparticles |
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