CN112679741B - Polydopamine polyethyleneimine nanoparticle, and preparation and application thereof - Google Patents

Abstract

The invention discloses a polydopamine polyethyleneimine nanoparticle (PDA/PEI NPs) and preparation and application thereof. The polydopamine polyethyleneimine nanoparticle disclosed by the invention can be used for loading nucleic acid (DNA, siRNA, microRNA, shRNA and the like). The invention also discloses a polydopamine polyethyleneimine nanoparticle (PDA/PEI NPs/NA) and a preparation method and application thereof.

Description

Polydopamine polyethyleneimine nanoparticle, and preparation and application thereof

Technical Field

The invention relates to a drug delivery technology in the technical field of drugs, and in particular relates to a polydopamine polyethyleneimine nanoparticle, and preparation and application thereof.

Background

Glaucoma is the leading cause of irreversible blindness in the world. Management of intraocular pressure (IOP) is the most effective method of limiting the progression of glaucoma. The current treatment strategy for glaucoma is to restore the balance between Aqueous Humor (AH) production and drainage. In Primary Open Angle Glaucoma (POAG), the classical outflow pathway is the major resistance site for AH drainage. However, only a few anti-glaucoma drugs have been designed in the prior art to target the conventional outflow pathway.

With the progress of technology, it has become possible to treat diseases by exogenous gene transfer. Viral vectors tend to have a higher gene transfer capacity but may be hampered by complications related to their intrinsic immunogenicity. Non-viral methods of gene delivery, including polymer, lipid-based and inorganic nanoparticles, as well as physical delivery techniques, have also been extensively studied.

Ocular gene therapy has entered a rapidly growing era, however, unless Nucleic Acids (NA) are complexed with other chemical molecules or physically forced into cells and introduced into the nucleus, the ability of naked nucleic acid molecules to induce cells is often ineffective. Although viral vectors are currently the best choice for replacing and/or correcting genes, there has been much research in treating ocular fundus genetic diseases, but improvements will still be sought to improve transduction efficiency and reduce iatrogenic risks. Compared with non-viral vectors (naked plasmid DNA, oligonucleotide and RNA), the viral vector has better safety (low immunogenicity and low risk of insertion mutation), larger drug loading capacity, repeated drug delivery potential and easier mass production.

Currently available vectors or transfection methods for nucleic acids include viral vectors, other non-viral vectors (e.g., liposomes, cationic polymers), liposomes; local electroporation transfection. Among them, viral vectors are widely adopted as a means for gene delivery, but there are limitations that viral vectors induce a certain degree of immune response in the body, and there are risks of tumorigenesis and toxicity such as insertional mutagenesis. Non-viral vectors: the gene transfer and expression efficiency is low, the targeting is also low, the expression effect of the introduced gene is short, and the curative effect is also reduced. The electroporation characteristics are influenced by various factors, such as voltage, electroporation times and time, temperature, DNA state and concentration, etc., and the damage to tissues caused by the poor parameter setting is certain. The adverse reactions caused by electroporation are mainly local cell damage and inflammatory reactions. Too high a voltage may cause cell death or tissue dysfunction, and too many electroporation events may cause irreversible damage to local tissues.

microRNA (miRNA) is an important regulation loop in a complex biological process, and miR-21-5p has the function of changing the resistance of the traditional outflow pathway. However, the important problem of transfection is faced by the research and future clinical transformation of nucleic acid which is transferred into in vitro eye cells and in vivo eyes.

Because the eyes are isolated from the whole body immune system by a barrier, and the blood flow and the metabolism of the eyes are kept at a higher level, the intraocular drug diffusion mode is more complicated and has more unknown property; in addition, the eyes are one of the finest structures of the whole body, and once damaged, the eyes are easy to cause irreversible blindness, which greatly influences the life quality of patients. Therefore, more careful vector selection is required for gene therapy. There is therefore a need to develop safer and more effective ocular delivery systems for nucleic acid drugs.

Disclosure of Invention

The invention provides a high-molecular polymer nano particle which can be applied to intraocular transfection therapy, the high-molecular polymer nano particle can be stably dispersed in a solution for a long time, surface bearing is not needed, the storage is convenient, the high-molecular polymer nano particle can simply and effectively load nucleic acid, the obtained polydopamine polyethyleneimine nano particle nucleic acid compound can be stably stored for a long time at 4 ℃, the polydopamine polyethyleneimine nano particle nucleic acid compound can be effectively delivered to eyes, the cell-targeted sustained-release administration is realized, and the delivery system has almost no immune reaction.

More specifically, the first technical problem to be solved by the present invention is: provides a preparation method of polydopamine polyethyleneimine nanoparticles (PDA/PEI NPs).

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a preparation method of polydopamine polyethyleneimine nanoparticles comprises the following steps:

(1) preparation of Polydopamine (PDA) solution: dissolving dopamine in water, and heating; adding an aqueous solution of alkali, and carrying out self-polymerization reaction to form a polydopamine solution;

(2) synthesis of poly-dopamine-polyethyleneimine nanoparticles (PDA/PEI NPs); adding polyethyleneimine into the polydopamine solution obtained in the step (1), performing Schiff base reaction and Michael addition reaction, and then purifying to obtain polydopamine polyethyleneimine nanoparticles;

in the step (1), the heating temperature is 40-60 ℃; the alkali is NaOH, and the molar concentration of the alkali in water is 5-8 mmol/L; the feeding mass ratio of the dopamine to the alkali is 5-10: 1; the time of the self-polymerization reaction is 1-6 hours; the temperature of the self-polymerization reaction is 20-70 ℃;

in the step (2), the polyethyleneimine is branched polyethyleneimine, the molecular weight of the polyethyleneimine is 5-30kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 10: 1-1: 10.

in a preferred embodiment of the invention:

in the step (1), the feeding mass ratio of the dopamine to the alkali is 7.4: 1; the molar concentration of the alkali in water is 6.8 mmol/L; the temperature of the self-polymerization reaction is 45-55 ℃;

in the step (2), the molecular weight of the polyethyleneimine is 25kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 2: 1-1: 2.

the second technical problem to be solved by the invention is to provide polydopamine polyethyleneimine nanoparticles.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: the polydopamine polyethyleneimine nanoparticle prepared by the method is provided.

The third technical problem to be solved by the invention is to provide the application of the polydopamine polyethyleneimine nanoparticle in preparing the medicines for treating the eye diseases.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: the invention relates to application of polydopamine polyethyleneimine nanoparticles in preparation of drugs for treating eye diseases. The eye diseases are various open angle glaucoma, angle closure glaucoma, high intraocular pressure, normal tension glaucoma, headache, optic atrophy and optic nerve degenerative changes which take intraocular pressure reduction as the only treatment means.

The fourth technical problem to be solved by the invention is to provide a polydopamine polyethyleneimine nanoparticle nucleic acid complex (PDA/PEINPs/NA).

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a method for preparing polydopamine polyethyleneimine nanoparticle nucleic acid complexes (PDA/PEINPs/NA), comprising the following steps:

(1) preparation of polydopamine solution: dissolving dopamine in water, and heating; adding an aqueous solution of alkali, and performing self-polymerization reaction to form a polydopamine solution;

(2) synthesizing polydopamine polyethyleneimine nanoparticles; adding polyethyleneimine into the polydopamine solution obtained in the step (1) to perform Schiff base reaction and Michael addition reaction, and then purifying to obtain polydopamine polyethyleneimine nanoparticles;

(3) synthesis of PDA/PEI NPs/NA: dissolving the polydopamine ethylene imine nano-particles obtained in the step (2) in sterilized water, adding nucleic acid, and uniformly mixing;

in the step (1), the heating temperature is 40-60 ℃; preferably 50 ℃; the alkali is NaOH, and the molar concentration of the alkali in water is 5-8 mmol/L; the feeding mass ratio of the dopamine to the alkali is 5-10: 1; the time of the self-polymerization reaction is 1-6 hours; the temperature of the self-polymerization reaction is 20-70 ℃;

in the step (2), the polyethyleneimine is branched polyethyleneimine, the molecular weight of the polyethyleneimine is 5-30kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 10: 1-1: 10;

in the step (3), the mass ratio of the polydopamine ethylene imine nanoparticles to the nucleic acid is 1: 1-100: 1; the concentration of the polydopamine ethylene imine nano-particles in the sterilized water is 0.1-1000 mg/ml.

In a preferred embodiment of the present invention, in step (1), the dopamine and base are fed in a mass ratio of 7.4: 1; the molar concentration of the alkali in water is 6.8 mmol/L; the temperature of the self-polymerization reaction is 45-55 ℃;

in the step (2), the molecular weight of the polyethyleneimine is 25kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 2: 1-1: 2;

in the step (3), the mass ratio of the polydopamine ethylene imine nanoparticles to the nucleic acid is 5: 1-20: 1; the concentration of the polydopamine ethylene imine nano-particles in the sterilized water is 1-10 mg/ml.

The nucleic acid of the invention can be selected from DNA, siRNA, microRNA or shRNA.

In some embodiments of the invention, the microRNA is miR-21-5 p.

The fifth technical problem to be solved by the invention is to provide a polydopamine polyethyleneimine nanoparticle nucleic acid complex.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: the polydopamine polyethyleneimine nanoparticle nucleic acid compound prepared by the method is provided.

The PDA/PEI NPs/NA prepared in some embodiments of the invention is PDA/PEI NPs/miR-21-5p, PDA/PEI NPs/NC, PDA/PEI NPs/pGL-3.

The sixth technical problem to be solved by the invention is to provide an application of the polydopamine polyethyleneimine nanoparticle nucleic acid complex in preparation of a medicine for treating eye diseases.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: the invention relates to application of a polydopamine polyethyleneimine nanoparticle nucleic acid complex in preparation of a medicine for treating eye diseases. The eye diseases include various open angle glaucoma, angle closure glaucoma, ocular hypertension, normal tension glaucoma, headache, optic atrophy and optic nerve degenerative disease caused by ocular hypertension.

The invention discloses PDA/PEI NPs as miRNA delivery nanoparticles for intraocular transfection for the first time. PDA/PEI NPs not only enhance the stability of target genetic materials, but also have transfection capacity equivalent to that of commercial vectors; there was no significant statistical difference in transfection efficiency with the same amount of nucleic acid transfected by liposomes (lipofectamin). More importantly, it showed a rather low cytotoxicity, 1 and 2 fold increase in cell activity compared to transfection of the same amount of nucleic acid with commercially available liposomes and PEI, respectively, which is a good choice for primary cell transfection.

The PDA/PEI NPs of the invention are easy to store, at least for one year in cold storage (4 ℃). The stability of the in vivo transfection liposome reagent which is commonly used at present is far lower than that of the PDA/PEI NPs prepared by the invention. For example, the commercial in vivo transfection liposome reagent, invivofectamin, must be stored frozen (-20 ℃), and the activity is affected when the number of thawing times exceeds 4. The more commonly used lentiviruses for intraocular transfection require ultra low temperature (-80 ℃) storage and likewise a 70% reduction in activity to the first every thaw.

The method for forming the compound by using the PDA/PEI NPs to carry the nucleic acid is relatively simple, and for nucleic acid fragments, the PDA/PEI NPs carrier and the nucleic acid are uniformly mixed and are kept still for about 1 hour, so that the compound can be immediately used for in vitro cells or in vivo tissues. While the commercial liposome needs to be incubated in an additional serum-free medium, the virus vector needs to be constructed in advance to be matched with the virus. The currently commonly used commercial reagent, invivofectamin, typically requires incubation at 50 degrees celsius for 30 minutes.

The nucleic acid of the invention can be selected from DNA, siRNA, microRNA or shRNA.

In some embodiments of the invention, the nucleic acid is miR-21-5p, and the control nucleic acid NC with the same length of non-functional sequence and the commonly used plasmid vector pGL-3.

Transfection of PDA/PEI NPs/miR-21-5p in porcine aqueous humor veins from endothelial cells (AAP) showed a decrease in monolayer permeability with concomitant redistribution of the cytoskeleton. In vivo studies showed that mice injected intracamerally with PDA/PEI NPs/NC had good biocompatibility with no evidence of significant toxicity or inflammatory response. Tissue distribution studies showed shed tissue and corneal accumulation. By promoting the traditional efflux pathway, overexpression of miR-21-5p in efflux tissues can significantly reduce IOP.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

Small RNA (MicroRNA, miRNA) is a small molecule highly conserved nucleic acid fragment (19-23bp), widely exists in organisms, and plays an important role in regulation of cell proliferation, differentiation, metabolism, functions and the like. One miRNA can be combined with various mRNAs to act, so that the corresponding mRNAs are degraded or silenced and expressed, the level of endogenous protein is changed to regulate the biological function, the aim of regulating different paths by multiple targets is fulfilled, and side effects caused by exogenous administration are reduced. At present, viruses or nano materials are used, so that miRNA can be stably up-regulated or down-regulated within a certain time, and can play a certain biological regulation function within a longer time. Therefore, the side effects caused by repeated administration for a plurality of times due to short effective time of the traditional glaucoma medicament can be solved.

Small interfering RNA (siRNA), sometimes referred to as short interfering RNA (short interfering RNA) or silencing RNA (silencing RNA), is a double stranded RNA of 20 to 25 nucleotides in length that has many different biological applications. It is known that siRNA is mainly involved in the phenomenon of RNA interference (RNAi) and regulates the expression of genes in a specific manner. In addition, they are involved in some response pathways related to RNAi, such as antiviral mechanisms or changes in chromatin structure. However, the reaction pathways of these complex mechanisms are not known at present.

Short hairpin RNA, (short hairpin RNA, shRNA is an abbreviation). The shRNA comprises two short inverted repeats separated by a stem loop (loop) sequence, forming a hairpin structure, controlled by a pol III promoter. Then 5-6T are connected as transcription terminator of RNA polymerase III.

In the invention, the PDA/PEI NPs/nucleic acid complex, the PDA/PEI NPs/NA, the polydopamine polyethyleneimine nucleic acid nano complex and the polydopamine polyethyleneimine nanoparticle nucleic acid complex have the same meaning.

pGL-3 is a DNA plasmid containing a luciferase reporter gene, and is a commonly used vector plasmid for carrying a target gene.

NC is RNA which is determined to be nonfunctional, has the same length as miR-21-5p and only has different sequences.

InviVofectamine is a commercially available in vivo transfection liposome reagent, used in the examples as InviVofectamine 3.0.

Lipofectamin is a commercially available cell transfection liposome reagent, Lipofectamin2000 being used in the examples, and is a cell transfection reagent commonly used in laboratories.

Has the advantages that:

1. the invention uses PDA/PEI NPs as independent nucleic acid vectors for intraocular therapy for the first time.

2. The PDA/PEI NPs prepared by the method can be stably dispersed in the solution without surface bearing, so that foreign matters are not required to be implanted in eyes. The PDA/PEI NPs prepared by the method can form a spherical polymer more stably and adsorb more nucleic acid.

3. The PDA/PEI NPs/nucleic acid compound is simple to prepare, and the PDA/PEI NPs and the nucleic acid can be used after being uniformly mixed; the PDA/PEI NPs/nucleic acid complex prepared by the invention can be stored stably at 4 ℃ for a long time, and has high stability; low toxicity, biodegradability and almost no immune response.

Drawings

FIG. 1 is a diagram of the synthesis reaction of the present invention.

FIG. 2 scanning electron microscope of example 1, wherein FIG. 2a shows the PDA/PEI NPs prepared in step (2) of example 1 or 2, and FIG. 2b shows the PDA/PEI NPs/pGL-3 complex in step (3) of example 1; FIG. 2c is the PDA/PEI NPs/NC complex prepared in step (3) of example 2; under the observation of an electron microscope, the complexes of the vector PDA/PEI NPs, the vector PDA/PEI NPs/pGL-3 full of nucleic acid and the PDA/PEI NPs/NC show circular dispersion distribution.

FIG. 3 comparison of cell Activity after transfection of cells with different concentrations of PDA/PEI NPs/NC complexes in example 4

FIG. 4 Effect of different transfection reagents on cell Activity of transfection of the same amount of NC in example 4

FIG. 5 transfection ability of PDA/PEI NPs/pGL-3 with the commonly used cell transfection reagent lipofectamin2000 in example 5

FIG. 6 particle diameters of PDA/PEI NPs/pGL-3 and PDA/PEI NPs/NC in example 6 within seven days

FIG. 7 comparison of transfection efficiency after 2, 4, 6 days storage of PDA/PEI NPs/pGL-3 in example 6 with 0 days

FIG. 8 comparison of transfection efficiencies after 0, 2, 4, and 6 days of storage of PDA/PEI NPs/pGL-3 in example 6 with those after the same time period as lipofectamine storage

FIG. 9 mapping of fluorescent particles in the cytoplasm of cells after transfection of AAP cells in example 7

FIG. 10 intracellular fold overexpression corresponding to different transfection concentrations in example 7

FIG. 11 is a graph of transmembrane resistance after transfection of PDA/PEI NPs/miR-21-5p in example 7.

FIG. 12 graph of the absence of inflammatory cell infiltration 1 day after intraocular transfection in example 8.

FIG. 13 fluorescence expression pattern of no inflammatory factors 1 day after intraocular transfection in example 8.

FIG. 14 is a graph showing the expression of mRNA without inflammatory factors 1 day after intraocular transfection in example 8.

FIG. 15 in an in vivo experiment in example 9, FIG. 15a shows that the PDA/PEI NPs distribution is concentrated in the cornea and shed tissue in the anterior chamber, and FIG. 15b shows that miR-21-5P is significantly overexpressed in vivo.

Figure 16 post-miRNA vector in intraocular transfection IOP measurement in example 9. FIG. 16a shows that PDA/PEI NPs/miR-21-5p has a significant ocular hypotensive effect, and FIGS. 16b, 16c show that this effect is exerted by increasing outflow through the traditional pathway.

FIG. 17 stability testing of PDA/PEI NPs/miRNA solutions.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.

The starting materials or reagents used in the examples of the present invention are commercially available unless otherwise specified. Mainly purchased from the following companies:

reagent	Raw materials supplier
		Dopamine	Sigma-Aldrich
Polyethylene imine	Sigma-Aldrich
		mirVana miRNA mimics	Thermo
Lipofectamine
	2000	Thermo
pGL-3			Kinseruit

The abbreviations used in the present invention have the usual meaning in the art, for example the following abbreviations have the following meanings:

example 1: preparation of PDA/PEI NPs/pGL-3

(1) Preparation of Polydopamine (PDA) solution.

Dopamine (100.6mg) was dissolved in 50mL of water and heated to 50 ℃ while stirring. In the alkaline or neutral pH environment, dopamine will self-polymerize into polydopamine, 0.34mL NaOH (1mol/L) is added, and the reaction is carried out for 3.5 hours.

(2) Synthesis of Polydopamine polyethyleneimine nanoparticles (PDA/PEI NPs)

PEI (100mg, molecular mass 25000) was added to the solution obtained in step (1) and the reaction was continued for 2 hours. Raw material dopamine: the mass ratio of PEI is 1: 1. PDA/PEI NPs were then obtained by ultrafiltration at 4500rpm using an ultrafiltration tube (50k) and washed several times. PDA/PEI NPs were lyophilized and then further characterized by observation.

As shown in FIG. 2a, under a scanning electron microscope, the obtained PDA/PEI NPs are spherical in morphology and well dispersed (FIG. 2 a). The average size of PDA/PEI NPs was determined to be 209 nm. Due to the modification of PEI, these nanoparticles were positively charged with a surface potential of about 26 mV. Meanwhile, carbon was measured by elemental analysis: the mass fraction of nitrogen was 2.2. From this, the actual mass ratio of PDA to PEI in PDA/PEI NPs can be calculated as 1: 2.5. based on the FT-IR spectrum structural analysis, it was shown that C ═ N bonds (peak at 1660 cm-1) were formed in PDA/PEI NPs due to the reaction between amino groups in PDA and PEI, indicating the successful synthesis of PDA/PEI NPs.

(3) Synthesis of PDA/PEI NPs/pGL-3 plasmid (DNA)

Preparing the polydopamine ethylene imine nanoparticles obtained in the step (2) into a mother solution with the concentration of 5mg/ml, and mixing PDA/PEI NPs and nucleic acid pGL-3 plasmid (DNA) in a mass ratio of 10: 1, mixing, ultrasonically mixing for 30 minutes, standing for 2 hours, and diluting to a proper concentration by using sterilized PBS.

Characterization of the PDA/PEI NPs/pGL-3 plasmid

Since PDA/PEI NPs are positively charged, the negatively charged nucleic acids can be condensed by electrostatic forces. The PDA/PEI NPs/pGL-3 plasmid was also well dispersed in spherical form (FIG. 2 b). The size and surface charge of those PDA/PEI NPs/nucleic acid complexes were studied using DLS.

The size of the PDA/PEI NPs/pGL-3 plasmid was about 240nm, with a narrow size distribution, slightly larger than the size of the PDA/PEI NPs, indicating that the nucleic acid has been loaded in these complexes. Also, the results show that the complexes obtained remain dispersed after loading with nucleic acid and they do not aggregate during complex formation, as evidenced by TEM observation. In addition, the potential of these complexes was lower than that of PDA/PEI NPs, which was approximately 21 mV. The positive surface charge of these complexes can help them interact with negatively charged cell membranes, promoting cell internalization and gene transfection.

Example 2: preparation of PDA/PEI NPs/NC

(1) Preparation of Polydopamine (PDA) solution.

Dopamine (100.6mg) was dissolved in 50mL of water and heated to 50 ℃ while stirring. Under alkaline pH environment, dopamine will self-polymerize into poly-dopamine, 0.34mL NaOH (1mol/L) is added, and reaction is carried out for 6 hours.

(2) Synthesis of Polydopamine polyethyleneimine nanoparticles (PDA/PEI NPs)

PEI (100mg, molecular mass 25000) was added to the solution obtained in step (1) and the reaction was continued for 2 hours. Raw material dopamine: the mass ratio of PEI is 1: 1. PDA/PEI NPs were then obtained by ultrafiltration at 4500rpm using an ultrafiltration tube (50k) and washed several times. PDA/PEI NPs were lyophilized and then further characterized by observation.

(3) Synthesis of PDA/PEI NPs/NC

Preparing the polydopamine ethylene imine nanoparticles obtained in the step (2) into a mother solution with the concentration of 5mg/ml, and mixing PDA/PEI NPs and NC in a mass ratio of 10: 1, mixing, ultrasonically mixing for 30 minutes, standing for 2 hours, and diluting to a proper concentration by using sterilized PBS.

Also, the PDA/PEI NPs/NC are well dispersed in spherical form (FIG. 2c), about 240nm, have a narrower size distribution, slightly larger than the PDA/PEI NPs, and are the same size as the PDA/PEI NPs/pGL-3 plasmids. Also, the results show that the complexes obtained after loading with RNA-based nucleic acids remain dispersed and do not aggregate, as evidenced by TEM observations. In addition, the potential of PDA/PEI NPs/NC, which is the same as the PDA/PEI NPs/pGL-3 plasmid, is lower than that of PDA/PEI NP.

Example 3: preparation of PDA/PEI NPs/miR-21-5p

(1) Preparation of Polydopamine (PDA) solution.

Dopamine (100.6mg) was dissolved in 50mL of water and heated to 50 ℃ while stirring. Under alkaline pH environment, dopamine will self-polymerize into poly-dopamine, 0.34mL NaOH (1mol/L) is added, and reaction is carried out for 6 hours.

(2) Synthesis of Polydopamine polyethyleneimine nanoparticles (PDA/PEI NPs)

PEI (100mg, molecular mass 25000) was added to the solution obtained in step (1) and the reaction was continued for 2 hours. Raw material dopamine: the mass ratio of PEI is 1: 1. PDA/PEI NPs were then obtained by ultrafiltration at 4500rpm using an ultrafiltration tube (50k) and washed several times. PDA/PEI NPs were lyophilized and then further characterized by observation.

(3) Synthesis of PDA/PEI NPs/miR-21-5p

Preparing the polydopamine ethylene imine nano-particles obtained in the step (2) into mother liquor with the concentration of 5mg/ml, and mixing PDA/PEI NPs and miR-21-5p in a mass ratio of 10: 1, sonicate for 30 minutes, rest for 2 hours, dilute to the appropriate concentration with sterile PBS for use in the subsequent examples.

Example 4: in vitro toxicity test

The cytotoxicity of PDA/PEI NPs/NC was studied by the 5-diphenyl-2-H-tetrazole bromide (MTT) method. Commercial transfection reagents lipofectamine 2000 and polyethyleneimine (PEI, Mw ═ 25kD) were used as references. In addition, the viability of corneal epithelial and endothelial cells, which are important in the homeostasis of the ocular surface and dioptric system and susceptible to topical administration, was studied.

As shown in the results of fig. 3, in the concentration gradient cell viability test, even though the nucleic acid concentration reached 50 μ g/mL (n ═ 5), cell viability remained higher than 80% after treatment with various concentrations of PDA/PEI NPs/NC complexes. Furthermore, as shown in the results of fig. 4, AAP cell viability was still higher than 88% after treatment with PDA/PEI NPs/NC complexes, much higher than with PEI/NC (45%) and lipofectamine 2000/NC complexes (37%) (n ═ 5 per group, mean p <0.001, t test). With respect to the effects on corneal endothelial and epithelial cells, viability did not drop significantly until a maximum of 50 μ g/mL cells was reached in corneal epithelial cells. These results indicate that the PDA/PEI NPs/nucleic acid complexes have relatively low cytotoxicity, which confirms their safety for further use.

Example 5: in vitro gene transfection assay

The gene transfection ability of PDA/PEI NPs in AAP cells was evaluated by luciferase reporter gene analysis using pGL-3 nucleic acids, and Lipofectamine 2000 and PEI were used as references. PEI has the highest transfection efficiency as a gold standard for gene delivery. As a result, as shown in FIG. 5, PDA/PEI NP and lipofectamine 2000 showed similar slightly lower transfection ability, indicating that PDA/PEI NP had sufficient gene transfection ability. Compared with PEI and lipofectamine 2000, the PDA/PEI NPs have obviously lower cytotoxicity and acceptable transfection efficiency, and the PDA/PEI NPs have huge application potential as nucleic acid vectors.

Example 6: in vitro stability assay

After loading with nucleic acids, the ideal gene vector must remain highly efficient in transfection while retaining the biological activity of the genetic material it carries. Therefore, we investigated the stability of PDA/PEI NPs/nucleic acid complexes using DLS and tested the gene transfection in vitro after storing these complexes for different days. The size of the PDA/PEI NPs/nucleic acid complexes remained stable 7 days after complex preparation (FIG. 6), indicating that these complexes did not aggregate during storage. And in the gene transfection assay, although the transfection results of the PDA/PEI NPs/pGL-3 complex slightly decreased after 4 days of storage (FIG. 7), they still showed similar transfection ability to lipofectamine 2000/pGL-3 (FIG. 8). These results indicate that the PDA/PEI NPs/nucleic acid complex can retain its efficient transfection capacity, which is crucial for further applications.

Example 7: intracellular drug distribution and function

To visualize the distribution of particles in the cells, Fluorescein Isothiocyanate (FITC) -labeled PDA/PEI NP was used. In the AAP cells of FIG. 9, both granules accumulated within the cytoplasm and near the nucleus 24 hours after incubation with PDA/PEI NPs/FITC, indicating that these granules had been successfully taken up by the cells.

To further quantify the cell transfection efficiency, three different concentrations (2, 4 and 8 μ g/ml) of PDA/PEI NPs/miR-21-5p were used to study the efficacy of miRNA overexpression (figure 10). At 24 hours post-transfection, 2, 4 and 8 μ g/ml PDA/PEI NPs/miR-21-5P significantly increased intracellular miR-21-5P levels by 14.92, 20.24 and 32.58 fold (n ═ 3, P <0.001, t-test). It is often difficult to transfer nucleic acids into primary cells. However, in this study, PDA/PEI NPs showed excellent transfection performance in primary AAP cells.

We further investigated whether rearranged cytoskeleton after transfection of PDA/PEI NPs/miR-21-5p eventually leads to altered AAP cell permeability. Transendothelial electrical resistance (TEER) was measured, a physical concept reflecting endothelial barrier permeability. A monolayer of endothelial cells can act as a resistor in the complete circuit. Thus, the electrical resistance value may reflect the permeability of endothelial cells. Lower TEER indicates higher cell permeability. In FIG. 11, TEER decreased significantly from 25.20. + -. 0.62. omega. cm2 to 17.50. + -. 0.27. omega. cm2 (n. about.5, p <0.001, t-test) 24 hours after miR-21-5p transfection. This indicates that the permeability of AAP cells is significantly increased by miR-21-5p overexpression.

Example 8: in vivo biotoxicity assay

We further investigated the biocompatibility of PDA/PEI NPs in vivo after intracameral injection. The reaction of PDA/PEI NPs/NC treated eyes was evaluated by slit lamp, Hematoxylin Eosin (HE) staining, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). By using untreated contralateral eyes as controls, no significant intraocular inflammation was observed under the slit lamp. In particular, local corneal haze indicates transient edema near the puncture site and disappears within 72 hours. Dilated vessels appeared near the limbus at 24 hours and returned to normal appearance at 48 hours. During the observation, the anterior chamber, pupil and lens were unaffected. As shown in fig. 12HE staining, no gross structural abnormalities or inflammatory cell infiltration were found in the anterior chamber tissue.

To further investigate the biocompatibility of NC loaded PDA/PEI NPs solutions, inflammatory markers of anterior segment uveitis were detected by immunofluorescence and qRT-PCR. S100A8 and S100a9 are active intraocular inflammation biomarkers. One of S100A8 was demonstrated to promote inflammatory cell migration and infiltration in the anterior segment tissues, suggesting acute inflammation. The anterior segment of the inflammatory marker S100A8/A9 stained negative compared to the positive control (mouse skin) (FIG. 13). Meanwhile, PCR results showed that mRNA of S100A8 had no statistical significance in corneal, iris and anterior chamber angle tissues compared to the control group (fig. 14, n-3, p >0.05, paired t-test). The two are combined, and our results show that PDA/PEI NPs are relatively safe for intraocular use in mice.

Example 9: in vivo transfection and biological function

Since the safety and transfection ability of PDA/PEI NPs were confirmed by in vitro studies, the transfection ability and function in vivo was investigated by delivering loaded PDA/PEI NPs particles by intracameral injection. First, the distribution profile of PDA/PEI NPs particles in the eye showed that fluorescence signals were observed in the anterior chamber angle, cornea and ciliary body 24 hours after injection of FITC-labeled PDA/PEI NPs, indicating successful endocytosis of the nanoparticles by cells in vivo (figure 15 a). Specifically, the fluorescence intensity is higher in the effusive tissue, corneal endothelium and stroma, and lower in the iris and ciliary body. To quantify the over-expression capacity of miR-21-5p in regular efflux tissues, qRT-PCR was performed. In dissected shed tissues, miR-21-5P increased significantly by a factor of 2.08 after 24 hours of transfection (FIG. 15b, 0.3. mu.g miR-21-5P or NC per eye, n. gtoreq.3, P <0.05, paired t-test).

After verifying whether PDA/PEI NPs are miRNA vectors in intraocular transfection, we subsequently measured IOP to explore functional changes. Figure 16a results show that the mean IOP of the control and contralateral PDA/PEI NPs/miR-21-5p injected eyes before treatment were (14.2 ± 0.7) mmHg and (14.2 ± 0.9) mmHg (n ═ 10, paired t-test), respectively. At 6 hours post-injection, the average IOP for PDA/PEI NPs/miR-21-5p was significantly reduced by 12.9% of PDA/PEI NPs/NC eyes. A statistically significant IOP reduction persists for at least 48 hours, with the greatest reduction occurring at 8 hours (13.7%) post-injection. After 72 hours, the intraocular pressure in the contralateral eye was restored to the same level. Our data indicate that IOP can be reduced by intracameral injection of PDA/PEI NPs/miR-21-5 p.

Outflow rate was then studied by eye perfusion of the enucleated eye. As in fig. 16b, c, flow rate and conventional efflux were significantly increased in miR-21-5p over-expressed eyes (n ═ 10, paired t test). The conventional efflux data is consistent with an increase in mouse AAP cell permeability and a decrease in IOP. These results therefore support the following notions: intracameral injection of PDA/PEI NPs/miR-21-5p successfully lowers IOP by targeting Schlemm's endothelial cells via conventional efflux pathways.

The stability of the PDA/PEI NPs/miRNA solutions was tested in vivo by IOP measurement using the stored solutions (3 days and 7 days). The results in figure 17 show that the stored solutions also have the ability to lower IOP (n-6 for both sets, paired t-test). The maximum intraocular pressure reduction 8 hours after administration was 13.77% (3 days) and 13.08% (7 days), almost the same as the compound prepared on site. The significant IOP reduction for at least 24 hours for both storage solutions demonstrated the ideal preservation effect of PDA/PEI NPs on miR-21-5 p.

In summary, we successfully designed and produced PDA/PEI NPs as miRNA delivery nanoparticles for intraocular transfection. PDA/PEI NPs not only enhance the stability of the target genetic material, but also have transfection capabilities comparable to commercial vectors. More importantly, it shows a rather low cytotoxicity, a good choice for transfection of primary cells compared to commercially available liposomes and PEI. Transfection of PDA/PEI NPs/miR-21-5p in AAP cells showed a decrease in monolayer permeability with a concomitant redistribution of the cytoskeleton. In vivo studies showed that mice injected intracamerally with PDA/PEI NPs/NC had good biocompatibility with no evidence of significant toxicity or inflammatory response. Tissue distribution studies showed shed tissue and corneal accumulation. By promoting the traditional efflux pathway, overexpression of miR-21-5p in efflux tissues shows lower IOP. Therefore, PDA/PEI NPs/miR-21-5p may be a successful anti-glaucoma drug that increases aqueous humor drainage. PDA/PEI NPs may be promising nucleic acid nanocarriers in other intraocular applications.

Claims (8)

1. The application of polydopamine polyethyleneimine nanoparticles in preparing a medicament for treating eye diseases comprises the following steps:

(1) preparation of polydopamine solution: dissolving dopamine in water, and heating; adding an aqueous solution of alkali, and performing self-polymerization reaction to form a polydopamine solution;

(2) synthesizing polydopamine polyethyleneimine nanoparticles; adding polyethyleneimine into the polydopamine solution obtained in the step (1), performing Schiff base reaction and Michael addition reaction, and then purifying to obtain polydopamine polyethyleneimine nanoparticles;

in the step (1), the heating temperature is 40-60 ℃; the alkali is NaOH, and the molar concentration of the alkali in water is 5-8 mmol/L; the feeding mass ratio of the dopamine to the alkali is 5-10: 1; the time of the self-polymerization reaction is 1-6 hours, and the temperature of the self-polymerization reaction is 20-70 ℃;

in the step (2), the polyethyleneimine is branched polyethyleneimine, the molecular weight of the polyethyleneimine is 5-30kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 10: 1-1: 10.

2. the use according to claim 1, wherein in step (1), the dopamine and base are fed in a mass ratio of 7.4: 1; the molar concentration of the alkali in water is 6.8 mmol/L; the temperature of the self-polymerization reaction is 45-55 ℃;

in the step (2), the molecular weight of the polyethyleneimine is 25kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 2: 1-1: 2.

3. use according to claim 1 or 2, characterized in that the ocular diseases are open angle glaucoma due to ocular hypertension, angle-closure glaucoma, ocular hypertension, headache, optic atrophy and optic neurodegeneration.

4. Use according to claim 1 or 2, wherein the ocular disease is normal tension glaucoma.

5. The application of a polydopamine-polyethyleneimine nanoparticle nucleic acid complex in preparing a medicament for treating eye diseases comprises the following steps:

(1) preparation of polydopamine solution: dissolving dopamine in water, and heating; adding an aqueous solution of alkali, and carrying out self-polymerization reaction to form a polydopamine solution;

(2) synthesis of poly-dopamine polyethyleneimine nanoparticles: adding polyethyleneimine into the polydopamine solution obtained in the step (1) to perform Schiff base reaction and Michael addition reaction, and then purifying to obtain polydopamine polyethyleneimine nanoparticles;

(3) synthesis of PDA/PEINPs/nucleic acid complexes: dissolving the polydopamine polyethyleneimine nanoparticles obtained in the step II in sterilized water, adding nucleic acid, and uniformly mixing;

in the step (1), the heating temperature is 40-60 ℃; the alkali is NaOH, and the molar concentration of the alkali in water is 5-8 mmol/L; the feeding mass ratio of the dopamine to the alkali is 5-10: 1; the time of the self-polymerization reaction is 1-6 hours, and the temperature of the self-polymerization reaction is 20-70 ℃;

in the step (2), the polyethyleneimine is branched polyethyleneimine, the molecular weight of the polyethyleneimine is 5-30kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 10: 1-1: 10;

in the step (3), the mass ratio of the polydopamine polyethyleneimine nanoparticles to the nucleic acid is 1: 1-100: 1; the concentration of the polydopamine polyethyleneimine nanoparticles in sterilized water is 0.1-1000 mg/ml;

the nucleic acid is miR-21-5P.

6. Use according to claim 5,

in the step (1), the feeding mass ratio of the dopamine to the alkali is 7.4: 1; the molar concentration of the alkali in water is 6.8 mmol/L; the temperature of the self-polymerization reaction is 45-55 ℃;

in the step (2), the molecular weight of the polyethyleneimine is 25kDa, and the mass ratio of the polydopamine to the polyethyleneimine is 2: 1-1: 2;

in the step (3), the mass ratio of the polydopamine polyethyleneimine nanoparticles to the nucleic acid is 5: 1-20: 1; the concentration of the polydopamine polyethyleneimine nanoparticles in the sterilized water is 1-10 mg/ml.

7. Use according to claim 5 or 6, characterized in that the ocular diseases are open angle glaucoma due to ocular hypertension, angle-closure glaucoma, ocular hypertension, headache, optic atrophy and optic neurodegeneration.

8. Use according to claim 5 or 6, wherein the ocular disease is normal tension glaucoma.

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