CN116211825A - Ferroferric oxide nanoparticle for adsorbing miR-141-3p and application thereof - Google Patents

Abstract

The purpose of the present invention is to provide ferric oxide nanoparticles adsorbing miR-141-3p and its application. Prepared iron ferric oxide nanoparticles adsorbing miR‑141‑3p, constructed a rat retinal injury model induced by optic nerve clamp, and then confirmed that ferric oxide nanoparticles adsorbing miR‑141‑3p can be used in the treatment of optic nerve clamp cause retinal damage.

Description

Ferric oxide nanoparticles adsorbing miR-141-3p and its application

technical field

The invention belongs to the field of nano-biotechnology, and relates to iron ferric oxide nanoparticles adsorbing miR-141-3p and its application.

Background technique

microRNA (miRNA) is a type of single-stranded endogenous RNA, about 18-24 nucleotides in length, which does not code for protein, but can pass through the 3'-untranslated region (3'-untranslated region, 3 '-UTR), reduce the stability of target mRNA or inhibit its translation, 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.

Serum microRNA-141-3p (miR-141-3p) is a mature miRNA, and miR-141-3p is one of the members of the miR-200 family, which is expressed in tumor tissues such as prostate cancer, colorectal cancer and bladder cancer It showed a down-regulation trend, and it played a tumor suppressor role. Other roles of miR-141-3p are also under further investigation.

Contents of the invention

The object of the present invention is to provide iron ferric oxide nanoparticles adsorbing miR-141-3p and its application. The ferroferric oxide nanoparticles that adsorb miR-141-3p absorb miR-141-3p through ferric oxide nanoparticle-level materials, and release miR-141-3p by acting on the retinal damage site caused by optic nerve clamping. The expression of miR-141-3p is affected, so as to have a therapeutic effect on retinal damage caused by optic nerve clamping.

In order to realize the purpose of the present invention, the technical scheme adopted in the present invention is:

The invention provides a preparation method of iron ferric oxide nanoparticles adsorbing miR-141-3p, comprising:

Add NHS and EDC.HCl to iron ferric oxide nanoparticles, activate at room temperature for 1 hour, dialyze in ultrapure water for 24 hours to remove unreacted NHS and EDC.HCl, collect the liquid after dialysis, add folic acid, stir and mix well , incubated overnight at room temperature, and dialyzed again for 24 hours to remove unreacted folic acid. The collected liquid was added to miR-141-3p, mixed evenly, and incubated overnight at 4°C up and down to obtain nanoparticles with modified folic acid and adsorbed miR-141-3p.

Preferably, a 5000D dialysis bag is used for dialysis for 24 hours, and the fluid is changed every 4 hours.

The present invention also provides the iron ferric oxide nanoparticles adsorbing miR-141-3p prepared by the above preparation method.

Preferably, the iron ferric oxide nanoparticles can absorb miR-141-3p 4 times its own mass at most.

The present invention further provides the application of the above iron ferric oxide nanoparticles adsorbing miR-141-3p in the preparation of a medicament for treating retinal damage.

Preferably, the application includes injecting ferric oxide nanoparticles adsorbing miR-141-3p into retinal damage sites for 14 days.

Preferably, it also includes constructing a rat retinal injury model caused by optic nerve clamping.

More preferably, model building includes:

a. Anesthesia: After the rats were anesthetized with 3% pentobarbital sodium at a dose of 3ml/kg, the operated eye was facing the operator, and lidocaine was instilled in the eye, and the rats did not respond after touching the eyelids for subsequent operations;

b. Expose the optic nerve: take the inner canthus as the 9 o'clock direction, and the outer canthus as the 3 o'clock direction, cut the conjunctiva from the 3-6 o'clock direction of the operated eye, separate it inward, and find the white optic nerve;

c. Optic nerve clamp: use toothed fiber hemostatic forceps to accurately clamp the position 0.5cm behind the optic nerve ball of the rat

Set it for 15s, and the clamp strength is to maximize the buckle of the hemostat;

d. After clamping, the conjunctiva was sutured with an 8-0 suture needle, and erythromycin ointment was applied to the operated eye, once a day for 3 days after the operation;

e. 14 days after operation, the eyeballs were taken and stored in 10% paraformaldehyde for paraffin section and HE staining. The retinal structure of the model was disordered, wavy, and the outer nuclear layer was thickened, indicating that the model was successful.

The beneficial effects of the present invention are:

Prepared iron ferric oxide nanoparticles adsorbing miR-141-3p, constructed a rat retinal injury model induced by optic nerve clamp, and then confirmed that ferric oxide nanoparticles adsorbing miR-141-3p can be applied to the treatment of optic nerve clamp cause retinal damage.

Description of drawings

Figure 1 is the transmission electron microscope characterization results of nanoparticles (PEI@Fe3O4) and folic acid modification and miR-141-3p adsorption in the embodiment of the present invention.

Fig. 2 is the results of particle size and Zeta potential after adsorption of nanoparticles (PEI@Fe3O4) and folic acid modification and miR-141-3p in the embodiment of the present invention.

Fig. 3 is the gel electrophoresis result of miR-141-3p adsorbed by iron ferric oxide nanoparticles in the embodiment of the present invention. The mass ratios corresponding to 1-10 in the figure are: 0:1, 0.05:1, 0.1:1, 0.2:1, 0.25:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1.

Fig. 4 is a schematic diagram of exposing the optic nerve in an embodiment of the present invention.

Fig. 5 is the HE staining result of the model validation of the present invention.

Fig. 6 is the HE staining result of the injection of miR-141-3p adsorbed by iron ferric oxide nanoparticles of the present invention into the retinal injury site.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described in detail below in conjunction with the embodiments and with reference to the accompanying drawings. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

Example

1. Preparation of Fe3O4 nanoparticles that adsorb miR-141-3p (1) Material (article number, manufacturer): Fe3O4 nanoparticles (PEI@Fe ₃ O ₄ ) (Beijing Zhongke Leiming); miR-141 -3p (Universal Biosynthesis); Folic Acid (IF0180 Solebol); Fluorescein Isothiocyanate (FITC) (IF8070 Solebol); EDC.HCl (N808856 McLean); NHS (N811124 Meclean). (2) Instruments used (model, manufacturer): magnetic stirring; 5000D dialysis bag; Zetasizer Pro (Malvern Panalytical); transmission electron microscope ((JEM-1230 (80KV), JEOL)); SEM (FEI Quanta250, American FEI company)

(3) Experimental method:

1. Nanoparticles (PEI@Fe ₃ O ₄ ) folate modification and miR-141-3p adsorption

Add 1.44mg NHS and 1.4mg EDC.HCl to 5ml of 1mg/ml iron ferric oxide nanoparticles (purchased from Zhongke Leiming, the stock solution is 1mg/ml aqueous solution), activate at room temperature for 1h, and use a 5000D dialysis bag in an ultrapure water environment Dialyze for 24 hours, change the medium every 4 hours to remove unreacted NHS and 1.4 mg EDC.HCl, collect the liquid after dialysis, add 2 mg of folic acid, stir and mix well, incubate overnight at room temperature, and dialyze again for 24 hours to remove unreacted Folic acid (method as above). The collected liquid was added with 4OD of miR-141-3p, mixed evenly, and incubated overnight at 4°C up and down to obtain the nanoparticles (Fe-Fa/miR-141) with modified folic acid and adsorbed miR-141-3p. Using the same method, the nanoparticles that did not adsorb miR-141-3p in the last step were Fe-Fa nanoparticles.

2. Characterization of Nanoparticles

(1) TEM characterization

After mixing the sample evenly, drop it on the copper grid and let it dry naturally, then observe it with a transmission electron microscope, as shown in Figure 1, the outline of the nanoparticles can be seen clearly.

(2) Characterization of particle size and Zeta potential

After mixing the sample, dilute to an appropriate concentration and use Zetasizer Pro for particle size and Zeta potential detection. The results are shown in Figure 2. The particle size results show that the hydrated particle size of the nanoparticles (PEI@Fe3O4) is about 40nm, and the particle size of the modified folic acid (PEI@Fe3O4-Fa) hardly changes. After further adsorption of miR-141-3p (PEI @Fe3O4-Fa/miR-141-3p), the hydrated particle size is slightly increased, about 50nm. The surface charge of nanoparticles (Zeta potential) shows a downward trend with modification and adsorption, from about +46mV at the beginning to about +30mV.

(3) Gel electrophoresis of miR-141-3p adsorbed by Fe3O4 nanoparticles

The iron ferric oxide nanoparticles and miR-141-3p were mixed according to 0:1, 0.05:1, 0.1:1, 0.2:1, 0.25:1, 0.5:1, 1:1, 1.5:1, 2:1 , mixed at a mass ratio of 2.5:1 (the total volume is 12 μL, the quality of control miR-141-3p is 3 μL and 1 μg/μL, the mass of nanoparticles is added according to the above mass ratio, if it is less than 12 μL, add DEPC water to make up), Incubate for 30 min at room temperature in the dark. Gel electrophoresis observed each band to determine the combination of nanoparticles and miR-141-3p (miR-141-3p molecular weight is 7066).

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

Weigh 0.45 g of agarose, add it into a conical flask containing 30 ml of 1×TAE electrophoresis solution, and mix well. Place the Erlenmeyer flask in a microwave oven until it boils, then take it out, shake it evenly, and repeat 3 times until the agarose is completely dissolved. Add 3 μL of Glodview to the agarose solution cooled to 60°C, shake 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 put the gel block into the electrophoresis solution. Mix the above-incubated sample with 1 μL 6×loading Buffer evenly at a ratio of 5:1, take 5 μL and add it to the sample tank (record the order of sample addition), set the electrophoresis instrument to 80V, and observe miR-141 on the gel imaging system after 20 minutes -3p band condition, that is, the adsorption condition of nanoparticles and miR-141-3p.

According to the electrophoresis results (see Figure 3), the mass concentration of 0.25:1 is at the critical position, and the adsorption and binding are just complete at this time, so the maximum embedding amount of ferric oxide nanoparticles is 1:4, that is, the maximum adsorption capacity is 4 times of its own mass miR-141-3p.

2. Optic nerve clamp induced retinal damage in rats

Model Introduction: Glaucoma is a group of diseases characterized by optic disc atrophy and depression, visual field defect and vision loss. Tolerance is also associated with the onset and progression of glaucoma. Obstacles in any part of the aqueous humor circulation pathway can lead to pathological changes caused by elevated intraocular pressure, but some patients also present with normal intraocular pressure glaucoma. Glaucoma is one of the three major blindness diseases that cause blindness in humans, 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 etiology, anterior chamber angle, and intraocular pressure tracings. In this procedure, the optic nerve is clamped to induce pressure injury to the optic nerve, thereby inducing retinal lesions.

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

2. Experimental materials: lidocaine hydrochloride injection, stereo microscope, surgical scissors, small tweezers, micro tweezers, suture thread, retractor, 8-0 suture with needle, needle holder, micro hemostat, etc.

3. Procedures:

3.1 Anesthesia: After rats were anesthetized with 3% pentobarbital sodium at a dose of 3ml/kg, the operated eye was facing the operator, lidocaine was instilled in the eye, and the rats did not respond after touching the eyelids, followed by subsequent operations.

3.2 Expose the optic nerve: take the inner canthus at 9 o'clock and the outer canthus at 3 o'clock, cut the conjunctiva from the 3-6 o'clock direction of the operated eye, separate it inward, find the white optic nerve, and slightly separate it, as shown in Figure 4 Show.

3.3 Optic nerve clamp: Use toothed fiber hemostats to precisely clamp the position about 0.5 cm behind the optic nerve ball of the rat for 15 seconds, and the clamp strength is to maximize the buckle of the hemostat.

3.4 After clamping, the conjunctiva was sutured with an 8-0 suture needle, and erythromycin ointment was applied to the operated eye (once a day for 3 days after the operation).

4. Model Validation

Fourteen days after operation, the eyeballs were preserved in 10% paraformaldehyde for paraffin section and HE staining.

result:

The retinal structure of the model was disordered, wavy, and the outer nuclear layer was thickened, indicating that the model was successful, as shown in Figure 5.

3. The adsorption of miR-141-3p by iron ferric oxide nanoparticles to retinal damage caused by optic nerve clamping

Inject 2 μl of miR-141-3p adsorbed by iron ferric oxide nanoparticles into the retinal injury site for 14 days (these 14 days only need to be injected once).

Result detection:

HE staining: the structure of the model retina was disordered, wavy, and the outer nuclear layer was thickened; the retina of the treatment group was significantly improved, as shown in Figure 6.

Apparently, the above-mentioned embodiments of the present invention are only examples for illustrating the present invention more clearly, and are not intended to limit the implementation of the present invention. Those of ordinary skill in the art can also Changes or changes in other different forms cannot be exhaustively listed here, and all obvious changes or changes derived from the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims (8)

1. The preparation method of the ferroferric oxide nano-particles for adsorbing miR-141-3p comprises the following steps:

adding NHS and EDC.HCl into the ferroferric oxide nano particles, activating for 1h at room temperature, dialyzing for 24h in an ultrapure water environment, removing unreacted NHS and EDC.HCl, collecting liquid after the dialysis is finished, adding folic acid, fully stirring and uniformly mixing, incubating overnight at room temperature, dialyzing for 24h again, removing unreacted folic acid, collecting liquid, adding miR-141-3p, uniformly mixing, and overturning and incubating overnight at 4 ℃ to obtain the nano particles for adsorbing miR-141-3p.

2. The method of claim 1, wherein the dialysis is performed for 24 hours using a 5000D dialysis bag, and the fluid is changed every 4 hours.

3. The ferroferric oxide nanoparticle adsorbing miR-141-3p prepared by the preparation method of claim 1 or 2.

4. The miR-141-3 p-adsorbed ferroferric oxide nanoparticle of claim 3, wherein the ferroferric oxide nanoparticle is capable of adsorbing a maximum of 4 times its own mass of miR-141-3p.

5. Use of the miR-141-3 p-adsorbed ferroferric oxide nanoparticle of claim 3 in the preparation of a medicament for treating retinal injury.

6. The use of claim 5, comprising injecting miR-141-3 p-adsorbed ferroferric oxide nanoparticles into a retinal injury site for 14 days.

7. The use of claim 6, further comprising constructing a model of optic nerve-clamped rat retinal damage.

8. The use of claim 7, wherein the model construction comprises:

a. anesthesia: after the rats are anesthetized by 3% sodium pentobarbital at a dose of 3ml/kg, the operation eyes face to operators, and the lidocaine drops into eyes, and the rats with the eyelids are subjected to subsequent operation after no reaction;

b. exposing the optic nerve: cutting conjunctiva from 3-6 points of eye with inner canthus of 9 o 'clock and outer canthus of 3 o' clock, separating inwards to find white optic nerve;

c. optical nerve clamp: the toothed fiber hemostatic forceps are adopted, the position of the rat optic nerve sphere is accurately clamped for 15s at the position 0.5cm behind the rat optic nerve sphere, and the clamping strength is that the hemostatic forceps are clamped to the maximum;

d. after the clamping is finished, an 8-0 suture needle with a thread is adopted to suture conjunctiva, erythromycin ointment is smeared on the operation eye, and the operation is carried out once a day for 3 days;

e. 14 days after operation, eyeballs are taken and placed in 10% paraformaldehyde for preservation, and are used for paraffin section and HE staining, so that model retina structural disorder appears in a wavy manner, and the outer nuclear layer is thickened, which indicates that the model is successful.

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