Delivery systems containing silicon nanoparticles

Abstract

To provide effective, safe and non-invasive means of delivering siRNA and mRNA to body sites including but not limited to the cornea.SOLUTION: A composition for controlled release of a nucleic acid such as short interfering RNA or messenger RNA comprises silicon nanoparticles, at least one amino acid, and at least one lipid, where the silicon nanoparticles comprise at least 50 wt.% of silicon. Also disclosed are related compositions and methods.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a delivery system for the delivery of bioactive agents. More specifically, but not exclusively, the invention relates to the use of silicon nanoparticles for the delivery of short interfering RNA, messenger RNA, or other nucleic acids. The invention also relates to related compositions. [Background technology]

[0002] Due to its specificity, gene therapy has attracted considerable interest as an approach to treating genetic diseases. Short interfering RNAs (also known as small interfering RNAs or siRNAs) can be designed to target virtually any gene, holding great potential for disease treatment. It is clear that siRNA technology has great therapeutic potential in medicine. However, one of the major limitations in its in vivo and in vitro applications is that siRNA molecules cannot cross the cell membrane and reach the cytoplasm. The negative charges arising from the phosphate groups on the siRNA backbone electrostatically repel the negatively charged cell membrane, preventing siRNA from diffusing across the cell membrane. Furthermore, delivery of siRNA across the cell membrane is hindered by its short in vitro half-life, due to effective cellular and systemic defense mechanisms that degrade naked siRNA molecules. Other obstacles to the successful application of siRNA therapy in medicine exist, including the high molecular weight of siRNA, low specificity and low uptake in target tissues, cytotoxicity, and undesirable off-target effects.

[0003] Moreover, mRNA shows promise as a therapeutic agent. mRNA has several advantages over DNA, including no need for any nuclear transport and much lower risk of damaging genome integration. While siRNA shows great potential for treating disorders caused by high or inappropriate gene expression, mRNA may be used to treat disorders caused by low gene expression. Furthermore, mRNA may be used in vaccines, including antitumor vaccines. mRNA-based therapy faces the same challenges as any therapeutic nucleic acid, namely, the challenges of delivery safety, specificity, and efficiency.

[0004] Additionally, RNA therapeutics (including siRNA and mRNA therapeutics) are applicable to the treatment of ocular disorders, but endogenous physical barriers, effective drug clearance mechanisms, and the complexity of other ocular tissues pose significant challenges for ocular RNA delivery. The ocular surface is one of the more complex biological barriers for drug delivery due to the combined effects of short contact time, the presence of tears, and corneal cell penetration. One of the major challenges in the topical treatment of ocular disorders is the corneal structure, which adapts to form an effective barrier against fluid loss and pathogen invasion.

[0005] While all ocular tissues are accessible via injection, the frequent treatment regimens required for siRNA-induced gene silencing and mRNA-mediated treatments make topical application preferable. Traditionally, drug delivery to the cornea is problematic because eye drops are rapidly drained via the nasolacrimal duct. Rapid tear drainage, combined with the blink reflex, shortens the precorneal residence time available for transcorneal absorption of active agents. It is estimated that 95% of the active compound contained in eye drop formulations is lost because it fails to cross the conjunctiva or is lost via drainage via the nasolacrimal duct. Despite the protective mechanisms of the eye, topical formulations have met with some success but still face challenges due to the barrier properties of the cornea.

[0006] Direct instillation of siRNA onto the ocular surface has been used to treat ocular surface and anterior segment disorders in vitro.However, the local administration of any compound, including mRNA and siRNA, into the eye is limited by the anatomical constraints and physiological protective mechanisms of the eye.The following lists some chemically modified siRNA therapeutics that are currently undergoing clinical trials, and the majority of them involve intravitreal injection. SYL1001, Sylenthis SA, Phase 1 and 2 completed, Phase 3 in progress - eye drops SYL040012 (bamosiran), Sylenthis SA, Phase 1 and 2 completed - eye drops Sirna-027 (AGN211745), Allergan, Phase 1 and 2 completed - intravitreal injection ·Bevasiranib (Cand5), OPKO Health Inc., Phase 2 completed - Intravitreal injection QPI-1007, Quark Pharmaceuticals, Phase 1 completed - Intravitreal injection PF-04523655, Quark Pharmaceuticals, Phase 1 completed - Intravitreal injection

[0007] RNA-based therapies for ocular diseases require frequent treatment regimens, increasing the likelihood of cataracts, retinal detachment, vitreous hemorrhage, and endophthalmitis due to the need for multiple injections.

[0008] Other approaches to RNA delivery include ocular injections containing modified viral particles. These particles are modified to deliver nucleic acid payloads to the eye. These viral types (adenovirus, adeno-associated virus, and lentivirus) are being investigated as vectors for RNAi therapy. Modified viral vectors, such as self-complementary AAV or helper-dependent adenovirus, are the current state-of-the-art in viral delivery. Several ophthalmic applications are currently being developed. However, viral vectors have disadvantages, including potential for mutagenesis, limited loading capacity, proper targeting, insertion predictability, high production costs, and adverse immune reactivity.

[0009] Delivery of plasmid vectors expressing mRNA or siRNA has been attempted with some success, but such DNA-based expression vectors can integrate into the host genome, increasing the probability of insertional mutagenesis. The advantage of modified non-viral RNA delivery systems is that they are relatively safe and can be easily modified with targeting ligands. Furthermore, encapsulating RNA into nanocarriers can be useful for providing a sustained release form of RNA over a long period of time, thus improving treatment regimens.

[0010] Numerous nonviral carriers have been proposed for RNA delivery, including natural and synthetic polymers, polyplexes, liposomes, lipoplexes, peptides, and dendrimer nanomaterials. Most of these strategies have been attempted in vivo and in vitro with varying degrees of success, but none have been used in clinical applications of RNA therapy in ocular and other tissue disorders.

[0011] Although many types of polymers have been used to deliver oligonucleotides, much attention has focused on the use of cationic polymers because they can electrostatically bind RNA without the need for covalent bonding or encapsulation, and amine-functionalized cationic polymers can provide endosomal buffering, allowing for escape for intracytosolic RNA delivery. Although numerous materials have been investigated, the development of biodegradable nanocarriers for improved RNA delivery without safety concerns remains a challenge.

[0012] Despite the many available RNA delivery strategies, the lack of safe and effective in vivo delivery has limited the translation of RNA therapeutics into clinics. A robust carrier system that can effectively condense oligonucleotides, be stable under physiological conditions, facilitate cellular uptake regardless of ocular barrier mechanisms, and promote RNA release into the cytoplasm is required for RNA delivery. Summary of the Invention [Problem to be solved by the invention]

[0013] Thus, there remains a need for effective, safe, and non-invasive means of delivering siRNA and mRNA to body sites, including but not limited to the cornea. [Means for solving the problem]

[0014] In a first aspect of the present invention, there is provided a composition for the controlled release of nucleic acids, such as short interfering RNA or mRNA, comprising silicon nanoparticles, at least one amino acid, and at least one lipid, wherein the silicon nanoparticles comprise at least 50% silicon by weight.

[0015] Advantageously, the presence of lipid in such composition provides the necessary zeta potential, which can enhance the loading of nucleic acid such as siRNA or mRNA, and has a beneficial effect on the surface charge of silicon nanoparticles, so as to control the release rate of nucleic acid at target site.In addition, the presence of at least one lipid in the formulation can control the hydrolysis rate of silicon, so that silicon nanoparticles hydrolyze into bioavailable orthosilicic acid (OSA) decomposition products.In certain embodiments, nucleic acid is RNA.In certain embodiments, RNA is siRNA.In certain embodiments, RNA is mRNA.

[0016] The above-mentioned composition further comprises at least one amino acid. Advantageously, the presence of an amino acid in the composition has been found to affect the release rate of the nucleic acid carried by the silicon nanoparticles over time.

[0017] Preferably, the above-mentioned composition further comprises a gene transfer reagent.

[0018] In a second aspect of the present invention, there is provided a composition according to the first aspect of the present invention for use as a medicament.

[0019] In a third aspect of the present invention, there is provided a composition according to the first aspect of the present invention for delivery to the eye.

[0020] It will be appreciated that features described with respect to one embodiment of the invention may be incorporated into other embodiments of the invention, for example, a method of the invention may incorporate any feature described with respect to an apparatus of the invention, and vice versa. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows a comparison of the zeta potential of siRNA-loaded and unloaded compositions of the present invention, with each formulation labeled F1 to F5. [Figure 2] FIG. 2 shows a comparison of the zeta potential of siRNA-loaded and non-loaded compositions of the invention when silicon nanoparticles are surface treated with stearylamine (FIG. 2), where the amine is arginine. [Figure 3] FIG. 3 shows a comparison of the zeta potential of siRNA-loaded and unloaded compositions of the invention when silicon nanoparticles are surface-treated with PC and the amine is arginine. [Figure 4] FIG. 4 shows a comparison of the zeta potential of siRNA-loaded and unloaded compositions of the invention when the silicon nanoparticles are surface-treated with lecithin and the amine is arginine. [Figure 5] Figure 5 shows the results of gene transfer efficiency of the compositions of the present invention in HCE cells. Cells in column 1 were stained with DAPI and fluoresced blue to show the nuclei. Cells in column 2 fluoresced green, indicating successful gene transfer of formulations F2 to F5 of the present invention. [Figure 6] Figure 6 shows the results of gel electrophoresis experiments, demonstrating that the silicon nanoparticles prepared in the present invention successfully encapsulate mRNA, especially at ratios of silicon nanoparticles to mRNA of 2:1 or greater (e.g., ratios ranging from 2:1 to 8:1). [Figure 7]FIG. 7 shows the results of spectrophotometric experiments, confirming that the silicon nanoparticles prepared in the present invention successfully encapsulate mRNA, especially at ratios of silicon nanoparticles to mRNA of 2:1 or greater (e.g., ratios ranging from 2:1 to 8:1). [Figure 8] FIG. 8 shows the results of an experiment measuring the gene transfer efficiency of an siRNA-loaded silicon nanoparticle delivery system of the present invention. [Figure 9] FIG. 9 shows the results of an experiment measuring the post-transfection viability of cells treated with an siRNA-loaded silicon nanoparticle delivery system of the present invention. [Figure 10] FIG. 10 shows the results of an experiment measuring the degree of siRNA-induced gene silencing when siRNA is delivered to cells using the silicon nanoparticle delivery system of the present invention. [Figure 11] FIG. 11 shows the results of experiments evaluating in vivo ocular siRNA delivery by topical silicon nanoparticle formulations. [Figure 12] FIG. 12 shows the results of an experiment measuring mouse corneal luciferase expression in vivo by live animal imaging when treated with an siRNA-loaded silicon nanoparticle delivery system of the present invention. [Figure 13] FIG. 13 shows the results of an experiment measuring mouse corneal luciferase expression in vivo by live animal imaging when treated with an siRNA-loaded silicon nanoparticle delivery system of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] In a first aspect of the present invention, there is provided a composition for the controlled release of nucleic acids, such as siRNA or mRNA, comprising silicon nanoparticles, at least one amino acid, and at least one lipid, wherein the silicon nanoparticles comprise at least 50% silicon by weight.

[0023] (definition) In the present disclosure, a derivative of a compound can be a compound that has substantially the same structure but one or more substitutions. For example, one or more chemical groups can be added, deleted, or replaced with other groups. In certain preferred embodiments, a derivative retains at least a portion of the pharmaceutical or cosmetic activity of the compound from which it is derived, for example, at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the activity of the compound from which it is derived. In some embodiments, a derivative can exhibit increased pharmaceutical or cosmetic activity compared to the compound from which it is derived. For example, in the case of a peptide, a peptide derivative can include a peptide in which one or more amino acid residues have been added, deleted, or replaced with another amino acid residue. In the case of substitutions, the substitutions can be non-conservative or conservative, preferably conservative.

[0024] In the context of the present disclosure, PC denotes phosphatidylcholine, SA denotes stearylamine, DOPE denotes dioleoylphosphatidylethanolamine, and DC-cholesterol denotes cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride.

[0025] (silicon nanoparticles) In all aspects of the present invention, the composition comprises silicon nanoparticles. The silicon nanoparticles have a nominal diameter of 5 to 400 nm, e.g., 50 to 350 nm, e.g., 80 to 310 nm, e.g., 100 to 250 nm, e.g., 120 to 240 nm, e.g., 150 to 220 nm, e.g., about 200 nm. The silicon nanoparticles are made from either pure silicon or hydrolyzable silicon-containing materials. The silicon nanoparticles are preferably porous. The nominal diameters indicated above may refer to average diameters, and at least 90% of the total particles in a silicon nanoparticle sample may fall within the specified size range. The silicon nanoparticles are made from either pure silicon or hydrolyzable silicon-containing materials. The silicon nanoparticles can be made porous by standard techniques, such as exposing the particles to a hydrofluoric acid (HF) / ethanol mixture and applying an electric current. The pore density and its size can be controlled by varying the HF concentration and current density and exposure time, and monitored by scanning electron microscopy and / or nitrogen adsorption-desorption volume isotherm measurements.

[0026] Silicon nanoparticles can be pure silicon or other hydrolyzable silicon-containing materials. If silicon nanoparticles are not pure silicon, they contain at least 50% silicon by weight. For example, silicon nanoparticles can contain at least 60, 70, 80, 90, or 95% silicon. Silicon nanoparticles preferably exhibit a hydrolysis rate (e.g., in PBS buffer at room temperature) that is at least 10% of the hydrolysis rate of pure silicon particles of the same size. Assays for the hydrolysis of silicon-containing materials are widely known in the art, for example, WO 2011 / 001456.

[0027] Nanoparticles in all aspects of the present invention (e.g., nanoparticles formulated with one or more of arginine, glycine, and histidine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives) are preferably porous. For example, their porosity can increase their surface area by at least 1.5, 2, 2.5, 3, 3.5, or 4 times compared to the surface area of ​​a non-porous material with comparable dimensions. In some embodiments (e.g., when nanoparticles are formulated with one or more of arginine, glycine, and histidine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives), their total area is preferably increased by at least 50% or at least 100% compared to the surface area of ​​a corresponding non-porous particle due to their porosity. In many cases, porous silicon nanoparticles actually have a much greater increase in total surface area due to their porosity.

[0028] Preferably, the silicon nanoparticles have an average diameter of 2 to 300 nm, e.g., 20 to 290 nm, 20 to 280 nm, e.g., 20 to 270 nm, 20 to 260 nm, 20 to 250 nm, 20 to 240 nm, 20 to 230 nm, 20 to 220 nm, 20 to 210 nm, especially 20 to 200 nm (e.g., when the nanoparticles are formulated with one or more of arginine, glycine, and histidine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof, and such nanoparticles are formulated for topical delivery, such as delivery to the skin surface or topical delivery to the eye). Advantageously, silicon nanoparticles of this size are suitable for dermal delivery, as they are small enough to block the pilosebaceous ostra or sweat ducts (pores), yet their small size allows the particles to actively penetrate to the base of the hair follicle rather than acting solely as a surface drug reservoir.

[0029] (Fat) In all aspects of the present invention, silicon nanoparticles are surface-treated with at least one lipid (for example, PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives, such that the nanoparticles formulated in this way can be suitable for delivery to the eye) (for example, when the nanoparticles are formulated with one or more of glycine, arginine, and histidine).It has been found that surface-treating silicon nanoparticles with lipids helps control the release rate of bioactive agents.Depending on the properties of bioactive agents (for example, nucleic acids such as siRNA or mRNA), the type of lipid used to treat the nanoparticle surface affects its release rate.In particular, surface-treating silicon nanoparticles with lipids has a beneficial effect on the surface charge of silicon nanoparticles, providing the necessary zeta potential that can increase the loading of short interfering RNA or messenger RNA, thereby controlling its release rate at target sites.

[0030] (silicon to lipid ratio) Preferably, the lipid to silicon ratio (e.g. when the lipid is selected from one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof) is 1:1 to 15:1, such as 1:1 to 13:1, 1:1 to 12:1, 1:1 to 11:1, 1:1 to 10:1, 1:1 to 9:1, 1:1 to 8:1, 1:1 to 13:1, 2:1 to 12:1, 2:1 to 11:1, 2:1 to 10:1, 2:1 to 9:1, 2:1 to 8:1, such as 1:1 to 7:1, 2:1 to 7:1, 3:1 to 6:1, 4:1 to 5:1. Advantageously, this lipid to silicon ratio provides a multilamellar vesicle system that can control and stabilize the release of bioactive agents (e.g., nucleic acids such as siRNA or mRNA) carried by the silicon nanoparticles, as well as control the release of OSA, a bioavailable degradation product of silicon.

[0031] Advantageously, lipid compounds can have a significant effect on the surface charge of silicon nanoparticles. Silicon nanoparticles treated with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lecithin exhibited negative surface charges when subjected to zeta potential analysis (ranging from -60 to -20 mV at silicon:lipid ratios ranging from 1:1 to 1:3). Silicon nanoparticle surfaces treated with stearylamine exhibited positive zeta potentials (ranging from 0 to 40 mV at silicon:lipid ratios ranging from 1:1 to 1:3).

[0032] In one embodiment, the composition of the first aspect of the present invention comprises silicon nanoparticles surface-treated with at least 5 wt.% lipid, for example at least 20 wt.%, typically at least 30 wt.%, and in particular at least 50 wt.% lipid, based on the total weight of the coated nanoparticles. Molar ratios of lipid to silicon of 0.8:1 to 3:1, such as 1:1, 1.5:1, 2:1, or 2.5:1, have been found to be particularly advantageous.

[0033] In certain embodiments (e.g., when the nanoparticles are formulated with one or more of arginine, glycine, and histidine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives, making them suitable for delivery to the eye), the composition of the first aspect of the present invention comprises silicon nanoparticles surface-treated with at least 5% by weight of phospholipid, for example at least 20% by weight, typically at least 30% by weight, and particularly at least 50% by weight of phospholipid, based on the total weight of the coated nanoparticles. A lipid to silicon molar ratio of 0.8:1 to 3:1, such as 1:1, 1.5:1, 2:1, or 2.5:1, has been found to be particularly advantageous.

[0034] In one embodiment, the phospholipid has a number-average molecular weight in the range of 500 to 1000. Particularly suitable phospholipids are glycerophospholipids. Particularly suitable phospholipids include those with a polar head group linked to a quaternary ammonium moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. The type of phospholipid can be selected depending on the properties of the formulation; neutral or negatively charged lipids are preferred for aprotic formulations, while positively charged and short CH3 chain lipids are preferred for protic formulations. Preferably, the side chain is an aliphatic side chain with 15 or more carbon atoms, such as a polyethylene glycol or polypropylene glycol chain, or an ether side chain with 6 or more repeating ether units.

[0035] Preferably (e.g., in formulations that include one or more of arginine, glycine, and histidine and may be suitable for delivery to the eye), the lipid is selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), lecithin, or any combination thereof.

[0036] In further embodiments, the silicon nanoparticle surface can be treated with phosphatidylcholine, hydrogenated phosphatidylcholine, stearylamine, lecithin, or a combination thereof, which can be particularly advantageous if the siRNA / mRNA is charged.

[0037] In certain embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, glycine, and histidine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives, and such nanoparticles can be formulated for delivery to the eye), the composition of the first aspect of the present invention comprises silicon nanoparticles surface-treated with at least 5% by weight of hydrogenated phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, and particularly at least 50% by weight of hydrogenated phosphatidylcholine, based on the total weight of the coated nanoparticles. A molar ratio of hydrogenated phosphatidylcholine to silicon of 0.8:1 to 5:1, such as 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, or 4.5:1, has been found to be particularly advantageous.

[0038] In certain embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, glycine, and histidine, and these nanoparticles can be formulated for delivery to the eye), the composition of the first aspect of the present invention comprises silicon nanoparticles surface-treated with at least 5% by weight of phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, and particularly at least 50% by weight of phosphatidylcholine, based on the total weight of coated nanoparticles.A molar ratio of phosphatidylcholine to silicon of 0.8:1 to 5:1, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, or 4.5:1, has been found to be particularly advantageous.

[0039] In certain embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, glycine, and histidine, and such nanoparticles can be formulated for delivery to the eye), the composition of the first aspect of the present invention comprises silicon nanoparticles surface-treated with at least 5% by weight of lecithin, for example at least 20% by weight, typically at least 30% by weight, and particularly at least 50% by weight of lecithin, based on the total weight of the coated nanoparticles.A molar ratio of lecithin to silicon of 0.8:1 to 3:1, for example 1:1, 1.5:1, 2:1, or 2.5:1, has been found to be particularly advantageous.

[0040] In certain embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, glycine, and histidine, and these nanoparticles can be formulated for delivery to the eye), the composition of the first aspect of the present invention comprises the silicon nanoparticles surface-treated with at least 5 wt.% stearylamine, for example at least 20 wt.%, typically at least 30 wt.%, and particularly at least 50 wt.% stearylamine, based on the total weight of the coated nanoparticles. A lipid to silicon molar ratio of 0.8:1 to 3.5:1, for example 1:1, 1.5:1, 2:1, 2.5:1, 2:0.75, 2:1.5, or 3:1, has been found to be particularly advantageous.

[0041] In certain embodiments (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, glycine, and histidine, and such nanoparticles can be formulated for delivery to the eye), the composition of the first aspect of the present invention comprises silicon nanoparticles surface-treated with PC and SA, preferably in a weight ratio of PC:SA of 1:1 to 20:1, more preferably 7:1 to 10:1, for example a weight ratio of PC:SA of 72:8.

[0042] In certain embodiments (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, glycine, and histidine, and such nanoparticles can be formulated for delivery to the eye), the composition of the first aspect of the present invention comprises silicon nanoparticles surface-treated with DOPE, SA, and DC-cholesterol. The weight ratio of DOPE:SA can be in the range of 1:1 to 10:1, such as 4:1 to 8:1. The weight ratio of DOPE:DC-cholesterol can be in the range of 1:1 to 5:1, such as 1:1 to 3:1. The weight ratio of SA:DC-cholesterol can be in the range of 1:1 to 1:5, such as 1:2 to 1:4. In some embodiments (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, glycine, and histidine, and such nanoparticles can be formulated for delivery to the eye), the weight ratio of DOPE:SA:DC-cholesterol can be 48:8:24.

[0043] (amino acid) In all aspects of the present invention (e.g., when the nanoparticles of the present invention are formulated with one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives), the lipid-treated silicon nanoparticles are further treated with an amino acid. In its broadest sense, the term "amino acid" encompasses any artificial or naturally occurring organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group. This includes α, β, γ, and δ amino acids. This includes amino acids of any chiral configuration. In some embodiments (e.g., when the nanoparticles of the present invention are formulated with one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives), the amino acid is preferably a naturally occurring α-amino acid. This can be a proteinogenic or non-proteinogenic amino acid (such as carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline). In preferred embodiments (e.g., when the nanoparticles of the present invention are formulated with one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof), the amino acid comprises arginine, histidine, or glycine, or a mixture of arginine and glycine. In particularly preferred embodiments, the amino acid comprises glycine.

[0044] In preferred embodiments (e.g., when the nanoparticles are formulated with one or more of arginine, glycine, and histidine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof), at least 70% by weight, such as at least 80% by weight, for example at least 90% by weight, of the siRNA or mRNA present in products of all aspects of the invention is associated with the surface-treated nanoparticles.

[0045] The molecular association of nucleic acid such as siRNA or mRNA with lipid-treated silicon nanoparticles advantageously ensures that when surface-treated silicon nanoparticles (for example, nanoparticles treated with PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives, and / or one or more of glycine, arginine, and histidine) decompose, nucleic acid such as siRNA or mRNA becomes bioavailable.The decomposition rate of the composition is determined by the hydrolysis of silicon nanoparticles.Since this rate can be controlled, the rate at which nucleic acid such as siRNA or mRNA becomes bioavailable can also be controlled to avoid dose dumping and / or ensure that nanoparticles are only released when they are directed away from the application site.For example, this may be when nanoparticles move from the skin surface to the base position where they are applied.

[0046] It has been found that treating lipid-processed silicon nanoparticles (for example, nanoparticles treated with one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives) with amino acid (for example, one or more of glycine, arginine, and histidine, preferably glycine) can provide beneficial stabilization effect to nucleic acid such as RNA (for example, mRNA or siRNA) loaded on silicon nanoparticles.In particular, treating lipid-processed silicon nanoparticles with amino acid has been shown to stabilize nucleic acid such as RNA in biological fluid, for example, in ocular tissue.The lipid-processed silicon nanoparticles formulated with amino acid in this way can be particularly suitable for delivery to the ocular region, and can provide a delivery system for delivering nucleic acid (for example, siRNA or mRNA) to the ocular region.

[0047] In certain embodiments of all aspects of the present invention, the lipid-treated silicon nanoparticles are further treated with arginine, glycine, or a combination thereof (e.g., when the nanoparticles are formulated with one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof). Silicon nanoparticles surface-treated with one or more of glycine and arginine and loaded with nucleic acids such as RNA exhibited better bioactivity stability in biological fluids and effectively delivered charged nucleic acids such as RNA, including siRNA or mRNA, to the ocular cellular and cytoplasmic environment.

[0048] (Amino acid to silicon ratio) Preferably, the ratio of amino acids to silicon (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof) is 0.05:1 to 2:1, for example 0.05:1 to 1.8:1, 0.05:1 to 1.6:1, 0.05:1-1.4:1, 0.05:1-1.2:1, 0.05:1-1:1, 0.05:1-0.9:1, 0.05:1-0.8:1, 0.05:1-0.6:1, 0.05:1-0.5:1, 0.05:1-0.4:1, 0.05:1-0.3:1, 0.05:1-0.2:1, preferably 0.2:1-0.8:1, particularly 0.3:1-0.7:1. Advantageously, this amino acid to silicon ratio also affects the release rate of the RNA molecules transported by silicon nanoparticles and stabilizes the RNA molecules.

[0049] In all aspects of the present invention, silicon nanoparticles (which may be formulated for delivery to the eye) are treated with a lipid (e.g., one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof) and an amino acid (e.g., one or more of glycine, arginine, and histidine, such as glycine, or a mixture of glycine and arginine). The amino acid can be any amino acid. Preferably, the amino acid is arginine or glycine, or a combination of glycine and arginine. The lipid can be any lipid. Preferably, the lipid is a phospholipid. More preferably, the lipid is selected from one or more of hydrogenated PC, PC, DOPE, lecithin, stearylamine, and derivatives thereof. Optionally, the lipid comprises DC-cholesterol and / or a derivative thereof. Preferably, the ratio of amino acid to silicon is 0.05:1 to 0.4:1, for example 0.08:1 to 0.35:1, in particular 0.09:1 to 0.32:1. In some embodiments, (for example, when the lipid is selected from one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives), the amino acid is a combination of arginine and glycine, and the ratio of Arg:Gly is 1:0.6-3:1, for example, 1:0.8-2.5:1, for example, 1:1-2:1.Advantageously, such a ratio has been found to achieve high-speed RNA release and effectively deliver RNA (such as siRNA or mRNA) in the ocular cell and cytoplasmic environment.In this way, the lipid-processed silicon nanoparticles of the present invention formulated with amino acids can be particularly suitable for delivery to the ocular region, and can provide a delivery system for delivering nucleic acids (such as siRNA or mRNA) to the ocular region.

[0050] In another embodiment of all aspects of the present invention, the lipid-processed silicon nanoparticles are processed with arginine (e.g., when the nanoparticles of the present invention are formulated for ocular delivery). The lipid can be any lipid. Preferably, the lipid is selected from one or more of hydrogenated PC, PC, DOPE, lecithin, stearylamine, and derivatives thereof. Optionally, the lipid comprises DC-cholesterol and / or its derivatives. Preferably, the ratio of arginine to silicon is 0.05:1 to 0.4:1, e.g., 0.08:1 to 0.35:1, particularly 0.09:1 to 0.32:1. Advantageously, such a ratio has been found to provide rapid RNA release and effective delivery of RNA (e.g., siRNA or mRNA) in the ocular cellular and cytoplasmic environment. Thus, the lipid-processed silicon nanoparticles of the present invention formulated with amino acids may be particularly suitable for ocular delivery and can provide a delivery system for delivering nucleic acids (e.g., siRNA or mRNA) to the eye.

[0051] In another embodiment of all aspects of the present invention, the lipid-processed silicon nanoparticles are processed with glycine (e.g., when the nanoparticles are formulated for ocular delivery). The lipid can be any lipid. Preferably, the lipid is selected from one or more of hydrogenated PC, PC, DOPE, lecithin, stearylamine, and derivatives thereof. Optionally, the lipid comprises DC-cholesterol and / or a derivative thereof. Preferably, the ratio of glycine to silicon is 0.05:1 to 0.5:1, e.g., 0.08:1 to 0.45:1, particularly 0.09:1 to 0.42:1. Advantageously, such a ratio has been shown to provide rapid RNA release and effective delivery of charged RNA, such as siRNA and mRNA, in the ocular cellular and cytoplasmic environment, supporting cellular internalization. Thus, the lipid-processed silicon nanoparticles of the present invention, formulated with amino acids, may be particularly suitable for ocular delivery and can provide a delivery system for delivering nucleic acids (e.g., siRNA or mRNA) to the eye.

[0052] (RNA) In preferred embodiments of all aspects of the present invention (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives, making such formulations suitable for ocular delivery), the lipid-treated silicon nanoparticles are loaded with RNA, which may be siRNA or mRNA. In its broadest sense, the term "siRNA" encompasses small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, and includes double-stranded RNA molecules of 5-50 base pairs in length that operate within the RNA interference (RNAi) pathway, e.g., 10-45 base pairs, 15-40 base pairs, 20-30 base pairs, and particularly 20-25 base pairs in length. The term "mRNA" encompasses messenger RNA, and may include mRNA that optionally includes a 5-prime cap and / or polyadenylated end, or may lack one or both of these features.

[0053] In preferred embodiments of all aspects of the present invention, the RNA can be naturally occurring or chemically modified to enhance therapeutic properties such as improved activity, increased serum stability, reduced off-targeting, and reduced immunological activation (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives, making such formulations suitable for delivery to the eye). Chemical modifications of RNA can include any modifications commonly known in the art.

[0054] In certain embodiments, (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine and glycine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol and one or more of their derivatives, and this formulation can be suitable for delivery to the eye), RNA is siRNA.In other embodiments, (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine and glycine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol and one or more of their derivatives, and this formulation can be suitable for delivery to the eye), RNA is mRNA.

[0055] In other embodiments, the nucleic acid is DNA or a DNA / RNA hybridization product (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof, making such formulations suitable for delivery to the eye).

[0056] (Ratio of silicon to nucleic acid) Preferably (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives, and such formulations are suitable for delivery to the eye), the ratio of silicon to nucleic acid (such as siRNA or mRNA) is 1:1 to 8:1, for example, 1:1 to 6:1, 1:1 to 5:1, 1:1 to 4:1, or 1:1 to 3:1. Preferably, the ratio of silicon to nucleic acid is 1:1 to 3:1. Advantageously, this ratio of silicon to nucleic acid also affects the release rate of the nucleic acid molecule (such as siRNA molecule or mRNA molecule) transported by the silicon nanoparticle, and stabilizes the nucleic acid molecule. Formulations with such a silicon to nucleic acid ratio are particularly suitable for delivery to the eye, and can provide a delivery system for delivering nucleic acid to ocular tissues.

[0057] (Other ingredients) (Gene transfection reagent) In preferred embodiments of all aspects of the present invention (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives, making such formulations suitable for delivery to the ocular region), the lipid-treated silicon nanoparticles can be surface-treated with a transfection agent. In its broadest sense, "transfection" is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Transfection can also refer to other methods and cell types. mRNA can be transfected into cells to translate its sequence. siRNA can be transfected to achieve RNA silencing (i.e., loss of RNA and protein in the target gene).

[0058] In the broadest sense, a "transfection reagent" is an agent that facilitates the introduction of naked or purified nucleic acids into eukaryotic cells. For example, some transfection reagents are agents that facilitate the introduction of naked or purified siRNA or mRNA into eukaryotic cells.

[0059] In other embodiments of all aspects of the invention (e.g., when the silicon nanoparticles of the invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives, making such formulations suitable for delivery to the eye), the gene transfer reagent can be a lipofection (liposomal gene transfer) reagent, a dendrimer, HEPE-buffered saline (HeBS) containing phosphate ions combined with calcium chloride solution, or a cationic polymer such as diethylaminoethyl-dextran (DEAE-dextran) or polyethyleneimine (PEI).

[0060] In a preferred embodiment (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof), the gene transfer reagent is a lipofection reagent such as lipofectamine.

[0061] Nucleic acids such as RNA (such as siRNA or mRNA) for use in various aspects of the present invention can be provided in various forms.For example, in some embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives), nucleic acids such as RNA are provided in a solution such as buffer (alone or in combination with various other nucleic acids).In some embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives), nucleic acids such as RNA are provided as salts, alone or in combination with other isolated nucleic acids. In some embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives), nucleic acids such as RNA are provided in a reconstitutable lyophilized form.For example, in some embodiments, nucleic acids such as RNA can be provided as lyophilized pellets alone or in lyophilized pellets containing other isolated nucleic acids.In some embodiments (for example, when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives), nucleic acids such as RNA are provided by immobilizing them on a solid material such as beads or a membrane. In some embodiments (e.g., when the silicon nanoparticles of the present invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof), nucleic acids such as RNA are provided in host cells, for example cell lines containing a plasmid or cell lines containing stably integrated sequences.

[0062] Nucleic acids such as RNA for use in accordance with the present invention include double and single stranded DNA, RNA, DNA:RNA hybrids, and PNA (peptide nucleic acid) or hybrids between RNA or DNA. The term also encompasses known types of modifications, such as labels known in the art, methylation, "capping," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and positively charged linkages (e.g., aminoalkyl phosphoramidates, aminoalkyl phosphotriesters), those containing pendant moieties such as proteins (nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralens, etc.), chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), alkylators, modified linkages (e.g., α-anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides or oligonucleotides.

[0063] As used herein, the terms "nucleoside" and "nucleotide" are understood to include moieties containing not only purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications to the sugar moiety, e.g., one or more of the hydroxyl groups are replaced with halogens, aliphatic groups, or functionalized as ethers, amines, or the like. Other modifications of nucleotides or polynucleotides involve rearranging, adding, substituting, or altering the functional groups of the purine or pyrimidine base that form hydrogen bonds with the respective complementary pyrimidines or purines, e.g., isoguanine and isocysteine. In some embodiments, the oligonucleotides and / or probes contain at least one, two, three, or four modified nucleotides.

[0064] In some embodiments, nucleic acids, such as RNA, disclosed herein comprise one or more universal bases. As used herein, the term "universal base" refers to a nucleotide analog that can hybridize to more than one nucleotide selected from A, U / T, C, and G. In some embodiments, the universal base can be selected from the group consisting of deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, and 5-nitroindole.

[0065] (Preparation of silicon nanoparticles) The silicon nanoparticles of the present invention can be easily prepared by conventional techniques in the art, such as by milling processes or other known particle size reduction techniques. Silicon-containing nanoparticles can be made from sodium silicate particles, colloidal silica, or silicon wafer materials. Macro- or micro-scale particles can be pulverized in a ball mill, a planetary ball mill, or other size reduction mechanisms. The resulting particles can be air-classified to recover the nanoparticles. Plasma methods and laser ablation can also be used to prepare nanoparticles.

[0066] The porous particles can be made by methods conventional in the art, including those described herein.

[0067] (Preparation of creams and gels) Creams and gels can be formulated by simply dispersing (i.e., mixing) the silicon nanoparticles of the present invention into a cream base or gel base.For example, silicon nanoparticles can be mixed into a medicinal cream base.For gels, powder can be mixed into a powder-form gel matrix, and then the gel can be hydrated, or powder can be mixed into a pre-hydrated gel.

[0068] (Delivery to the eye) In a third aspect of the present invention, there is provided a composition according to the first aspect of the present invention (e.g., a composition formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof), for delivery to the eye.

[0069] Although all ocular tissues are accessible by injection, the frequent treatment regimens required for siRNA-induced gene silencing treatments make topical application preferable. However, the ocular surface is one of the more complex biological barriers for drug delivery due to the combined effects of short contact time, dilution by tears, and poor corneal cell penetration.

[0070] The composition of the present invention provides an effective, clinically safe, and non-invasive means of delivering siRNA to ocular tissues, such as one or more of the tissues of the fibrous layer, the vascular layer, and the retina. For example, the composition of the present invention provides an effective, clinically safe, and non-invasive means of delivering siRNA to one or more tissues selected from the tissues of the cornea, the sclera, the iris, the ciliary body, the choroid, the zonular fiber, the lens capsule, the lens nucleus, the vitreous body, and the retina.

[0071] The compositions of the present invention can encapsulate and stabilize active pharmaceutical agents, particularly nucleic acids such as siRNA and mRNA. The nanoparticles of the present invention can then deliver these active pharmaceutical agents, such as nucleic acids (e.g., siRNA or mRNA), to ocular cells (e.g., to one or more cells of the cornea, sclera, iris, ciliary body, choroid, zonular fibers, lens capsule, lens nucleus, vitreous body, and retina). When the composition is applied to one or more ocular tissues, the ocular cells internalize the nanoparticles. This allows these active pharmaceutical agents, such as nucleic acids (e.g., siRNA or mRNA), to penetrate ocular cells, ensuring controlled release of the nucleic acids at the target site.

[0072] The composition of the first aspect of the present invention can be formulated for delivery to the eye. For example, the composition can be formulated with one or more excipients compatible with the eye. The composition can be used to treat eye disorders. For example, the composition of the first aspect of the present invention can be used to treat macular degeneration, conjunctivitis, glaucoma, diabetic retinopathy, diabetic macular edema, keratoconus, cataract, retinitis, and uveitis, especially macular degeneration. Macular degeneration can include age-related macular degeneration.

[0073] In a further aspect, the present invention provides methods and medicaments relating to the composition of the first aspect of the present invention. For example, a method is provided that includes delivering the composition of the first aspect of the present invention, optionally in combination with one or more ocular-compatible excipients, to one or more ocular tissues (e.g., to one or more tissues selected from the cornea, sclera, iris, ciliary body, choroid, zonular fibers, lens capsule, lens nucleus, vitreous body, and retinal tissues). A method is also provided for treating an ocular disorder, including delivering the composition of the first aspect of the present invention, optionally in combination with one or more ocular-compatible excipients, to one or more ocular tissues. For example, such a method can include treating macular degeneration, conjunctivitis, glaucoma, diabetic retinopathy, diabetic macular edema, keratoconus, cataract, retinitis, or uveitis, particularly macular degeneration. [Example]

[0074] The invention is further described by the following non-limiting examples.

[0075] (material) (Silicon Preparation) Single-side polished P-type or N-type silicon wafers were purchased from Si-Mat (Germany). All cleaning and etching reagents were cleanroom grade. The etching was performed at 80 mA / cm in 1:1 (v / v) pure ethanol and 10% aqueous HF acid. 2 Etched silicon was prepared by anodic etching of p-type Si at anodic current densities for 2–10 min. After etching, the samples were washed with pure ethanol and dried under a stream of high-purity dry nitrogen before use.

[0076] Etched silicon wafers (P+ or N-) were ground using a milling ball and / or a pestle and mortar. The fine powder was sieved using a shaker as200 with Retsch brand sieves (gauge 38 μm). The opening size of the sieves allowed for the selection of uniform size (20-100 μm). Particle size was measured using a quantachrome system and a PCS from a Malvern instrument. Samples were stored in a sealed container until further use.

[0077] Nanosilicon powders were also obtained from Sigma and Hefel Kaier (China). Particle size was measured by PCS and recorded (ranging from 20 to 100 nm) before loading and etching. Silicon wafers were ground using a milling ball or a pestle and mortar. The fine powder was sieved using a Retsch brand sieve (gauge 38 μm) on a shaker as200 to recover uniform nanoparticles with the desired size.

[0078] (Activation of silicon nanoparticles) 250 ml of ethanol and 500 mg of 100 nm diameter porous silicon nanoparticles were mixed and stirred for 30 minutes using a magnetic bar. The solution was then centrifuged at 3000 rpm for 30 minutes. The supernatant was discarded, and the nanoparticles were washed with 5 ml of distilled water and transferred to a round-bottom flask. The contents of the flask were frozen (-25°C for 2 hours). The frozen nanoparticles were lyophilized overnight using a freeze dryer. The resulting dry powder is the activated nanoparticles.

[0079] Preparation of amine-, lipid-, and lipofectamine-loaded siRNA nanoparticles Both formulations, SIS005-PS91 and SIS005-DS61 (with glycine), were prepared by dissolving a thin film of liposome-forming material in an aqueous suspension of siRNA-containing silicon nanoparticles. The mixtures were then freeze-thawed three times and tested for gene transfer efficiency in HCES cells in vitro. In parallel, the SIS005-PDS1051 formulation was tested after further modification aimed at introducing an additional positive charge (since it was shown to have a negative zeta potential), which may be preferable for a corneal siRNA delivery system. To this end, the ratio of cationic lipid in the formulation was increased.

[0080] Colloidal stability was assessed by dynamic light scattering in parallel with zeta potential measurements for both unloaded and loaded formulations. The encapsulation efficiency of siRNA was measured by spectrophotometry. Gene transfer (internalization) efficiency was assessed in human corneal epithelial cells by flow cytometry followed by a dual luciferase assay to measure in vitro knockdown after treatment with siRNA-loaded formulations.

[0081] (Preparation procedures for gene transfer formulations (SI005-PS91, SI005-DS61, SI005-DSC613, etc.)) The ingredients of these and other formulations are shown in the table below. [Table 1]

[0082] (material) Silicon nanoparticles, nuclease-free water, chloroform Lipid: Stearylamine (SA), catalog number 305391 (Sigma-Aldrich) Egg yolk L-α-phosphatidylcholine (PC), catalog number 61755 (Sigma-Aldrich) Dioleoyl L-α-phosphatidylethanolamine (DOPE), catalog number P1223 (Sigma-Aldrich) N-(2-Dimethylaminoethyl)carbamate cholesterol (DC-Chol), catalog number 92243 (Sigma-Aldrich) 100 μM (1.33 μg / μl) nonspecific NSC4 (customized siRNA duplex, Eurogentec) 100 μM (1.33 μg / μl) targeting siLUC (customized siRNA oligonucleotide duplex, Eurogentec) 100 μM (1.33 μg / μl) FAM-tagged siRNA (green siGLO, Dharmacon)

[0083] (device) Rotary evaporation systems, vortexers, ultrasonic bath processors, water baths, round-bottom flasks, general test tubes, Eppendorf tubes, micropipettes, freeze-drying systems, Zetasizers, high-speed centrifuges, NanoDrop spectrophotometers, fluorescence readers

[0084] (procedure) It is noted that the fluorescent siRNA is not exposed to light during the procedure (eg, flasks and test tubes are covered with aluminum foil). A - siRNA Loading in Silicon Nanoparticle / Lipid Compositions Step I - Component 1, Preparation of siRNA-SiNP Mixture 1. Prepare a 0.2 mg / ml filtered silicon nanoparticle solution in nuclease-free water 2. Aliquot (700 μl) of the above solution into eight Eppendorf microtubes, each containing 140 μg of SiNPs. 3. To each aliquot, add siRNA and glycine and adjust the volume to 1.4 ml with nuclease-free water. [Table 2] 4. Mix the tube thoroughly and incubate the sample with agitation at room temperature for 1 hour.

[0085] Step II - Component 2, Preparation of the Lipid Film 1. Dissolve each lipid component (SA, PC, DOPE, DC-Chol) in chloroform to a concentration of 0.2 mg / ml. 2. Transfer the desired amount of each lipid to a small round-bottom flask and mix thoroughly. Prepare eight replicates of each lipid mixture (in eight flasks). a. (Lipid base DS61) 68 μg of DOPE (340 μl) 12 μg of SA (60 μl) b. (Lipid base PS91) 72 μg of PC (360 μl) 8 μg of SA (40 μl) c. (Lipid base DSC613) 48 μg of DOPE (240 μl) 8 μg of SA (40 μl) 24 μg DC-Chol (120 μl) d. (Lipid base PDS1051) 50 μg of PC (250 μl) 25 μg of DOPE (125 μl) 5 μg of SA (25 μl) e. (Lipid base PDS1052) 48 μg of PC (240 μl) 23 μg of DOPE (115 μl) 9 μg of SA (45 μl) f. (Lipid base PDSC10514) 40 μg of PC (200 μl) 20 μg of DOPE (100 μl) 4 μg of SA (20 μl) 16 μg of DC-Chol (80 μl) 3. Carefully evaporate the solvent using a rotary evaporator to form a thin lipid film. Place the dried lipid under vacuum to remove any remaining solvent.

[0086] (Step III—Encapsulation of siRNA-SiNPs (component 1) in liposomes (component 2)) 1. Dissolve the thin lipid film (from step II) along with the siRNA-loaded or empty Si nanoparticle samples (from step I). Separately, add 200 μl of 1, 2, 3, or 4 Si nanoparticle / siRNA mixtures to each flask containing the lipid film (a, b, c, d, e, f). [Table 3] 2. Add 200 μl of nuclease-free water to each flask to adjust the volume of the solution to 400 μl. 3. Cover each flask with parafilm, mix contents thoroughly and incubate at room temperature for 1 hour. 4. Vortex to completely dissolve the lipid film. Sonicate the flask for 15 seconds to aid in dissolving the lipids. 5. Transfer the entire contents of each flask separately to an Eppendorf microtube. 6. Place all tubes in the freezer (-20°C) for at least 3 hours (or overnight). 7. Remove samples from the freezer and place in a 30°C water bath for 10 minutes. Cool to room temperature and vortex thoroughly. 8. Repeat the freeze-thaw cycle (steps 5 and 6) two more times. 9. All samples are stored in a freezer (-20°C) until assayed. 10. Each loaded sample contains [10 μg siRNA: 20 μg SiNP: 80 μg lipid base: 10 μg glycine] in a volume of 400 μl.

[0087] (B - Characteristics of silicon nanoparticle / lipid formulation) (Encapsulation efficiency) The siRNA encapsulation efficiency was investigated by separating the loaded particles from free (unbound) siRNA by high-speed centrifugation, followed by measuring the encapsulation efficiency. 1.50 μl of each sample is collected in a microcentrifuge tube. 2. Centrifuge all samples at 21,000 x g for 30 minutes. 3. Transfer 25 μl (top half) of the supernatant from each sample to a separate tube (labeled S1) and store at 4° C. (do not expose to light in the case of siGlo-loaded samples) until assayed. 4.25 μl of 2% SDS is added to the pellet sample (including the remaining supernatant) to disrupt the lipid bilayer and release the bound siRNA. 5. Centrifuge the tube again at 21,000 x g for 30 minutes and collect the supernatant (labeled S2). 6. Measure the optical density (OD) at 260 nm in all supernatant S1 and S2 samples using a NanoDrop spectrophotometer. For siGlo-loaded samples, measure the fluorescence intensity (FI) in both S1 and S2 supernatant samples. 7. Calculate the encapsulation efficiency (EE%).

number

[0088] (Zetasizer measurement) Colloidal stability was assessed by dynamic light scattering in parallel with zeta potential measurements. Measurements were performed on unloaded (empty) and siLUC / NSC4-loaded formulation samples. 1. Take 200 μl of each sample and dilute to a total volume of 1 ml with nuclease-free ultrapure water. 2. Load the sample into the bent capillary cell. 3. Read the samples for particle size, polydispersity index, and zeta potential using a Zetasizer. Perform each measurement in triplicate. The data are shown in Figures 1, 2, and 3.

[0089] (Gene transduction efficiency in HCES cells) The efficiency of gene transfer (internal internalization) was evaluated in human corneal epithelial cells by flow cytometry. For the purpose of this study, a preparation sample loaded with a fluorescently tagged siRNA probe (siGlo) was used. 1. 2 x 10 cells per well (in a 12-well plate) in 1 ml of DMEM enriched with 10% FBS 5 HCES cells are seeded and grown in standard conditions for 24 hours (to 80% confluency). 2. Replace the medium with 950 μl per well of fresh DMEM enriched with 10% FBS. 3. Allow the siGlo-loaded silicon nanoparticle / lipid sample to warm to room temperature. 4. Prepare a control sample containing Lipofectamine transfection reagent by mixing 6 μl of Lipofectamine RNAiMAX reagent with 3 μl of siGlo stock solution (100 μM) in 151 μl of OptiMEM and incubate at room temperature for 15 minutes. 5. Treat three replicate cells by adding 53.3 μl per well of each siGlo loading formulation or LPF control to obtain a siGlo concentration of 0.1 μM. 6. Grow cells for 24 hours under standard conditions (37°C with 5% CO2). 7. Discard the medium and wash the wells with 500 μl of PBS. 8. Add 300 μl of trypsin-EDTA and incubate the plate at 37° C. for 10 minutes. 9. Immediately add 300 μl of DMEM enriched with 10% FBS to stop trypsinization. 10. Transfer the cells separately to a microtube and centrifuge at 1000-2000 rpm for 5 minutes. 11. Discard the supernatant and carefully suspend the cells in 500 μl of PBS. 12. Centrifuge the sample at 1000-2000 rpm for 5 minutes. 13. Discard the supernatant and carefully resuspend the cells in 600 μl of FACS buffer containing PI dye. 14. Analyze the samples using a flow cytometer.

[0090] (Knockdown efficiency) To measure the efficiency of knockdown induction, dual luciferase assays are performed on test formulation samples loaded with specific (siLUC), non-specific (NSC4), and the unloaded formulation. 1. 6.5 x 10 cells per well (in a 96-well plate) in 100 μl of DMEM enriched with 10% FBS 3 HCES cells are seeded and grown for 24 hours under standard conditions. Two plates are prepared to study every formulation sample in five replicates each. 2. Transfect the cells with the Renilla and Luc2p plasmids using Lipofectamine 2000 according to the manufacturer's protocol: Add 50 μl of a reagent mixture containing 0.3 μl of Lipofectamine 2000, 1 ng of Renilla plasmid, and 5 ng of Luc2p plasmid diluted in OptiMEM medium to a total volume of 50 μl to each well. 3. Grow cells at 37° C. in 5% CO 2 for 24 hours. 4. Replace the medium with 90 μl of fresh DMEM per well. 5. Allow formulation samples to come to room temperature. Mix 32 μl of each sample with 28 μl of OptiMEM. 6. Prepare a control sample containing Lipofectamine transfection reagent. Mix 1.2 μl of Lipofectamine RNAiMAX Reagent into 55.8 μl of OptiMEM and incubate at room temperature for 5 minutes, then add 3 μl of siLUC stock solution and continue incubation for 10 minutes. Mix 1.2 μl of Lipofectamine RNAiMAX Reagent into 55.8 μl of OptiMEM and incubate at room temperature for 5 minutes, then add 3 μl of NSC4 stock solution and continue incubation for 10 minutes. Mix 1.2 μl of Lipofectamine RNAiMAX Reagent into 55.8 μl of OptiMEM and incubate at room temperature for 15 minutes. 7. Five replicates of cells are treated by adding 10 μM of loading or blank formulation or LPF control per well. 8. Cells are grown under standard conditions (37°C in 5% CO2). 9. After 48 hours, discard the medium and wash the wells with PBS. 10. Add 20 μl of Passive Lysis Buffer per well. 11. Incubate the plate at room temperature for 15 minutes on an orbital shaker (900 rpm). 12. Read luminescence levels using the Dual Luciferase Reporter Assay Kit and LUMIstar OPTIMA plate reader according to the manufacturer's protocol.

[0091] (Silicone-loaded nanoparticles combined with lipofectamine (SIS005-LPF)) 1. Aliquot the silicon nanoparticle suspension into six Eppendorf microtubes, each containing 20 μg of SiNPs. 2. Add 10 μg of siRNA to the above SiNP aliquot. a. Add 37 μl of 20 μM siLuc2p (targeting siRNA) to two microtubes. b. Add 37 μl of 20 μM NSC4 (non-specific siRNA) to two microtubes. c. (No-loading control) Set aside the remaining two microtubes containing SiNPs without siRNA to prepare an empty control. 3. Incubate the sample at room temperature with stirring for 1 hour, then vortex and place the tube in the freezer (-20°C) for 2-3 hours. 4. Connect the sample to a freeze-drying system overnight to evaporate the water. 5. Prior to further analysis, the above siRNA-loaded (or unloaded control) SiNP samples are dissolved in Lipofectamine solution (Lipofectamine RNAiMAX Reagent, Cat. No. 13778075, ThermoFisher Scientific), diluted in nuclease-free water to a total volume of 150 μl, vortexed, and incubated at room temperature for 1 hour. [Table 4]

[0092] (Results shown in Figure 3) The change in surface charge of silicon nanoparticles of the present invention upon loading was evaluated. Normally, the surface charge of particles decreases (changes from a more positive value to a more negative value) after binding with negatively charged siRNA molecules on the surface. This effect was observed in approximately half of the formulation samples tested, while the remaining samples did not show any significant differences. The change in ZP after siRNA loading is shown in Figure 3. The zeta potential of all samples containing PE was not affected by siRNA loading. Similarly, most formulations containing lecithin did not show significant differences in ZP. Only the lecithin-Si-NP (F31) sample at a ratio of 75 μg:200 μg showed a decrease in surface charge when loaded with siRNA (p=0.0001). Furthermore, for both PC-loaded nanoparticles (F01, F02), we observed a significant decrease in ZP after siRNA loading (p = 0.0002 for the Si:PC ratio of 75 μg:200 μg and p = 0.0005 for the Si:PC ratio of 150 μg:200 μg), and for PC-SiNPs (F04, F07) also loaded with arginine, we observed a significant decrease in ZP regardless of the amino acid content (p = 0.0196 for the Si:PC:Arg ratio of 75 μg:200 μg:20 μg and p = 0.0490 for the Si:PC:Arg ratio of 75 μg:200 μg:40 μg).

[0093] siRNA loading had the greatest effect on the surface charge of stearylamine-treated silicon nanoparticles, inducing a high negative charge. As shown in previous analyses, SA- and SA+arginine-loaded empty Si-NPs had positive or approximately neutral surface charges and therefore effectively attracted anionic siRNA molecules.

[0094] (Effect of Loading Ratio on mRNA Encapsulation Efficiency) The samples were prepared according to the protocol described above in SIS005-DSC613G. Each sample consisted of a weight ratio of silicon nanoparticles: dioleoylphosphatidylethanolamine (DOPE): stearylamine: cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC-cholesterol): glycine of 10:24:4:12:5. Samples were prepared with various ratios of nucleic acid to silicon nanoparticles, as shown in the table below. Table 5 shows ProSilic-DSC613G formulation samples with various mRNA loading ratios. [Table 5] *This is the weight ratio of all other components (silicon nanoparticles, lipids, and amino acids) to mRNA. ** Approximate charge ratio (known as N / P ratio) is calculated based on the length of the mRNA, the average molecular mass of the RNA (approximately 325 Da), and the molecular weight of the cationic lipid (stearylamine: 269.5 Da, DC-cholesterol: 500.8 Da).

[0095] (Evaluation of mRNA encapsulation in silicon nanoparticle formulations using gel electrophoresis) The effect of the silicon nanoparticle to mRNA loading ratio on mRNA encapsulation efficiency was examined by gel electrophoresis. A 1% E-Gel EX precast agarose gel was used. The gel was visualized using a Gel Logic 100 imaging system (Kodak). The results are shown in Figure 6.

[0096] Figure 6 shows unloaded mRNA (U0) in column 1. The mRNA-loaded silicon nanoparticles (L0.5, L1, L2, L3, L4, L5, L6, and L8) are shown in columns 2–9, from left to right, in order of increasing silicon nanoparticle to mRNA ratio. Equal amounts of mRNA (100 ng) were loaded in each of columns 1–9. Invitrogen's E-Gel 1Kb Plus Express DNA Ladder (80 ng) was loaded in column M as a marker. Column 10 was left empty (as a blank solution).

[0097] Electrophoresis showed that the SIS005-DSC613G formulation of the present invention successfully encapsulated mRNA, and that encapsulation was particularly successful at higher silicon nanoparticle to mRNA ratios, such as ratios greater than 2:1. The control column (unloaded mRNA, U0) in Figure 6 shows a single, fast-moving band, as expected. This band was also observed in columns 2 (loaded L0.5) and 3 (loaded L1). This observed band corresponds to unbound mRNA. The intensity of the bands in columns 2 and 3 (especially column 3) was lower than that of the control (U0). This indicates that even in samples with very low silicon nanoparticle to mRNA ratios, some mRNA is still encapsulated by the silicon nanoparticles and is therefore not observed in the band.

[0098] Efficiently encapsulated mRNA is unable to migrate through the gel pores and remains in the well (no band appears, unlike U0, L0.5, and L1). Figure 6 shows that samples L2 to L8, which contain increased amounts of silicon nanoparticles compared to columns 1 to 3, successfully encapsulate mRNA. This is evidenced by the absence of bands seen in U0, L0.5, and L1. This indicates that the silicon nanoparticle formulations of the present invention can successfully encapsulate mRNA. These results suggest that the optimal loading ratio (the ratio that maximizes mRNA encapsulation but minimizes the amount of nanoparticles used) is L2, which corresponds to 2:1 (silicon nanoparticles:mRNA) and 11:1 (all other components of the delivery system:mRNA). The same loading ratio has been found to be effective for siRNA loading. This ratio corresponds to an N / P charge ratio of approximately 2.5.

[0099] (Evaluation of Encapsulation Efficiency Using Spectrophotometry) To estimate the efficiency of mRNA encapsulation (EE, expressed as a percentage), SIS005-DSC613G samples (U0 to L8) were also centrifuged to separate unbound mRNA. The nucleic acid content in the supernatant liquid was measured spectrophotometrically, and the encapsulation efficiency was calculated using the following formula:

number

[0100] The results are shown in Figure 7 and confirm those of the gel electrophoresis experiments. As the ratio of silicon nanoparticles to mRNA increases, the encapsulation efficiency also increases until it reaches a plateau and levels off at silicon to mRNA ratios above 2:1.

[0101] (Evaluation of the activity of silicon nanoparticle formulations in vivo) The samples were prepared according to the protocols in SIS005-PS91G and SIS005-DSC613G described above, and contained the following weight ratios of siRNA, silicon nanoparticles, lipids, and glycine: [Table 6] SA is stearylamine, DOPE is dioleoylphosphatidylethanolamine, PC is phosphatidylcholine, and DC-Chol is cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride.

[0102] Preparation of siRNA-loaded silicon nanoparticle formulations Samples containing specific siRNA (siLUC) and nonspecific siRNA (NSC4) were prepared. Both siRNAs (siLUC and NSC4) were designed as 21mers with a 19-bp central double-stranded region and symmetric dTdT dinucleotide overhangs at each 3' end. The siRNAs were provided by Eurogentec (Shanghai).

[0103] To prepare the samples, siRNA dissolved in nuclease-free water was added to the aqueous nanoparticle solution and incubated at room temperature for 60 minutes. A 2:1 silicon nanoparticle to mRNA ratio was used (as this was found to be the optimal ratio in the gel electrophoresis and spectrophotometric experiments described above).

[0104] (Live animal imaging) Ethical approval was obtained from the Home Office (Scotland) and the Department of Health, Social Care and Public Safety (Northern Ireland), and animals were used in the following experiments in accordance with the UK Animal Welfare Act. Experiments evaluating the delivery of fluorescent siRNA (DY-547-labeled siGLO, Dharmacon, UK) to the cornea were performed in wild-type C57BL / 6 mice. To evaluate the bioavailability and silencing activity of the siRNA formulation, a reporter knock-in mouse strain (Krt12+ / luc2) was used to express firefly luciferase specifically in the corneal epithelium (under the control of the endogenous Krt12 promoter). This animal model was developed on a C57BL / 6 background as previously reported and provides a reliable model for the in vivo evaluation of siRNA delivery methods using reporter gene expression monitoring. For in vivo imaging, mice were anesthetized with 1.5–2% isoflurane (Abbott Laboratories Ltd., UK) in an oxygen flow of approximately 1.5 L / min. Fluorescence of siGlo was detected at predetermined time points after topical application using a Xenogen IVIS Spectrum (both Perkin Elmer, UK) with LivingImage 3.2 software, using a DsRed filter combination (excitation 535 nm, absorption 570 nm). To measure luciferase reporter gene expression, luciferin (30 mg / mL D-luciferin potassium salt, Gold Biotechnology, USA) mixed 1:1 w / w with Viscotears gel (Novartis, UK) was instilled into the eyes of anesthetized mice immediately before imaging. Bioluminescence readings were taken over approximately 10 minutes using the IVIS Spectrum to ensure that the signal remained stable within the acquisition time, and then quantified using LivingImage software. For signal intensity quantification, regions of interest (ROIs) were selected separately in each eye, and ROI parameters (size and shape) were kept constant throughout the experiment. A split body control measurement regime was used and values ​​are expressed as right eye / left eye ratio (RE / LE).

[0105] In vivo siRNA treatment Experiments were performed using split-body controls by comparing test treatment in one eye with a negative control in the other eye of the same animal. During treatment, mice were anesthetized as described above. Silicon nanoparticle formulations containing 25 μM siRNA complexed at a 2:1 SiNP-to-mRNA weight ratio were prepared and applied topically to the eye as droplets onto the intact cornea in a total volume of 4 μL. After application, mice were kept anesthetized for an additional 15 minutes to allow absorption and maximize uptake. Following treatment, fluorescence and luminescence experiments were performed as follows.

[0106] (Evaluation of siRNA penetration into the cornea) To investigate corneal delivery of siRNA, in vivo fluorescence studies were performed in wild-type mice using eye drops containing siGlo. A fluorescent siRNA-silicon nanoparticle formulation was applied to the right eye, and the same amount of naked siGlo was topically applied to the left eye of each mouse as a control. Live fluorescence imaging was acquired by IVIS Spectrum at 15 minutes (i.e., immediately after the treatment procedure) and 3, 6, and 24 hours after siGlo application. Signal intensity was normalized to background fluorescence measured before treatment (i.e., in the untreated eye) and quantified as described above. After measurements were taken at either 3 or 24 hours, the mice were sacrificed, and the eyes were removed and fixed in 4% paraformaldehyde in PBS at room temperature for 30 minutes, immersed in PolyFreeze (Sigma-Aldrich, UK), and immediately frozen at -80°C. Five-micrometer sections were cut using a cryostat (CM 1850, Leica) and mounted on APES-coated slides (3-aminopropyltriethoxysilane, Sigma Aldrich, UK) with DAPI-containing mounting medium (DAPI I, Vysis, USA), and fluorescence was visualized by an AxioScope A1 microscope (Carl Zeiss, Germany) equipped with an AxioCam MRc camera and 20× / 40×N Archoplan lenses.

[0107] (Evaluation of siRNA-mediated gene silencing) Luciferase reporter mice (n=7) were used to measure the bioavailability of siRNA in the cornea after topical delivery via silicon nanoparticles of the present invention. In a split-body control experiment, luciferase-targeted siLuC complexed with a silicon nanoparticle formulation was topically applied as droplets to the intact cornea of ​​the right eye (RE) of anesthetized mice, while the left eye (LE) was correspondingly treated with NSC4 complexed with a silicon nanoparticle formulation as a negative control. Treatment was repeated daily for 8 consecutive days, with in vivo ocular luminescence measurements performed approximately 4-5 hours later. The effect of treatment on luciferase reporter gene expression was determined by measuring luciferase bioluminescence activity (as described above) daily during the treatment regimen and for an additional 8 days after cessation of treatment to monitor the washout period. Baseline luminescence was defined for each experimental animal by monitoring ocular luciferase activity at 24-hour intervals for 4 days prior to treatment. Relative RE / LE luciferase bioluminescence activity was quantified using IVIS LivingImage software and plotted as mean values ​​± standard deviation.

[0108] (statistical analysis) Data are presented as mean ± standard deviation (SD) unless otherwise noted and represent at least three independent measurements. Statistical significance was assessed by one- or two-way analysis of variance followed by Tukey's post-hoc test at the 95% confidence level. For in vitro dual-luciferase assays, a two-tailed Student's t-test was performed separately for each formulation to analyze knockdown levels (siLuc vs. NSC4 control). For in vivo luciferase experiments, statistical analysis was performed by comparing the mean right / left ratio of all seven mice on the first four days before treatment initiation (set as baseline) with the right / left ratio measured on subsequent days. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, USA).

[0109] (result) (Characteristics of silicon-based siRNA delivery systems) Two variants of the silicon nanoparticle delivery system of the present invention (SIS005-PS91G and SIS005-DSC613G) were formulated by surface functionalizing silicon with cationic lipids commonly used for nucleic acid delivery, stearylamine and DC-cholesterol, resulting in hybrid particles with similar hydrodynamic dimensions of approximately 350 nm and comparable positive zeta potential values. Complexation of the cationic silicon nanoparticle formulations with siRNA, as examined by gel electrophoresis, demonstrated complete encapsulation of the nucleic acid at a minimum SiNP-siRNA w / w ratio of 2:1. The percentage of complexed siRNA at various w / w ratios was measured spectrophotometrically and calculated from the difference between the amount of siRNA loaded on the carrier and the siRNA concentration in solution after particle separation. Higher siRNA encapsulation efficiency was observed in complexes with nanoparticles containing cationic cholesterol derivatives compared to particles functionalized with stearylamine. However, both variants exhibited siRNA loading capacities ranging from 13 to 48 nmol per mg of silicon nanoparticles. After the siRNA loading studies described above, a fixed SiNP / siRNA ratio of 2:1 was chosen for all further experiments.

[0110] Because the physicochemical properties of nanoparticles play an important role in drug delivery, we measured the particle size and surface charge of siRNA-loaded complexes. While SIS005-DSC613G showed no significant differences in size or zeta potential when compared with the empty and loaded carriers, SIS005-PS91G exhibited an increase in mean particle size and a negative surface charge when complexed with siRNA, suggesting encapsulation of siRNA along with the absorption of nucleic acid molecules on the hybrid particle surface (see table below). The formulations investigated in this study were compared with Lipofectamine RNAiMAX, the gold standard for gene silencing, a commercially available lipid-based carrier specifically designed for siRNA delivery. Zetasizer analysis of empty and siRNA-loaded RNAiMAX also demonstrated an increase in particle size and a reversal of surface charge from positive to negative after complexation with nucleic acid. Table 7 shows the particle properties characteristics. [Table 7]

[0111] Measurements were performed in nuclease-free water and data represent the mean ± standard deviation (n=3).

[0112] (Evaluation of siRNA delivery to corneal cells in vitro) For initial in vitro screening, a human corneal epithelial cell line (HCE-S) was used to evaluate the effectiveness of the silicon nanoparticle delivery system of the present invention in cellular gene transfer, along with potential cytotoxicity. Gene transfer efficiency was quantified by flow cytometry analysis performed 24 hours after treatment with fluorescent oligonucleotide duplexes loaded into the carrier system. See Figure 8. Compared to 84±1% FAM-positive cells observed with the RNAiMAX reagent, SIS005-DSC613G showed 55±2% FAM-positive cells, and SIS005-PS91G showed 65±6% FAM-positive cells.

[0113] Cell viability after transfection was assessed based on live / dead staining with propidium iodide (PI), a common indicator of membrane disruption. This demonstrated that, in contrast to Lipofectamine, the silicon nanoparticle formulation was well tolerated by corneal epithelial cells. See Figure 9. Although Lipofectamine is highly effective in delivering exogenous nucleic acids to cells in vitro, it is not suitable for clinical use. While more than 50% of cells transfected with Lipofectamine RNAiMAX showed membrane damage and internalization of the membrane-impermeable PI dye, more than 86% and 98% of intact cells were observed after treatment with the silicon nanoparticle formulation.

[0114] The bioavailability of siRNA was evaluated in gene expression studies using a dual-luciferase reporter assay. After treating HCE-S cells with 0.1 μM siLuc complexed with a silicon-based delivery system, knockdown of 46 ± 5% (p<0.01, siLuc vs. NSC4 control) and 38 ± 8% (p<0.01) was achieved in SIS005-PS91G and SIS005-DCS613G, respectively, whereas siLuc delivered in RNAiMAX reduced luciferase reporter gene expression by 66 ± 9% (p<0.001). See Figure 10. Thus, the silicon nanoparticle delivery system was safe and well-tolerated by cells, while demonstrating up to 70% of the efficacy of commercially available siRNA delivery reagents.

[0115] Evaluation of in vivo ocular siRNA delivery by topical silicon nanoparticle formulations After demonstrating successful siRNA delivery and gene knockdown in vitro, two silicon nanoparticle formulations (SIS005-PS91G and SIS005-DCS613G) were evaluated in vivo for topical administration to the anterior segment of the eye. First, SIS005-PS91G and SIS005-DCS613G were conjugated with fluorescent siGlo and applied as eye drops to wild-type mice after unilateral treatment in which a naked siGlo control was instilled in the contralateral eye. Ocular fluorescence was monitored for up to 24 hours using an in vivo imaging system. The first measurement was taken 15 minutes after administration, while the mice were still anesthetized after treatment, and subsequent measurements were repeated at 3, 6, and 24 hours after administration. Equal amounts of siGlo were topically applied to each eye, but the highest fluorescence intensity measured after 15 minutes was observed in eyes treated with SIS005-DCS613G, slightly less in SIS005-PS91G, and twofold less in naked siGlo (p<0.05). See Figure 11. This demonstrates increased ocular surface adhesion of the formulated drug and improved residence time for the two silicon nanoparticle formulations compared to naked oligonucleotides. After 3 hours, the in vivo fluorescence signal decreased threefold in eyes treated with siGlo-SIS005-DSC613G but returned to baseline levels for siGlo-SIS005-PS91G and unformulated siGlo eye drops due to active ocular clearance mechanisms. Although a further gradual decrease in in vivo signal intensity was observed, fluorescence in eyes treated with siGlo-SIS005-DSC613G persisted up to 24 hours and was significantly higher than that in eyes treated with unformulated naked siGlo at all time points (p<0.01 at 3 and 6 hours, and p<0.05 at 24 hours). This suggests effective uptake of topically administered siRNA agents formulated with the silicon nanoparticles of the present invention. To verify the tissue penetration of the nanoparticles, the distribution of siRNA in the corneal layers was examined by fluorescence microscopy of corneal sections after treatment.Red siGlo fluorescence was detected throughout all corneal layers in all sections of ProSilic-treated eyes taken 3 hours after eye drop application, whereas no fluorescence above background was observed in the naked siGlo control. 24 hours after administration of the siRNA formulation, fluorescence was observed only in corneal sections treated with SIS005-DSC613G.

[0116] Following in vivo uptake studies, SIS005-DSC613G siRNA delivery was further investigated in functional assays using a mouse reporter model in which luciferase expression was restricted to the corneal epithelium. Prior to in vivo treatment, basal corneal luciferase activity in reporter mice was quantified every 24 hours for 4 days to confirm consistent right-to-left ratios in split-body control experiments. SIS005-DSC613G complexed with siLuc or control siRNA was applied topically as eye drops to the opposite eye of the same animal 8 times daily, and corneal luciferase expression was assessed daily by live-animal imaging during the treatment regimen and for the following 8 days. A reduction in luciferase expression was observed within 24 hours of treatment initiation, with maximum inhibition (41% ± 13%, p < 0.001) achieved on day 11. Significant gene silencing activity persisted throughout the treatment regimen and continued for 4 days after treatment termination. As expected, the decline in ocular bioluminescence levels slowly returned to baseline after treatment was stopped, indicating normal recovery from gene silencing. See Figures 12 and 13 for these results. Importantly, gross examination of the treated eyes and daily visual inspection of the animals after topical treatment revealed no adverse effects from the eye drops, suggesting that the silicon nanoparticle formulation was well tolerated in vivo.

Claims

1. A composition for the controlled release of a nucleic acid that is RNA, comprising: silicon nanoparticles that have been surface-treated with at least one lipid, including a phospholipid, and further treated with at least one amino acid, including arginine and / or glycine; the silicon nanoparticles comprise at least 50% silicon by weight and are loaded with the RNA; A composition having a silicon to nucleic acid ratio of 2:1 to 8:

1.

2. The ratio of silicon to nucleic acid is 2:1 to 3:1; The composition of claim 1.

3. Further comprising a gene transfer reagent and / or further comprising at least one disaccharide; The composition of claim 1.

4. The gene transfer reagent is lipofectamine. The composition of claim 3.

5. the lipid to silicon ratio is 1:1 to 15:1 and / or the amino acid to silicon ratio is 0.05:1 to 2:1; The composition according to any one of claims 1 to 4.

6. The silicon nanoparticles have an average diameter of 20 to 200 nm. The composition according to any one of claims 1 to 4.

7. The phospholipid is selected from phosphatidylcholine, hydrogenated phosphatidylcholine, didecanoylphosphatidylcholine, myristoyl phosphatidylcholine, lecithin, phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or a combination thereof. The composition according to any one of claims 1 to 4.

8. the phospholipid is selected from phosphatidylcholine, hydrogenated phosphatidylcholine, lecithin, phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or a combination thereof; The composition according to any one of claims 1 to 4.

9. Use of a composition in the manufacture of a medicament for the controlled release of a nucleic acid that is RNA, comprising: the composition comprises silicon nanoparticles that have been surface-treated with at least one lipid, including a phospholipid, and further treated with at least one amino acid, including arginine and / or glycine; the silicon nanoparticles comprise at least 50% silicon by weight and are loaded with the RNA; Use wherein the ratio of silicon to nucleic acid is 2:1 to 8:

1.

10. In the manufacture of a medicament for use in treating an eye disorder, Use of the composition according to any one of claims 1 to 8.

11. In the manufacture of a drug for delivery to the eye, Use of the composition according to any one of claims 1 to 8.

12. In the manufacture of a medicament for the treatment of ocular tissues, Use according to any one of claims 1 to 8.

13. The ocular tissue is selected from the cornea, sclera, iris, ciliary body, choroid, zonular fibers, lens capsule, lens nucleus, vitreous body, and retina.

13. The use according to claim 12.

14. In the manufacture of a medicament for the treatment of an ocular disorder, Use of the composition according to any one of claims 1 to 8.

15. The ocular disorder is selected from macular degeneration, conjunctivitis, glaucoma, diabetic retinopathy, diabetic macular edema, keratoconus, cataract, retinitis, or uveitis.

15. The use according to claim 14.

16. the ocular disorder is macular degeneration; 16. The use according to claim 15.

17. The lipid and the amino acid are (a) stearylamine (SA), phosphatidylcholine (PC) and glycine, or (b) stearylamine (SA), dioleoyl L-α-phosphatidylethanolamine (DOPE), N-(2-dimethylaminoethyl)carbamate cholesterol (DC-Chol), and glycine; The composition according to any one of claims 1 to 8.

18. The lipid and the amino acid are (a) stearylamine (SA), phosphatidylcholine (PC) and glycine, or (b) stearylamine (SA), dioleoyl L-α-phosphatidylethanolamine (DOPE), N-(2-dimethylaminoethyl)carbamate cholesterol (DC-Chol), and glycine; Use according to any one of claims 9 to 16.

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