CN112791194A - Preparation method of PEG/PEI modified magnetic nanoparticles - Google Patents

Preparation method of PEG/PEI modified magnetic nanoparticles Download PDF

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CN112791194A
CN112791194A CN202110024603.5A CN202110024603A CN112791194A CN 112791194 A CN112791194 A CN 112791194A CN 202110024603 A CN202110024603 A CN 202110024603A CN 112791194 A CN112791194 A CN 112791194A
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pei
peg
magnetic
magnetic nanoparticle
drug
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刘艳
李翔
陆云姝
顾芬芬
李若男
达先鸿
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XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
University of Shanghai for Science and Technology
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University of Shanghai for Science and Technology
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Abstract

The invention provides a preparation method of magnetic nanoparticles, which is characterized by comprising the following steps of S1, adding alcohol into vortex iron particles, and then dispersing the vortex iron particles by ultrasonic waves; s2, removing alcohol through magnetic adsorption separation; s3, adding PEG and/or PEI and water, and performing ultrasonic emulsification; and S4, after the supernatant is discarded, washing with water and alcohol respectively, collecting the precipitate, and drying to obtain the PEG and/or PEI modified magnetic nanoparticles. The nano carrier has low toxicity and good effect.

Description

Preparation method of PEG/PEI modified magnetic nanoparticles
Technical Field
The invention relates to the field of biomedicine, in particular to a nano carrier, and more particularly relates to a preparation method and application of PEG/PEI modified magnetic nanoparticles.
Background
Magnetic nanoparticles, due to the nano-size effect, exhibit distinct physicochemical properties from macroscopic magnetic materials, such as: the magnetic material has the characteristics of large specific surface area, super-paramagnetic property, diversified topological magnetic structure and the like, and the surface of the magnetic material is easy to functionalize, and the like, so that the magnetic material can be gathered and positioned in a constant magnetic field, and can be widely applied to biomedical fields such as targeted drug carriers, magnetic control drug release, tumor thermotherapy by absorbing electromagnetic waves and generating heat in an alternating magnetic field and the like.
In the prior art, a liver cancer specific promoter and a hypoxic reaction sequence are utilized to guide the expression of downstream cancer suppressor genes to construct liver cancer specific therapeutic genes, and magnetic nanoparticles PEI-Fe3O4 are used as gene carriers to transport the therapeutic genes, so that tumor targeted gene therapy and magnetofluid thermotherapy are combined, and the safety, specificity and effectiveness of therapy are greatly improved.
In addition, Yang et al have published that the superparamagnetic nanoparticles are successfully applied to tumor targeted therapy by coating a layer of organic matter such as biocompatible protein bodies on the surface of the superparamagnetic nanoparticles as a drug carrier, and the results show the feasibility of the superparamagnetic nanoparticles in clinical application.
Research shows that the magnetic nano Fe3O4The particles have the characteristics of excellent biocompatibility, no toxicity, strong targeting property and the like, so the particles can be used as a slow-release targeting drug carrier, and the targeting drug can be directly positioned and acted on a diseased region through the guiding action of an external magnetic field, thereby achieving the purposes of reducing the dosage of a medicament, reducing the toxic and side effects of the drug and improving the therapeutic index of the drug.
Vivek et al constructed breast cancer HER2 targeted nanoparticles wrapping anticancer drugs by using superparamagnetic nano-iron, and the results showed that the nanoparticles have good breast cancer targeting property and breast cancer cell proliferation resistance.
However, such current vector systems have the following disadvantages:
1. the active targeting of the carrier system is insufficient, and the movement of the magnetic nanoparticles in blood is influenced by the blood flow velocity and the magnetic field strength, which means that a magnetic field strength large enough to resist the linear flow velocity of blood in the tissue needs to be established to enable the drug carrier to reach and maintain the treatment area.
2. Superparamagnetic particles are too small in size, are easily phagocytosed by endoplasmic reticulum, and are not easily positioned and retained in tumor tissues.
3. Superparamagnetic particles tend to agglomerate.
Therefore, a novel low-toxicity and high-efficiency biomedical magnetic nanoparticle is urgently needed to be found to make up for the defects of superparamagnetic particles, so that better biomedical application performance is realized.
Disclosure of Invention
In order to solve the problems, the invention discovers a unique magnetic structure with magnetization closed distribution, namely a vortex magnetic domain, by modulating the size and the geometric structure of the nano particles. The vortex magnetic nanoparticles with the magnetic vortex structure reduce stray fields due to closed distribution of magnetic moments, and can effectively weaken magnetic interaction among particles, thereby avoiding the occurrence of particle agglomeration. Meanwhile, the eddy magnetic nanoparticles exhibit higher magnetic susceptibility and saturation magnetization than the superparamagnetic particles due to the larger particle size. In addition, the larger particle size of the vortex magnetic nanoparticles can wrap more biomolecules (drug molecules, fluorescent agents and the like) outside the particles to form medical nanocluster particles, and the particles can have high selective permeability and retentivity at tumor positions, so that the enrichment in tumor tissues and the improvement of tumor treatment effects are facilitated.
The invention provides a preparation method of magnetic nanoparticles, which is characterized by comprising the following steps:
s1, adding alcohol into the vortex iron particles, and then dispersing the vortex iron particles by ultrasonic waves;
s2, removing alcohol through magnetic adsorption separation;
s3, adding PEG and/or PEI and water, and performing ultrasonic emulsification;
and S4, after the supernatant is discarded, washing with water and alcohol respectively, collecting the precipitate, and drying to obtain the PEG and/or PEI modified magnetic nanoparticles.
Further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
the blending ratio of the vortex iron particles to the alcohol in the S1 is as follows: 0.1-1ml alcohol per 1mg of the vortex iron particles was added.
Further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
the preparation method of the vortex iron particles comprises the following steps:
further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
repeating S1-S2 at least twice.
Further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
in S1, the ultrasonic treatment time is 10-30 min.
Further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
in S3, the amount of PEG was 10mg or more per 1mg of the vortex iron particles:
the dosage of the PEI is more than 10ul of PEI per 1mg of vortex iron particles;
the amount of water used was: adding 0.1-1ml of water to 1mg of the vortex iron particles;
the PEI is a PEI aqueous solution, and the mass percentage concentration of the PEI is 40-90%.
Further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
the PEG is selected from one or more PEG with the molecular weight of more than 1000;
the PEI is one or more selected from PEI with molecular weight more than 800.
Further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
in S3, the number of sonications is two or more.
Further, the preparation method of the magnetic nanoparticle provided by the invention is characterized by comprising the following steps:
in S3, the time of each ultrasonic treatment is 1-5 min.
In addition, the research of the invention shows that the magnetic nano-particles prepared by the method are also characterized by having any one or more of the following applications:
as a pharmaceutical carrier;
as a drug carrier with slow release effect;
as a drug carrier with targeting effect.
In addition, the invention also provides a drug-loaded magnetic nanoparticle, which is characterized in that:
coupling the drug I/factor I with the magnetic nanoparticle prepared by the method through PEG;
and/or
Drug II/factor II was electrostatically adsorbed onto one of the magnetic nanoparticles prepared as described above by PEI.
The above drugs and factors can be any drug or silver seed with targeting or therapeutic effects.
The preparation method of the drug-loaded carrier comprises the following steps: adding a specified amount of drugs/factors into the dried PEG and/or PEI modified magnetic nanoparticles, and performing ultrasonic emulsification.
The mass ratio of the nanoparticles to the drug/factor is 5: 0.1-10.
In addition, the invention also provides a drug-loaded magnetic nanoparticle which is characterized by being prepared by the following preparation method:
s1, coupling a drug I/factor I with the magnetic nanoparticle prepared by the method through PEG;
and/or
Electrostatically adsorbing the medicine II/the factor II on the magnetic nano-particles prepared by the method through PEI;
s2, wrapping the cell membrane for simulation on the composite nano material of S1 by using ultrasound.
The preparation method of the drug-loaded thin film carrier comprises the following steps: adding a specified amount of drugs/factors into the dried PEG and/or PEI modified magnetic nanoparticles, carrying out ultrasonic emulsification, adding a cell membrane solution, carrying out ultrasonic treatment, centrifuging, washing, and carrying out resuspension by using deionized water to obtain the DOX-carrying PEG and/or PEI modified magnetic nanoparticles wrapped by the cell membrane.
The mass ratio of the nanoparticles to the drug/factor is 5: 0.1-10.
The addition amount of the cell membrane solution is 1-20ul per mg of the nanoparticle.
Drawings
FIG. 1 is a TEM morphology of magnetic nanoparticles;
wherein, FIG. 1a is a TEM morphology of magnetic nanorings;
FIG. 1b is a magnetic nanoring TEM morphogram;
FIG. 1c shows the results of magnetic property measurements;
FIG. 2 is an infrared spectrum of a nanoring modified with different amounts of PEG;
FIG. 3 is an infrared spectrum of the nanorods modified with PEG at different dosages;
FIG. 4 is a graph comparing stability of PEG-modified nanorings;
wherein, FIG. 4a is an initial state diagram;
FIG. 4b is a state diagram after 20 min;
FIG. 4c is a state diagram after 40 min;
FIG. 5a is an infrared spectrum of the nanorod modified with different amounts of PEG after DOX loading;
FIG. 5b ultraviolet map of PEG-Fe3O4 carrying DOX;
FIG. 6 is a nanoring infrared spectrum modified with different amounts of PEI;
FIG. 7 is a potential diagram of PEI modified nanorods;
wherein, FIG. 7a is a bare rod potential diagram;
FIG. 7b is a potential map of 500ul PEI;
FIG. 7c is a potential diagram of 1000ul PEI.
FIG. 8 is an infrared spectrum of PEG and PEI coated rod-shaped Fe3O 4;
FIG. 9 is a potential diagram of PEG and PEI coated rod-shaped Fe3O 4;
FIG. 10 PEG/PEI-Fe3O4 carries DOX drug loading;
FIG. 11 PEG/PEI-Fe3O4 carries DOX encapsulation efficiency;
FIG. 12 shows that the annular and rod-shaped PEG/PEI-Fe3O4 coated with the film carries 2mgDOX encapsulation efficiency and drug loading efficiency;
FIG. 13 shows that the annular and rod-shaped PEG/PEI-Fe3O4 coated with the film carries a drug release rate of 2 mgDOX.
FIG. 14 is a drug release rate line graph of 2mgDOX carried by annular, rod-shaped PEG/PEI-Fe3O4 after coating.
Detailed Description
Example 1. preparation of vortex iron particles:
example 1-1 preparation of annular vortex iron particles:
hydrothermal synthesis of alpha-Fe2O3Nanorings, the reactant being FeCl3·6H2O, the surfactant introduced being NH4H2PO4And Na2SO4. 0.43g of FeCl was weighed3·6H2O, 0.00092g NH4H2PO4And 0.00625g of Na2SO4Dissolving in distilled water, placing the mixture on a magnetic stirrer, stirring vigorously until the mixture is uniform, transferring to stainless steel under high pressureAnd (3) putting the mixture into a 220 ℃ oven, reacting for 48 hours at constant temperature, cooling to room temperature, washing and centrifuging to obtain the required precipitate. Finally drying to obtain the required alpha-Fe2O3A nano-ring particle. Then the prepared alpha-Fe2O3As a raw material, Fe is obtained by a hydrogen reduction method3O4Nano-ring to obtain final product of ring-shaped Fe3O4And (3) nanoparticles. The TEM is shown in FIG. 1 a.
Examples 1-2 preparation of rod-shaped vortex iron particles:
weigh 0.432g of FeCl3·6H2O and 0.0046gNH4H2PO4Adding a small amount of distilled water for dissolving, stirring vigorously, transferring the mixture into a stainless steel autoclave when the solute is completely dissolved, performing hydrothermal treatment at 205 deg.C for 48 hr, cooling to room temperature, washing, and drying at 60 deg.C for 24 hr to obtain desired rod-shaped alpha-Fe2O3And (3) nanoparticles. Reuse of the prepared alpha-Fe2O3Production of Fe by hydrogen reduction as a starting Material3O4And (4) nanorods. Drying the alpha-Fe2O3Annealing the powder in a continuous hydrogen/argon flow at 500 ℃, cooling to room temperature, and simultaneously maintaining the continuous hydrogen flow to obtain the final product rod-like Fe3O4And (3) nanoparticles. The TEM is shown in FIG. 1 b.
Examples 1-3 spherical vortex iron particles.
As shown in FIG. 1c, the magnetic performance test results of various forms of rings and bars show that the magnetic performance is relatively good
Example 2 preparation of PEG-modified magnetic nanoparticles:
20mg of the vortex iron particles prepared in the example 1 are weighed, 4ml of absolute ethyl alcohol is added, and the particles are dispersed in the absolute ethyl alcohol by ultrasonic treatment for 20 minutes (the ultrasonic treatment time can be adjusted and controlled within a range of 10-30min according to the requirements of different material quantities). Then the magnetic adsorption separation is carried out, the absolute ethyl alcohol is discarded, and the process is repeated twice.
Weighing different amounts of PEG4000 for experiment (in this embodiment, 200mg, 500mg, 1000mg, 2000mg are selected for preparation of the experimental example, PEG with molecular weight more than 1000 can be selected for PEG, preferably PEG2000 and PEG4000), adding 10ml of deionized water (the adjustable range of the water amount is 5-20ml according to the requirements of different material amounts), performing ultrasonic treatment for 20 minutes (the adjustable range of the ultrasonic treatment time is 10-30min according to the requirements of different material amounts), and performing ultrasonic emulsification for 3 minutes each time (the adjustable range of the ultrasonic treatment time is 1-5min according to the requirements of different material amounts). Centrifuging, removing supernatant, washing with deionized water for three times, and washing with ethanol for three times. Collecting the precipitate and drying to obtain the PEG modified magnetic nano-particles.
Experimental example 2-1. Infrared analysis results:
as shown in fig. 2, compared to bare iron. PEG-modified iron at 580cm-1The strong characteristic absorption peak is shown nearby and is 1104cm-1A relatively strong C-O-C absorption peak appears at 1623cm-1The C-O bending vibration peak is also nearby and is 2884cm-The C-H vibration peak appears at 3385cm-1Stretching vibration peaks of hydroxyl O-H are also shown nearby, and the appearance of the obvious characteristic absorption peaks of the groups indicates that PEG is successfully modified in the particles.
As shown in FIG. 3, also, when the nanorod is coated with PEG, the characteristic peak of iron is reduced, and there is an absorption peak of the group in PEG, indicating that PEG successfully modifies the iron surface.
Experimental examples 2-2. stability experiment:
as shown in FIGS. 4a, 4b and 4c, when the amount of PEG added is 200mg, a significant solid-liquid separation phenomenon appears after settling for 20 min;
when the addition amount of PEG is 500mg, the obvious solid-liquid separation phenomenon appears after settling for 40 min;
obviously, when the addition amount of PEG is 2000mg, the stability is obviously better than that of other dosage.
EXAMPLES 2-3 toxicity test(using CCK8 kit):
A. the results of the nanorod toxicity to 231 cells for 24h are shown in the following table (PEG dosage 2000 mg):
Figure RE-GDA0003003444050000071
B. the toxicity results of the nanorings on 231 cells for 24h are shown in the following table:
Figure RE-GDA0003003444050000072
from the above data, it can be seen that the nanomaterials, whether cyclic or rod-shaped, are substantially non-toxic.
Example 3. preparation of DOX-loaded particles with the magnetic nanoparticles of example 2 (PEG in 2000mg) as carrier:
and weighing 5mg of the dried PEG modified magnetic nanoparticles, adding 2mgDOX, and performing ultrasonic emulsification.
Experimental example 3-1 Infrared analysis results:
as shown in fig. 5a, it can be seen from the figure that the peak fluctuation is strong and the characteristic peak of iron is reduced, indicating that DOX is present on the iron surface.
Experimental example 3-2. UV analysis results (see FIG. 5 b).
Example 4 preparation of PEI modified magnetic nanoparticles:
20mg of the prepared iron particles were weighed, 4ml of absolute ethanol was added, and dispersed in the absolute ethanol by sonication for 20 minutes. Then the magnetic adsorption separation is carried out, the absolute ethyl alcohol is discarded, and the process is repeated twice.
Different amounts of PEI (molecular weight 1300, 50% aqueous solution density of 0.54g/ml concentration of 1.08 g/ml) were weighed out for experiments (in this example, 200ul, 500ul, 1000ul were selected for the preparation of the experimental examples, PEI with a molecular weight above 1000 is preferred), 10ml of deionized water was added, and the mixture was sonicated for 20 minutes and several further 3 minutes each. Centrifuging, removing supernatant, washing with deionized water for three times, and washing with ethanol for three times. Collecting the precipitate and drying to obtain the PEI modified magnetic nano-particles.
Experimental example 4-1. Infrared analysis results: (from bottom to top 200, 500, 1000)
As shown in FIG. 6, there is a distinct peak characteristic of PEI, indicating the successful modification of PEI on the iron surface.
Experimental example 4-2. potential results:
as shown in FIG. 7, the potential of 500ul PEI in the wand: 8.7mV
Potential of 1000ul PEI rod: 35.6mV
Therefore, the potential is better than that of a bare rod after PEI is loaded, and the potential can reach the best expected value obviously when the dosage of PEI is 1000 ul.
Example 5 preparation of PEG/PEI co-modified magnetic nanoparticles:
20mg of the prepared iron particles were weighed, 4ml of absolute ethanol was added, and dispersed in the absolute ethanol by sonication for 20 minutes. Then the magnetic adsorption separation is carried out, the absolute ethyl alcohol is discarded, and the process is repeated twice. Weighing a proper amount of 2000mgPEG4000 and 1000ul PEI (molecular weight 1300, 50% aqueous solution density is 1.08g/ml, concentration is 0.54g/m), adding 10ml of deionized water, carrying out ultrasonic treatment for 20 minutes, and carrying out ultrasonic emulsification for several times, wherein each time lasts for 3 minutes. Centrifuging, removing supernatant, washing with deionized water for three times, and washing with ethanol for three times. Collecting the precipitate and drying to obtain the PEG/PEI modified magnetic nano-particles.
Experimental example 5-1 Infrared analysis results (bar shape):
as shown in FIG. 8, PEG, PEI modified nanoparticles except at 1045cm compared to bare iron-1、1408cm-1The PEG characteristic absorption peak is shown nearby, and is 1623cm-1The peak appears in the N-H bending vibration absorption range, which is 2857cm-1、2922cm-1The C-H stretching vibration peak appears at 3445cm-1The N-H stretching vibration peak appears nearby, and the appearance of the obvious characteristic absorption peak of the groups indicates that PEG and PEI are successfully modified in the particles.
Experimental example 5-2. potential results (bars):
as shown in FIG. 9, PEI-PEG-Fe3O4The rod potentials were: 50.8 mV.
Example 6. preparation of DOX-loaded particles with the magnetic nanoparticles of example 5 as support:
after drying, 5mg of the PEG/PEI modified magnetic nanoparticles prepared in example 5 were weighed, and a specific amount of DOX (the amount of the drug added can be adjusted according to the target drug) was added, followed by ultrasonic emulsification.
Experimental example 6-1. drug Loading results:
as shown in FIG. 10, 5mg of PEG/PEI-Fe 3041, 2 and 3mg of DOX were added, respectively.
And (2) ring-shaped: the drug loading rate is 26.35%; the drug loading rate of the rod-shaped iron is as follows: 28.436 percent.
Experimental example 6-2. encapsulation efficiency results:
as shown in FIG. 11, the amount of the cyclic iron was about 56% at the time of the DOX addition of 1mg and 2mg, which was almost the same;
the amount of the iron rod-like DOX added is preferably about 62 mg.
Example 7. preparation of DOX-loaded particles with the coated magnetic nanoparticles of example 5 as the carrier:
the preparation method is shown in fig. 12, and the processes of coating and drug loading are as follows: and weighing 5mg of the dried PEG/PEI modified magnetic nanoparticles, adding 2mgDOX, and performing ultrasonic emulsification. 50ul of the cell membrane solution previously extracted was added and sonicated for three minutes. Then centrifuging, washing, and then resuspending with deionized water to obtain the DOX-carrying PEG/PEI modified magnetic nanoparticles wrapped by the cell membrane.
Experimental example 7-1 drug loading and encapsulation efficiency results:
as shown in fig. 12, the cyclic iron encapsulation rate is 70%, and the drug loading is 28%;
the rod-shaped iron encapsulation rate is 78 percent, and the drug loading rate is 31 percent;
compared with magnetic nanoparticles without cell membranes.
Experimental example 7-2. sustained Release results:
as shown in fig. 13, the sustained release rate of the ring was: 65.79 percent;
the sustained release rate of the stick was: 59.21 percent;
and then the slow release is carried out, which indicates that the sustained-release tablet can meet the requirements of drug release as a drug carrier.

Claims (10)

1. The preparation method of the magnetic nano-particles is characterized by comprising the following steps:
s1, adding alcohol into the vortex iron particles, and then dispersing the vortex iron particles by ultrasonic waves;
s2, removing alcohol through magnetic adsorption separation;
s3, adding PEG and/or PEI and water, and performing ultrasonic emulsification;
and S4, after the supernatant is discarded, washing with water and alcohol respectively, collecting the precipitate, and drying to obtain the PEG and/or PEI modified magnetic nanoparticles.
2. The method of claim 1, wherein the magnetic nanoparticle comprises:
the blending ratio of the vortex iron particles to the alcohol in the S1 is as follows: 0.1-1ml alcohol per 1mg of the vortex iron particles was added.
3. The method of claim 1, wherein the magnetic nanoparticle comprises:
repeating S1-S2 at least twice.
4. The method of claim 1, wherein the magnetic nanoparticle comprises:
in S1, the ultrasonic treatment time is 10-30 min.
5. The method of claim 1, wherein the magnetic nanoparticle comprises:
in S3, the amount of PEG was 10mg or more per 1mg of the vortex iron particles:
the dosage of the PEI is more than 10ul of PEI per 1mg of vortex iron particles;
the amount of water used was: adding 0.1-1ml of water to 1mg of the vortex iron particles;
the PEI is a PEI aqueous solution, and the mass percentage concentration of the PEI is 40-90%.
6. The method of claim 1, wherein the magnetic nanoparticle comprises:
in S3, the number of sonications is two or more.
7. The method of claim 1, wherein the magnetic nanoparticle comprises:
in S3, the time of each ultrasonic treatment is 1-5 min.
8. A magnetic nanoparticle produced by the method of any one of claims 1 to 7, having any one or any of the following uses:
A. as a pharmaceutical carrier;
B. as a drug carrier with slow release effect;
C. as a drug carrier with targeting effect.
9. A drug-loaded magnetic nanoparticle, characterized in that:
coupling drug I/factor I via PEG to a magnetic nanoparticle prepared according to any one of claims 1-7;
and/or
Electrostatically adsorbing drug II/factor II by PEI onto a magnetic nanoparticle prepared according to the method of any one of claims 1-7.
10. A drug-loaded magnetic nanoparticle is characterized by being prepared by the following preparation method:
s1, coupling drug I/factor I with a magnetic nanoparticle prepared by any one of the methods of claims 1-7 through PEG;
and/or
Electrostatically adsorbing drug II/factor II by PEI onto a magnetic nanoparticle prepared by the method of any one of claims 1-7;
s2, wrapping the cell membrane for simulation on the composite nano material of S1 by using ultrasound.
CN202110024603.5A 2021-01-08 2021-01-08 Preparation method of PEG/PEI modified magnetic nanoparticles Pending CN112791194A (en)

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