Bioresponsive Particles

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.

Claims

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 O.sub.2 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1. Schematic representation of the preparation of hybrid enzyme-silica nanoparticles using catalase as a model enzyme (CAT-HES-NP).

[0033] FIG. 2. Representative TEM images of HES-NP produced under aqueous (left) or reverse emulsion (right) conditions.

[0034] FIG. 3A. Characterization of HES-NP obtained by reverse emulsion: TRPS and intensity-weighted DLS size distribution. FIG. 3B. Number-weighted size distribution.

[0035] FIG. 4. Activity of free catalase and CAT-HES-NP after incubation at 37 C. for 16 h with or without proteinase K.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0036] 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.

[0037] 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.

[0038] General procedure for the preparation and characterization of hybrid enzyme-silica nanoparticles (HES-NP):

[0039] Preparation of HES-NP

[0040] 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 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 L) 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).

[0041] Before particle formulations, 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) yields nanoparticles around 100 nm and the second method (reverse emulsion) yield ultrasmall nanoparticles around 50 nm.

[0042] A] Aqueous conditions

[0043] 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.sub.4OH 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.

[0044] B] Reverse emulsion conditions

[0045] 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.sub.4OH 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.

[0046] Characterization of HES-NP:

[0047] 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)

[0048] 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 1.710.sup.12 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.

[0049] Stability Measurements of HES-NP:

[0050] 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 H.sub.2O.sub.2) 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 CaCl.sub.2 (10 mM, 50 L) and proteinase K (50 L 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.

[0051] 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.