TWI508746B - Preparation of superparamagnetic iron oxide nanoclusters - Google Patents

Description

Method for preparing superparamagnetic iron oxide nano cluster

The invention relates to a method for preparing a Magnetic Resonance Imaging (MRI) contrast agent, in particular to a preparation of a water-soluble superparamagnetic iron Oxide (SPIO) nanocluster. method.

Magnetic resonance imaging is a very important diagnostic technique in clinical medicine and has been regarded as a non-invasive and excellent imaging contrast in soft tissue, with high spatial resolution and the ability to have faults. In recent years, scientists have made many efforts to improve their resolution and contrast ratio, and the targeted delivery of contrast agents to the target site is a very important method in clinical medical testing, which can improve the contrast effect and reduce the negative impact. And toxicity. The magnetic resonance imaging contrast agent to be used, which is often used as a superparamagnetic nanoparticle, has been proved to be a non-invasive cell marker in clinical experiments and has excellent performance in tumor detection (Non-Patent Document 1).

Nanoparticles attract the interest of scientists in the field of biomedical research, functionalizing the properties of magnetic nanoparticles, and simultaneously responding to a magnetic field, making it a useful tool, as well as a combination of diagnostic and therapeutic techniques (Non-Patent Document 2) ). In nanoparticle, surface chemistry is a key factor that changes the physical and chemical properties of magnetic nanoparticles, including their size, solubility, dispersion state and magnetization value. Surface chemistry greatly affects the magnetic nanoparticles in biological systems. Properties, including mechanisms of cell recognition, biodistribution, and immune responses, to reduce potential nanotoxicity (Non-Patent Document 3).

Magnetic iron oxide nanoparticles have attracted much attention in the field of biomedical research due to their special magnetic properties, such as magnetic resonance imaging, magnetic hyperthermia, biomagnetic separation, magnetic targeting drug carriers. The particle size, size distribution, monodispersity and magnetic responsiveness of magnetic iron oxide nanoparticles are critical for their biomedical applications. Since small-sized magnetic nanoparticles have a size comparable to that of viruses (20-450 nm), proteins (5-50 nm), DNA or genes (2 nm wide and 10-100 nm long), they are suitable for combination with these biological units. Magnetic marker. The monodispersity and narrow particle size distribution of magnetic nanoparticles can provide a targeted drug carrier with almost the same physical, chemical and biological properties for the study of drug distribution in vivo and pharmacokinetics. The high saturation magnetization is advantageous for improving the operability of the magnetron and reducing the magnetic loss caused by the surface modification of the magnetic nanoparticles, and the superparamagnetic nano particles can completely eliminate the magnetic agglomeration.

Due to its high tissue specificity and high safety, superparamagnetic iron oxide has been used as a magnetic resonance contrast agent in foreign countries for early diagnosis of certain cancers, and even in vivo monitoring at the molecular and cellular levels. At present, research on superparamagnetic nanoparticles mainly focuses on ferroferric oxide (Fe ₃ O ₄ ) and ferric oxide (γ-Fe ₂ O ₃ ). However, the preparation of small size, monodisperse, narrow particle size distribution, high saturation magnetization and superparamagnetic magnetic iron oxide nanoparticles has been a bottleneck restricting its biomedical applications.

At present, the preparation method of the magnetic nanoparticle includes a physical method and a chemical method, and the magnetic nanoparticle prepared by a physical method such as an evaporation condensation method, a grinding and pulverization method, or a mechanical alloy method generally has a large particle size and a wide distribution. Chemical methods such as the commonly used coprecipitation method of magnetic nanoparticles have a wide particle size distribution, the size and stability of which are strongly dependent on the pH value of the system; the particle size comparison of magnetic nanoparticles prepared by sol-gel method It is uniform, but has poor crystallinity and weak magnetic responsiveness. Although the hydrothermal synthesis method has good crystallinity and magnetic responsiveness, it requires high temperature and high pressure and it is difficult to obtain magnetic nanoparticles having a small particle size and monodispersion. Therefore, in order to solve the above problems, scientists have developed a method of organic phase synthesis to develop a metal-organic precursor thermal decomposition synthesis method of magnetic nanoparticles. For example, scientists have stabilized oleic acid and oleylamine. a 1,2-hexadecanediol (1,2-hexadecanediol) as a reducing agent, which is synthesized by thermally decomposing iron acetonitrile iron (Fe(acac) ₃ ) in an organic phase and by a seed crystal growth method. Monodisperse Fe ₃ O ₄ magnetic nanoparticles having an adjustable particle size in the range of 5 to 20 nm (Non-Patent Document 4).

The magnetic nanoparticle obtained by the above method has only a small molecule with a long alkyl chain modified on its surface, and the modification allows the obtained magnetic nanoparticle to be dissolved or dispersed only in a non-polar or weakly polar organic medium. It is difficult to apply to the field of biomedicine. Although it is possible to pass through a complicated surface modification procedure, the magnetic nanoparticles having a hydrophobic structure on the surface have water solubility, but the modification procedure is complicated, which is not suitable for practical use. For example, Taiwan Patent Publication No. 200724904 discloses a multifunctional magnetic nanoparticle, and the magnetic nanoparticle is modified by a biocompatible polymer, and the biocompatible polymer coupling specific identification molecule and the fluorescent dye are disclosed. A biocompatible macromolecule for modifying magnetic nanoparticles is disclosed in US Patent Publication No. US 2006/0216239. In addition, currently available products such as Feridex (modified with dextran) or Resovist (Modified with carboxydextran), the superparamagnetic iron oxide nanoparticles were also modified with a biocompatible polymer.

Therefore, how to invent a method for preparing water-soluble superparamagnetic iron oxide nano-clusters so as to effectively reduce the preparation time and simplify the preparation procedure will be actively disclosed in the present invention.

Non-Patent Document 1: Nasongkla N. et al., Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems, Nano Letters, 6, 2008, 2427-2430.

Non-Patent Document 2: Ozdemir V. et al., Shifting emphasis from pharmacogenomics to theragnostics, Nature Biotechnology, 24, 2006, 942-946.

Non-Patent Document 3: Gupta A.K. et al., Recent advances on surface engineering of magnetic iron oxide nanoparticles and their biomedical applications, Nanomedicine, 2, 2007, 23-39.

Non-Patent Document 4: Sun S. et al., Size-controlled synthesis of magnetite nanoparticles, Journal of the American Chemical Society, 124, 2002, 8204-8205.

In view of the shortcomings of the above-mentioned prior art, the inventors felt that they had not perfected their efforts, exhausted their mental research and overcoming, and based on their accumulated experience in the industry for many years, developed a preparation of water-soluble superparamagnetic iron oxide. The method of SPIO nanocluster, in order to reduce the preparation time and simplify the preparation process.

SUMMARY OF THE INVENTION A primary object of the present invention is to provide a process for preparing a water-soluble superparamagnetic iron oxide nanocluster which is effective in reducing the preparation time and simplifying the preparation procedure, and increasing the monodispersity and stability in an aqueous solution.

In order to achieve the above object, a method for preparing a water-soluble superparamagnetic iron oxide nano-clustered cluster of the present invention comprises the steps of:

a) dissolving the superparamagnetic iron oxide nanoparticles and the biocompatible polymer in an organic solvent to form an organic phase solution;

b) adding the organic phase solution to the aqueous phase solution and emulsifying using ultrasonic dispersion;

c) removing the organic solvent.

The above method, wherein the biocompatible polymer is polyethylene glycol (PEG), polylactic acid-polyethylene glycol (PLA-PEG), polylactide (PLA), polyglycolide (PGA), poly Caprolactone (PCL) or polymethyl methacrylate (PMMA).

In the above method, the biocompatible polymer can be modified by a functional group to couple the specificity of the molecule to achieve the function of the water-soluble superparamagnetic iron oxide cluster.

The above method, wherein the specificity identifies a molecular protein or a peptide.

The above method, wherein the protein is a CD133 monoclonal antibody.

The above method, wherein the functional group is maleimide.

The above method wherein the coupling is carried out using Traut's reagent.

The above method, wherein the biocompatible polymer is a polyethylene glycol having a molecular weight of from 200 to 5,000, preferably a polyethylene glycol having a molecular weight of from 200 to 600.

In the above method, the superparamagnetic iron oxide nanoparticles are prepared by a thermal decomposition synthesis method and have a particle diameter of 5 to 20 nm.

The above method, wherein the aqueous phase solution is water, PBS buffer, cell culture solution or biocompatible buffer solution.

The above method, wherein the organic solvent is n-hexane, cyclohexane, ethyl acetate, tetrahydrofuran or chloroform, preferably n-hexane.

The above method, wherein the organic phase solution further comprises an oil phase surfactant which is selected from long chain oil phase surfactants of 15 to 25 carbons, such as oleic acid.

In the above method, the particle size of the superparamagnetic iron oxide nano-clusters is controlled by adjusting the amount of the oil phase surfactant added. The above method, wherein the superparamagnetic iron oxide nanoclusters have a particle diameter of about 5 to 400 nm, preferably about 20 to 200 nm.

In the above method, the step c) is heated at 70 to 120 ° C for 15 to 60 minutes.

In the above method, the magnet adsorption sedimentation method is further used to remove the uncoated biocompatible polymer and most of the aqueous phase solution.

Thereby, the method for preparing a water-soluble superparamagnetic iron oxide nano-clustered cluster of the present invention can effectively reduce the preparation time and simplify the preparation procedure, and increase the monodispersity and stability in an aqueous solution.

In order to fully understand the objects, features and advantages of the present invention, the present invention will be described in detail by the following specific embodiments and the accompanying drawings.

Example 1: Preparation of superparamagnetic iron oxide nanoparticles

Ethylacetone iron (Fe(acac) ₃ ) and 1,2-hexadecanediol (1,2-hexadecanediol) were mixed according to a mole ratio of 1:10, using phenyl ether 20 mL, and then added to the reaction. The bottle was heated to about 60 ° C. After the mixture was completely dissolved in phenyl ether, 1.4 mL of each of oleic acid and oleylamine was added as an oil phase surfactant. Nitrogen gas was introduced and the temperature was slowly raised to 250 ° C. The reaction was continued for 3 hours. Then, excess ethanol can be added and centrifuged at 6000 rpm for 15 min to obtain monodisperse superparamagnetic iron oxide nanoparticles with a diameter of about 6 nm.

Example 2: Preparation of superparamagnetic iron oxide nanoclusters

The biocompatible polymer used in the examples is polyethylene glycol (PEG) having molecular weights of 200, 400 and 600 respectively. Because of the surface activity of PEG, it is often used as a solubilizing agent, an emulsifier and a fat-soluble drug carrier. The pharmaceutical preparation belongs to a pharmaceutical grade surfactant, so that the superparamagnetic iron oxide nano particles can be converted into water-soluble and stably dispersed in a biological buffer solution, and further applied to the field of biomedicine.

Superparamagnetic iron oxide is encapsulated in PEG using emulsification and solvent evaporation. The advantage of emulsification is that two mutually incompatible systems can be ultrasonically oscillated to form small oil droplets stably in aqueous solution, and then oil phase The solvent volatilizes so that small oil droplets originally dispersed in the aqueous solution form water-soluble nano-cluster particles.

The superparamagnetic iron oxide (SPIO) nanoparticles prepared in Example 1 were mixed in n-hexane (0.5 mL) at room temperature, and oleic acid (5 μL) was optionally added as the amount shown in Table 1. Oil phase surfactant. Then PEG (0.245 mmol) was dissolved in deionized water (10 ml), then the oil phase solution was added to the aqueous phase containing PEG, and emulsified by ultrasonic vibration for 10 minutes at room temperature to form an emulsified state. Or even nano-sized chylomicrons (small oil droplets). The n-hexane in the mixed solution was evaporated under heating at 95 ° C for 15 minutes to ensure complete evaporation of the solvent. After the preparation of the product is completed, it is cooled to room temperature, and finally the magnetic nano-clusters are adsorbed and sedimented by a magnet, and excess water is removed to concentrate the product, and the concentrated product can be dispersed and dissolved in water or a biological buffer solution (PBS). The advantage of removing the aqueous phase by the magnet adsorption sedimentation method is that the properties of the prepared nanoclusters and the particle size distribution are less likely to change due to the concentration process.

The monodisperse superparamagnetic iron oxide nanoclusters prepared in Example 1 had a particle size of about 70 to 140 nm.

Example 3: Controlling the particle size of superparamagnetic iron oxide nanoclusters using oleic acid

According to the preparation method of Example 2, different amounts of oleic acid (5 to 30 μL) were added as shown in Table 2, and the particle size of the superparamagnetic iron oxide nano-clusters was effectively controlled.

Example 4: Preparation of superparamagnetic iron oxide nanoclusters with target function

0.016 g of superparamagnetic iron oxide (SPIO) nanoparticles prepared in Example 1 was dissolved in n-hexane (0.5 mL), and PEG (0.245 mmol) was dissolved in deionized water (10 ml). The SPIO-containing oil phase solution was then added to the PEG-containing aqueous phase and vortexed for 10 minutes at room temperature to form an emulsified state. Separately, PEG (3.675 x 10-3 mmol) having a maleimide modification and having a molecular weight of 3,500 was added to the aqueous phase in an emulsified state, and subjected to ultrasonic vibration for 10 minutes at room temperature. The n-hexane in the mixed solution was evaporated under heating at 95 ° C for 15 minutes to ensure complete evaporation of the solvent. After the preparation of the product is completed, it is cooled to room temperature. Finally, the magnetic nano-clusters are adsorbed and sedimented by a magnet, and excess water is removed to concentrate the product to form a superparamagnetic iron oxide nano-clustered cluster with functional group modification.

Next, 0.00588 g of Traut's reagent was added to 1 mL of an EDTA solution (2 mM) having a pH of 8, and diluted to 100,000 times. Subsequently, 50 μL of CD133 monoclonal antibody was added and reacted in the dark at room temperature for 1 hour. The above-mentioned functional group-modified superparamagnetic iron oxide nano-clusters are added, and the reaction is protected from light at 24 ° C for 24 hours to complete the superparamagnetic iron oxide nano-clusters with target function, wherein the target is targeted. The coupling ratio of the functional CD133 monoclonal antibody was about 89.4%.

As described above, the present invention fully complies with the three requirements of the patent: novelty, advancement, and industrial applicability. In terms of novelty and advancement, the invention adopts ultrasonic emulsification method and organic solvent evaporation method to prepare superparamagnetic iron oxide nano-clusters, which can effectively reduce preparation time and simplify the preparation process, and is added to the aqueous solution. The efficacy of monodispersity and stability in terms of industrial availability; the products derived from the present invention can fully satisfy the needs of the current market.

The invention has been described above in terms of the preferred embodiments, and it should be understood by those skilled in the art that the present invention is not intended to limit the scope of the invention. It should be noted that variations and permutations equivalent to those of the embodiments are intended to be included within the scope of the present invention. Therefore, the scope of the invention is defined by the scope of the following claims.

Claims (14)

A method for preparing a water-soluble superparamagnetic iron oxide nano cluster (SPIO nanocluster), the steps comprising: a) dissolving superparamagnetic iron oxide nanoparticles and a biocompatible polymer in an organic solvent to form an organic phase a solution; b) adding the organic phase solution to the aqueous phase solution, and emulsifying using ultrasonic dispersion; c) removing the organic solvent; and wherein the biocompatible polymer is a polyethylene glycol having a molecular weight of 200 to 5000 (PEG), wherein the biocompatible polymer is a polyethylene glycol containing a maleimide modified coupling specific identification molecule, the aqueous phase solution being water, PBS buffer, cell culture solution or organism A compatibility buffer solution of n-hexane, cyclohexane, ethyl acetate, tetrahydrofuran or chloroform. The method according to claim 1, wherein the biocompatible polymer is a maleimide-modified coupling-specific molecular polyethylene glycol having a molecular weight of 200 to 3,500. The method of claim 1, wherein the specificity identifies a molecular protein or a peptide. The method of claim 3, wherein the protein is a CD133 monoclonal antibody. For example, the method described in claim 1 is to use Traut's. The reagent is coupled. The method of claim 1, wherein the biocompatible polymer is a polyethylene glycol having a molecular weight of from 200 to 600. The method of claim 1, wherein the superparamagnetic iron oxide nanoparticles are prepared by a thermal decomposition synthesis method and have a particle diameter of 5 to 20 nm. The method of claim 1, wherein the organic solvent is n-hexane. The method of claim 1, wherein the organic phase solution further comprises an oil phase surfactant of 15 to 25 carbons. The method of claim 9, wherein the particle size of the superparamagnetic iron oxide nanoclusters is controlled by adjusting the amount of the oil phase surfactant added. The method of claim 1, wherein the superparamagnetic iron oxide nanoclusters have a particle size of about 5 to 400 nm. The method of claim 11, wherein the superparamagnetic iron oxide nanoclusters have a particle size of about 20 to 200 nm. The method of claim 1, wherein the step c) is heated at 70 to 120 ° C for 15 to 60 minutes. The method of claim 1, further using a magnet adsorption sedimentation method to remove the aqueous phase solution.