Delivery system comprising 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 present invention relates to the use of silicon nanoparticles for the delivery of short interfering RNA, messenger RNA, or other nucleic acids. The present invention also relates to related compositions. [Background technology]

[0002] Gene therapy has attracted considerable interest as an approach to treat genetic diseases due to its specificity. Short interfering RNA (also known as small interfering RNA or siRNA) can be designed to target virtually any gene, and has great potential for disease treatment. It is clear that siRNA technology has great therapeutic potential in medicine. However, one of the main limitations in its in vivo and in vitro applications is that siRNA molecules cannot cross the cell membrane and reach the cytoplasm. siRNA cannot diffuse across the cell membrane because the negative charges arising from the phosphate groups of the siRNA backbone are electrostatically repelled by the negatively charged cell membrane. Delivery of siRNA across the cell membrane is also hindered by its short half-life in vitro, due to effective cellular and systemic defense mechanisms that degrade naked siRNA molecules. There are other obstacles to the proper application of siRNA therapy in medicine, such as the high molecular weight of siRNA, low specificity and low uptake in target tissues, cytotoxicity, and undesirable off-target effects.

[0003] Also, 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 possible damage to genome integration. While siRNA shows great potential for treating disorders caused by high or inappropriate expression of genes, mRNA may be used to treat disorders caused by low gene expression. Also, mRNA may be used in vaccines, including antitumor vaccines. mRNA-based therapeutics face the same challenges as any therapeutic nucleic acid, namely safety, specificity and efficiency of delivery.

[0004] RNA therapeutics (including siRNA and mRNA therapeutics) are also applicable to the treatment of ocular disorders, but the intrinsic physical barriers, effective drug clearance mechanisms, and the complexity of other ocular tissues pose great 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 structure of the cornea, which adapts to form an effective barrier against fluid loss and pathogen invasion.

[0005] Although all ocular tissues are accessible by injection, topical application would be preferred for the frequent treatment regimes required for siRNA-induced gene silencing and mRNA-mediated treatment. Traditionally, drug delivery to the cornea is problematic because eye drops are rapidly drained through the nasolacrimal duct. Rapid tear drainage, in conjunction with the blink reflex, shortens the precorneal residence time available for the active agent to be absorbed through the cornea. It is estimated that 95% of the active compound contained in eye drop formulations is lost because it cannot cross the conjunctiva or is lost through 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 on 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, to the eye is limited by the anatomical constraints and physiological protective mechanisms of the eye.Below are listed some chemically modified siRNA therapeutics that are undergoing clinical trials, the majority of which 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 in ocular diseases require frequent treatment regimens, increasing the likelihood of cataracts, retinal detachment, vitreous hemorrhage and endophthalmitis by requiring multiple injections.

[0008] Other approaches to RNA delivery include ocular injections containing modified viral particles. The particles are modified to deliver nucleic acid payloads to the eye. Such virus 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 mutagenesis, limited loading capacity, proper targeting, insertion predictability, high cost of production, 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 may 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, encapsulation of RNA into nanocarriers can be useful to provide a sustained release form of RNA over a long period of time, thus improving treatment regimens.

[0010] A number of non-viral 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] Many kinds of polymers have been used to deliver oligonucleotides, but much attention has been 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 to escape intracytosolic RNA delivery. Although many materials have been investigated, the development of biodegradable nano-carriers to enhance RNA delivery without safety concerns remains a challenge.

[0012] Despite the many available RNA delivery strategies, the lack of safe and effective delivery in vivo has limited the translation of RNA therapeutics into the clinic. A robust carrier system that can effectively condense oligonucleotides, be stable in physiological conditions, facilitate cellular uptake regardless of ocular barrier mechanisms, and promote release of RNA 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 a nucleic acid, such as a 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 has a beneficial effect on the surface charge of silicon nanoparticles, so as to provide the necessary zeta potential that can enhance the loading of nucleic acid such as siRNA or mRNA, and control the release rate of nucleic acid at target site.The presence of at least one lipid in the formulation can also control the hydrolysis rate of silicon, so that silicon nanoparticles hydrolyze into bioavailable orthosilicic acid (OSA) decomposition products.In certain embodiments, the nucleic acid is RNA.In certain embodiments, the RNA is siRNA.In certain embodiments, the 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 transported 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 invention there is provided a composition according to the first aspect of the invention for use as a medicament.

[0019] In a third aspect of the invention, there is provided a composition according to the first aspect of the 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 in 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 description of the drawings]

[0021] [Figure 1] 1 shows a comparison of the zeta potential of siRNA-loaded and non-loaded compositions of the invention, with each formulation labeled F1-F5. [Diagram 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. [Diagram 3] FIG. 3 shows a comparison of the zeta potential of siRNA-loaded and non-loaded 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 non-loaded compositions of the invention when the silicon nanoparticles are surface treated with lecithin and the amine is arginine. [Diagram 5] 5 shows the results of gene transfer efficiency of the compositions of the present invention in HCE cells. The cells in the first column are stained with DAPI and fluoresce in blue to show the nuclei. The cells in the second column fluoresce in green to show successful gene transfer of the formulations F2 to F5 of the present invention. [Figure 6] FIG. 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 more (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 a 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 a siRNA-loaded silicon nanoparticle delivery system of the invention. [Figure 10] FIG. 10 shows the results of an experiment measuring the extent of siRNA-induced gene silencing when siRNA is delivered to cells using a silicon nanoparticle delivery system according to 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 a 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 a siRNA-loaded silicon nanoparticle delivery system of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In a first aspect of the present invention, there is provided a composition for the controlled release of a nucleic acid, 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 has 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 a substitution, the substitution can be a non-conservative substitution or a conservative substitution, preferably a conservative substitution.

[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 invention, the composition comprises silicon nanoparticles. The silicon nanoparticles have a nominal diameter of 5-400 nm, such as 50-350 nm, such as 80-310 nm, such as 100-250 nm, such as 120-240 nm, such as 150-220 nm, such as 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 given above may refer to average diameters, and at least 90% of the total particles in a silicon nanoparticle sample may be 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 contacting the particles with 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 volumetric 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 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 one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and 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 ​​nonporous material with comparable dimensions. In some embodiments (e.g., when 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), their total area is preferably increased by at least 50% or at least 100% compared to the surface area of ​​the corresponding nonporous particle due to their porosity. In many cases, porous silicon nanoparticles actually have a much larger increase in total surface area due to their porosity.

[0028] Preferably, (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, and such nanoparticles are formulated for topical delivery, such as delivery to the skin surface or topical delivery to the eye) the silicon nanoparticles have an average diameter of 2-300 nm, e.g. 20-290 nm, 20-280 nm, e.g. 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220 nm, 20-210 nm, in particular 20-200 nm. Advantageously, silicon nanoparticles of this size are suitable for dermal delivery, since they are small enough to block the pilosebaceous ostra or sweat ducts (pores), but their small size allows the particles to actively penetrate to the base of the hair follicle rather than acting only as a surface drug reservoir.

[0029] (Fats) In all aspects of the present invention, silicon nanoparticles are surface-treated with at least one lipid (e.g., PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives, such that nanoparticles formulated in this way can be suitable for delivery to the eye) (e.g., when 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. Subject to the properties of bioactive agents (e.g., 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, so as to provide the necessary zeta potential that can enhance the loading of short interfering RNA or messenger RNA, and control 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) delivered 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 (range of -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 (range of 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% by weight of lipid, for example at least 20% by weight, typically at least 30% by weight, and in particular at least 50% by weight of lipid, based on the total weight of the coated nanoparticles. Molar ratios of lipid to silicon of 0.8:1 to 3:1, for example 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, which may make such nanoparticles 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 in particular at least 50% by weight of phospholipid, 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.

[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 are those in which a polar head group is 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, with neutral or negatively charged lipids being 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 combinations thereof. This can be particularly advantageous when the siRNA / mRNA is charged.

[0037] 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 / or one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol and 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% hydrogenated phosphatidylcholine by weight, for example at least 20%, typically at least 30%, and in particular at least 50% hydrogenated phosphatidylcholine by weight, based on the total weight of the coated nanoparticles. It has been found that 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, is particularly advantageous.

[0038] 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 at least 5% by weight of phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, and in particular at least 50% by weight of phosphatidylcholine based on the total weight of coated nanoparticles.It has been found that the 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, is particularly advantageous.

[0039] 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 surface-treated silicon nanoparticles with at least 5% by weight of lecithin, for example at least 20% by weight, typically at least 30% by weight, and in particular at least 50% by weight of lecithin, based on the total weight of 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 (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 at least 5% by weight of stearylamine, for example at least 20% by weight, typically at least 30% by weight, and in particular at least 50% by weight of stearylamine, based on the total weight of the coated nanoparticles.A lipid to silicon molar ratio of 0.8:1 to 3.5:1, such as 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 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 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 invention, the lipid-treated silicon nanoparticles are further treated with amino acids (e.g., when the nanoparticles of the invention are formulated with PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives). In the broadest sense, the term "amino acid" encompasses any organic compound of artificial or natural origin that contains amine (-NH2) and carboxyl (-COOH) functional groups. This includes alpha, beta, gamma, and delta amino acids. This includes amino acids of any chiral configuration. In some embodiments, the amino acid is preferably an alpha amino acid of natural origin (e.g., when the nanoparticles of the invention are formulated with PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives). This can be a proteinogenic or non-proteinogenic amino acid, such as carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline. In a preferred embodiment (e.g., when the nanoparticles of the 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 a particularly preferred embodiment, 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 the 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 nucleic acid such as siRNA or mRNA becomes bioavailable when surface-treated silicon nanoparticles (e.g. nanoparticles treated with PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and one or more of their derivatives, and / or glycine, arginine, and histidine) degrade.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 release only when nanoparticles move to a location away from application site.For example, this may be when nanoparticles move from the skin surface to which they are applied to a basal location.

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

[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 their derivatives). Silicon nanoparticles surface-treated with one or more of glycine and arginine and loaded with nucleic acid such as RNA showed better bioactivity stability in biological fluids and effectively delivered charged nucleic acid such as RNA, such as siRNA or mRNA, in ocular cell 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 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, e.g. 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, in particular 0.3:1-0.7:1. Advantageously, this amino acid to silicon ratio also influences the release rate of the RNA molecule transported by the silicon nanoparticles and stabilizes said RNA molecule.

[0049] In all aspects of the invention, the 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, such as 0.08:1 to 0.35:1, in particular 0.09:1 to 0.32:1. In some embodiments, (e.g., when lipid is selected from one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and their derivatives), amino acid is a combination of arginine and glycine, and the ratio of Arg:Gly is 1:0.6-3:1, such as 1:0.8-2.5:1, such as 1:1-2:1. Advantageously, such ratio has been found to provide high-speed RNA release and effective delivery of RNA (such as siRNA or mRNA) in ocular cell and cytoplasmic environment. Thus, the lipid-processed silicon nanoparticles of the present invention formulated with amino acid can be particularly suitable for delivery to the eye, and can provide a delivery system for delivery of nucleic acid (such as siRNA or mRNA) to the eye.

[0050] In another embodiment of all aspects of the invention, the lipid-processed silicon nanoparticles are processed with arginine (e.g., when the nanoparticles of the invention are formulated for delivery to the eye). 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, such as 0.08:1 to 0.35:1, in particular 0.09:1 to 0.32:1. Advantageously, such ratios have been found to provide a high rate of RNA release and effective delivery of RNA (such as siRNA or mRNA) in the ocular cell and cytoplasmic environment. Thus, the lipid-processed silicon nanoparticles of the invention, formulated with amino acids, can be particularly suitable for delivery to the eye and can provide a delivery system for delivery of nucleic acids (such as siRNA or mRNA) to the eye.

[0051] In another embodiment of all aspects of the invention, the lipid-processed silicon nanoparticles are processed with glycine (e.g., when the nanoparticles are formulated for delivery to the eye). 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, such as 0.08:1 to 0.45:1, in particular 0.09:1 to 0.42:1. Advantageously, such ratios have been found to provide a high rate of RNA release and to effectively deliver charged RNA, such as siRNA and mRNA, in the ocular cellular and cytoplasmic environment, and such ratios support cellular internalization. Thus, the lipid-processed silicon nanoparticles of the invention, formulated with amino acids, can be particularly suitable for delivery to the eye and can provide a delivery system for delivering nucleic acids, such as siRNA or mRNA, to the eye.

[0052] (RNA) In a preferred embodiment 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 derivatives thereof, making such formulations suitable for delivery to the eye), 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, operating within the RNA interference (RNAi) pathway, e.g., 10-45 base pairs, 15-40 base pairs, 20-30 base pairs, and especially 20-25 base pairs in length. The term "mRNA" encompasses messenger RNA, and may include mRNAs that optionally include a 5-prime cap and / or polyadenylated end. Alternatively, one or both of these features may be absent.

[0053] In a preferred embodiment of all aspects of the present invention, the RNA can be of natural origin or chemically modified to enhance therapeutic properties such as improved activity, increased serum stability, less 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, such that the formulation is suitable for delivery to the eye). Chemical modification of RNA can include any modification generally known in the art.

[0054] In certain embodiments, RNA is siRNA (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, and such formulations can be suitable for delivery to the eye).In other embodiments, RNA is mRNA (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, and such formulations can be suitable for delivery to the eye).

[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, such formulations may be 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, such formulations can be 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 ratio of silicon to nucleic acid can be particularly suitable for delivery to the eye, and can provide a delivery system for delivering nucleic acid to ocular tissue.

[0057] (Other ingredients) (Gene transfer reagent) In a preferred embodiment 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, such formulations may be suitable for delivery to the eye), the lipid-treated silicon nanoparticles may be surface-treated with a transfection agent. In the broadest sense, "transfection" is the process of deliberately introducing naked or purified nucleic acid into eukaryotic cells. Transfection may also refer to other methods and cell types. mRNA may be transfected into cells to translate the sequence. siRNA may be transfected to perform 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 acid 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 derivatives thereof, 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 (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), nucleic acids such as RNA are provided in a solution, such as a buffer (alone or in combination with various other nucleic acids).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 their derivatives), nucleic acids such as RNA are provided as a salt, alone or in combination with other isolated nucleic acids. 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 their derivatives), nucleic acid such as RNA is provided in a reconstitutable lyophilized form.For example, in some embodiments, nucleic acid such as RNA can be provided in a lyophilized pellet alone or in a lyophilized pellet containing other isolated nucleic acid.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 their derivatives), nucleic acid such as RNA is provided immobilized on a solid material such as beads or 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), a nucleic acid such as RNA is provided in a host cell, for example a cell line containing a plasmid or a cell line containing a stably integrated sequence.

[0062] Nucleic acids such as RNA for use according to the present invention include double and single stranded DNA, RNA, DNA:RNA hybrids, as well as PNA (peptide nucleic acid) or hybrids between RNA or DNA. The term also includes known types of modifications, such as, for example, labels known in the art, methylation, "capping," replacement 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.), those containing intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified linkages (e.g., alpha 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 that contain not only purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides may also include modifications of the sugar moiety, e.g., one or more of the hydroxyl groups are replaced with halogens, aliphatic groups, or functionalized as ethers, amines, and the like. Other modifications of nucleotides or polynucleotides involve rearranging, adding, substituting, or altering the functional groups of the purine or pyrimidine bases that form hydrogen bonds with the respective complementary pyrimidines or purines, e.g., isoguanine and isocysteine, and the like. In some embodiments, the oligonucleotides and / or probes include at least one, two, three, or four modified nucleotides.

[0064] In some embodiments, the nucleic acids, such as RNAs, 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) Silicon nanoparticles according to the present invention can be prepared simply by techniques conventionally used in the art, for example, by milling process or other known techniques for reducing particle size.Silicon-containing nanoparticles can be made from sodium silicate particles, colloidal silica, or silicon wafer materials.Macro- or micro-scale particles are ground in a ball mill, planetary ball mill, or other size-reducing mechanism.The resulting particles can be air classified to recover nanoparticles.Plasma methods and laser ablation can also be used to make nanoparticles.

[0066] The porous particles can be made by methods conventional in the art, including the methods 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 in a cream or gel base.For example, silicon nanoparticles can be mixed into a medicinal cream base.For gels, powders can be mixed into a gel matrix in powder form and then the gel can be hydrated, or powders can be mixed into a pre-hydrated gel.

[0068] (Delivery to the eye) In a third aspect of the invention, there is provided a composition according to the first aspect of the invention (e.g., 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 tissue, such as one or more of the tissues of fibrous layer, vascular layer and 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 of the tissues selected from the tissues of cornea, sclera, iris, ciliary body, choroid, zonular fiber, lens capsule, lens nucleus, vitreous body and 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 of the 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 of the 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 into ocular cells, ensuring the controlled release of 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 addition, the present invention provides, in a further aspect, a method and a medicament 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 excipients compatible with the eye, to one or more ocular tissues (for example, to one or more tissues selected from the tissues of the cornea, sclera, iris, ciliary body, choroid, zonular fiber, lens capsule, lens nucleus, vitreous body, and retina).A method is provided for treating an eye disorder, including delivering the composition of the first aspect of the present invention, optionally in combination with one or more excipients compatible with the eye, 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, in particular macular degeneration. EXAMPLES

[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. 80 mA / cm 2 in 1:1 (v / v) pure ethanol and 10% aqueous HF acid was used. 2 Etched silicon was prepared by anodic etching of p-type Si at anodic current density 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 milling balls and / or pestle and mortar. The fine powder was sieved using a shaker as200 with Retsch brand sieves (gauge 38 μm). The opening size of the sieve resulted in a selected uniform size (20-100 μm). Particle size was measured by a quantachrome system and PCS of a Malvern instrument. Samples were stored in airtight containers until further use.

[0077] Nano silicon powders were also obtained from Sigma and Hefel Kaier (China). Particle size was measured by PCS and recorded (size ranged from 20-100 nm) before loading and etching. Silicon wafers were ground using milling balls or with a pestle and mortar. Fine powders were sieved using a Retsch brand sieve (gauge 38 μm) shaker as200 to collect uniform nanoparticles with the desired dimensions.

[0078] (Activation of silicon nanoparticles) 250 ml of ethanol and 500 mg of 100 nm diameter porous silicon nanoparticles were mixed and stirred with a magnetic bar for 30 minutes. 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 silicon nanoparticles containing siRNA. 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 would be favorable for a corneal siRNA delivery system. For this purpose, the ratio of cationic lipids in the formulation was increased.

[0080] Colloidal stability was evaluated by dynamic light scattering in parallel with zeta potential measurement for both unloaded and loaded formulations. siRNA encapsulation efficiency was also measured by spectrophotometry. Gene transfer (internal internalization) efficiency was evaluated in human corneal epithelial cells by flow cytometry followed by 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 No. 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) non-specific 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, standard test tubes, Eppendorf tubes, micropipettes, freeze-drying systems, Zetasizer, 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 solution of filtered silicon nanoparticles in nuclease-free water 2. Aliquot (700 μl) the above solution into 8 Eppendorf microtubes, each containing 140 μg of SiNPs. 3. To each aliquot, add siRNA and glycine and adjust volume to 1.4 ml with nuclease free water. [Table 2] 4. Mix the tube thoroughly and incubate the sample with agitation for 1 hour at room temperature.

[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 into a small round bottom flask and mix thoroughly. Prepare 8 replicates (in 8 flasks) of each lipid mixture. a. (Lipid base DS61) 68 μg DOPE (340 μl) 12 μg SA (60 μl) b. (Lipid base PS91) 72 μg PC (360 μl) 8 μg SA (40 μl) c. (Lipid-based DSC613) 48 μg DOPE (240 μl) 8 μg SA (40 μl) 24 μg DC-Chol (120 μl) d. (Lipid-based PDS1051) 50 μg PC (250 μl) 25 μg DOPE (125 μl) 5 μg SA (25 μl) e.(Lipid-based PDS1052) 48 μg PC (240 μl) 23 μg DOPE (115 μl) 9 μg SA (45 μl) f.(Lipid base PDSC10514) 40 μg PC (200 μl) 20 μg DOPE (100 μl) 4 μg SA (20 μl) 16 μg DC-Chol (80 μl) 3. Carefully evaporate the solvent using a rotary evaporation system 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 blank Si nanoparticle samples (from step I). Add 200 μl of 1, 2, 3, or 4 silicon nanoparticle / siRNA mixtures separately 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 freezer and place in a 30°C water bath for 10 minutes. Cool at 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 examined by separating the loaded particles from free (unbound) siRNA by high speed centrifugation, followed by measurement of the encapsulation efficiency. 1. Take 50 μl of each sample into a centrifuge microtube. 2. Centrifuge all samples at 21,000 x g for 30 minutes. 3. Transfer 25 μl (top half) of the supernatant of 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 remaining supernatant) to disrupt the lipid bilayer and release 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. Each measurement is performed in triplicate. The data are shown in Figures 1, 2, and 3.

[0089] (Gene transfer 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 × 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 h (to 80% confluency). 2. Replace 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 gene transfer 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 in 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 unloaded formulations. 1. 6.5 × 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 Renilla and Luc2p plasmids using Lipofectamine 2000 according to the manufacturer's protocol: Add 50 μl of a reagent mixture to each well, containing 0.3 μl Lipofectamine 2000, 1 ng Renilla plasmid, and 5 ng Luc2p plasmid, diluted in OptiMEM medium to a total volume of 50 μl. 3. Cells are grown for 24 hours at 37° C. in 5% CO2. 4. Replace the medium with 90 μl of fresh DMEM per well. 5. Allow formulation samples to reach 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 Lipofectamine RNAiMAX Reagent into 55.8 μl OptiMEM and incubate at room temperature for 5 minutes, then add 3 μl siLUC stock solution and continue incubation for 10 minutes. Mix 1.2 μl Lipofectamine RNAiMAX Reagent into 55.8 μl OptiMEM and incubate at room temperature for 5 minutes, then add 3 μl NSC4 stock solution and continue incubation for 10 minutes. Mix 1.2 μl Lipofectamine RNAiMAX Reagent into 55.8 μl OptiMEM and incubate at room temperature for 15 minutes. 7. Five replicates of cells are treated by adding 10 μM 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 for 15 minutes at room temperature on an orbital shaker (900 rpm). 12. Read luminescence levels using the Dual Luciferase Reporter Assay Kit and a LUMIstar OPTIMA plate reader according to the manufacturer's procedures.

[0091] (siRNA-loaded silicon nanoparticles combined with Lipofectamine (SIS005-LPF)) 1. Aliquot the silicon nanoparticle suspension into 6 Eppendorf microtubes, each containing 20 μg SiNPs. 2. To the above SiNP aliquot, add 10 μg of siRNA. 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 blank controls. 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 samples 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 when loaded was evaluated. Usually, the surface charge of a particle 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 almost half of the formulation samples tested, while the remaining samples did not show any significant difference. The change in ZP after loading with siRNA 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 difference in ZP. Only the lecithin-Si-NP (F31) sample with a ratio of 75μg:200μg showed a decrease in surface charge when loaded with siRNA (p=0.0001). We also observed a significant decrease in ZP after siRNA loading for both nanoparticles loaded with PC (F01, F02) (p=0.0002 for Si:PC ratio of 75μg:200μg and p=0.0005 for Si:PC ratio of 150μg:200μg) and for PC-SiNPs also loaded with arginine (F04, F07) regardless of the amino acid content (p=0.0196 for Si:PC:Arg ratio of 75μg:200μg:20μg and p=0.0490 for Si:PC:Arg ratio of 75μg:200μg:40μg).

[0093] siRNA loading had the greatest effect on the surface charge of stearylamine-surface-treated silicon nanoparticles, inducing a high negative charge. As shown in previous analyses, SA and SA + arginine-loaded blank 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 loading ratio of silicon nanoparticles to mRNA on the efficiency of mRNA encapsulation was examined by gel electrophoresis. 1% E-Gel EX precast agarose gels were used. The gels were visualized using a Gel Logic100 imaging system (Kodak). The results are shown in Figure 6.

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

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

[0098] Efficiently encapsulated mRNA cannot 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-L8, which have an increased content of silicon nanoparticles compared to columns 1-3, successfully encapsulate mRNA. This is evidenced by the absence of bands seen in U0, L0.5, and L1. This indicates that the silicon nanoparticle formulation of the present invention can successfully encapsulate mRNA. This result suggests that the optimal loading ratio (the ratio that shows the greatest 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 about 2.5.

[0099] (Evaluation of Encapsulation Efficiency Using Spectrophotometric Method) To estimate the efficiency of mRNA encapsulation (EE, expressed as a percentage), SIS005-DSC613G samples (U0-L8) were also centrifuged to separate unbound mRNA. The nucleic acid content in the supernatant liquid was measured by spectrophotometry, 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, containing siRNA, silicon nanoparticles, lipids, and glycine in the following weight ratios: [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 non-specific siRNA (NSC4) were prepared. Both siRNAs (siLUC and NSC4) were designed as 21mers with a 19bp central double-stranded region and symmetric dTdT dinucleotide overhangs at each 3' end. 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 for 60 min at room temperature. A 2:1 ratio of silicon nanoparticles to mRNA 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 by 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 delivery of fluorescent siRNA (DY-547-labeled siGLO, Dharmacon, UK) to the cornea were performed in wild-type C57BL / 6 mice. To evaluate the siRNA bioavailability and silencing activity of the formulations, a reporter knock-in mouse line (Krt12+ / luc2) was used to express firefly luciferase (under the control of the endogenous Krt12 promoter) specifically in the corneal epithelium. This animal model was developed from 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. siGlo fluorescence was detected at the indicated time points after topical application by Xenogen IVIS Spectrum with LivingImage3.2 software (both Perkin Elmer, UK) 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 by IVIS Spectrum for approximately 10 min to ensure that the signal remained stable within the acquisition time, and then quantified using LivingImage software. For quantification of signal intensity, 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 The experiment was performed with a split-body control by comparing the test treatment in one eye with a negative control in the other eye of the same animal. During the treatment, the 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 on the intact cornea in a total volume of 4 μL. After application, the mice were kept anesthetized for an additional 15 minutes to allow absorption and maximize uptake. After 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. Fluorescent siRNA-silicon nanoparticle formulations were applied onto the right eye, and the same amount of naked siGlo was applied topically to the left eye of each mouse as a control. Fluorescent live imaging was acquired by IVIS Spectrum at 15 min (i.e. immediately after the treatment procedure), and 3, 6, and 24 h after siGlo application, and signal intensity was normalized to background fluorescence measured before treatment (i.e. untreated eye) and quantified as described above. After measurements were made at either 3 or 24 h, mice were sacrificed, and eyes were removed and fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, 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 by silicon nanoparticles of the invention. In split-body control experiments, luciferase-targeted siLuC complexed with silicon nanoparticle formulations was topically applied as droplets to the intact cornea of ​​the right eye (RE) of anesthetized mice, whereas the left eye (LE) was correspondingly treated with NSC4 complexed with silicon nanoparticle formulations as a negative control. Treatments were repeated daily for 8 consecutive days, with in vivo ocular luminescence measurements taken approximately 4-5 hours later. The effect of treatment on luciferase reporter gene expression was determined by measuring luciferase bioluminescence activity (as described above) every day of the treatment regimen and for an additional 8 days after cessation of treatment to monitor the drug holiday period. Baseline luminescence was defined in 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 unless otherwise stated and represent at least three independent measurements. Statistical significance was assessed by one-way or two-way ANOVA followed by Tukey's HSD 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 the knockdown level (siLuc vs. NSC4 control). For in vivo luciferase experiments, statistical analysis was performed by comparing the mean right / left ratio of all seven mice in the first 4 days before the start of treatment (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 functionalization of silicon with cationic lipids, stearylamine and DC-cholesterol, commonly used for nucleic acid delivery, resulting in hybrid particles with similar hydrodynamic dimensions of about 350 nm and comparable positive zeta potential values. Complexation of the cationic silicon nanoparticle formulations with siRNA, examined by gel electrophoresis, showed 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 by spectrophotometry and calculated from the difference between the amount of siRNA added to the carrier and the siRNA concentration in the solution after particle separation. A higher efficiency of siRNA encapsulation was observed in complexation with nanoparticles containing cationic cholesterol derivatives compared to particles functionalized with stearylamine. However, both variants showed 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] Since the physicochemical properties of nanoparticles play an important role in drug delivery, the particle size and surface charge of the siRNA-loaded complexes were measured. SIS005-DSC613G showed no significant differences in size and zeta potential when comparing the empty and loaded carriers, whereas SIS005-PS91G showed an increase in average 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 to the gold standard for gene silencing, Lipofectamine RNAiMAX, a commercially available lipid-based carrier specifically designed for siRNA delivery. Zetasizer analysis of empty and siRNA-loaded RNAiMAX also showed an increase in particle size and a reversal of the surface charge from positive to negative values ​​after complexation with nucleic acids. Table 7 characterizes the particle properties. [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, human corneal epithelial cell line (HCE-S) was used to evaluate the effect of the silicon nanoparticle delivery system of the present invention in cell gene transfer, together with the potential for cytotoxicity. Gene transfer efficiency was quantified by flow cytometry analysis performed 24 hours after treatment with fluorescent oligonucleotide duplexes loaded into carrier system. See Figure 8. Compared to 84±1% FAM-positive cells observed with 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 evaluated based on live / dead staining with propidium iodide (PI), a common indicator of membrane disruption. This showed that, in contrast to lipofectamine, silicon nanoparticle formulations were well tolerated by corneal epithelial cells. See FIG. 9. Although lipofectamine is highly effective in delivering exogenous nucleic acids to cells in vitro, it is not suitable for clinical use. More than 50% of cells transfected with lipofectamine RNAiMAX showed membrane damage and internalization of membrane-impermeable PI dye, whereas more than 86% and 98% of intact cells were observed after treatment with silicon nanoparticle formulations.

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

[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 complexed with fluorescent siGlo and applied as eye drops to wild-type mice after unilateral treatment in which naked siGlo control was instilled in the opposite 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 under anesthesia after treatment, and subsequent measurements were repeated at 3, 6, and 24 hours after administration. Equal amounts of siGlo were applied topically to each eye, but the highest fluorescence intensity measured after 15 minutes was observed in the SIS005-DCS613G treatment, slightly less in SIS005-PS91G, and 2-fold less in naked siGlo (p<0.05). See FIG. 11. This demonstrated increased ocular surface adhesion of the formulated drug compared to the naked oligonucleotide, and improved residence time in the two silicon nanoparticle formulations. After 3 hours, the in vivo fluorescence signal was reduced 3-fold in eyes treated with siGlo-SIS005-DSC613G, but returned to baseline levels in 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, the fluorescence in the eyes treated with siGlo-SIS005-DSC613G persisted until 24 hours and was significantly higher than that in the eyes treated with unformulated naked siGlo at all time points (p<0.01 at 3 hours and 6 hours, and p<0.05 at 24 hours). This suggests effective uptake of the siRNA agent formulated with silicon nanoparticles of the present invention administered topically. To verify the penetration of nanoparticles into tissues, 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] After in vivo uptake studies, SIS005-DSC613G siRNA delivery was further explored in functional assays using a mouse reporter model that restricted luciferase expression 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 animals 8 times per day, 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) obtained on day 11. Significant gene silencing effects persisted throughout the treatment regimen and continued for 4 days after treatment was terminated. 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 treated eyes and visual inspection of animals daily after topical treatment showed no adverse effects from the eye drops, suggesting that the silicon nanoparticle formulations were well tolerated in vivo.

Claims

1. A composition for the controlled release of a nucleic acid that is RNA, comprising: The present invention relates to a method for producing a surface-treated silicon nanoparticle comprising the steps of: surface-treating the surface of the silicon nanoparticles with at least one lipid, including a phospholipid, and further treating the surface of the silicon nanoparticles with at least one amino acid, including arginine and / or glycine; the silicon nanoparticles comprise at least 50% by weight silicon and are loaded with the RNA; A composition having a silicon to nucleic acid ratio of from 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, didecanoyl phosphatidylcholine, 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, the nucleic acid being RNA, comprising: The composition comprises silicon nanoparticles that are 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% by weight silicon 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 a composition according to any one of claims 1 to 8.

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

12. In the manufacture of a medicament for the treatment of ocular tissue, The 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 a 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, cataracts, 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; The use according to any one of claims 9 to 16.

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