CN107929262A - Ethylenediamine cationized albumin anti-tumor nano grain and its preparation method and application - Google Patents
Ethylenediamine cationized albumin anti-tumor nano grain and its preparation method and application Download PDFInfo
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- CN107929262A CN107929262A CN201711222796.5A CN201711222796A CN107929262A CN 107929262 A CN107929262 A CN 107929262A CN 201711222796 A CN201711222796 A CN 201711222796A CN 107929262 A CN107929262 A CN 107929262A
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- albumin
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- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims abstract description 69
- 229930002330 retinoic acid Natural products 0.000 claims abstract description 68
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/203—Retinoic acids ; Salts thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
Abstract
The invention discloses ethylenediamine cationized albumin anti-tumor nano grain and its preparation method and application.The albumin nano preparation, preferably using all-trans retinoic acid as anti-tumor active substance, using ethylenediamine cationized albumin as pharmaceutical carrier, is added dispersant, solvent, is prepared using albumin combination nanoparticle technology, average grain diameter is less than 220nm.Its preparation method is：Water phase is configured to by the dispersant is soluble in water, the all-trans retinoic acid, which is dissolved in the solvent, is configured to oil phase；Homogeneous after the water phase is mixed high speed shear with the oil phase, then through evaporation of the solvent, freeze-drying, is made the albumin nano preparation.The present invention reduces albumin nano particle diameter to nano-scale, solves solubility, makes human serum albumin surface positively charged, the absorption between the tumour cell negatively charged with surface is added, strengthens its targeting, reduces adverse reaction, curative effect is improved, improves the applicability of Clinical practice.
Description
Technical field
The invention belongs to the pharmaceutical preparation of pharmaceutical technology field, it is related to that a kind of ethylenediamine cationized albumin is antitumor to be received
Grain of rice and its preparation method and application, particularly ethylenediamine cationized albumin are being prepared as the carrier of all-trans retinoic acid
Purposes in nano injection agent.
Background technology
All-trans retinoic acid (All-trans-retinoic acid, ATRA) is the derivative of vitamin A, is to control at present
The choice drug of acute promyelocytic leukemia is treated, there is good effect, complete remission rate height, do not cause diffusivity intravascular solidifying
The advantages that blood.Research in recent years finds that ATRA has the function that to suppress tumor stem cell (CSC).In addition, document report, ATRA energy
Enough express breast carcinoma stem cell tumour ball number and CSC surface markers CD44 to significantly reduce, significantly inhibit breast cancer cell and
The growth of breast carcinoma stem cell.But since ATRA stability is poor, removing is fast in vivo, bioavilability is low and right in vivo
The problems such as tumor tissues are without specific selectivity, greatly limit the clinical practice of ATRA.
In order to reduce the dosage of ATRA and number, targeting accumulations of the increase ATRA in tumor tissues, it is anti-to strengthen it
Tumor efficiency reduces toxic side effect at the same time, and energy is more and more put into exploitation ATRA novel formulations, especially by researchers
Come on nanometer formulation.Soo-Jeong Lim etc. contain ATRA in solid lipid nano granule (SLN), it is found that SLN can increase
The stability of ATRA, but carrying drug ratio is relatively low, only 2.4%.Zhang Jingru etc. is prepared for ATRA liposomes by reverse phase evaporation,
Each formulation stability is preferable, but envelop rate is below 70%.The research of ATRA novel nanos preparation is in laboratory at present
In the stage, not yet obtain that envelop rate is good, carrying drug ratio is high, particle diameter and stability all meet the new formulation of clinical requirement.
Albumin nano granular has many advantages, such as non-immunogenicity, good biocompatibility, spy as a kind of pharmaceutical carrier
Be not with nabTM technologies by medicine and human serum albumins with reference to and the albumin nano granular for preparing, make use of tumor physiology
The endogenous path of feature, albumin and EPR effects (enhanced permeability and retention effect), have
Dual-target, substantially increases the tumour distribution of medicine；Simultaneously because greatly reduce the regular dosage form based on solvent transmission
In allergic reaction and toxicity, the compliance of patient to avoid pretreatment, can be added.
The present invention explores the carrier of tumour medicine, and obtains a kind of outstanding anti-tumor nano grain.
The content of the invention
Present invention firstly provides a kind of cationized albumin anti-tumor nano grain, including ethylenediamine cationized albumin
With at least one anti-tumor active substance, load of the ethylenediamine cationized albumin as the anti-tumor active substance
Body.
There are the functional groups such as abundant carboxyl on albumin surface, and carry out modification to it easy to us more contributes to so as to reach
The new function of drug delivery.Ethylenediamine (ethylenediamine) can with carboxyl reaction and albumin covalent bond, while not
The space for influencing albumin combines, so as to remain albumin biological function.It is more meaningful, due in infinite multiplication
Tumour cell is because of the functional protein of expressed in abundance and acceptor and surface carries more negative electrical charges, using after ethylene diamine-modified
The nanoparticle that is prepared into of albumin by with certain positive charge, this is beneficial to nanoparticle and the negatively charged tumour in surface
Cell combination, improves tumor-selective and transmission efficiency of the nanoparticle to anti-tumor active substance transmission.Therefore, ethylenediamine is utilized
The albumin of modification prepares the novel nano preparation for containing anti-tumor active substance as carrier, can effectively improve antitumor work
On the premise of the stability and bioavilability of property material, by the distinctive EPR effects of nanoparticle, albumin Inner source paths and
The collective effect that positive and negative charge attracts, achievees the purpose that to improve the antitumous effect of ATRA, reduces toxic side effect.
In a specific embodiment, the anti-tumor active substance is fat-soluble medicine, selected from total trans dimension first
One or more in acid, taxol, docetaxel, Cabazitaxel, vincristine, camptothecine and Carmustine.
In a preferred embodiment, the anti-tumor active substance is all-trans retinoic acid.
In a specific embodiment, the cationized albumin is water-soluble albumin, preferably is selected from human serum
One or more in albumin and bovine serum albumin(BSA).
In a preferred embodiment, the dosage form of the cationized albumin anti-tumor nano grain is lyophilized
Powder-injection.
In a further preferred embodiment, the average grain diameter of the cationized albumin anti-tumor nano grain is less than
220nm。
The present invention also provides purposes of the cationized albumin anti-tumor nano grain in antitumor drug is prepared.
In a specific embodiment turns, the preparation method of the cationized albumin anti-tumor nano grain, bag
Include following steps：
A, ethylenediamine cationized albumin is dissolved in the water, obtains a liquid；
B, anti-tumor active substance is dissolved in organic solvent, obtains b liquid；
C, a liquid and b liquid are mixed, by ultrasonication, high-pressure homogeneous and rotary evaporation, resisted up to cationized albumin
The dispersion liquid of tumour nanoparticle.
In a more particular embodiment, the organic solvent is that can dissolve anti-tumor active substance and be easy to be evaporated
The solvent of removing, the one or more preferably being selected from absolute ethyl alcohol, dichloromethane, chloroform, ethyl acetate, acetone.
The present invention also provides a kind of preparation method of the freeze drying powder injection of cationized albumin anti-tumor nano grain, it is special
Sign is, on the basis of above-mentioned preparation method, further includes the dispersion liquid of the cationized albumin anti-tumor nano grain
The step of being freeze-dried.
The present invention also provides purposes of the ethylenediamine cationized albumin as the carrier of anti-tumor active substance.More specifically
Ground, the present invention also provides carrier of the ethylenediamine cationized albumin as anti-tumor active substance to prepare anti-tumor nano grain
Purposes in preparation, especially as purposes of the carrier in anti-tumor nano injection is prepared of all-trans retinoic acid.
The present invention combined using ethylenediamine cationized albumin as carrier material with ATRA, can prepare one kind it is easy to use,
The good nano-drug preparation of safe and tumor-targeting, it can reassemble into the mixture of injectable with physiological solution.
Wherein, ethylenediamine cationized albumin forms a kind of positively charged load medicine in surface with ATRA for carrier material and receives
The grain of rice, the average grain diameter of nanoparticle are less than 0.22 micron.This drug-carrying nanometer particle acts not only as various water not capacitive medicine
Means of delivery, and pharmaceutical carrier good biocompatibility itself, it is biodegradable, have no toxic side effect.By using nano drug-carrying
System, adds the solubility and stability of ATRA, improves the bioavilability and oncotherapy curative effect of ATRA, reduces ATRA's
Adverse reaction.One kind is provided for clinical cancer therapy to prepare simply, and stablizes height, safely and effectively ATRA nano injection formulations.
Nano-medicament carrier of the present invention based on ethylenediamine cationized albumin, mainly injected by solvent-
It is prepared by the method for ultrasonication-high-pressure homogeneous-rotary evaporation.
It is stable, high encapsulation rate it is another object of the present invention to prepare, while there is good targeting anti-tumor effect
ATRA- ethylenediamine cationized albumin nanoparticles (ATRA-eBSA-NPs), solve ATRA ejection preparations used in clinic at present and deposit
Unstable, inconvenient to use, the problem of adverse reaction is more, and bioavilability is low.
In one embodiment, the preparation of ATRA-eBSA-NPs comprises the following steps：
(1) ATRA is dissolved in absolute ethyl alcohol (and/or chloroform), is re-introduced into a certain amount of ethylenediamine cation
Change in albumin aqueous solution；
(2) resulting solution is placed under cell Ultrasonic Cell Disruptor ultrasonic；It is placed in again high-pressure homogeneous under nanometer high pressure homogenizer；
Rotary evaporation is depressurized again, removes organic solvent, that is, obtains the dispersion liquid of albumin nano granular.
Further, as a kind of preparation method of preferable novel multifunctional nano pharmaceutical carrier, comprise the following steps：
A, the ATRA of recipe quantity is weighed；
B, ATRA is dissolved in 1 volume absolute ethyl alcohol, be re-introduced into 0~50 DEG C of ethylenediamine sun of 5~100 volumes from
In sonization albumin aqueous solution；
C, step b resulting solutions are immediately placed on 0~50 DEG C of water bath sonicator 5-30min under cell Ultrasonic Cell Disruptor；
Step c resulting solutions are placed in high-pressure homogeneous under nanometer high pressure homogenizer, pressure 9000-18000psi, extremely d,
5 circulations are carried out less；
E, step d resulting solutions are subjected to decompression rotary evaporation, remove organic solvent, revolving temperature is 25~50 DEG C, i.e.,
Obtain ATRA-eBSA-NPs.
Benefit of the present invention
Novel cation albumin drug-carrying nanometer particle prepared by the present invention, the transmission that can both make insoluble drug are put down
Platform, improves the solubility, stability and bioavilability of contained insoluble drug, reduces adverse reaction；Also medicine pair can be improved
The selectivity and affinity of tumor tissues, strengthen the cancer target ability of medicine.Specifically, the ATRA-eBSA- that prepared by the present invention
NPs, for particle diameter between 150-300nm, most of particle concentrates on 180nm or so, and average zeta current potentials are 47mV, relatively
Stablize, can be stabilized under 4 DEG C of environment more than 4 weeks.
Brief description of the drawings
Fig. 1 is transmission electron microscope (TEM) figure of ATRA-eBSA-NPs prepared by the present invention.
Fig. 2 is differential scanning calorimetry (DSC) figure of ATRA-eBSA-NPs prepared by the present invention.
Fig. 3 is the release in vitro picture of ATRA-eBSA-NPs prepared by the present invention.
The preparation stability that Fig. 4 is ATRA-eBSA-NPs prepared by the present invention is investigated.
The cell in vitro that Fig. 5 is ATRA-eBSA-NPs prepared by the present invention absorbs ability and investigates.
Fig. 6 is the vitro cytotoxicity result of ATRA-eBSA-NPs prepared by the present invention.
Fig. 7 is the direct result that ATRA-eBSA-NPs prepared by the present invention suppresses one-tenth knurl ability in tumour cell body.
Fig. 8 is ATRA-eBSA-NPs Biodistribution in mice features prepared by the present invention.
Embodiment
Following embodiments are only being expanded on further and illustrate to the present invention, without limiting the scope of the present invention.This
Field technology personnel should be appreciated that the present invention is not limited to these embodiments and the preparation method used.Moreover, this area skill
Description can carry out equivalent substitution, combination, improvement or modification to art personnel to the present invention according to the present invention, but these will all include
Within the scope of the invention.
Embodiment 1
The preparation of ATRA-eBSA-NPs：Weigh 4.5mg ATRA to be placed in 1.5ml centrifuge tubes, add the anhydrous second of 0.9ml
Alcohol, fully dissolving, obtain drug containing organic phase (5mg/ml)；Weigh 50mg eBSA and be placed in glass XiLin with the dissolving of 10ml purified waters
In bottle, as water phase；Organic phase is injected into water phase, carries out ultrasound under condition of ice bath using cell Ultrasonic Cell Disruptor immediately
Broken, ultrasonic power 50W, super 5s stop 5s, altogether ultrasound 20min；Then solution in cillin bottle is transferred to high pressure homogenizer,
High-pressure homogeneous 15 circulations under 18000psi pressure；Then solution in cillin bottle is transferred in 100ml round-bottomed flasks, 35 DEG C of water
Bathe lucifuge vacuum rotary steam and remove organic solvent, that is, obtain ATRA-eBSA-NPs solution.The solution appearance to be faint yellow, transparent and
General to have opalescence, laser irradiation has Tyndall phenomenon.Average grain diameter is 180nm, and average zeta current potentials are 47.1mV, and HPLC is detected
The envelop rate of ATRA is 94.93%.
By dispersion liquid freeze-drying 48 it is small when, be not added with any antifreezing agent.After gained cake block adds sterile water or physiological saline
Original dispersion liquid is easily reconstructed very much, the size for reconstructing particle is identical with before freeze-drying.
Embodiment 2
The preparation of ATRA-eBSA-NPs：Weigh 10mg ATRA to be placed in 5ml centrifuge tubes, add the absolute ethyl alcohol of 2ml, fill
Divide dissolving, obtain drug containing organic phase (5mg/ml)；100mg eBSA are weighed to be placed in glass cillin bottle with the dissolving of 10ml purified waters,
As water phase；Organic phase is injected into water phase, ultrasonication is carried out under condition of ice bath using cell Ultrasonic Cell Disruptor immediately,
Ultrasonic power is 50W, and super 5s stops 5s, altogether ultrasound 20min；Then solution in cillin bottle is transferred to high pressure homogenizer,
High-pressure homogeneous 15 circulations under 18000psi pressure；Then solution in cillin bottle is transferred in 100ml round-bottomed flasks, 35 DEG C of water
Bathe lucifuge vacuum rotary steam and remove organic solvent, that is, obtain ATRA-eBSA-NPs solution.The solution appearance to be faint yellow, transparent and
General to have opalescence, laser irradiation has Tyndall phenomenon.Average grain diameter is 200nm, and average zeta current potentials are 45.2mV, and HPLC is detected
The envelop rate of ATRA is 92.56%.
By dispersion liquid freeze-drying 48 it is small when, be not added with any antifreezing agent.After gained cake block adds sterile water or physiological saline
Original dispersion liquid is easily reconstructed very much, the size for reconstructing particle is identical with before freeze-drying.
Embodiment 3
The preparation of ATRA-eBSA-NPs：Weigh 50mg ATRA to be placed in 10ml centrifuge tubes, add the absolute ethyl alcohol of 10ml,
Fully dissolving, obtains drug containing organic phase (5mg/ml)；Weigh 500mg eBSA and be placed in glass cillin bottle with the dissolving of 50ml purified waters
In, as water phase；Organic phase is injected into water phase, it is broken to carry out ultrasound under condition of ice bath using cell Ultrasonic Cell Disruptor immediately
Broken, ultrasonic power 50W, super 5s stop 5s, altogether ultrasound 20min；Then solution in cillin bottle is transferred to high pressure homogenizer,
High-pressure homogeneous 15 circulations under 18000psi pressure；Then solution in cillin bottle is transferred in 100ml round-bottomed flasks, 35 DEG C of water
Bathe lucifuge vacuum rotary steam and remove organic solvent, that is, obtain ATRA-eBSA-NPs solution.The solution appearance to be faint yellow, transparent and
General to have opalescence, laser irradiation has Tyndall phenomenon.Average grain diameter is 210nm, and average zeta current potentials are 44.3mV, and HPLC is detected
The envelop rate of ATRA is 92.15%.
By dispersion liquid freeze-drying 48 it is small when, be not added with any antifreezing agent.After gained cake block adds sterile water or physiological saline
Original dispersion liquid is easily reconstructed very much, the size for reconstructing particle is identical with before freeze-drying.
Embodiment 4
The preparation of ATRA-eBSA-NPs：Weigh 5mg ATRA to be placed in 1.5ml centrifuge tubes, add the anhydrous second of 0.025ml
The chloroform of alcohol and 0.225ml fully dissolve, and obtain drug containing organic phase (5mg/ml)；50mg eBSA are weighed with 10ml to be purified
Water dissolving is placed in glass cillin bottle, as water phase；Organic phase is injected into water phase, is existed immediately using cell Ultrasonic Cell Disruptor
Ultrasonication is carried out under condition of ice bath, ultrasonic power 50W, super 5s stop 5s, altogether ultrasound 20min；Then by solution in cillin bottle
High pressure homogenizer is transferred to, high-pressure homogeneous 15 circulations under 18000psi pressure；Then solution in cillin bottle is transferred to
In 100ml round-bottomed flasks, 35 DEG C of water-bath lucifuge vacuum rotary steams remove organic solvent, that is, obtain ATRA-eBSA-NPs solution.This is molten
Liquid appearance for it is faint yellow, transparent and it is general have an opalescence, laser irradiation has Tyndall phenomenon.Average grain diameter is 175nm, average zeta electricity
Position is 47.5mV, and the envelop rate of HPLC detections ATRA is 95.50%.
By dispersion liquid freeze-drying 48 it is small when, be not added with any antifreezing agent.After gained cake block adds sterile water or physiological saline
Original dispersion liquid is easily reconstructed very much, the size for reconstructing particle is identical with before freeze-drying.
Embodiment 5
The preparation of ATRA-eBSA-NPs：Weigh 10mg ATRA to be placed in 5ml centrifuge tubes, add the absolute ethyl alcohol of 0.05ml
Fully dissolved with the chloroform of 0.45ml, obtain drug containing organic phase (5mg/ml)；Weigh 100mg eBSA 10ml purified waters
Dissolving is placed in glass cillin bottle, as water phase；Organic phase is injected into water phase, immediately using cell Ultrasonic Cell Disruptor in ice
Ultrasonication is carried out under the conditions of bath, ultrasonic power 50W, super 5s stop 5s, altogether ultrasound 20min；Then solution in cillin bottle is turned
High pressure homogenizer is moved to, high-pressure homogeneous 15 circulations under 18000psi pressure；Then solution in cillin bottle is transferred to 100ml
In round-bottomed flask, 35 DEG C of water-bath lucifuge vacuum rotary steams remove organic solvent, that is, obtain ATRA-eBSA-NPs solution.Outside the solution
See for it is faint yellow, transparent and it is general have an opalescence, laser irradiation has Tyndall phenomenon.Average grain diameter is 185nm, and average zeta current potentials are
The envelop rate of 45.6mV, HPLC detection ATRA are 95.80%.
By dispersion liquid freeze-drying 48 it is small when, be not added with any antifreezing agent.After gained cake block adds sterile water or physiological saline
Original dispersion liquid is easily reconstructed very much, the size for reconstructing particle is identical with before freeze-drying.
Embodiment 6
Carry the preparation of taxol eBSA nanoparticles (PTX-eBSA-NPs)：Weigh 5mg taxols (PTX) be placed in 1.5ml from
In heart pipe, the absolute ethyl alcohol of 1ml is added, fully dissolving, obtain drug containing organic phase (5mg/ml)；Weigh 50mg eBSA 10ml
Purified water dissolving is placed in glass cillin bottle, as water phase；Organic phase is injected into water phase, immediately using cell ultrasonication
Instrument carries out ultrasonication under condition of ice bath, and ultrasonic power 50W, super 5s stop 5s, altogether ultrasound 20min；Then by cillin bottle
Solution is transferred to high pressure homogenizer, high-pressure homogeneous 15 circulations under 18000psi pressure；Then solution in cillin bottle is shifted
Into 100ml round-bottomed flasks, 35 DEG C of water-bath lucifuge vacuum rotary steams remove organic solvent, that is, obtain PTX-eBSA-NPs solution.Should
Solution appearance for it is faint yellow, transparent and it is general have an opalescence, laser irradiation has Tyndall phenomenon.Average grain diameter is 120nm, average zeta
Current potential is 40.5mV, and the envelop rate of HPLC detections PTX is 95%.
By dispersion liquid freeze-drying 48 it is small when, be not added with any antifreezing agent.After gained cake block adds sterile water or physiological saline
Original dispersion liquid is easily reconstructed very much, the size for reconstructing particle is identical with before freeze-drying.
Embodiment 7
The preparation of ATRA-eBSA-NPs：Weigh 50mg ATRA to be placed in 5ml centrifuge tubes, add the absolute ethyl alcohol of 0.25ml
Fully dissolved with the chloroform of 2.25ml, obtain drug containing organic phase (5mg/ml)；Weigh 500mg eBSA 30ml purified waters
Dissolving is placed in glass cillin bottle, as water phase；Organic phase is injected into water phase, immediately using cell Ultrasonic Cell Disruptor in ice
Ultrasonication is carried out under the conditions of bath, ultrasonic power 50W, super 5s stop 5s, altogether ultrasound 20min；Then solution in cillin bottle is turned
High pressure homogenizer is moved to, high-pressure homogeneous 15 circulations under 18000psi pressure；Then solution in cillin bottle is transferred to 100ml
In round-bottomed flask, 35 DEG C of water-bath lucifuge vacuum rotary steams remove organic solvent, that is, obtain ATRA-eBSA-NPs solution.Outside the solution
See for it is faint yellow, transparent and it is general have an opalescence, laser irradiation has Tyndall phenomenon.Average grain diameter is 190nm, and average zeta current potentials are
The envelop rate of 46.6mV, HPLC detection ATRA are 93.50%.
By dispersion liquid freeze-drying 48 it is small when, be not added with any antifreezing agent.After gained cake block adds sterile water or physiological saline
Original dispersion liquid is easily reconstructed very much, the size for reconstructing particle is identical with before freeze-drying.
Characterization and pharmacodynamic experiment
1st, transmission electron microscope characterizes
Fig. 1 is the transmission electron microscope picture of ATRA-eBSA-NPs prepared by embodiment 1.It can be seen from the figure that ATRA-eBSA-
NPs nano-particles are spherical structure, and particle diameter is more uniform, particle diameter substantially 180nm or so.
2nd, differential calorimetric scan characterizes
Fig. 2 is the differential scanning calorimetry figure of BSA, ATRA and the ATRA-eBSA-NPs of the preparation of embodiment 1.It is wherein negative
Absworption peak represents endothermic peak.ATRA occurs an endothermic peak at about 190 DEG C, and then occurs an exothermic peak at 460 DEG C；
BSA respectively has an endothermic peak at 220,320 and 450 DEG C；And in the heat analysis collection of illustrative plates of ATRA-eBSA-NPs, only in 110 and 450
DEG C nearby respectively there is an exothermic peak, ATRA absworption peaks are wholly absent, illustrate ATRA be substantially completely wrapped into nanoparticle in shape
Into new thing phase, it is highly dispersed in molecular forms
In ATRA-eBSA-NPs.
3rd, release in vitro is tested
ATRA-eBSA-NPs carries out the detection of preparation in the phosphate buffer solution containing 20% ethanol.Prepare
The phosphate buffer solution 200ml of 20% ethanol is divided into two equal portions, as dialysis medium.Take what 2ml was prepared by embodiment 1 respectively
ATRA-eBSA-NPs solution (ATRA containing 0.9mg) and ATRA ethanol solutions (0.45mg/ml) are injected into bag filter and seal,
It is then immersed in dialysis medium and is discharged.Discharging environment is:In the gas bath constant temperature oscillator of 37C, with 50 revs/min of speed
Lucifuge shakes.In set time point, 1ml dialysis media are respectively taken to carry out HPLC detections, then every group of supplement 1ml Fresh dialysate is situated between
Matter.Accumulation calculates the release amount of medicine of each time point.Fig. 3 is the releasing curve diagram of medicine, it can be seen that free ATRA exists
91.39% is released during 16h, and during the 48h of ATRA-eBSA-NPs upon discharge, only release 80.08% ATRA.Explanation
ATRA-eBSA-NPs can significantly control the release of ATRA, extend the action time of medicine in vivo, can increase the biology profit of medicine
Expenditure.
4th, the preparation stability of ATRA-eBSA-NPs nanoparticles is investigated.
The ATRA-eBSA-NPs prepared by embodiment 1 is placed in 4 DEG C of refrigerators, observes the change of size after surrounding (28 days).
From the grain size distribution (Fig. 4) of ATRA-eBSA-NPs, under 4 DEG C of environment, it is stabilized in the nanoparticle surrounding, particle diameter
And surface potential illustrates that the preparation stability of ATRA-eBSA-NPs is good without significant changes.
5th, the distribution of confocal laser scanning microscope nanoparticle in the cell
The B16F10 cells in exponential phase are collected in digestion, are added suitable 1640 complete mediums of RPMI and are made
Single cell suspension.By cell inoculation in being placed in advance in 6 orifice plates of sterile cover slips, it is all 2 × 10 to make cell inoculation amount5A/
Hole, is placed in 37 DEG C, 5%CO2Incubator culture is to cell attachment.Cell is divided into two groups, is separately added into coumarin 6 (coumarin-
6) (ATRA-eBSA-NPs is prepared the ATRA-BSA-NPs and ATRA-eBSA-NPs of mark by embodiment 1, ATRA-BSA-NPs ginsengs
Prepared according to embodiment 1, but eBSA therein be changed to BSA), 5 μM of dosage ATRA is incubated 4h jointly with cell.After stopping culture,
Hole inner liquid medicine is discarded, cell is gently washed 1-2 times with the PBS buffer of precooling, 4% paraformaldehyde fixes 10min.Again with cold
PBS buffer is washed 1 time, adds suitable DAPI dyeing liquors, room temperature avoid light place 5min.Anti- fluorescence is quenched after fishing for cell climbing sheet
Go out mounting fluid-tight piece.The results are shown in Figure 5 for observation under laser confocal microscope,
ATRA-BSA-NPs and ATRA-eBSA-NPs can be taken in by tumour cell, but the latter's green fluorescence is significantly strong
In the former, and the visible high brightness green phosphor dot in dispersed distribution, illustrates the ingestion efficiency of nanoparticle after cationization modification
Greatly improve.
6th, the extracorporeal anti-tumor ability of ATRA-eBSA-NPs nanoparticles is investigated.
Using B16F10 cells as model, using mtt assay detection ATRA-eBSA-NPs (being prepared by embodiment 1) and free medicine
Propagation toxicity of the ATRA to B16F10 cells.Take the logarithm growth period B16F10 cells with 1 × 105The even density in a/hole connects
Kind is in 96 orifice plates, CO2gas incubator overnight incubation.The culture medium in hole is sopped up, is then respectively adding 100 μ l series concentrations
ATRA-eBSA-NPs, ATRA of gradient, each concentration set 5 multiple holes, and set 5 holes for giving fresh culture as negative
Control group.Continue after cultivating 48h, suction out the liquid in 96 orifice plates, with PBS cleaning 3 times, 100 μ l serum-frees are then added per hole
Culture medium and the pre-configured MTT solution (5mg/ml) of 10 μ l, continue to be put in carbon dioxide incubator culture.After 4h, 96 are taken out
Orifice plate, careful inhale abandon nutrient solution, 150 μ l DMSO are then added per hole, low speed on shaking table is placed in and shakes 10min, surveyed with microplate reader
Determine the absorbance (OD values) in each hole at 492nm, and by following equation calculate inhibitory rate of cell growth (Inhibition Rate,
IR)。
IR=(1-OD experimental groups/OD control groups) × 100%
It will be appreciated from fig. 6 that effects of the ATRA-eBSA-NPs in low concentration to tumour cell nothing compared with free drug is notable
Gender gap, but the anti-tumor capacity of the ATRA-eBSA-NPs under highly concentrated is significantly higher than the free ATRA of same concentrations, explanation
ATRA-eBSA-NPs can significantly improve the antitumor curative effect of ATRA.
7th, ATRA-eBSA-NPs nanoparticles to B16F10 cells in animal body Tumor formation can influence.
The present invention uses C57BL/6 female mices as animal model, and B16F10 cell subcutaneous injections will be treated through different pharmaceutical
To the different parts of mouse, then see look into each treatment group into knurl situation.Concrete operations are as follows：Logarithmic phase will be grown into
B16F10 cells with after 0.25% Trypsin Induced press 2 × 105The even density in a/hole is inoculated in six orifice plates, is placed in dioxy
Change carbon incubator overnight incubation to fusion rate up to more than 80%.Then each hole is separately added into free ATRA (5 μM), the ATRA- of 1ml
BSA-NPs(ATRA:5 μM) and ATRA-eBSA-NPs (ATRA:5 μM) solution co-cultured, and control group adds isometric serum-free
Culture medium (ATRA-eBSA-NPs is prepared by embodiment 1, and ATRA-BSA-NPs is prepared with reference to embodiment 1, but will be therein
EBSA is changed to BSA).After 48h, digestion collect cell simultaneously cleaned 2 times with PBS (PH=7.2), be then resuspended in respectively serum-free without
In dual anti-culture medium, it is 1 × 10 to control cell density4/ml.Blank control group and each administration group cell are subcutaneously noted respectively again
Same mouse four limbs oxter is mapped to, and carries out mark, cellular control unit is expelled to another mouse oxter (n=5).Real Time Observation
Mouse into knurl situation, the 22nd day group mouse has obvious trend into knurl situation after inoculated tumour cell, and then cervical vertebra takes off
Mortar method puts to death each group mouse, and vernier caliper measurement gross tumor volume, finally chooses one group of control group and one group of experimental mice carries out
Dissection, observes and photographs to record.As a result, all mouse grow tumour, ATRA-BSA-NPs groups 5 in control group and ATRA groups
2 mouse grow tumour in mouse, and 5 mouse do not grow tumour in ATRA-eBSA-NPs groups, compared with control group, each medicine
Thing group can substantially suppress one-tenth knurl ability of the B16F10 cells in Mice Body.As shown in Figure 7, blank control group, ATRA groups,
The knurl volume of ATRA-BSA-NPs groups and ATRA-eBSA-NPs groups is obviously reduced successively.The tumor size of control group is each with other
There are significant difference (P for administration group<0.05).Compared with ATRA, ATRA-eBSA-NPs nanoparticles can significantly inhibit
One-tenth knurl ability of the B16F10 cells in Mice Body.
8th, influence of the ATRA-eBSA-NPs nanoparticles to intravenous injection ATRA Biodistribution in mice features.
The present invention uses B16F10 cells pulmonary metastases C57BL/6 female mices to be divided to two groups of difference tails for animal model (n=5)
(ATRA-eBSA-NPs is by real for ATRA-BSA-NPs the and ATRA-eBSA-NPs nanoparticles solution of intravenous injection DID fluorescent markers
Apply example 1 to prepare, ATRA-BSA-NPs is prepared with reference to embodiment 1, but eBSA therein is changed to BSA), dosage ATRA
5mg/kg, distribution situations (Fig. 8) of the 8h with small animal living body imaging nanoparticle in Mice Body after administration.Can by Fig. 8
Know, compared with ATRA-BSA-NPs groups, stronger fluorescence signal is presented in ATRA-eBSA-NPs group mouse-borne tumors lung, and prompting is received
The grain of rice is enriched in lung tissue, prompts the treatment that can be used for lung tumors.Mouse dissection is subjected to fluorescence sight to main organs
Examine, further confirm this result.It is therefore believed that ATRA-eBSA-NPs nanoparticles can be effectively by ATRA targeted deliveries
To lung, the ATRA concentration of tumor locus is improved, strengthens tumor killing effect, reduces toxic side effect.
The ATRA (all-trans retinoic acid) used in embodiment is purchased from Sa grace chemical technology Shanghai Co., Ltd.BSA (ox bloods
Pure albumen) it is purchased from Sinopharm Chemical Reagent Co., Ltd..EBSA (ethylenediamine cationized albumin) makes by oneself：By 40mL
The ethylenediamine solution of 1.4M salt acid for adjusting pH to 4.75, is slowly added dropwise in 5mL BSA aqueous solutions (10%, v/v).With
Afterwards, a certain amount of condensing agent 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCI, 100mg) are added, are continued
Reaction 2h is stirred at room temperature, is terminated and reacted with 400 μ L 4M acetate buffers (pH=4.75).Dialysis (molecular cut off
14000) unreacted ethylenediamine and impurity are removed, freezes and obtains eBSA.
Although embodiments of the present invention are illustrated in specification, these embodiments are intended only as prompting,
It should not limit protection scope of the present invention.It is equal that various omission, substitution, and alteration are carried out without departing from the spirit and scope of the present invention
It should include within the scope of the present invention.
Claims (10)
1. a kind of cationized albumin anti-tumor nano grain, it is characterised in that including ethylenediamine cationized albumin and extremely
A kind of few anti-tumor active substance, carrier of the ethylenediamine cationized albumin as the anti-tumor active substance.
2. cationized albumin anti-tumor nano grain according to claim 1, it is characterised in that the antitumor activity
Material is fat-soluble medicine, selected from all-trans retinoic acid, taxol, docetaxel, Cabazitaxel, vincristine, camptothecine and
One or more in Carmustine.
3. cationized albumin anti-tumor nano grain according to claim 2, it is characterised in that the antitumor activity
Material is all-trans retinoic acid.
4. cationized albumin anti-tumor nano grain according to claim 1, it is characterised in that the cationization is white
Albumen is water-soluble albumin.
5. according to claim 1-4 any one of them cationized albumin anti-tumor nano grains, it is characterised in that the sun
The dosage form for ionizing albumin anti-tumor nano grain is freeze drying powder injection.
6. use of the claim 1-5 any one of them cationized albumin anti-tumor nano grains in antitumor drug is prepared
On the way.
7. the preparation method of claim 1-5 any one of them cationized albumin anti-tumor nano grains, including following step
Suddenly：
A, ethylenediamine cationized albumin is dissolved in the water, obtains a liquid；
B, anti-tumor active substance is dissolved in organic solvent, obtains b liquid；
C, a liquid and b liquid are mixed, it is antitumor up to cationized albumin by ultrasonication, high-pressure homogeneous and rotary evaporation
The dispersion liquid of nanoparticle.
8. preparation method according to claim 7, it is characterised in that the organic solvent is selected from absolute ethyl alcohol, dichloromethane
One or more in alkane, chloroform, ethyl acetate, acetone.
9. a kind of preparation method of the freeze drying powder injection of cationized albumin anti-tumor nano grain, it is characterised in that in right
It is required that on the basis of 7, the step for being freeze-dried the dispersion liquid of the cationized albumin anti-tumor nano grain is further included
Suddenly.
10. ethylenediamine cationized albumin as anti-tumor active substance carrier in anti-tumor nano grain preparation is prepared
Purposes.
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Cited By (2)
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| CN109125291A (en) * | 2018-08-28 | 2019-01-04 | 南通大学 | Compound siRNA nano-carrier and its preparation method and application |
| CN112089704A (en) * | 2020-09-27 | 2020-12-18 | 中国药科大学 | Bionic nano-carrier and preparation method and application thereof |
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2017
- 2017-11-29 CN CN201711222796.5A patent/CN107929262A/en active Pending
Non-Patent Citations (1)
| Title |
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| HYEONG JUN BYEON,ET AL: "Doxorubicin-loaded nanoparticles consisted of cationic- and mannose-modified-albumins for dual-targeting in brain tumors", 《JOURNAL OF CONTROLLED RELEASE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109125291A (en) * | 2018-08-28 | 2019-01-04 | 南通大学 | Compound siRNA nano-carrier and its preparation method and application |
| CN109125291B (en) * | 2018-08-28 | 2020-12-22 | 南通大学 | Composite siRNA nano-carrier and preparation method and application thereof |
| CN112089704A (en) * | 2020-09-27 | 2020-12-18 | 中国药科大学 | Bionic nano-carrier and preparation method and application thereof |
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Application publication date: 20180420 |