CN102240403B - Method for preparing CaCO3 nanotube/podophyllum composite material - Google Patents

Abstract

The invention relates to a method for preparing a CaCO3 nanotube/podophyllum composite material. The method comprises the following steps of: dissolving 4.0 grams of phenanthroline carrier in 100 milliliters of chloroform, and stirring violently; cleaning and drying a transparent film of ordered molecular (TFOM), putting into a solution system, immersing for a certain period of time, taking the TFOM out, cleaning by using filter paper, and putting into the middle of a gas reaction generator (GRC) container to form an isolation system of which the middle is provided with TFOM and left and right sides have solution; preparing 0.53 to 0.57 percent of sodium carbonate solution and 2.13 to 2.17 percent of calcium chloride solution respectively, putting the sodium carbonate solution and the calcium chloride solution on the left and right sides of the TFOM, adding a crystal growth modifier M according to different conditions, and regulating the pH value to 8; stirring at a low speed, taking solution on one side of the sodium carbonate solution, and centrifuging to obtain precipitate products; and cleaning twice by using water, acetone and absolute ethanol respectively; and storing the products in the absolute ethanol. The method is stable in reaction, is easy to operate and control and low in cost and does not have pollution, appearance and a structure are easy to control, and the products are distributed uniformly, and are difficult to agglomerate, high in purity and easy to industrialize.

Description

A kind of CaCO 3The preparation method of nanotube/podophyllum composite material

Technical field

The present invention relates to a kind of novel C aCO ₃The preparation method of nanotube/podophyllum composite material.

Background technology

The target that one of material science receives people's concern day by day is exactly to make up the nano material with symmetrical labyrinth, function admirable, and micrometer/nanometer hollow ball, Guan Ze are the representative substances of this spacelike cavity material, they are at biotechnology, photocatalysis, photic device, high-energy electrode material, there is important using value in the fields such as air-sensitive, although a lot of reports have been arranged about utilizing initial crystal unit assembling to form one dimension, two dimension or three-dimensional hollow-core construction, for example stratiform electrostatic attraction, self-assembling technique, template removal method etc.Yet the technology of conventional preparation hollow nano-material needs the operating procedure of relative complex such as template and Ostwald ripening growth etc., and versatility is bad, and certain limitation is arranged.In processing procedure, be limited by the integrity that will keep product (final crystal) and be subject to a large amount of template of paying close attention to, although there have been a lot of methods to be attempted, how easy and have method or a difficult problem of certain versatility." crystal structure " also remains a challenge, and attracts people's concern always, and development easily, the gentle method for preparing hollow-core construction remains unusual Worth Expecting.

Hollow material being used for the biomedical uses such as medicine carrying, is bold innovation of field of nanometer technology.Recent years; based on novel pharmaceutical carrier; research and comparison such as microcapsule, nanotube, hollow Nano particle is popular; thereby carried object and can be resisted the damage that extraneous physics, chemistry and biotic environment cause by material protection; be easy to again distributed by capillarity and lymphatic capillary, be easy to be absorbed by cells in vivo.Up to the present, can be divided into two large classes on medicine carrying mechanism for the nanometer system of medicine carrying: first class is to carry out drug conveying with surface adsorption (by physics or chemical action), and second largest class is to utilize the cavity system to carry out the load of shipping formula.Latter's research and comparison is many is the hybrid material that is combined into of macromolecular material, bioceramic class, biologically-derived thing class, bioactive substance and non-living material etc., all be potential biomaterial such as polylactic acid, ortho esters, polyamino acid and amino acid whose copolymer, poly-own Inner ester etc., but the complexity of organic synthesis has limited their clinical use greatly, and the general enzymatic degradation characteristics of macromolecular material also can limit their use position and method.And inorganic matter is relatively organic, stable in properties, preparation process good reproducibility.The comparative maturity of at present inanimate matter cavity investigation of materials be the nanotubes such as carbon, zinc oxide, silicon dioxide, but there is again biocompatibility issues in various degree in vivo in this class material.Also having the problem of a commonplace existence, is exactly the leakage of receptor in the lift-launch process, causes drug side effect and curative effect to reduce.In general, existing nano material is at aspects such as bioavailability, untoward reaction, Half-life in vivo, medicine stability, dissolubility, absorption, enzymatic degradation and barrier cells, and is more or less all also not fully up to expectations.So people expect the exploitation of new material, even re-recognize traditional material, their nano-structured may being with are later served breakthrough.

Nanotube (nanotubes.NTs) material has special hollow and loose structure, and its axial dimension is that micron number Seedling level, radial dimension are an end closure of nanometer scale, pipe.The internal cavities of nanotube can be used as the container that medicine stores, and the open end of pipe then can be used as the passage of drug release.Compare with the nanoparticle of homogenous material, the surfaces externally and internally of nanotube can carry out respectively different modifications, and this multifunction for nano-tube material provides platform, and becomes the ideal chose of drug carrier material.

In recent years, the nanotube of various materials is such as CNT (CNTs), Si0 ₂Nanotube (SNTs), Ti0 ₂Nanotube (TNTs) and Magnetic nano-pipe (MNTs) etc. have the slow controlled release that is in the news as medicine.Wherein, with the research of CNTs and be most widely used.Bianco etc. (Wu W, Benincasa M, Bianco A, et a1.Targeted Delivery of Amphotericin B to Cells byUsing Functionalized Carbon Nanotubes[J]. Angew.Chem.Int.Ed ., 2005,44 (39): 6358_6362.) take amphotericin B (AmB) as model drug, fluorescein is tracer, AmB is connected on the sidewall of CNTs, and fluorescein is connected to the top of CNTs.AmB is a kind of effectively antibiotic, but excessively low dissolubility makes it produce very large toxicity to mammiferous cell.Research finds that employing is tied platform with medicine significantly reduces the toxicity of Amb to the mode on the support C NTs.

Calcium carbonate (Calcium Carbonate, CaCO ₃) be a kind of for human body can metabolism material, calcium itself is a kind of indispensable element of needed by human body simultaneously, therefore, CaCO ₃Particle can be used as a kind of drug delivery and is applied to field of medicaments.Nanometer CaCO ₃The characteristics little with its particle diameter, that specific surface area is large have the not available property of common CaCO3 particle, just more and more widely as medicine carrying.

Inanimate matter crystal CaCO ₃There are three kinds of crystal formations: calcite, vaterite, aragonite, each crystal formation has common crystal habit, and calcite always grows up to the rhombohedron form, because crystal face is the most stable crystal face of calcite.And vaterite always grows up to sphere, and aragonitic always grows up to acicular crystal.If but there is foeign element to disturb crystallization process, the stable crystal face of some times also can appear in the last pattern of crystal.Laboratory prepares CaCO ₃, according to the phase transition law, product mostly is thermodynamically stable calcite, and other two kinds of forms also are not easy preparation.And nature, particularly biosphere are then on the contrary, prepare CaCO so simulate for some time biomineralization ₃Be subject to concentrating and pay close attention to, these class methods at first will be carried out template and be made up, this template can be molecular sieve, also can be a compounds (such as parents' water block copolymer), next is to have the junior unit of nano-scale to offer the template reunion of leading, although first requirement is easy to accomplish, make template etc. such as the microemulsion droplet, but often second requirement is difficult to accomplish, because it requires colloid to keep certain stability, the maintenance nanoparticle is that the temporary transient stable of colloid of primitive is not an easy thing in water environment.

The material preparation of comparing traditional, the use probability of this traditional material has been expanded in the research of cavity-like nano-calcium carbonate (Cavity-shaped Nano Calcium Carbonate, CNCC) undoubtedly greatly, has become at present CaCO ₃The study hotspot in field.Yu Q etc. (Yu Q., Ou H.D., Song R.Q., Xu A.W., J. Cryst. Growth 2006,286:178) at CaCO ₃Crystallization process in add polyacrylamide and obtain hollow vaterite, the people such as Tremel Wolfgang (Loges N., Graf K., Nasdala L., Tremel W., Langmuir , 2006,22:3073) utilize bionical abductive approach to obtain the CaCO of micron-scale ₃Hollow ball.S.Mann etc. (Walsh D., Mann S., Nature 1995,377:320.) reported and utilize the direction micelle to prepare CaCO ₃The hollow shell structure.These researchs are CaCO ₃Further develop and made contribution, still, on the angle of using from biological medicine, the requirement with a certain distance from biomaterial has these products at aspects such as crystalline size, morphology control homogeneity, productive rates.

From present existing bibliographical information, the people such as Ma G.H. (Wei W., Ma G.H., Hu G., Yu D., Mcleish T., Su Z.G., Shen Z.Y., J. Am. Chem. Soc. 2008,130:15808) once prepared the CaCO of hollow ₃Nanoparticle is used for anti-cancer medicament carrier, the people such as Mizushima Y. (Ueno Y., Futagama H., Takagi Y., Ueno A. .Mizushima Y, Journal of Controlled Release , 2005,103:93) utilize porous C aCO ₃The surface adsorption activated protein, the people such as Cao C.Y. (Zhao Q, Zhang S., Tong W.J., Gao C.Y., Shen J.C., European Polymer Journal , 2006,42:3341) utilize CaCO ₃Decorative material carries and discharges antitumor drug, and the skin effect that also has the solid nano material of some research and utilizations is by physical absorption or weak chemical bond and carry out covering of some albumen and DNA and carry.These researchs are CaCO ₃Biological medicine use and to have established certain basis, still, also do not have bibliographical information systematically to study the CNCC of different crystal forms, different-shape in general character and the difference of biological medicine application facet, lack especially further breakthrough in the biological medicine application facet.Analyze its reason, the one, be subjected to chemical preparation and crystal-controlled restriction, the 2nd, be subjected to the restriction of the specific (special) requirements of complexity that biological medicine uses and interdisciplinary research.Consider CaCO ₃The advantageous property that itself has if improved on its performance, size, can make it become bio-carrier a kind of highly compatible, multiduty fully.This material can as efficient pharmaceutical carrier, also can further be attempted in many-side science of carrying out such as gene transfection, lift-launch vaccines.The object that carries not only can be the large medicine of some toxic and side effects, can also be the protein molecular, DNA (deoxyribonucleic acid), virus, polypeptide etc. of different size size.

Pattern by the control material can be improved its physical property, such as surface area, crystalline size, stability etc., particularly nano-scale particle excellent performance especially.Adopt the penetrating film of our this ordered moleculars (Transparent Film of Ordered Molecular, TFOM) new method of preparation material nano pipe, can prepare expediently good dispersion, the tubular attitude CNCC of size homogeneous has significant application value in technical field of biological material.Because as medicine carrying material, CNCC can carry the medicine greater than the common material amount of carrying owing to have the hollow characteristics on the structure, in addition, the little and homogeneous of its particle diameter can fine dispersion, have better biocompatibility, be expected to be utilized in a large number in biological medicine carrying field.

Podophyllotoxin (Podophyllotoxin, PPT) be the main effective ingredient of Rhizoma Dysosmae Versipellis class plant. 1942, Kaplan topical application PPT treats condyloma acuminatum, obtained satisfied effect. 1947, scientist has confirmed the destruction of PPT to the animal cancerous cell, caused that people study widely to this, but because the toxicity of PPT is too large, it is limited by very large in application as antitumor drug. now, people are making great efforts to explore, expectation finds therapeutic effect better, the podophillotoxines cancer therapy drug that toxic and side effects is less or new podophyllotoxin form of administration.

It is to separate the natural antitumor active component nJ that obtains from Rhizoma Dysosmae Versipellis class plant that podophyllotoxin (Podophyllotoxin 1) is again podophyllin, podophyllotoxin, Podophyllinic Acid Lactone, and Rhizoma Dysosmae Versipellis class plant mainly is distributed in the ground 12l such as Gansu, Qinghai, Tibet, Shaanxi, Sichuan in China.The application of podophyllotoxin can be traced back to 1820, at that time the dry rhizome of America Rhizoma Dysosmae Versipellis and Himalayas Rhizoma Dysosmae Versipellis by the locals as choleretic and cathartic, 1820-nineteen forty-two the water-insoluble extract podophyllin of rhizome of Rhizoma Dysosmae Versipellis class plant be put into the anti-tumor activity .1880 Podwyssotzki that American Pharmacopeia .1861 Bentley found podophyllotoxin and isolated first podophyllotoxin.Borsche and Niemann and Spath have proposed respectively the structure of podophyllotoxin independently.Nineteen forty-two Kaplanti has confirmed that podophyllin has inhibitory action to growth of tumour cell.1946.1947 it is the growth that suppresses tumor by the formation that stops cell mitogen microtubule fasolculus in mid-term that year King and Sullivan have illustrated podophyllotoxin, with colchicine similar Antitumor Mechanism is arranged.Caused people's extensive attention, and confirmed that further it has active anticancer.But because its serious toxic and side effects has limited its application clinically.Nineteen fifty-one Hartwell and Schrecker have provided the structure of podophyllotoxin.Von Wartburg in 1954 and his partner have found the carbohydrate ligands of podophyllotoxin.The seventies, Switzerland mountain pass scholar company has successively synthesized etoposide (etoposide, VP16) and teniposide (teniposide, VM26), the prodrug Etopophos. of the VP.16 that the mid-90 is released and prove highly active antitumor drug .1983 etoposide by U.S. FDA authentication .VP16 as topoisomerase .II (topoisomerase.II, TOPII) inhibitor, now become one of the most frequently used clinically antitumor drug, to small cell lung cancer, Fei Hejieshi is sick, acute monocytic leukemia, the myeloid monocyte leukemia, breast carcinoma, bladder cancer, numerous tumors such as carcinoma of testis have preferably therapeutic effect, simultaneously Kaposi sarcoma and acquired immunodeficiency disease are also had certain curative effect.

Summary of the invention

The object of the present invention is to provide a kind of novel C aCO ₃The preparation method of nanotube/podophyllum composite material.

The present invention utilizes the penetrating film of ordered molecular (Transparent Film of Ordered Molecular, TFOM) reaction system is made the cross-film transmission medium, add an amount of dressing agent by the opposite side at TFOM, produce synergism, come the organic media intervention in analog cell film and the biomineralization forming process.Wherein, the dressing agent that adds is in a kind of weak effect of the nucleation interface formation of TFOM reaction system, so that product forms a kind of nano-particle of tubulose under a kind of like this condition by soft template crystallization control nucleation, solve the general chemistry reaction and be difficult to produce unique crystal result of tubulose and the difficult problem of multilevel ordered crystal, and this is a kind of gentleness, efficient and green chemical reaction.Reaction occurs in a kind of gas reaction generator (Gas Reaction Generator, GRC) in the reaction system of our design like this, and the organic carboxyl acid root of one of reactant is being met one of reactant Na ₂CO ₃Can react, produce CO ₂Gas, because reaction system is a closed system, these CO ₂Gas has again one and generates HCO in solution ₃ ^-Reversible reaction (chemical equation is as follows),

2RCOOH?+?CO ₃ ^2-

?2RCOO ^-?+?H ₂O?+?CO ₂↑

CO ₂?+?H ₂O?

?HCO ₃ ^-?+?H ⁺

Because the organic carboxyl acid root is to CaCO ₃Crystal generate and to have a crystal face adsorption effect, by molecular recognition optionally with after the crystal one party combines to veil, can suppress the growth of this veil, make the speed of growth of crystal on vertical this veil direction relatively and the speed of growth of other veil can weaken or stop, from but this veil is relatively stable and finally show the last pattern of crystal.So on the whole, our experimental technique has three outstanding large characteristics: the one, and control ion transfer speed, thereby control crystal nucleation speed; The 2nd, have the gas molecule of relative stiffness in the crystal formation process as induction pattern plate; The 3rd, also having a soft organic carboxyl acid root template in the crystal maturing process affects its crystal development.

A kind of novel C aCO provided by the invention ₃The preparation method of nanotube/podophyllum composite material, Concrete steps are as follows:

(1) the Phen carrier is dissolved in the organic solvent, after the high-speed stirred, put into the penetrating film of the clean ordered molecular of surface treatment (Transparent Film of Ordered Molecular, TFOM), soaked 20-25 hour, then TFOM is taken out, with filter paper suction being wiped on the TFOM surface processes clean, packing into and be fixed in the middle of the gas reactor, be TFOM in the middle of forming, and left and right is that the system of being separated by that is provided with agitating device is the GRC system;

(2) preparation 2.13-2.17 % CaCl ₂Solution, 0.53-0.57 % Na ₂CO ₃Solution places respectively the left and right system of being separated by of gas reaction generator (Gas Reaction Generator, GRC) of step (1), at Na ₂CO ₃Solution one side adds dressing agent M, stirs lower reaction after 24-48 hour, takes out Na ₂CO ₃Precipitation in solution one side is centrifugal, water, acetone, dehydrated alcohol respectively clean twice successively, products therefrom is kept in the dehydrated alcohol, with FITR, XRD and SEM structure and the shape of product is characterized respectively, and product is pure, pattern is hollow tubular, and particle diameter is below micron order; The addition of dressing agent M is Na ₂CO ₃The 0.75-0.85% of quality;

(3) with step (2) products therefrom through ultraviolet detection, determine that nano-calcium carbonate can carry behind podophyllotoxin, before the reaction, phase Na in centrifuge tube ₂CO ₃Solution one side adds and is equivalent to CaCO ₃The podophyllotoxin of the 10-15% of theoretical yield, material is slowly wrapped up podophyllotoxin wherein in forming process, after reaction is finished, to remain the podophyllotoxin eluting out with acetone and dehydrated alcohol, with reference to the standard curve of podophyllotoxin, respectively with ultraviolet-uisible spectrophotometer and two kinds of methods of HPLC to nanometer CaCO ₃The podophyllotoxin of load carries out quantitative analysis.

Nanometer CaCO ₃Drug loading=(nanometer CaCO ₃The gross mass of the quality/composite of Chinese medicine) * 100%

Nanometer CaCO ₃Envelop rate=(nanometer CaCO ₃Quality/the institute of Chinese medicine weighs the medicine gross mass) * 100%

Among the present invention, described dressing agent M is citric acid.

Among the present invention, described organic solvent is chloroform.

The CaCO of gained of the present invention ₃The normal cell toxicity of nanotube/podophyllum composite material and inhibition rate of tumor growth can adopt mtt assay to detect, and concrete steps are as follows:

Selecting HEK 293T cell is the normal cell model, and the Hela cell is tumor models.The DMEM(Dulbecco's Modified Eagle Medium that contains 10% calf serum) culture fluid is with 2.0 * l0 ⁴The concentration in individual/hole is inoculated in 96 orifice plates, every pore volume 100 μ l.Used MTT(bromination-3-(4,5 dimethylthiazoles-2)-2,5 diphenyl tetrazoliums) be Sigma company product.After preculture 24 h made cell attachment grow to logarithmic (log) phase, every group of 3 holes added respectively the nanometer CaCO of variable concentrations ₃Solution, Rhizoma Dysosmae Versipellis solution and Rhizoma Dysosmae Versipellis/nanometer CaCO ₃Outstanding mixed liquid (concentration is respectively 5,10,20,40 μ g/ml) and the contrast of blank culture medium, cultivate certain hour after, every hole adds MTT solution 20 μ l, hatches abandoning supernatant behind 4 h, every hole adds 150 μ l DMSO cessation reactions.With culture plate level 15 min that vibrate, in 490 nm places mensuration trap, be calculated as follows cell survival rate with enzyme connection detector:

Cell survival rate %=A _{The 490(sample)}/ A _{The 490(contrast}) * 100 %

(A _{The 490(sample)}: add the sample sets absorbance; A _{The 490(contrast)}: the absorbance of the blank culture medium cell of matched group)

The present invention has the following advantages:

One, this method has proposed a kind of CaCO that can control a kind of uniform morphology of formation first ₃The new method of nanotube.The product amount is large, the little and distribution homogeneous of particle diameter, and monodispersity is good, and the present invention has filled up cast nanometer CaCO ₃The blank of preparation.

Two, the technology of the present invention operation is simple and easy, and reaction system is gentle, stable, interference is few, and product is processed and is easy to process, and it is synthetic on a large scale to be convenient to material.

Three, the CaCO of this method preparation ₃Nanotube has the drug loading height, medicine carrying is stable and can be absorbed the characteristics of degrading by Bio-ontology, is the effective pharmaceutical carrier of a class, can be applied to field of medicaments as a kind of active drug carrier.

Four, the standby CaCO of TFOM legal system ₃The nanotube process not only reaction temperature and, also can avoid causing residue to pollute to environment, the wasting of resources, and low production cost are that the nano material of a kind of ideal, environmental protection prepares novel method.

Description of drawings

Fig. 1 is the novel C aCO of the embodiment of the invention 1 ₃The transmission electron microscope observation figure of nanotube/podophyllum composite material, hollow tubular structures.

Fig. 2 is that the composite of the embodiment of the invention 2 is to the toxicity test of normal cell HEK 293T.

Fig. 3 is that the composite of the embodiment of the invention 2 is to the suppression ratio experiment of tumor cell.

Fig. 4 is the structural diagrams of gas reaction generator.

Number in the figure: 1 is constant speed mixing control device, and 2 is iron stand, and 3 is magnetic stir bar, 4 electric blender, and 5 is the first glass container, and 6 is the penetrating film of ordered molecular, and 7 is constant temperature blender with magnetic force, and 8 is sodium carbonate liquor, 9 is calcium chloride solution.

The specific embodiment

The invention is further illustrated by the following examples.

Embodiment 1:

The structure of described self-control gas reaction generator as shown in Figure 4, by constant speed mixing control device 1, iron stand 2, magnetic stir bar 3, electric blender 4, the first glass container 5, the penetrating film 6 of ordered molecular and constant temperature blender with magnetic force 7 form, wherein: the first glass container 5 is positioned over constant temperature blender with magnetic force 7 tops, the second glass container one end is put into the first glass container 5, and the second glass container bottom opening, this opening is provided with the penetrating film 6 of ordered molecular, between the second glass container bottom and the first glass container 5 bottoms certain distance is arranged, the second glass container is fixing by iron stand 2, the puddler of electric blender 4 inserts in the second glass container, electric blender 4 connects constant speed mixing control device 1, be placed with sodium carbonate liquor in the second glass container, be placed with calcium chloride solution in the first glass container 5.First 4.0 g Phen carriers are dissolved in the 100ml chloroform, with the rotating speed vigorous stirring 15-20min of 3000 rpm, make the penetrating film of the ordered molecular that contains carrier (Transparent Film of Ordered Molecular, TFOM); Then put into above-mentioned liquid film after the TFOM drying of cleaning treatment being crossed and soak taking-up after 20-25 hour, with filter paper suction being wiped on the TFOM surface processes clean, pack into and be fixed in the middle of the reaction vessel, be TFOM in the middle of forming, left and right for the solution phase isolated system that is provided with agitating device be gas reaction generator (Gas Reaction Generator, GRC).

The first step, 4.0 g Phens are dissolved in the pure chloroform of 100 ml, with about 15 min of vigorous stirring under the rotating speed of 3000 rpm, make the TFOM that contains carrier, put into above-mentioned TFOM system reaction after the TFOM drying that cleaning treatment is crossed and soak after 24 hours and take out, with filter paper that the TFOM surface treatment is clean, self-control gas reaction generator (Gas Reaction Generator packs into, GRC) device is TFOM in the middle of forming, about be the isolated system of solution phase.Respectively there is a magnetic agitation instrument film both sides.

Second step is with 2.17%CaCl ₂Solution, 0.53% Na ₂CO ₃Each 80ml of solution joins respectively the TFOM left and right sides, and the stirring at low speed reaction is after 5 minutes, with Na ₂CO ₃The product C aCO of solution one side ₃After the centrifugalize, successively water, acetone, dehydrated alcohol respectively clean twice, at last product are kept in the dehydrated alcohol.Product is white powder, exquisiteness, evenly.(above-mentioned all reagent are analytical pure).

Embodiment 2:

The first step: maximum absorption wavelength: take by weighing the 0.003g podophyllotoxin, be placed in the 10mL volumetric flask with the distilled water dissolving and carry out standardize solution.Scans by ultraviolet-uisible spectrophotometer in 200 ~ 800 nm scopes, 285 nm are the maximum absorption wavelength of podophyllotoxin, so experimental selection concentration of podophyllotoxin in the test solution under this wavelength.

Make the ultraviolet detection Rhizoma Dysosmae Versipellis/nanometer CaCO that uses the same method ₃The ultraviolet of nano material is composed entirely, observes its ultraviolet spectra absorbing state, judges whether this material exists interference in the responsive Absorption Wave strong point of Rhizoma Dysosmae Versipellis.

The drafting of Rhizoma Dysosmae Versipellis working curve: prepare respectively 1,2,3,4,5,6,8,10 μ g/ml Rhizoma Dysosmae Versipellis standard solution, survey its absorbance at the 285nm place.The match working curve.

Rhizoma Dysosmae Versipellis/nanometer CaCO ₃The mensuration of composite nano materials drug loading: the preparation sample, with the Rhizoma Dysosmae Versipellis of 0.003 g/nanometer CaCO ₃Dried powder places 10 ml volumetric flasks, drips 6 M hydrochloric acid solutions, 100 μ L, and the phosphate buffer of 0.02 M, pH 7.45 is settled to 10 ml, after ultrasonic 30 minutes, places 37 ℃ of water-baths to spend the night again.Survey its ultraviolet absorptivity value next day at 295 nm places, contrast is the Rhizoma Dysosmae Versipellis working curve of drafting before, can draw the concentration of Rhizoma Dysosmae Versipellis in the sample.The employing following formula calculates:

Nanometer CaCO ₃Drug loading=(nanometer CaCO ₃The gross mass of the quality/composite of Chinese medicine) * 100%

Nanometer CaCO ₃Envelop rate=(nanometer CaCO ₃Quality/the institute of Chinese medicine weighs the medicine gross mass) * 100%

As seen from Figure 2, CaCO ₃The lower cell survival rate of suspension concentration is higher, as 100% cell proliferation rate, calculates the average survival rate of cell more than 80% with the OD value of negative control group, and cytotoxicity is 1 grade, can think the CaCO that the TFOM legal system is standby ₃Has good cell compatibility.Work as CaCO ₃Can reduce the toxicity of simple PPT after compound with PPT.Although purer CaCO ₃, the cytotoxicity behind the medicine carrying is bigger, is 1 grade of scope but also belong to cytotoxicity.The result can prove CaCO on the one hand ₃Be that a toxicity is very little, the safety barrier of good biocompatibility can prove CaCO on the other hand ₃-PPT nano complex is a safe drugs transport system to normal cell.Rhizoma Dysosmae Versipellis/nanometer CaCO ₃The composite nano materials drug loading is 38.5 %, and envelop rate is 64.4 %.

Show from Fig. 3 experimental result, along with PPT, CaCO ₃-PPT and CaCO ₃The increase three of adding concentration is significantly improved to the inhibition of Hela cell, simultaneously CaCO ₃The inhibition degree that-PPT compound system and simple PPT compare tumor cell has had certain raising, obviously greater than the activity of tumor cells suppression ratio of drug molecule PPT itself, and CaCO ₃Itself the Hela cell also there is certain toxicity.This shows CaCO ₃A good pharmaceutical carrier, and because CaCO ₃With the synergism of PPT, so that medicine inhibition tumor cell better.

Embodiment 3:

The first step: use normal cell strain HEK 293T that its cytotoxicity is detected, the inhibition of tumor cell is selected the Hela tumor cell.

Second step: after the growth of preculture 24 h cell attachments, with the negative matched group of cell culture fluid, every group of 3 holes add respectively PPT solution, the CaCO of variable concentrations ₃-PPT suspension and CaCO ₃Suspension (concentration is respectively 5,10,20,40 μ g/ml).

The 3rd step: after cultivating 24 h, every hole adds MTT solution 20 μ l, hatches abandoning supernatant behind 4 h, and every hole adds DMSO 150 μ l cessation reactions.

The 4th step: with culture plate level 30 min that vibrate, in 490 nm places mensuration trap, be calculated as follows cell survival rate with enzyme connection detector:

Cell survival rate %=A _{490 (samples)}/ A _{490 (contrasts)}* 100 %

Claims (1)

1. CaCO ₃The preparation method of nanotube/podophyllum composite material is characterized in that concrete steps are as follows:

(1) the Phen carrier is dissolved in the organic solvent, after the high-speed stirred, put into the penetrating film of the clean ordered molecular of surface treatment, soaked 20-25 hour, then the penetrating film of ordered molecular is taken out, with filter paper the penetrating film of ordered molecular surface is wiped to inhale and process totally, pack into and be fixed in the middle of the gas reaction generator, be the penetrating film of ordered molecular in the middle of forming, left and right for being provided with the system of being separated by of agitating device; Described organic solvent is chloroform;

(2) preparation 2.13-2.17 % CaCl ₂Solution, 0.53-0.57 % Na ₂CO ₃Solution places respectively the left and right system of being separated by of gas reaction generator of step (1), at Na ₂CO ₃Solution one side adds dressing agent M, stirs lower reaction after 24-48 hour, takes out Na ₂CO ₃Precipitation in solution one side is centrifugal, water, acetone, dehydrated alcohol respectively clean twice successively, products therefrom is kept in the dehydrated alcohol, with FITR, XRD and SEM structure and the shape of product is characterized respectively, and product is pure, pattern is hollow tubular, and particle diameter is below micron order; The addition of dressing agent M is Na ₂CO ₃The 0.75-0.85% of quality; Described dressing agent M is citric acid;

(3) with step (2) products therefrom through ultraviolet detection, determine that nano-calcium carbonate can carry behind podophyllotoxin, before the reaction, phase Na in centrifuge tube ₂CO ₃Solution one side adds and is equivalent to CaCO ₃The podophyllotoxin of the 10-15% of theoretical yield, material is slowly wrapped up podophyllotoxin wherein in forming process, after reaction is finished, to remain the podophyllotoxin eluting out with acetone and dehydrated alcohol, with reference to the standard curve of podophyllotoxin, respectively with ultraviolet-uisible spectrophotometer and two kinds of methods of HPLC to nanometer CaCO ₃The podophyllotoxin of load carries out quantitative analysis.

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