Compositions Comprising Doped Silicon Particles and Related Methods

Abstract

A pharmaceutical composition comprising particles comprising hydrolyzably doped silicon and one or more lipids complexed with an active pharmaceutical ingredient, said particles having a diameter of less than 1 cm 3 1x10 16 The pharmaceutical composition is doped with at least one dopant atom, and related products, methods, and medical uses.

Description

[Technical field]

[0001] The present invention relates to improved particles for use in compositions containing nucleic acids and / or other pharma-ceutically active compounds, and related production and methods. Such methods, products and compositions are particularly useful, but not limited to, for the delivery of nucleic acids in gene therapy and vaccine compositions. The particles of the present invention comprise silicon doped with one or more additional elements. The present invention also relates to pharmaceutical compositions comprising the particles of the present invention, and related methods and uses. [Background technology]

[0002] Improved delivery vectors and vehicles for active pharmaceutical agents are needed if advances in biomedical research are to be fully translated into effective, safe and cost-effective treatments.

[0003] As an illustrative example, nucleic acids such as RNA have been proposed as therapeutic agents. Small interfering RNA (siRNA) has been proposed for use in gene therapy. Gene delivery for therapeutic or other purposes is well known, particularly for the treatment of diseases such as cystic fibrosis and certain cancers, and mRNA has recently been used in an effective vaccine against SARS-CoV-2. As used herein, the term "gene therapy" refers to the delivery of a gene or part of a gene to a cell to correct some defect. As used herein, the term "nucleic acid therapy" is also used to refer to any introduction of nucleic acid material into a target cell, and includes genetic vaccination. The term "nucleic acid delivery" may also encompass the in vitro production of commercially useful proteins in so-called cell factories.

[0004] There are three broad classes of delivery systems for delivering nucleic acids to cells: those involving direct injection of naked nucleic acids, those using viruses or genetically modified viruses, and those using non-viral delivery agents. Each has advantages and disadvantages. Viruses as delivery agents have the advantages of high efficiency and high cell selectivity, but the disadvantages of toxicity, generation of inflammatory responses, and the difficulty of delivering large nucleic acid fragments. Thus, mRNA vaccines may include injectable naked mRNA or non-viral delivery systems, such as lipid nanoparticle vectors. Unfortunately, it has been observed that non-viral delivery systems have low transfection efficiency. mRNA also has well-known stability issues.

[0005] Non-viral gene delivery systems are based on the compression of genetic material into nanometer particles by electrostatic interactions between the negatively charged phosphate backbone of the nucleic acid, cationic lipids, and optionally peptides or other compounds (Erbacher, P. et al, Gene Therapy, 1999, 6, 138-145). The mechanism of payload delivery by lipid nanoparticle non-viral vectors has been proposed to involve the binding of a complex formed between the nucleic acid and the lipid to the cell surface, followed by endocytosis of the intact complex, which then enters the cell by endocytosis. The complex then remains localized within vesicles or endosomes for some time, and the nucleic acid components are subsequently released into the cytoplasm. Production of the protein encoded by the nucleic acid, followed by expression of another genetic modification, can then occur.

[0006] The components of the non-viral delivery system associate electrostatically to form a vector complex. The lipid component shields both the nucleic acid and, to some extent, any peptide component(s) from degradation, endosomes, or otherwise. Cationic lipids for such use were developed by Felgner in the late 1980s and reported in Proc. Natl. Acad. Sci. USA 84, 7413-7417, 1987, and U.S. Pat. No. 5,264,618. Felgner developed the currently commercially available cationic liposomes known by the trademark "Lipofectin." "Lipofectin" liposomes are spherical vesicles with a lipid bilayer of the cationic lipid DOTMA (2,3-dioleyloxypropyl-1-trimethylammonium) and the neutral phospholipid DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) in a 1:1 ratio. Since then, various other cationic liposome formulations have been devised, most of which combine synthetic cationic lipids with neutral lipids. In addition to DOTMA analogues, one may mention complex alkylamines / alkylamides, cholesterol derivatives such as DC-cholesterol, as well as synthetic derivatives of dipalmitol, phosphatidylethanolamine, glutamate, imidazole, and phosphonate. However, cationic vector systems vary greatly in their transfection efficiency in the presence of serum, which clearly affects their potential use for in vivo gene therapy and vaccination. Ionizable lipids, such as positively charged lipids, are useful transfection agents because their positive charge tends to allow complexation with negatively charged nucleic acids. It is understood that different lipids have different levels of positive charge. Unfortunately, there are many lipids that are not sufficiently positively charged to allow sufficient complexation of nucleic acids to adequately protect the nucleic acid from degradation, for example during long-term storage.Conversely, lipids with very high positive charges may be very efficient in binding nucleic acids and therefore may be promising candidates for use in protecting nucleic acids during long-term storage, but may have toxicity issues that prevent their clinical use. For example, polyethyleneimine (PEI) is highly cationic and is used as an efficient in vitro transfection lipid. However, they are toxic, preventing their use in clinical treatment. Lipid transfection agents may also degrade (or "age") during storage, which reduces their ability to protect the nucleic acids they are complexed with and may require the use of excess lipids to mitigate expected loss of activity over time.

[0007] There are a surprisingly small number of lipid transfection agents suitable and approved for clinical use that have both an acceptable toxicity profile and result in efficient transfection. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) is currently the transfection agent of choice for many applications (including Pfizer's SARS-CoV-2 mRNA vaccine), but it has supply issues and few alternatives are available.

[0008] Turning to the optional peptide components of the non-viral delivery system mentioned above, peptides for optional use with lipids in transfection complexes typically have two functional groups: a "head group" that contains a cell surface receptor (e.g., integrin-) recognition sequence, and a "tail" that can non-covalently bind to nucleic acids (e.g., mRNA). Such peptide components can be designed to be cell type-specific or cell surface receptor-specific to some degree. Specificity results from targeting to cell surface receptors. A degree of integrin specificity, for example, can confer a degree of cell specificity to the complex. Transfection efficacies comparable to some adenoviral vectors can be achieved (Jenkins et al. Gene Therapy 7, 393-400, 2000). However, there remains a need for compositions that have the ability to target specific cells or specific tissues without relying on such peptides.

[0009] The present invention seeks to increase the efficiency of pharmaceutical compositions for delivering drug substances, particularly lipid transfection agents, to achieve one or more of the following advantages: (1) increasing the effectiveness of the lipids' ability to protect nucleic acids from degradation during storage; (2) increased transfection efficiency; (3) increased stability of lipids used to protect nucleic acids from degradation during storage; (4) the ability to use lower levels of lipids, particularly lower levels of cationic lipids such as DOTMA or DOTAP, in the transfection composition while still maintaining reasonable transfection capacity and / or good storage stability; (5) the ability to use a wide range of lipids in the transfection composition while still maintaining reasonable transfection capacity and / or good storage stability; and (6) More targeted delivery of active drug substances, such as nucleic acids, to specific types of tissues or specific types of cells.

[0010] So far, non-viral delivery of messenger RNA (mRNA) to cells has been particularly problematic and limited by the lack of efficient vectors. Attempts to deliver mRNA using known non-viral vehicles may result in suboptimal levels of protein expression, for example, due to insufficient targeting of mRNA to specific tissues or specific cells. Furthermore, known non-viral vehicles have poor storage stability when packaged with mRNA. Overcoming lipid bilayers to deliver RNA to cells has remained a major obstacle to the widespread development of RNA therapeutics.

[0011] Therefore, there is a need for a vector that is specifically tailored for the delivery of mRNA, which can optionally deliver high levels of mRNA specific to a certain type of tissue or a certain type of cell, and can lead to good levels of protein expression.There is also a need for a composition that is tailored for the delivery of mRNA, which has good stability during storage, and in particular mRNA delivery complexes that retain their structure and functionality during storage at moderate temperatures.Similar considerations apply to the delivery of siRNA therapeutics.

[0012] Several mRNA vaccines against SARS-CoV-2, including the Pfizer BioNTech vaccine BNT162b2 ("Comirnaty") and the Moderna CX-024414 vaccine, require ultra-cold chain storage and transportation. This limits access to the vaccine in low-income countries and adds cost and logistical complexity in all markets. It would be advantageous if the vaccine could be stored and transported at standard refrigerator temperatures (approximately -4°C) or room temperature (approximately 20°C). It would also be beneficial if the vaccine could tolerate higher temperatures (e.g., 30°C, 40°C, or 50°C) for storage or at least in the short term during transportation and distribution. Specifically, it would be useful if existing mRNA vaccine formulations, including mRNA and lipids, could be modified to increase their transfection efficacy. This has the advantage of allowing the use of lower doses, potentially reducing side effects, and increasing the total number of doses available. It would also be useful if existing mRNA vaccine formulations, including mRNA and lipids, could be modified to improve their storage stability, thereby allowing distribution and storage at higher temperatures and / or for longer periods.It would also be useful if existing mRNA vaccine formulations could be modified to require lower amounts of lipids (particularly lower amounts of cationic lipids, such as DOTAP) and / or to work with a wider range of lipids, in order to reduce the pressure on the availability of certain lipids, particularly cationic lipids, such as DOTAP.

[0013] Maintaining mRNA stability in injectable compositions, such as mRNA vaccine compositions, by low temperature, and also poses logistical challenges, has the technical limitations that mRNA must be thawed before injection, and must remain stable in the body at high temperatures for a sufficient time to show sufficient biological activity after injection.This may require maintaining stability during transit to the body environment and / or during escape from endosomal compartments.In vivo stability must also be maintained for a sufficient length of time for sufficient translation into protein to occur.

[0014] mRNA is vulnerable to enzymatic and chemical degradation. Enzymes that can degrade RNA such as mRNA are present in the biological culture systems used to produce mRNA and are difficult to completely remove. Enzyme activity can be slowed down by low temperature and / or lyophilization of the RNA, but each of these solutions has its drawbacks.

[0015] Lipid encapsulation has been used in the prior art to protect RNA (e.g., mRNA for gene therapy or vaccination) from degradation.This approach can be effective, but it presents the problem that it uses a relatively large amount of specific lipid, which can be expensive and / or in short supply, and the lipid itself degrades over time, thus losing its protective qualities.There is also a need for improved excipients to enhance the stability of multiple components of compositions that include nucleic acid and lipid.

[0016] Although the compositions and methods of the invention are currently believed to hold most promise in improving pharmaceutical compositions containing nucleic acids and lipids, they are also suitable for stabilizing and protecting from degradation non-nucleic acid active ingredients, including small organic compounds and peptides (e.g., peptide antigens). Summary of the Invention

[0017] The present invention relates to doped, particularly 1×10 15 Amounts of 10 or more, especially 1×10 16 Dopant atoms / cm or more 3The present invention is based on the recognition that elemental silicon, especially doped with boron, is useful for stabilizing drug substances, especially nucleic acids, in compositions, such as transfection compositions containing one or more lipids. Such compositions show enhanced transfection efficacy, enhanced tissue or cell targeting ability, reduced dependency on cationic lipids, and / or enhanced storage stability. The doped silicon can both stabilize the drug substance (especially the nucleic acid) itself, and can also stabilize one or more lipids, so that the one or more lipids can retain properties that allow them to protect the drug substance, especially the nucleic acid, for extended periods of time. The stabilization of the lipid and / or nucleic acid can allow the composition to be stored without problematic degradation for longer periods of time and / or at higher temperatures than was possible with conventional compositions having a drug substance that is a nucleic acid (e.g., at room temperature or 4° C.) but does not contain hydrolyzable doped silicon particles. The stabilization of the lipid and / or nucleic acid can also allow the composition to be more efficient in transfecting cells, for example by providing improved targeting to a particular tissue type and / or a particular cell type, and subsequently more efficient in providing one or more therapeutic effects by the drug substance, especially the nucleic acid. Although some of the stability and efficient delivery challenges are particularly acute for nucleic acid (e.g., mRNA) therapeutics, it has further been found that the stabilizing and protective properties of the composition comprising hydrolyzable doped silicon particles and one or more lipids are applicable to other non-nucleic acid drug substances, such as peptides, proteins, and small molecules. Doping with hydrolyzable silicon may allow different amphiphilic molecules with various functional groups to be fitted into the matrix crystal structure of the hydrolyzable silicon material; thus, hydrolyzable doped silicon not only counteracts the instability of organic molecules such as lipids, but also enhances the stability of drug substances, particularly nucleic acid (e.g., mRNA) molecules. The advantages of the present invention are believed to come solely from the binding or loading of organic compounds, which may occur, for example, within the pores of the hydrolyzable doped silicon particles, but also include intentionally introduced impurities (dopants) that may be used as part of the structural backbone for secondary binding of organic materials, such as one or more lipids.

[0018] According to a first aspect of the present invention there is provided a pharmaceutical composition comprising particles comprising hydrolyzably doped silicon and at least one lipid complexed with an active pharmaceutical ingredient. Optionally the particles are of a size of less than 1 cm 3 1x10 16 The doped layer is doped with an amount of dopant atoms equal to or greater than 1000000.

[0019] According to a second aspect of the invention there is provided the use of particles comprising hydrolyzably doped silicon (optionally hydrolyzably boron doped silicon) to enhance the efficacy of a pharmaceutical composition comprising an active pharmaceutical ingredient. Again, optionally the particles are of a size of 1 cm 3 1x10 16 The doped layer is doped with an amount of dopant atoms equal to or greater than 1000000.

[0020] According to a third aspect of the invention there is provided a pharmaceutical composition according to the first aspect of the invention for use as a medicament.

[0021] According to a fourth aspect of the invention there is provided the use of a pharmaceutical composition according to the first aspect of the invention in the manufacture of a medicament, such as a vaccine.

[0022] According to a fifth aspect of the present invention there is provided a method of treating or preventing a disease or disorder in a subject comprising administering to a subject in need of such treatment or prevention a pharmaceutical composition according to the first aspect of the present invention.

[0023] According to a sixth aspect of the present invention there is provided a method of providing a vaccine to a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition according to the first aspect of the present invention.

[0024] According to a seventh aspect of the present invention there is provided a method of increasing the storage stability of a drug substance (e.g. a nucleic acid, such as mRNA or saRNA or shRNA or siRNA), the method comprising contacting the nucleic acid with hydrolyzable doped silicon particles and one or more lipids.

[0025] According to an eighth aspect of the invention there is provided a pharmaceutical composition according to the first aspect of the invention for use in targeting a drug substance to a cell or tissue.

[0026] According to a ninth aspect of the present invention there is provided a pharmaceutical composition according to the first aspect of the present invention for use in the manufacture of a medicament for cell or tissue targeting of an active ingredient.

[0027] According to a tenth aspect of the present invention there is provided a method of targeting a pharmaceutical agent to a cell or tissue comprising administering a pharmaceutical composition according to the first aspect of the present invention to a subject in need thereof. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows silicon after boron doping. [Diagram 2] FIG. 2 shows the boron doped silicon of FIG. 1 after it has been ground into a powder. [Diagram 3] FIG. 3 shows ClCn7G213R expression in mouse PMBC. [Figure 4] FIG. 4 shows bone expression of ClCn7G213R. [Diagram 5] FIG. 5 shows the CTX-blood test results. [Figure 6] FIG. 6 shows gel electrophoresis images of pDNA-loaded Biocouriers freshly prepared and after 6 hours of storage at room temperature (RT). [Figure 7] FIG. 7 shows gel electrophoresis images of pDNA-loaded Biocouriers after 24 and 48 hours of storage at room temperature (RT). [Figure 8] FIG. 8 shows gel electrophoresis images of pDNA-loaded Biocouriers after 72 hours and 8 days of storage at room temperature (RT). [Figure 9]FIG. 9 shows gel electrophoresis images of pDNA-loaded Biocouriers freshly prepared and after 6 hours of storage at 4° C. [Figure 10] FIG. 10 shows gel electrophoresis images of pDNA-loaded Biocouriers after 24 and 48 hours of storage at 4° C. [Figure 11] FIG. 11 shows gel electrophoresis images of pDNA-loaded Biocouriers after 72 hours and 8 days of storage at 4° C. [Figure 12] Figure 12 shows an agarose gel retardation assay of RNA from baker's yeast loaded onto SIS0012 at different concentrations and volume ratios. Naked RNA was used as a control. In the first three columns of images, boxes around some loading wells indicate control samples. In the last column of images, lanes 2, 3, and 4 contain SIS0113 at a 10-fold dilution, V / V:2.5; lanes 5, 6, and 7 contain SIS0113 at a 5-fold dilution, V / V:2.5; and lanes 8, 9, and 10 contain SIS0113 at a 5-fold dilution, V / V:5. [Figure 13] Figure 13 shows an agarose gel retardation assay of DNA from herring sperm loaded into SIS0012 at selected concentrations and volume ratios at different time points (0-5 h) after storage at different temperatures (room temperature (RT), 4 °C and -20 °C). Naked DNA was used as a control. In the top left image, boxes around some loading wells indicate control samples. In all other images, lanes 2, 3, 4 hold samples stored at room temperature, lanes 5, 6, 7 hold samples stored at 4 °C and lanes 8, 9, 10 hold samples stored at 20 °C. [Figure 14]FIG. 14 shows agarose gel retardation assays of DNA from herring sperm loaded onto SIS0012 at selected concentrations and volume ratios at different time points (24-72 h) after storage at different temperatures (room temperature (RT), 4°C and -20°C). Naked DNA was used as a control. In the left and center columns of the images, lanes 2, 3, 4 hold samples stored at room temperature, lanes 5, 6, 7 hold samples stored at 4°C, and lanes 8, 9, 10 hold samples stored at 20°C. In the two right-most images, lanes 2, 3, 4 hold samples stored at room temperature, lanes 5, 6, 7 hold samples stored at 4°C, lanes 8, 9 hold samples stored at 20°C, and lane 10 control holds samples stored at 20°C. [Figure 15] Figure 15 shows agarose gel retardation assay of ADO-siRNA loaded in SIS0012 at selected concentrations and volume ratios at different time points (0-6 hours) after storage at different temperatures (room temperature (RT), 4°C and -20°C). Naked siRNA was used as a control (lane 1 in all images). In all images, lanes 2, 3, 4 hold samples stored at room temperature, lanes 5, 6, 7 hold samples stored at 4°C, and lanes 8, 9, 10 hold samples stored at 20°C. [Figure 16] Figure 16 shows agarose gel retardation assay of ADO-siRNA loaded in SIS0012 at selected concentrations and volume ratios at different time points (24-120 hours) after storage at different temperatures (room temperature (RT), 4°C and -20°C). Naked siRNA was used as a control (lane 1 in all images). In all images, lanes 2, 3, 4 hold samples stored at room temperature, lanes 5, 6, 7 hold samples stored at 4°C, and lanes 8, 9, 10 hold samples stored at 20°C. [Figure 17]FIG. 17 shows agarose gel retardation assay of ADO-siRNA loaded into SIS0012 without silicon nanoparticles at selected concentrations and volume ratios at different time points (0-6 h) after storage at different temperatures (room temperature (RT) and 4° C.). Naked siRNA was used as a control. In the six rightmost images, lanes 5, 6, and 7 hold samples stored at room temperature, while lanes 8, 9, and 10 hold samples stored at 4° C. [Figure 18] Figure 18 shows agarose gel retardation assay of ADO-siRNA loaded in SIS0012 without silicon nanoparticles at selected concentrations and volume ratios at different time points (24-120 h) after storage at room temperature (RT) or 4°C. Naked siRNA was used as a control (lane 1 in all images). In all images, lanes 5, 6, 7 hold samples stored at room temperature, while lanes 8, 9, 10 hold samples stored at 4°C. [Figure 19] Figure 19 shows the agarose gel retardation assay of ADO-siRNA loaded into SIS0013 at selected concentrations and volume ratios at different time points (0-6 hours) after storage at room temperature. Naked siRNA was used as a control. [Figure 20] Figure 20 shows agarose gel retardation assay of ADO-siRNA loaded into SIS0013 at selected concentrations and volume ratios at different time points (24-120 hours) after storage at room temperature. Naked siRNA was used as a control. [Figure 21] FIG. 21 shows an agarose gel retardation assay of ADO-siRNA loaded onto SIS0013 at selected concentrations and volume ratios at different time points (2-120 h) after storage at 4° C. Naked siRNA was used as a control. In the two rightmost images, lanes 2, 3, and 4 are labeled "72 h." In the image immediately to their left, lanes 2, 3, and 4 are labeled "24 h," and lanes 5, 6, and 7 are labeled "48 h." [Figure 22]FIG. 22 shows a standard curve of UV-Vis absorbance at 405 nm as a measure of enzyme activity for various concentrations of alkaline phosphatase. [Figure 23] FIG. 23 shows UV-Vis absorbance curves at 405 nm as a measure of enzyme activity for free alkaline phosphatase, alkaline phosphatase loaded in SIS0012, and alkaline phosphatase loaded in SIS0013 after incubation at 50° C. [Figure 24] FIG. 24 shows gel electrophoresis images of SIS0012 and SIS0013, which contain NAD, TYR, and QUE, when loaded with siRNA. [Diagram 25] FIG. 25 shows a gel electrophoresis image of the DPPC / PAL-KTTKS-DOPE formulation of Example 6 when loaded with siRNA. [Figure 26] FIG. 26 shows further gel electrophoresis images of the DPPC / PAL-KTTKS-DOPE formulation of Example 6 when loaded with siRNA. [Figure 27] FIG. 27 shows further gel electrophoresis images of the DPPC / PAL-KTTKS-DOPE formulation of Example 6 when loaded with mRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The particles of the composition of all aspects of the invention comprise hydrolytically doped silicon. The silicon is doped; advantageously, the particles are 1 cm 3 1x10 15 More than 10, especially 1×10 16 doped with dopant atoms in an amount of 1 cm or more 3 1x10 17 pcs or more, 1×10 18 pcs or more, 1×10 19 10 or more, or 1×10 20or more dopant atoms). The silicon may be n-doped or p-doped. All aspects of the invention include embodiments in which the silicon is doped with one or more elements selected from Mg, P, Cu, Ga, Al, In, Bi, Ge, Li, Xe, N, Au, Pt.

[0030] Most preferably, the dopant is a p-type dopant; preferably, the dopant comprises boron. Thus, most preferably, the dopant is boron. P-doped silicon may be particularly suitable for stabilizing negatively charged nucleic acids and other negatively charged pharma- ceutical active ingredients. In this way, optionally, doping hydrolyzable silicon particles according to all aspects of the present invention allows for less use, and potentially no use, of cationic lipids such as DOTMA or DOTAP, compared to conventional transfection compositions that do not contain hydrolyzable doped silicon, while still maintaining reasonable transfection capacity (e.g., good tissue targeting capacity or good cell targeting capacity) and / or good storage stability.

[0031] N-doped silicon may be particularly useful in stabilizing positively charged pharma- ceutically active ingredients and in protecting lipids, such as positively charged lipids, from degradation, which indirectly increases the stabilization and protection of drug substances, such as nucleic acids.

[0032] In embodiments of the various aspects of the invention where the drug substance is a nucleic acid, the pharmaceutical composition optionally further comprises a polycationic nucleic acid binding component. The term "polycationic nucleic acid binding component" is well known in the art and refers to a polymer having at least three repeating cationic amino acid residues or other cationic units carrying positively charged groups, such polymers being capable of forming complexions with nucleic acids under physiological conditions. An example of a nucleic acid binding polycationic molecule is an oligopeptide comprising one or more cationic amino acids. Such an oligopeptide may be, for example, an oligolysine molecule, an oligohistidine molecule, an oligoarginine molecule, an oligoornithine molecule, an oligodiaminopropionic acid molecule or an oligodiaminobutyric acid molecule, or a complex oligomer comprising or consisting of any combination of histidine, arginine, lysine, ornithine diaminopropionic acid, and diaminobutyric acid residues. Further examples of polycationic components include dendrimers and polyethyleneimines.

[0033] Particles containing hydrolyzable silicon According to all aspects of the present invention, the doped silicon particles can be pure doped silicon or another hydrolyzable doped silicon-containing material. If the particles are not pure doped silicon, they contain at least 50% silicon by weight, i.e., they contain at least 50% silicon atoms by weight based on the total mass of atoms in the particle. For example, the silicon particles can contain at least 60%, 70%, 80%, 90%, or 95% silicon. The silicon particles preferably exhibit a hydrolysis rate of at least 10% of the hydrolysis rate of a pure silicon particle of the same size, for example in PBS buffer at room temperature. Assays for hydrolysis of silicon-containing materials are widely known in the art (see, for example, WO 2011 / 001456, which is incorporated herein by reference). The particles of the present invention may contain some silica, but the silica is not hydrolyzable silicon, and at least half of the silicon atoms in the particles are in the form of elemental silicon (or doped elemental silicon).

[0034] According to all aspects of the invention, the particles containing hydrolytically doped silicon may be nanoparticles. The 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 nanoparticles may be made of either pure silicon or hydrolytically doped silicon-containing materials. They are preferably porous, more preferably mesoporous. The above nominal diameter may refer to the average diameter, and at least 90% of the total mass of the particles in a sample of the particles may fall within the specified size range. The particles containing hydrolytically doped silicon may be made porous by standard techniques such as contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and applying an electric current. By varying the HF concentration as well as the current density and exposure time, the density of the pores and their size can be controlled and monitored by scanning electron microscopy and / or nitrogen adsorption-desorption volumetric isotherm measurements.

[0035] In all aspects of the present invention, it is preferred that the particles are porous. When the particles are porous, their total surface area is increased due to their porosity. For example, the surface area can be increased by at least 50% or at least 100% over the surface area of ​​the corresponding non-porous particle. In many circumstances, the porous particles according to all aspects of the present invention actually have a much larger increase in total surface area due to their porosity. Preferably, the particles are mesoporous.

[0036] According to certain embodiments, the porosity is at least 30%, 40%, 50%, or 60%, meaning that 30%, 40%, 50%, or 60%, respectively, of the particle volume is in the pore space. Preferred pore sizes range from 1 nm to 50 nm, for example from 5 nm to 25 nm.

[0037] doping All aspects of the present invention relate to doped silicon-containing materials. The production of doped silicon is well understood in the semiconductor industry and includes ion implantation and diffusion methods. Thus, doped silicon is readily available. Alternatively, silicon can be doped by using diffusion methods to increase the amount of dopant present in the silicon. As an example of the diffusion method, silicon powder and doping reagent (e.g., B2O3 for boron doping) are placed in a bowl, mixed, placed under N2 atmosphere, and the dopant (e.g., boron) is diffused into the silicon at temperatures between 1050°C and 1175°C for several minutes. Figures 1 and 2 show boron-doped silicon produced by this method.

[0038] In one particular embodiment, the doping of the silicon is a heavy doping, which is at least 1×10 15 dopant atoms / cm 3 In some preferred embodiments, the dopant is 1×10 16 Dopant atoms / cm 3 Thus, in a particularly preferred embodiment, boron is present in an amount of 1×10 16 Boron atoms / cm 3 is present in amounts greater than or equal to

[0039] For example, 1×10 17 ≥ 10 dopant atoms / cm 3 , 1×10 18 ≥ 10 dopant atoms / cm 3 , or 1 × 10 19 ≥ 10 dopant atoms / cm 3 may exist.

[0040] Optionally, up to 1×10 20 dopant atoms / cm 3 For example, 1×10 16 ≥ 100 boron atoms / cm 3 and up to 1×10 20 Boron atoms / cm 3There may be boron present at levels of 0.1 to 0.5.

[0041] When boron is used as a dopant, 1×10 15 dopant atoms / cm 3 , and 1 × 10 20 dopant atoms / cm 3 doping amounts of 13.6 ohm-cm and 1.3 mohm-cm preferably correspond to resistivities of 13.6 ohm-cm and 1.3 mohm-cm, respectively. The various aspects of the invention in which boron is the preferred dopant do not exclude silicon that, in addition to being doped, e.g., heavily doped, with boron, is also doped with other elements. According to preferred embodiments of all aspects of the invention, the majority dopant is boron.

[0042] Lipids Lipids in the art are generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides.

[0043] As used in this application, the term "lipid" may also encompass lipidated oligopeptides (a term used interchangeably herein with the term lipopeptide) in which a short peptide sequence (e.g., a peptide sequence having 3-20 amino acid residues, such as 5-15 amino acid residues, in particular 3, 4 or 5 amino acid residues, most in particular 5 amino acid residues) is conjugated to one or more fatty acid chains (in particular fatty acid chains having a carbon chain length of 10-24, preferably 12-18 carbon chain length; e.g., 14, 15 or 16 carbon chain lengths; for example, the peptide moiety may be optionally lipidated with palmitoyl, cetyl or myristoyl moieties).

[0044] The lipidated oligopeptide may optionally be a lipidated tetrapeptide, lipidated pentapeptide, or lipidated hexapeptide. Preferably, the amino acid residues include at least one amino acid residue (e.g., 2 or 3 amino acid residues) that is cationic at a pH of 7.4 (physiological pH), such as lysine or arginine. For example, the lipidated oligopeptide may include one or more (e.g., 2) lysine residues. Thus, a specific example is palmitoyl-pentapeptide-4 (CAS number 214047-00-4; abbreviated as PAL-KTTKS). [ka] Palmitoyl Pentapeptide-4

[0045] Thus, in certain preferred embodiments according to all aspects of the invention, the one or more lipids comprise one or more lipidated oligopeptides, particularly those having one or more amino acid residues that are cationic at pH 7.4 (physiological pH; examples include lysine and arginine).

[0046] The lipidated oligopeptide may be used in combination with one or more phospholipids, such as DOPE or DPPC. The alkyl chains of the lipopeptide may be advantageously assimilated into the phospholipid bilayer, while the surface of the bilayer may be decorated with the peptide moiety. In this manner, the peptide may provide tissue and / or cell targeting, for example, when the peptide carries a cationic charge at physiological pH, it may stabilize a negatively charged API, such as a nucleic acid, such as mRNA.

[0047] According to all aspects of the invention, one or more lipids are present in the pharmaceutical composition, preferably said one or more lipids being provided in association with the hydrolysable doped silicon particles of the invention.

[0048] According to certain embodiments, the lipid is or comprises at least one cationic lipid; a helper lipid, such as a phospholipid; a structural lipid, such as a cholesterol-based lipid; and / or a polyethylene glycol (PEG) lipid.

[0049] The lipid may include one or more of phosphatidylcholine (PC), hydrogenated PC, stearylamine (SA), dioleoylphosphatidylethanolamine (DOPE), cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC)-cholesterol, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and derivatives thereof. In certain embodiments, the lipid includes or consists of DOTAP. Surface treatment of particles with lipids has been found to be useful in controlling the release rate of nucleic acids or other pharma-ceutically active agents. The type of lipid used to treat the surface of silicon-containing particles can affect their release rate. In particular, surface treatment of hydrolyzable doped silicon particles with lipids has a beneficial effect on the surface charge of the particles, which provides the necessary zeta potential to improve the loading of small interfering RNA, or small activating RNA, or small hairpin RNA, or messenger RNA, and controls their release rate at the target site. The presence of at least one lipid may also allow the hydrolysis rate of the doped silicon to be controlled so that it hydrolyzes into bioavailable orthosilicic acid (OSA) degradation products rather than insoluble polymer hydrolysis products. Controlling the hydrolysis rate of the doped silicon affects the rate at which nucleic acids or other drug substances associated with the doped silicon are released. Controlling the release rate affects the period of time that protection of the drug substance is sustained.

[0050] Nevertheless, doped silicon as used herein provides the ability to use less lipid (particularly lower amounts of cationic lipid such as DOTAP) than conventional compositions that contain drug substance but do not contain hydrolyzable doped silicon particles, particularly conventional transfection compositions that contain nucleic acids, and / or the ability for such compositions to be formulated with a broader range of lipids while still providing transfection efficacy, storage stability, and / or targeted delivery to a particular type of tissue or a particular type of cell. Thus, a method is provided that reduces the reliance on certain lipids, particularly cationic lipids such as DOTAP. Thus, in certain embodiments of all aspects of the invention, the one or more lipids are selected from the group consisting of cationic lipids; helper lipids, such as phospholipids; structural lipids, such as cholesterol-based lipids; and / or polyethylene glycol (PEG) lipids. In such embodiments, optionally, none of the one or more lipids is a cationic lipid, such as DOTAP.

[0051] In certain embodiments of all aspects of the invention, the lipid is DOTAP or the lipid present in the formulation comprises DOTAP. DOTAP exists in S and R enantiomeric forms and may exist as S, R or racemic according to all aspects of the invention. According to certain embodiments of the total DOTAP present in the composition of the invention, the R and S forms may be approximately equal (i.e., 60% or less of either form). In other embodiments, at least 80%, 90%, 95%, 98%, or 99% of the total DOTAP is in the R form. In other embodiments, at least 80%, 90%, 95%, 98%, or 99% of the total DOTAP is in the S form. When one or more lipids are or comprise cationic lipids such as DOTAP, doping silicon particles according to all aspects of the invention preferably allows the use of less cationic lipid such as DOTAP than pharmaceutical compositions for delivery of drug substances such as nucleic acids that do not comprise hydrolyzable doped silicon particles.

[0052] According to all aspects of the invention, the lipid or lipids may have an average molecular weight in the range of 500-1000, etc.

[0053] Preferably, according to all aspects of the present invention (e.g., when the lipid contains one or more of cationic lipids, helper lipids, structural lipids, and PEG lipids, or when the lipid is selected from one or more of PC, hydrogenated PC, SA, DOPE, DOTAP, DC-cholesterol, and derivatives thereof), the lipid to be doped (i.e., by filtration or sterilization process) is used before any further processing (i.e., by filtration or sterilization process). , total lipid components) is 1:1 to 45:1, for example 1:1 to 20:1, 1:1 to 16:1, 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, for example 1:1 to 7:1, 2:1 to 7:1, 3:1 to 6:1, 4:1 to 5:1. Ratios of lipid components to silicon moles of 0.8:1 to 20:1 have proven to be particularly advantageous, for example 16:1, 12:1, 8:1 or 2.5:1.

[0054] Advantageously, this ratio of lipid to doped silicon can provide a multilamellar vesicle system that can control the release of a drug substance (e.g., a nucleic acid) in contact with the particles of hydrolyzable doped silicon, and can stabilize this drug substance and promote the controlled release of OSA, a bioavailable degradation product of silicon.

[0055] Advantageously, lipid compounds can have a significant effect on the surface charge of doped silicon nanoparticles. Particles containing hydrolyzable pure silicon treated with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lecithin demonstrate a negative surface charge when subjected to zeta potential analysis (ranging from -60 to -20 mV, with various preferred ratios of silicon:lipid). Particle surfaces treated with stearylamine or DOTAP demonstrate a positive zeta potential (ranging from 0 mV to +40 mV, with various preferred ratios of silicon:lipid). Doping of silicon changes the surface charge. The use of a p-dopant such as boron (preferred in many embodiments of the invention in all its various aspects) makes the zeta potential more positive (i.e., less negative). A typical value of -40 mV for pure silicon becomes approximately -25 mV when silicon is doped with boron. Thus, boron-doped silicon can more easily achieve a positive zeta potential when treated with a cationic lipid. For example, treatment with stearylamine or DOTAP can achieve values ​​of about +20 mV to +60 mV. This means that a positive surface zeta potential can be achieved with less cationic lipid or with a wider range of cationic lipids, including those that are less cationic than stearylamine and DOTAP. This also means that the surface zeta potential of the particles will remain positive for longer periods of time, even if the cationic lipids degrade ("age") during storage, resulting in a partial loss of the lipid's positive charge.

[0056] Ratios of lipid components to silicon moles between 0.8:1 and 20:1 have proven to be particularly advantageous, for example 16:1, 12:1, 8:1 or 2.5:1.

[0057] The lipid or lipid component may be or may include a phospholipid in some embodiments. The term "phospholipid" refers to a lipid that includes a fatty acid chain and a phosphate group. Phospholipids are typically neutral molecules in that they have no overall charge or may carry a negative charge, unlike positively charged cationic lipids. Phospholipids are typically zwitterionic compounds that include both positively and negatively charged components, but do not include an overall charge. Thus, phospholipids are typically classified as neutral lipids. Particularly suitable phospholipids are glycerophospholipids. Particularly suitable phospholipids are those in which the polar head group is attached to a quaternary ammonium moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. Another example of a phospholipid is DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). The type of lipid may be selected depending on the nature of the formulation, with neutral or negatively charged phospholipids being preferred for aprotic formulations, while positively charged cationic lipids and small CH3 chain lipids are preferred for protic formulations. The phospholipid may be or be derived from lecithin.

[0058] Preferably, the side chain(s) of the phospholipid is an aliphatic side chain(s) having 15 or more carbon atoms, or an ether side chain having 6 or more repeating ether units, such as a polyethylene glycol or polypropylene glycol chain. A lipid having an ether side chain may be referred to as a "PEG lipid" or a "PEGylated" lipid. Thus, as used in this application, the term "lipid" may thus encompass a PEG lipid. Thus, according to certain embodiments, the lipid is or comprises one or more polyethylene glycol (PEG) lipids.

[0059] The lipid or lipid component may be or include a cationic lipid in some embodiments. The term "cationic lipid" refers to a positively charged molecule with a cationic head group attached to a hydrophobic tail via some spacer. Examples include DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleyloxypropyl-1-trimentylammonium), DHDTMA (dihexadecyltrimethylammonium), and stearylamine (SA). The positive charge is typically stabilized by a negative counterion. In a preferred embodiment, the cationic lipid is or includes DOTAP. As described herein, doping of silicon according to all aspects of the present invention may provide the ability to use lower amounts of cationic lipid such as DOTAP compared to conventional compositions, such as transfection compositions formulated without hydrolyzable doped silicon, but the compositions provided herein that include hydrolyzable doped silicon maintain reasonable transfection ability (e.g., good tissue targeting ability or good cell targeting ability) and / or good storage stability.

[0060] In certain embodiments, the lipid is selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), or any combination thereof.

[0061] In certain embodiments, the lipid may consist essentially of phosphatidylcholine, hydrogenated phosphatidylcholine, stearylamine, or combinations thereof.

[0062] In certain embodiments, the lipid may be composed of at least 5% by weight of hydrogenated phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, in particular at least 50% by weight of hydrogenated phosphatidylcholine, based on the total weight of the particle. Molar ratios of hydrogenated phosphatidylcholine to doped silicon of 0.8:1 to 5:1 have been found to be particularly advantageous, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1.

[0063] In certain embodiments, the lipid may consist of at least 5% by weight of phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, in particular at least 50% by weight of phosphatidylcholine, based on the total weight of the particle. Molar ratios of phosphatidylcholine to doped silicon of 0.8:1 to 5:1 have been found to be particularly advantageous, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1.

[0064] In certain embodiments, the lipid may comprise at least 5% by weight of stearylamine, for example at least 20% by weight, typically at least 30% by weight, in particular at least 50% by weight of stearylamine, based on the total weight of the particle. A molar ratio of stearylamine to doped silicon of 0.8:1 to 5:1 has been found to be particularly advantageous, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1.

[0065] In certain embodiments, the lipids may consist of PC and SA, preferably in a weight ratio of PC:SA of 1:1 to 20:1, more preferably 7:1 to 10:1, such as a weight ratio of PC:SA of 72:8.

[0066] In certain embodiments, the lipids may consist of DOPE, SA, and DC-cholesterol. The weight ratio of DOPE:SA may be in the range of 1:1 to 10:1, such as 4:1 to 8:1. The weight ratio of DOPE:DC-cholesterol may be in the range of 1:1 to 5:1, such as 1:1 to 3:1. The weight ratio of SA:DC-cholesterol may be in the range of 1:1 to 1:5, such as 1:2 to 1:4. In some embodiments, the weight ratio of DOPE:SA:DC-cholesterol may be 48:8:24.

[0067] In certain preferred embodiments, the lipids may consist of DOTAP, DOPE, and PEG lipids (such as mPEG2000-DSPE). The weight ratio of DOTAP:DOPE may be 1:2 to 2:1, such as about 1:1. The ratio of DOTAP:PEG lipid and DOPE:PEG lipid may be 10:1 to 5:1, such as about 7:1. The total weight ratio of total lipid to silicone may be 20:1 to 10:1, such as about 16:1.

[0068] amino acid All aspects of the present invention may include the additional optional presence or use of one or more amino acids.

[0069] In its broadest sense, the term "amino acid" encompasses any artificial or naturally occurring organic compound containing amine (-NH2) and carboxyl (-COOH) functional groups. The term "amino acid" includes alpha amino acids, beta amino acids, gamma amino acids, and delta amino acids. The term "amino acid" includes amino acids of any chiral configuration. According to some embodiments (e.g., when the doped silicon particles of the invention are formulated with one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof), the amino acid is preferably a naturally occurring alpha amino acid. It can be a proteinogenic or non-proteinogenic amino acid (such as carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline). In preferred embodiments, 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. Such amino acids may function to stabilize the doped silicon particles and to control hydrolysis of the doped silicon, both during storage and in vivo.

[0070] In addition to the amino acids listed above, all embodiments may include peptides that contain cell surface receptor, e.g., integrin recognition sequences, to confer some degree of cell specificity to the particle. The peptides may have a "head group" that contains the cell surface receptor recognition sequence and an additional "tail" that can non-covalently bind to an active pharmaceutical agent, e.g., a nucleic acid, and / or bind to doped silicon.

[0071] Active Pharmaceutical Agent-Particle Association According to a preferred embodiment (e.g., when the particles containing hydrolyzable boron-doped silicon are formulated with one or more amino acids, such as arginine, glycine, and histidine, and / or one or more lipids, such as PC, hydrogenated PC, SA, DOPE, DOTAP, DC-cholesterol, and derivatives thereof, or other ionic or cationic lipids), at least 70% by weight, such as at least 80% by weight, such as at least 90% by weight, of the active pharmaceutical agent (e.g., nucleic acid, saRNA, shRNA, siRNA, or mRNA) present in the product of all aspects of the invention is associated with the particle. By this, it is meant that the active pharmaceutical agent is non-covalently bound to the doped silicon. Without wishing to be bound by theory, it is hypothesized that when this occurs, the random Brownian motion of the active pharmaceutical agent, such as nucleic acid, may be reduced, enhancing the efficiency of the pharmaceutical composition. For example, the chance of it being degraded by, for example, degradative enzymes present in the formulation is reduced. Also, without wishing to be bound by theory, it is believed that there may be a reduction in the number of water molecules available for enzyme-catalyzed reactions with active pharmaceutical agents (e.g., nucleic acids and especially mRNA). For example, water molecules may be removed by reaction of water with hydrolyzable doped silicon.

[0072] The rate of degradation of the particles, and the end of the association with the active pharmaceutical agent resulting from degradation, is governed by the hydrolysis of the doped silicon in the particles. Because this rate can be controlled, the rate at which the active pharmaceutical agent becomes bioavailable can also be controlled to avoid dose dumping and / or ensure gradual release over a suitable extended period of time.

[0073] When the active pharmaceutical agent is a nucleic acid, it has been found that treating lipid-treated boron-doped silicon particles (e.g., nanoparticles treated with PC, hydrogenated PC, SA, DOPE, DOTAP, DC-cholesterol, and one or more of their derivatives) with amino acids (e.g., one or more of glycine, arginine, and histidine, preferably glycine) has a beneficial stabilizing effect on nucleic acids such as RNA (e.g., mRNA, saRNA, shRNA, or siRNA). In particular, treating lipid-treated particles with amino acids has been shown to stabilize nucleic acids such as RNA in biological fluids, such as ocular tissue or plasma. Lipid-treated particles formulated with amino acids in this manner may be particularly suitable for delivery to the body, for example, by transdermal injection or intravitreal injection.

[0074] Ratio of amino acid(s) to doped silicon Preferably (e.g., when the particles of the invention are formulated with one or more amino acids, such as one or more of arginine, histidine, and glycine; and / or one or more lipids, such as PC, hydrogenated PC, SA, DOTAP, DOPE, DC-cholesterol, and derivatives thereof), the amino acid (e.g., glycine, or a mixture of glycine and lysine) may optionally be present in a weight ratio to the silicon of at least 500:1, at least 50:1, at least 5:1, at least 2.5:1, at least 1:1, or at least 0.5:1 or 0.05:1. Preferably, the amino acid is glycine, which is optionally present in a weight ratio to the silicon of at least 500:1, at least 50:1, at least 5:1, at least 2.5:1, at least 1:1, or at least 0.5:1, or 0.05:1.

[0075] Advantageously, this ratio of amino acid to doped silicon further influences and stabilizes the release rate of active pharmaceutical agents, such as RNA molecules, associated with the particles.

[0076] According to all aspects of the invention, the particles may be treated with lipids (e.g., one or more of PC, hydrogenated PC, SA, DOTAP, DOPE, DC-cholesterol, and derivatives thereof) and amino acids (e.g., one or more of glycine, arginine, and histidine, such as glycine, or a mixture of glycine and arginine). The amino acid may be any amino acid. Preferably, the amino acid is arginine, or glycine, or a combination of glycine and arginine. The lipid may be any lipid. Preferably, the lipid is a phospholipid. Optionally, the lipid further comprises a cationic lipid. More preferably, the lipid or lipids are selected from one or more of hydrogenated PC, PC, DOTAP, DOPE, lecithin, stearylamine, and derivatives thereof. Optionally, the lipid comprises DOTAP and / or a derivative thereof.

[0077] Preferably, the ratio of amino acid to doped 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., where the lipid is selected from one or more of PC, hydrogenated PC, SA, DOTAP, DOPE, DC-cholesterol, and derivatives thereof), the amino acid is a combination of arginine and glycine, where the ratio of Arg:Gly is 1:0.6 to 3:1, such as 1:0.8 to 2.5:1, for example 1:1 to 2:1.

[0078] According to another embodiment of all aspects of the invention, the particles are formulated with arginine, preferably in a ratio of arginine to boron doped silicon of 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.

[0079] According to another embodiment of all aspects of the invention, the particles are formulated with glycine, preferably in a ratio of glycine to boron doped silicon of 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.

[0080] Preferred amino acids for use in all aspects of the present invention include arginine, glycine, proline, lysine, and histidine, and mixtures of two or more thereof.

[0081] Active Pharmaceutical Agents According to all aspects of the present invention, the active pharmaceutical agent (also referred to as drug substance) can be any pharma- ceutical active compound. In some embodiments, the active pharmaceutical agent can be a prodrug. In preferred embodiments of all aspects of the present invention, the active pharmaceutical agent is a nucleic acid.

[0082] Nucleic acids such as RNA for use according to the present invention include double-stranded and single-stranded DNA, RNA, DNA:RNA hybrids, and PNA (peptide nucleic acid) or hybrids between RNA or DNA. The term also includes 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.), those with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and those with positively charged linkages (e.g., aminoalkyl phosphoramidates, aminoalkyl phosphotriesters), those containing pendant moieties, such as proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing interfering substances (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.

[0083] As used herein, the terms "nucleoside" and "nucleotide" are understood to include moieties that contain not only known 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 also include modifications to the sugar moiety, for example, where one or more of the hydroxyl groups have been replaced with halogens, aliphatic groups, or functionalized as ethers, amines, and the like. Other modifications to nucleotides or polynucleotides involve rearranging, adding, substituting, or otherwise changing the functional groups on the purine or pyrimidine bases that form hydrogen bonds to the respective complementary pyrimidines or purines, e.g., isoguanine, isocysteine, and the like. In some embodiments, the oligonucleotides and / or probes include at least one, two, three, or four modified nucleotides.

[0084] In some embodiments, a nucleic acid, such as an RNA, disclosed herein comprises one or more universal bases. As used herein, the term "universal base" refers to a nucleotide analog that can hybridize to two or more nucleotides selected from A, U / T, C, and G. In some embodiments, the universal base may be selected from the group consisting of deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, 5-nitroindole.

[0085] According to preferred embodiments of all aspects of the present invention, the nucleic acid may be DNA or RNA. In preferred embodiments, the nucleic acid is RNA. RNA includes mRNA, saRNA, shRNA, and siRNA in various embodiments of all aspects of the present invention. Preferably, the nucleic acid is mRNA. In preferred embodiments, the nucleic acid may be the mRNA of an mRNA vaccine.

[0086] In its broadest sense, the term saRNA encompasses small activating RNAs, including double-stranded RNA molecules of 5-50 base pairs in length that operate within the RNA activating (RNAa) pathway. For example, saRNAs can be 10-45 base pairs, 15-40 base pairs, 20-30 base pairs, and particularly 20-25 base pairs in length.

[0087] In its broadest sense, the term shRNA encompasses single-stranded RNA molecules that have base pairing and are 10-100 bases in length that operate within the RNA interference (RNAi) pathway. For example, shRNAs can be 15-95 base pairs, 20-80 base pairs, or 25-75 base pairs, particularly 30-70 base pairs in length.

[0088] In its broadest sense, the term "siRNA" encompasses small interfering RNA (siRNA), sometimes known as small interfering RNA or silencing RNA, which comprises a double-stranded RNA molecule of 5-50 base pairs in length and operates within the RNA interference (RNAi) pathway. For example, siRNAs can be 10-45 base pairs, 15-40 base pairs, 20-30 base pairs, and particularly 20-25 base pairs in length.

[0089] The term "mRNA" encompasses messenger RNA, and may optionally include mRNAs that include a 5 prime cap and / or a polyadenylated end. Alternatively, one or both of these features may be absent. An mRNA may, in certain embodiments, be at least 100 base pairs, at least 200 base pairs, at least 300 base pairs, at least 500 base pairs, or at least 1000 base pairs in length.

[0090] RNA according to preferred embodiments of all aspects of the invention (e.g., when the particles of the invention are formulated with one or more of arginine, histidine, and glycine, and / or one or more of PC, hydrogenated PC, SA, DOTAP, DOPE, DC-cholesterol, and derivatives thereof) may be naturally occurring or chemically modified to enhance their therapeutic properties, such as enhanced activity, increased serum stability, reduced off-targeting, and reduced immunological activity. Chemical modifications to RNA may include any modifications commonly known in the art.

[0091] According to some embodiments (e.g., when the particles 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, DOTAP, DC-cholesterol, and derivatives thereof), the RNA is siRNA. According to some other embodiments (e.g., when the particles 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, DOTAP, DOPE, DC-cholesterol, and derivatives thereof), the RNA is mRNA.

[0092] According to another embodiment, the mRNA encodes an antigen, thereby providing a pharmaceutical composition that is a vaccine. The antigen may be a viral antigen, such as an antigen of SARS-CoV-2, such as an antigen derived from the spike protein of SARS-CoV-2.

[0093] In certain embodiments, the mRNA may encode multiple proteins. For example, the mRNA may encode a viral antigen and an adjuvanting protein, or more. In other embodiments, an adjuvant may alternatively or additionally be provided as an additional component of the pharmaceutical composition in addition to the active pharmaceutical agent.

[0094] Ratio of doped silicon to active pharmaceutical agent Preferably (e.g. when the particles of the invention are formulated with one or more of arginine, histidine and glycine, and / or one or more of PC, hydrogenated PC, SA, DOTAP, DOPE, DC-cholesterol and their derivatives), the ratio of doped (e.g. boron doped) silicon to active pharmaceutical agent such as nucleic acid (e.g. 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 doped (e.g. boron doped) silicon to active pharmaceutical agent such as nucleic acid is 1:1 to 3:1. Advantageously, this ratio of doped (e.g. boron doped) silicon to pharmaceutical agent such as nucleic acid further influences and stabilizes the release rate of the active pharmaceutical agent carried by the particle, e.g. nucleic acid molecule (e.g. siRNA, saRNA, shRNA or mRNA molecule).

[0095] It should be noted that in all of these ratios (and other ratios throughout this specification) the ratios are weight ratios and the ratio attributed to "silicon" is the total weight of the hydrolyzable doped silicon-containing particles and is measured prior to any additional preparation steps, such as sterilization or filtration processes, that may alter the subsequent ratios.

[0096] Favourable combinations According to all aspects of the invention, a particularly preferred embodiment relates to a doping that is boron doping (particularly high boron doping as defined above) and an active ingredient that is a nucleic acid (particularly an mRNA, and in particular an mRNA encoding an antigen for an mRNA vaccine). Optionally, the lipid is or comprises DOTAP. However, as shown in the examples herein below, in particular in Example 6, it has been found that DOTAP is not necessary for the formulation. Thus, according to the examples, in a particularly preferred embodiment, the one or more lipids are or comprise one or more of a phospholipid (such as DPPC and / or DOPE) and a lipidated oligopeptide having one or more amino acid residues that are cationic at pH 7.4 (physiological pH; examples include lysine and arginine). Optionally, one or more sugars (particularly trehalose) and / or one or more amino acids (particularly glycine) are also present.

[0097] Alternatively, in other preferred embodiments, the one or more lipids are or include one or more phospholipids (such as DPPC and / or DOPE) and are formulated with one or more coenzymes (e.g., NAD); one or more flavanols (e.g., quercetin), and / or one or more amino acids (e.g., tyrosine). Optionally, one or more sugars (especially trehalose) and / or one or more amino acids (especially glycine) are also present.

[0098] Enhancement of the efficacy of pharmaceutical compositions In some aspects, the present invention relates to the recognition that particles comprising hydrolyzably doped (eg, boron doped) silicon can be effective in enhancing the efficacy of pharmaceutical compositions comprising active pharmaceutical ingredients.

[0099] Thus, in a seventh aspect, the present invention provides the use of particles comprising hydrolyzably doped silicon, optionally hydrolyzably boron doped silicon, to enhance the efficacy of a pharmaceutical composition comprising a drug substance, as well as a method of enhancing the efficacy of a pharmaceutical composition comprising a drug substance by incorporating particles comprising hydrolyzably doped silicon into the pharmaceutical composition. The efficacy of the pharmaceutical composition may be enhanced by the particles increasing the stability of the drug substance at room temperature (or 4°C) and / or by the particles enhancing the uptake of the drug substance by the target cells or tissues, as the particles comprising hydrolyzably doped silicon enhance tissue or cell targeting ability. The efficacy of the pharmaceutical composition may also or alternatively be enhanced by the particles increasing the intracellular stability of the drug substance and / or by the particles protecting the drug substance from degradation, e.g. enzymatic degradation. The pharmaceutical composition is optionally as defined herein with reference to all aspects of the invention. In this way, one may optionally provide the ability to use lower amounts of cationic lipid, e.g., DOTMA or DOTAP, compared to conventional transfection compositions, such as transfection compositions that do not contain hydrolyzable doped silicon, while still maintaining reasonable transfection capacity (e.g., good tissue targeting capacity or good cell targeting capacity) and / or good storage stability.

[0100] Further components and features According to a preferred embodiment of all aspects of the present invention, the active pharmaceutical agent is a nucleic acid, and one or more additional components may additionally be present, including additional transfection reagents.

[0101] In its broadest sense, a "transfection agent" is an agent that facilitates the introduction of naked or purified nucleic acid into a eukaryotic cell. For example, some transfection agents are agents that facilitate the introduction of mRNA into a eukaryotic cell.

[0102] According to other embodiments of all aspects of the invention (e.g., when the boron doped 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, DOTAP, DOPE, DC-cholesterol, and derivatives thereof), the transfection reagent may be, according to lipofection (liposomal transfection), a lipofection (liposomal transfection) reagent, a dendrimer, HEPES buffered saline (HeBS) containing phosphate ions combined with calcium chloride solution, or a cationic polymer such as diethylaminoethyl-dextran (DEAE dextran) or polyethyleneimine (PEI).

[0103] According to a preferred embodiment (e.g., when the boron-doped silicon particles 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, DOTAP, DOPE, DC-cholesterol, and derivatives thereof), the transfection reagent is a lipofection reagent, such as lipofectamine.

[0104] Nucleic acids according to preferred embodiments of all aspects of the present invention, such as RNA (such as siRNA, saRNA, shRNA, 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 particles 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, DOTAP, DC-cholesterol, and their derivatives), nucleic acids such as RNA are provided in solution (either alone or in combination with various other nucleic acids), for example in a buffer solution.In some embodiments (e.g., when the boron-doped 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, DOTAP, DOPE, DC-cholesterol, and their derivatives), nucleic acids such as RNA are provided as salts, either alone or in combination with other isolated nucleic acids. In some embodiments (e.g., when the boron-doped 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, DOTAP, DOPE, DC-cholesterol, and derivatives thereof), the nucleic acid such as RNA is provided in a lyophilized form that can be reconstituted. For example, in some embodiments, the nucleic acid such as RNA can be provided in a lyophilized pellet alone or together with other isolated nucleic acids. In some embodiments (e.g., when the particles 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, DOTAP, DOPE, DC-cholesterol, and derivatives thereof), the nucleic acid such as RNA is provided attached to a solid material, such as, for example, a bead, a membrane, etc. In some embodiments (e.g., when the particles 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, DOTAP, DC-cholesterol, and derivatives thereof), a nucleic acid such as RNA is provided to a host cell, e.g., a host cell of a cell line harboring a plasmid or a host cell of a cell line harboring a stably integrated sequence.

[0105] The pharmaceutical compositions of the present invention may further comprise excipients, including, but not limited to, preservatives, cryoprotectants, and adjuvants.

[0106] The pharmaceutical composition of the present invention containing siRNA may optionally include siRNA produced synthetically by chemical synthesis outside of a biological system. Such siRNA may be produced without the presence of nucleases.

[0107] However, a preferred method for producing longer nucleic acids such as mRNA (e.g., for use in vaccine formulations) involves the use of biological systems. mRNA is typically purified from the biological system to reduce the amount of degradative enzymes (e.g., ribonucleic acid nuclease (RNase)). It can be difficult to completely eliminate all degradative enzymes. This usually requires storage at low temperatures to minimize enzyme activity. Furthermore, during entry into the body and / or escape from endosomal compartments, the nucleic acid is typically exposed to physiological and intracellular conditions, including contact with degradative enzymes.

[0108] However, it has been found that by formulating a nucleic acid (such as an mRNA) with particles comprising hydrolyzable boron doped silicon and an ome or more lipids (and optionally a non-reducing disaccharide) according to the invention, in its various embodiments, the shelf life of the mRNA in the formulation can be extended, which may negate, alleviate or reduce the need for cold storage; and / or stabilize the mRNA during its transit into the body and / or during its escape from the endosomal compartment.

[0109] Thus, the methods of the invention include methods of protecting a drug substance that is a nucleic acid (e.g., mRNA) from degradation due to enzymes present in the nucleic acid preparation. The products of embodiments of the invention can also be such that the nucleic acid is protected from degradation due to enzymes present in the nucleic acid-containing composition.

[0110] Preferably, enzymatic degradation at room temperature (20° C.) is reduced by at least half, more preferably one-fold, or at least 5-fold, 10-fold, 35-fold, 50-fold, 100-fold, 500-fold, or 1000-fold, as compared to an equivalent composition that does not include particles of hydrolyzably doped (e.g., boron-doped) silicon-containing material. Preferably, the nucleic acid (such as mRNA) in the composition has a half-life at 4° C. of at least 3 months, at least 6 months, or at least 12 months.

[0111] Certain embodiments of pharmaceutical compositions according to the first and other aspects of the invention contain a nucleic acid (such as mRNA) from a biological source as the active pharmaceutical agent, and a low but measurable amount of a degradative enzyme derived from that biological source. For example, at least 1 μmol min on a nucleic acid substrate at pH 7.4 and a temperature of 25° C. -1 There may be one or more degradative enzymes present, including one or more enzymes having the activity of

[0112] Proposed mechanism of action Without wishing to be bound by theory, it is proposed that the process of "ensilication" may be part of the explanation why boron-doped silicon can extend the shelf-life of mRNA.

[0113] Silication consists of forming a protective and resistant silica "cage" around the mRNA, which is the product of partial hydrolysis of doped silicon particles. Silication can provide protection against temperature-induced loss of structure and function without the need for freezing or refrigeration. In the silicification process, the negatively charged silanol groups can participate in direct non-covalent interactions with either the pharma- ceutically active agent or with the lipids in the composition.

[0114] The resulting combination can physically prevent thermal denaturation of nucleic acids or other drugs.

[0115] To obtain further specific improvements in the silicification process, some properties of the silica material can be tailored to the compound to be protected. In particular, the use of boron-doped silicon allows the creation of permanent cationic sites capable of forming electrostatic interactions with nucleic acids, thus protecting them from degradation.

[0116] The interaction of boron with silicon clusters is responsible for the formation of silica cages. Three boron atoms are bonded to the Si cluster as the number of silicon atoms increases. n There is a tendency to form B3 triangles encapsulated in cages.

[0117] Doping boron into the silicon matrix creates a positive charge within the crystalline structure, allowing binding of nucleic acids within the crystalline structure of the silicon itself, which alleviates the current problems caused by lipid degradation or "aging", thus extending the lifespan of the nucleic acid complexed delivery system.

[0118] Preparation of particles containing doped hydrolyzable silicon

[0119] The particles of the present invention can be conveniently prepared by conventional techniques in the art, such as by milling processes or other known techniques for reducing particle size. Doped silicon-containing particles can be made from sodium silicate particles, magnesium reduction of silica, colloidal silica, or silicon wafer material. Macro- or micro-scale particles can be ground in a ball mill, a planetary ball mill, or other size reduction mechanisms. The resulting particles can be sieved or air classified to recover the particles. It is also possible to use plasma methods and laser ablation for the generation of particles.

[0120] The porous particles may be prepared by methods conventional in the art, including the methods described herein.

[0121] Exemplary specifications for boron doped silicon for use in accordance with the present invention are as follows: Single-sided polished wafer, CZ Diameter: 150±0.2mm Orientation: (100)±1° Type: p / boron Resistivity: 0.014±25%Ohm cm. 5×10 18 atoms / cm 3 Close to. Primary flat: 57.50±2.5mm Primary flat 1 position: D <100> ~{110} Thickness: 675±15μm Packaging: Ultrapak Shipping Cassette TTV:≦18μm TIR:≦5μm

[0122] Such boron doped silicon is commercially available, for example, from Nanografi, Jena, Germany or Si-Mat, Germany.

[0123] Various aspects and embodiments of the invention are described with reference to the following non-limiting examples. Where the examples use undoped silicon, they may be outside the scope of some aspects of the invention, but are included as comparative examples. EXAMPLES

[0124] Example 1: Preparation of boron-doped silicon particles Single-side polished boron-doped silicon wafers were purchased from Si-Mat, Germany. All cleaning and etching reagents were cleanroom grade. Silicon wafers were etched by subjecting the Si to etch in a 1:1 (v / v) mixture of pure ethanol and 10% aqueous HF acid at 80 mA / cm for 2–10 min. 2The samples were prepared by anodic etching at an anodic current density of 1000 . After etching, the samples were rinsed with pure ethanol and dried under a stream of dry high purity nitrogen before use. Etching can also be performed in a mixture of up to 1:3 solvent to 50% HF. Typical solvents are ethanol and isopropyl alcohol, but other apriotic solvents such as DMSO or cyclopentanone can also be used to achieve the desired particle morphology.

[0125] Etched silicon wafers were crushed using a milling ball and / or a pestle and mortar. The fine powder was sieved using a Retsch™ sieve shaker AS 200, 38 μm gauge. A uniform particle size selection (20-100 μm) was achieved depending on the opening size of the sieve. Particle size was measured by a quantachrome system and PCS from Malvern Instruments. Samples were kept in a closed container until further use.

[0126] Nanosilicon powders were also obtained from Sigma and Hefei Kaier, China. Before loading and etching, the particle size was measured by PCS and the size of the particles was recorded (sizes ranging from 20 to 100 nm).

[0127] 500 mg of porous silicon nanoparticles with a diameter of 100 nm were mixed with 250 mL of ethanol 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 activates the silicon nanoparticles.

[0128] Example 2: Preparation of boron-doped silicon particles Wafers of boron doped silicon were obtained from BS Silicon prepared to the following specifications: Single-sided polished wafer, CZ Diameter: 150±0.2mm Orientation: (100)±1° Type: p / boron Resistivity: 0.014±25%Ohmcm. 5×10 18 Boron atoms / cm 3 Close to. Primary flat: 57.50±2.5mm Primary flat 1 position: D <100> ~{110} Thickness: 675±15μm Packaging: Ultrapak Shipping Cassette TTV:≦18μm TIR:≦5μm

[0129] Undoped silicon was obtained from American Elements (Manchester, UK).

[0130] Silicon wafers were electrically etched (as in Example 1) to achieve a porosity of at least 40% and then crushed using milling balls and / or a pestle and mortar. The fine powder was sieved using a Retsch™ sieve shaker AS 200, 38 μm gauge. Uniform particles in size between 20 μm and 100 μm were selected by sieving.

[0131] The powder was then activated by treating it with methanol (50 mg in 5 mL) and left under a fume hood followed by a slow evaporation process. The resulting powder was dispersed in nuclease-free water at a concentration of 1 mg / mL in the presence of a non-reducing disaccharide (trehalose) and an amino acid (glycine).

[0132] DOTAP solution: DOTAP was solubilized in methanol at a concentration of 5 mg / mL. In particular, 50 mg of DOTAP was solubilized in 10 mL of methanol and sonicated until completely solubilized.

[0133] DOPE solution: DOPE was solubilized in methanol at a concentration of 5 mg / mL. In particular, 60 mg of DOPE was solubilized in 12 mL of methanol and sonicated until completely solubilized.

[0134] mPEG2000-DSPE solution: mPEG2000-DSPE was solubilized in methanol at a concentration of 5 mg / mL. In particular, 40 mg of mPEG2000-DSPE was solubilized in 8 mL of methanol and sonicated until completely solubilized.

[0135] Trehalose was used as provided by Sigma Aldrich.

[0136] Glycine was used as provided by Sigma Aldrich.

[0137] The activated silicon particles were suspended with glycine and trehalose in nuclease-free water. Specifically, 50 mg of activated silicon particles, 50 mg of trehalose, and 25 mg of glycine were suspended in 50 mL of nuclease-free water and sonicated for 60 minutes.

[0138] siRNA specific for ClCn7G213R (for details see Capulli et al., 2015, Clin. Molec. Therap. 4, e248) was then loaded into the transfection vehicle.

[0139] In vivo study - head to head transfection comparison using boron doped and undoped silicon particles prepared in Example 2 in a formulation for gene therapy.

[0140] ADO2 mutant mice, whose cells can be treated with siRNA specific for ClCn7G213R if the siRNA is successfully transfected, are used to model osteopetrosis type 2. Thus, this model can be used to evaluate transfection efficacy.

[0141] A transfection vehicle was prepared using boron-doped particles according to the invention or control undoped particles, DOTAP, DOPE, mPEG2000-DSPE, glycine, trehalose, and nuclease-free water. The vehicle was administered either empty or together with siRNA specific for ClCn7G213R.

[0142] The samples were coded as follows: ADO2+SIS0012-empty-undoped Si-containing vehicle ADO2+SIS0012-siRNA-Undoped Si-containing vehicle with siRNA specific for ClCn7G213R ADO2+SIS0013-Empty-Boron-Doped Si-Containing Vehicle ADO2+SIS0013-siRNA-ClCn7G213R specific siRNA-containing boron-doped Si vehicle

[0143] Ten-day-old ADO2 mice were injected intraperitoneally with one of these three constructs, three times a week for two weeks (n=5 mice per group).

[0144] result ClCn7G213R expression in mouse PMBC was assayed for each treatment group and the results are presented in Figure 3. Figure 4 shows bone expression of ClCn7G213R and Figure 5 shows the CTX-blood test results (CTX is a marker of bone turnover).

[0145] The ADO2SIS0012-siRNA complex was able to significantly downregulate the expression of a related gene (ClCn7G213R) in the target organ (femur, p<0.05) when compared to its relevant control group (ADO2SIS0012 empty, no siRNA administration). In addition, a significant downregulation of the same gene was observed in peripheral blood mononuclear cells (PBMC, p<0.005 when comparing ADO2SIS0012-siRNA complex with ADO2SIS0012 empty, no siRNA). In the analyzed period and proposed administration regimen (only 2 weeks of treatment), bone resorption was also slightly elevated, and the changes are expected to be more substantial over a longer period.

[0146] When comparing the performance of ADO2SIS0012-siRNA complexes (made with undoped porous silicon particles) with ADO2SIS0013-siRNA complexes (made with boron-doped silicon particles) in the target organ ADO2SIS0013, both compositions provided significant efficacy (Figure 4, p<0.02). The use of boron-doped silicon resulted in faster bone turnover when compared with the performance of non-doped porous silicon (Figure 5, CTX evaluation). Therefore, it can be said that the superior storage properties provided by using boron-doped silicon do not compromise the transfection efficacy or therapeutic efficacy of the siRNA formulation.

[0147] Example 3: Preparation of biological pDNA-containing formulations Formulation type 1 (referred to as the "Biocourier" formulation) was prepared using silicone (porous, activated, average particle size <100 nm, doped and undoped as in Example 2) according to the ingredients in Table 2.

[0148] Table 2: Composition of Biocourier Type 1 [Table 1]

[0149] Lipid film preparation The lipids listed in Table 2 above were transferred to a clean glass round bottom flask and mixed in a solvent, which was evaporated using a rotary evaporator in a water bath at 40° C. and under vacuum.

[0150] Rehydration of lipid films Silicon particles, glycine, and trehalose in solution were added to the lipid film, and the final volume was adjusted to 10 mL with nuclease-free water, if necessary. The flask was covered with parafilm and stirred in a water bath (60°C) for 5 minutes. Aliquots of 1 mL were stored in RNA-free Eppendorf tubes in the refrigerator. Before use, the suspension was passed through membrane filters with pore sizes of 0.4 μm and 0.1 μm at 60°C, 10 times for 0.4 μm and then 10 times for 0.1 μm.

[0151] Loading pDNA into the vehicle A frozen pDNA sample of 6768 bp in size was thawed from -40°C to room temperature.

[0152] The vehicle and pDNA were placed together in a sterile Eppendorf tube and gently vortexed for 15 seconds, then left at room temperature for 30 minutes and then stored in the refrigerator until use.

[0153] Example 4: Storage stability study using pDNA the purpose To evaluate the storage stability of the different formulations produced as described in Example 2 using both doped and undoped silicon by agarose gel electrophoresis.

[0154] Preparations used "Biocourier SIS0012" - Formulation containing undoped silicon particles onto which pDNA is loaded

[0155] "Biocourier SIS0013" - Formation containing boron-doped silicon particles onto which pDNA is loaded

[0156] "Biocourier MV10010" - Same as Biocourier SIS0012 but from a different batch made by the contract manufacturer.

[0157] method A. Preparation of pDNA-Biocourier complexes The pDNA-Biocourier formulation was prepared by mixing pDNA solution (2 mg / mL) with Biocourier with a lipid / DNA ratio of 7.2. The final concentration of DNA in the mixture was 0.275 mg / mL. The mixture was incubated at room temperature for 30 minutes to allow complete complexation and then stored either at room temperature or at 4° C. Samples were prepared in 20 μL aliquots for each measurement time point and stored separately to minimize the risk of cross-contamination during storage and analysis.

[0158] B. Agarose gel electrophoresis The pDNA-Biocourier complexes were analyzed by agarose gel electrophoresis at the indicated time points (0 h, 6 h, 24 h, 48 h, 72 h, and 8 days). Naked pDNA stored in similar conditions was used as a control in all cases. The samples were loaded onto E-Gel™ agarose gels (1%) in an E-Gel™ Power Snap electrophoresis device. The amount of each sample and the total content of pDNA loaded onto the gel at different time points are presented in the table below. The total amount of naked pDNA loaded onto the gel at each time point was also the same as the pDNA-Biocourier complexes. The gels were transilluminated and imaged at 3 and 7 min using an E-Gel™ Power Snap electrophoresis camera.

[0159] Table showing the amount of pDNA-Biocourier complexes loaded onto the agarose gel and the total amount of pDNA per well at different measurement time points. [Table 2]

[0160] result Test 1. Stability of pDNA-Biocourier complexes at room temperature. Agarose gel electrophoresis images of pDNA-Biocourier formulations immediately after preparation and after storage at room temperature for 8 days are presented in Figures 6-8. All three Biocourier formulations in their liquid form completely retained the pDNA in the gel wells, indicating complete complexation. However, in the lyophilized form, a small amount of pDNA passed through the gel, indicating the presence of some unbound DNA. The higher band intensity of SIS0012 and SIS0013 compared to MVI0010 indicates a higher amount of unbound DNA. Nevertheless, comparing the band intensity of Biocourier formulations to the bands from naked pDNA containing the same total amount of DNA loaded onto the Biocourier, shows that only a small percentage of the total DNA loaded onto the Biocourier was unbound, demonstrating high binding efficiency. After storage at room temperature for 6 hours, the amount of unbound pDNA released from the lyophilized formulations into the gel was small and could be attributed to the degradation of pDNA at room temperature. Migration of unbound pDNA through the gel containing the lyophilized formulations was continued for up to 24 hours. However, no significant signal was observed for pDNA after 48 hours, indicating a lack of DNA migration through the gel. From these data, it can be inferred that although the lyophilized formulations did not completely complex with the loaded pDNA, their binding efficiency was still very high, as demonstrated by the negligible amount of DNA passing through the gel compared to the large amount of DNA loaded into the Biocourier. Also, the pDNA-Biocourier complexes were stable over 8 days at room temperature, as pDNA was no longer released into the gel during a time that would have indicated dissociation.

[0161] Test 2. Stability of pDNA-Biocourier complexes at 4°C. Agarose gel electrophoresis images of pDNA-Biocourier formulations immediately after preparation and after storage at 4° C. for 8 days are presented in FIG. 9-FIG. 11. As observed, after 6 hours of storage at 4° C., only a small amount of pDNA passed through the gel with the lyophilized forms of SIS0012 and SIS0013. However, the amount of unbound pDNA passing through the gel was low compared to the total amount of pDNA loaded on the Biocourier, indicating high binding efficiency. The liquid form of the formulation, and the lyophilized form of MVI0010, also did not release any pDNA into the gel, indicating complete complexation of the loaded pDNA. The amount of pDNA passing through the gel of the lyophilized forms of SIS0012 and SIS0013 decreased after 24 hours, indicating some trace degradation of pDNA. After 48 hours, no pDNA was observed with lyophilized SIS0012, whereas with lyophilized SIS0013, there was still some trace of pDNA passing through the gel, which continued for up to 8 days. This could either be the same unbound pDNA that was present from the beginning of the experiment, or new pDNA that dissociated from the complex. However, due to the very small amount of DNA, it is difficult to comment with certainty on its source, other than to note that most of the pDNA remains associated with the complex. The liquid forms of all formulations were able to retain all the loaded pDNA in the wells for up to 8 days, indicating complete binding and stability in this storage condition.

[0162] conclusion The liquid forms of all Biocourier formulations fully complexed with pDNA and were stable for up to 8 days at both room temperature and 4° C. However, it was found that the lyophilized formulations, despite having high binding efficacy, were unable to fully complex with all the loaded pDNA and small amounts of excess / unbound pDNA in these samples. Despite the lower binding efficacy of the lyophilized formulations compared to their liquid counterparts, they were stable for up to 8 days at both room temperature and 4° C.

[0163] Example 4: Storage stability study using DNA or RNA the purpose Stability of Biocourier formulations loaded with DNA or siRNA stored at different temperatures to be assessed using agarose gel electrophoresis.

[0164] Preparations used The following formulations (prepared as detailed above) were loaded with DNA, mRNA, or siRNA as described above. "Biocourier SIS0012" - Formation containing undoped silicon particles "Biocourier SIS0013" - Formation containing boron-doped silicon particles

[0165] method A. Preparation of Nucleic Acid-Biocourier Complexes Prepare nucleic acid stock solution by dissolving the mRNA / siRNA / DNA powder in nuclease-free water according to the table below:

[0166] [Table 3]

[0167] The stock solution was then diluted with nuclease-free water to reach the required concentration according to the following table:

[0168] [Table 4]

[0169] Biocourier stock solutions were also diluted with nuclease-free water to obtain the concentrations required for the complexation assay. Nucleic acid-Biocourier complexes were made by mixing the required amounts of Nucleic Acid Working Solution and Biocourier Working Solution listed in the table below, and then incubated at room temperature for 40 minutes to ensure complete complexation: [Table 5]

[0170] The prepared nucleic acid-Biocourier complexes were then subjected to analysis by gel electrophoresis.

[0171] B. Agarose gel electrophoresis The gel electrophoresis system used for these experiments consisted of a gel electrophoresis device, a precast agarose gel (with electrophoresis buffer and electrodes embedded within the gel), and a camera. Samples, controls (naked DNA or RNA), and DNA ladder were mixed with the required amount of loading solution to a total volume of 20 μL according to the table above and then loaded onto the gel. The gel (1%) was then inserted into the device chamber, samples, controls, and DNA ladder were loaded, and run for 7-8 min. The gel was transilluminated and imaged at 3 and 7 min with a camera (the camera was mounted on the device through a docking port available on the camera and device). The same procedure was repeated at the designated time points (indicated in the table above) after DNA-SIS0012, siRNA-SIS0012, and siRNA-SIS0013 were stored at different storage conditions (room temperature (20°C), 4°C, and -20°C).

[0172] result Figure 12 shows the results from a gel retardation assay of RNA from baker's yeast loaded onto SIS0012 at different concentrations of RNA, different concentrations of SIS0012 formulations, and different volume ratios. It was observed that 100 ng / well of RNA, recommended by the manufacturer for this type of gel, resulted in very weak luminescence of RNA, while a stronger signal was obtained when the amount of RNA was increased to 200 ng / well. Therefore, the amount of nucleic acid was fixed at 200 ng / well for all future experiments. SIS0012 formulations were mixed with RNA at different dilutions from the stock solution and different mixing ratios (v / v) to find the minimum concentration of SIS0012 solution and the lowest mixing ratio that could form a complex with RNA and inhibit its migration through the gel. It was observed that a 10-fold dilution from the SIS0012 stock solution at a mixing ratio of 2.5 partially blocked the migration of the RNA through the gel, since the intensity of the luminescence (brightness of the band) was reduced compared to the naked RNA used as a control, and a complete block of the RNA migration was obtained when a 5-fold diluted SIS0012 solution was mixed with the RNA at a ratio of 2.5 or 4. Lower concentrations of SIS0012 preparations or lower mixing ratios were not able to bind efficiently to the RNA and inhibit its migration through the gel. Based on these findings, a 5-fold diluted SIS0012 solution and a mixing ratio of 2.5 (V / V) were selected for future experiments. The RNA used in these experiments was able to provide a proof-of-concept for the complexation assay, but the purity of the samples seemed low, since the bands observed for the RNA were not sharp and not as intense as the DNA ladder. Furthermore, the size of the RNA fragments was found to be very small (smaller than the smallest fragment of the DNA ladder, which is 100 bp), indicating the presence of oligomers rather than full-length mRNA.

[0173] SIS0012: A study to evaluate the complexation efficiency and stability of DNA from herring sperm loaded onto Biocourier

[0174] Figure 13 shows images from an agarose gel retardation assay of DNA from herring sperm loaded onto SIS0012 formulations at selected concentrations and mixing ratios based on previous studies immediately after complexation and up to 5 hours. The DNA-SIS0012 complexes were stored at three different conditions: room temperature, 4°C, and -20°C. It could be observed that the carrier efficiently captured the DNA and inhibited its migration through the gel (Figure 13A). No signal from DNA was observed in any of the gels, indicating that no DNA was released by any of the three formulations (storage at different temperatures) within the first 5 hours. Therefore, the experiment was continued further to study the stability of the DNA-SIS0012 complexes over a longer period of time. It is noteworthy that the DNA control (naked DNA) did not give a degradation band of a specific size, but rather showed a mixture of DNA fragments of different sizes. Nevertheless, since these experiments were aimed simply at assessing the complex formation between DNA and the carrier and the stability of the corresponding complexes, for the purposes of these experiments it was not necessary to obtain complete separation of the DNA fragments or to obtain single bands of a specific size, which could be achieved by further treating the DNA by sonication before using it in the complex formation experiments.

[0175] Figure 14 shows the results of gel retardation assay of DNA-SIS0012 complexes stored at different temperatures for up to 72 hours. There was no evidence of DNA release from any of the three samples. Therefore, from these observations, it could be inferred that DNA-SIS0012 complexes were stable for 72 hours in all three different storage conditions.

[0176] Study to evaluate the complexation efficacy and stability of ADO-siRNA loaded on Biocourier-SIS0012 Following the encouraging results from the DNA-SIS0012 experiment, the stability of ADO-siRNA-SIS0012 complexes produced by the same method (same concentration and mixing ratio) and stored under the same conditions (room temperature, 4°C and -20°C) was tested. The results are presented in Figures 15 and 16. As is evident from these figures, the SIS0012 formulations were able to efficiently capture siRNA and block its migration through the gel at all measured time points, regardless of storage conditions showing stability of siRNA-SIS0012 complexes in all three storage conditions up to 120 hours.

[0177] A study to evaluate the complexation efficacy and stability of ADO-siRNA loaded onto Biocourier-SIS0012 without silicon nanoparticles Figures 17 and 18 show the gel retardation assay results of ADO siRNA loaded onto SIS0012 formulation without silicon nanoparticles. Biocourier was able to complex with siRNA and block its migration through the gel. Neither the samples stored at room temperature nor at 4°C released any amount of associated siRNA at any of the measurement time points up to 6 hours, since no siRNA bands were observed. However, after 24 hours, some traces of siRNA were detected in the gel, which resulted in a very weak signal (indicating a small amount of siRNA) that was enhanced at 120 hours. Nevertheless, it should be noted that the 72 and 120 hour samples were run without gel loading solution and were only diluted with water before loading onto any gel. Therefore, the intensity of the signal observed would be less than that observed if the sample was diluted with gel loading solution. Another observation of note is the presence of two bands of the control siRNA, indicating degradation of the siRNA. The siRNA solution (66 μg / mL) used as a control in these experiments was stored at −20° C. and thawed before the start of each experiment, therefore, any degradation of the siRNA was likely the result of repeated short-term freeze-thaw cycles.

[0178] Study to evaluate the complexation efficacy and stability of ADO-siRNA loaded on Biocouriers-SIS0013 Figures 19 and 20 show gel retardation assays performed on ADO-siRNA loaded into SIS0013 formulations immediately after complexation and up to 6 hours after storage at room temperature. Although the Biocouriers were able to capture the siRNA and some traces of siRNA could be observed throughout the gel from the beginning of the experiment, the signal was not significant before 120 hours. At 120 hours after storage, the signal was stronger, indicating increased release of siRNA from the complexes. However, even at this point, the signal was significantly weaker compared to the signal from naked siRNA, indicating a very small amount of siRNA passing through the gel. Also note that the 72 hour and 120 hour samples were run without gel loading fluid and were only diluted with water before loading on any gel. Therefore, the intensity of the signal observed would be less than that observed if the sample was diluted with gel loading fluid. Similar to that shown in Figure 16, the naked siRNA exhibited two bands indicating degradation of the siRNA due to multiple freeze-thaw cycles.

[0179] Unlike storage at room temperature, siRNA-SIS0013 complexes stored at 4° C. remained stable for up to 120 hours, as indicated by the lack of siRNA migration through the gel (FIG. 21).

[0180] conclusion The gel retardation assay developed with the reported experimental parameters is effective to detect the complex formation between nucleic acids and Biocourier and to test the stability of the complex formed with very small amount (2-3 μL / well) and low concentration (100 ng / μL) of nucleic acid over time. It could be applied to different types of nucleic acids (DNA, mRNA, siRNA). The optimal complexation condition of nucleic acids and Biocourier was found to be 2.5 V / V mixing ratio of 5-fold diluted Biocourier solution with DNA / mRNA (100 μg / mL) or siRNA (66.5 μg / mL). Biocourier formulation SIS0012 was able to retain associated siRNA up to 120 hours, while SIS0012 formulation without silicon nanoparticles started releasing siRNA after 24 and 120 hours, and the amount of siRNA released through the gel was significant. The SIS0013 formulation released small amounts of siRNA into the gel even at the start, suggesting a lower entrapment efficiency compared to SIS0012, and a significant amount of siRNA passed through the gel after 120 h. However, SIS0013-siRNA complexes were stable for up to 120 h at 4 °C, demonstrating excellent storage stability when the silicon particles comprise boron-doped silicon.

[0181] Example 5: SIS0012 (undoped Si) vs. SIS0013 (boron-doped Si; 5×10 18 Boron atoms / cm 3 Stabilization of alkaline phosphatase by Alkaline phosphatase is an enzyme that exists in various forms, catalyzes the decomposition of various proteins, and can be found in all tissues of the human body. Alkaline phosphatase is mainly concentrated in bone, kidney, liver, intestine, and placenta. Alkaline phosphatase contributes to, among other things, the protection of the intestinal tract against bacteria; digestive function; the decomposition of fat and vitamin B; and bone formation. Alkaline phosphatase exhibits loss of activity at low pH and high temperature.

[0182] Alkaline phosphatase activity can be monitored in an in vitro assay by measuring changes in concentration of one or more of its substrates or products, as proxied by UV-Vis absorbance. For example, the concentration of the substrate 4-nitrophenylphosphatase (PNPP), whose structure is shown below, may be monitored to follow the following reaction: [ka]

[0183] Materials, methods, and results Alkaline phosphatase, supplied as a 56 kD recombinant enzyme isolated from bovine intestine and expressed in the yeast Pichia pastoris, was obtained from Sigma Aldrich / Merck (The Old Brickyard, New Rd, Gillingham, Dorset, SP8 4XT). A stock aqueous solution of alkaline phosphatase (ALP) was prepared at a concentration of 1 U / mL. 1 U (μmol / min) is defined as the amount of ALP that catalyzes the conversion of 1 μmole of PNPP per minute at 37°C and pH 7.4.

[0184] A 20 mM solution of 4-nitrophenylphosphatase (PNPP) was prepared in Tris buffer (100 mM / L) at pH 7.4.

[0185] Following the protocol of Example 2, SIS0012 (undoped Si) and SIS0013 (boron-doped Si; 5×10 18 Boron atoms / cm 3 ) samples were provided.

[0186] ALP solutions were prepared at the following concentrations:

[0187] 0.1, 0.5, 1, 5, 10, 50, and 100mU / mL

[0188] From a 1U / mL stock solution in a 15mL tube.

[0189] These were then mixed with the prepared 20 mM solution of PNPP in Tris buffer in an Eppendorf tube.

[0190] The tubes were incubated in a 37° C. water bath for 30 min, after which UV-Vis absorbance measurements were taken at 405 nm as shown in FIG.

[0191] ALP was loaded into SIS0012 and SIS0013 as follows.

[0192] 1.3 sets of 8 Eppendorf tubes were prepared:

[0193] Eight Eppendorf tubes were prepared, each containing 50 μL of ALP (50 mU / mL) and 500 μL of SIS0012 (undoped Si). After these components were added to the tubes, they were mixed, vortexed, and refrigerated overnight.

[0194] B Eight Eppendorf tubes were prepared, each containing 50 μL of ALP (50 mU / mL) and 500 μL of SIS0013 (boron-doped Si). After these components were added to the tubes, they were mixed, vortexed, and refrigerated overnight.

[0195] C Eight Eppendorf tubes were prepared, each containing 50 μL of ALP (50 mU / mL) and 500 μL of Tris buffer. After these components were added to the tubes, they were mixed, vortexed, and refrigerated overnight.

[0196] 2. After these preparations, all Eppendorf tubes were placed in a water bath at 50° C. Each of the eight tubes in each of the three sets of tubes A to C was removed from the water bath after 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 40 minutes, or 60 minutes.

[0197] Then, 3.300 μL of PNPP was added to all Eppendorf tubes of all Sets A to C. The tubes were mixed and vortexed. They were then placed in a 37° C. water bath for 30 minutes, during which time dephosphorylation of PNPP occurred.

[0198] 4. Following this, UV-Vis analysis was performed (405 nm) on all samples from Sets A to C. The results are shown in FIG.

[0199] Consideration Surprisingly, the activity of ALP loaded on SIS0013 (boron-doped Si) was approximately 20–30% higher than that of ALP loaded on SIS0012, which may be attributed to the improved protection of ALP by doped Si.

[0200] Free ALP showed significant degradation compared to both SIS0012 and SIS0013. The activity of free ALP decreased with increasing incubation time at 50°C. On the other hand, the activity of ALP loaded in SIS0012 and SIS0013 remained relatively constant regardless of incubation time. Free ALP appears to denature over time at 50°C, whereas ALP loaded in SIS0012 and SIS0013, especially SIS0013, is protected from denaturation and remains stable.

[0201] Example 6: Complexation of further silicon-containing formulations with mRNA or siRNA Alternatives were investigated to replace the cationic lipids such as DOTAP used in SIS0012 and SIS0013.

[0202] One such alternative is lipopeptides. These are amphiphiles consisting of a lipid chain (generally 12-18 carbon atoms long) conjugated to a peptide sequence (generally 3-20 amino acid residues). A specific example of such a lipopeptide is palmitoyl pentapeptide-4 (abbreviated as PAL-KTTKS). [ka] Palmitoyl Pentapeptide-4

[0203] PAL-KTTKS is believed to be a good candidate for an alternative to cationic lipids due to its cationic lysine residues that aid in binding to negatively charged RNA.

[0204] Other candidates investigated in SIS0012 and SIS0013 include NAD, tyrosine (TYR), and quercetin (QUE). It is believed that these ligands can enhance organ-specific uptake of RNA and improve cellular internalization and targeting. Furthermore, these ligands can help provide a positively charged environment for binding of negatively charged RNA.

[0205] Nicotinamide adenine dinucleotide (NAD) is a coenzyme. In its oxidized form, NAD+, it has the following structure: [ka] NAD+

[0206] Tyrosine is a naturally occurring amino acid that has the following structure at physiological pH (pH 7.4): [ka] Tyrosine at physiological pH

[0207] Quercetin is a flavanol having the following structure: [ka] Quercetin

[0208] NAD, TYR, and QUE - Investigation of the addition or substitution of DOTAP in SIS0012 and SIS0013

[0209] Modified SIS0012 and modified SIS0013 compositions were prepared by adding 0.2 mg of NAD, TYR, and QUE to the normal composition described above (i.e., in addition to DOTAP). Then, complexation with siRNA was evaluated. Figure 24 shows the gel electrophoresis results, which show that siRNA was successfully and completely bound in these formulations.

[0210] Dynamic light scattering measurements were also performed using a Zetasizer (available from Malvern Instruments) to assess size and charge both before and after siRNA complexation, as shown in the table below. As the table shows, an increase in size was observed after siRNA complexation, along with a 10-15 mV decrease in surface charge.

[0211] [Table 6]

[0212] Next, replacement of DOTAP with NAD, TYR, or QUE (rather than simply adding NAD, TYR, or QUE to a DOTAP-containing composition) was investigated.

[0213] In this study, DPPC and DOPE were chosen for use with NAD, TYR, or QUE because they are zwitterionic lipids that do not play a significant role in surface charge at neutral pH.

[0214] Therefore, DPPC / DOPE formulations were prepared using (i) 0.2 mg and (ii) 1 mg of NAD, TYR, and QUE. The formulations are shown in the table below.

[0215] Table: Composition of DPPC / DOPE LNP Biocourier functionalized with beta-nicotinamide adenine dinucleotide (NAD) [Table 7]

[0216] Table: Composition of DPPC / DOPE LNP Biocourier functionalized with quercetin (QUE). [Table 8]

[0217] Table: Composition of DPPC / DOPE LNP Biocourier SIS0012 functionalized with tyrosine (TYR) [Table 9]

[0218] As shown in the table below, Zetasizer measurements were obtained and revealed negative zeta potential across all formulations, regardless of the amount of NAD, TYR, or QUE.

[0219] Table: DPPC / DOPE LNPs functionalized with 0.2 mg and 1 mg of NAD, TYR, and QUE. [Table 10]

[0220] Investigation of DOTAP replacement in lipopeptides-SIS0012 and SIS0013 Lipopeptides, also known as peptide amphiphiles (PAs), were investigated as another alternative to DOTAP.

[0221] It is believed that lipopeptides may provide a solution to the problem of replacing or reducing the amount of cationic lipids such as DOTAP in transfection compositions. Lipopeptides consist of alkyl chains conjugated to peptide sequences. These alkyl chains can be assimilated into lipid bilayers while the surface of the bilayer is decorated with peptide moieties.

[0222] An exemplary PA is the molecule palmitoyl pentapeptide-4 (abbreviated as PAL-KTTKS, see above). The two cationic lysine residues may function similarly to cationic lipids such as DOTAP, which exhibit electrostatic interactions with negatively charged RNA.

[0223] DPPC and DOPE were selected as neutral lipids to formulate PAL-KTTKS. Various formulations were prepared with different silicon nanoparticles or without silicon at all; and with pH 4 buffer (to investigate the effect of pH).

[0224] Complete details of the DPPC, DOPE, and PAL-KTTKS containing formulations are provided in the table below.

[0225] Table - Various formulation compositions of DPPC, DOPE, and Pal-KTTKS with silicon nanoparticles, boron doped silicon, and boric acid / silicon nanoparticles. The formulation counterparts without silicon nanoparticles were also formulated as controls. All formulations were prepared up to a final volume of 10 ml.

[0226] [Table 11]

[0227] All eight formulations had a positively charged surface and the zeta potential (measured with a Zetasizer available from Malvern Instruments) is presented in the table below.

[0228] Based on these results, it is believed that during lipid film assembly, PAL-KTTKS positions itself in the lipid bilayer with the peptide moiety exposed to the nanoparticle surface, and furthermore, the lysine residues on its surface contribute to the positively charged formulation.

[0229] Table: Zeta potential measurements of all eight DPPC / DOPE / PAL-KTTKS formulations. [Table 12]

[0230] All formulations were evaluated for their ability to electrostatically bind to siRNA and mRNA.

[0231] Gel electrophoresis analysis was performed. No complete complexation was observed for siRNA. However, complete complexation was observed for mRNA. Figure 25 shows the gel electrophoresis results for siRNA, and Figure 26 shows the gel electrophoresis results for mRNA.

[0232] To address the partial complexation of siRNA with DPPC / DOPE / PAL-KTTKS, an alternative loading method was employed. Figure 27 shows gel electrophoresis of the complex after using the alternative loading method, which indicates successful complete complexation of siRNA. (The bright spot in lane 3 of Figure 27 is an artifact of the imaging device.)

[0233] In an alternative loading method, compared to the protocol hereinabove, the following steps were adopted:

[0234] 1. A thin lipid film was prepared by dissolving DPPC, DOPE, and Pal-KTTKS in methanol and evaporating using a rotary evaporator.

[0235] 2. The lipid film was rehydrated with a suspension containing activated silicon (SIS0012) or activated boron-doped silicon (SIS0013) with trehalose and glycine and either siRNA or mRNA. Reydration was performed at 40° C. for 10 min to ensure that no lipid-silicon film remained on the walls of the round-bottom rotary evaporator flask.

[0236] Lipopeptides are very versatile molecules, and they can be fine-tuned by modifying their alkyl chains and / or their peptide sequences. It is believed that peptide customization can enhance cell and / or tissue targeting. In the field of gene therapy, peptide customization can result in electrostatic interactions with nucleic acids, such as RNA, especially mRNA. As an example, PAL-KTTKS, when formulated with DPPC and DOPE, resulted in a positively charged surface, as confirmed by the zeta potential.

[0237] Lipopeptides, which are amphiphilic molecules, have properties very similar to surfactants that can self-assemble to form micelles. This is at least partially due to the fact that the peptide sequence can form intermolecular hydrogen bonds, while the alkyl chains are suitable for hydrophobic interactions. Phospholipids such as DPPC and DOPE can also self-assemble into liposomes. Thus, when incorporating PA, of which PAL-KTTKS is a representative example (although other lipopeptides may be used), the alkyl chains can form hydrophobic interactions with DPPC and DOPE to obtain liposomal structures.

[0238] On the one hand, silicon nanoparticles provide structural stability to the complex as a whole and can interact with lipids and other ligands, such as lipopeptides, NAD, QUE, or TYR, via non-covalent (electrostatic) interactions, thus promoting long-term stability and binding of nucleic acids.

Claims

1. A pharmaceutical composition comprising particles containing a hydrolyzable doped silicon and one or more lipids complexed with a drug substance, The particles are 1 cm ３ per hit with 1 × 10 １６ or more amounts of dopant atoms, a pharmaceutical composition.

2. The hydrolyzable doped silicon particles are at most 1 × 10 ３ per cm ２０ doped with a quantity of dopant atoms and the pharmaceutical composition according to claim 1.

3. The pharmaceutical composition according to claim 1, wherein the drug substance is a nucleic acid.

4. The pharmaceutical composition according to claim 3, wherein the nucleic acid is RNA.

5. The pharmaceutical composition according to claim 4, wherein the nucleic acid is small interfering RNA (siRNA), small activating RNA (saRNA), or small hairpin RNA (shRNA).

6. The pharmaceutical composition according to claim 4, wherein the nucleic acid is messenger RNA (mRNA).

7. The pharmaceutical composition according to claim 6, wherein the mRNA encodes a protein of a pathogen.

8. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a vaccine composition.

9. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises an amino acid.

10. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a non-reducing disaccharide.

11. The pharmaceutical composition according to claim 10, wherein the non-reducing disaccharide is trehalose.

12. The pharmaceutical composition according to claim 1, wherein the doped silicon particles contain boron-doped silicon.

13. The pharmaceutical composition according to claim 1, wherein the doped silicon particles contain phosphorus-doped silicon.

14. The pharmaceutical composition according to claim 1, wherein the doped silicon particles are doped for the purpose of intentionally introducing impurities to regulate nucleic acid binding properties.

15. The pharmaceutical composition according to claim 1, wherein the doped silicon particles are doped for the purpose of intentionally introducing impurities to regulate lipid binding properties.

16. The pharmaceutical composition according to claim 1, wherein the one or more lipids contain ionizable lipids.

17. The pharmaceutical composition according to claim 1, wherein the one or more lipids contain cationic lipids.

18. The pharmaceutical composition according to claim 17, wherein the one or more lipids contain DOTAP.

19. The pharmaceutical composition according to claim 17, wherein the one or more lipids contain DOTAP in a racemic mixture.

20. The pharmaceutical composition according to claim 17, wherein the one or more lipids contain DOTAP in the S enantiomeric form.

21. The pharmaceutical composition according to claim 17, wherein the one or more lipids contain DOTAP in the R enantiomeric form.

22. The pharmaceutical composition according to claim 1, wherein the lipid or lipids of 1 or more contain 1 or more lipid-added oligopeptides.

23. The pharmaceutical composition according to claim 22, wherein each of the lipid-added oligopeptides contains an oligopeptide moiety having 3 to 20 amino acid residues and a fatty acid chain having 12 to 18 carbon atoms.

24. The pharmaceutical composition according to claim 23, wherein at least one of the amino acid residues is positively charged at pH 7.

4.

25. Use of particles containing hydrolyzable doped silicon for enhancing the effectiveness of a pharmaceutical composition containing a drug substance, wherein the particles are used, doped with a dopant atom in an amount of 1×10 ３ or more per 1 cm １６ .

26. The use according to claim 25, wherein the hydrolyzable doped silicon is hydrolyzable boron-doped silicon.

27. The use according to claim 25, wherein the pharmaceutical composition is as defined in any one of claims 1 to 24.

28. The use according to claim 25, wherein the drug substance is as defined in any one of claims 3 to 7.

29. The use according to claim 25, wherein the effectiveness of the pharmaceutical composition is enhanced by the particles that increase the stability of the drug substance at room temperature.

30. The use according to claim 25, wherein the effectiveness of the pharmaceutical composition is enhanced by the particles that increase the intracellular stability of the drug substance.

31. The use according to claim 25, wherein the effectiveness of the pharmaceutical composition is enhanced by the particles that protect the drug substance from degradation.

32. The use according to claim 31, wherein the degradation is enzymatic degradation.

33. The use according to claim 25, wherein the effectiveness of the pharmaceutical composition is enhanced by the particles that enhance the uptake of the drug substance by target cells or target tissues.

34. The pharmaceutical composition according to any one of claims 1 to 24, for use as a medicament.

35. The pharmaceutical composition according to claim 34, wherein the medicament is a vaccine.

36. Use of the pharmaceutical composition according to claim 1 in the manufacture of a medicament.

37. The use according to claim 36, wherein the medicament is a vaccine.

38. A method for increasing the storage stability of the nucleic acid as a drug substance, comprising contacting the nucleic acid with hydrolyzable doped silicon particles and 1 or more lipids. The hydrolyzable doped silicon particles are doped with a maximum of 1 × 10 ３ dopant atoms per cm １６ quantity, method.

39. The method according to claim 38, wherein the nucleic acid is mRNA or siRNA.

40. The method according to claim 39, wherein the nucleic acid is as defined in any one of claims 4 to 6, and / or the hydrolyzable doped silicon particles are hydrolyzable boron-doped silicon particles.

41. The method according to claim 38 or claim 39, wherein the contacting is additionally carried out in the presence of an amino acid and / or in the presence of a non-reducing disaccharide.

42. The method according to claim 41, wherein the non-reducing disaccharide is trehalose.

43. The pharmaceutical composition according to claim 1, wherein the particles target the prodrug to cells or tissues.

44. The pharmaceutical composition according to claim 1, for use in the manufacture of a medicament for targeting a prodrug to cells or tissues.

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