CN115120736A - Multifunctional gene vector and application thereof in miRNA delivery - Google Patents
Multifunctional gene vector and application thereof in miRNA delivery Download PDFInfo
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- CN115120736A CN115120736A CN202210751176.5A CN202210751176A CN115120736A CN 115120736 A CN115120736 A CN 115120736A CN 202210751176 A CN202210751176 A CN 202210751176A CN 115120736 A CN115120736 A CN 115120736A
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Abstract
A multifunctional gene vector and application thereof in miRNA delivery. The invention belongs to the technical field of biomedicine, and particularly relates to a multifunctional gene vector of a magnetic nanoparticle modified by Polyethyleneimine (PEI) coated with fucoidin and application of the multifunctional gene vector in miRNA delivery. The inner core of the nano-particle is Fe modified by Polyethyleneimine (PEI) 3 O 4 The surface of the nanoparticle is coated with fucoidan through electrostatic adsorption, and the nanoparticle can be further combined with miRNA through self-assembly and used for the targeted delivery of miRNA. Wherein, fucoidin and miRNA are self-assembled to PEI modified magnetic Fe layer by layer 3 O 4 The preparation method of the core is simple, the conditions are mild, and the operation and the repetition are easy. In addition, the fucose composite nano-carrier can realize tissue targeted delivery by means of the magnetic nano-inner core in the carrier, thereby realizing systematic targeted therapy of tumors. The nanoparticle has the characteristics of no immunogenicity, high targeting property and the like, and has good application prospects in function research, disease treatment and clinical application based on miRNA.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to preparation of a multifunctional gene vector and application of the multifunctional gene vector in miRNA delivery.
Background
Different from traditional chemotherapy drugs and biological drugs (antibodies and cell drugs), nucleic acid drugs (such as siRNA and miRNA) can take molecules which cannot be targeted by chemical drugs or antibody drugs as action targets, and are expected to produce breakthrough progress on diseases with poor curative effects of traditional drugs, especially genetic diseases, cancers and the like which are difficult to treat. However, delivery of nucleic acid drugs faces significant problems. The entry of nucleic acid drugs into cells faces two major challenges, one is that RNA exposed in blood is easily degraded by RNase in plasma and tissues and can trigger unwanted immune responses; the other is that negatively charged RNAs are difficult to enter into the cell through the membrane, so a delivery system is required to deliver them into the cell for functional function. The development of delivery platforms is an important point of the whole chain of nucleic acid therapy industry and is also a necessary condition for the success of nucleic acid therapy.
With the development of nanotechnology, nanomaterials are becoming new options for delivery of mirnas. For example, Hosseinbour et al encapsulates miRNA-26a-5p in mesoporous silica nanoparticles (MSN-CC-PEI), wraps the mesoporous silica nanoparticles on polyethyleneimine (MSN-CC-PEI), and is used as a system for promoting osteogenic differentiation of rat bone marrow mesenchymal cells, and the delivery system shows stability and high efficiency in vivo.
Fucoidan is a sulfated polysaccharide of marine origin, and has been incorporated into the field of nanomedicine applications. Fucoidin can be used in a nano drug delivery system, can stabilize a nano carrier, and can increase the anticancer activity of a chemotherapeutic drug. Patent publication No. CN 108451931A discloses nanoparticles prepared from polyacrylamide hydrochloride and fucoidan by polyelectrolyte complexation, which can increase the anticancer activity of the chemotherapeutic drug methotrexate. The design and preparation of the fucoidin multifunctional gene vector for delivering miRNA can realize the targeted delivery of miRNA, and the miRNA is used as an immune activator and can improve the tumor microenvironment of immunosuppression. This design will provide new therapeutic strategies for the treatment of cancer.
Disclosure of Invention
The invention aims to provide a multifunctional gene vector loaded with miRNA with anti-tumor activity so as to realize the targeted transportation of miRNA in vivo and the synergistic anti-cancer effect of miRNA and fucoidin.
The specific technical scheme of the invention is as follows:
a multifunctional gene vector. The nanoparticles comprise Polyethyleneimine (PEI) -modified Fe 3 O 4 Nanometer magnetic core and fucoidin assembled on the outer layer. Fucoidin is combined to the outer layer of the magnetic core in a self-assembly manner through electrostatic adsorption. The carrier has an average particle diameter of 50 to 150 nm.
The invention provides a preparation method of the multifunctional gene vector, which comprises the following steps:
uniformly mixing 10-15 mmol of anhydrous ferric chloride, preferably 15mmol, 3-5 mmol of sodium hydroxide, preferably 5mmol, 45-55 mL of ethylene glycol, preferably 50mL and 3.6mL of sterile water, transferring the mixture into a three-neck bottle, heating to boil, keeping boiling and refluxing for 8-12 h, and stopping reactionCooling to room temperature, magnetic separating and washing to obtain Fe 3 O 4 Nano particles for later use;
(2) cross-linking of sodium tripolyphosphate and modification of Polyethyleneimine (PEI)
Dispersing the ferroferric oxide nano particles obtained in the step (1) into sterile water, and adding sodium tripolyphosphate and Fe 3 O 4 The mass ratio of the nano particles to the sodium tripolyphosphate is 1: 1 to 1: 1.5, preferably 1: 1.2, uniformly mixing, and reacting at room temperature for 30-50min to obtain ferroferric oxide phosphate nano particles; magnetically separating the reacted ferroferric oxide phosphate nano particles, and then re-dispersing the nano particles in 50-80mL of ultrapure water; slowly adding the solution into 50-80mL of Polyethyleneimine (PEI) aqueous solution with the concentration of 80-100 mg/L, stirring for 20-30min, uniformly mixing, performing magnetic separation (the magnetic separation is strong magnet separation), and suspending to 100-120mL of sterile water to obtain a Polyethyleneimine (PEI) -coated magnetic iron oxide nanoparticle solution;
(3) self-assembly of fucoidan
And (2) mixing and adsorbing the nanoparticle solution obtained in the step (1) and a fucoidin solution at room temperature for 30min, centrifuging at 7000-8000 rpm for 10min, removing supernatant, and adding sterile water for re-suspension to prepare magnetic nanoparticles (Fuc-NPs) combined with fucoidin.
Wherein the molecular weight range of the fucoidin is 5 kDa-130 kDa, and the preferred molecular weight range is 80 kDa-130 kDa.
Wherein the mass ratio of the nanoparticle solution to the fucoidan is 3: 1-1: 1, wherein the most preferred is 2: 1.
the invention also provides application of the multifunctional gene vector in delivering the miRNA with the anti-tumor activity. The combination method of the miRNA and the magnetic nanoparticles comprises the following steps:
mixing the Fuc-NPs solution with miRNA (20 mu M) dissolved in DEPC (sterile ultrapure water) at room temperature for 30min, assembling the miRNA to magnetic nuclei, centrifuging at 7000-8000 rpm for 10min, discarding supernatant, and resuspending with sterile water to obtain miRNA-loaded magnetic nanoparticles (Fuc-5-FU-miRNA NPs).
Wherein the volume ratio of the nanoparticle solution to the miRNA is 10: 1 to 10: 3, wherein the most preferred is 10: 2.
the invention also provides a compound obtained by loading the miRNA molecules with the anti-tumor activity by the multifunctional gene vector.
The delivered mirnas of the present invention have the following characteristics:
uracil in the sense strand and/or uracil in the antisense strand of miRNA mics is/are completely replaced by fluorouracil, namely 5-FU-miRNA mics.
Furthermore, the invention also provides application of the compound in preparing medicines and preparations for preventing or treating tumor diseases.
The invention has the advantages that:
the invention provides a multifunctional gene vector. The nano-particles have the following advantages:
(1) the preparation process is simple: the fucoidin and the miRNA are combined to the nano magnetic core in a layer-by-layer self-assembly mode, and the preparation method is simple, mild in condition and easy to operate and repeat.
(2) Double targeting effect: magnetic targeting and the targeting effect of fucoidin on p-selectin highly expressed in tumor can efficiently deliver miRNA into tumor, reduce toxic and side effects on other organ tissues, and increase the effective dose of miRNA at tumor part.
(3) Triple drug delivery systems: the fucoidin loaded by the nano-particles can activate an immune system as an effector molecule, improve the tumor microenvironment of immunosuppression and generate a synergistic anti-tumor effect with 5-FU-miRNA mimics; the carried miRNA can target oncogene to inhibit tumor growth, and the released free 5-FU can directly kill tumor cells and tumor interstitial cells, thereby realizing systemic treatment of tumors. The one-arrow three-carving nano drug delivery system designed by the project provides a new collaborative design strategy for a tumor targeted nano drug delivery system.
Drawings
FIG. 1 comparison of the immunomodulatory Activity of fucoidan with different molecular weights
FIG. 2 morphology and characterization of Fuc-5-FU-miRNA NPs
FIG. 3 mean particle size and Zeta potential of Fuc-5-FU-miRNA NPs
FIG. 4 uptake of Fuc-5-FU-miRNA NPs
FIG. 5 confocal laser microscopy characterization of intracellular uptake of Fuc-5-FU-miRNA NPs
FIG. 6 Effect of Fuc-5-FU-miRNA NPs on target Gene expression
FIG. 7 cytotoxic Effect of Fuc-5-FU-miRNA NPs
FIG. 8.Transwell coculture System characterization of the Effect of Fuc-5-FU-miRNA NPs and 5-FU-miRNA NPs on cell viability
FIG. 9 in vivo antitumor activity of Fuc-5-FU-miRNA NPs. (a) Tumor tissue photographing images;
(b) tumor volume and weight; (c) representative images of tumor tissues Ki67, CD68, CD206 immunohistochemistry. All images were on a scale of 50 μm.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the examples.
Example 1: comparison of immunomodulatory Activity of fucoidan and Low molecular weight fucoidan
The present invention compares the immunomodulatory activity of low molecular weight fucoidan LF2(7.2kDa) and fucoidan FPS1M (130 kDa).
Raw264.7 was seeded at 7000 cells/well in 96-well plates. Uninduced raw264.7 cells were used as M0 macrophages. After incubation for 24h with 20ng/mL IFN-. gamma. (PEPROTECH) and 100ng/mL LPS (Leptobiosis), M0 macrophages were polarized to M1 macrophages. M0 macrophages were incubated for 24h with 20ng/mL IL-4(PEPROTECH) and 20ng/mL IL-13(PEPROTECH) to give M2 type macrophages. The cells were cultured for 12h and the culture medium supernatant was collected. At the same time, FPS1M (200. mu.g/mL) and LF2 (200. mu.g/mL) were incubated with M0 macrophages for 36h, respectively, and the supernatants of the cells were collected to determine the levels of NO and cytokines (IL-6 and TNF-. alpha.). The content of IL-6 and TNF-alpha in the supernatant of Raw264.7 cell culture broth was measured by ELISA (doctor's laboratories). Nitric Oxide (NO) was detected using a nitric oxide assay kit according to the instructions (petun sky).
FIG. 1 shows that FPS1M and LF2 both promote release of proinflammatory factors (NO, IL-6 and TNF α) from macrophages. Among them, FPS1M exerts a better effect than LF2 in promoting the differentiation of macrophage M1 into M1 phenotype. This indicates that the higher molecular weight fucoidan exerts better immunostimulatory activity than the lower molecular weight fucoidan. Therefore, high molecular weight fucoidan (80-130 kDa) is preferred for the preparation of Fuc-5-FU-miR-15a NPs.
Example 2: preparation and characterization of miRNA-loaded multifunctional gene vector
2.1 preparation of miR-15 a-loaded multifunctional Gene vector (Fuc-5-FU-miR-15a NPs)
Uniformly mixing 15mmol of anhydrous ferric chloride, 5mmol of sodium hydroxide, 50mL of ethylene glycol and 3.6mL of deionized water, transferring the mixture into a three-necked bottle, heating the mixture to boiling, keeping the boiling and refluxing for 8 hours, stopping reaction, cooling the mixture to room temperature, separating and washing the mixture to obtain Fe 3 O 4 And (3) nanoparticles.
Dispersing 0.2g of the obtained ferroferric oxide nano particles into 100mL of deionized water, adding 200mg of sodium tripolyphosphate, uniformly mixing, magnetically separating, and then dispersing again into 100mL of water; slowly adding the solution into 50mL of polyethyleneimine water solution with the concentration of 100mg/L, uniformly mixing, and carrying out magnetic separation or centrifugal separation to obtain the PEI-coated magnetic iron oxide nanoparticles.
Adsorbing the nanoparticle solution 100 μ L and the fucoidin solution 10 μ L (1mg/mL) at room temperature for 30min to allow the fucoidin to self-assemble to the outer layer of the magnetic core. The solution was centrifuged at 7000rpm for 10min, the supernatant was discarded, and sterilized water was added for resuspension to prepare fucoidan-conjugated magnetic nanoparticles (Fuc-NPs).
Mixing Fuc-NPs solution 100 μ L with sterile water dissolved 5-FU-miR-15a mimics (20 μ M) 20 μ L, adsorbing the two at room temperature for 30min to make miRNA adsorb and assemble to magnetic core, centrifuging at 7000rpm for 10min, discarding supernatant, resuspending with sterile water,
FIG. 2 shows the simulated combination mode of Fuc-5-FU-miR-15a NPs, and the finally obtained Fuc-5-FU-miR-15a NPs are stable magnetic nanoparticle systems.
2.2 characterization of Fuc-5-FU-miR-15a NPs
The size distribution and Zeta potential of the micelles at 25 ℃ were analysed by Dynamic Light Scattering (DLS) using a Malvern Zetasizer nano ZS device (Malvern, UK) at scattered light detection angles of 90 and 15 respectively.
The morphology of the nanoparticles was analyzed by Transmission Electron Microscopy (TEM). The sample solution (10. mu.L; 1mg/mL) was dropped onto a copper grid coated with amorphous carbon and allowed to dry naturally in a desiccator. An aqueous solution of 1 wt% uranyl acetate was dropped on a copper grid and the sample was stained for 1 min. After blotting with filter paper, the sample was thoroughly dried in a desiccator and then subjected to TEM observation. The samples were finally observed on a Philips CM120 transmission electron microscope (Philips, Netherlands).
Transmission electron microscopy revealed that the multifunctional gene vector was uniformly dispersed round particles (FIG. 2).
FIG. 3 shows that the average particle diameter of the nano-carrier is 58nm and the Zeta potential is +38.6 mV. The PEI-magnetic iron oxide nanoparticles and the fucoidin solution are mixed to form Fuc-NPs through self-assembly, wherein the particle size of the nano-composite is about 72.3nm, and the Zeta potential is-17.7 mV. Finally, the nano-composite is compounded with 5-FU-miR-15a mimics to form Fuc-5-FU-miR-15a NPs, wherein the particle size of the nano-composite is 143.6nm, and the Zeta potential is-33.1 mV. Along with gradual combination, the charge is gradually reduced, and the particle size is gradually increased, which indicates that the 5-FU modified miRNA-15a mics and the fucoidin are successfully combined to the nano-iron oxide magnetic core, and the Fuc-5-FU-miR-15a-NPs magnetic nano-particle is successfully prepared.
Example 3 cellular uptake and targeting of Fuc-5-FU-miR-15a NPs
2.1 cellular uptake of Fuc-5-FU-miR-15a NPs
2.1.1qRT-PCR method for detecting release of miR-15a
Three pancreatic cancer cells (sw1990, ASPC-1, Paca-mia-2) at 1x10 5 The cells were seeded in 6-well plates at a density of/mL and 24 hours later, the cancer cells were treated with Fuc-5-FU-miR-15a NPs for 6 hours. Total RNA was extracted using Trizol method. PrimeScript TM RT reagent Kit (Taraka, Japan) was used to synthesize cDNA. Quantitative RT-PCR was performed using SYBR Green Master Mix and Light Cycler PCR detection system (ABI PRISM-7900). The 5' primer sequence of miR-15a is as follows: CGCCTAGCAGCACATAATGGTTTGTG, the 3 'end is mRQ 3' universal sequence provided by the kit. The expression level of the U6 gene is used as an internal reference. Calculating relative miRNA water according to formulaFlat delta CT is equal to delta CT Test sample -ΔCT Control sample . Use 2 -ΔΔCT The method calculates the change in gene expression.
2.1.2HPLC method for detecting 5-FU Release
The amount of 5-FU released was determined by HPLC method. Three pancreatic cancer cells at 1x10 5 The cells were seeded in 6-well plates at a density of/mL and 24 hours later, the cancer cells were treated with Fuc-5-FU-miR-15a NPs for 6 hours. The cells were collected, sonicated for 30min, and centrifuged to determine the 5-FU content using CBM-L20 liquid chromatography system (Shimadzu, Japan).
Specifically, using a C18 reverse phase column, mobile phase, methanol: water 10: 90, the column temperature is 30 ℃, the sample injection amount is 20 mu L, and the detection wavelength is 265 nm.
2.1.3 confocal detection of uptake of Fuc-5-FU-miR-15a NPs @ Cy3 magnetic nanoparticles in cells by laser confocal detection
Cy3 mark is added during synthesis of 5-FU-miR-15a mimics to prepare Fuc-5-FU-miR-15a NPs @ Cy3 magnetic nanoparticles. Fuc-5-FU-miR-15a NPs @ Cy3 magnetic nanoparticles (5-FU-miR-15a final concentration is 50nM) and ASPC-1 cells seeded on a cell slide were incubated for 0 and 6 hours, washed 3 times with PBS, stained with CellMask green stain at 37 ℃ for 10min, and after washing 3 times with PBS, cell nuclei were stained with DAPI at room temperature for 10 min. The encapsulated tablets were encapsulated and observed using a confocal laser microscope.
FIG. 4 shows that Fuc-5-FU-miR-15a-NPs can be efficiently taken up by cells through gene expression analysis, and miR-15a and 5-FU are released in the cells to play a role in inhibiting cell growth.
FIG. 5 shows the intracellular accumulation of 5-FU-miR-15a @ Cy3, indicating that Fuc-5-FU-miR-15a NPs can successfully deliver 5-FU-miR-15a into cells to exert antitumor activity.
2.2Fuc-5-FU-miR-15a NPs target gene expression
The expression condition of Fuc-5-FU-miR-15a NPs target gene is verified by using a qRT-PCR method. Three pancreatic cancer cells at 1x10 5 The cells were seeded in 6-well plates at a density of/mL and 24 hours later, the cancer cells were treated with DMEM containing Fuc-5-FU-miR-15a NPs for 24 hours. Cells were collected and total RNA was extracted. Experiment of the inventionThe process is referred to example 2.1.1. The sequence of the primers YAP-1, 5 '-primer: CAGAACCGTTTCCCAGACTACCTTG, 3' -primer: GCAGACTTGGCATCAGCTCCTC; BCL-2,5 '-primercTCGCCCTGTGTGGATGACTGAGTAC, 3' -primercACAGCCAGGAGAAATCAAACAAGAGAG; TS, 5 '-primerctttcagcgaACCCAGACCTACTTC, 3' -primeraggTTGGATGCGGATTGTACCCTTC.
Fuc-5-FU-miR-15a-NPs successfully release 5-FU-miR-15a into cells, and remarkably reduces the expression of target genes Bcl-2, Yap-1 and TS (figure 6).
Example 4 cytotoxic Effect of Fuc-5-FU-miR-15a NPs
3.1 cytotoxic Effect of Fuc-5-FU-miR-15a NPs
Cell viability was determined by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Cells were seeded in 96-well plates (7X 10) 3 One cell/well), Fuc-5-FU-miR-15a NPs nanoparticles were added after 12 hours, and after 48 hours of incubation, 10 μ Ι _ of MTT solution (5mg/mL) was added to each well, and the cells were then incubated at 37 ℃ for an additional 4 hours. The medium was gently discarded, 150 μ L DMSO was added per well to dissolve insoluble formazan, and the solution was assayed using a microplate reader (Tecan,
switzerland) was measured for absorbance at 570 nm.
FIG. 7 shows that Fuc-5-FU-miR-15a NPs significantly reduced cell viability of sw1990 and Mia-2 cells. And compared with the single use of 5-FU-miR-15a mimics, the composition shows better activity of killing tumor cells.
Further, we compared the effect of Fuc-5-FU-miR-15a NPs on cell viability with nanoparticulate 5-FU-miR-15a NPs not loaded with fucoidan.
A co-culture system of SW1990 cells and macrophages was constructed using the Transwell system (0.4 μm). 5X10 4 One Raw264.7 cell was seeded on the bottom of a 24-well plate cell culture dish. And induced macrophages to a different phenotype according to the method of example 1. Fuc-5-FU-miR-15a NPs and 5-FU-miR-15a NPs are added into the medicinal composition respectively to treat the lower layer cells. 7000 SW1990 cells were seeded in the upper layer of the Transwell chamber, and macrophage-thinlyThe cells were co-cultured for 48 h. The NPs and macrophage-released' cytokines in the lower chamber affect the survival of SW1990 cells in the upper chamber. The SW1990 cells in the upper chamber were washed 3 times with PBS, fixed in 4% paraformaldehyde for 10min, stained with 0.2% crystal violet for 15min, and washed 3 times with PBS. Stained cells were photographed under an optical microscope (come) and quantified by Image J software.
The fucoidan-loaded nanoparticles (Fuc-5-FU-miR15a NPs) exerted better anti-tumor activity than 5-FU-miR15a NPs, which is presumed to be related to the loaded fucoidan promoting macrophages to exhibit a pro-inflammatory phenotype (FIG. 8).
Example 5 in vivo anti-tumor Activity Studies of Fuc-5-FU-miR-15a NPs
Male BALB/C mice, 6-8 weeks old, were purchased from Peking Wittingle laboratory animals Co., Ltd and were bred at SPF animal centers. All animals were cared for according to the recommendations of the guidelines for animal Care and use of the institute of Marine research, national academy of sciences. Taking HCT116 cells in logarithmic growth phase at 1 × 10 7 cells/mL were injected subcutaneously with 100. mu.L to the upper right limb of the mouse. When the tumor grows to about 100mm 3 When mice are randomly divided into a CK group, a 5-FU-miR-15a group, a Fuc-5-FU-miR-15a NPs group, a CK group tail vein injection physiological saline, a 5-FU-miR-15a group tail vein injection 80 mu g of 5-FU-miR-15a mimics (in vivo-jetPEI prepackaged), and a Fuc-5-FU-miR-15a NPs group tail vein injection Fuc-5-FU-miR-15a NPs (NPs contain 80 mu g of 5-FU-miR-15a), and magnets are placed at tumor positions. The injection is given once every 3 days for 7 times. According to the formula V-W 2 X L x 0.5 the estimated tumor volume (V) was calculated, where W represents the largest tumor diameter (cm) and L represents the second largest tumor diameter. After anesthesia, serum from mice was taken for biochemical analysis (Qingdao gold medical testing center). Excised tumors were fixed in 4% paraformaldehyde for at least 24h, and then 4 μm paraffin sections were prepared. The fixed tumor tissue was used for immunohistochemical studies, and the remaining tumors were frozen at-80 ℃ for use.
Fuc-5-FU-miR-15a NPs significantly reduced the tumor volume compared to the blank. Both the 5-FU-miR-15a mimics and the Fuc-5-FU-miR-15a NPs significantly reduce the weight of tumors, but the anti-tumor activity of the Fuc-5-FU-miR-15a NPs is better than that of JetPEI pre-packaged 5-FU-miR-15a mimics (FIGS. 9 a-b). Immunohistochemistry showed that, compared with JetPEI prepackaged 5-FU-miR-15a mimics, Fuc-5-FU-miR-15a NPs can remarkably increase infiltration of M1 macrophages in tumor tissues (CD86 expression is increased, CD206 expression is reduced) due to carrying fucoidan, and improve immunosuppressive tumor microenvironment, which possibly promotes the antitumor activity of Fuc-5-FU-miR-15a NPs (FIG. 9 c).
Supplemental content
The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A multifunctional gene vector, which is characterized in that: consists of magnetic ferroferric oxide nano-particles modified by polyethyleneimine coated with fucoidin.
2. The multifunctional gene vector of claim 1, wherein: the average particle size of the magnetic ferroferric oxide nano particles is 50-150 nm.
3. A method for preparing the multifunctional gene vector of claim 1, wherein the magnetic nanoparticles are prepared by the following steps:
(1) preparation of nano magnetic core
Uniformly mixing 10-15 mmol of anhydrous ferric chloride, preferably 15mmol, 3-5 mmol of sodium hydroxide, preferably 5mmol, 45-55 mL of ethylene glycol, preferably 50mL and 3.6mL of sterile water, transferring the mixture into a three-neck flask, heating to boil, keeping boiling and refluxing for 8-12 h, stopping reaction, cooling to room temperature, and carrying out magnetic separation and washing to obtain Fe 3 O 4 Nano particles for later use;
(2) cross-linking of sodium tripolyphosphate and modification of Polyethyleneimine (PEI)
Fe obtained in the step (1) 3 O 4 Dispersing the nano particles in 50mL of ultrapure water, adding sodium tripolyphosphate and Fe 3 O 4 The mass ratio of the nano particles to the sodium tripolyphosphate is 1: 1 to 1: 1.5, preferably 1: 1.2, uniformly mixing, and reacting at room temperature for 30-50min to obtain ferroferric oxide phosphate nanoparticles; magnetically separating the reacted ferroferric oxide phosphate nanoparticles, and then dispersing the ferroferric oxide phosphate nanoparticles into ultrapure water again; slowly adding the obtained solution into 50-80mL of Polyethyleneimine (PEI) aqueous solution with the concentration of 80-100 mg/L, stirring at room temperature for 20-30min to uniformly mix, carrying out magnetic separation, and suspending to 100-120mL of sterile ultrapure water to obtain a Polyethyleneimine (PEI) -coated magnetic iron oxide nanoparticle solution;
(3) self-assembly of fucoidan
And (3) mixing and adsorbing the nanoparticle solution obtained in the step (2) and a fucoidin solution at room temperature for 30-40 min, centrifuging at 7000-8000 rpm for 10-20 min, removing the supernatant, and adding sterile ultrapure water for resuspension to prepare magnetic nanoparticles (Fuc-NPs) combined with fucoidin.
4. The production method according to claim 3, characterized in that: the molecular weight range of the fucoidin is 5-130 kDa, and preferably 80-130 kDa.
5. The production method according to claim 3, characterized in that: the method is characterized in that: the mass ratio of the nanoparticle solution to the fucoidan is 3: 1-1: 1, wherein the most preferred is 2: 1.
6. use of the multifunctional gene vector of claim 1 for delivering miRNA having anti-tumor activity.
7. Use of the multifunctional gene vector of claim 6 for the delivery of mirnas with anti-tumor activity, characterized in that: the combination method of miRNA and magnetic nanoparticles comprises the following steps:
mixing a magnetic nanoparticle (Fuc-NPs) solution combined with fucoidin with miRNA (20 mu M) dissolved in sterile water, adsorbing the two at room temperature for 30-40 min to assemble the miRNA to magnetic nuclei, centrifuging at 7000-8000 rpm for 10-20 min, discarding supernatant, and carrying out resuspension with sterile water to obtain the miRNA-loaded magnetic nanoparticles (Fuc-miRNA NPs).
8. Use of the multifunctional gene vector according to claim 7 for the delivery of mirnas with antitumor activity, characterized in that:
wherein the volume ratio of Fuc-NPs solution to miRNA is 20: 1-10: 3, wherein the most preferred is 10: 1.
9. the multifunctional gene vector of any one of claims 1 to 6, which is loaded with a miRNA molecule having anti-tumor activity.
10. Use of a complex according to claim 8 for the preparation of a medicament or formulation for the prevention or treatment of a neoplastic disease.
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| KR20220082138A (en) * | 2020-12-09 | 2022-06-17 | 영남대학교 산학협력단 | Compositions comprising Fucoidan from Ecklonia cava as an active ingredient |
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