CN111281984A - Ferroferric oxide magnetic nano probe based on surface modification and preparation and application thereof - Google Patents
Ferroferric oxide magnetic nano probe based on surface modification and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a ferroferric oxide magnetic nano probe based on surface modification and preparation and application thereof, wherein the preparation method comprises the following steps: (1) uniformly dispersing ferroferric oxide nanoparticles dissolved in dichloromethane and grafted with oleic acid on the surface in a DMF (dimethyl formamide) solvent, adding EDC (EDC) and Sulfo-NHS (sulfolane-N-hydroxysuccinimide) activated ferroferric oxide nanoparticles, reacting at room temperature, and adding an acetic acid solution for full resuspension; (2) adding chitosan fully dissolved in the acetic acid solution into the resuspended solution, and stirring at room temperature for reaction; (3) dialyzing and purifying; (4) taking KGN solvent in DMSO, adding into ferroferric oxide-chitosan solution, fully shaking, dialyzing and purifying to obtain the target product. Compared with the prior art, the preparation method is simple to operate, the prepared magnetic nano probe fills the blank that no nano magnetic bead which is applied to diagnosis and treatment and can be used for cartilage repair is available at present, and the magnetic nano probe has wide application prospect in diagnosis and treatment of cartilage injury.
Description
Technical Field
The invention belongs to the technical field of nano-drug preparation, and relates to a ferroferric oxide magnetic nano-probe based on surface modification, and preparation and application thereof.
Background
Joint diseases are often accompanied by pain, which limits people's mobility. Cartilage damage due to injury, degenerative disease or other pathological causes often leads to the development of osteoarthritis, ultimately leading to progressive total joint destruction. While current advances indicate that biologies can slow the progression of deterioration, such drugs do not promote tissue repair. Since articular cartilage lacks a blood supply and has a limited ability to regenerate articular cartilage due to its specific structure, it is difficult to perform a self-repair process once it is damaged. Furthermore, local joint damage may cause degeneration of the entire articular cartilage, which in turn leads to the development of arthritis, seriously affecting the quality of life of humans.
The current clinical treatment progress shows: early proper intervention can delay the progressive destruction of joint disease. However, the cartilage is thin in section, and the damage of the articular cartilage is difficult to effectively identify and treat, so that more precise imaging technology is required to clearly show the position of the articular lesion. Magnetic Resonance Imaging (MRI) has been widely used as a classical tool in clinical joint damage diagnosis for the past decades, and despite significant advances such as high field strength, coil and improved pulse sequence improvements, cartilage level defect detection has not been able to be correctly differentiated[4]. Superparamagnetic nanoparticles, such as ferroferric oxide and other contrast agents, are widely used in reliable diagnosis, and provide a tool for imaging deep tissues of human or animals.
The ferroferric oxide nano-particle is a safe nano-probe, the safety evaluation of which is approved by the FDA, and is considered as a nano-particle which can be metabolically absorbed by the organism. Ferroferric oxide-based nanoparticles have applications in many fields, however, there is no report on ferroferric oxide magnetic nanoparticles for imaging of fine structures and treatment of cartilage defects.
Chinese patent ZL201010512272.1 discloses a preparation method of ferroferric oxide nano particles modified by surface dendritic polymers, wherein carboxylated dendritic polymers are modified on the surfaces of the nano particles, and the particle size of the carboxylated dendritic polymers is 6-10 nm. But the preparation conditions are relatively harsh, and the treatment effect of the obtained nano-particles is also poor.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a ferroferric oxide magnetic nano probe based on surface modification and preparation and application thereof. The nuclear magnetic imaging shows that the prepared nanoprobe can well display the outline of a specific damaged area and has a good cartilage repair effect. Therefore, the nano probe has wide application prospect in cartilage damage detection and treatment.
Compared with the common traditional method, firstly, the method disclosed by the invention is milder, and the novel magnetic nanoparticles with uniform appearance and large drug-loading capacity can be prepared without adopting a high-temperature reaction step; secondly, chitosan with good biological safety is used as a grafting molecule, and KGN is loaded and then is further self-assembled to generate particles with the diameter of 80-150nm, so that the drug uptake of cells can be effectively increased, and the treatment effect is further achieved; finally, compared with other traditional ferroferric oxide nanoparticles, the magnetic nanoparticles prepared by the method disclosed by the invention can be used as a forward contrast agent for imaging, and the MRI imaging effect can be observed more clearly.
The purpose of the invention can be realized by the following technical scheme:
one of the technical schemes of the invention provides a preparation method of a ferroferric oxide magnetic nano probe based on surface modification, which comprises the following steps:
(1) uniformly dispersing ferroferric oxide nano particles which are dissolved in dichloromethane and have oleic acid grafted on the surface in a DMF solvent, adding EDC and Sulfo-NHS activated ferroferric oxide nano particles in batches, reacting at room temperature, removing the redundant DMF solvent, and adding an acetic acid solution for full resuspension;
(2) adding chitosan fully dissolved in the acetic acid solution into the resuspended solution obtained in the step (1), and stirring at room temperature for reaction;
(3) dialyzing and purifying the reaction solution obtained in the step (2);
(4) and (3) dropwise adding a KGN solvent into the purified ferroferric oxide-chitosan solution obtained in the step (3) in DMSO, fully shaking, and dialyzing and purifying in pure water to obtain the target product.
Further, in the step (1), the volume ratio of dichloromethane to DMF is 1: 5.
Further, in the step (1), the mass ratio of the ferroferric oxide nanoparticles with the surfaces grafted with the oleic acid, EDC and Sulfo-NHS is 1:3:3-1:1: 1.
Further, in the step (1), the reaction time at room temperature is 24 hours.
Further, in step (1), after removing DMF solvent, washing with 0.1% acetic acid solution for 2-5 times, each time 1-5 mL.
Further, in the step (1) and the step (2), the acetic acid solution used is 0.1 wt% acetic acid solution.
Further, in the step (2), the concentration of chitosan in the acetic acid solution is 1-5mg/L, and the mass ratio of chitosan to ferroferric oxide nanoparticles with surfaces grafted with oleic acid is 2-10: 1.
Further, in the step (3) and the step (4), dialysis purification is carried out in pure water, the purification time is 48-72h, and the pure water is replaced every 6 h.
Further, in the step (4), the mass ratio of the added KGN to the chitosan is 1: 5-5: 2.
Further, in the step (4), the oscillation time is 24 hours, and the rotation speed during oscillation is 100-200 rpm.
In the invention, the purchased ferroferric oxide magnetic nanoparticles with the surface grafted with oleic acid are dissolved in dichloromethane, and DMF with good solubility on general compounds is selected as a reaction solvent to activate oleic acid in order to enable EDC/Sulfo-NHS as a coupling agent to be better dissolved and further enable the oleic acid on the surfaces of magnetic beads to be more fully activated. In the invention, the purpose of selecting Sulfo-NHS is to improve the water solubility of the oleic acid modified ferroferric oxide, so that the oleic acid modified ferroferric oxide is loaded with sulfonic acid groups, the water solubility can be greatly increased, and conditions are provided for further modification of the subsequent water-soluble chitosan.
Meanwhile, the addition amount of each raw material reagent, treatment process conditions and the like are limited, the main reaction and post-treatment are mainly carried out at room temperature, the activation of magnetic beads and the coupling reaction of chitosan, and the reaction reagents have high reaction activity and can carry out complete reaction at room temperature, so the experiment is safe and simple, and the operability is high. The reason why the operation step of loading KGN needs to be dropwise added in the invention is that KGN has poor water solubility, so that KGN is not easy to uniformly disperse in the process of diffusing from an organic phase to a water phase if the dropwise adding speed is too high, and the subsequent loading rate is greatly influenced, so the loss can be reduced by selecting dropwise adding.
The second technical scheme of the invention provides a ferroferric oxide magnetic nano probe based on surface modification, which is prepared according to the preparation method.
The third technical scheme of the invention provides application of a ferroferric oxide magnetic nano probe based on surface modification in preparation of medicines for cartilage imaging and soft tube defect treatment.
Furthermore, the prepared magnetic nanoparticles can be incubated with adipose-derived mesenchymal stem cells, and the survival and proliferation of the cells are not influenced;
further, the prepared magnetic nanoparticles can be used for nuclear magnetic imaging of articular cartilage and treatment of articular cartilage defects together with adipose-derived mesenchymal stem cells;
furthermore, the prepared magnetic nanoparticles can be used as a forward contrast agent and show a bright signal in the magnetic imaging of the inner core of an experimental rabbit body;
further, the relaxation rate of the magnetic nanoparticles prepared as described above in vitro was 66.59mM-1S-1；
Further, the magnetic nanoparticles prepared above were completely metabolized after 12 weeks in the experimental rabbits, and no signal remained in the experimental rabbits.
According to the invention, superparamagnetic ferroferric oxide nanoparticles are used as a carrier, carboxyl on the surface of ferroferric oxide is coupled with amino of chitosan through a condensation reaction, and finally a small molecular drug KGN with negative charges is adsorbed on the surface of chitosan through the action of electrostatic adsorption, so that the magnetic assembly with a larger particle size of 80-150nm is further self-assembled. The nanoprobe can be uniquely used as a forward contrast agent of nuclear magnetic imaging, presents a bright signal at a focus and is distinguished from surrounding tissues, so that the damaged cartilage tissue and the normal cartilage tissue are simply distinguished, and the repair and real-time diagnosis of the cartilage defect disease repair are carried out.
Compared with the prior art, the invention has the following advantages:
(1) the preparation method of the nano probe is simple, and the preparation condition is mild;
(2) the prepared nanoprobe has good biocompatibility;
(3) the prepared nanoprobe has the imaging of a forward contrast agent, and can well distinguish a focus from normal cartilage tissues;
(4) the prepared nano probe can be metabolized and discharged in vivo, and has good biological safety.
Drawings
FIG. 1 is a flow chart of preparation of a ferroferric oxide nanoprobe;
FIG. 2 is a TEM representation diagram of a ferroferric oxide nanoprobe;
FIG. 3 is a diagram of in vitro induction of adipose-derived mesenchymal stem cells into cartilage by a ferroferric oxide nanoprobe;
FIG. 4 is an in vivo nuclear magnetic imaging diagram of a ferroferric oxide nanoprobe.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
In the following examples, ferroferric oxide nanoparticles surface-grafted with oleic acid were purchased from Ocean NanoTech, USA (Cat. No.: SN 0101).
The rest of the raw material reagents or treatment techniques, if not specifically mentioned, indicate that the raw materials are all conventional commercial raw materials or conventional treatment techniques in the field.
Example 1:
the preparation of the chitosan surface modified ferroferric oxide nano particles is shown in figure 1:
uniformly dispersing 0.5mL of ferroferric oxide nanoparticles (20 mg/mL) which are dissolved in dichloromethane and have oleic acid grafted on the surface into 2.5mL of DMF solvent, adding EDC (EDC) with the mass of 20mg and Sulfo-NHS (Sulfo-NHS) activated ferroferric oxide nanoparticles in batches at room temperature, and reacting for 24 hours; removing redundant DMF solvent from the activated ferroferric oxide-sulfo-NHS nano particles, adding 2mL of 0.1% (V/V) acetic acid, washing for 3 times, and finally fully suspending with 1mL of 0.1% acetic acid solution for later use; 2mL of chitosan (2mg/mL) fully dissolved in 0.1% acetic acid solution was added to the above solution in portions, and after continuously stirring at room temperature and continuing the reaction for 24 to 48 hours, excess chitosan and coupling agent were removed by dialysis for 72 hours with pure water in a dialysis bag having a molecular weight of 300 kDa. Thus, the ferroferric oxide nano-particles with chitosan modified on the surfaces are preliminarily prepared.
Example 2:
preparing a ferroferric oxide nano particle with a chitosan surface modified and loaded KGN assembly, and referring to the figure 1 again:
1mg KGN was dissolved in 0.1mL DMSO, the KGN solution in DMSO was added dropwise to 0.5mL ferroferric oxide nanoparticles (5mg/mL) obtained in example 1 and shaken for 24h on a shaker at 100 rmp. Thereafter, the above solution was dialyzed in pure water for 48 hours, during which pure water was replaced every 6 hours. Finally preparing the chitosan surface modified ferroferric oxide nano particles and loading KGN assemblies, namely Fe3O4-CS/KGN nanoprobes.
The final chitosan surface-modified ferroferric oxide nanoparticles and KGN-loaded assembly nanoprobes can be prepared through the embodiments 1-2, and the flow is shown in fig. 1.
FIG. 2 is Fe prepared3O4A TEM representation picture of a CS/KGN nano probe, wherein the KGN-loaded ferroferric oxide magnetic nano particles are in a core-shell structure, a thicker shell polysaccharide layer is arranged on the surface, a plurality of ferroferric oxide nano particles with higher contrast are arranged inside the magnetic nano particles, and the magnetic nano particles are attracted by negative charges on the KGN surface from a monodisperse state to form large nano particles.
Example 3:
Fe3O4-in vitro evaluation test of CS/KGN nanoprobes as forward contrast agents:
0.5mL of Fe with the concentration of 0, 0.01, 0.025, 0.05 and 0.1mM are respectively taken3O4CS/KGN nanoprobes, placed in 1.5mL centrifuge tubes, and the relaxation rate was determined in the T2-weighted mode in a low-field NMR spectrometer (0.5T). And at the same concentration, the relaxation rates were measured, respectively, under the conditions set as follows: the flip angle is 90 °, TR 1800ms, TE 18.2ms, RG 26 dB.
FIG. 3 shows: as the Fe content in the nanoprobe is increased, the signal of the magnetic bead in vitro is brighter, which indicates that the nanoprobe can be used as a forward contrast agent in a T2 weighting mode.
Example 4:
in order to confirm the safety of the nano magnetic beads in vitro, the adipose-derived mesenchymal stem cells are cultured at a cell density of 3X 104cells/mL were seeded in 96-well plates, prepared in 5 duplicate wells, 100 μ L per well, and cultured overnight. The next day, the supernatant was discarded, nanoparticles containing different concentrations and KGN at the corresponding concentrations were added, PBS was added as a blank control, and after incubation for 24 hours, the supernatant was discarded, 100 μ L of 10% CCK-8 solution was added to each well, incubation for 2 hours, and uv absorption was detected at a wavelength of 450nm using a microplate reader.
The results in FIG. 4 show that: KGN and the addition of the nanoprobe set do not show obvious cytotoxicity at different concentrations, which indicates that the nanoprobe has good biocompatibility.
Example 5:
evaluation of Fe3O4Ability of CS/KGN to induce chondrogenic differentiation of adipose mesenchymal stem cells:
in order to verify the capacity of the nano-particle to induce the differentiation of mesenchymal stem cells in a chondrogenic direction in vitro. Adipose-derived mesenchymal stem cells were seeded in 6-well plates (corning) (7X 10 per well)4cells/mL), incubated overnight. Then the cell culture medium was removed and 10. mu.M Fe was added3O4Chondrogenic induction solution of CS/KGN, with replacement every 3 days, for 2 weeks of continuous culture. 4% or more after useAfter fixing the cells at room temperature for 10min with paraformaldehyde, they were washed 3 times with PBS and blocked with 5% BSA at 37 ℃ for 2 h. Cell determination Novus type II collagen (col II) primary antibody diluted at a ratio of 1:400 was added to each well at 300. mu.L, incubated overnight at 4 ℃, primary antibody was removed, PBS was washed twice, Cy 3-labeled secondary antibody was diluted at a ratio of 1:400, 300. mu.L was added to each well, incubated at room temperature for 1 hour, and nuclei were counterstained with 1. mu.g/mL DAPI.
The results show that: after 2 weeks of induction, immunofluorescence staining shows that red fluorescence is attached to the surface of the original adipose mesenchymal stem cells, which indicates that type II collagen is generated and has the characteristics of chondrocytes.
Example 6:
MR imaging of nanoprobes at cartilage damage in rabbit knee joints was evaluated.
The right leg of a New Zealand white rabbit weighing 2.5-2.8kg was debrided of superficial hair, the knee joint was fixed and sterilized with iodophor three times. The post-sterilization surgery was used to scratch open the articular surface of the rabbits and carefully expose the femoral and tibial plateaus. Subsequently, a volume of 4X 3mm was taken at the femoral trochlear using a trephine3Followed by debridement of the wound. Mixing 10 μ M Fe3O4After the CS/KGN and the adipose-derived mesenchymal stem cells are incubated together for one week, the cells are gently scraped by a cell scraper and filled to the cartilage injury part, the patella is sutured after reduction, the experiment is carried out for 4 and 12 weeks for repair observation, and the repair condition is observed by using a T2 weighting mode for MR imaging.
The results show that: at 4 weeks, the magnetic beads can be effectively enriched in the damaged area, and the outline of the wound can be clearly seen; until 12 weeks, the nuclear magnetic signals of the magnetic beads disappear, which shows that the magnetic beads can be digested and metabolized in vivo, and have good safety and diagnostic and therapeutic significance.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A preparation method of a ferroferric oxide magnetic nano probe based on surface modification is characterized by comprising the following steps:
(1) uniformly dispersing ferroferric oxide nano particles which are dissolved in dichloromethane and have oleic acid grafted on the surface in a DMF (dimethyl formamide) solvent, adding EDC (EDC) and Sulfo-NHS (sulfolane-N-hydroxysuccinimide) activated ferroferric oxide nano particles, reacting at room temperature, removing the redundant DMF solvent, and adding an acetic acid solution for full resuspension;
(2) adding chitosan fully dissolved in the acetic acid solution into the resuspended solution obtained in the step (1), and stirring at room temperature for reaction;
(3) dialyzing and purifying the reaction solution obtained in the step (2);
(4) and (3) dropwise adding a KGN solvent into the purified ferroferric oxide-chitosan solution obtained in the step (3) in DMSO, fully shaking, and dialyzing and purifying in pure water to obtain the target product.
2. The preparation method of the ferroferric oxide magnetic nanoprobe based on the surface modification according to claim 1, characterized in that in the step (1), the volume ratio of dichloromethane to DMF is 1: 5.
3. The preparation method of the ferroferric oxide magnetic nanoprobe based on the surface modification of claim 1, wherein in the step (1), the mass ratio of the ferroferric oxide nanoparticles with the oleic acid grafted on the surface to EDC to Sulfo-NHS is 1:3:3-1:1: 1.
4. The preparation method of the ferroferric oxide magnetic nanoprobe based on the surface modification as claimed in claim 1, wherein in the step (1), the reaction time is 24 hours at room temperature.
5. The preparation method of the ferroferric oxide magnetic nanoprobe based on the surface modification according to claim 1, characterized in that in the step (1) and the step (2), the acetic acid solution is 0.1 wt% acetic acid solution.
6. The preparation method of the ferroferric oxide magnetic nanoprobe based on the surface modification as claimed in claim 1, wherein in the step (2), the concentration of chitosan in the acetic acid solution is 1-5mg/L, and the mass ratio of chitosan to ferroferric oxide nanoparticles with oleic acid grafted on the surface is 2-10: 1.
7. The preparation method of the ferroferric oxide magnetic nanoprobe based on the surface modification as claimed in claim 1, wherein in the step (3) and the step (4), dialysis purification is carried out in pure water, the purification time is 48-72h, and the pure water is replaced every 6 h.
8. The preparation method of the ferroferric oxide magnetic nanoprobe based on the surface modification according to claim 1, wherein in the step (4), the mass ratio of KGN to chitosan is 1: 5-5: 2;
the oscillation time is 24h, and the rotation speed during oscillation is 100-200 rpm.
9. A ferroferric oxide magnetic nanoprobe based on surface modification, which is prepared according to the preparation method of any one of claims 1-8.
10. The application of the ferroferric oxide magnetic nanoprobe based on surface modification of claim 9 in preparation of drugs for cartilage imaging and soft tube defect treatment.
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