US20190365869A1 - Bioresponsive Particles - Google Patents
Bioresponsive Particles Download PDFInfo
- Publication number
- US20190365869A1 US20190365869A1 US16/428,986 US201916428986A US2019365869A1 US 20190365869 A1 US20190365869 A1 US 20190365869A1 US 201916428986 A US201916428986 A US 201916428986A US 2019365869 A1 US2019365869 A1 US 2019365869A1
- Authority
- US
- United States
- Prior art keywords
- enzyme
- silica
- nanoparticles
- groups
- silyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- 239000002245 particle Substances 0.000 title description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 100
- 108090000790 Enzymes Proteins 0.000 claims abstract description 100
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 47
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 28
- 229940088598 enzyme Drugs 0.000 claims description 97
- 238000000034 method Methods 0.000 claims description 29
- 239000002105 nanoparticle Substances 0.000 claims description 28
- 102000016938 Catalase Human genes 0.000 claims description 20
- 108010053835 Catalase Proteins 0.000 claims description 20
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical group [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 claims description 15
- 239000000839 emulsion Substances 0.000 claims description 13
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 claims description 11
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 10
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims description 10
- 102000015790 Asparaginase Human genes 0.000 claims description 9
- 108010024976 Asparaginase Proteins 0.000 claims description 9
- 125000003277 amino group Chemical group 0.000 claims description 9
- 229960003272 asparaginase Drugs 0.000 claims description 9
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 claims description 9
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical group ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 8
- -1 acrylic compound Chemical class 0.000 claims description 8
- 125000003647 acryloyl group Chemical class O=C([*])C([H])=C([H])[H] 0.000 claims description 8
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 7
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 7
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 claims description 7
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 108010043135 L-methionine gamma-lyase Proteins 0.000 claims description 6
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- 239000005089 Luciferase Substances 0.000 claims description 6
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 6
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 6
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- YXMISKNUHHOXFT-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) prop-2-enoate Chemical compound C=CC(=O)ON1C(=O)CCC1=O YXMISKNUHHOXFT-UHFFFAOYSA-N 0.000 claims description 5
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 claims description 5
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 claims description 5
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 4
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 239000000908 ammonium hydroxide Substances 0.000 claims description 4
- 229960001230 asparagine Drugs 0.000 claims description 4
- 235000009582 asparagine Nutrition 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
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- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 3
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- 230000004071 biological effect Effects 0.000 abstract description 2
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- 238000000151 deposition Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 3
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- 238000009826 distribution Methods 0.000 description 3
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
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- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 description 2
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- 210000000987 immune system Anatomy 0.000 description 2
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- 239000012465 retentate Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
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- 238000003917 TEM image Methods 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000006731 degradation reaction Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 238000007421 fluorometric assay Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
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Images
Classifications
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/446—Superoxide dismutase (1.15)
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
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- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
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- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
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- A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
- A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
- A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01006—Catalase (1.11.1.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
- C12Y113/12005—Renilla-luciferin 2-monooxygenase (1.13.12.5), i.e. renilla-luciferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17011—Glutamate carboxypeptidase (3.4.17.11)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01001—Asparaginase (3.5.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y404/00—Carbon-sulfur lyases (4.4)
- C12Y404/01—Carbon-sulfur lyases (4.4.1)
- C12Y404/01011—Methionine gamma-lyase (4.4.1.11)
Definitions
- ALL Acute lymphoblastic leukemia
- our invention provides for incorporating this treatment enzyme in ultra-small particles that are porous to asparagine, but at the same time, prevent the entry of large components of the immune system, will protect a child from immune reactions, but will effectively deplete asparagine. This will not only increase the enzyme's functional life in the child's body, but will also eliminate the harmful immune responses. By creating this nanoscale “force field” around the cancer treatment enzyme, it can do its job to cure the children of cancer much more safely.
- Prior enzyme shielding strategies include the silica coating of single enzymes for industrial use (US2014/0127778) and the passive accumulation of enzymes inside hollow silica shells (US2016/0243262). Neither approach is optimal. The first produces ultra-small nanoparticles; however, shielding is suboptimal as enzyme activity decreases by 90 percent at room temperature in one week, and even faster in vivo and at body temperature, since plasma contains protein-cleaving enzymes to weaken the shield and deactivate the enzyme. The second approach has particle size and enzyme loading limitations. Since it is produced using two templates of markedly different sizes to create hollow silica shells with relatively large pores to allow entry of enzymes prior to closing the pores, particles cannot be made smaller than 100-200 nanometers.
- Our invention allows continuous fine-tuning of enzyme activity per particle and particle size. Because we begin by attaching anchors on each enzyme molecule without affecting its function that serve as seeds upon which silica can deposit, we can control how many enzyme molecules we can put together in each particle to provide full control of number of enzyme molecules per particle and ultimately particle size.
- the invention provides methods and compositions for shielding enzymes with silica. We use enone groups to decorate enzymes, which then allow the facile reaction of the silyl amine derivative to obtain silyl groups on the enzyme which acts as the seed for the growth of the siloxane scaffold around the enzyme (nanoporous silica network/shell that protects the enzymes).
- the invention provides a silica modified enzyme comprising an enzyme covalently decorated with enone groups, around which is grown a siloxane scaffold, to form a hybrid enzyme-silica nanoparticle (HES-NP). Loading the silica modified enzyme into a silica nanoshell protects the enzyme, such as from inactivation by proteolysis
- the invention provides a method of making a silica-modified enzyme comprising the steps of: a) reacting an acrylic compound (acryloyl derivative) with amine groups of an enzyme to covalently decorate the enzyme with enone groups; and b) coupling a silyl amine to the enone groups to covalently decorate the enzyme with silyl groups, forming a silica-modified enzyme.
- the invention provides a method of making hybrid enzyme-silica nanoparticles (HES-NPs) comprising the steps of: (i) growing a siloxane scaffold around a silica-modified enzyme wherein the silyl groups seed the growth of the siloxane scaffold (e.g., in an emulsion or aqueous medium) to form hybrid enzyme-silica nanoparticles (HES-NPs); and (ii) isolating (e.g. from the emulsion or medium) the hybrid enzyme-silica nanoparticles.
- HES-NPs hybrid enzyme-silica nanoparticles
- the invention includes all combinations of the recited particular embodiments as if each combination had been laboriously separately recited.
- FIG. 1 Schematic representation of the preparation of hybrid enzyme-silica nanoparticles using catalase as a model enzyme (CAT-HES-NP).
- FIG. 2 Representative TEM images of HES-NP produced under aqueous (left) or reverse emulsion (right) conditions.
- FIG. 3A Characterization of HES-NP obtained by reverse emulsion: TRPS and intensity-weighted DLS size distribution.
- FIG. 3B Number-weighted size distribution.
- FIG. 4 Activity of free catalase and CAT-HES-NP after incubation at 37° C. for 16 h with or without proteinase K.
- our method to encapsulate catalase and also to encapsulate asparaginase.
- Our novel approach is not enzyme-specific and can be applied to any enzymes.
- Other exemplary enzymes include but are not limited to superoxide dismutase, methioninase, carboxypeptidase G2 and luciferase.
- the invention provides a method for coating enzymes in nanoporous silica that allows free access to small molecules substrates, but not larger molecules such as antibodies or immune cells to be used as a treatment or imaging tool without interacting with the immune system. This approach extends the enzyme's activity in vivo and limits or prevents immune reactions.
- Enzyme i.e., catalase, superoxide dismutase, asparaginase etc.
- 36 mg was dissolved in sodium carbonate buffer (7.2 mL, 20 mM, pH 9.15) and a solution of N-acryloxysuccinimide (36 mg, in DMSO (72 ⁇ L) was added.
- the silica-modified enzyme was filtered through a syringe filter (0.2 ⁇ m) to remove large aggregates.
- the silica-modified enzyme was then formulated into particles using two different formulations.
- the first method aqueous conditions
- the second method reverse emulsion
- Tetraethoxysilane (240 ⁇ L) was added to the silica-modified enzyme solution in water (1.5 mg/mL, 2 mL). The resulting mixture was stirred vigorously for 10 minutes and ammonium hydroxide (7.2 ⁇ L of 28% NH 4 OH solution) was added to hydrolyze silane groups and start the silica particle growth. The resulting emulsion was stirred vigorously for 2 hours at room temperature particles were collected by high speed centrifugation at 20,000 g for 15 minutes. After this time, supernatant was discarded and pellets were redispersed in water (4 mL) for a second wash and centrifugation. The supernatant was discarded a second time and pelleted particles were dispersed in water for storage and characterization.
- Tetraethoxysilane (142 ⁇ L) was added to the silica-modified enzyme solution (1.5 mg/mL, 500 ⁇ L) under reverse emulsion conditions with decane (oil phase, 28.409 mL), IGEPAL® CO-520 (surfactant, 2.318 mL) n-hexanol (co-surfactant, 784 ⁇ L). The resulting mixture was stirred vigorously for 10 minutes and ammonium hydroxide (71 ⁇ L of 28% NH 4 OH solution) was added to hydrolyze silane groups and start the silica particle growth. The resulting emulsion was stirred vigorously overnight at room temperature and ethanol (16 mL) was added to remove surfactants and precipitate the particles.
- decane oil phase, 28.409 mL
- IGEPAL® CO-520 surfactant, 2.318 mL
- n-hexanol co-surfactant, 784 ⁇ L
- the resulting bottom layer was extracted and submitted to high speed centrifugation at 20,000 g for 15 minutes. After this time, supernatant was discarded and pellets were redispersed in water (4 mL) for a second wash and centrifugation. The supernatant was discarded a second time and pelleted particles were dispersed in water for storage and characterization.
- Nanoparticles were sonicated at 10° C. for three minutes in a bath sonicator before size measurements to prevent aggregation.
- Transmission electron microscopy (TEM, FEI Tecnai G2 Spirit transmission electron microscope equipped with a Gatan camera operating at 120 kV with Digital Micrograph software) was performed with negative staining (2% uranyl acetate in water) and TEM pictures were taken and showed monodisperse particles with sizes between 30 and 60 nm ( FIG. 2 )
- DLS Dynamic Light Scattering
- TRPS Resistive Pulse Sensing
Abstract
Shielding enzymes are made by modifying the enzyme surface with silica precursors and then depositing silica to a desired thickness while retaining biological activity of the enzyme.
Description
- Priority: This application claims priority to Ser No. 62/679,762, filed: Jun. 01, 2018.
- This invention was made with government support under Grant Number UL1TR001105 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
- Acute lymphoblastic leukemia (ALL) is the most common childhood cancer accounting for more than 25 percent of all pediatric cancers in the U.S. Unfortunately, 30 percent of children have immune responses to one of the most effective treatments for ALL that is highly allergenic. These immune responses either render the treatment completely ineffective, particularly when children relapse, or worse, immediately threaten the life of the child, or both. Because this treatment is essential for permanently curing children of ALL, it is critical that novel strategies be devised to completely eliminate these immune reactions.
- In an aspect, our invention provides for incorporating this treatment enzyme in ultra-small particles that are porous to asparagine, but at the same time, prevent the entry of large components of the immune system, will protect a child from immune reactions, but will effectively deplete asparagine. This will not only increase the enzyme's functional life in the child's body, but will also eliminate the harmful immune responses. By creating this nanoscale “force field” around the cancer treatment enzyme, it can do its job to cure the children of cancer much more safely.
- Prior enzyme shielding strategies include the silica coating of single enzymes for industrial use (US2014/0127778) and the passive accumulation of enzymes inside hollow silica shells (US2016/0243262). Neither approach is optimal. The first produces ultra-small nanoparticles; however, shielding is suboptimal as enzyme activity decreases by 90 percent at room temperature in one week, and even faster in vivo and at body temperature, since plasma contains protein-cleaving enzymes to weaken the shield and deactivate the enzyme. The second approach has particle size and enzyme loading limitations. Since it is produced using two templates of markedly different sizes to create hollow silica shells with relatively large pores to allow entry of enzymes prior to closing the pores, particles cannot be made smaller than 100-200 nanometers. This relatively large size forces them to stay in blood speeding their removal by the liver and spleen, and limiting their ability to reach the cellular microenvironment in sufficient quantities to adequately fight cancer or provide enzymes to cells that so desperately need them. Further, since they are filled by suspending them in aqueous solutions of the enzyme, they can only trap the amount that can be dissolved without precipitation limiting enzyme loading. Low enzyme activity requires higher dosages to achieve the enzyme activity needed for the desired application, increasing toxicity.
- Our invention allows continuous fine-tuning of enzyme activity per particle and particle size. Because we begin by attaching anchors on each enzyme molecule without affecting its function that serve as seeds upon which silica can deposit, we can control how many enzyme molecules we can put together in each particle to provide full control of number of enzyme molecules per particle and ultimately particle size.
- Trogler et al., US20150273061
- Yang et al., In situ synthesis of porous silica nanoparticles for covalent immobilization of Enzymes, Nanoscale 2012, 4, 414
- Ortac I, Simberg O, Yeh Y S, Yang J, Messmer B, Trogler W C, Tsien R Y, Esener S. Dualporosity hollow nanoparticles for the immunoprotection and delivery of nonhuman enzymes. Nano Lett. 2014;14(6):3023-32. doi: 10.1021/nl404360k. PubMed PMID: 24471767; PMCID: PMC4059531.
- Olson E S, Ortac I, Malone C, Esener S, Mattrey R. Ultrasound Detection of Regional Oxidative Stress in Deep Tissues Using Novel Enzyme Loaded Nanoparticles. Adv Healthc Mater. 2017;6(5). doi: 10.1002/adhm.201601163. PubMed PMID: 28081299; PMCID: PMC5516546.
- Aspects of this disclosure were presented at Bioengineering Seminar at UT Arlington. Bioresponsive Particles for the Detection of Disease by Ultrasound. Nov. 1, 2017.
- The invention provides methods and compositions for shielding enzymes with silica. We use enone groups to decorate enzymes, which then allow the facile reaction of the silyl amine derivative to obtain silyl groups on the enzyme which acts as the seed for the growth of the siloxane scaffold around the enzyme (nanoporous silica network/shell that protects the enzymes).
- The invention provides a silica modified enzyme comprising an enzyme covalently decorated with enone groups, around which is grown a siloxane scaffold, to form a hybrid enzyme-silica nanoparticle (HES-NP). Loading the silica modified enzyme into a silica nanoshell protects the enzyme, such as from inactivation by proteolysis
- In an aspect the invention provides a method of making a silica-modified enzyme comprising the steps of: a) reacting an acrylic compound (acryloyl derivative) with amine groups of an enzyme to covalently decorate the enzyme with enone groups; and b) coupling a silyl amine to the enone groups to covalently decorate the enzyme with silyl groups, forming a silica-modified enzyme.
- In embodiments:
-
- the acrylic compound comprises an acryloyl group and an N-hydroxysuccinimide group, such as N-acryloxysuccinimide or acrylate-polyethyleneglycol N-hydroxysuccinimide; and/or
- the silyl amine is comprises a silyl ether group and an amine group, such as 3-aminopropyl trimethoxysilane (APTMS) or 3-aminopropyl triethoxysilane (APTES).
- In another aspect the invention provides a method of making hybrid enzyme-silica nanoparticles (HES-NPs) comprising the steps of: (i) growing a siloxane scaffold around a silica-modified enzyme wherein the silyl groups seed the growth of the siloxane scaffold (e.g., in an emulsion or aqueous medium) to form hybrid enzyme-silica nanoparticles (HES-NPs); and (ii) isolating (e.g. from the emulsion or medium) the hybrid enzyme-silica nanoparticles.
- In embodiments:
-
- step (i) comprises contacting the silica-modified enzyme with tetraethoxysilane under aqueous conditions and hydrolyzing (e.g., with ammonium hydroxide) silane groups to start the growth of the siloxane scaffold;
- step (i) comprises contacting the silica-modified enzyme with tetraethoxysilane under reverse emulsion conditions and hydrolyzing silane groups to start the growth of the siloxane scaffold;
- the method further comprises the antecedent steps of: a) reacting an acrylic compound (acryloyl derivative) with amine groups of an enzyme to covalently decorate the enzyme with enone groups; and b) coupling a silyl amine to the enone groups to covalently decorate the enzyme with silyl groups, forming a silica-modified enzyme, wherein embodiment: the acrylic compound comprises an acryloyl group and a N-hydroxysuccinimide group, such as N-acryloxysuccinimide or acrylate-polyethyleneglycol N-hydroxysuccinimide; and/or the silyl amine comprises a silyl ether group and an amine group, such as 3-aminopropyl trimethoxysilane (APTMS) or 3-aminopropyl triethoxysilane (APTES);
- the enzyme is selected from catalase, superoxide dismutase, asparaginase, methioninase, carboxypeptidase G2 and luciferase;
- the nanoparticles are of average size 20-100 nm or 20-50 nm diameter;
- the method further comprises the step of administering the nanoparticles to a person in need thereof, and particularly wherein:
- the enzyme is catalase;
- the nanoparticles provide a bioresponsive ultrasound contrast agent, and imaging the patient by ultrasound, such as wherein the enzyme is catalase, effective to generate O2 bubbles;
- the person has or is at (imminent, demonstrable) risk of reperfusion injury and the enzyme is catalase, effective to scavenge reactive oxygen species (ROS);
- the person has leukemia (e.g. acute lymphoblastic leukemia, ALL) and the enzyme is asparaginase, effective to deplete asparagine; and/or
- the person is, has been or will be administered a prodrug, and the enzyme is prodrug converting enzyme, effective to convert the prodrug to a therapeutic drug.
- The invention includes all combinations of the recited particular embodiments as if each combination had been laboriously separately recited.
-
FIG. 1 . Schematic representation of the preparation of hybrid enzyme-silica nanoparticles using catalase as a model enzyme (CAT-HES-NP). -
FIG. 2 . Representative TEM images of HES-NP produced under aqueous (left) or reverse emulsion (right) conditions. -
FIG. 3A . Characterization of HES-NP obtained by reverse emulsion: TRPS and intensity-weighted DLS size distribution.FIG. 3B . Number-weighted size distribution. -
FIG. 4 . Activity of free catalase and CAT-HES-NP after incubation at 37° C. for 16 h with or without proteinase K. - We disclose a novel hybrid approach to shielding enzymes. We first modify the enzyme surface with silica precursors and then proceed to deposit silica to a desired thickness while retaining its biological activity. An advantage of this approach is that we can control final nanoparticle size and desired enzyme activity per particle by incorporating one or more or different enzyme molecules to optimize delivery and efficacy. Unlike passive trapping of enzymes in hollow silica spheres that utilize templates ≥100 nanometers, our nanoparticles can be made as small as 20-50 nanometer to achieve optimal delivery and enzyme activity. In an embodiment we exemplify the method with catalase as a model enzyme because it can be used to detect tissues in oxidative stress using ultrasound imaging, can be used as an anti-oxidant, and its activity is easily measured using commercial assay kits. In another embodiment example, we used our method to encapsulate catalase and also to encapsulate asparaginase. Our novel approach is not enzyme-specific and can be applied to any enzymes. Other exemplary enzymes include but are not limited to superoxide dismutase, methioninase, carboxypeptidase G2 and luciferase.
- The invention provides a method for coating enzymes in nanoporous silica that allows free access to small molecules substrates, but not larger molecules such as antibodies or immune cells to be used as a treatment or imaging tool without interacting with the immune system. This approach extends the enzyme's activity in vivo and limits or prevents immune reactions.
- General procedure for the preparation and characterization of hybrid enzyme-silica nanoparticles (HES-NP):
- Preparation of HES-NP
- Enzyme (i.e., catalase, superoxide dismutase, asparaginase etc.) (36 mg) was dissolved in sodium carbonate buffer (7.2 mL, 20 mM, pH 9.15) and a solution of N-acryloxysuccinimide (36 mg, in DMSO (72 μL) was added. The resulting mixture was stirred for 1 hour at room temperature and was purified by spin filtration in Amicon spin filters (Molecular weight Cutoff=10 kDa) at 4,000 g for 10 min. The filtrate was discarded, and the retentate was washed with water and spin filtered again at 4,000 g for 10 more minutes to yield the enone-modified enzyme (
FIG. 1 , step a). The purified enone-modified enzyme (3 mL, 12 mg/mL) was diluted down to a concentration of 1.5 mg/mL in 1M phosphate buffer pH 6.0 and water. (3-aminopropyl) trimethoxysilane (96 μL) was then added the resulting mixture was stirred for 1 hour at room temperature and purified by spin filtration in Amicon spin filters (Molecular weight Cutoff=10 kDa) at 4,000 g for 10 min. The filtrate was discarded, and the retentate was washed with water and spin filtered again at 4,000 g for 10 more minutes to yield the silica-modified enzyme (FIG. 1 . Step b). - Before particle formulations, the silica-modified enzyme was filtered through a syringe filter (0.2 μm) to remove large aggregates. The silica-modified enzyme was then formulated into particles using two different formulations. The first method (aqueous conditions) yields nanoparticles around 100 nm and the second method (reverse emulsion) yield ultrasmall nanoparticles around 50 nm.
- A] Aqueous conditions
- Tetraethoxysilane (240 μL) was added to the silica-modified enzyme solution in water (1.5 mg/mL, 2 mL). The resulting mixture was stirred vigorously for 10 minutes and ammonium hydroxide (7.2 μL of 28% NH4OH solution) was added to hydrolyze silane groups and start the silica particle growth. The resulting emulsion was stirred vigorously for 2 hours at room temperature particles were collected by high speed centrifugation at 20,000 g for 15 minutes. After this time, supernatant was discarded and pellets were redispersed in water (4 mL) for a second wash and centrifugation. The supernatant was discarded a second time and pelleted particles were dispersed in water for storage and characterization.
- B] Reverse emulsion conditions
- Tetraethoxysilane (142 μL) was added to the silica-modified enzyme solution (1.5 mg/mL, 500 μL) under reverse emulsion conditions with decane (oil phase, 28.409 mL), IGEPAL® CO-520 (surfactant, 2.318 mL) n-hexanol (co-surfactant, 784 μL). The resulting mixture was stirred vigorously for 10 minutes and ammonium hydroxide (71 μL of 28% NH4OH solution) was added to hydrolyze silane groups and start the silica particle growth. The resulting emulsion was stirred vigorously overnight at room temperature and ethanol (16 mL) was added to remove surfactants and precipitate the particles. The resulting bottom layer was extracted and submitted to high speed centrifugation at 20,000 g for 15 minutes. After this time, supernatant was discarded and pellets were redispersed in water (4 mL) for a second wash and centrifugation. The supernatant was discarded a second time and pelleted particles were dispersed in water for storage and characterization.
- Characterization of HES-NP:
- Nanoparticles were sonicated at 10° C. for three minutes in a bath sonicator before size measurements to prevent aggregation. Transmission electron microscopy (TEM, FEI Tecnai G2 Spirit transmission electron microscope equipped with a Gatan camera operating at 120 kV with Digital Micrograph software) was performed with negative staining (2% uranyl acetate in water) and TEM pictures were taken and showed monodisperse particles with sizes between 30 and 60 nm (
FIG. 2 ) - The hydrodynamic diameter of HES-NP was measured at 122.6 nm with a PdI of 0.168 by Dynamic Light Scattering (DLS,
FIG. 3A ) (Zetasizer ZS, Malvern Instruments). HES-NP were measured with a mean diameter of 117 nm (StdDev=40.5) with a concentration of 1.7×1012 NPs/mL by Tunable Resistive Pulse Sensing (TRPS, IZON, qNano Gold). The difference in size between the real size (TEM) and the size measured by DLS is explained by the higher scattering from larger molecules increasing the overall hydrodiameter of the particle population. This is demonstrated by displaying the size distribution of the particle population weighted by number, which provides a mean diameter around 50 nm (FIG. 3B ). In the case of TRPS the overestimation of the size is due to the physical limitations of the instrument which uses a nanopore that cannot detect particles under the size of 50 nm, which means that we overestimate the size and underestimate the count of our nanoparticles using this method. - Stability Measurements of HES-NP:
- To confirm that enzyme-loaded silica nanoshells protect enzymes from inactivation by proteolysis, I evaluated the activity of free enzyme and encapsulated enzyme in the presence of proteinase K, a serine protease that cleaves a wide range of proteins. In this experiment, we used catalase as a model protein, as it is not expensive, allows facile observation of activity by naked eye (bubbles generated upon addition of H2O2) and quantitative measurement of the enzymatic activity using a fluorometric assay. Specifically, we incubated free catalase and CAT-HES-NP overnight at 37° C. in pure water in the presence of CaCl2 (10 mM, 50 μL) and proteinase K (50 μL at 1 mg/mL). After 16 h, free catalase kept only 6% of its activity, while CAT-HES-NP kept 87% of its activity (
FIG. 4 ). This slight activity loss is most likely due to the degradation of catalase attached at the surface of the particle. - It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.
Claims (20)
1. A method of making a silica-modified enzyme, comprising the steps of:
a) reacting an acrylic compound (acryloyl derivative) with amine groups of an enzyme to covalently decorate the enzyme with enone groups; and
b) coupling a silyl amine to the enone groups to covalently decorate the enzyme with silyl groups, forming a silica-modified enzyme.
2. The method of claim 1 wherein the acrylic compound comprises an acryloyl group and a N-hydroxysuccinimide group, such as N-acryloxysuccinimide or acrylate-polyethyleneglycol N-hydroxysuccinimide.
3. The method of claim 1 wherein the silyl amine is comprises a silyl ether group and an amine group, such as 3-aminopropyl trimethoxysilane (APTMS) or 3-aminopropyl triethoxysilane (APTES).
4. The method of claim 2 wherein the silyl amine is comprises a silyl ether group and an amine group, such as 3-aminopropyl trimethoxysilane (APTMS) or 3-aminopropyl triethoxysilane (APTES).
5. The method of claim 1 wherein the enzyme is selected from catalase, superoxide dismutase, asparaginase, methioninase, carboxypeptidase G2 and luciferase.
6. The method of claim 4 wherein the enzyme is selected from catalase, superoxide dismutase, asparaginase, methioninase, carboxypeptidase G2 and luciferase.
7. A method of making hybrid enzyme-silica nanoparticles (HES-NPs) using a silica-modified enzyme synthesizable by the method of claim 1 , comprising the steps of:
i) growing a siloxane scaffold around the silica-modified enzyme, wherein the silyl groups seed the growth of the siloxane scaffold (e.g., in an emulsion or aqueous medium) to form hybrid enzyme-silica nanoparticles (HES-NPs); and
ii) isolating (e.g. from the emulsion or medium) the hybrid enzyme-silica nanoparticles.
8. The method of claim 7 wherein step (i) comprises contacting the silica-modified enzyme with tetraethoxysilane under surfactant-free aqueous conditions and hydrolyzing (e.g. with ammonium hydroxide) silane groups to start the growth of the siloxane scaffold.
9. The method of claim 7 wherein step (i) comprises contacting the silica-modified enzyme with tetraethoxysilane under reverse emulsion conditions and hydrolyzing silane groups to start the growth of the siloxane scaffold.
10. The method of claim 7 further comprising the antecedent steps of:
a) reacting an acrylic compound (acryloyl derivative) with amine groups of an enzyme to covalently decorate the enzyme with enone groups; and
b) coupling a silyl amine to the enone groups to covalently decorate the enzyme with silyl groups, forming a silica-modified enzyme.
11. The method of claim 10 wherein:
the acrylic compound comprises an acryloyl group and a N-hydroxysuccinimide group, such as N-acryloxysuccinimide or acrylate-polyethyleneglycol N-hydroxysuccinimide; and
the silyl amine is comprises a silyl ether group and an amine group, such as 3-aminopropyl trimethoxysilane (APTMS) or 3-aminopropyl triethoxysilane (APTES).
12. The method of claim 7 wherein the enzyme is selected from catalase, superoxide dismutase, asparaginase, methioninase, carboxypeptidase G2 and luciferase.
13. The method of claim 11 wherein the enzyme is selected from catalase, superoxide dismutase, asparaginase, methioninase, carboxypeptidase G2 and luciferase.
14. The method of claim 7 wherein the nanoparticles are of average size 20-100 nm or 20-50 nm diameter.
15. The method of claim 7 further comprising the step of administering the nanoparticles to a person in need thereof.
16. The method of claim 7 further comprising the steps of administering the nanoparticles to a person in need thereof, the enzyme is catalase.
17. The method of claim 7 further comprising the step of administering the nanoparticles to a person in need thereof, wherein the nanoparticles provide a bioresponsive ultrasound contrast agent, and imaging the patient by ultrasound, such as wherein the enzyme is catalase, effective to generate O2 bubbles.
18. The method of claim 7 further comprising the step of administering the nanoparticles to a person in need thereof, wherein the person has or is at (imminent, demonstrable) risk of reperfusion injury and the enzyme is catalase, effective to scavenge reactive oxygen species (ROS).
19. The method of claim 7 further comprising the step of administering the nanoparticles to a person in need thereof, wherein the person has leukemia (e.g. acute lymphoblastic leukemia, ALL) and the enzyme is asparaginase, effective to deplete asparagine.
20. The method of claim 7 further comprising the step of administering the nanoparticles to a person in need thereof, wherein the person is, has been or will be administered a prodrug, and the enzyme is prodrug converting enzyme, effective to convert the prodrug to a therapeutic drug.
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