US20160038608A1 - Silica-based mesoporous carrier and delivery method of using the same - Google Patents

Silica-based mesoporous carrier and delivery method of using the same Download PDF

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US20160038608A1
US20160038608A1 US14/819,438 US201514819438A US2016038608A1 US 20160038608 A1 US20160038608 A1 US 20160038608A1 US 201514819438 A US201514819438 A US 201514819438A US 2016038608 A1 US2016038608 A1 US 2016038608A1
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Prior art keywords
targets
carriers
silica nanoparticles
mesoporous silica
cell
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US14/819,438
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Chung-Yuan Mou
Yi-Ping Chen
Si-Han Wu
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National Taiwan University NTU
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National Taiwan University NTU
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Priority to US14/819,438 priority Critical patent/US20160038608A1/en
Assigned to NATIONAL TAIWAN UNIVERSITY reassignment NATIONAL TAIWAN UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHEN, YI-PING, MOU, CHUNG-YUAN, WU, Si-han
Priority to AU2016303039A priority patent/AU2016303039B2/en
Priority to CN201680046902.6A priority patent/CN108368303A/zh
Priority to CA2994809A priority patent/CA2994809C/en
Priority to EP16833417.5A priority patent/EP3331947A4/en
Priority to US15/750,759 priority patent/US11666662B2/en
Priority to PCT/US2016/014194 priority patent/WO2017023358A1/en
Priority to JP2018506167A priority patent/JP2018533543A/ja
Priority to TW105103067A priority patent/TWI611812B/zh
Publication of US20160038608A1 publication Critical patent/US20160038608A1/en
Priority to IL257337A priority patent/IL257337A/en
Abandoned legal-status Critical Current

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    • A61K47/48869—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A61K47/48015—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/163—Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx

Definitions

  • the present invention generally relates to a mesoporous carrier, in particular, to silica-based mesoporous carriers and the delivery methods by using the silica-based mesoporous carriers.
  • porous materials can be classified as microporous materials having pore sizes of less than 2 nm, mesoporous materials having pore sizes of 2-50 nm and macroporous materials having pore sizes of greater than 50 nm.
  • the size of the pores and the large surface area of the pores allow the mesoporous materials to be ideal vehicles for carrying chemicals or drugs.
  • mesoporous carriers for delivering targets into a cell.
  • the mesoporous carriers comprise hollow silica nanospheres (HSN) or mesoporous silica nanoparticles (MSN) and the targets bound to or encapsulated by the hollow silica nanospheres or the mesoporous silica nanoparticles.
  • the targets includes first targets and second targets, and the first and second targets are different.
  • the targets may include peptides, proteins, enzymes and/or enzymatic mimetics.
  • a method of delivering targets into a cell is proposed.
  • mesoporous carriers are prepared and provided.
  • the mesoporous carriers comprises hollow silica nanospheres (HSN) or mesoporous silica nanoparticles (MSN) and the targets bound to or encapsulated by the hollow silica nanospheres or the mesoporous silica nanoparticles.
  • the targets include first targets and second targets, and the first and second targets are different.
  • the mesoporous carriers contact with the cell by incubating the cell with the mesoporous carriers.
  • the first targets and the second targets are co-delivered into the cell at the same time, as the mesoporous carriers and the targets carried by the hollow silica nanospheres or the mesoporous silica nanoparticles enter into the cell.
  • FIGS. 1A-1B show the reaction schemes for the PEI-modification of the enzymes SOD and CAT and the encapsulation of PEI-SOD and PEI-CAT within HSN.
  • FIG. 2 shows the adsorption and desorption isotherms of HSN according to one embodiment of the present invention.
  • FIGS. 3A-3D show transmission electron microscopy (TEM) images of various enzyme encapsulated HSN.
  • FIG. 4A shows the relative enzyme activity for different enzyme-encapsulated HSN.
  • FIG. 4B shows the fluorescence intensity over the wavelength for different enzyme-encapsulated HSN.
  • FIG. 5 shows the cell viability results using WST-1 assay when exposed to HSN or PEI-SOD/CAT@HSN.
  • FIG. 6 shows the quantification of fluorescence intensity of oxidized DHE from HeLa cells were treated with various enzyme encapsulated HSN.
  • FIG. 7 describes the reaction scheme for the conjugation of NF- ⁇ B p65 antibody and Cys-TAT peptide to the surface functionalized MSN.
  • FIGS. 8A-8D show transmission electron microscopy (TEM) images of various functionalized MSN nanoparticles.
  • FIG. 9A-9C show the results of in vitro pull-down assay of various functionalized MSN nanoparticles.
  • FIG. 10A shows the conjugation of FMSN-PEG/PEI nanoparticles.
  • FIG. 10B shows the TEM images of FMSN-PEG/PEI nanoparticles.
  • FIGS. 11A-11C show the protection effects of co-delivery of TAT-SOD and TAT-GPx into Hela cells.
  • silica-based mesoporous carrier materials are described.
  • the silica-based mesoporous materials may be classified as hollow silica nanospheres (HSN) and mesoporous silica nanoparticles (MSN) and the targets may be carried on the surface of the silica-based mesoporous materials or be encapsulated within the silica-based mesoporous materials.
  • HSN hollow silica nanospheres
  • MSN mesoporous silica nanoparticles
  • the targets may be carried on the surface of the silica-based mesoporous materials or be encapsulated within the silica-based mesoporous materials.
  • the ingredients, the reaction conditions or parameters illustrated in the examples are merely for illustration purposes, but it is not intended to limit the material or the preparation method by the exemplary embodiments described herein.
  • the target suitable for being carried by the silica-based mesoporous materials may include an enzymes containing cysteine (thiol group), lysine (amino group), aspartate or glutamate (carboxyl group), a peptide containing cysteine (thiol group), lysine (amino group), aspartate or glutamate (carboxyl group) or an antibody containing cysteine (thiol group), lysine (amino group), aspartate or glutamate (carboxyl group).
  • the target suitable for being carried by the silica-based mesoporous materials may include an enzymes containing polyhistidine-tag (His-tag), a peptide containing polyhistidine-tag or an antibody containing polyhistidine-tag.
  • the polyhistidine-tag consists of at least six histidine (His) residues.
  • hollow silica nanospheres with porous silica shells and large interior spaces (cavities) have been synthesized, which is suitable for loading enzymes within the cavities of HSN for intracellular catalysis.
  • a microemulsion method has been developed for synthesizing hollow silica nanospheres (HSN), of which one or more enzymes may be loaded within the cavities.
  • the enzymes may be antioxidant enzymes, including horseradish peroxidase (HRP), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, glutathione reductase and their enzymatic mimetics.
  • the enzymes may be enzymes involved in biochemical enzymatic cascades, which refers to a series of biochemical reactions involving enzymes, such as blood coagulation, metabolism pathways, and signal transduction pathways.
  • HSN may be synthesized by silica sol-gel process of water-in-oil microemulsion, and polyethyleneimine (PEI) modified enzymes in aqueous phase are then encapsulated inside HSN. It has been demonstrated that encapsulation of HRP in the cavities of HSN (HRP@HSN) is feasible and the intracellular delivery of HRP@HSN showing its function as a catalytic nanoreactor inside Hela cells for converting prodrug into toxic agent to kill the cancer cells.
  • PEI polyethyleneimine
  • PEI grafted superoxide dismutase (PEI-SOD) and catalase (PEI-CAT) are prepared and then co-encapsulated within HSN as the loaded enzymes.
  • FIGS. 1A-1B describe the reaction schemes for the PEI-modification of the enzymes (SOD and CAT) and the encapsulation of the positively charged PEI-SOD and PEI-CAT within HSN.
  • SOD and CAT the enzymes
  • the silica shell of HSN shields off PEI from contacting with cellular machineries, but the pores of the silica shells allow protons to diffuse inside the hollow spheres of HSN and keep the proton sponge effects of PEI.
  • FIG. 1A the PEI-enzyme conjugation is achieved through the amidation of the carboxyl group with PEI.
  • FIG. 1B illustrates the synthesis of PEI-SOD and PEI-CAT co-encapsulated HSN (denoted as PEI-SOD/CAT@HSN).
  • PEI is covalently linked to the enzymes by the conventional EDC/NHS coupling reaction.
  • PEI-grafted enzymes (PEI-SOD, PEI-CAT), either individually or together, are encapsulated inside HSN by an one-pot water-in-oil (w/o) microemulsion approach as shown in FIG. 1B .
  • an aqueous solution containing PEI-grafted enzyme is emulsified in an oil system containing surfactant (isooctylphenyl ether, CA-520), co-surfactant (n-hexanol) and organic solvent (decane).
  • surfactant isooctylphenyl ether, CA-520
  • co-surfactant n-hexanol
  • organic solvent decane
  • TEOS tetraethoxysilane
  • APIMS aminopropyltrimethoxy silane
  • aqueous ammonia aqueous ammonia
  • the water-in-oil (w/o) mixture was stirred for 10 h at 20° C. to form enzyme-encapsulated silica nanoparticles.
  • Enzymes-loaded particles were foiiiied after the nanoparticles were further suspended in waiin deionized water (40° C.) for 40 min and washed with water, which is critical for the transformation to hollow nanospheres.
  • SOD and CAT Modification of SOD and CAT with PEI: PEI-conjugation of enzymes was carried using NHS/EDC crosslinking reaction. Briefly, 3 mg of SOD and CAT were dissolved in 50 mM sodium phosphate buffer (pH 7.8) at a concentration of 3 mg/mL. Amine-reactive NHS esters and EDC ⁇ HCl were prepared in 200 ⁇ L sodium phosphate buffer (50 mM, pH 7.8) and then added to the enzyme solution. The SOD solution contained 17 mg NHS/14 mg EDC ⁇ HCl and CAT solution contained 15 mg NHS/13 mg EDC ⁇ HCl.
  • PEI-SOD PEI-SOD
  • CAT PEI-CAT
  • Synthesis of PEI-SOD@HSN, PEI-CAT@HSN, and PEI-SOD/CAT@HSN Hollow silica nanospheres (HSN) were synthesized by a reverse microemulsion method. Typically, 20 mL of decane, 1.63 mL of CA-520, 550 ⁇ L of n-hexanol and 350 ⁇ L of H 2 O with concentrated PEI-enzymes (PEI-SOD, PEI-CAT, or a mixture of PEI-SOD and PEI-CAT) were mixed at room temperature to generate the water-in-oil microemulsion. Then, silica sources (100 ⁇ L of TEOS and 25 ⁇ L of ethanolic APTMS solution) were added with stirring.
  • silica sources 100 ⁇ L of TEOS and 25 ⁇ L of ethanolic APTMS solution
  • the ethanolic APTMS solution was prepared by adding 200 mL of APTMS to 1.4 mL of absolute ethanol. After 10 min, 250 ⁇ L of aqueous ammonia (35 wt %) was introduced to the mixture which was stirred for 10 h at 20° C. Then 95% ethanol was added to destabilize the microemulsion system and solid products were centrifuged at 11000 rpm for 30 min.
  • the samples were suspended in 40 mL warm water (40° C.), stirred for 40 minutes, and isolated by centrifugation to obtain PEI-SOD encapsulated HSN (denoted as PEI-SOD@HSN), PEI-CAT encapsulated HSN (denoted as PEI-CAT@HSN), PEI-SOD/CAT@HSN or HSN only (without adding enzymes). Finally, all nanoparticles were washed several times with ethanol and deionized water to extract the surfactants in the pores.
  • the amount of PEI-enzymes used to prepare enzyme-loaded HSN 36 nmole of PEI-SOD for PEI-SOD@HSN; 4 nmole of PEI-CAT for PEI-CAT@HSN; 36 nmole of PEI-SOD and 4 nmole of PEI-CAT for PEI-SOD/CAT@HSN.
  • FIGS. 3A-3D show transmission electron microscopy (TEM) images of enzyme encapsulated silica nanospheres; FIG. 3A : PEI-SOD/CAT@HSN, FIG. 3B : PEI-SOD/CAT@HSN stained with uranyl acetate (UA); FIG. 3C : PEI-SOD@HSN and FIG. 3D : PEI-CAT@HSN.
  • TEM transmission electron microscopy
  • PEI-SOD The loading efficiency of PEI-SOD (12.2%) and PEI-CAT (8.7%) were much higher than that of SOD (2.8%) and CAT (2.8%). This is due to the positively charged PEI-SOD and PEI-CAT are effective in attracting the negatively charged hydrolyzed silica precursors for encapsulation. The higher loading will improve the enzyme activity for biomedical use and thus PEI-coated enzymes are chosen for further study thereafter.
  • the loading yields of PEI-SOD@HSN and PEI-CAT@HSN were 44.7 and 29.5 ⁇ g enzymes/mg HSN. Compared with single enzyme loading, about the same level of efficiency and loading yields were observed when two enzymes, PEI-SOD and PEI-CAT, were co-encapsulated in HSN.
  • the loading efficiency of PEI-SOD and PEI-CAT were 12.3% and 9.4%.
  • PEI-SOD/CAT@HSN contains about 44.5 and 26.1 ⁇ g enzymes/mg HSN (Table
  • PEI-SOD/CAT@HSN and PEI-SOD@HSN maintain 46.6% and 18.6% of the native SOD activity (Table 2). Also, as seen in Table 2, when compared with the reaction rate of H 2 O 2 decomposition between native CAT and CAT-loaded particles, PEI-SOD/CAT@HSN and CAT@HSN keep 57.9% and 62.7% of the native CAT activity.
  • FIG. 4A shows the stability of CAT-loaded particles and native CAT after treated with KO 2 solution.
  • KO 2 solution which generates 0 2 • ⁇
  • native CAT lost almost all its activity
  • CAT activity of PEI-SOD/CAT@HSN and PEI-CAT@HSN still remained at about 80% and 70%.
  • SOD and CAT are antioxidant enzymes and work together as primary defence system against free radical damage in living cells.
  • PEI-SOD and PEI-CAT could be simultaneously encapsulated in HSN and the final nanoparticle system showed activities in both enzymes.
  • fluorescence assay was performed in which the resorufin formation was recorded by monitoring the fluorescence at 570 nm.
  • the O 2 • ⁇ generated by KO 2 was unstable and converted to H 2 O 2 in the presence or absence of SOD in aqueous solution.
  • the dismutation rate of O 2 • ⁇ in the presence of SOD is faster.
  • H 2 O 2 could be either reduced by CAT or further involved in peroxidase/Amplex red oxidation reaction.
  • the fluorescence intensity of PEI-SOD@HSN was significantly higher than that of blank (control), HSN (negative control), and PEI-SOD/CAT@HSN.
  • the cascade reaction occurs. Hydrogen peroxide produced by SOD could be further decomposed by CAT and the system showed weaker fluorescence.
  • FIG. 4B shows the cascade reactions within PEI-SOD/CAT@HSN.
  • the fluorescence intensity reflects the amount of H 2 O 2 in the assay buffer.
  • the same SOD units of PEI-SOD/CAT@HSN and PEI-SOD@HSN were used for comparison.
  • Cell proliferation assay 2 ⁇ 10 4 cells were seeded in 24-well plates and allowed to attach for 24 h. To determine the particle toxicity, cells were incubated in fresh serum-free medium containing different amounts of BSA-stabilized particles (0-400 ⁇ g/mL) for 2.5 h. After washing twice with phosphate-buffered saline (PBS), particle-treated cells were cultured in regular growth medium. After 24 h, cells were washed twice with PBS and incubated with 200 ⁇ L WST-1 (10%) in Dulbecco's modified eagles medium (DMEM). Cell viability was determined by the fornazan dye generated by the live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680).
  • PBS phosphate-buffered saline
  • DMEM Dulbecco's modified eagles medium
  • FIG. 5 shows the cell viability results for Hela cells exposed to HSN or PEI-SOD/CAT@HSN using WST-1 assay.
  • FIG. 6 shows the quantification of fluorescence intensity of oxidized DHE from HeLa cells were treated with various nanoparticles.
  • the PQ-induced ROS productions are the same for the un-protected cells (positive control and HSN only). All enzyme-nanoparticle treated cells obviously inhibit PQ-induced ROS production.
  • PEI-SOD@HSN and PEI-CAT@HSN show similar levels of reduction of DHE-positive cells by about 25% (compared to HSN only).
  • PEI-SOD/CAT@HSN shows a much higher level of reduction of DHE-positive cells by about 60%.
  • the PEI-SOD/CAT@HSN nanosystem clearly shows a synergetic effect in ROS reduction.
  • DHE-positive cells and fluorescence intensity quantitated indicates that PEI-SOD/CAT@HSN having the synergetic effect of both antioxidant enzymes displays the weakest fluorescence intensity under microscopic observation.
  • the enzyme-loaded particles, especially PEI-SOD/CAT@HSN show better cell attaching morphology, which implies the treatment by PEI-SOD/CAT@HSN detoxify the imposed ROS.
  • the nanoparticle-treated cells after incubation with PQ were further assayed with Western blotting to check the level of p-p38 and COX2 expression.
  • Down-regulation of PQ-induced activation of p-p38 and COX2 is observed for cells pre-treated with PEI-SOD/CAT@HSN, PEI-SOD@HSN and PEI-CAT@HSN.
  • the present invention provides a mesoporous hollow silica nanosphere (HSN) materials (about 40 nm size) based on templating water-in-oil with sol-gel condensation.
  • the enzyme SOD and/or catalase (CAT) can be individually or jointly encapsulated within HSN, with the help of the polyamine PEI.
  • the mesopores on the silica shell allow easy access of small molecules while keeping the enzymes inside the nanosphere from undesirable protein-protein interaction.
  • PEI-SOD and PEI-CAT@HSN can complete cascade transformation of superoxide through hydrogen peroxide to water with synergism.
  • HSN Downstream reactive oxygen species (ROS) production and COX-2/p-p38 expressions show that co-encapsulated SOD/CAT inside HSN give the highest cell protection against the toxicants.
  • ROS reactive oxygen species
  • PEI-SOD/CAT@HSN may be applied as antioxidants for medical treatments, such as inflammation, ischemia, stroke and other strong oxidative stress situation.
  • the sizes of many proteins or enzymes are significantly bigger than the inner space of HSN or MSN or certain proteins are not suitable to be encapsulated within the nanomaterials and the loading efficiency is limited. In this case, it is feasible to link or conjugate the enzymes or proteins to the exterior or outer surface of the nanoparticles or nanomaterials for better delivery efficiency.
  • mesoporous silica nanoparticle (MSN) materials are synthesized and functionalized to carry peptides and/or antibodies.
  • the peptide may be any peptide containing cysteine or a polyhistidine tag, including nucleus localization sequence (NLS)-peptide, cancer-targeting peptide and lysosomal targeting peptide.
  • the antibody may be any antibody containing cysteine or a polyhistidine tag, including signal transduction antibody and cancer-targeting antibody.
  • the present invention provides nanoparticles consisting of mesoporous silica nanoparticle (MSN) with surface functionalization of NF- ⁇ B (nuclear factor-kappa B) p65 antibody and TAT transducing peptide (i.e., HIV trans-activator of transcription (TAT) protein transduction domain).
  • TAT transducing peptide i.e., HIV trans-activator of transcription (TAT) protein transduction domain.
  • TAT transducing peptide i.e., HIV trans-activator of transcription (TAT) protein transduction domain.
  • TAT transducing peptide i.e., HIV trans-activator of transcription (TAT) protein transduction domain.
  • TAT transducing peptide i.e., HIV trans-activator of transcription (TAT) protein transduction domain
  • FIG. 7 describes the reaction scheme for the conjugation of NF- ⁇ B p65 antibody and Cys-TAT peptide to the surface functionalized MSN.
  • amine groups are formed on the surface of MSN by reacting with 3-aminopropyltrimethoxysilane (APTMS) to foim MSN-APTMS with an average loading of nitrogen content of APTMS at 2.6 wt % by elemental analysis.
  • APTMS 3-aminopropyltrimethoxysilane
  • PEG polyethylene glycol
  • MAL maleimide
  • PEG 2k or PEG 3.4k polyethylene glycol (PEG) having an average molecular weight of 2000 or 3400
  • SCM succinimidyl carboxymethyl.
  • the MAL-PEG-SCM crosslinkers containing a succinimidyl moiety react with the amine groups of MSN-APTMS through an active succinimidyl link to obtain the MSN-PEGs (MSN-PEGs: MSN-PEG 2k , MSN-PEG 34k ).
  • the MAL-end of MSN-PEG reacted with the thiol groups of the antibody and Cys-TAT peptide.
  • FITC-APTMS N-1-(3-trimethoxysilyl propyl)-N′-fluoreceylthiourea
  • FITC fluorescein isothiocyanate
  • APIMS 3-aminopropyltrimethoxysilane
  • 200 mg of as-synthesized samples were redispersed in 25 mL of 95% ethanol with 0.5 g of 37 wt % HCl.
  • Surfactant was extracted by heating the ethanol suspension at 60° C. for 24 h.
  • the product, called FITC-MSN (MSN) was collected by centrifugation and washed with ethanol several times and stored in ethanol.
  • MSN The surface of MSN was functionalized with amine groups by treatment with APTMS.
  • MSNs 200 mg were first dispersed in 50 mL of ethanol, and then the solution was refluxed for 18 h after the addition of 500 ⁇ L of APTMS. After centrifugation and washing with ethanol, amine-functionalized MSNs were redispersed in ethanol. To remove the surfactants, the amine-functionalized MSNs were suspended in acidic ethanol (1 g of HCl in 50 mL of EtOH) and refluxed for 24 h. After centrifugation and washing with ethanol, amine-functionalized MSN (MSN-APTMS) were redispersed in ethanol.
  • TEM images were taken using a Hitachi H-7100 instrument with an operating voltage of 75 KV. Samples were sonicated to disperse in ethanol and 10 ⁇ L of the suspension was dropped to fix on a microgrid.
  • the p65 antibody was covalently immobilized with the MAL-end of MSN-PEG 3.4k in different ratios (1:6, 1:12, 1:24) via C-S binding.
  • the Cys-TAT peptide was conjugated to fill up the free MAL-end of MSN-PEG 3.4k .
  • MSN-PEG 3.4k without antibody coupling was also directly conjugated with Cys-TAT as a control.
  • the physical properties of the nanoparticles were characterized by nitrogen adsorption-desorption isotherms, powder X-ray diffraction (XRD), FT-IR, TEM, dynamic light scattering (DLS) and zeta potential.
  • TEM 8A-8D show transmission electron microscopy (TEM) images of various functionalized MSN. From the TEM images, it is shown that the MSN particles possess well-ordered mesoporous structures and the average particle size obtained from TEM images is about 40 nm.
  • the WST-1 assay was applied to measure the cell viability and growth inhibition assay: 2 ⁇ 10 4 HeLa cells per well were seeded in 24-well plates for 16 h for HeLa cell viability assay. HeLa cells were incubated in serum-free medium containing different amounts of MSN-PEG 3.4k -Ab-TAT (100 ⁇ g/L) for 4 h.
  • HNSCC growth inhibition assay HNSCC cells were seeded in 24-well plates with a density of 4 ⁇ 10 4 cells/well for 16 h and incubated with 200 ⁇ g/mL of MSN-PEG 3.4k -Ab(1:24)-TAT, MSN-PEG 3.4k -TAT or anti-TNF antibody (100 ng/mL) in serum-free medium for 4 h. Following medium replacement with culture medium, HNSCC cells were incubated for further 72 h.
  • WST-1 assay HeLa or HNSCC cells were allowed to grow in culture medium containing WST-1 (Clontech) for 4 h at 37° C. The dark red formazan dye generated by the live cells was proportional to the number of live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680).
  • the cell viability of the MSN-PEG 34k -Ab-TAT was examined by using WST-1 assay and MSN-PEG 3.4k -Ab-TAT shows no significant cytotoxicity.
  • PVDF polyvinylidene difluoride
  • MSN-APTMS 100 ⁇ g/mL of MSN-APTMS, MSN-PEG 2k and MSN-PEG 3.4k were treated in HeLa cells for 4 h, and then incubation without or with TNF- ⁇ (50 ng/mL), a NF- ⁇ B activator, for another 0.5 h.
  • TNF- ⁇ 50 ng/mL
  • a NF- ⁇ B activator 50 ng/mL
  • the p65 expression level in either cytosol or nucleus was determined by western blotting experiments.
  • the MSN-PEG 3.4k -Ab-TAT 100 ⁇ g/mL was mixed and incubated with total lysate of HeLa cell at 4° C. for 18 h in vitro. Then, the mixture was centrifuged at 12,000 rpm for 20 mins and the supernatant (10 ⁇ L) was assayed for the free p65 expression level by Western blotting.
  • FIGS. 9A-9C show the results of in vitro pull-down assay of various functionalized MSN nanoparticles.
  • MSN-PEG 3.4k -Ab-TAT blocks NF- ⁇ B p65 nuclear translocation and thus inhibits the NF- ⁇ B p65 downstream protein expression.
  • HeLa cells were treated with MSN-PEG 3.4k -TAT or MSN-PEG 3.4k -Ab-TAT for 4 h at different doses (100 ⁇ ug/mL for FIG. 9B , 50-200 ⁇ g/mL for FIG. 9C ). After the delivery, the cells were stimulated with or without 5Ong/mL TNF- ⁇ for another 0.5 h.
  • Dose-dependence study of the blockage as shown in FIG. 9C indicates that nuclear p65 level decreases with the increasing concentration of MSN-PEG 3.4k -Ab(1:24)-TAT.
  • MSN-PEG 3.4k -Ab(1:12)-TAT and MSN-PEG 3.4k -Ab(1:24)-TAT show obvious suppression of the p65 translocation to nucleus, whereas MSN-PEG 3.4k -TAT did not prevent the TNF- ⁇ inducing nuclear p65 translocation.
  • MSN-PEG 3.4k -Ab-TAT shows the specificity and effectiveness to block NF- ⁇ B p65 nuclear translocation through immunogenic binding.
  • a nanoparticle/antibody complex targeting NF- ⁇ B is employed to catch the Rel protein p65 in perinuclear region and thus blocking the translocation near the nuclear pore gate.
  • TAT peptide conjugated on mesoporous silica nanoparticles (MSN) help non-endocytosis cell-membrane transducing and converge toward perinuclear region, where the p65 specific antibody performed the targeting and catching against active NF- ⁇ B p65 effectively.
  • a protein delivery system combining MSN nanoparticle carriers and one or more denatured fusion proteins has been developed.
  • Such combination of the nanomaterial and one or more fusion proteins not only solves the problems of protein delivery, including chemical solvents, stability, and permeability, but also provide a new approach for protein therapy.
  • TAT-SOD and TAT-GPX protein conjugation we constructed and overexpressed the His-tag human Cu, Zn-superoxide dismutase (SOD) and human glutathione peroxidase (GPx) which contain a human immunodeficiency virus (HIV) transducing domain (TAT, residues 49-57).
  • the sequence of TAT transducing peptide RKKRRQRRR.
  • the genes of TAT-SOD and TAT-GPx were cloned and inserted into prokaryotic protein expression vector of pQE-30 to form pQE-TAT-SOD and pQE-TAT-GPx. The vectors were transformed into JM109 E.
  • TAT-SOD and TAT-GPx with high protein overexpression were displayed in accordance with increasing induction time in 10% SDS-PAGE electrophoresis. Finally, the supernatants of pellets of E. coli crude lysates expressed TAT-SOD or TAT-GPx were tried to further directly conjugate in 8M urea.
  • FITC solution was prepared by dissolving 1 mg of FITC in 5 ml of anhydrous ethanol. 100 L of APTMS was added with rapid stirring at room temperature in darkness for 24 hours. 0.58 g of C 16 TAB was dissolved in 300 g of 0.17 M NH 3 solution, and 5 mL of dilute TEOS solution (5% v/v TEOS/ethanol) was added with stirring for 5 h. FITC-APTMS solution added before 5 ml of concentrate TEOS solution (25% v/v TEOS/ethanol) was added dropwise with vigorous stirring for 1 h. The solution was then aged at 40° C. for 24 hours to complete the silica condensation. As-synthesized products was collected by centrifugation and washed with 95% ethanol three times. The products called FITC-MSN (FMSN) were stored in absolute ethanol.
  • FMSN-NTA-Ni were obtained and stored in absolute ethanol. FMSN-NTA-Ni with an average loading of Ni content is 0.6 wt % by ICP-MS analysis.
  • FITC solution was prepared by dissolving 1 mg of FITC in 5 mL of anhydrous ethanol. 100 of APTMS was added with rapid stirring at room temperature in darkness for 24 hours. 0.58 g C 16 TAB was dissolved in 300 g of 0.17 M NH 3 solution, and 5 mL of dilute TEOS solution (5% v/v TEOS/ethanol) was added with stirring for 5 h. FITC-APTMS solution added before 5 mL of concentrate TEOS solution (25% v/v TEOS/ethanol) was added dropwise with vigorous stirring for 1 h.
  • TEM images were taken on a JEOL JSM-1200 EX II operating at 120 kV.
  • the nickel amount of sample was determined by inductively coupled plasma mass spectrometry (ICP-MS) using Agilent 7700e instrument. Size measurements were performed using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern, UK). Zeta potential was determined by the electrophoretic mobility and then applying the Henry equation on Malven Zetasizer Nano ZS (Malvern, UK).
  • Table 3 shows dynamic light scattering (DLS) data for average particle size of FMSN-PEG/PEI nanoparticles in different solutions.
  • FIG. 10A shows the conjugation of FMSN-PEG/PEI nanoparticles
  • FIG. 10B shows the TEM images of FMSN-PEG/PEI nanoparticles.
  • the TEM images show that these FMSN-PEG/PEI particles possess well-ordered mesoporous structure with an average particle size of about 60-70 nm. DLS-determined size indicates very little aggregation in biological solutions (Table 3).
  • FMSN-PEG/PEI 20 mg was dispersed in 2.5 mL of PBS buffer, and then 6.8 mg of NHS-PEG-MAL(3.4k) was dissolved in 2.5 mL of PBS and then added to FMSN-PEG/PEI solution. The solution was stirred for 2 hours at room temperature.
  • Thiolated Na,Na-Bis(carboxymethyl)-L-lysine hydrate (BCLH) solution was prepared by added 400 ⁇ L of Traut's reagent (100 ⁇ M) and 5.24 mg of Na,Na-Bis(carboxymethyl)-L-lysine hydrate in 5 mL of PBS buffer and stirred for 30 mins.
  • the lysate of E.coli containing His-TAT-SOD or His-TAT-GPx was mixed with FMSN-NTA-Ni at 4° C. overnight. Based on the metal affinity between the Ni (II) and His-tag protein offered a tight linkage with a very low dissociation constant, the FMSN-NTA-Ni was directly mixed with TAT-SOD or TAT-GPx proteins from the supernatants of pellets of E. coli crude lysates under 8M urea without purifying. The protein-conjugated particles were isolated by centrifugation and washed by ethanol. The protein-functionalized particles were denoted as FMSN-TAT-SOD or FMSN-TAT-GPx.
  • samples were prepared in 300 ⁇ L and monitored using a microplate reader (Bio Tek, SynergyTM H1). Firstly, a stock of cocktail reagents contained EDTA (10 ⁇ 4 M), cytochrome c (10 ⁇ 5 M), and xanthine (5 ⁇ 10 ⁇ 5 M) in 1 mL of 50 mM K 3 PO 4 was prepared. Then, 280 ⁇ L of cocktail reagent was added with various samples, xanthine oxidase (10 ⁇ L of 58 mU/mL) and completed with D.I. water up to 300 ⁇ L total volume. Finally, 200 ⁇ L of each sample was transferred to microplate reader and the absorbance at 550 nm was detection.
  • cocktail reagents contained EDTA (10 ⁇ 4 M), cytochrome c (10 ⁇ 5 M), and xanthine (5 ⁇ 10 ⁇ 5 M) in 1 mL of 50 mM K 3 PO 4 was prepared. Then, 280 ⁇ L of cocktail reagent
  • SOD specific activity was expressed as unit per milligram (U/mg) of total lysate proteins (The Journal of Biological Chemistry, 1969, 244, 6049-6055.).
  • GPx activity in HeLa cell was measured using the Glutathione Peroxidase Assay Kit (Cayman Chemical), based on the method of Paglia and Valentine, with hydrogen peroxide as substrate.
  • the method was based on an NADPH-coupled reaction, whereby GPx reduces hydrogen peroxide while oxidizing GSH to GSSG.
  • the generated GSSG is reduced to GSH with consumption of NADPH by GR.
  • Enzyme activity was measured at 340 nm and expressed in units representing oxidation of 1 ⁇ mole NADPH per minute per mL sample.
  • GPX specific activity was expressed as unit per milligram (U/mg) of protein.
  • Cell Viability Assay 3 ⁇ 10 4 cells per well were seeded in 24-well plates for proliferation assays. After incubation with different amounts of nanoparticles suspended in serum-free medium for 4 h, respectively, then the 500 ⁇ M N, N′-dimethyl-4, 4′-bipyridinium dichloride (paraquat) was added to the culture medium for 24 h. Particle-treated cells were then washed twice with PBS and incubated with 200 ⁇ L WST-1 (10%) in DMEM. Cells viability was estimated by a formazan dye generated by the live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680).
  • FIGS. 11A-C show the protection effects of co-delivery of TAT-SOD and TAT-GPx into Hela cells.
  • FIG. 11A shows the enhanced cell viability results for various nanoparticles by using WST-1 assay.
  • FIG. 3B shows the results of ROS detection for various nanoparticles. The levels of ROS were stained by DHE assays and quantified by flow cytometry.
  • FIG. 11C shows the results of Western blotting assays to show the levels of COX II and p-p38.
  • the concentration of PQ and co-delivery of FMSN-TAT-SOD and FMSN-TAT-GPx (1:1 ratio) are 500 ⁇ M and 25 ⁇ g/mL, respectively.
  • the denatured TAT-SOD or TAT-GPx fusion protein can be co-delivered into Hela cells and the denatured fusion proteins can be refolded and exhibit the specific enzymatic activities after delivering into the cells.
  • the TAT-SOD or TAT-GPx fusion protein functionalized FMSN named as FMSN-TAT-SOD or FMSN-TAT-GPx, still has the enzymatic activity by the refolding mechanism of the cells.
  • the mesoporous carriers of the present disclosure embodiments can deliver peptides, proteins, enzymes or enzymatic mimetics into the cells as needed and the native activities of the peptides, proteins, enzymes or enzymatic mimetics being delivered into the cell are maintained.
  • the mesoporous carriers can function as nanoreactors located within the cells and the delivered peptides, proteins, enzymes or enzymatic mimetics can work together to provide multiple functions.

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