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.

Description

INTRODUCTION

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.

Relevant Literature

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.

SUMMARY OF THE INVENTION

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.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

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

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; andb) 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); andii) 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; andb) 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; andthe 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.