CN109758581A - Antioxidant magnetic iron oxide nanoparticles and preparation method thereof - Google Patents
Antioxidant magnetic iron oxide nanoparticles and preparation method thereof Download PDFInfo
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- CN109758581A CN109758581A CN201910114141.9A CN201910114141A CN109758581A CN 109758581 A CN109758581 A CN 109758581A CN 201910114141 A CN201910114141 A CN 201910114141A CN 109758581 A CN109758581 A CN 109758581A
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Abstract
本发明一种抗氧化磁性氧化铁纳米颗粒，包括Fe3O4内核表面包覆介孔二氧化硅构成的核壳结构的磁性介孔二氧化硅纳米颗粒，还包括抗氧化剂N‑乙酰半胱氨酸，抗氧化剂N‑乙酰半胱氨酸负载在介孔二氧化硅中。还提供了一种抗氧化磁性氧化铁纳米颗粒的制备方法，一个配制抗氧化剂N‑乙酰半胱氨酸溶液的步骤；称量核壳结构的磁性介孔二氧化硅纳米颗粒溶液，离心后加入抗氧化剂N‑乙酰半胱氨酸溶液；超声后用封口膜封好，放入摇床中振荡，结束后，取出样品离心，干燥，获得抗氧化磁性氧化铁纳米颗粒。本发明的抗氧化磁性氧化铁纳米颗粒到达靶部位后，通过释放抗氧化剂，减轻氧化铁所介导的氧化应激损伤，从而改善磁性氧化铁纳米颗粒的组织相容性。
An anti-oxidation magnetic iron oxide nanoparticle of the present invention comprises a magnetic mesoporous silica nanoparticle with a core-shell structure composed of Fe3O4 inner core surface coated with mesoporous silica, and also includes an antioxidant N - acetylcysteine Amino acid, the antioxidant N‑acetylcysteine, is supported on mesoporous silica. Also provided is a preparation method of antioxidative magnetic iron oxide nanoparticles, a step of preparing an antioxidant N-acetylcysteine solution; weighing the magnetic mesoporous silica nanoparticle solution of the core-shell structure, and adding Antioxidant N-acetylcysteine solution; sealed with a parafilm after ultrasonication, placed in a shaker for oscillation, after the end, the sample was taken out and centrifuged, dried to obtain antioxidative magnetic iron oxide nanoparticles. After the anti-oxidation magnetic iron oxide nanoparticle of the present invention reaches the target site, it releases antioxidants to alleviate oxidative stress damage mediated by iron oxide, thereby improving the histocompatibility of the magnetic iron oxide nanoparticle.
Description
Technical field
The invention belongs to materialogy fields, are related to a kind of nano material, specifically a kind of anti-oxidant magnetic iron oxide
Nano particle and preparation method thereof.
Background technique
Superparamag-netic iron oxide (MNPs) is a kind of emerging functional material with nanometer diameter and superparamagnetism, more
It is raw to be applied to more cardiovascular field, including magnetic resonance imaging, Cellular tracking, pharmaceutical carrier, gene and cell-targeting etc.
The even clinical research of object medical experiment.Therefore, MNPs has broad application prospects in cardiovascular disease diagnosis and therapy field
With huge clinical Transformation Potential.
However, numerous studies confirm that MNPs has the cytotoxicity of time and concentration dependant in recent years, in cardiovascular field
When applied to diagnosing or treat disease, it is be easy to cause the chronic iron overloading of cardiac muscular tissue, is degraded to free iron in target area, led to
It crosses Fenton's reaction and generates a large amount of reactive oxygen species (ROS), cause silicosis oxidant and anti-oxidant system unbalance, mediate target organ oxidative stress damage
Wound, induces cell apoptosis, and aggravates the negativity reconstruct of target organ, function is caused to deteriorate.
Superparamag-netic iron oxide (MNPs) has broad application prospects in cardiovascular disease diagnosis and therapy field
With huge clinical Transformation Potential, but its oxidative stress toxicity just increasingly causes to worry in recent years, its clinic is seriously hindered to turn
Change.
Its title of M-MSNs are as follows: the magnetic mesoporous silica dioxide nano particle (Magnetic of core-shell structure (Core-shell)
Mesoporous silica nanoparticles is that (there is pin in Shanghai Suo Fei biological medicine Science and Technology Ltd. for commercial product
Sell), it is with Fe3O4Nano particle is as kernel, composite nanometer particle of the mesoporous silicon oxide as clad.M-MSNs has
The growth of magnetic core surface mesoporous layer is uniformly complete, composite particles particle diameter distribution is uniform, water dispersible is good, superelevation specific surface area,
The advantages that relatively narrow pore-size distribution and big pore volume.Internal small particle Fe3O4Nanometer core has superparamagnetism, can be used as T2 and makes
Shadow agent carries out MRI imaging, and magnetic thermotherapy effect can be also generated under external magnetic field；The mesopore silicon oxide shell of outside cladding can fill
Carrying all kinds of guest molecules, the M-MSNs such as fluorescent molecule, chemotherapeutics, DNA/siRNA, albumen is to have examining for huge applications prospect
Treat Integral rice grain platform.But there is safety, toxicity main source in cardiovascular field use in M-MSNs
In Fe3O4Myocardial tissue oxidizing stress damage is mediated after kernel degradation.
Summary of the invention
For above-mentioned technical problem in the prior art, the present invention provides a kind of anti-oxidant superparamag-netic iron oxides
And preparation method thereof, described this anti-oxidant superparamag-netic iron oxide and preparation method thereof will solve prior art synthesis
The MNPs time and concentration dependant cytotoxicity, be easy to cause the chronic iron overloading of cardiac muscular tissue, cause oxidation and antioxygen
Change system imbalance, mediate target organ oxidativestress damage, induce cell apoptosis, aggravates the negativity reconstruct of target organ, lead to function
The technical issues of deterioration.
The present invention provides a kind of anti-oxidant superparamag-netic iron oxides, by the magnetic mesoporous silica of core-shell structure
Nanoparticle is constituted, and the magnetic mesoporous silica dioxide nano particle of the core-shell structure is with Fe3O4Nano particle is situated between as kernel
Composite nanometer particle of the hole silica as clad further includes antioxidant NAC, and the antioxidant NAC is supported on
In the mesoporous silicon oxide.
The present invention also provides a kind of preparation methods of above-mentioned anti-oxidant superparamag-netic iron oxide, including walk as follows
It is rapid:
1) the step of preparation antioxidant NAC solution, the antioxidant NAC is dissolved in chloroform, so that described
Antioxidant NAC concentration be 0.1-1.5mg/mL；
2) the magnetic mesoporous silica nanoparticle solution of core-shell structure, the magnetic mesoporous silica nanometer are prepared
The concentration of grain solution is 0.1-50mg/mL, removes supernatant after centrifugation, and it is molten that antioxidant NAC described in step 1) is then added
Liquid, the volume of the magnetic mesoporous silica nanoparticle solution of the core-shell structure and the antioxidant NAC solution
Than being sealed with sealing film after ultrasound, being put into shaking table and vibrate 1-3h, hunting speed is for 0.082-2.05 mL:0.5-7.5mL
200 ~ 300rmp, oscillation terminate, and take out sample centrifugation, and precipitating is placed on drying in 60 ~ 75 DEG C of vacuum ovens, obtains anti-oxidant magnetic
Property ferric oxide nanometer particle.
Further, the magnetic mesoporous silica nanoparticle solution and the antioxidant of the core-shell structure
The volume ratio of NAC solution is 1.968 mL:0.75 mL.
Purposes the present invention also provides a kind of above-mentioned anti-oxidant superparamag-netic iron oxide as pharmaceutical carrier.
The present invention also provides a kind of above-mentioned anti-oxidant superparamag-netic iron oxides to compare as magnetic resonance imaging
The purposes of agent.
Purposes the present invention also provides a kind of above-mentioned anti-oxidant superparamag-netic iron oxide as Cellular tracking agent.
The present invention is by first preparing NAC titer, using M-MSNs nano particle as the skill for improving MNPs biocompatibility
Then NAC is loaded into the mesoporous SiO 2 on the surface M-MSNs by art platform in chloroform phase, have superparamagnetism with synthesis
With the M-MSN@NAC of anti-oxidant double grading.
The present invention has the superparamag-netic iron oxide (M-MSN@NAC) of antiopxidant effect by building；M-MSN@NAC
After reaching target area, by discharging antioxidant NAC, mitigate the oxidativestress damage that iron oxide is mediated, so as to improve MNPs
Histocompatbility.Present invention alleviates the oxidative stress toxicity of MNPs, significantly improve the biocompatibility of MNPs.
Good biocompatibility and safety are the premises of biomaterial clinic conversion.In view of the core of MNPs poisonous effect
Thimble section is oxidativestress damage, the present invention by load antioxidant in constructing anti-oxidant magnetic nanoparticle on MNPs, can
To realize magnetic nanoparticle and the accurate common location of antioxidant in target area, while retaining its superparamagnetism, release is lived
Property form antioxidant, pass through play antioxidation mitigate MNPs mediate oxidativestress damage, improve ferric oxide nano
The biocompatibility of particle and cardiac muscular tissue improves MNPs safety, is conducive to actively promote superparamag-netic iron oxide
Clinical conversion.
For the present invention compared with existing, technological progress is significant.The present invention loads successful M-MSN NAC and remains
The superparamagnetism of MNPs, and there are anti-oxidation characteristics.After M-MSN@NAC reaches lesion target area, NAC is to play for dissociation release
Antioxidation mitigates the oxidativestress damage that MNPs is mediated, significantly improves cardiac muscular tissue's compatibility of MNPs.
Detailed description of the invention
The TG that Fig. 1 shows that M-MSNs loads NAC in chloroform phase schemes.
Fig. 2 shows M-MSN@NAC synthesis schematic diagram, characterization and functionalization analysis；A is synthesis schematic diagram；B is transmission
Electric microscopic observation result；C is nano particle hydrodynamic diameter；D is the N2 adsorption curve of nano particle；E is nano grain surface
Mesoporous pore size；F is release profiles of the NAC in PBS (pH=7.4) in nano particle under the conditions of 37 DEG C of room temperature；G is nanometer
The Zeta potential of particle.
Fig. 3 shows that cardiac muscle cell detects from ROS after the incubation of different nano particles 24 hours；A be cardiac muscle cell from it is different
DHE detection ROS is horizontal after nano particle is incubated for 24 hours；B is each group cardiac muscle cell ROS fluorescence intensity statistical analysis.
Fig. 4 shows each group cardiac muscle cell apoptosis GAP-associated protein GAP and damage criterion detection；A is that Western Blot detects the heart
Pro apoptotic protein (Cleaved caspase 3 and Bax) and suppression apoptosis egg after myocyte is incubated for 24 hours from different nano particles
White (Bcl-2) expression；B-D is each group cardiac muscle cell pro apoptotic protein and suppression apoptotic proteins sxemiquantitative statistical analysis；E is each group heart
Lactic dehydrogenase (LDH) result statisticallys analyze after myocyte is incubated for 24 hours from different nano particles.
Fig. 5 shows that ischemia-reperfusion rat infarct week sideband cardiac muscle ROS compares；A and B is respectively ischemia-reperfusion rat
Infarct week sideband injects 24 hours DHE dyeing detection ROS levels and fluorescence intensity statistical analysis after different nano particles.
Fig. 6 shows that myocardial ischemia reperfusion in rats apoptotic index and LVEF compare；A is that each group rat injects different nanometers
1 week infarct week, sideband cardiac muscular tissue apoptotic index compared after particle；B is to inject 4 weeks each group rat LVEF after different nano particles
Compare.
Specific embodiment
The experimental data statistical method that following embodiment uses: multiple-group analysis uses ANOVA method.
Embodiment 1
Prepare Antioxidant N-acetyl-cysteine (NAC) standard solution
50mg NAC is weighed in 100mL beaker, 20mL chloroform ultrasonic dissolution is added, the volumetric flask of 100mL is transferred to after dissolution
In.With chloroform beaker 3 times, and the chloroform of washing is poured into volumetric flask, chloroform is added into volumetric flask to graduation mark, obtains
To solution A, concentration 0.5mg/mL.The standard solution of various concentration: B:0.1mg/mL is prepared respectively；C:0.02mg/mL；D:
0.004mg/mL；E:0.0008mg/mL；F:0.00016mg/mL；G:0.000032mg/mL.
Embodiment 2
The magnetic mesoporous silica dioxide nano particle (M-MSNs) of core-shell structure loads NAC
It takes 0.410mL M-MSNs solution (4 parts, every part of 2mg) in the centrifuge tube of 2mL, the concentration of M-MSNs solution is 4.8mg/
ML is centrifuged 25min at 12800rpm, removes supernatant after centrifugation；1.5mL 0.5mg/mL is added into 4 centrifuge tubes respectively
NAC, ultrasonic 2min；It is sealed with sealing film after ultrasound, is put into shaking table (25 DEG C, 250rmp) and vibrates 2h；Oscillation terminates, and takes out sample
Product centrifugation, is centrifuged 30min at 12800rmp, supernatant is poured into centrifuge tube, and precipitating is placed in 70 DEG C of vacuum ovens and does
It is dry, the product after drying is subjected to thermogravimetric analysis (TGA)；It takes 1mL supernatant that 4mL chloroform is added, rocks uniformly, obtained supernatant
The concentration of liquid is 0.5mg/mL, tests UV-Vis.
Judged by phenetic analysis, NAC is successfully loaded on M-MSNs in chloroform phase.
TG analysis: it will be seen from figure 1 that at 65 DEG C -130 DEG C, two curves all decline, this is because moisture
It is lost；At 130 DEG C -700 DEG C, the decline of TG curve is mainly the loss of organic matter；The curve of black indicates unloaded NAC's
Thermogravimetric weight loss, the ratio of thermogravimetric weight loss are about 7%；Dotted curve indicates that M-MSNs loads the thermogravimetric weight loss of NAC in chloroform phase,
Thermogravimetric weight loss ratio is about 17%, and discovery NAC in chloroform phase is successfully loaded on M-MSNs.
By transmission electron microscope observing to M-MSN@NAC favorable dispersibility, typical spherical nucleocapsid is presented in even particle size
Spline structure, kernel is by single Fe3O4Nano particle is constituted, kernel average diameter about 15nm, and it is radial that surface coats worm hole sample
The mesoporous silicon oxide of arrangement；Dynamic light scattering method measures M-MSN@NAC particle diameter about 80 ± 20nm(as also illustrated in figs. 2 a-b),
Unimodal (Fig. 3 C) of 100nm is presented in hydraulic diameter detection；The measurement display of M-MSN@NAC Nitrogen adsorption isotherm has H1 hysteresis loop
IV type thermoisopleth, in relative pressure p/p0=0.9, it will be apparent that nitrogen condensing steps reflect relatively narrow pore size distribution curve (Fig. 2 D-
E), spike concentrates at 5nm, supports the meso-hole structure observed in Fig. 2 B.There is time condensation in relatively high force region
Step, the space formed between the M-MSN@NAC particle of aggregation.The surface area and pore volume of M-MSNs is respectively 696
m2/ g and 0.44 cm3/ g shows that this structure has the ability for loading sufficient amount NAC.Under the conditions of 37 DEG C of room temperature, M-MSN@
NAC 2 hours i.e. release 72%NAC, 5 hours release 82%NAC, then into plateau, release time in PBS (pH=7.4)
Up to 48h(Fig. 2 F)；About -34.63 ± -1.19mV(Fig. 2 G of Zeta potential of M-MSN@NAC).
3 M-MSN@NAC of embodiment mitigates the short-term antiopxidant effect that MNP mediates anoxia-reoxygenation myocardial cells toxicity in vitro
SD neonatal rat cardiomyocytes exposed is separated by tissue block digestion method, and is identified；Neonatal rat myocardial cell is lured through anoxic box
It leads anoxic 3 hours, then puts back to incubator (condition of culture 95%O2And 5%CO2) in reoxygenation 3 hours, establish hypoxia-reoxygenation (H/R) heart
Myocyte model detects oxygen after then co-culturing MNPs, M-MSNs and M-MSN@NAC 24 hours with H/R cardiac muscle cell respectively
Change stress, mitochondrial membrane potential, cellular damage and apoptosis index.
Compared with H/R cardiac muscle cell, DHE detection display MNP group and M-MSN group ROS generation significantly increase (Fig. 3), cause
Cellular oxidation and antioxidant system are unbalance；ELASA and biochemical method detection prompt, MNP group and M-MSN group lipid peroxidation product
MDA and 8-iso-PGF2 α (P < 0.01) and DNA Peroxidation Product 8-OHDG are significantly raised (P < 0.0001), and Antioxidant Enzymes
It unites SOD, CAT, GSH-Px (P < 0.0001) and important non-enzyme antioxidant GSH significantly reduces (P < 0.0001), JC-1 is glimmering
Light probe shows that mitochondrial membrane potential (MMP) significantly reduces (P < 0.05)；Western Blot detection prompt, MNP group and M-MSN
The significant up-regulation (p < 0.05) of group pro apoptotic protein Caspase 3 and Bax expression, inhibits apoptotic proteins Bcl-2 expression is significant to lower
(p < 0.05)；MNP group and M-MSN group cardiac muscle cell LDH significantly increase (P < 0.05) (Fig. 4), and result above is prompted with Fe3O4For
The magnetic nanoparticle (MNPs and M-MSNs) of core dramatically increases H/R cardiac muscle cell's oxidative stress, leads to cellular oxidation
It is unbalance with oxidation resistance, anoxia-reoxygenation myocardial cells damage is aggravated, more cardiac muscle cell apoptosis can be can induce.
It is amazing to be, M-MSN@NAC group compared with H/R group, ROS level, Peroxidation Product, oxidation resistance,
Cellular damage and apoptotic proteins detection etc. without marked difference (Fig. 3 and Fig. 4), prompt M-MSN@NAC that can discharge activity
The antioxidant NAC of form, plays effective antioxidation, can completely inhibit Fe in the observation period for 24 hours in vitro3O4It mediates
The oxidativestress damage and apoptosis of anoxia-reoxygenation myocardial cells.
4 M-MSN@NAC of embodiment mitigates the long-term antiopxidant effect that MNP aggravates the reconstruct of ischemic myocardium negativity
Acute Myocardial Ischemia in Rats re-perfusion model is established, MNPs, M-MSNs, M-MSN NAC are injected in infarct week sideband, with note
It penetrates PBS rat as a control group, detects cardiac muscle cell's oxidative stress for 24 hours after injection.Compared with the control group, MNP group and
M-MSN group ischemic myocardium in rat ROS level dramatically increases (p < 0.0001) (Fig. 5), leads to ischemic myocardium oxidative and anti-oxidative system
It unites unbalance (p < 0.001), it is consistent with results of in vitro studies；1 week after injection, MNP group and M-MSN group ischemic myocardium in rat apoptosis
Index dramatically increases (p < 0.01) (Fig. 6)；4 weeks after injection, prussian blue staining prompted MNP group, M-MSN group rat infarct
A large amount of iron accumulations occur in all sidebands, prompt Fe3O4Chronic iron load mistake in ischemic myocardium can be caused after nano particle topical application
Weight；MNP group and Left Ventricular Ejection Fraction (LVEF) is substantially less than control group (p < 0.05) (Fig. 6) at M-MSN group rat 4 weeks.
After injection for 24 hours, cardiac muscle cell ROS is horizontal and control group is without marked difference (p > 0.05) for M-MSN@NAC group rat,
It is substantially less than MNP or M-MSN group (p < 0.01) (Fig. 5)；1 week after injection, M-MSN@NAC group rat apoptosis index was significantly low
In MNP and M-MSN group (p < 0.001) (Fig. 6)；4 weeks after injection, prussian blue staining prompted M-MSN@NAC group rat ischemia
Chronic iron overloading equally has occurred in myocardium；But LVEF is all remarkably higher than MNP group and M- at M-MSN@NAC group rat 4 weeks
MSN group (p < 0.05)；Result above prompt, it is negative that M-MSN NAC can lead to the chronic iron of ischemic myocardium after the injection of infarct week sideband
Lotus is overweight, but it discharges antioxidant NAC by part and significantly mitigates Fe3O4The ischemic myocardium oxidativestress damage and the heart of mediation
Muscle cell apoptosis, so as to improve Fe3O4The ischemic myocardium negativity of induction reconstructs.
The above result of study shows that M-MSN@NAC can significantly mitigate Fe3O4The oxidativestress damage and the ischemic heart of mediation
The reconstruct of flesh negativity, magnetic nanoparticle load this technology of preparing of antioxidant can significantly improve iron oxide and ischemic myocardium
Biocompatibility is conducive to improve magnetic nanoparticle in cardiovascular field diagnosis or the safety of therapeutic application, has wide
Wealthy application prospect and huge Transformation Potential.
Claims (6)
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| CN114601818A (en) * | 2022-03-09 | 2022-06-10 | 五邑大学 | Novel mesoporous nano material, preparation method and application thereof |
| CN115477329A (en) * | 2022-09-14 | 2022-12-16 | 北京信息科技大学 | Preparation method of a carbon-based core-shell structure pinecone-shaped nanoflower magnetic composite material |
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| CN114601818A (en) * | 2022-03-09 | 2022-06-10 | 五邑大学 | Novel mesoporous nano material, preparation method and application thereof |
| CN115477329A (en) * | 2022-09-14 | 2022-12-16 | 北京信息科技大学 | Preparation method of a carbon-based core-shell structure pinecone-shaped nanoflower magnetic composite material |
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