CN105535989A - Nano-carbon crystal drug carrier for target identification of tumor cells and preparation method of drug carrier - Google Patents
Nano-carbon crystal drug carrier for target identification of tumor cells and preparation method of drug carrier Download PDFInfo
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- CN105535989A CN105535989A CN201510899719.8A CN201510899719A CN105535989A CN 105535989 A CN105535989 A CN 105535989A CN 201510899719 A CN201510899719 A CN 201510899719A CN 105535989 A CN105535989 A CN 105535989A
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Abstract
The invention discloses a nano-carbon crystal drug carrier material for target identification of tumor cells and a preparation method of the drug carrier material. The drug carrier material consists of nano-carbon crystals, and adriamycin and transferrin are sequentially linked to C atoms on the surfaces of the nano-carbon crystals. The preparation method comprises the following steps: 1) preprocessing the nano-carbon crystals; 2) preparing a transferrin-adriamycin physical composite solution; and 3) assembling the nano-carbon crystals and the transferrin-adriamycin physical composite solution, so that the drug carrier material is obtained. The obtained nano-drug carrier material of the invention is strong in targeting property and is high in modifying rate; and moreover, active transport protein can be modified on the surface of the drug carrier material in an immobilized mode, and the drug carrier material has the advantage of universal applicability.
Description
Technical field
The present invention relates to nano-medicament carrier material of a kind of target recognition of tumor cell and preparation method thereof.
Background technology
Current antitumor drug mainly contains cytotoxic drugs and hormones and hormone antagonist class medicine.Cytotoxic drugs mainly acts on propagation and the vital metabolic that DNA and RNA carrys out tissue tumor cells; If hormones and hormone antagonist class drug main act on life entity and kill or the propagation of inhibition tumor cell and vital movement to reach to regulate hormonal readiness.These antitumor drug also can act on normal cell while acting on tumor cell, and to the damage of body normal structure not second to the damage to tumor tissue cell.Medicine overflows blood vessel also can cause tissue ulcer and necrosis outward.In addition, traditional chemotherapeutic antineoplastics thing exists without selective guide effect, and the transportation in body is complicated and to reach the drug molecule quantitative proportion of pathological area low, therefore needs a large amount of and SM for a long time, while increase treatment cost, also irreversible damage is caused to the health of patient.
In cellular morphology, function and metabolism, there is huge different feature based on tumor cell from normal cell, people have developed the medicine with magnetic target therapy cancer.Early stage scientists application serum albumin,
gelatinthe macromolecular material of the natural degradables such as hemoglobin ossein and degradable synthesis base polymer are as target medicine carrier, but it exists the problems such as preparation technology is extremely complicated, output is uncontrollable, thus finally cannot extensive use clinically.
In recent years, along with the develop rapidly of nanometer biotechnology, the small size of nano-particle brings its many advantages.The advantage of nanoparticle is that particle diameter is superfine and surface activity is high: the superfine high-activity nano particle surface of particle diameter can initiative recognition enter inside tumor cells by the drug molecule carried release to kill tumor cell after functionalization.Current existing fullerene and inorganic nano-particle etc. start to be used as pharmaceutical carrier and transport medicine in vivo.But, existing nano-medicament carrier just can consider to carry out the modification of Active transport albumen when carrying out feature drug molecule and modifying, this modification efficiency is low and be not easily permanently effectively attached to nano-medicament carrier surface, and this modification does not have general applicability.
Summary of the invention
The object of the present invention is to provide a kind of nanometer carbon crystal pharmaceutical carrier of target recognition of tumor cell, provide its corresponding preparation method to be another goal of the invention of the present invention simultaneously.
Based on above-mentioned purpose, the present invention takes following technical scheme: the nano-medicament carrier material of target recognition of tumor cell, comprises nanometer carbon crystal, and the C atom on the surface of described nanometer carbon crystal connects amycin and transferrins successively.
This carbon crystalline substance of described nanometer carbon crystal is class spherical morphology, the brilliant C atom on surface of carbon and the dimeric structure of the C atomic building C atom of internal layer diamond phase, two carbon atoms in the dimeric structure of C atom are asymmetric distribution, spacing of lattice is 0.21nm, mean diameter is R, 0 < R≤10nm, the C content of this carbon crystalline substance is 99 ~ 100%.
Described nanometer carbon crystal is obtained by following methods: a) pickling is purified: fine powder diamond raw material powder being broken into more than 10000 orders, adopt concentrated sulphuric acid and concentrated nitric acid mixed liquor, dilute hydrochloric acid, Fluohydric acid. to this fine powder pickling successively, then use washed with de-ionized water to cleanout fluid pH close to 7; B) sorting: the material after cleaning is carried out centrifugalize, gets supernatant liquid and carries out the sorting of 4-7 days precipitations, remove the supernatant, by after the drying of lower sediment thing and get final product.Described nanometer carbon crystal is class spherical morphology, the dimeric structure of the C atom on nanometer carbon crystal surface and the C atomic building C atom of internal layer diamond phase, and two carbon atoms in the dimeric structure of C atom are asymmetric distribution; Spacing of lattice is 0.21nm; Mean diameter is R, 0 < R≤10nm; The C content of this carbon crystalline substance is 99 ~ 100%.
The preparation method of the nano-medicament carrier material of target recognition of tumor cell, comprises the following steps:
1) pretreatment of nanometer carbon crystal: dense H nanometer carbon crystal being placed in mass ratio 2:1 ~ 4:1
2sO
4/ dense HNO
3mixed liquor in, reflux, centrifugation; Get precipitation and be first washed with distilled water to pH4.0 ~ 5.0, then use 0.1 ~ 0.4molL
-1naOH washs, and ultrasonic disperse, then use 0.1 ~ 0.4molL
-1hCl washs, and ultrasonic disperse, then dry with deionized water wash;
2) transferrins-amycin physics complex liquid is prepared:
1. the configuration of PBS buffer: by NaCl6 ~ 10g, KCl0.2 ~ 0.8g, Na
2hPO
412H
2o2 ~ 5g, KH
2pO
40.2 ~ 0.8g takes each raw material, adds 1500 ~ 2000mL distilled water, adjusts pH to 7.0 ~ 7.5 until completely dissolved;
2. transferrins liquid: by transferrins and the PBS obtained transferrins liquid of 1.5:1 ~ 3.5:1 mixing in mass ratio;
3. amycin liquid: amycin and distilled water are obtained amycin liquid in 10 ~ 20mg:5 ~ 10mL ratio mixing;
4. transferrins-amycin physics complex liquid: by the transferrins liquid in 2. and amycin liquid in mass ratio 2:1 ~ 4:1 mix and obtain transferrins-amycin physics complex liquid.
3) assembling of nanometer carbon crystal and transferrins-amycin physics complex liquid: by nanometer carbon crystal treated in step 1) and step 2) in transferrins-amycin physics complex liquid in mass ratio 0.01:1 ~ 0.08:1 mix, through the emulsion that ultrasonic emulsification obtains, after deionized water wash to pH7.0 ~ 7.5, centrifugation, the precipitate frozen centrifugation, the vacuum drying that obtain and get final product.
Step 1|) in the temperature of reflux be 88 ~ 92 DEG C, the time of reflux is 24 ~ 48h; Centrifugal rotating speed is 1000 ~ 2000rpm.
Step 1|) middle NaOH washs, ultrasonic disperse process repeats at least 3 times, and each ultrasonic disperse time is 1 ~ 3h; HCl washing, ultrasonic disperse process repeat at least 3 times, and each ultrasonic disperse time is 1 ~ 3h.
Step 2) 4. in mixing concrete operations be: at 10 ~ 15 DEG C with the speed of 50 ~ 100rpm concussion 2 ~ 4h.
In step 3), phaco time is 2 ~ 10min, and the power of ultrasonic employing is 50 ~ 150W.
The present invention adopts gas to grind separating method and prepares nanometer carbon crystal, its technique is simple, production cost is low and the C content of the nanometer carbon crystal prepared between 99 ~ 100%, purity is high, particle size range narrowly distributing, substantially between 0-10nm, crystallinity is comparatively strong and surface activity is strong, high adsorption capacity, regular appearance is controlled, and loss rate is low, and loss rate can be controlled within 1%; The nanometer carbon crystal surface of preparing has the dimeric structure of the C atom of asymmetric distribution, has very high surface activity, is easy to by surface-functionalized process; Particularly, the nanometer carbon crystal adopted in the present invention, it is advantageous that:
1, high surface and high-specific surface area: the surface of nanometer carbon crystal also exists a large amount of two kinds of multi-form C atoms, and these two kinds of carbon atoms are all in activated state, surface-functionalized process is very easily carried out to it in the site that these activity are very high, the ingredient that the nanometer carbon crystal after surface-functionalized process can access or compound is nearly all; And the nanometer carbon crystal after activation processing has high specific surface area and makes its surface can access more ingredient and molecular indicator, greatly will increase the targets identification rate of pharmaceutical carrier like this, make medicine be entered pathological area by more effective transport;
2, superfine granularity and good biocompatibility: traditional nano-medicament carrier eliminates in human body or by biodegradation that it is residual or get rid of external by thin exopinocytosis, but all can have certain residual and irreversible infringement is produced to human body; And nanometer carbon crystal is due to the superfine and good biocompatibility of granule, can free in and out cell completely thus thoroughly be got rid of external;
3, surface-functionalized process: traditional nano-medicament carrier, using nano-particle as kernel, is first polymerized one deck proton sponge body at nano-particle skin, then carry out surface-functionalized process (see
fig. 1).The specific surface area of nano-particle is declined and the quantity reduction of avtive spot and uncontrollable in the result of nano-particle outer layers of polymer one deck proton sponge body.Just there is profuse avtive spot on its surface of nanometer carbon crystal itself, and other have the film body of surface active function not need layers of polymer outside, only need carry out simple pretreatment, just make the avtive spot " exposure " on its surface out (see
fig. 2);
4, can at the fixing modification in its surface Active transport albumen.Conventional nano pharmaceutical carrier only just can consider to carry out the modification of Active transport albumen when carrying out feature drug molecule and modifying, and this modification efficiency is low and be not easily permanently effectively attached to nano-medicament carrier surface.The present invention can carry out the generally modification of Active transport protein factor before nanometer carbon crystal carries out surface drug functionalization, and this modification has general applicability; At the active surface fixing modification Active transport albumen of nanometer carbon crystal, nanometer carbon crystal after treatment has the effect of Active transport, concentration difference inside and outside cell membrane can be overcome and entered tumor cell tissue by cell active absorption " reverse ", reaching the effect freed in and out in tumor cell tissue inside.
Based on this, know-why of the present invention is: first transferrins and amycin are carried out physics compound; Again nanometer carbon crystal is carried out pretreatment, obtain more avtive spot (
fig. 2); Finally transferrins-adriamycin composite is adsorbed on nanometer carbon crystal surface with make there is targeting Active transport nano-medicament carrier (
fig. 3).Wherein, transferrins is as the targeting factor identifying cancerous cell; Amycin is a class nucleic acid cumulative interference medicine, and its effect is that in the intercalation of DNA, RNA interfering copies transcribing of cancerous cell gene, utilizes nanometer carbon crystal to adsorb the complex of transferrins-amycin, finally reaches the object stoping cancer cell division propagation.Particularly:
(1) transferrins is as the targeting factor identifying cancerous cell.First, ferrum is one of necessary trace element of matrix, has important effect to a series of cell activities, and it is also the collaborative molecule of some important enzymes simultaneously.But iron content is too much or very fewly all can produce irreversible damage to matrix, and organism has developed the regulatory mechanism of self, comprises the picked-up of ferrum, storage and output, to control intracellular ferrum beinthebalancestate for this reason.TfR (TfR) in blood plasma is the important albumen in cellular uptake ferrum process, and cell constantly obtains ferrum by the circulation of transferrins.Because the growth of cancerous cell and split speed can be faster than normal cell, therefore also just larger to the demand of ferrum, according to the study, the affinity of cancer cell surfaces TfR and transferrins is Normocellular 10 ~ 100 times.Therefore, the present invention utilizes the difference of cancerous cell and normal cell surface TfR, by medicine and transferrins coupling, can make it have targeting;
(2) amycin is as a class nucleic acid cumulative interference medicine, itself has toxicity more weak, gentle, neutral, and the feature easily excreted (is recorded according to pharmacology: amycin is got rid of from bile and urine with form that is circular and alcohol shape metabolite.Therefore can not in Remained mass).After being compounded to form couplings with transferrins, amycin due to molecule little and first adsorb with the avtive spot on nanometer carbon crystal and combine compared with transferrins, amycin as much as possible can be ensured adsorbed, the modification rate of drug molecule improves, the modification rate also corresponding raising of targeting factor (transferrins), more easily identifies cancerous cell;
Therefore, compared with prior art, beneficial effect of the present invention is: its targeting is strong, modification rate is high, and at the fixing modification in its surface Active transport albumen, can have the advantage of general applicability.
Accompanying drawing explanation
fig. 1it is the structural representation of traditional nano-medicament carrier
figure;
fig. 2it is the structural representation of the treated nanometer carbon crystal of the present invention
figure;
fig. 3it is the structural representation of nano-medicament carrier of the present invention
figure;
fig. 1-3in: 1-drug molecule; 2-functionalization group; 3-proton sponge body polymer; 4-targeting factor; 5-nanoparticle core; 6-surface activity C atom; 7-nanometer carbon crystal nucleome; 8-transferrins; 9-amycin; 10-transferrins-adriamycin composite;
fig. 4it is the technological process frame of nanometer carbon crystal of the present invention
figure;
fig. 5the test result of the transferrins-adriamycin composite residual quantity that is untreated nanometer carbon crystal with process nanometer carbon crystal when soak time is 60min in filtrate;
fig. 6it is the test result of transferrins-adriamycin composite residual quantity in the nanometer carbon crystal that the processed filtrate after different soak time process;
fig. 7it is the test result of transferrins-adriamycin composite residual quantity in the nanometer carbon crystal of the unprocessed mistake filtrate after different soak time process;
fig. 8it is the liveness test result of the brilliant pharmaceutical carrier of targeted nanometer carbon;
fig. 9it is the liveness test result of the brilliant pharmaceutical carrier of non-targeted nanometer carbon;
figure 10that the residual quantity of 10 groups of parallel tests compares result;
figure 11that the residual quantity of 10 groups of controlled trials compares result.
Detailed description of the invention
Embodiment is only for further illustrating the present invention below, does not limit the present invention in any form.
embodiment 1
A preparation technology for nanometer carbon crystal, its technological process frame
figure is as Fig. 4shown in, the steps include:
(1) tentatively broken: first diamond raw material to be sent in the crusher chamber of mechanical crusher, broken through the pivotal tup effect of disintegrating machine inner high speed, material after fragmentation is through the screen cloth of different meshes, it is again broken that disintegrating machine inside stayed by the material being greater than screen cloth diameter, particulate material through the thinnest screen filtration of disintegrating machine lower floor collects (sieve number is not less than 32 orders) through sieve plate, and can to sell as coarse fodder or for the preparation of the material of other granularity through the material of other order number sieve net filtration; Granules more than 32 orders utilizes multilamellar vibrating sieving machine to carry out granularity just to divide, and the sieve time general control that shakes is at 30min ~ 3h, and sieve number is arranged from 40 orders to 500 orders successively, the coarse fodder that 40 eye mesh screens sub-elect return mechanical crusher carry out again broken (
fig. 4 A), the material that 40 order to 500 object screen clothes are collected is for the preparation of the material of other granularity, and the fines collected after crossing 500 eye mesh screens, for the preparation of nanometer carbon crystal, is the accuracy ensureing sorting granularity, repeats more than screening choosing once to the fines collected.
(2) again broken: airflow crash crusher machine intracavity sent into by fines step (1) sieved out, high pressure draught is utilized to carry out fragmentation to fines, the material after sieved through sieve is collected at airflow crash machine rewinding mouth, sieve number is arranged from 1000 orders from high to low successively to 10000 orders, collect the fine powder of 10000 eye mesh screen screenings for the preparation of nanometer carbon crystal, the material collected of other order number sieve net for the preparation of other material or still Returning flow disintegrating machine carry out again broken, the coarse fodder auto-returned airflow crash machine that 1000 eye mesh screens are collected carry out again broken (
fig. 4 B).
(3) pickling is purified: 10000 object fine powders after the airflow crash of collection are placed in concentrated sulphuric acid and concentrated nitric acid mixed liquor pickling 1 ~ 5h, filtration, cleaning, and described concentrated sulphuric acid and concentrated nitric acid mixed liquor are that 7:1.2 mixes by mass fraction to be 98% concentrated sulphuric acid and mass fraction be 10% concentrated nitric acid according to mass ratio; Then fine powder is placed in dilute hydrochloric acid 4 ~ 11h, filtration that 120 DEG C ~ 180 DEG C mass fractions are 5 ~ 10%; Again this fine powder is placed in Fluohydric acid. 5 ~ 20h, the impurity removed the metal impurities in material and be attached in Mechanical Crushing, airflow crash process on material, then uses washed with de-ionized water to cleanout fluid pH close to 7.
(4) sorting: the material after cleaning is carried out centrifugalize, and centrifugation time is 30min-2h, and rotating speed is 8000rpm-20000rpm; Point centrifuge tube top mixed liquor and two kinds, centrifuge tube bottom deposit material after centrifugal classification; Get upper strata mixed liquor and carry out the sorting of 4-7 days precipitations, the sedimentation time is longer, and the particle diameter being sorted into material is thinner, and remove the supernatant after precipitation sorting, lower sediment thing can obtain finished product nanometer carbon crystal after the qualified post-drying of granularity Detection.Centrifuge tube bottom deposit material can be sold compared with the finished product material of coarseness Returning flow disintegrating machine continuation broken also can treating as.
Adopt the nanometer carbon crystal that said method is prepared, C content is between 99 ~ 100%, and purity is very high, particle size range narrowly distributing, basic between 0-10nm, the stronger surface activity of product crystallinity is strong, high adsorption capacity, regular appearance is controlled, and loss rate is low, and loss rate can be controlled within 1%.
When sorting, centrifugation time is 30 ~ 50min, and rotating speed is 8000rpm, the sorting of 7 days precipitations, and the nanometer carbon crystal mean diameter obtained is 9-10nm; Centrifugation time is 30min, and rotating speed is 20000rpm, and the precipitation sorting time is 4 days, and the granularity of the nanometer carbon crystal prepared is 6 ~ 8nm; Centrifugation time is 1h, and rotating speed is 10000rpm, and the precipitation sorting time is 6 days, and the granularity of the nanometer carbon crystal prepared is 4 ~ 6nm; Centrifugation time is 2h, and rotating speed is 20000rpm, and the precipitation sorting time is 7 days, and the granularity of the nanometer carbon crystal prepared is 0 ~ 4nm.
In the present embodiment during sorting, centrifugation time is 30 ~ 50min, and rotating speed is 8000rpm, and the sorting of 7 days precipitations, the nanometer carbon crystal mean diameter obtained is 9-10nm.
embodiment 2
The nano-medicament carrier material of target recognition of tumor cell, comprises nanometer carbon crystal, and the C atom on the surface of described nanometer carbon crystal connects amycin and transferrins successively.The preparation method of nanometer carbon crystal is with embodiment 1.
The preparation method of described nano-medicament carrier material, comprises the following steps:
1) nanometer carbon crystal pretreatment: dense H nanometer carbon crystal being placed in mass ratio 3:1
2sO
4/ dense HNO
3mixed liquor in, 88 ~ 92 DEG C of reflux 36h, centrifugation, centrifugal rotating speed is 1500rpm; Get precipitation and be first washed with distilled water to pH4.0 ~ 5.0, then use 0.2molL
-1naOH washs, and ultrasonic disperse 1h, this process in triplicate, then uses 0.24molL
-1hCl washs, and ultrasonic disperse 1h, this process in triplicate, then is dried with deionized water wash;
2) transferrins-amycin physics complex liquid is prepared:
1. the configuration of PBS buffer: by NaCl8g, KCl0.6g, Na
2hPO
412H
2o4g, KH
2pO
40.6g takes each raw material, adds 1800mL distilled water, adjusts pH to 7.0 ~ 7.5 until completely dissolved;
2. transferrins liquid: by transferrins and the PBS obtained transferrins liquid of 2:1 mixing in mass ratio;
3. amycin liquid: amycin and distilled water are obtained amycin liquid in the mixing of 15mg:8mL ratio;
4. transferrins-amycin physics complex liquid: by the transferrins liquid in 2. and amycin liquid in mass ratio 3:1 mix and obtain transferrins-amycin physics complex liquid; The concrete operations of mixing are: with the speed of 80rpm concussion 3h at 10 ~ 15 DEG C.
3) nanometer carbon crystal and transferrins-amycin physics complex liquid assembling and get final product: by nanometer carbon crystal treated in step 1) and step 2) in transferrins-amycin physics complex liquid in mass ratio 0.04:1 mix, through the emulsion that ultrasonic emulsification 6min obtains, the power of ultrasonic employing is 100W, after deionized water wash to pH7.0 ~ 7.5, centrifugation, the precipitate frozen centrifugation, the vacuum drying that obtain and get final product.
embodiment 3
The nano-medicament carrier material of target recognition of tumor cell, comprises nanometer carbon crystal, and the C atom on the surface of described nanometer carbon crystal connects amycin and transferrins successively.The preparation method of nanometer carbon crystal is with embodiment 1.
The preparation method of described nano-medicament carrier material, comprises the following steps:
1) pretreatment of nanometer carbon crystal: dense H nanometer carbon crystal being placed in mass ratio 2:1
2sO
4/ dense HNO
3mixed liquor in, 88 ~ 92 DEG C of reflux 24h, centrifugation, centrifugal rotating speed is 1000rpm; Get precipitation and be first washed with distilled water to pH4.0 ~ 5.0, then use 0.1molL
-1naOH washs, and ultrasonic disperse 2h, this process repeats 4 times, then uses 0.1molL
-1hCl washs, and ultrasonic disperse 2h, this process repeats 4 times, then dries with deionized water wash;
2) transferrins-amycin physics complex liquid is prepared:
1. the configuration of PBS buffer: by NaCl6g, KCl0.2g, Na
2hPO
412H
2o2g, KH
2pO
40.2g takes each raw material, adds 1500mL distilled water, adjusts pH to 7.0 ~ 7.5 until completely dissolved;
2. transferrins liquid: by transferrins and the PBS obtained transferrins liquid of 1.5:1 mixing in mass ratio;
3. amycin liquid: amycin and distilled water are obtained amycin liquid in the mixing of 10mg:5mL ratio;
4. transferrins-amycin physics complex liquid: by the transferrins liquid in 2. and amycin liquid in mass ratio 2:1 mix and obtain transferrins-amycin physics complex liquid; The concrete operations of mixing are: with the speed of 50rpm concussion 2h at 10 ~ 15 DEG C.
3) nanometer carbon crystal and transferrins-amycin physics complex liquid assembling and get final product: by nanometer carbon crystal treated in step 1) and step 2) in transferrins-amycin physics complex liquid in mass ratio 0.01:1 mix, through the emulsion that ultrasonic emulsification 2min obtains, the power of ultrasonic employing is 50W, after deionized water wash to pH7.0 ~ 7.5, centrifugation, the precipitate frozen centrifugation, the vacuum drying that obtain and get final product.
embodiment 4
The nano-medicament carrier material of target recognition of tumor cell, comprises nanometer carbon crystal, and the C atom on the surface of described nanometer carbon crystal connects amycin and transferrins successively.The preparation method of described nanometer carbon crystal is with embodiment 1.
The preparation method of described nano-medicament carrier material, comprises the following steps:
1) pretreatment of nanometer carbon crystal: dense H nanometer carbon crystal being placed in mass ratio 4:1
2sO
4/ dense HNO
3mixed liquor in, 88 ~ 92 DEG C of reflux 48h, centrifugation, centrifugal rotating speed is 2000rpm; Get precipitation and be first washed with distilled water to pH4.0 ~ 5.0, then use 0.4molL
-1naOH washs, and ultrasonic disperse 3h, this process repeats 5 times, then uses 0.4molL
-1hCl washs, and ultrasonic disperse 3h, this process repeats 5 times, then dries with deionized water wash;
2) transferrins-amycin physics complex liquid is prepared:
1. the configuration of PBS buffer: by NaCl10g, KCl0.8g, Na
2hPO
412H
2o5g, KH
2pO
40.8g takes each raw material, adds 2000mL distilled water, adjusts pH to 7.0 ~ 7.5 until completely dissolved;
2. transferrins liquid: by transferrins and the PBS obtained transferrins liquid of 3.5:1 mixing in mass ratio;
3. amycin liquid: amycin and distilled water are obtained amycin liquid in the mixing of 20mg:10mL ratio;
4. transferrins-amycin physics complex liquid: by the transferrins liquid in 2. and amycin liquid in mass ratio 4:1 mix and obtain transferrins-amycin physics complex liquid; The concrete operations of mixing are: with the speed of 100rpm concussion 4h at 10 ~ 15 DEG C.
3) nanometer carbon crystal and transferrins-amycin physics complex liquid assembling and get final product: by nanometer carbon crystal treated in step 1) and step 2) in transferrins-amycin physics complex liquid in mass ratio 0.08:1 mix, through the emulsion that ultrasonic emulsification 10min obtains, the power of ultrasonic employing is 150W, after deionized water wash to pH7.0 ~ 7.5, centrifugation, the precipitate frozen centrifugation, the vacuum drying that obtain and get final product.
contrast test 1: untreated nanometer carbon crystal and process nanometer carbon crystal are on the impact of the residual concentration of transferrins-amycin in complex liquid
EXPERIMENTAL DESIGN principle: the transferrins-amycin complex liquid untreated nanometer carbon crystal of equal in quality and process nanometer carbon crystal (processing method is with reference to embodiment 2) being put into same mass concentration, after identical soak time, filter out nanometer carbon crystal, adopt ultraviolet spectrophotometer to measure the residual concentration of transferrins-amycin in complex liquid (filtrate).Each sample does five groups of parallel tests respectively, and it the results are shown in
fig. 5-7shown in.
in figurethe data of vertical coordinate are transferrins-adriamycin composite residual quantity in the filtrate after filtering.
fig. 5the test result of the transferrins-adriamycin composite residual quantity that is untreated nanometer carbon crystal with process nanometer carbon crystal when soak time is 60min in filtrate, by
fig. 5known, in filtrate after nanometer carbon crystal after treatment soaks, transferrins-adriamycin composite residual concentration will well below undressed nanometer carbon crystal, this shows, the surface activity of the nanometer carbon crystal after process is higher, more easily accesses more transferrins-adriamycin composite.
fig. 6with
fig. 7the test result of transferrins-adriamycin composite residual quantity in process and the nanometer carbon crystal of the unprocessed mistake filtrate after different soak time process respectively, by
fig. 6with
fig. 7known, along with the increase of soak time, in complex liquid, the residual quantity of complex is all in reduction, there is a limit in the reducing amount of untreated nanometer carbon crystal, namely soak time reaches necessarily, and continue to extend soak time, in complex liquid, the residual quantity of complex substantially no longer reduces; And treated nanometer carbon crystal sample is after reaching same soak time, in its complex liquid the residual quantity of complex still after significantly reducing amount, this also shows, the surface activity of the nanometer carbon crystal after process is than before process, and activity significantly improves.
contrast test 2: targeting is tested:
This contrast test utilizes isotope-labelling method to characterize targeting effect.N isotopic labeling is carried out to transferrins, because N isotope nitrogen stable isotope does not have radioactivity, synthesizes at label and process all fairly simple, do not decay, non-radiating is decomposed, free from environmental pollution, do not need to take safeguard procedures, there is no toxicity, impact is not existed to final result.
Test is divided into two groups, and one group containing the isotope-labeled targeting factor transferrins of N
,i.e. non-targeted nanometer carbon crystal pharmaceutical carrier group; One group contains the isotope-labeled targeting factor transferrins of N, i.e. the brilliant pharmaceutical carrier group of targeted nano carbon.
Concrete grammar is: 1. the treated nanometer carbon crystal of equal in quality is put into respectively equal in quality concentration containing N isotope-labeled targeting factor transferrins Doxorubicin solution and not containing the pure Doxorubicin solution having N isotope-labeled targeting factor transferrins, concussion mixing same time in mixing oscillator, filtration also dry obtaining is adsorbed with the nanometer carbon crystal carrier (the brilliant pharmaceutical carrier of targeted nano carbon) of amycin and targeting factor and is only adsorbed with the nanometer carbon crystal carrier (non-targeted nanometer carbon crystal pharmaceutical carrier) of amycin; 2. on agar block, cultivate normal cell group and the cancerous cell bacterium colony of equal number.And labelling is carried out to the fluorescent agent of normal population of cells and cancerous cell bacterium colony different colours; 3. the brilliant pharmaceutical carrier of targeted nano carbon and the non-targeted nanometer carbon crystal pharmaceutical carrier that take equal in quality 1. are also dissolved in serum respectively.Agar block is immersed in above-mentioned serum, after same time, takes out agar block and use deionized water rinsing agar block; 4. observe under instrument and isotopicly in these agar blocks occur position; Measure the liveness of normal cell and cancer cell; 6. often organize test and do 10 groups of parallel tests.It the results are shown in
fig. 8with
fig. 9shown in.
fig. 8with
fig. 9the liveness test result of the brilliant pharmaceutical carrier of targeted nanometer carbon and non-target tropism nanometer carbon crystal pharmaceutical carrier respectively.
From
fig. 8with
fig. 9can find out, the brilliant pharmaceutical carrier of targeted nanometer carbon has good specific aim effect, does not almost damage, but do not have the nanometer carbon crystal pharmaceutical carrier of targeting then also to have larger to normal cell to normal population of cells
kill and woundproperty.
contrast test 3: modification effect
Modification effect is characterized with isotope-labelling method in this contrast test.N isotopic labeling is carried out to transferrins, because N isotope nitrogen stable isotope does not have radioactivity, synthesizes at label and process all fairly simple, do not decay, non-radiating is decomposed, free from environmental pollution, do not need to take safeguard procedures, there is no toxicity, impact is not existed to final result.
Test divides two groups: one group of use without the nanometer carbon crystal of surface-functionalized process, one group uses treated nanometer carbon crystal, is placed with in the solution of the isotope-labeled transferrins-adriamycin composite of N by identical in quality two groups of samples and carries out moditied processing.Carry out filtration cleaning after moditied processing, get the debris of two groups of same volumes, measure the residual quantity of transferrins-adriamycin composite in two groups of debris and the ratio of both calculating, obtain residual quantity ratio.The ratio of residual quantity is lower, shows that modification effect is better.This project test point two parts a: part is 10 groups of parallel tests, and the processing time often organized is 30min; A part is 10 groups of controlled trials, and the processing time of each test, from 30min, increases the processing time with 20min successively.It the results are shown in
figure 10with
figure 11shown in.
figure 1010 groups of parallel test data
figure,
figure 1110 groups of controlled trial data
figure.
From
figure 10can find out with 11, along with the increase in processing time, the modification rate of two kinds of samples increases all to some extent, but the residual quantity for the treatment of samples is lower, and namely modification rate is higher.Final result is that the two all has one to modify the limit, and the finally reduction of the residual ratio of the two is slowed down all to some extent.
Claims (8)
1. the nano-medicament carrier material of target recognition of tumor cell, is characterized in that, comprises nanometer carbon crystal, and the C atom on the surface of described nanometer carbon crystal connects amycin and transferrins successively.
2.
as claimthe nano-medicament carrier material of the target recognition of tumor cell described in 1, it is characterized in that, this carbon crystalline substance of described nanometer carbon crystal is class spherical morphology, the brilliant C atom on surface of carbon and the dimeric structure of the C atomic building C atom of internal layer diamond phase, two carbon atoms in the dimeric structure of C atom are asymmetric distribution, and spacing of lattice is 0.21nm, and mean diameter is R, 0 < R≤10nm, the C content of this carbon crystalline substance is 99 ~ 100%.
3.
as claimthe nano-medicament carrier material of the target recognition of tumor cell described in 1, it is characterized in that, described nanometer carbon crystal is obtained by following methods: a) pickling is purified: fine powder diamond raw material powder being broken into more than 10000 orders, adopt concentrated sulphuric acid and concentrated nitric acid mixed liquor, dilute hydrochloric acid, Fluohydric acid. to this fine powder pickling successively, then use washed with de-ionized water to cleanout fluid pH close to 7; B) sorting: the material after cleaning is carried out centrifugalize, gets supernatant liquid and carries out the sorting of 4-7 days precipitations, remove the supernatant, by after the drying of lower sediment thing and get final product.
4. the preparation method of the nano-medicament carrier material of target recognition of tumor cell, is characterized in that, comprises the following steps:
1) pretreatment of nanometer carbon crystal: dense H nanometer carbon crystal being placed in mass ratio 2:1 ~ 4:1
2sO
4with dense HNO
3mixed liquor in, reflux, centrifugation; Get precipitation and be first washed with distilled water to pH4.0 ~ 5.0, then use 0.1 ~ 0.4molL
-1naOH solution washing, and ultrasonic disperse, then use 0.1 ~ 0.4molL
-1hCl washs, and ultrasonic disperse, then dry with deionized water wash;
2) transferrins-amycin physics complex liquid is prepared:
1. the configuration of PBS buffer: by NaCl6 ~ 10g, KCl0.2 ~ 0.8g, Na
2hPO
412H
2o2 ~ 5g, KH
2pO
40.2 ~ 0.8g takes each raw material, adds 1500 ~ 2000ml distilled water, adjusts pH to 7.0 ~ 7.5 until completely dissolved;
2. transferrins liquid: by transferrins and the PBS buffer obtained transferrins liquid of 1.5:1 ~ 3.5:1 mixing in mass ratio;
3. amycin liquid: amycin and distilled water are obtained amycin liquid in 10 ~ 20mg:5 ~ 10ml ratio mixing;
4. transferrins-amycin physics complex liquid: by the transferrins liquid in 2. and amycin liquid in mass ratio 2:1 ~ 4:1 mix and obtain transferrins-amycin physics complex liquid;
3) nanometer carbon crystal is assembled with transferrins-amycin physics complex liquid: by nanometer carbon crystal treated in step 1) and step 2) in transferrins-amycin physics complex liquid in mass ratio 0.01:1 ~ 0.08:1 mix, through the emulsion that ultrasonic emulsification obtains, after deionized water wash to pH7.0 ~ 7.5, centrifugation, the precipitate frozen centrifugation, the vacuum drying that obtain and get final product.
5.
as claimthe preparation method of the nano-medicament carrier material of the target recognition of tumor cell described in 4, is characterized in that, in step 1), the temperature of reflux is 88 ~ 92 DEG C, and the time of reflux is 24 ~ 48h; Centrifugal rotating speed is 1000 ~ 2000rpm.
6.
as claimthe preparation method of the nano-medicament carrier material of the target recognition of tumor cell described in 4, is characterized in that, in step 1), NaOH solution washing, ultrasonic disperse process repeat at least 3 times, and each ultrasonic disperse time is 1 ~ 3h; HCl washing, ultrasonic disperse process repeat at least 3 times, and each ultrasonic disperse time is 1 ~ 3h.
7.
as claimthe preparation method of the nano-medicament carrier material of the target recognition of tumor cell described in 4, is characterized in that, step 2) 4. in mixing concrete operations be: at 10 ~ 15 DEG C with the speed of 50 ~ 100rpm concussion 2 ~ 4h.
8.
as claimthe preparation method of the nano-medicament carrier material of the target recognition of tumor cell described in 4, is characterized in that, in step 3), phaco time is 2 ~ 10min, and the power of ultrasonic employing is 50 ~ 150W.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106236936A (en) * | 2016-08-30 | 2016-12-21 | 河南省豫星华晶微钻有限公司 | A kind of efficient sterilizing containing nanometer carbon crystal dredges intestinal granule and preparation method thereof |
| WO2017050263A1 (en) * | 2015-09-25 | 2017-03-30 | 郑州华晶金刚石股份有限公司 | Novel nanocarbon crystal |
| CN107929242A (en) * | 2017-11-26 | 2018-04-20 | 郑州大学 | Pharmaceutical carrier, drug composite based on Nano diamond and its preparation method and application |
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| CN104382919A (en) * | 2014-10-29 | 2015-03-04 | 山西大学 | Preparation method and application of nano diamond targeted medicine |
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| CN104382919A (en) * | 2014-10-29 | 2015-03-04 | 山西大学 | Preparation method and application of nano diamond targeted medicine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017050263A1 (en) * | 2015-09-25 | 2017-03-30 | 郑州华晶金刚石股份有限公司 | Novel nanocarbon crystal |
| CN106236936A (en) * | 2016-08-30 | 2016-12-21 | 河南省豫星华晶微钻有限公司 | A kind of efficient sterilizing containing nanometer carbon crystal dredges intestinal granule and preparation method thereof |
| CN107929242A (en) * | 2017-11-26 | 2018-04-20 | 郑州大学 | Pharmaceutical carrier, drug composite based on Nano diamond and its preparation method and application |
| CN107929242B (en) * | 2017-11-26 | 2020-01-07 | 郑州大学 | Drug carrier based on nano-diamond, drug complex, preparation method and application thereof |
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