Fusion lipidosome-coated porous silicon nanoparticles

Abstract

The present disclosure describes a fusion lipid-coated porous silicon nanoparticle for high loading efficiency of anionic payloads (small molecules, dyes, nucleic acids) and for non-endocytic delivery of hydrophilic and lipophilic payloads via membrane fusion. The lipid coating can be further modified with targeting peptides or antibodies via covalent binding chemistry between a ligand and functionalized poly(ethylene glycol). The surface moiety can be transferred to the cell membrane surface by fusion uptake. The compositions of the present disclosure can be applied to disease treatment by delivering the entrapped / encapsulated payloads.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201680047665.5, application date July 8, 2016, and invention name “Fusion liposome-coated porous silicon nanoparticles” after the PCT international application with PCT application number PCT / US2016 / 041639 entered the Chinese national phase. Technical Field

[0002] The present invention relates to delivery systems, and more particularly, fusogenic liposome nanoparticle compositions for delivering drugs, nucleic acids, and peptides to target cells or tissues. Background Art

[0003] In vivo gene delivery remains a challenge due to low efficiency or cytotoxicity. Endocytosis is the primary uptake pathway for most nanoplatforms, which leads to lysosomal degradation of genetic materials and low therapeutic efficacy. Summary of the Invention

[0004] The present disclosure provides a biodegradable liposomal porous silicon nanoparticle system that can bypass endocytic uptake via liposome-plasma membrane fusion. Unlike most currently studied nanoparticle delivery systems that allow endocytic uptake of all their payloads, membrane fusion allows hydrophilic payloads to be released directly from the core of the liposome into the cytoplasm; hydrophobic molecules to be transferred from the liposome bilayer to the cell membrane bilayer; and conjugated moieties (antibodies, small molecules, peptides, etc.) on the outer surface of the liposome to be transferred to the cell membrane surface.

[0005] Liposomal porous silicon particles, their synthesis methods and applications are disclosed. Liposomes can be loaded with lipophilic payloads and functionalized with poly(ethylene glycol) (PEG) and other surface moieties (targeting peptides, antibodies, aptamers, etc.) to transfer them into and onto cell membranes via fusion. The porous silicon-based core can trap large amounts of payloads (small molecules, proteins, nucleic acids) and deliver them directly to the cytoplasm of target cells; bypassing cellular endocytosis can increase the delivery and therapeutic efficacy of the treatment. In vitro knockdown efficiencies comparable to Lipofectamine (liposomes) have been demonstrated.

[0006] The present disclosure provides a fusogenic liposome-coated porous silicon nanoparticle, the nanoparticle comprising a silicon-containing core material having a nanostructure with a plurality of pores, the material comprising (a) a silicon-containing core; (b) a porous surface chemically connected to the core; (c) a plurality of carried molecules physically associated with the silicon-containing core material; and (d) a metal silicate; and a fusogenic liposome coating around the silicon-containing core material. In one embodiment, the fusogenic liposome comprises 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In another embodiment, the silicon-containing core is substantially oxidized silicon or fully oxidized silicon. In yet another embodiment of any of the foregoing embodiments, the metal silicate comprises calcium silicate or magnesium silicate. In another embodiment, the metal silicate forms a shell on the silicon-containing core. In yet another embodiment, no gaps exist between the silicon-containing core and the calcium or magnesium silicate shell. In another embodiment, the plurality of pores are chemically or physically configured to accommodate a transported molecule. In yet another embodiment, chemical oxidation of the pore surface combined with formation of the calcium or magnesium silicate shell results in physical entrapment of the plurality of transported molecules. In yet another embodiment, the plurality of pores accommodates a drug. In another embodiment, the plurality of pores accommodates a non-drug substance. In another embodiment, the silicon-containing core comprises a molecule that is physically adsorbed or covalently entrapped, or attached to the plurality of pores, by use of the plurality of pores, and wherein the silicon-containing core is coated with a calcium or magnesium-containing shell formed by the action of an aqueous solution of calcium or magnesium ions added to the host material in the presence of the molecule. In yet another embodiment, the fusogenic liposomes comprise a mixture of DMPC, DOTAP, and DSPE-PEG(methoxy). In another embodiment, the fusogenic liposomes comprise a mixture of DMPC, DOTAP, and DSPE-PEG(carboxyl). In another embodiment, the fusion liposome comprises a mixture of DMPC, DOTAP and DSPE-PEG (maleimide). In another embodiment again, the silicon-containing core material has a hydrodynamic diameter of about 10nm-100nm scope. In another embodiment, the fusion liposome has a hydrodynamic diameter of about 100nm-400nm scope. In another embodiment again, the targeting molecule is conjugated to the fusion liposome coating. In one embodiment, an antibody is conjugated to the fusion liposome coating. In another embodiment, the hydrophobicity carried molecule is loaded in the fusion liposome coating.In yet another embodiment, a hydrophilic cargo molecule is trapped within the pores of the silicon-containing core. In yet another embodiment, a hydrophobic agent / cargo molecule is present in the fusogenic coating and a hydrophilic agent / cargo is present in the pores of the silicon-containing core. In another embodiment, a nucleic acid cargo molecule is trapped within the pores of the silicon-containing core. In another embodiment, a small molecule cargo molecule is trapped within the pores of the porous silicon-containing core.

[0007] The present disclosure also provides a method of delivering a nucleic acid payload to a cell, the method comprising contacting the cell with the fusogenic liposome-coated porous silicon nanoparticles of the present disclosure containing the nucleic acid.

[0008] The present disclosure also provides a method of treating a disease or condition of the eye, the method comprising delivering the fusogenic liposome-coated porous silicon nanoparticles of the present disclosure into the eye or onto the surface of the eye.

[0009] The present disclosure also provides a method for treating cancer, comprising delivering the fusogenic liposome-coated porous silicon nanoparticles of the present disclosure into the body.

[0010] The present disclosure also provides a method for treating bacterial infection, comprising delivering the fusogenic liposome-coated porous silicon nanoparticles of the present disclosure into the body.

[0011] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A-H shows schematic diagrams representing embodiments of the present disclosure. (A) Schematic diagram illustrating the function of fusogenic liposome-coated porous silicon nanoparticles of the present disclosure. (B) Schematic diagram illustrating the loading of calcium-coated porous silicon (pSi) with anionic payloads. The anionic payload and the cationic metal silicate-deposited pSiNPs form clusters through electrostatic interactions. (C) Schematic diagram illustrating the loading of "loaded" pSi into cationic liposomes. The payload-pSi clusters are encapsulated into cationic fusogenic liposomes via electrostatic interactions and mechanical extrusion. (D) Schematic diagram illustrating the modification of liposomes with targeting peptides. Targeting peptides or antibodies can be conjugated to functional PEG via chemical conjugation. (E) Schematic diagram illustrating a low-magnification image of the particle. Imaged using a Tecnai TEM. Negatively stained with uranyl acetate. Scale bar represents 500 nm. (F) Schematic diagram illustrating a high-magnification image of the particle, showing turbid liposomes coated around a dark and dense porous silicon-based core. Imaged using a JEOL 1200 EX TEM. Negatively stained with 2% PTA. Scale bar represents 200 nm. (G) Table of particle size and zeta potential measured by DLS (n=3). (H) Schematic diagram showing particles of the present disclosure.

[0013] Figure 2 Schematic diagram showing drug loading of porous silicon nanoparticles.

[0014] Figure 3 Schematic diagram showing calcium silicate-coated porous silicon nanoparticles (Ca-pSiNPs) loaded with cargo.

[0015] Figure 4A-IConfocal microscopy and transmission electron microscopy are shown. (AF) Confocal microscopy of J771A.1; (A) J771A.1 after incubation with DiI-loaded F-pSi for 10 minutes; (B) J774a.1 after incubation with Lysotracker Red (lysosomal red tracking probe) for 1 hour and with F-pSi with calcein for 10 minutes; (C) J774a.1 after incubation with DiI-loaded F-pSi conjugated with MTP-FAM for 5 minutes; (D) J771A.1 after incubation with DiI-loaded NF-pSi for 10 minutes; (E) (I) Transmission electron microscopy (TEM) of HeLa cells after 1 hour of incubation with DiI-loaded NF2-pSi conjugated to MTP-FAM. (G) Non-fusogenic liposome-coated particles are localized in vesicles (endosomes / lysosomes). The inset shows the pinocytic uptake of particles. The scale bar represents 1 μm. (H) Fusogenic liposome-coated particles are localized in the cytoplasm. The scale bar represents 500 nm. (I) Fusogenic liposome-coated particles are localized in the cytoplasm. The scale bar represents 1 μm.

[0016] Figure 5A-B Shown are confocal microscopy of Neuro2a cells after 1 hour incubation with particles; (A) Fusogenic liposomes show DiI signal in the cell membrane, indicating fusogenic uptake; (B) Non-fusogenic liposomes show DiI signal in the cytoplasm as distinct focal points, indicating endocytic uptake.

[0017] Figure 6 Shown are confocal microscopy images of HeLa cells incubated with fusogenic liposome-coated particles for up to 8 hours.

[0018] Figure 7 Shown are confocal microscopy images of HeLa cells incubated with non-fusogenic liposome-coated particles for up to 8 hours.

[0019] Figure 8A-B shows (A) mouse survival rate after infection on day 0 and after injection of therapeutic agents (PBS, NF-siIRF5-MTP, F-siLuc-MTP, and F-siIRF5-MTP) on day 1. Each group has n = 6 mice. (B) Mean survival days of mice after infection on day 0 and after injection of therapeutic agents on day 1. Error bars represent standard deviation. One-way ANOVA and Tukey's HSD post hoc test (α = 0.05) revealed significant differences between F-siIRF5-MTP and three control groups (PBS, NF-siIRF5-MTP, and F-siLuc-MTP).

[0020] Figure 9 Shown are month-long observations of the average hydrodynamic diameters of fusogenic liposome-coated calcium silicate porous silicon nanoparticles (F-CapSi) and RVG-conjugated fusogenic liposome-coated calcium silicate porous silicon nanoparticles (RVG-F-CapSi) by DLS.

[0021] Figure 10 Shown are the absorbance (dashed line) and emission (solid line) spectra of calcein loaded in fusogenic liposome-coated calcium silicate porous silicon nanoparticles (F-Ca-pSi) and uncoated calcium silicate porous silicon nanoparticles (Ca-pSi).

[0022] Figure 11 Shown are the photoluminescence spectra obtained during the reaction time of pSiNPs and CaCl2 (or MgCl2) solution.

[0023] Figure 12 Confocal microscopy of Neuro2a cells and HeLa cells after incubation with particles is shown; upper left panel: Neuro2a cells after 1 hour incubation with fusogenic liposome-coated particles loaded with the lipophilic fluorescent dye DiI; upper right panel: HeLa cells after 8 hours incubation with fusogenic liposome-coated particles loaded with the cell-impermeable dye calcein; lower left panel: Neuro2a cells after 1 hour incubation with non-fusogenic liposome-coated particles loaded with the lipophilic fluorescent dye DiI; lower right panel: HeLa cells after 8 hours incubation with non-fusogenic liposome-coated particles loaded with the cell-impermeable dye calcein.

[0024] Figure 13A-BConfocal microscopy of Neuro2a cells after 1 hour incubation with particles is shown; (A) RVG-conjugated fusogenic liposomes (RVG-F) show successful targeting of RVG to cells and high levels of fusion staining; (B) Fusogenic liposomes without RVG conjugation (F) show fusion at a relatively lower level compared to RVG-conjugated particles.

[0025] Figure 14 Shown are siRNA gene knockdown results from incubation of particles in Neuro2a mouse neuroblastoma cells for 48 hours. F-Ca-pSi-siPPIB: fusogenic liposome-coated calcium silicate porous silicon nanoparticles loaded with siRNA targeting PPIB; NF-Ca-pSi-siPPIB: non-fusogenic liposome-coated calcium silicate porous silicon nanoparticles loaded with siRNA targeting PPIB; F-Ca-pSi-siLuc: fusogenic liposome-coated calcium silicate porous silicon nanoparticles loaded with siRNA targeting luciferase.

[0026] Figure 15A-K Figure 2 shows FACS analysis of calcein accumulation in homogenized Staphylococcus aureus-infected Balb / C lungs. (A) PBS compared to NF-pSi loaded with calcein (Cal) and conjugated with a targeting peptide (MTP); (B) PBS compared to F-pSi loaded with calcein without a targeting peptide. (C) PBS compared to F-pSi loaded with calcein and conjugated with MTP. (DK) Unstained healthy or infected lung tissue sections imaged for fluorescence detection of DiI-loaded particles. DETAILED DESCRIPTION

[0027] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a well" includes a plurality of such wells, and reference to "the antigen" includes reference to one or more antigens known to those skilled in the art, and so forth.

[0028] Additionally, the use of "or" means "and / or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0029] It should be further understood that while the term "comprising" is used in the description of various embodiments, those skilled in the art will appreciate that in some specific cases, the language "consisting essentially of" or "consisting of" may alternatively be used to describe an embodiment.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, devices, and materials are described herein.

[0031] The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors of the present application are not entitled to antedate such publication by virtue of prior disclosure.

[0032] Gene delivery systems for in vivo therapy remain a challenge due to low efficiency or cytotoxicity. Endocytosis is the primary uptake pathway for most nanoplatforms, which leads to lysosomal degradation of genetic material and low therapeutic efficacy.

[0033] Porous silicon and silicon oxide have been studied as the candidate drug delivery carrier material in many applications because of their inorganic and biodegradable character.Porous nanostructures can accommodate therapeutic agents, diagnostic agents or other useful substances (sometimes referred to as "payloads" herein).However, the early release of these payloads before or after using may be undesirable for the intended purpose.In addition, porous silicon or porous silicon oxide have been degraded under aqueous conditions for continuous drug delivery, in vivo or in vitro imaging and biosensor applications in the site except the target site, and this can benefit from the protective coating around the porous silicon core.

[0034] Lipid-based nanoparticles were first developed three decades ago as The liposomal formulation of vincristine sulfate for acute lymphoblastic leukemia is approved by the Food and Drug Administration (FDA) in the form of (liposomes encapsulating doxorubicin). It was recently approved in 2012, and several paclitaxel and cisplatin formulations, such as EndoTag-1 and SPI-077, are in clinical trials. The reason for this continued investment in liposomal formulations is due to their major advantages: biocompatibility, ease of synthesis, and surface modifiability.

[0035] Lipid nanoparticles are difficult to deliver genetic material due to low loading efficiency and high lysosomal degradation rate. The lipid nanoparticles of load small interfering RNA (siRNA) are taken up by cells via clathrin-mediated endocytosis and macropinocytosis / micropinocytosis in the case of proton pump, mTOR and cathepsin activation. After uptake, after the lipid comprising nanoparticles is regulated by C1 type Niemann-Pick protein (Niemann-Pick type C1, NPC1) in the late endosome / lysosome stage and is recycled, about 70% of the internalized siRNA is exocytosed from the cell. Only 1%-2% of siRNA can escape from the early endosome to the cytoplasm. Therefore, the delivery system that bypasses the lipid recycling in the late endosome / lysosome is crucial in achieving high therapeutic efficacy.

[0036] Preparation of nanoporous silicon, microporous silicon, mesoporous silicon and macroporous silicon by electrochemical means or chemical means is already mature, and the material is partially or completely converted into corresponding nanoporous silicon oxide, microporous silicon oxide, mesoporous silicon oxide and macroporous silicon oxide.In addition, liposomes are known and recognized in the art.

[0037] The present disclosure provides a biodegradable liposomal porous silicon (pSi) nanoparticle system that can bypass endocytic uptake via liposome-plasma membrane fusion (see e.g. Figure 1A ). Membrane fusion allows hydrophilic payloads to be released directly from the core of the liposome into the cytoplasm and hydrophobic molecules to be transferred from the liposome bilayer to the cell membrane bilayer. In addition, the liposomal porous silicon nanoparticle system allows the conjugated moieties (antibodies, small molecules, peptides, etc.) on the outer surface of the liposome to be transferred to the cell membrane surface. The porous silicon core has photoluminescent properties, thereby allowing these particles to be used as tracking tools by using time-gated luminescence imaging. In addition, porous silicon allows highly anionic genetic material to condense into small clusters, thereby allowing liposomes to easily encapsulate particles and any payload contained in the pores of the particles. The present disclosure confirms that fusion liposome-coated pSi particles fuse with cell membranes and transfer various payloads into cells. For example, the present disclosure confirms that fusion liposome-coated pSi particles successfully fuse with Neuro2a mouse neuroblasts and transfer lipophilic DiI dye from the liposome membrane to the cell membrane. In contrast, non-fusion pSi particles are found in small groups in cells that indicate endosomal uptake and lysosomal localization. Furthermore, surface conjugation of the Neuro2a targeting moiety, rabies virus glycoprotein (RVG), allowed for an accelerated fusion rate of liposomal pSi. Overall, liposomal porous silica nanoparticles demonstrated potential as highly efficient delivery vehicles.

[0038] As used herein, the terms "microparticles" or "nanoparticles," "microparticles and / or nanoparticles," "LPSiNPs," and "pSiNPs" refer to porous silicon materials that are at least partially comprised of silicon dioxide and have a size range from a few nanometers to hundreds of micrometers. Typically, the size ranges from about 10 nm to 20 nm to 1 micrometer. The geometric shape of the porous silicon material / particles can be spherical, elliptical, square, rectangular, cubic, etc.

[0039] It should be understood that, as used herein, "porous silicon oxide" refers to a material containing silicon and oxygen of the general stoichiometric formula SiOx, where x can be as small as 0.01 and as large as 2, and that "porous silicon" refers to a material composed of elemental silicon (in its crystalline or amorphous state) having a surface containing hydrogen-containing, oxygen-containing, or carbon-containing species. As used herein, the terms "porous silicon" or "porous silicon oxide" refer to a material containing micropores (pore diameters typically less than about 2 nm), mesopores (pore diameters typically in the range of about 2 nm to 50 nm), or macropores (pore diameters greater than about 50 nm), or a combination of any two or all three pore types. Furthermore, it should be understood that the surface of a porous material, including the surface of the interior pore walls, can contain hydrogen-containing, oxygen-containing, or carbon-containing species.

[0040] The present disclosure provides porous silicon microparticles and / or nanoparticles (pSiNPs) that can carry one or more molecules to be delivered to cells or tissues. The molecules can be diagnostic and / or therapeutic agents. In some embodiments, the molecules are anticancer agents, anti-inflammatory agents, small molecule drugs, peptides, polypeptides, nucleic acids (e.g., siRNA), and the like.

[0041] Furthermore, in contrast to many micro- and nanomaterials (e.g., carbon nanotubes (CNTs), gold nanoparticles (GNs), and quantum dots (QDs), pSiNPs degrade into renal clearance components in a relatively short period of time with little or no signs of toxicity. Furthermore, in contrast to many bioderived delivery systems, nanoparticles alone (without added activating complexes or molecules) do not induce an immune response.

[0042] Porous silicon microparticles and / or nanoparticles containing desired payloads (e.g., nucleic acids, peptides, small molecules, etc.) can be encapsulated into liposomes (fusogenic or non-fusogenic). Liposomes can be modified to have target specificity or can be unmodified (see, e.g., FIG1 ).

[0043] Therefore, the present disclosure provides a kind of biodegradable porous microstructure and / or nanostructure, it comprises the silicon material that is encapsulated in liposome vesicle.In one embodiment, described silicon material comprises silicon dioxide material.In another embodiment, described silicon material comprises silicon material and silicon dioxide material both.In another embodiment, described biodegradable / biocompatible porous nanostructure comprises the particle size of about 0.01 μm to 1 μm.In another embodiment again, described biodegradable / biocompatible porous structure can be characterized as nontoxic.In another embodiment again, described porous silicon material is loaded with " effective load " material.Described effective load material can be medicine, small molecule, diagnostic agent, therapeutic agent, peptide, antibody, antibody fragment, polypeptide, nucleic acid (such as siRNA) etc.

[0044] The present disclosure also provides a method for preparing porous silicon particles, the method comprising: (1) electrochemically etching a silicon wafer to produce a porous structured film; (2) peeling the porous structured film from the silicon wafer substrate; (3) fragmenting the porous film to produce microparticles and / or nanoparticles having a size of 10 to 1000 nanometers; and (4) activating the structure in an aqueous solution. In one embodiment, the aqueous solution comprises pure water. In one embodiment, the aqueous solution comprises sodium hydroxide, hydrogen peroxide, or a borate.

[0045] For example, porous silicon nanoparticles (pSiNPs) can be prepared as described by Qin et al. (Part. Part. Syst. Charact. 31(2): 252-256, 2014; the disclosure of which is incorporated herein by reference). Briefly, porous silicon is prepared by constant current anodic etching of crystalline silicon wafers. Perforations along the etched plane are introduced by short periodic pulses of high current during long periods of low current etching, resulting in alternating layers of high porosity and low porosity. The porous silicon layer is removed from the wafer by applying low current density pulses in dilute HF aqueous solution, and the resulting free-standing film is broken up by ultrasonic treatment. This produces porous nanoparticles with a predetermined porosity and average pore size. The tunability of porosity and pore size can be used to determine the efficiency of subsequent payload loading processes.

[0046] In embodiments with a payload, the porous silicon particles can be placed in an aqueous solution containing the payload and a 1 M or greater concentration of, for example, calcium chloride (CaCl2) solution. The solutions are mixed and purified by centrifugation to produce porous silicon nanoparticles coated with calcium silicate loaded with the payload (Ca-pSiNP-carried). As a control, porous silicon nanoparticles coated with calcium silicate without a payload (Ca-pSiNP) can be prepared in the same manner as described above, but without the added payload solution. Figure 1B 、 Figure 2-Figure 3The loading of cargoes by calcium silicate-coated porous silicon nanoparticles was demonstrated.

[0047] It will be apparent to those skilled in the art that other embodiments can be used to produce Ca-pSiNPs, one example involving replacing the electrochemical etching for producing a porous silicon core with a chemical dye etch, and another example utilizing a porous silicon core prepared by chemically reducing nanostructured silicon oxide. The dye etch uses silicon powder instead of silicon wafers as a silicon precursor, and uses a chemical oxidant instead of a power source to drive the electrochemical reaction. In some cases, it may be desirable to replace calcium chloride with calcium nitrate, calcium nitrite, calcium gluconate, or other calcium ion sources. Calcium nitrate or calcium nitrite can oxidize porous silicon faster than calcium chloride due to the oxidizing properties of nitrate and nitrite ions.

[0048] In one embodiment, the porous silicon particles may be luminescent. The present disclosure provides a method for producing luminescent porous Si nanoparticles (LPSiNPs). The method comprises applying about 200 mA / cm in an aqueous HF / ethanol electrolyte. 2 The p-type silicon wafer was electrochemically etched at a constant current density of 100 nm. The p-type silicon wafer was then electrochemically etched by applying about 4 mA / cm in an aqueous HF / ethanol electrolyte. 2 The resulting free-standing porous silicon nanostructured membrane is removed from the crystalline silicon substrate by a current pulse of 100 s.h.i.+1 .h.i.+2 ...

[0049] In another embodiment, LPSiNP materials can be produced by first creating a silicon layer with a pore size range of 2 nm to 100 nm (e.g., 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, etc.). The silicon layer is etched down to a single crystal silicon substrate in an HF ethanol solution. The entire porous nanostructure is removed from the Si substrate by applying a current pulse. The free-standing hydrogen-terminated porous silicon membrane is then placed in an aqueous solution and broken into multi-sized particles by, for example, overnight sonication. If desired, the particles can then be filtered (e.g., through a 0.22 μm porous filter or other size separation device) to obtain porous silicon nanoparticles. For example, separation or size control of the LPSiNPs can be achieved by passing the colloidal suspension through a physical filter, by centrifuging the suspension, by electrophoresis, by size exclusion chromatography, or by electrostatic precipitation. The nanoparticles are incubated in an oxidizing aqueous solution to activate their luminescence.

[0050] The activation of luminescence is carried out in aqueous solution. During activation, silicon oxide grows on the surface of hydrogen-terminated porous silicon, thereby producing significant luminescence due to quantum confinement effects and defects located at the Si / SiO2 interface. The preparation conditions of the nanoparticles can be optimized to provide a pore volume and surface area suitable for loading therapeutic agents and a desired in vivo circulation time, while maintaining an acceptable degradation rate.

[0051] The thickness, pore size, and porosity of a given membrane are controlled by the current density, the duration of the etching cycle, and the composition of the etchant solution. Furthermore, porous silicon membranes can be used as templates to create imprints of biologically compatible or bioresorbable materials. The porous silicon membrane or its imprint has a sinusoidally varying porosity gradient, providing a sharp feature in the optical reflectivity spectrum that can be used to monitor the presence or absence of chemicals trapped in the pores.

[0052] For in vivo applications, it is often desirable to prepare porous Si in the form of particles. The porous layer can be removed from the Si substrate using a procedure commonly referred to as "electropolishing" or "stripping". The etching electrolyte is replaced with an etching electrolyte containing a lower concentration of HF and a current pulse is applied for a few seconds. The lower concentration of HF causes a diffusion-limited situation, which removes silicon from the crystalline Si / porous Si interface at a speed faster than the pores can propagate. The result is an undercut of the porous layer, which is released from the Si substrate. The independent porous Si film can then be removed with tweezers or severe rinsing. The film can then be converted into microparticles by ultrasonic disruption. If particles with a more uniform shape are desired, conventional photolithography or droplet patterning methods can also be used.

[0053] The ability to easily adjust pore size and pore volume during electrochemical etching is a unique property of porous Si that is very useful for drug delivery applications. Other porous materials generally require more complex design schemes to control pore size, and even then, the pore sizes that can be obtained tend to span a limited range. Using electrochemically prepared porous Si, control over porosity and pore size is obtained by adjusting the current setting during etching. Generally, greater current density produces larger pores. Macropores are desirable when relatively large molecules or drugs are incorporated into the pores. Pore size and porosity are not only important for drug loading; it also determines the degradation rate of the porous Si host matrix.

[0054] Smaller pores provide greater surface area and expose more sites to attack by aqueous media. Smaller porous filaments within the membrane result in a greater dissolution rate, thus providing a convenient means to control the degradation rate of the porous Si host.

[0055] Due to its high surface area, porous Si is particularly susceptible to oxidation by air or water. Once oxidized, nanophase SiO2 readily dissolves in aqueous media, and surfactants or nucleophiles accelerate the process. Si-O bonds are readily formed on porous Si by oxidation, and a variety of chemical or electrochemical oxidants can be used. Thermal oxidation in air tends to produce relatively stable oxides, especially if the reaction is carried out at temperatures >600°C. Ozone oxidation, typically performed at room temperature, forms more hydrated oxides that dissolve rapidly in aqueous media.

[0056] Slow oxidation of the porous Si surface by dimethyl sulfoxide (DMSO) - when combined with dissolution of the newly formed oxide by HF - is a mild means of enlarging the pores in the porous Si membrane. Aqueous solutions of bases such as KOH can also be used to enlarge the pores after etching. Electrochemical oxidation, in which porous Si samples are anodized in the presence of a mineral acid such as H2SO4, produces a fairly stable oxide. Oxidation imparts hydrophilicity to the porous structure, thereby enabling the incorporation and adsorption of hydrophilic drugs or biomolecules within the pores. In the presence of Ca 2+ Liquid-phase oxidation in the presence of various ions produces calcified forms of porous Si that have been shown to be bioactive and particularly interesting for in vivo applications. Calcification can be enhanced by applying a DC current.

[0057] The nanoparticles of the fusion liposome coating of the present disclosure provide devices and methods for drug delivery and tissue and disease (e.g., tumor) monitoring. Depending on the type of the patient's condition to be treated, tissue, or cancer, the nanoparticle compositions of the drug delivery fusion liposome coating can include many drug candidates. The drug candidate can be "physically" trapped in the hole of the silicon particles, or the hole itself can be chemically modified to combine the drug candidate. Such medicine can include peptides, polypeptides, small molecule reagents, nucleic acids, and combinations thereof in a general sense.

[0058] More precisely, "physical entrapment" is analogous to building a boat in a bottle, where the "boat" is the drug candidate and the "bottle" is the nanoscale pores within the porous Si particles. Small molecules can be entrapped within the porous matrix by oxidizing the porous Si surrounding the molecule. Because oxidation of silicon adds two oxygen atoms per Si atom to the material, the volume of the matrix increases significantly after oxidation. This has the effect of expanding the pore walls and reducing the free volume within the pores. Under appropriate conditions, molecules present in the pores during oxidation are entrapped within the oxide matrix. One embodiment of the entrapment process is the increase in concentration of the active ingredient that occurs during the entrapment process. Crystals can present a negatively charged environment, and active ingredients—such as proteins and other drugs—can be concentrated within the crystals to levels much higher than the free concentration of the active ingredient in solution. Upon association with the crystals, this can increase the active ingredient concentration by 10- to 100-fold or more. Oxidation can be performed at repeated intervals by performing layered oxidation. For example, biologics or drugs can be entrapped within the pores through the controlled addition of an oxidizing agent. Oxidation of freshly prepared (hydride-terminated) porous Si materials results in an effective reduction in the size of the pores. This occurs because the silicon oxide formed has a larger volume than the Si starting material. If the drug is also present in the solution containing the oxidant, the drug is trapped in the pores. Furthermore, porous silicon oxide can contain higher concentrations of biological agents or drugs than non-oxidized silicon hydride materials.

[0059] The free volume in porous Si membranes is typically 50% to 80%. Oxidation should reduce this value slightly, but the free volume is expected to still be quite high. Most current drug delivery materials are dense solids and can deliver small weight percentages of drugs.

[0060] Various methods for loading molecular payloads into porous Si hosts have been investigated, and they can be divided into the following general categories: covalent attachment, physical entrapment, and adsorption.

[0061] Covalent attachment provides a convenient means to attach biomolecule capture probes to the inner pore walls of porous silicon for biosensor applications, and this approach can also be used to attach drug molecules, peptides, etc. As described elsewhere herein, attaching biomolecules via Si-C bonds tends to be a more stable approach than using Si-O bonds because Si-O species are susceptible to nucleophilic attack.

[0062] One of the more common approaches is to graft an organic molecule containing a carboxyl group onto the distal end of a terminal olefin. The olefin terminus participates in a hydrosilylation reaction, thereby bonding to the Si surface and leaving the carboxyl terminus free for further chemical modification. One such linker molecule is undecylenic acid, which provides a hydrophobic 10-carbon aliphatic chain to isolate the linker from the porous Si surface. The drug payload can be directly attached to the carboxyl group of the olefin, or it can be further separated from the surface using a PEG linker. Due to the stability of the Si-C bond, hydrosilylation is a good way to attach the payload to porous Si. The payload is only released when the covalent bond breaks or the supporting porous Si matrix degrades.

[0063] In yet another embodiment, electrostatic adsorption, essentially an ion exchange mechanism that holds molecules more weakly, can be used. Electrostatics is a useful means of achieving more rapid drug delivery, as opposed to covalent or physical entrapment methods that release drugs over a period of days, weeks, or months.

[0064] The affinity of porous Si particles for specific molecules can be controlled using surface chemistry. The surface of oxidized porous Si has a point of zero charge at a pH of about 2, so it presents a negatively charged surface to most aqueous solutions of interest. At the appropriate pH, porous SiO2 spontaneously adsorbs positively charged proteins such as serum albumin, fibrinogen, protein A, immunoglobulin G (IgG), or horseradish peroxidase, concentrating them in the process.

[0065] The calcium silicate porous silica core can be used to provide the dual functions of concentrating anionic genetic payloads with high loading efficiency and emitting photoluminescence to allow particle tracking. Calcium silicate is cationic, which allows strong electrostatic interactions with anionic nucleotides to form stable clusters of genes and particles. Another aspect of calcium silicate pSiNPs is their rapid dispersion and degradation in the intercellular environment when the liposome coating is shed through membrane fusion after uptake. Delayed separation or degradation of the concentrate from the nucleotide payload can lead to excretion of the entire cluster because the cell recognizes the foreign material as inaccessible. Data have shown that fusogenic pSi particles containing a calcium silicate core rapidly degrade in the cytoplasm without liposome protection and lose photoluminescence. On the other hand, non-fusogenic particles remain intact within endosomes and lysosomes, maintaining pSi photoluminescence. The rapid degradation of calcium silicate pSiNPs allows the release of, for example, siRNA into the cytoplasm for RNA interference and gene silencing.

[0066] Porous Si can also be made hydrophobic, and hydrophobic molecules, such as the steroid dexamethasone or serum albumin, can be loaded into these nanostructures. Hydrophilic molecules can also be loaded into such materials with the help of appropriate surfactants. The natural hydride surface of porous Si is hydrophobic. Such techniques have been used for short-term loading and release. Because water is excluded from these hydrophobic surfaces, water degradation and leaching reactions tend to be slow. Grafting alkanes onto surfaces by hydrosilylation is often used to prepare materials that are stable in biological media; this stability is largely due to the ability of the hydrophobic moiety to locally exclude water or dissolved nucleophiles.

[0067] Other drugs (e.g., cargo) or "active ingredients" that can be used with the porous silicon particles of the present disclosure include, but are not limited to, any one or any combination of the following: anti-angiogenic compounds, such as bevacizumab, ranibizumab, pegaptanib, and other compounds in the angiogenic cascade. Anti-cancer drugs, such as chemotherapeutic compounds and / or their derivatives (e.g., 5-fluorouracil, vincristine, vinblastine, cisplatin, doxorubicin, adriamycin, tamoxifen, etc.). Also included are glucocorticoids, such as dexamethasone, triamcinolone acetonide, fluocinolone acetonide, and other similar compounds in the corticosteroid and cortisone families. Also included are compounds such as antacids, anti-inflammatory substances, coronary vasodilators, cerebral vasodilators, peripheral vasodilators, anti-infectives, psychotropics, antimanics, stimulants, antihistamines, laxatives, decongestants, vitamins, gastrointestinal sedatives, antidiarrheal preparations, antianginals, vasodilators, antiarrhythmics, antihypertensives, vasoconstrictors and migraine treatments, anticoagulants and antithrombotics, analgesics, antipyretics, hypnotics, sedatives, antiemetics, antinauseants, anticonvulsants, neuromuscular drugs, hyperglycemic and hypoglycemic agents, thyroid and antithyroid preparations, diuretics, antispasmodics, uterine relaxants, minerals and nutritional supplements, antiobesity drugs, anabolic drugs, erythropoiesis-stimulating drugs, antiasthmatics, bronchodilators, expectorants, antitussives, mucolytics, drugs affecting calcification and bone turnover, and anti-uricotic drugs. Specific medications include gastrointestinal sedatives such as metoclopramide and propantheline bromide; antacids such as aluminum trisilicate, aluminum hydroxide, ranitidine, and cimetidine; anti-inflammatory drugs such as phenylbutazone, indomethacin, naproxen, ibuprofen, flurbiprofen, diclofenac, dexamethasone, prednisone, and prednisolone; and coronary vasodilators such as glyceryl trinitrate, isosorbide dinitrate, and pentaerythritol tetranitrate.Peripheral and cerebral vasodilators, such as soloctidilum, vincamine, naftidrofuryl oxalate, co-dergocrine mesylate, cyclandelate, papaverine, and niacin; anti-infectives, such as erythromycin stearate, cephalexin, nalidixic acid, tetracycline hydrochloride, ampicillin, flucloxacillin sodium, hexamine mandelate, and phenoxyethanol. mandelate, and methenamine hippurate; neuroleptics such as flurazepam, diazepam, temazepam, amitryptyline, doxepin, lithium carbonate, lithium sulfate, chlorpromazine, thioridazine, trifluperazine, fluphenazine, piperothiazine, haloperidol, maprotiline hydrochloride, imipramine, and desmethylimipramine; central nervous system stimulants such as methylphenidate, ephedrine, epinephrine, isoproterenol, amphetamine sulfate, and amphetamine hydrochloride. hydrochloride); antihistamines, such as diphenhydramine, diphenylpyraline, chlorpheniramine, and brompheniramine; antidiarrheals, such as bisacodyl and magnesium hydroxide; laxatives, such as sodium succinate; nutritional supplements, such as ascorbic acid, alpha-tocopherol, thiamine, and pyridoxine; antispasmodics, such as dicyclomine and diphenoxylate;Medications that affect heart rhythm, such as verapamil, nifedipine, diltiazem, procainamide, disopyramide, bretylium tosylate, quinidine sulfate, and quinidine gluconate; medications used to treat high blood pressure, such as propranolol hydrochloride, guanethidine monosulphate, methyldopa, oxprenolol hydrochloride, captopril, and hydralazine; medications used to treat migraines, such as ergotamine; medications that affect blood clotting, such as epsilon-aminocaproic acid and protamine sulfate; and pain relievers, such as acetylsalicylic acid, acetaminophen, and codeine phosphate. phosphate), codeine sulfate, oxycodone, dihydrocodeine tartrate, oxycodeinone, morphine, heroin, nalbuphine, butorphanol tartrate, pentazocine hydrochloride, cyclazacine, pethidine, buprenorphine, scopolamine, and mefenamic acid; anticonvulsants such as phenytoin sodium and sodium valproate; neuromuscular drugs such as dantrolene sodium; substances used to treat diabetes, such as tolbutamide, disbenase, glucagon, and insulin; and drugs used to treat thyroid dysfunction, such as triiodothyronine, thyroxine, and propylthiouracil;Diuretics, such as furosemide, chlorthalidone, hydrochlorothiazide, spironolactone, and triamterene; uterine relaxants, such as ritodrine; appetite suppressants, such as fenfluramine hydrochloride, phentermine, and diethylproprion hydrochloride; and antiasthmatics and bronchodilators, such as aminophylline, theophylline, salbutamol, orciprenaline sulfate, and terbutaline sulfate. expectorants, such as guaiphenesin; cough suppressants, such as dextromethorphan and noscapine; mucolytics, such as carbocisteine; antiseptics, such as cetylpyridinium chloride, tyrothricin, and chlorhexidine; decongestants, such as phenylpropanolamine and pseudoephedrine; hypnotics, such as dichloralphenazone and nitrazepam; and antinauseants, such as promethazine. theoclate); hematopoietic agents such as ferrous sulfate, folic acid, and calcium gluconate; uricosuric agents such as sulfinpyrazone, allopurinol, and probenecid; and agents that affect calcification, such as bisphosphonates, e.g., etidronate, pamidronate, alendronate, residronate, teludronate, clodronate, and alondronate.

[0068] Within the scope of the present disclosure, which encompasses an almost unlimited number of drugs, in vitro pharmacokinetic studies can be used to determine the appropriate configuration of porous silicon particles for each drug. Once delivered to a subject, the drug-conjugated silicon particles can be monitored. For example, the light intensity from the luminescent silicon nanoparticles (LPSiNPs) can be measured using a low-power spectrophotometer. Using such methods, the half-life, delivery, and collection of drugs and / or LPSiNPs can be monitored.

[0069] Luminescence spectra for particle identification can be easily measured using inexpensive and portable instruments such as CCD spectrometers or diode laser interferometers. Removal of drug from LPSiNPs can cause a change in their luminescence, which is a wavelength shift in the spectrum. Such techniques can be used to enable noninvasive sensing through opaque tissue.

[0070] In any of the foregoing embodiments, silicon particles carrying a cargo (e.g., a load or in combination with a drug or reagent) or containing no pharmaceutical agent and / or wherein the particles are luminescent or non-luminescent particles are encapsulated in liposomes suitable for fusion with cell membranes (e.g., fusogenic liposomes). In addition, these liposomes can be modified or unmodified. If modified, the modification can include a targeting moiety as further described herein.

[0071] Fusion liposome preparation can be prepared by many known lipids.Liposomal vesicle can be made up of multiple lipids, and wherein liposome has the diameter of 10nm to 500nm or any diameter therebetween (for example 20nm-400nm, 50nm-300nm, 60nm-250nm, 100nm-200nm, 120nm-180nm, 140nm-160nm etc.). In another embodiment, liposome is unilamellar liposome or micelle. In another embodiment, liposome is multilamellar liposome. In another embodiment, multiple lipid comprises the phospholipid selected from the following: phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, lysophosphatidylcholine and / or any derivative thereof. In yet another embodiment, the phospholipid derivative is selected from 1,2-bis-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphocholine, 1,2-didecanoyl-sn-glycero-3-phosphocholine, 1,2-dierucoyl-sn-glycero-3-phosphate, 1,2-dierucoyl-sn-glycero-3-phosphocholine, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1 ,2-Dilauroyl-sn-glycerol-3-phosphate, 1,2-Dilauroyl-sn-glycerol-3-phosphocholine, 1,2-Dilauroyl-sn-glycerol-3-phosphoethanolamine, 1,2-Dilauroyl-sn-glycerol-3-phospho-(1'-racemic-glycerol), 1,2-Dimyristoyl-sn-glycerol-3-phosphate, 1,2-Dimyristoyl-sn-glycerol-3-phosphocholine, 1,2-Dimyristoyl-sn-glycerol-3-phosphoethanolamine amine, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, 1,2-dimyristoyl-sn-glycero-3-phosphoserine, 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, L-α-phosphatidyl-DL-glycerol, 1,2-dioleoyl-sn-glycero-3-phosphoserine, 1,2-dipalmitoyl-sn-glycero- 3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoserine, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-2-Distearoyl-sn-glycero-3-phosphoglycerol, egg sphingomyelin, egg PC, hydrogenated egg PC, hydrogenated soybean PC, 1-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, In another embodiment, the liposome further comprises cholesterol. In another embodiment, the liposome further comprises polyethylene glycol. For example, in one embodiment, a fusogenic liposome formulation can be prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) at a molar ratio of 76.2:3.8:20. For experimental controls, a non-fusogenic formulation can be synthesized in the same manner, but at a molar ratio of 80:0:20 (i.e., lacking PEG). DSPE-PEG(methoxy) can be replaced with carboxylic acid- (DSPE-PEG(carboxyl)) or maleimide- (DSPE-PEG(maleimide)) functionalized PEG lipids at the same molar ratio for further surface modification, such as with a targeting moiety. For example, the use of DSPE-PEG (carboxyl) or DSPE-PEG (maleimide) can be used for modification with antibodies or targeting peptides, respectively. In another embodiment, the liposomes further comprise one or more site-targeting moieties. Examples of site-targeting moieties include, but are not limited to, peptides, aptamers, antibodies, and antibody fragments (e.g., F(ab')2, Fab, and scFv).

[0072] The term "lipid" refers to any suitable material that produces a bilayer so that the hydrophobic portion of the lipid material is oriented toward the bilayer, while the hydrophilic portion is oriented toward the aqueous phase. Amphiphilic lipids are used as the main structural elements of lipid vesicles. The hydrophilicity characteristic is derived from the presence of phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro, and other similar groups. Hydrophobicity can be imparted by including the following groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted by one or more aromatic groups, alicyclic groups, or heterocyclic groups. Typical amphiphilic compounds are phosphoglycerides and sphingolipids, and their representative examples include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine, which can be used. Other compounds lacking phosphorus, such as the sphingolipid and glycosphingolipid families, also fall within the group designated as lipids. Additionally, the amphiphilic lipids described above may be mixed with other lipids including triglycerides and sterols.

[0073] The term "neutral lipid" refers to any of a number of lipid substances that exist in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, and cerebrosides.

[0074] The term "non-cationic lipid" refers to any neutral lipid as described above as well as anionic lipids. Examples of anionic lipids include cardiolipin, diacylphosphatidylserine and diacylphosphatidic acid.

[0075] The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol, and DMRIE. In addition, many commercial preparations of cationic lipids are available that can be used in the present disclosure. These carriers are improved by adding PEG-modified lipids, particularly PEG-modified ceramide lipids. Adding PEG-modified lipids prevents particle aggregation and provides a means for extending circulation life and increasing delivery of lipid-nucleic acid particles to target cells. In addition, it has been found that cationic lipids are more likely to fuse with target cells and, therefore, the addition of electrically neutral PEG-modified ceramide lipids does not mask or reduce the positive charge of the carrier liposomes.

[0076] Exemplary lipids that can be used to formulate liposomes include, but are not limited to:

[0077]

[0078] 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);

[0079]

[0080] 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP);

[0081]

[0082] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG2000(methoxy));

[0083]

[0084] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000(maleimide));

[0085]

[0086] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG2000(carboxy)).

[0087] In general, lipids dissolved in chloroform are mixed and the solvent is evaporated to produce a dry film. In one embodiment in which a hydrophobic payload is embedded in the liposome membrane, the hydrophobic payload solution is mixed with the lipid solution to be dried together to form a film. The fused liposome membrane is hydrated by gently pipetting using a porous silicon core that entraps the payload. The mixture will become turbid to indicate liposome formation. In some embodiments, the mixture can be heated at about 40°C for 10 minutes and then mechanically extruded, for example, through a polycarbonate membrane with 200 nm pores 20 times. Excess payload is washed off by centrifugation three times with deionized water. Figure 1B -D shows a schematic diagram of the synthesis of liposomal porous silica nanoparticles. Figure 1E Schematic diagrams depicting fusogenic liposomal porous silicon microparticle and / or nanoparticle compositions of the present disclosure.

[0088] Typically, the liposome coating comprises a fusogenic lipid and a moiety. For example, DMPC can be used as the structural backbone of liposomes having a relatively low phase transition temperature (T m =24°C). The low transition temperature provides L α Liquid crystal phase. αThe phase is a more fluid, dynamic and permeable structure that allows for a wide size range of extruded liposome coatings (100 nm-400 nm) and easier fusion potential. DOTAP is a cationic lipid that can be used for electrostatic attraction to anionic plasma membranes. PEGylated lipids can also be used for fusion; although the exact mechanism of action of PEG is unknown, it is hypothesized that PEG electrostatically binds water molecules to dehydrate the lipid head groups, which induces structural asymmetry in the lipid arrangement and drives bilobal fusion into unilobal fusion as an energetically favorable pathway, similar to how SNARE proteins anchor the plasma membrane and pull vesicles to fuse endogenously with the plasma membrane; indeed, neuronal SNAREs have been observed to promote PEG-mediated fusion.

[0089] It is further contemplated herein that the liposome layer encapsulating the silicon particles can be adapted for site targeting by tethering targeting moieties (peptides, aptamers, antibodies, antibody fragments, sugars or glycolipids) to or within the liposome layer, which can selectively direct the silicon particles and their cargo to the desired site, thereby promoting local drug delivery and therapeutic effects. For example, various reactive groups can be used to tether targeting groups to the lipids comprising the liposomes disclosed herein, such as sulfhydryl reactive groups, maleimides, haloacetyl groups, pyridyl disulfides, thiosulfonates, and vinyl sulfones; carboxyl to amine reactive groups, such as carbodiimides (e.g., EDC); amine reactive groups, such as NHS esters, imido esters, pentafluorophenyl esters, hydroxymethylphosphine; aldehyde reactive groups, such as hydrazides and alkoxyamines; photoreactive groups, such as diazines and aryl azides; and hydroxyl (non-aqueous) reactive groups, such as isocyanates.

[0090] The targeting moiety associated with the surface of the liposome can range from a small hapten of about 125-200 Daltons to a hapten of at least about 6 kD, but generally less than 10 6 The kD molecular weight of the liposomes may vary from that of the much larger antigens. Protein ligands and receptors are of particular relevance. Since the agent / cargo incorporated into the silica particles contained in the liposomes can be indiscriminate with respect to the cell type on which it acts, the targeted delivery system offers a significant improvement over random injection of nonspecific liposomes.

[0091] A number of procedures can be used to covalently link polyclonal or monoclonal antibodies to the liposome bilayer. Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof, such as scFv, Fab, or F(ab')2, as long as they effectively bind to an antigenic epitope on the target cell.

[0092] As mentioned above, the present disclosure also provides embodiments in which the nanoparticles coated with fusion liposomes include targeting moieties / molecules. Targeting moieties / molecules can include, but are not limited to, ligands or antibodies (including antibody fragments) that specifically bind to their corresponding targets, such as receptors or antigens on the cell surface. Therefore, for example, when the targeting molecule is an antibody or its fragment, the nanoparticles coated with fusion liposomes will specifically bind (target) cells and tissues with the epitope to which the antibody or antibody fragment is directed. Therefore, targeting molecules generally refer to all molecules that can react with receptors or polypeptides (such as their homologous pairs) on target cells or otherwise recognize or bind to the receptors or polypeptides. Any known ligand or targeting molecule can be used. There are many examples of targeting peptides that can be manipulated and cloned or connected to produce nanoparticles coated with fusion liposomes in the literature. In general, any peptide ligand or a fragment thereof based on the receptor binding sequence of the ligand can be used. In immunology, such a peptide domain is referred to as an epitope, and the term epitope can be used herein to refer to a ligand that a receptor can recognize. For example, a ligand comprises a sequence of a protein or peptide that is recognized by a binding partner on the surface of a target cell, which is referred to as a receptor for convenience. However, it should be understood that for the purposes of this disclosure, the term "receptor" encompasses signal transduction receptors (e.g., receptors for hormones, steroids, cytokines, insulin, and other growth factors), recognition molecules (e.g., MHC molecules, B cell receptors, or T cell receptors), nutrient uptake receptors (e.g., transferrin receptors), lectins, ion channels, adhesion molecules, extracellular matrix binding proteins, etc., which are located and accessible at the surface of the target cell.

[0093] Various cell types can be targeted. For example, antigen presenting cells (APCs), which include leukocytes, can be targeted by preparing fusogenic liposome-coated nanoparticles containing targeting molecules that recognize targets on APCs. In operation, the fusogenic liposome-coated nanoparticles bind to the target, fuse with the membrane, are internalized by the cell, and release the contents of the nanoparticles into the cell. Examples of receptors that can be targeted or used as targeting moieties include, e.g., receptors for or receptors for E-selectin, CD3, CD 4, CD8, CD11, CD 14, CD 34, CD 123, CD 45Ra, CD64, E-cadherin, ICAM-1, interleukins, interferons, tumor necrosis factor, E-cadherin, Fc, MCH, CD 36 and other integrins, chemokines, macrophage mannose receptor and other lectin receptors, B7, CD 40, CD 50, CD 80, CD 86 and other co-stimulatory molecules, Dec-205, scavenger receptors, and toll receptors, see also Guermonprez et al. (Annu. Rev. Immunol., 2002).

[0094] Various embodiments provided herein generally relate to systems and methods for producing drug delivery devices that can deliver cargo to treat or diagnose various diseases or conditions, including viral and bacterial infections, cancers, tumors, and other cell proliferative diseases and conditions, inflammatory diseases and conditions, and tissue damage. In addition, the present disclosure provides immunological techniques that enhance drug delivery or promote drug action or improve immunogen processing in conjunction with silicon nanoparticles of the present disclosure. Such methods may include activating dendritic cells and other inflammatory cells and stimulating an immune response.

[0095] Compositions comprising liposomes encapsulating silicon nanoparticles and / or microparticles can be formulated for enteral delivery, parenteral delivery, topical delivery, or inhalation.The liposome-containing silicon particles of the present disclosure can be formulated for in vitro and in vivo administration using techniques known in the art.

[0096] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration can include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and an agent for regulating tension, such as sodium chloride or dextrose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0097] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to administer by syringe. It must be stable under the conditions of manufacture and storage and must prevent contamination by microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is useful to include an isotonic agent in the composition, such as a sugar, a polyol such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0098] Sterile injectable solutions can be prepared by mixing the desired amount of a liposomal silicon particle composition, such as a composition disclosed herein, into an appropriate solvent along with one or a combination of the ingredients listed above, as needed, followed by filtration sterilization. In general, dispersions are prepared by mixing the active compound into a sterile vehicle containing a basic dispersion medium and the other ingredients required (from those listed above). In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0099] In a specific embodiment, one or more liposome formulations of the present disclosure are prepared using a carrier that will protect the compound from rapid elimination in the body, such as a controlled release formulation, including the use of polyethylene glycol, implants, and microencapsulation delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. It will be apparent to those skilled in the art that the method for preparing such preparations should be readily apparent. The material can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc.

[0100] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. A dose can be formulated in animal models to achieve an IC determined in cell culture. 50The present invention also provides a method for determining the circulating plasma concentration range of the test compound (e.g., the concentration of the test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. The methods and compositions of the present disclosure are applicable to a wide range of species, such as humans, non-human primates, horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.

[0101] The liposomal silica particle compositions and formulations disclosed herein, including pharmaceutical compositions comprising the formulations, can be used to treat a variety of diseases or conditions requiring the delivery of peptides, small molecules, nucleic acids (eg, siRNA) into cells.

[0102] The following working examples are provided to illustrate but not to limit the present invention. Various parameters of the scientific methods used in these examples are described in detail below and generally provide guidance for practicing the present invention.

[0103] Example

[0104] Fusion silicon nanoparticle synthesis.

[0105] A lipid solution of a mixture of DMPC:DSPE-PEG:DOTAP at a molar ratio of 76.2:3.8:20 was used, and 21 μL of 1.25 mg / ml (2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindole-1- -2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole (DiI) was used to prepare fusogenic liposome membranes.

[0106] Porous silicon nanoparticles (pSiNPs) were prepared by electrochemically etching single-crystalline silicon wafers in HF ethanol aqueous solution, followed by removal of the porous layer and ultrasonication.

[0107] The liposome mixture was dried into a film and hydrated with a solution of porous silicon or porous silicon nanoparticles deposited from calcium silicate. The hydrated suspension was heated to 40°C for 20 minutes under magnetic stirring and mechanically extruded twenty times through a polycarbonate membrane with 200 nm pores.

[0108] Figure 1B -H shows a schematic diagram of particle synthesis and characterization data. Scattering and microscopy data of fusogenic liposome-coated pSi (F-pSi) confirmed a hydrodynamic diameter of approximately 190 nm. Figure 1E -F shows particle characterization by transmission electron microscopy (TEM).

[0109] Drugs are loaded into porous silicon particles.

[0110] The siRNA effective load is loaded into the pSi core cluster via calcium silicate sealing chemical method and reaches the loading efficiency of about 20 % by weight.The nano platform of other load oligonucleotide, as nanoparticle based on lipid and mesoporous silica-polymer hybrid system have the average loading efficiency (table 1) of 1 % by weight-14 % by weight.Specifically, in these materials, the particle of 200nm that size is suitable can only load less than 5 % by weight.Therefore, the fusion porous silicon particle of the present disclosure demonstrates the increase of oligonucleotide loading efficiency four times, and this can then enhance cell gene to knock down efficiency.

[0111]

[0112]

[0113] In vitro uptake behavior of fusion particles

[0114] Neuro2a cells were incubated with fusogenic and non-fusogenic liposomes for 1 hour and visualized under a confocal microscope. Fusogenic particles transferred lipophilic DiI from the liposome membrane to the plasma membrane to stain the cell outline, while non-fusogenic liposomes were found in distinct groups in the cytoplasm, characteristic of endosomal or lysosomal compartmentalization.

[0115] Make particle load hydrophilic dye (calcein) or lipophilic dye (DiI) to evaluate the difference of dye location and therefore particle uptake (Fig. 4).Select calcein as model siRNA effective load, this is because it is anionic dye that can not pass cell membrane usually, is very similar to siRNA behavior.On the other hand, use DiI as the mode of confirming fusion uptake, this is because DiI is the lipophilic dye that is loaded into liposome bilayer.If particle merges when uptake, then DiI will be transferred from liposome bilayer and diffuse into plasma membrane bilayer.

[0116] F-pSi loaded with DiI in the liposome membrane successfully transferred DiI to the cell membrane and dispersed the calcein signal throughout the cytoplasm, indicating uptake via membrane fusion. In addition, minimal photoluminescence signal was detected from pSiNPs, due to rapid degradation after fusion without liposome protection. On the other hand, non-fusogenic liposome pSi (NF-pSi) loaded with DiI or calcein were located in dense clusters within the cytoplasm, indicating endocytosis. In addition, the calcein signal co-localized with the pSi photoluminescence signal in concentrated spots. The pSi signal indicates that due to the protection of the liposome, the core remains intact and is in the endosome / lysosome together with the calcein dye. Since membrane fusion did not occur, we assume that the liposome-coated pSi particles are endocytosed.

[0117] Using LysoTracker red, the lysosomal intracellular compartment is stained, as shown in Figure 4 (b) and (e). Fusion particles and non-fusion particles are loaded with calcein and applied to cells. The results show that when fusion particles show no dispersed calcein signal co-located with lysosomes, non-fusion particles show that calcein is co-located with the lysosomal compartment. In addition, fusion particles and non-fusion particles of the load DiI conjugated to macrophage targeting peptide (MTP) are prepared and macrophages are processed. MTP is also labeled with 6-FAM dye to allow fluorescence monitoring. It is shown that fusion particles fuse along the specific part of the cell membrane, wherein the MTP-FAM signal is also co-located, which means that MTP-FAM successfully anchors particles to specific macrophage membrane receptors to allow for localization fusion. It is also worth noting that MTP seems to accelerate the fusion process by making particles quickly anchor the cell membrane surface, because only half the incubation time is needed to achieve a considerable fusion level. Non-fusion particles conjugated with MTP-FAM show the same positioning as the same particles without MTP. The MTP-FAM signal and the DiI signal colocalized in clusters within the cytoplasm, indicating endosomal and lysosomal compartmentalization.

[0118] Particles loaded with cell-impermeable dyes for cytoplasmic staining

[0119] Calcein is an anionic cell-impermeable fluorescent dye. Due to its strong negative charge, it can be trapped in porous silicon nanoparticles using the calcium chloride interaction identical with the nucleic acid payload. Porous silicon particles are placed in an aqueous solution containing calcein and 1M or greater concentrations of CaCl2 solution. The solution is mixed and purified by centrifugation to obtain porous silicon nanoparticles or pSiNPs (Ca-pSi-calcein) coated with calcium silicate loaded with calcein. Unloaded calcein is washed off by centrifugation three times in deionized water.

[0120] HeLa cells were incubated with fusogenic and non-fusogenic liposome-coated particles and visualized under a confocal microscope at the following time intervals: 30 minutes, 1 hour, 2 hours, 4 hours, and 8 hours. The fusogenic liposome-coated particles were observed to release calcein directly into the cytoplasm and disperse it throughout the cells. The absence of red luminescence signal from the porous silicon indicates that it rapidly dissolves in the intracellular environment due to loss of liposome coating protection due to fusogenic uptake ( Figure 6 In contrast, non-fusogenic liposome-coated particles colocalized with calcein signal at focal points in the cytoplasm. These observations suggest that non-fusogenic liposome-coated particles are endocytosed whole into cells and that maintenance of the liposome coating around the porous silicon-based core prevents core dissolution and luminescence loss ( Figure 7 ).

[0121] In vivo gene knockdown and therapeutic effects

[0122] Infected mice were tested with different formulations to test the effectiveness of IRF5 knockdown as an antibacterial treatment and fusion. Six-week-old female Balb / C mice were infected intratracheally with Staphylococcus aureus bacteria 24 hours before intravenous tail vein injection of saline (PBS), targeted non-fusion particles loaded with therapeutic siRNA (NF-siIRF5-MTP), targeted fusion particles loaded with control non-therapeutic siRNA (F-siLuc-MTP), or targeted fusion particles loaded with therapeutic siRNA (F-siIRF5-MTP). The survival rate of mice was observed for 7 days after treatment and recorded to obtain Figure 8A The results shown in .

[0123] Figure 8B The mean survival days from each treatment group are shown, along with statistical significance. From one-way ANOVA and post hoc comparisons using the Tukey HSD test, data showed that mice injected with F-siIRF5-MTP particles had significantly higher mean survival days than all other formulations, i.e., PBS, NF-siIRF5-MTP, and F-siLuc-MTP (p level < 0.05, [F(3,20) = 8.78, p = 0.001]).

[0124] In all treatment groups, mice that survived beyond 4 days displayed normal healthy behaviors and had no obvious outward signs of distress or illness.

[0125] Fusion silicon particle stability

[0126] The stability of the particles in phosphate buffered saline (PBS) was observed by measuring the change in hydrodynamic diameter via DLS for 28 days ( Figure 9 Fusogenic liposome-coated particles (F-CapSi) were extruded to an initial average hydrodynamic diameter of approximately 140 nm. Conjugation of RVG with maleimide-terminated PEG (RVG-F-CapSi) resulted in an increase in size to approximately 180 nm. This size remained highly stable for the first 7 days, followed by a gradual increase over the next 3 weeks.

[0127] Antibody-conjugated particles

[0128] The antibody can be conjugated to the surface of the liposome by interacting with a similar PEG used for targeting peptide conjugation. The fusion liposome membrane is made of a mixture of DMPC: DSPE-PEG (carboxyl): DOTAP in a 76.2: 3.8: 20 molar ratio. The mixture is dried into a film and hydrated with a porous silicon nanoparticle solution deposited with porous silicon or metal (Ca) silicate. The hydrated suspension is heated to 40°C under magnetic stirring for 20 minutes and mechanically extruded twenty times through a polycarbonate membrane with 200 nm pores. The amine groups on the lysine residues in the Fc region of the antibody are conjugated to carboxyl-terminated PEG to form amide bonds via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide / sulfo-N-hydroxysuccinimide (EDC / sulfo-NHS) chemistry.

[0129] To verify cellular uptake via membrane fusion, the lipophilic (2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindole-1- The liposome membrane was loaded with a fluorescent dye containing [-2-yl] prop-2-enyl]-3,3-dimethyl-1-octadecyl indole (DiI). Neuro2a cells were treated with the particles and incubated for 1 hour for cellular uptake. Fusogenic particles were able to successfully transfer the lipophilic DiI from the liposome membrane to the plasma membrane, while particles coated with non-fusogenic liposomes were found in a different group in the cytoplasm, which is characteristic of endosomal / lysosomal uptake ( Figure 12 , left picture).

[0130] Subsequently, the differences in the intracellular localization of hydrophilic payloads between fusogenic and non-fusogenic liposome coating were investigated. The highly anionic cell-impermeable dye calcein was loaded into the porous silica core using calcium chloride entrapment and encapsulated in both fusogenic and non-fusogenic liposomes. Figure 10 Shown are the absorbance and fluorescence spectra of calcein loaded in liposome-coated particles.Liposome encapsulation of calcein loaded particles allows for dequenching of fluorescence emission.

[0131] The photoluminescence of calcium silicate coated particles was also studied; either calcium silicate / silicon oxide shells or magnesium silicate / silicon oxide shells showed a strong ability to passivate the surface of silicon nanostructures, resulting in an increase in the intrinsic photoluminescence from the material. Figure 11Photoluminescence spectra obtained at different times during the reaction between pSiNPs and CaCl2 (or MgCl2) solution are shown. During the reaction, the intensity of the photoluminescence gradually increases, which is due to the passivation of non-radiative carrier traps on the pSiNP surface. In addition, the peak wavelength of the photoluminescence shows a significant blue shift as the reaction progresses. Both phenomena (increase in photoluminescence intensity and blue shift in the photoluminescence spectrum) indicate the growth of a passivating surface layer on the silicon nanocrystals. The observed blue shift is a typical feature of quantum confined nanoparticles, whose emission wavelength is strongly dependent on size and is expected to blue shift as the quantum confined silicon domains become smaller. Because it is closely related to the host silicon matrix, the intrinsic photoluminescence of the nanoconstructs can be used to monitor the degradation of the matrix and, by inference, the release of the payload in in vitro or in vivo experiments.

[0132] HeLa cells were treated with particles and visualized over time under a confocal microscope. Figure 12 The right figure above shows the cellular uptake of the particles after 8 hours of incubation. The fusogenic liposome-coated particles were observed to release the calcein payload into the cytoplasm and disperse them throughout the cells. The lack of luminescent signal from the calcium silicate-coated porous silicon suggests that it rapidly dissolves in the intracellular environment due to the lack of protection from the fusogenic liposomes. In contrast, the non-fusogenic liposome-coated particles co-localize with the calcein signal at concentrated points in the cytoplasm. The observations suggest that the non-fusogenic liposome-coated particles are endocytosed into the cell in their entirety and that the maintenance of the liposome coating around the porous silicon-based core prevents the core from dissolving.

[0133] Targeting peptide-conjugated particles

[0134] Rabies virus glycoprotein (RVG, (CCGG)YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 1)) is a Neuro2a mouse neuroblast targeting peptide that was conjugated to liposome-coated maleimide-terminated PEG via a maleimide-thiol / cysteine ​​covalent binding interaction. To visualize the interaction with cells under confocal microscopy, the RVG peptide was labeled with 5-FAM dye and the liposome membrane was labeled with lipophilic DiI. Targeting peptide conjugation did not appear to affect the fusogenicity of the liposome coating, and only an accelerated fusion and uptake rate was observed compared to particles coated with fusogenic liposomes without the targeting peptide.

[0135] siRNA-loaded particles for gene knockdown

[0136] Small interfering RNA (siRNA) cargo was loaded into the porous silicon core by mixing the porous silicon particles with siRNA dissolved in deionized water and 3M CaCl2 and washing by centrifugation.

[0137] For siRNA gene knockdown test in Neuro2a cells, peptidyl prolyl isomerase B (PPIB / siPPIB) was selected as the target gene ( Figure 14 siPPIB-loaded F-pSi showed approximately 95% gene knockdown efficiency at 100 nM. In comparison, the common transfection agent Lipofectamine achieved only 90% gene knockdown efficiency under similar conditions. Non-fusion control nanoparticles, NF-pSi, showed only 30% gene knockdown at 100 nM, demonstrating high variability.

[0138] In vivo macrophage targeting and infection homing

[0139] Balb / C mice were infected intratracheally with Staphylococcus aureus to induce lung infection. Macrophage / monocyte targeting peptides were connected to the F / NF-pSi loaded with calcein and DiI, and the preparations were administered to mice infected with Staphylococcus aureus via intravenous injection to observe the targeting efficacy of macrophages and the homing of macrophages to the infected lungs (Figure 15). The lungs of the mice injected with the particles of the harvested mice loaded with calcein were homogenized for the accumulation of calcein in the infected lungs by FACS quantification, as shown in Figure 15 (ac). The lungs of mice injected with DiI loaded particles were fixed for fluorescence histological evaluation, as shown in Figure 15 (dk). Although the targeting peptide was connected, NF-pSi did not show obvious homing to the lungs in the FAC data of the loaded calcein and the fluorescence histological evaluation of the loaded DiI. F-pSi without the targeting peptide showed minimal accumulation in both assessments, whereas F-pSi with the targeting peptide showed clear targeting to macrophages and subsequent homing to infected lungs in both FACS and fluorescence histology. Therefore, the macrophage targeting peptide is essential for the homing of nanoparticles to macrophages in vivo.

[0140] Using MTP targeting peptide conjugation as described herein, nanoparticles loaded with calcein or DiI were able to successfully home to infected lungs, whereas particles without the MTP peptide and non-fusogenic particles were unable to do so. Healthy lungs also did not show significant homing due to fewer recruited macrophages. Having validated MTP peptide homing in infected lungs, the therapeutic efficacy of formulations loaded with therapeutic siIRF5 was tested.

[0141] This disclosure demonstrates that the fusion liposome-encapsulated pSi system is able to bypass endocytosis to achieve greater gene knockdown efficacy than non-fusion formulations. Furthermore, in vivo targeting of macrophages / monocytes was successfully demonstrated, and macrophages / monocytes delivered with payloads effectively homed to infected lungs. In the case of delivering M1 phenotype inhibitory siRNA to macrophages, the data show that the fusion system can achieve improved infection and effective immunogen clearance, likely due to higher phagocytic activity, reduced inflammation, and tissue healing.

[0142] A number of embodiments of the present invention have been described. However, it will be appreciated that various modifications may be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the following claims. Sequence Listing <110> Regents of the University of California <120> Fusogenic liposome-coated porous silica nanoparticles <130> 00015-293WO1 <140> Not yet allocated <141> 2016-07-08 <150> US 62 / 190,705 <151> 2015-07-09 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 33 <212> PRT <213> rabies virus <400> 1 Cys Cys Gly Gly Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Pro Gly 1 5 10 15 Thr Pro Cys Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Asn 20 25 30 Gly

Claims

1. A method for preparing porous silicon nanoparticles coated with fusogenic liposomes, which comprises the following steps: (a) Electrochemically etching a crystalline silicon wafer to produce a film with a porous structure; (b) Stripping the film with the porous structure from the crystalline silicon wafer; (c) Fragmenting the film with the porous structure to produce a silicon-containing core with a nanostructure having a plurality of pores and microparticles and / or nanoparticle cores with a size of 10 nanometers to 1000 nanometers; (d) Activating the silicon-containing core in an aqueous solution containing a plurality of cargo molecules and metal salts; and (e) Encapsulating the silicon-containing core with fusogenic lipids, wherein the fusogenic lipids comprise 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and polyethylene glycolated lipids; and wherein a metal silicate forms a shell on the silicon-containing core.

2. The method according to claim 1, wherein the silicon-containing core is oxidized silicon.

3. The method according to claim 1 or 2, wherein the metal salt forms a metal silicate comprising calcium silicate or magnesium silicate.

4. The method according to claim 1, wherein there is no gap between the silicon-containing core and the metal silicate shell.

5. The method according to claim 1, wherein the plurality of pores are chemically or physically configured to accommodate cargo molecules.

6. The method according to claim 1, wherein the silicon-containing core is chemically oxidized, and wherein the chemical oxidation combined with the formation of the metal silicate shell physically entraps the plurality of cargo molecules in the plurality of pores.

7. The method according to claim 1, wherein the cargo molecules are drug molecules.

8. The method according to claim 1, wherein the cargo molecules are non-drug substances.

9. The method according to claim 1, wherein the silicon-containing core of the nanoparticles comprises molecules that are physically adsorbed or covalently entrapped in the plurality of pores or attached to the plurality of pores, and wherein the silicon-containing core of the nanoparticles is coated with a shell containing calcium or magnesium, and the shell is formed by the action of an aqueous solution of calcium ions or magnesium ions added to the core in the presence of the molecules.

10. The method according to claim 1, wherein the fusogenic lipids comprise a mixture of DMPC, DOTAP and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-methoxy).

11. The method according to claim 1, wherein the fusogenic lipids comprise a mixture of DMPC, DOTAP and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-carboxy).

12. The method according to claim 1, wherein the fusogenic lipids comprise a mixture of DMPC, DOTAP and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide).

13. The method according to claim 1, wherein the silicon-containing core has a hydrodynamic diameter in the range of 10 nm - 100 nm.

14. The method according to claim 1, wherein the fused liposome has a hydrodynamic diameter in the range of 100 nm - 400 nm.

15. The method according to claim 1, wherein the targeting molecule is conjugated to the fused lipid.

16. The method according to claim 1, wherein the antibody is conjugated to the fused lipid.

17. The method according to claim 1, wherein the molecule to be carried is a hydrophobic molecule to be carried.

18. The method according to claim 1, wherein the molecule to be carried is a hydrophilic molecule to be carried.

19. The method according to claim 1, wherein the molecule to be carried is a nucleic acid.

20. The method according to claim 1, wherein the molecule to be carried is a small molecule.

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