Ferroferric oxide nanoparticles adsorbing miR-141-3p and their applications

Abstract

The purpose of the present invention is to provide ferroferric oxide nanoparticles adsorbing miR-141-3p and their application. Ferroferric oxide nanoparticles adsorbing miR-141-3p were prepared, and a rat retinal injury model induced by optic nerve clamping was constructed, thereby confirming that ferroferric oxide nanoparticles adsorbing miR-141-3p can be used to treat retinal injury induced by optic nerve clamping.

Description

Technical Field

[0001] The invention belongs to the field of nano-biotechnology and relates to ferrosoferric oxide nanoparticles capable of adsorbing miR-141-3p and applications thereof. Background Art

[0002] MicroRNA (miRNA) is a type of single-stranded endogenous RNA, about 18-24 nucleotides long, which does not encode proteins. However, it can reduce the stability of target mRNA or inhibit its translation by complementary binding to the 3'-untranslated region (3'-UTR) of target gene mRNA, participate in the regulation of gene post-transcriptional level, and play a regulatory role in various biological processes such as cell differentiation, proliferation, apoptosis and tumors.

[0003] Serum microRNA-141-3p (miR-141-3p) is a mature miRNA. MiR-141-3p is a member of the miR-200 family. It is downregulated in tumor tissues such as prostate cancer, colorectal cancer, and bladder cancer, and plays a role in suppressing tumors. Other effects of miR-141-3p are also under further study. Summary of the invention

[0004] The purpose of the present invention is to provide ferroferric oxide nanoparticles adsorbing miR-141-3p and applications thereof. The ferroferric oxide nanoparticles adsorbing miR-141-3p are encapsulated with ferroferric oxide nanoparticle-grade materials to adsorb miR-141-3p, release miR-141-3p by acting on the retinal damage site caused by optic nerve clamping, and affect the expression of miR-141-3p, thereby performing relevant therapeutic effects on retinal damage caused by optic nerve clamping.

[0005] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0006] The present invention provides a method for preparing ferroferric oxide nanoparticles that adsorb miR-141-3p, comprising:

[0007] NHS and EDC.HCl were added to the ferroferric oxide nanoparticles, activated at room temperature for 1 hour, dialyzed in ultrapure water for 24 hours to remove unreacted NHS and EDC.HCl, and the liquid was collected after dialysis and added with folic acid, stirred and mixed thoroughly, incubated at room temperature overnight, and dialyzed again for 24 hours to remove unreacted folic acid. The collected liquid was added with miR-141-3p and mixed, and incubated at 4°C overnight, thus obtaining nanoparticles modified with folic acid and adsorbed with miR-141-3p.

[0008] Preferably, a 5000D dialysis bag is used for 24 hours, and the liquid is changed every 4 hours.

[0009] The present invention also provides ferrosoferric oxide nanoparticles adsorbing miR-141-3p prepared by the above preparation method.

[0010] Preferably, the ferrosoferric oxide nanoparticles can adsorb miR-141-3p at a maximum of 4 times their own mass.

[0011] The present invention further provides the use of the above-mentioned ferroferric oxide nanoparticles adsorbing miR-141-3p in the preparation of a drug for treating retinal damage.

[0012] Preferably, the application comprises injecting ferrosoferric oxide nanoparticles adsorbing miR-141-3p into the site of retinal damage for 14 days.

[0013] Preferably, the method further comprises constructing a rat retinal injury model induced by optic nerve clamp.

[0014] More preferably, model building includes:

[0015] a. Anesthesia: The rats were anesthetized with 3% sodium pentobarbital at a dose of 3 ml / kg, with the operated eye facing the operator. Lidocaine was dripped into the eyelids. Subsequent operations were performed after the rats showed no reaction when the eyelids were touched.

[0016] b. Expose the optic nerve: With the inner canthus at 9 o'clock and the outer canthus at 3 o'clock, cut the conjunctiva from 3-6 o'clock of the operated eye and separate it inwards to find the white optic nerve;

[0017] c. Optic nerve clamp: Use a toothed fiber hemostatic forceps to precisely clamp the optic nerve of the rat 0.5 cm behind the bulb.

[0018] The clamp is set for 15 seconds, and the clamp strength is to clamp the hemostatic forceps to the maximum;

[0019] d. After clamping, suture the conjunctiva with 8-0 suture needle and apply erythromycin ointment to the operated eye once a day for 3 days after surgery;

[0020] e. 14 days after surgery, the eyeballs were collected and preserved in 10% paraformaldehyde for paraffin sectioning and HE staining. The retinal structure of the model was disordered and wavy, and the outer nuclear layer was thickened, indicating that the model was successful.

[0021] The beneficial effects of the present invention are:

[0022] Iron tetroxide nanoparticles adsorbing miR-141-3p were prepared, and a rat retinal injury model induced by optic nerve clamp was constructed. It was further confirmed that iron tetroxide nanoparticles adsorbing miR-141-3p can be used to treat retinal injury induced by optic nerve clamp. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the transmission electron microscopy characterization result of the nanoparticles (PEI@Fe3O4) and folic acid modification and miR-141-3p adsorption in the embodiment of the present invention.

[0024] Figure 2 These are the particle size and Zeta potential results of the nanoparticles (PEI@Fe3O4) and folic acid modification and miR-141-3p adsorption in the embodiments of the present invention.

[0025] Figure 3 This is the gel electrophoresis result of the adsorption of miR-141-3p by ferrosoferric oxide nanoparticles in an embodiment of the present invention. In the figure, 1-10 correspond to the mass ratios of 0:1, 0.05:1, 0.1:1, 0.2:1, 0.25:1, 0.5:1, 1:1, 1.5:1, 2:1, and 2.5:1, respectively.

[0026] Figure 4 Schematic diagram of exposing the optic nerve in an embodiment of the present invention.

[0027] Figure 5 This is the HE staining result verified by the model of the present invention.

[0028] Figure 6 This is the HE staining result of the ferroferric oxide nanoparticles of the present invention adsorbing miR-141-3p and injected into the damaged part of the retina. DETAILED DESCRIPTION

[0029] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0030] Example

[0031] 1. Preparation of Fe3O4 nanoparticles adsorbing miR-141-3p (I) Materials (article number, manufacturer): Fe3O4 nanoparticles (PEI@Fe3O4) (Beijing Zhongke Leiming); miR-141-3p (General Biosynthesis); folic acid (IF0180 Solebao); fluorescein isothiocyanate (FITC) (IF8070 Solebao); EDC.HCl (N808856 McLean); NHS (N811124 McLean). (II) Instruments used (model, manufacturer): magnetic stirring; 5000D dialysis bag; Zetasizer Pro (MalvernPanalytical); transmission electron microscope ((JEM-1230 (80KV), JEOL)); SEM (FEI Quanta250, FEI Company, USA)

[0032] (III) Experimental methods:

[0033] 1. Folic acid modification of nanoparticles (PEI@Fe3O4) and adsorption of miR-141-3p

[0034] 1.44 mg NHS and 1.4 mg EDC.HCl were added to 5 ml of 1 mg / ml ferroferric oxide nanoparticles (purchased from Zhongke Leiming, the stock solution was 1 mg / ml aqueous solution), activated at room temperature for 1 hour, dialyzed in a 5000D dialysis bag in an ultrapure water environment for 24 hours, and the liquid was changed every 4 hours to remove unreacted NHS and 1.4 mg EDC.HCl. After the dialysis was completed, the liquid was collected and added with 2 mg folic acid, stirred and mixed thoroughly, incubated at room temperature overnight, and dialyzed again for 24 hours to remove unreacted folic acid (the method is the same as above). The collected liquid was added with 4 OD of miR-141-3p and mixed, and incubated at 4°C overnight, and the modified folic acid and adsorbed miR-141-3p nanoparticles (Fe-Fa / miR-141) were obtained. Using the same method, the nanoparticles that did not adsorb miR-141-3p in the last step were Fe-Fa nanoparticles.

[0035] 2. Characterization of Nanoparticles

[0036] (1) Transmission electron microscopy characterization

[0037] After mixing the sample, drop it onto the copper grid and let it dry naturally before observing it under a transmission electron microscope. Figure 1 As shown, the outline of the nanoparticles can be clearly seen.

[0038] (2) Particle size and Zeta potential characterization

[0039] After mixing the sample, dilute it to an appropriate concentration and use Zetasizer Pro to measure the particle size and Zeta potential. Figure 2 The particle size results showed that the hydrated particle size of the nanoparticles (PEI@Fe3O4) was about 40nm. After modification with folic acid (PEI@Fe3O4-Fa), the particle size was almost unchanged. After further adsorption of miR-141-3p (PEI@Fe3O4-Fa / miR-141-3p), the hydrated particle size increased slightly to about 50nm. The surface charge (Zeta potential) of the nanoparticles showed a downward trend with modification and adsorption, from about +46mV at the beginning to about +30mV.

[0040] (3) Gel electrophoresis of miR-141-3p adsorbed by ferroferric oxide nanoparticles

[0041] The ferroferric oxide nanoparticles and miR-141-3p were mixed at a mass ratio of 0:1, 0.05:1, 0.1:1, 0.2:1, 0.25:1, 0.5:1, 1:1, 1.5:1, 2:1, and 2.5:1 (the total volume was 12 μL, and the mass of miR-141-3p was controlled to be consistent at 3 μL 1 μg / μL. The mass of the nanoparticles was added according to the above mass ratio. If it was less than 12 μL, DEPC water was added to make up), and incubated at room temperature in the dark for 30 minutes. Gel electrophoresis was used to observe each band to determine the binding of the nanoparticles to miR-141-3p (the molecular weight of miR-141-3p is 7066).

[0042] Note: 1OD miR-141-3p is 40μg, add 40μL DEPC water to make 1μg / μL; PEI@Fe3O4 nanoparticles are 1mg / ml, diluted to the corresponding concentration according to the mass required for 10 groups.

[0043] Weigh 0.45g agarose and add it to a conical flask containing 30ml 1×TAE electrophoresis solution, and mix thoroughly. Heat the conical flask in a microwave oven until it boils, then take it out and shake it immediately. Repeat 3 times until the agarose is completely dissolved. Add 3μL Glodview to the agarose solution cooled to 60°C and shake it well. Gently pour it into a small electrophoresis gel tank, insert a comb, wait for the agarose gel to solidify, pull out the comb, and place the gel block into the electrophoresis solution. Mix the above incubated samples with 1μL 6× loading Buffer at a ratio of 5:1, take 5μL and add it to the sample tank (record the order of sample addition), set the electrophoresis instrument to 80V, and after 20min, observe the miR-141-3p band on the gel imaging system, that is, the adsorption of nanoparticles and miR-141-3p.

[0044] According to the electrophoresis results (see Figure 3 ), the mass concentration of 0.25:1 is at the critical position, at which the adsorption and binding are just complete. Therefore, the maximum embedding amount of ferroferric oxide nanoparticles is 1:4, that is, the maximum amount of miR-141-3p that can be adsorbed is 4 times its own mass.

[0045] II. Retinal damage in rats caused by optic nerve clamp

[0046] Model introduction: Glaucoma is a group of diseases characterized by optic disc atrophy and depression, visual field defects and decreased vision. Pathological increase in intraocular pressure and insufficient blood supply to the optic nerve are the primary risk factors for the onset of glaucoma. The tolerance of the optic nerve to pressure damage is also related to the occurrence and development of glaucoma. Any obstruction in the aqueous humor circulation pathway can lead to pathological changes caused by increased intraocular pressure, but some patients also present with normal intraocular pressure glaucoma. Glaucoma is one of the three major blinding eye diseases that cause human blindness, with an incidence rate of 1% in the general population and 2% after the age of 45. Clinically, glaucoma is divided into three categories: primary, secondary and congenital according to the cause, chamber angle, intraocular pressure recording and other conditions. This procedure uses optic nerve clamping to cause pressure damage to the optic nerve, thereby inducing retinal lesions.

[0047] 1. Experimental animals: SD rats, adult, male or female.

[0048] 2. Experimental materials: lidocaine hydrochloride injection, stereo microscope, surgical scissors, small tweezers, micro forceps, suture needles and threads, retractors, 8-0 suture threads with needles, needle holders, micro hemostatic forceps, etc.

[0049] 3. Regulations:

[0050] 3.1 Anesthesia: Rats were anesthetized with 3% sodium pentobarbital at a dose of 3 ml / kg, with the operated eye facing the operator. Lidocaine was dripped into the eyelids, and subsequent operations were performed after the rats showed no reaction when the eyelids were touched.

[0051] 3.2 Expose the optic nerve: With the inner canthus at 9 o'clock and the outer canthus at 3 o'clock, cut the conjunctiva from 3-6 o'clock of the operated eye and separate it inwards to find the white optic nerve and separate it slightly. Figure 4 shown.

[0052] 3.3 Optic nerve clamping: Use a toothed fiber hemostatic forceps to accurately clamp the rat's optic nerve about 0.5 cm behind the bulb for 15 seconds. The clamping strength is to clamp the hemostatic forceps to the maximum.

[0053] 3.4 After clamping, use 8-0 suture needle to suture the conjunctiva and apply erythromycin ointment to the operated eye (once a day for 3 days after surgery).

[0054] 4. Model Validation

[0055] Fourteen days after surgery, the eyeballs were collected and preserved in 10% paraformaldehyde for paraffin sectioning and HE staining.

[0056] result:

[0057] The retinal structure of the model is disordered, wavy, and the outer nuclear layer is thickened, indicating that the model is successful. Figure 5 shown.

[0058] 3. Effect of FeO nanoparticles adsorbing miR-141-3p on retinal damage induced by optic nerve clamp

[0059] The ferroferric oxide nanoparticles adsorbed with miR-141-3p were injected into the damaged retina with a microinjector at a volume of 2 μl for 14 days (only one injection was required in these 14 days).

[0060] Results test:

[0061] HE staining: The retina of the model was disordered, wavy, and the outer nuclear layer was thickened; the retina of the treatment group was significantly improved, such as Figure 6 shown.

[0062] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing ferroferric oxide nanoparticles modified with folic acid and adsorbed with miR-141-3p, comprising: NHS and EDC.HCl were added to the polyethyleneimine-modified ferroferric oxide nanoparticles, activated at room temperature for 1 h, dialyzed in a 5000 D dialysis bag in an ultrapure water environment for 24 h, and the liquid was changed every 4 h to remove unreacted NHS and EDC.HCl. After the dialysis was completed, the liquid was collected and folic acid was added, stirred and mixed thoroughly, incubated at room temperature overnight, dialyzed again for 24 h to remove unreacted folic acid, and the liquid was collected and added to miR-141-3p to mix well. The mixture was incubated at 4°C with upside down overnight to obtain ferroferric oxide nanoparticles modified with folic acid and adsorbed with miR-141-3p.

2. The ferroferric oxide nanoparticles modified with folic acid and adsorbed with miR-141-3p prepared by the preparation method according to claim 1, wherein the ferroferric oxide nanoparticles can adsorb miR-141-3p of a maximum of 4 times their own mass.

3. Use of the modified folic acid and ferroferric oxide nanoparticles adsorbed with miR-141-3p as claimed in claim 2 in the preparation of a drug for treating retinal damage, the use comprising injecting the modified folic acid and ferroferric oxide nanoparticles adsorbed with miR-141-3p into the site of retinal damage for 14 days.

4. The use according to claim 3, characterized in that: It also includes the construction of a rat retinal injury model induced by optic nerve clamp.

5. The use according to claim 4, characterized in that: Model building includes: a. Anesthesia: The rats were anesthetized with 3% sodium pentobarbital at a dose of 3 ml / kg, with the operated eye facing the operator. Lidocaine was dripped into the eyelids, and subsequent operations were performed after the rats showed no reaction when the eyelids were touched; b. Expose the optic nerve: With the inner canthus at 9 o'clock and the outer canthus at 3 o'clock, cut the conjunctiva from 3-6 o'clock of the operated eye and separate it inwards to find the white optic nerve; c. Optic nerve clamping: Use a toothed fiber hemostatic forceps to precisely clamp the rat's optic nerve 0.5 cm behind the bulb for 15 seconds. The clamping strength is to clamp the hemostatic forceps to the maximum. d. After clamping, suture the conjunctiva with 8-0 suture needle and apply erythromycin ointment to the operated eye once a day for 3 days after surgery; e. 14 days after surgery, the eyeballs were collected and preserved in 10% paraformaldehyde for paraffin sectioning and HE staining. The retinal structure of the model was disordered and wavy, and the outer nuclear layer was thickened, indicating that the model was successful.

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