Multiple-enzyme nanocomplexes
Inventors
Lu, Yunfeng • Yan, Ming • Liu, Yang
Assignees
Defense Threat Reduction Agency
Publication Number
US-10016490-B2
Publication Date
2018-07-10
Expiration Date
2032-07-06
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Abstract
Provided are nanocomplexes having at least two different enzymes and a polymeric network anchored to at least one of the enzymes. In some embodiments, the activities of the enzymes catalyze a cascade reaction.
Core Innovation
The invention provides multiple-enzyme nanocomplexes containing at least two different enzymes and a polymeric network anchored to at least one of the enzymes. These nanocomplexes enable the enzymes to cooperatively carry out their enzymatic functions, including catalyzing cascade reactions. The polymeric network can form a permeable shell around the enzymes, allowing substrate and intermediate diffusion while spatially constraining the enzymes within the nanocomplex.
The problem addressed by the invention arises from natural and artificial enzyme systems. In vivo, enzymes are spatially organized within organelles or enzyme complexes to increase reaction efficiency and specificity, and to rapidly eliminate toxic intermediates, such as hydrogen peroxide. However, current enzyme-based applications largely use single enzymes or simple mixtures, lacking the structural organization and stability found in nature. Attempts to construct multiple-enzyme architectures using fusion proteins or post-translational assemblies have limitations including reduced enzyme activity or lack of general applicability. Inorganic or polymeric materials with multi-enzymes have been random and large in scale, unsuitable for therapeutic applications.
Claims Coverage
The claims include one independent claim detailing the structure and preparation method of a multiple-enzyme nanocomplex. It covers various features regarding enzyme linkage, polymer network composition, encapsulation, and functional cooperation of enzymes.
Multiple-enzyme nanocomplex with polymeric network encapsulation
A nanocomplex comprising at least two different enzymes with a polymeric network anchored to at least one enzyme, forming a permeable shell encapsulating the linked enzymes.
Enzyme linkage via hybridizable nucleic acid strands
Linking the first and second enzymes to one another via conjugation to single nucleic acid strands that hybridize, providing controlled assembly prior to polymer network formation.
Polymeric network formation by in situ polymerization
Polymerizable monomers are combined with acrylated linked enzymes and polymerized in situ on the enzyme complex surface to form a permeable polymeric shell.
Polymeric network anchored to all enzymes
In some embodiments, the polymeric network is anchored to all of the enzymes within the nanocomplex to provide spatial constraint and stability.
Cascade reaction enzymatic activity
The enzymes catalyze cascade reactions where the product of the first enzyme serves as substrate for the second enzyme within the nanocomplex.
Covalent or non-covalent enzyme linking with degradable linkage
Enzymes are linked covalently or non-covalently; covalent linkages may be degradable in vivo to allow functional flexibility.
Polymeric network composition
The polymeric network comprises polymers from at least one or multiple monomeric units, optionally including crosslinkers to control network properties.
Protection against degradation in vivo
The polymeric network encapsulating the enzymes inhibits degradation of the enzyme complex when disposed in an in vivo environment.
Specific enzyme systems featuring alcohol oxidase and catalase
Specific embodiments with a first enzyme generating hydrogen peroxide (alcohol oxidase) and a second enzyme (catalase) converting hydrogen peroxide into water and oxygen, with controlled substrate and product diffusion through the polymer shell.
Formulation for oral administration
The nanocomplex can be combined with one or more filling agents, binding agents, or buffering agents adapted for use in orally administered formulations.
The claims define multiple-enzyme nanocomplexes with controlled enzyme linkage via nucleic acid hybridization, encapsulated within a polymeric network formed by in situ polymerization. The inventive features include enzyme spatial constraint, cascade reaction facilitation, and enhanced stability and protection in vivo, with specific embodiments for alcohol detoxification and oral administration.
Stated Advantages
Enhanced catalytic efficiency due to spatial confinement and close proximity of multiple enzymes within the nanocomplex.
Increased stability of enzymes against high temperature, protease degradation, and non-physiological conditions.
Reduced release of toxic intermediates, such as hydrogen peroxide, mitigating cell and tissue damage.
Programmable function and composition of enzyme complexes through simple chemical approaches.
Ability to protect enzymes during in vivo administration, allowing versatile routes of delivery including oral and intravenous.
Documented Applications
Use of enzyme nanocomplexes to catalyze cascade enzymatic reactions with enhanced efficiency and stability.
Therapeutic applications including antidotes for alcohol overdose by enzymatic breakdown of alcohol and toxic intermediates.
In vivo detoxification of metabolites such as hydrogen peroxide to prevent cell and tissue damage.
Delivery of therapeutic enzymes to cells or subjects with improved stability and prolonged activity.
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