CN106344927A - Folate-modified medicine-carrying hydroxylapatite and preparation method thereof - Google Patents
Folate-modified medicine-carrying hydroxylapatite and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a folate-modified medicine-carrying hydroxylapatite and a preparation method thereof. The method comprises the following steps: firstly preparing medicine-carrying hydroxylapatite, performing surface amination treatment on the medicine-carrying hydroxylapatite by use of a silane coupling agent or an amino polymer, and modifying the surface-aminated medicine-carrying hydroxylapatite by use of folic acid. The folate-modified medicine-carrying hydroxylapatite, which is prepared by the method, is capable of sensitively recognizing and combining folate receptors highly expressed in most of tumors; the folate-modified medicine-carrying hydroxylapatite penetrates through cytomembranes in an endocytosis manner, so that slow targeted release of anti-cancer medicines can be realized. Not only can the coating rate of anti-cancer medicines be improved, but also the leakage rate of nano carriers in the anti-cancer medicines during a blood circulation process can be effectively reduced, further the release efficiency and local concentration of the anti-cancer medicines are improved, the dosage, toxic and side effects and the like of the anti-cancer medicines are reduced, and the folate-modified medicine-carrying hydroxylapatite has great research value and application prospects in the field of cancer treatment.
Description
Technical field
The present invention relates to Bio-Nano-Materials field is and in particular to the hydroxyl phosphorus of a kind of folate molecule targeting and acid-sensitive response is grey
Stone Nano medication complex and its preparation method and application.
Background technology
Cancer is one of main disease of harm whole mankind's health.The annual new cancer patient in the whole world up to 1,000 at present
Many ten thousand, more than dead 600 ten thousand, the arduousness of the seriousness of its harm and treatment has caused many country public, government and scientific circles
Very big attention.The means of this kind of disease can be more efficiently treated in development, be significant to promoting human health.
Cancer therapy drug chemotherapy is most important anti-cancer therapies in addition to operation.Most small molecule anti-cancer drug are water-soluble at present
Property is poor, and surfactant is clinically usually used carries out emulsifying to it, but blood stability is poor, does not substantially have slow release
Or controlled release properties, and there are numerous shortcomings such as blood halflife is short, toxic and side effects are big.Therefore, new high potency drugs are studied
Transport agent has become the task of top priority of biomedicine field.Wherein, hydroxyapatite nano-medicament carrier is with its excellent biology
Chemical property and application development potentiality, constantly attract the extensive concern of industry.
Generally, the method realizing active tumour targeting mainly includes intratumor injection and carrier modification two big class.Intratumor injection
Although easy, in most cases because actual tumor locus cannot accurately be found out, thus and inapplicable.At present, more is to use
It is capable of identify that the particular ligand of specific molecular signal on cancer cell membrane carrys out modified medicaments carrier outer layer, mainly include Folic Acid, contracting ammonia
Sour (as ring-type pentapeptide crgd), siderophillin and some antibody.Compared with other targeted moleculars, Folic Acid relative molecular mass
Little, non-immunogenicity, cheap and easy to get, the chemical bonds between good stability, and medicine or carrier are simple.Therefore Folic Acid is
The ideal selection of targeting aptamer molecule.
Folic Acid (folic acid, fa), also known as pteroylglutamic acid, vitamin b11, is a kind of vitamin needed by human,
Intracellular reducible the latter is the coenzyme of one carbon unit transferring enzyme for tetrahydrofolic acid, participate in one carbon unit metabolism and purine, thymus
The synthesis of pyrimidine, is cellular metabolism, dna synthesis and the ultimate constituent repaired.The fast-growth of tumor cell needs abundance
The synthesis to maintain dna for the Folic Acid.The enzyme of synthesis Folic Acid is lacked, the growth of cell and propagation rely on from extraneous ring in zooblast
Border obtains Folic Acid.Folacin receptor (folate receptor, fr) is surface of cell membrane glycosyl phosphatidylinositol base alcohol (gpi) grappling
Glycated polypeptides, including three kinds of isomers: α-fr, β-fr, γ-fr.β-fr is cell embrane-associated protein, is anchored on by gpi
On cell membrane, the two has 70% homology.α-fr and γ-fr lacks the carboxyl terminal signal peptide modifying the attachment of gpi anchor, group
Secreted protein is belonged on one-tenth.Folacin receptor has three distinguishing features: (1) fr is very high with the affinity of free Folic Acid, dissociation
Constant kd ＜ 1nmol/l；(2) fr is connected the parent of the complex being formed to folate molecule by chemical bond with other macromolecular substances
Suitable with free Folic Acid with power and endocytosis effect, there is the good dysuria with lower abdominal colic that enters and transport potential；(3) knot to Folic Acid and its derivant for the fr
Close, transhipment is that special receptor-ligand combines, and can be by free Folic Acid Competitive assays.Folic Acid and receptor binding, by internalization side
Formula passes through cell membrane, is that Folic Acid enters intracellular main path.Folacin receptor expression in the normal tissue is highly conserved, only
Kidney, lung, choroid, Placenta Hominiss have low to medium level expression, and based on β hypotype.But in most of malignant cell
In, such as all height such as ovarian cancer, carcinoma of endometrium, cervical cancer, nonsmall-cell lung cancer, colon cancer and nasopharyngeal carcinoma are expressed, and sometimes may be used
Exceed 100-300 times than normal structure.Folacin receptor transporting mechanism is as follows: ectogenic folic acid composite and surface of cell membrane
Folacin receptor specifically binds, and surrounding forms depression, enters intracellular formation endosome.In the presence of nucleome inner proton pump,
Ph within endosome drops to 5 by 7, makes complex conformational change, the medicine of modified with folic acid dissociates from complex, is discharged into
Intracellular, and folacin receptor can return surface of cell membrane, circulation transhipment medicine.
Due to the change with biological internal ph, organic drug carrier such as liposome, polymerizing microballoons etc. is unstable, easily occurs
The change of structure is it is impossible to effectively carry out drug release.So it is defeated to be used for targeted drug using inorganic nano material as pharmaceutical carrier
Send system, obtain fast development in recent years.Although the inorganic drug carrier such as drug carrying capacity such as silicon dioxide, ferroso-ferric oxide
Height, chemical stability and good biocompatibility, if but excess accumulation in vivo, can normal tissue cause to damage, existing at present
In document report body, the silicon dioxide of excess accumulation is particularly evident to the damage of lung regulating liver-QI.And hydroxyapatite (hap) be vertebra move
The host inorganic constituent of thing skeleton and tooth, has excellent biocompatibility and biological degradability, safety and low toxicity, stablizes
Chemical constitution and carry the property of medicine wide the advantages of.Additionally, in acid condition (ph3.5-6), hydroxyapatite facile hydrolysiss, and to normal
Cytotoxic side effect, is a kind of potential pharmaceutical carrier.
Content of the invention
Present invention is primarily targeted at providing the hydroxyapatite nanometer medicine of a kind of folate molecule targeting and acid-sensitive response
Thing carrier, for the tumor cell of targeting surface folacin receptor overexpression, thus realize the slow Targeting delivery of cancer therapy drug.
The present invention discloses a kind of preparation method of the load medicine hydroxyapatite (fa@d@hap) of modified with folic acid first, described
Method includes: prepares load medicine hydroxyapatite (d@hap) first, and carries out surface with silane coupler or amino polymer to it
The step that amination is processed, and the step load medicine hydroxyapatite of described surface amination modified with Folic Acid.
The object of the invention also includes providing the load medicine hydroxyapatite of the preparation-obtained modified with folic acid of method described above.
By the nano-medicament carrier of the surface modification Folic Acid of the method preparation of the invention described above, its role is to folacin receptor
In the expression of most of tumor camber, and folacin receptor is all very strong with the affinity of folic acid derivatives and free Folic Acid, and Folic Acid is spread out
Biology and receptor binding, and cell membrane can be passed through by endocytosis mode, thus the tumor of targeting surface folacin receptor overexpression is thin
Born of the same parents, realize the slow Targeting delivery of cancer therapy drug.
Compared with prior art, the load medicine hydroxyapatite of the modified with folic acid of the present invention have following aspects prominent
Advantage: (1), due to the change with biological internal ph, organic drug carrier such as liposome, polymerizing microballoons etc. is unstable, Yi Fa
The change of raw structure is it is impossible to effectively carry out drug release.Although the inorganic drug carrier such as medicine such as silicon dioxide, ferroso-ferric oxide
Load capacity is high, chemical stability and good biocompatibility, if but excess accumulation in vivo, can normal tissue cause to damage.
And hydroxyapatite (hap) is the host inorganic constituent of vertebrate skeletal and tooth, have excellent biocompatibility and
Biological degradability, safety and low toxicity, stable chemical constitution and carry the property of medicine wide the advantages of.Additionally, (ph3.5- in acid condition
6), hydroxyapatite facile hydrolysiss, and to normal cytotoxic side effect, be a kind of potential pharmaceutical carrier.(2) of the present invention
Pharmaceutical carrier be that cancer therapy drug and hydroxyapatite nanoparticles are carried out codope, rather than only by cancer therapy drug modify receiving
Rice grain surface, thus improve the clad ratio of cancer therapy drug.(3) the hydroxyapatite nanoparticles surface of doping cancer therapy drug is repaiied
After Folic Acid on decorations, cover last layer thin film just as on this nano-carrier surface, thus significantly reduce this nano-carrier existing
The slip of the cancer therapy drug during blood circulation.And then improve the release efficiency of cancer therapy drug and local concentration and reduce
The using dosage of cancer therapy drug and toxic and side effects etc., have great researching value and application prospect in field of cancer.(4)
Folacin receptor is expressed in most of tumor camber, and folacin receptor with folic acid derivatives and free Folic Acid affinity all very
By force, folic acid derivatives and receptor binding, and cell membrane can be passed through by endocytosis mode.Therefore, the fa@dox@of present invention preparation
Hap nano-medicament carrier there is good active response performance to tumor cell that is to say, that this nano-medicament carrier have good
Good folate molecule specific target tropism, thus the targeting realizing cancer therapy drug slowly discharges.
Brief description
Accompanying drawing 8 width of the present invention:
Fig. 1 is transmission electron microscope (tem) figure of embodiment 2.1fa@dox@hap nano-particle.Wherein, a) and b) it is mark respectively
Chi is the electron microscope under 1000nm and 500nm, is c) and d) electron microscope that scale is under 200nm.Its form is bar-shaped, averagely long
Degree is 50-100nm, average diameter 5-20nm, is difficult to assemble, dispersibility is preferable.
Fig. 2 is infrared spectrum (ftir) figure of embodiment 2.2.Wherein, a) be dox@hap, b) be amidized dox@
Hap, c) is fa@dox@hap.
Fig. 3 is embodiment 2.3hap, dox@hap, the zeta electricity of amidized dox@hap, fa@dox@hap nano-particle
Bitmap.Wherein hap, dox@hap, the concentration of amidized dox@hap, fa@dox@hap is 0.5mg/ml.
Fig. 4 is the xrd figure of embodiment 2.4.Wherein, a) be amidized dox@hap, b) be fa@dox@hap.
Fig. 5 is embodiment 3dox and the fluorescence spectra of fa@dox@hap nano-particle.Dox and fa@dox@hap concentration is divided
Not Wei 2.5mm and 5mg/ml, excitation wavelength be 480nm.
Fig. 6 is drug release process under different ph values for the embodiment 4fa@dox@hap.Wherein, a) under different ph values
Relative intensity of fluorescence figure, b) be different ph values under drug accumulation release rate figure.Excitation wavelength is 480nm.
Fig. 7 is the cytotoxicity figure of embodiment 5fa@dox@hap nano-particle.Wherein, a) for human liver cell (hl-
7702), b) be human liver cancer cell (hepg-2), c) be breast cancer cell (mcf-7), d) be cervical cancer cell (hela), e) be
The cytotoxicity broken line graph of four kinds of cells.Fa@dox@hap concentration is 5mg/ml, and excitation wavelength is 480nm.
Fig. 8 is the fluorescence co-focusing micro-imaging figure in different cells, different time for the embodiment 6fa@dox@hap.Wherein,
A) it is African green monkey kidney cell (cos-7), b) is human liver cell (hl-7702), c) be breast cancer cell (mcf-7), d) be palace
Neck cancer cell (hela).Fa@dox@hap concentration is 5mg/ml, and excitation wavelength is 480nm.
Specific embodiment
The present invention is intended to provide a kind of preparation method of the load medicine hydroxyapatite (fa d hap) of modified with folic acid, Yi Jiyou
The product that the method prepares.Described preparation method includes: prepare load medicine hydroxyapatite (d@hap) first, and with silicon
The step that alkane coupling agent or amino polymer carry out surface amination process to it, and with Folic Acid to described surface amination
Carry the step that medicine hydroxyapatite is modified.
If no special instructions, symbol fa as used in this specification all represents Folic Acid, and d represents contained medicine, dox generation
Epirubicin, hap representation hydroxy apatite, d@hap represents and carries medicine hydroxyapatite, and fa@d@hap represents the load medicine of modified with folic acid
Hydroxyapatite.
In specific embodiment, in methods described, carry medicine hydroxyapatite by gas-liquid precipitation hydro-thermal method preparation: water dissolvable
In ethanol-water system, prepared by reaction under the conditions of 100～120 DEG C for calcium salt, phosphate and medicine.Wherein, described solubility
Calcium salt is preferably but not limited to ca (no3)2·4h2o；Described soluble phosphate is preferably but not limited to (nh4)2hpo4.
On the other hand, this described carry contained medicine of addressing in medicine hydroxyapatite and is selected from: amycin, paclitaxel, cisplatin,
Gemcitabine, fluorouracil, Gai Nuo, epirubicin, cyclophosphamide, vincristine, bleomycin, Docetaxel or derivatives thereof.
Particularly preferably amycin (dox) or derivatives thereof, it can be illustrated but be not limited to: amycin, Perarubicin, epirubicin, rice
Support anthraquinone.
Prepared load medicine hydroxyapatite is bar-like material, and average length is 50-100nm, and average diameter is 5-20nm.
The another aspect of specific embodiment, to described carry medicine hydroxyapatite surface amination process by with silane
Coupling agent or amino polymer reaction complete, and wherein said silane coupler is selected from but is not limited to γ-aminopropyl three ethoxy
One of base silane or ethylenediaminepropyltriethoxysilane；Described amino polymer is selected from, but not limited to, polyethyleneimine
One of or polyacrylamide.
The another further aspect of specific embodiment, described is modified to the load medicine hydroxyapatite of surface amination with Folic Acid
Step be: Folic Acid (fa), 1- (- 3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride (edc) and n- hydroxysuccinimidyl acyl
Imines (nhs) is reacted with the load medicine hydroxyapatite of surface amination according to the mixture of mol ratio 1～2:1～2:1～3.
In more specifically complete specific embodiment, the preparation side of the load medicine hydroxyapatite of the modified with folic acid of the present invention
Method, comprises the steps:
(1) water dissolvable nitrate, phosphate and medicine, in ethanol-water system, react under the conditions of 100～120 DEG C
Preparation carries medicine hydroxyapatite；
(2) silane coupler or amino polymer react in ethanol-water system with carrying medicine hydroxyapatite, prepare surface
Amidized load medicine hydroxyapatite；
(3) Folic Acid (fa), 1- (- 3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride (edc) and n- hydroxysuccinimidyl
Acid imide (nhs) is reacted with the load medicine hydroxyapatite of surface amination according to the mixture of mol ratio 1～2:1～2:1～3,
Prepare the load medicine hydroxyapatite of modified with folic acid.
Preferably in technical scheme, the preparation method of the load medicine hydroxyapatite of the modified with folic acid of the present invention, including as follows
Step:
(1) to concentration be 15～30mg/ml, ph be 10.5 ± 0.5 ca (no3)2·4h2Slowly drip in the ethanol solution of o
Plus medicine-aqueous solution that concentration is 6～10mg/ml, after stirring 5～15min, Deca concentration is the (nh of 60～100mg/ml4)2hpo4Aqueous solution, continues stirring 1～2h；After mixture hydro-thermal reaction 12～24h under the conditions of 100～120 DEG C, high speed centrifugation,
Precipitate washs drying, obtains carrying medicine hydroxyapatite；
Wherein, adjust described ca (no using the ammonia of 25% (w/w)3)2·4h2The ph value of the ethanol solution of o.
(2) it is slowly added dropwise the silane that mass fraction is 90～98% in the ethanol-water solution of volume ratio 4:1～10:1 even
Connection agent or amino polymer, after stirring 15～30min, is slowly added to carry medicine hydroxyapatite, and gained mixed system is ultrasonic 1～
5min, stirs 1～3h, adjusts ph to 10～12, and continues to react 2～3h；High speed centrifugation, precipitate washs drying, obtains table
Face is amidized to carry medicine hydroxyapatite；
(3) by mol ratio be 1～2:1～2:4～8 Folic Acid (fa), 1- (- 3- dimethyl aminopropyl) -3- ethyl carbon two
Inferior amine salt hydrochlorate (edc) and deionized water stir 0.5～2h, will be sub- for the n- hydroxysuccinimidyl acyl with respect to 1～3 times of mole of Folic Acid
Amine (nhs) adds in fa-edc system, after continuing stirring 5～7h, is slowly added to the surface ammonia with respect to 1～3 times of Folic Acid quality
The load medicine hydroxyapatite of base, adjusts mixture ph to 7～8, after continuing stirring 2～3h, high speed centrifugation, and precipitate washing is dry
Dry, obtain the load medicine hydroxyapatite of modified with folic acid.
Further, the present invention also provides the load medicine hydroxyapatite of the modified with folic acid obtained by above-mentioned any preparation method.
It is based on folate molecule targeting and acid-sensitive response hydroxyapatite nano-medicament carrier is used for targeting delivery cancer therapy drug, gained
The key propertys such as the structure verification of nano-medicament carrier, size and form are respectively adopted FTIR spectrum analyser
(ftir), X-ray diffraction pattern spectrometer (xrd), zeta potentiometric analyzer, transmission electron microscope (tem) are measuring.It is right
The size of cancer therapy drug load capacity is then verified using ultraviolet-uisible spectrophotometer, fluorescence microscope, and passes through body
Outer cell experiment is detecting the targeted therapy effect of this nano-medicament carrier.
Additionally, this nano-medicament carrier to evaluate this orientable transmission cancer therapy drug also by cell experiment thin to tumor
The target system of born of the same parents, that is, adopt human liver cell (hl-7702), human liver cancer cell (hepg-2), breast cancer cell (mcf-7), palace
Neck cancer cell (hela) carrying out external cytotoxicity experiment, to measure surface modification folate molecule and to load cancer therapy drug
The cytotoxicity to different cells for the hydroxyapatite nano-carrier.Wherein hl-7702, hepg2, mcf-7 are to test as a comparison, because
For these three cell surface folacin receptor low expressions.
Following non-limiting examples are for the present invention is described further, but are not construed as in the present invention
Hold any type of restriction.
The preparation of embodiment 1.fa@dox@hap
1.1 synthesis carrying medicine hydroxyapatite (dox@hap)
Combined using gas-liquid precipitation hydro-thermal method, weigh 30mg doxorubicin hydrochloride, the ca (no of 1.490g3)2·4h2O and
(the nh of 0.495g4)2hpo4It is dissolved in the ultra-pure water of 2ml respectively, in the dehydrated alcohol of 30ml and the ultra-pure water of 2ml, and by ca
(no3)2·4h2The ethanol solution ph of o is adjusted to 10.5 about.Under this alkaline environment, be slowly added dropwise while stirring hydrochloric acid Ah
The aqueous solution of mycin, after stablizing 10 minutes, continues Deca (nh4)2hpo4Aqueous solution, after completion of dropping, continues stirring 1h, then
Turbid solution is poured in reactor, hydro-thermal reaction 24h under the conditions of 120 DEG C, then high speed centrifugation is many with ultra-pure water and dehydrated alcohol
Secondary washed product, is vacuum dried 12h at 60 DEG C, grinds, obtains final product sample.
The 1.2 load medicine hydroxyapatite (dox@hap surface amination) preparing surface amination
100mg gamma-aminopropyl-triethoxy-silane is added to 27ml dehydrated alcohol and the mixing of 3ml distilled water composition is molten
In agent, it is stirred at room temperature 30 minutes, be subsequently adding 300mg and carry medicine hydroxyapatite (dox@hap), and ultrasonic 5 minutes, make dox@
Hap is well-dispersed in the mixed solvent of second alcohol and water.Stir 2h under room temperature, then utilize ammonia to adjust the ph value of reaction system
Save to 10, continue reaction 3h, high speed centrifugation, then again will solid matter ultra-pure water and absolute ethanol washing repeatedly, removing does not have
Have and be bonded in nanometer silane coupler on dox hap surface, finally gained material is vacuum dried, obtains the surface of nano-scale
Amidized dox@hap.
The load medicine hydroxyapatite (fa@dox@hap) of 1.3 modified with folic acid
Weigh 50mg Folic Acid (fa) and 22mg1- (- 3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride (edc) is molten
In 140ml deionized water, and stir 30 minutes under dark room temperature environment, subsequently weigh 35mg n- hydroxysuccinimidyl acyl sub-
Amine (nhs) is added in edc-fa system, stirs 6h under dark room temperature environment.Then weigh 200mg surface amination
Dox@hap is added thereto, and adjusts ph to 7.4, continues stirring 3h, last high speed centrifugation, deionized water and anhydrous second respectively
Alcohol washing repeatedly, is vacuum dried 12h at 60 DEG C, obtains final product the load medicine hydroxyapatite (fa@dox@hap) of modified with folic acid.
The test carrying medicine hydroxyapatite basic nature energy of embodiment 2. modified with folic acid
The test carrying medicine hydroxyapatite size and form of 2.1 modified with folic acid
The form size of the load medicine hydroxyapatite of embodiment 1 gained modified with folic acid to be observed using transmission electron microscope
Determine, test result is shown in Fig. 1.From transmission electron micrograph it is apparent that passing through the side that co-precipitation and hydro-thermal combine
The fa@dox@hap nano-particle of method synthesis, its form is bar-shaped, and average length is 50-100nm, average diameter 5-20nm, no
Easily assemble, dispersibility is preferable.
2.2 senior Fourier transform infrared spectroscopies measure
Take 1-2mg modified with folic acid carries the potassium bromide powder with drying in agate mortar for the medicine hydroxyapatite sample
(a.r. level) mixes and grinds to form fine powder, loads in mould, tabletted on tablet machine after in 6700ftir (thermo
Fisher, usa) upper test.Test parameter is: spectral region 7800-350cm-1, resolution 0.09cm-1, 25 DEG C of temperature.Test
Result as shown in Fig. 2 and dox@hap, the ftir spectrogram of amidized dox@hap compares, and fa@dox@hap is in 1641cm-1,
1590cm-1And 1490cm-1The peak at place is stretching vibration and the plane bending vibration of carboxyl.Additionally, 612cm-1The peak at place is hap
The stretching vibration of carbonyl, 1002cm-1The peak at place is the po of hap4 3-Characteristic peak.Thus prove that Folic Acid is modified well in dox@hap
On the surface of nano-particle.
The mensure of 2.3zeta current potential
The measurement of zeta current potential is carried out on nano particle size and zeta potentiometric analyzer (nanozs90, uk).Test parameter
For: 25 DEG C of temperature, test is averaged several times.Test result as shown in figure 3, because hydroxyapatite is almost electroneutral, therefore
Survey its zeta current potential close to zero, and after surface amination, its current potential increases, after Folic Acid in surface modification, its current potential is again
It is changed into close to zero, this also illustrates that Folic Acid is modified well on the surface of dox@hap nano-particle.
The mensure of 2.4xrd
The measurement of xrd is carried out on x-ray diffraction instrument (xd-3a, japan), and test parameter is: the angle of diffraction is 5-80., temperature
Spend for 25 DEG C.Test result is as shown in figure 4, the xrd spectrogram of fa@dox@hap is substantially identical with standard spectrogram, when 2 θ=30.9.
When, (211) crystal face of the corresponding standard spectrogram of this nano-particle.
The load medicine hydroxyapatite spectral quality test of embodiment 3. modified with folic acid
Weigh fa@dox@hap and dox of certain mass, the material weighing up is moved in the brown volumetric flask of 1.5ml respectively
Face, uses dmso constant volume, is made into the mother solution that concentration is respectively 5mg/ml and 2.5mm.It is that 10 μ l microsyringes draw 3 using range
μ l mother solution, moves in 3ml test system quartz cell, stirs, and surveys it and absorbs and launch wavelength.Test result as shown in figure 5,
After Folic Acid in the surface modification of dox hap, the fluorescence of amycin is substantially quenched completely, and trace it to its cause possibly Ah mould
Element is coated completely by Folic Acid, and slip reduces.Spectrum test is in ultraviolet spectrophotometer (agiilent 8453, usa) and fluorescence
Carry out on spectrophotometer (agiilent cary ecliipse, usa).Test parameter is: the maximum absorption wave of cancer therapy drug
A length of 480nm, maximum emission wavelength is 590nm.
Drug release test in the load medicine hydroxyapatite of embodiment 4. modified with folic acid
Under different ph values, as shown in fig. 6, the burst size of medicine is different, and the continuous reduction with ph, its medicine is released
High-volume constantly increase, the fluorescence of amycin constantly strengthens.And in same ph value, in different time, the burst size of medicine is not yet
Disconnected increase, as ph=5.5, the maximum burst size of its medicine has reached more than 80%, thus illustrates, this nano-carrier of synthesis
Medicine can effectively be carried out slowly to discharge, thus reaching the effect for the treatment of tumor cell.The load medicine of embodiment 5. modified with folic acid
Hydroxyapatite cytotoxicity test
Tetrazolium bromide mtt (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide) analytic process is selected in this experiment,
The toxicity size to cell for the dyestuff is assessed with the survival rate of cell.Respectively by hl-7702, the hepg-2 of exponential phase,
Mcf-7, hela cell is inoculated in 96 orifice plates with every milliliter of 1 × 105 cell concentration, and every hole adds 10 μ l, in 37 DEG C, 5%
co2Under the conditions of cultivate 24h, be separately added into 10mg/l, the pharmaceutical carrier fa@dox@of 20mg/l, 30mg/l, 40mg/l, 50mg/l
Hap, discards original fluid after being further cultured for 24h under the same terms, after every hole adds mtt solution (10 μ l, 5mg/ml) incubation 4h, little
The heart sops up in the hole supernatant and adds the dmso of 200 μ l, after the crystallization of first a ceremonial jade-ladle, used in libation is completely dissolved, measures 570nm's in microplate reader
Light absorption value od, selects 490nm as reference wavelength, parallel assay three times.Cell survival rate=(odExperimental group-odBlank)/
(odPositive control-odBlank) × 100%, blank is to be not added with pharmaceutical carrier group.
Cytotoxicity is to weigh the important indicator that nano-carrier is used for living cells imaging performance quality.Test result is such as
Shown in Fig. 7, there it can be seen that 6 μ l nano-carriers and hl-7702, hepg-2, mcf-7, and hela co-culture of cells 24h it
Afterwards, cell survival rate, respectively 90%, 80%, 80% and 55% about, shows this nano-carrier to normal cell and surface leaf
The tumor cell of acid acceptor low expression has relatively low cytotoxicity, and the tumor cell of surface folacin receptor overexpression is had relatively
High cytotoxicity.This experimental result complies fully with experiment anticipation.
The cell imaging experiment of the load medicine hydroxyapatite Absorption in vitro of embodiment 6. modified with folic acid
The cell used in experimentation mainly has hela (human cervical carcinoma cell), mcf-7 (human breast cancer cell), cos-
7 (African green monkey kidney cells) and hl-7702 (human liver cell).
Cell incubation: treat that the cell of adherent growth covers with bottom of bottle, outwell the culture fluid in culture bottle, and buffered with pbs molten
Liquid is washed twice.Add appropriate pancreatin to be digested, be subsequently adding fresh culture medium and blow attached cell off, discard part cell and hang
Liquid, adds the culture medium of proper amount of fresh, is placed on 37 DEG C, 5%co2Cell incubation case in cultivate.Cell culture medium is to add
Enter the dmem culture medium of 10% hyclone and 0.1% gentamycin sulfate.During experimental implementation, in Tissue Culture Dish
In with cell in every milliliter of 2 × 105 cell density kinds, be placed in incubator culture, stand-by.
When carrying out cell imaging, 6 μ l mother solution (5mg/ml) are added in Tissue Culture Dish, at 37 DEG C and 5%co2Condition
Carefully rinse to remove with pbs solution after lower culture 30min and be introduced into the dyestuff of cell, add after 2ml new dmem culture medium
Carry out under olympus fv1000-ix81 laser confocal fluorescence microscope observing light field and fluoroscopic image.Test result such as Fig. 8
Shown, there it can be seen that almost do not enter into after nano-carrier 2h surface folacin receptor low expression cell (hl-7702,
Cos-7), only cover in surface of cell membrane.Mcf-7 cell has a small amount of nano-carrier to take in.And for surface folacin receptor
Overexpression hela cell, its nano-carrier intake just increases significantly, and fluorescence intensity also strengthens therewith.Above experimental result table
Bright, nano-carrier fa@dox@hap is capable of the tumor cell of specific recognition folacin receptor overexpression.
Claims (9)
1. the preparation method of the load medicine hydroxyapatite of modified with folic acid, comprising:
Prepare load medicine hydroxyapatite first, and surface amination process is carried out to it with silane coupler or amino polymer
Step, and,
The step load medicine hydroxyapatite of described surface amination modified with Folic Acid.
2. preparation method according to claim 1 is it is characterised in that described load medicine hydroxyapatite is by water dissolvable calcium
In ethanol-water system, prepared by reaction under the conditions of 100～120 DEG C for salt, phosphate and medicine.
3. preparation method according to claim 1 it is characterised in that with Folic Acid the load medicine hydroxyl to described surface amination
The step that apatite is modified is: Folic Acid (fa), 1- (- 3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride and n-
N-Hydroxysuccinimide is anti-according to the mixture of mol ratio 1～2:1～2:1～3 and the load medicine hydroxyapatite of surface amination
Should.
4. the preparation method of the load medicine hydroxyapatite of the modified with folic acid described in claim 1, comprises the steps:
(1) in ethanol-water system, prepared by reaction under the conditions of 100～120 DEG C for water dissolvable nitrate, phosphate and medicine
Carry medicine hydroxyapatite；
(2) silane coupler or amino polymer react in ethanol-water system with carrying medicine hydroxyapatite, prepare surface amino groups
The load medicine hydroxyapatite changed；
(3) Folic Acid (fa), 1- (- 3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride and n- N-Hydroxysuccinimide are pressed
React with the load medicine hydroxyapatite of surface amination according to the mixture of mol ratio 1～2:1～2:1～3, prepare modified with folic acid
Carry medicine hydroxyapatite.
5. preparation method according to claim 1 is it is characterised in that the medicine described in step (1) is selected from: amycin,
Paclitaxel, cisplatin, gemcitabine, fluorouracil, Gai Nuo, epirubicin, cyclophosphamide, vincristine, bleomycin, Docetaxel
Or derivatives thereof.
6. the load medicine hydroxyapatite of modified with folic acid according to claim 5 is it is characterised in that carry in medicine hydroxyapatite
Contained medicine is amycin or derivatives thereof.
7. preparation method according to claim 4 it is characterised in that the silane coupler described in step (2) be selected from γ-
One of aminopropyl triethoxysilane or ethylenediaminepropyltriethoxysilane；Amino polymer be selected from polyethyleneimine or
One of polyacrylamide.
8. the preparation method according to right goblin's gas 1, comprises the steps:
(1) to concentration be 15～30mg/ml, ph be 10.5 ± 0.5 ca (no3)2·4h2It is slowly added dropwise dense in the ethanol solution of o
Spend the medicine-aqueous solution for 6～10mg/ml, after stirring 5～15min, Deca concentration is the (nh of 60～100mg/ml4)2hpo4
Aqueous solution, continues stirring 1～2h；After mixture hydro-thermal reaction 12～24h under the conditions of 100～120 DEG C, high speed centrifugation, precipitation
Thing washs drying, obtains carrying medicine hydroxyapatite；
(2) it is slowly added dropwise the silane coupler that mass fraction is 90～98% in the ethanol-water solution of volume ratio 4:1～10:1
Or amino polymer, stirring 15～30min after, be slowly added to carry medicine hydroxyapatite, the ultrasonic 1～5min of gained mixed system,
Stirring 1～3h, adjusts ph to 10～12, and continues to react 2～3h；High speed centrifugation, precipitate washs drying, obtains surface amino groups
The load medicine hydroxyapatite changed；
(3) by mol ratio be 1～2:1～2:4～8 Folic Acid (fa), 1- (- 3- dimethyl aminopropyl) -3- ethyl carbodiimide
Hydrochlorate and deionized water stir 0.5～2h, and the n- N-Hydroxysuccinimide with respect to 1～3 times of mole of Folic Acid is added fa-
In edc system, after continuing stirring 5～7h, it is slowly added to the load medicine hydroxyl of the surface amination with respect to 1～3 times of Folic Acid quality
Apatite, adjusts mixture ph to 7～8, after continuing stirring 2～3h, high speed centrifugation, and precipitate washs drying, obtains Folic Acid and repaiies
The load medicine hydroxyapatite of decorations.
9. the load medicine hydroxyapatite of the modified with folic acid of method preparation according to claim any in claim 1～8.
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| CN116370705A (en) * | 2023-04-14 | 2023-07-04 | 洛兮生命科技(杭州)有限公司 | Folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair stent material and preparation method thereof |
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