U.S. patent application number 14/131159 was filed with the patent office on 2014-07-03 for oral delivery of enzymes by nanocapsules for targeted metabolism of alcohol or toxic metabolites.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is Cheng Ji, Yang Liu, Yunfeng Lu, Ming Yan, Otto O. Yang. Invention is credited to Cheng Ji, Yang Liu, Yunfeng Lu, Ming Yan, Otto O. Yang.
Application Number | 20140186436 14/131159 |
Document ID | / |
Family ID | 47437459 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186436 |
Kind Code |
A1 |
Yang; Otto O. ; et
al. |
July 3, 2014 |
ORAL DELIVERY OF ENZYMES BY NANOCAPSULES FOR TARGETED METABOLISM OF
ALCOHOL OR TOXIC METABOLITES
Abstract
The invention disclosed herein includes nanocomplexes that are
designed include enzymes that have complementary functional
attributes and methods for using these nanocomplexes. Illustrative
examples include nanocomplexes that comprise both an alcohol
oxidase enzyme as well as a catalase enzyme. These nanocomplexes
can be used in methods designed to lower blood alcohol levels in
vivo, and/or to break down the toxic byproducts of alcohol
metabolism. Consequently these nanocomplexes can be used to treat a
variety of conditions resulting from the consumption of alcohol,
including for example, acute alcohol intoxication.
Inventors: |
Yang; Otto O.; (Los Angeles,
CA) ; Lu; Yunfeng; (Culver City, CA) ; Ji;
Cheng; (Los Angeles, CA) ; Yan; Ming; (Encino,
CA) ; Liu; Yang; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yang; Otto O.
Lu; Yunfeng
Ji; Cheng
Yan; Ming
Liu; Yang |
Los Angeles
Culver City
Los Angeles
Encino
Los Angeles |
CA
CA
CA
CA
CA |
US
US
US
US
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
47437459 |
Appl. No.: |
14/131159 |
Filed: |
July 6, 2012 |
PCT Filed: |
July 6, 2012 |
PCT NO: |
PCT/US2012/045692 |
371 Date: |
March 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61504975 |
Jul 6, 2011 |
|
|
|
61529767 |
Aug 31, 2011 |
|
|
|
Current U.S.
Class: |
424/451 ;
424/94.2; 435/262.5 |
Current CPC
Class: |
A61K 47/549 20170801;
A61K 38/44 20130101; A61P 39/02 20180101; C12N 11/18 20130101; B82Y
5/00 20130101; A61K 38/443 20130101; A61K 47/64 20170801 |
Class at
Publication: |
424/451 ;
424/94.2; 435/262.5 |
International
Class: |
A61K 38/44 20060101
A61K038/44 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
Nos. R01 AA18612 and R01 AA018846 awarded by the National
Institutes of Health and National Institute on Alcohol Abuse and
Alcoholism. The Government has certain rights in this invention.
Claims
1. A method of decreasing the concentration of ethanol in an
individual comprising the steps of: (a) administering a
multiple-enzyme nanocomplex system to the individual, wherein the
multiple-enzyme nanocomplex system comprises: an alcohol oxidase
enzyme that generates hydrogen peroxide in a first enzymatic
reaction with ethanol; a catalase enzyme that converts the hydrogen
peroxide into water in a second enzymatic reaction; and a polymeric
network configured to form a shell that encapsulates the alcohol
oxidase and the catalase, wherein: the polymeric network exhibits a
permeability sufficient to allow the ethanol to diffuse from an
external environment outside of the shell to the alcohol oxidase so
that the hydrogen is peroxide is generated; the polymeric network
exhibits a permeability sufficient to allow the hydrogen peroxide
to diffuse away from the alcohol oxidase and to the catalase so
that the water is generated; and the alcohol oxidase is coupled to
the catalase; or the polymeric network is coupled to the alcohol
oxidase or the catalase; and (b) allowing the alcohol oxidase and
the catalase in the multiple-enzyme nanocomplex system to react
with ethanol in the individual and generate hydrogen peroxide and
water; so that the concentration of ethanol in the individual is
decreased.
2. The method of claim 1, wherein the multiple-enzyme nanocomplex
system is administered prior to the consumption of ethanol so as to
avoid ethanol intoxication.
3. The method of claim 1, wherein the multiple-enzyme nanocomplex
system is administered orally.
4. The method of claim 1, wherein the individual suffers from acute
ethanol intoxication.
5. The method of claim 1, wherein the multiple-enzyme nanocomplex
system is administered parenterally.
6. The method of claim 1, wherein the multiple-enzyme nanocomplex
system reduces blood ethanol concentrations in the individual by at
least 25, 50, 75 or 100 mg/dL within 90 minutes following
administration to the individual.
7. The method of claim 1, further comprising a third enzyme
encapsulated within the polymeric network, wherein the third enzyme
is an alcohol dehydrogenase, an aldehyde dehydrogenase or an
acetaldehyde oxidase.
8. The method of claim 1, wherein the first enzyme and the second
enzyme are coupled together by strands of complimentary
polynucleotides.
9. The method of claim 1, wherein the polymeric network
encapsulates the alcohol oxidase and the catalase in a manner that
inhibits degradation of the alcohol oxidase and the catalase when
the multiple-enzyme nanocomplex is disposed in an in vivo
environment.
10. A multiple-enzyme nanocomplex system for use in a patient for
the treatment of a condition resulting from the consumption of
alcohol, wherein the multiple-enzyme nanocomplex system comprises:
an alcohol oxidase enzyme that generates hydrogen peroxide in a
first enzymatic reaction with alcohol; a catalase enzyme that
converts the hydrogen peroxide into water in a second enzymatic
reaction; and a polymeric network configured to form a shell that
encapsulates the alcohol oxidase and the catalase, wherein: the
polymeric network exhibits a permeability sufficient to allow the
alcohol to diffuse from an external environment outside of the
shell to the alcohol oxidase so that the hydrogen peroxide is
generated; the polymeric network exhibits a permeability sufficient
to allow the hydrogen peroxide to diffuse away from the alcohol
oxidase and to the catalase so that the water is generated; and the
alcohol oxidase is coupled to the catalase; or the polymeric
network is coupled to the alcohol oxidase or the catalase.
11. The multiple-enzyme nanocomplex system for use according to
claim 1, wherein the multiple-enzyme nanocomplex system is
administered in an amount sufficient to reduce the blood alcohol
concentration of the patient by at least 10%, 20% or 30% within 90
minutes following administration.
12. The multiple-enzyme nanocomplex system for use according to
claim 1, wherein the condition in the patient resulting from the
consumption of alcohol is acute alcohol intoxication.
13. The multiple-enzyme nanocomplex system for use according to
claim 1, wherein the multiple-enzyme nanocomplex system is
administered orally.
14. The multiple-enzyme nanocomplex system for use according to
claim 1, wherein the alcohol oxidase enzyme and catalase enzyme are
disposed within the polymeric network at a distance from each other
of less than 50, 40, 30, 20 or 10 nm.
15. A method of decreasing a concentration of hydrogen peroxide in
an aqueous environment comprising the steps of: (a) introducing a
multiple-enzyme nanocomplex system into the aqueous environment,
wherein the multiple-enzyme nanocomplex system comprises: a first
enzyme that generates hydrogen peroxide in a first enzymatic
reaction with a first substrate present in the aqueous environment;
a second enzyme that converts the hydrogen peroxide into water in a
second enzymatic reaction; and a polymeric network configured to
form a shell that encapsulates the first enzyme and the second
enzyme, wherein: the polymeric network encapsulates the first
enzyme and the second enzyme in a manner that inhibits degradation
of the first enzyme and the second enzyme when the multiple-enzyme
nanocomplex is disposed in an in vivo environment; the polymeric
network exhibits a permeability sufficient to allow the substrate
to diffuse from an external environment outside of the shell to the
first enzyme so that the hydrogen peroxide is generated; the
polymeric network exhibits a permeability sufficient to allow the
hydrogen peroxide to diffuse away from the first enzyme and to the
second enzyme so that the water is generated; and the first enzyme
is coupled to the second enzyme; or the polymeric network is
coupled to the first enzyme or the second enzyme; and (b) allowing
the first enzyme and the second enzyme in the multiple-enzyme
nanocomplex system to react with the substrate and generate
hydrogen peroxide and water; so that the concentration of hydrogen
peroxide in the aqueous environment is decreased.
16. The method of claim 15, wherein at least one enzyme
encapsulated within the polymeric network is selected from the
group consisting of an alcohol oxidase, a catalase, a peroxidase,
an alcohol dehydrogenase, an aldehyde dehydrogenase or an
acetaldehyde oxidase.
17. The method of claim 15, wherein: the first enzyme is an alcohol
oxidase; and the second enzyme is a catalase.
18. The method of claim 17, wherein the aqueous environment
comprises an in vivo environment.
19. The method of claim 15, wherein: the first enzyme is a glucose
oxidase; and the second enzyme is a horseradish peroxidase.
20. The method of claim 19, wherein the aqueous environment
comprises an in vitro environment.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. Section
119(e) of co-pending U.S. Provisional Patent Application Ser. No.
61/504,975, filed on Jul. 6, 2011, entitled "ORAL DELIVERY OF
ENZYMES BY NANOCAPSULES FOR TARGETED METABOLISM OF ALCOHOL OR TOXIC
METABOLITES", and co-pending U.S. Provisional Patent Application
Ser. No. 61/529,767, filed on Aug. 31, 2011, entitled
"MULTIPLE-ENZYME NANOCOMPLEXES", the contents of each of which are
incorporated herein by reference.
FIELD OF THE DISCLOSURE
[0003] This disclosure relates to methods of administering enzyme
nanocomplexes designed to metabolize alcohol and/or alcohol
metabolites.
BACKGROUND
[0004] Ethyl alcohol is the most widely used psychoactive drug in
the world. Alcohol intoxication and alcohol associated diseases
represent a serious threat to human health and pose major medical,
social, and economic problems. In the United States alone, an
estimated 10% of the population is affected by alcoholism. The
physical, social and psychological harm which results from alcohol
abuse and dependence, such as acute alcohol poisoning, fetal
alcohol syndrome and cirrhosis of the liver. In addition to the
long-term deleterious effects of alcohol intoxication on various
organs and systems, alcohol consumption and intoxication can result
in short-term, but very unpleasant or inconvenient, effects. These
effects, commonly collectively referred to as a "hangover," can
include, for example, headaches, nausea, and fatigue. Many problems
associated with alcohol abuse including increased health care costs
and lost worker productivity are very costly to affected
individuals, their families and to society at large.
[0005] When alcohol is consumed, it is absorbed into the blood from
the stomach and intestines. Alcohol is then metabolized through the
action of various enzymes. In the liver, alcohol dehydrogenase
(ADH) mediates the conversion of alcohol to acetaldehyde.
Acetaldehyde is then quantitatively oxidized to acetic acid in the
presence of aldehyde dehydrogenase (ALDH) and nicotinamide-adenine
dinucleotide (NAD). Acetate is eventually metabolized to carbon
dioxide and water. Acetaldehyde and reactive oxygen species
accumulate upon chronic or high consumption of ethanol. Toxic
compounds generated during the metabolism of alcohol can cause cell
injury through lipid peroxidation, protein inactivation and DNA
damage. The rate of alcohol metabolism depends, in part, on the
amount of metabolizing enzymes in the liver, which varies among
individuals and has genetic determinants The liver can metabolize
only a certain amount of alcohol per hour, and alcohol is
metabolized more slowly than it is absorbed. Because the rate of
alcohol metabolism is relatively slow as compared to its rate of
absorption, alcohol can quickly accumulate in the body and lead to
acute alcohol poisoning and death.
[0006] Ethanol's acute effects are largely due to its nature as a
central nervous system depressant, and are dependent on blood
alcohol concentrations. At concentrations of approximately 200-299
mg/dL, individuals can exhibit marked ataxia, slurred speech, poor
judgment, labile mood, nausea and vomiting. At concentrations of
approximately 300-399 mg/dL, individuals can exhibit--Stage 1
anesthesia, memory lapse, labile mood. At concentrations above
approximately 400+ mg/dL, individuals can exhibit respiratory
failure and coma. After a very high level of consumption, the
respiratory system becomes depressed and the person will stop
breathing. In this context, individuals suffering from dangerous
levels of alcohol intoxication should get immediate and rapid
emergency medical treatment. Comatose patients may aspirate their
vomit (resulting in vomit in the lungs, which may cause "drowning"
and later pneumonia if survived). Moreover, CNS depression and
impaired motor co-ordination along with poor judgment increases the
likelihood of accidental injury occurring.
[0007] In view of the variety of problems associated with alcohol
consumption, intoxication and abuse, there is a need for methods
and materials that can reduce the concentrations of ethanol in
vivo. Such methods and materials are useful, for example, in
treating or ameliorating pathological conditions associated with
the consumption of alcohol, including acute alcohol intoxication as
well as treating alcohol abuse and dependence.
SUMMARY
[0008] The invention disclosed herein relates to nanocomplexes
designed to include a combination of different enzymes having
complementary functions, for example alcohol oxidase enzymes and
catalase enzymes. As discussed in detail below, embodiments of the
invention can be used to quickly lower blood alcohol levels in vivo
while simultaneously breaking down toxic metabolites. Such
embodiments of the invention can be used to treat a variety of
conditions resulting from the consumption of alcohol, including for
example, acute alcohol intoxication.
[0009] Embodiments of the invention include a multiple-enzyme
nanocomplex system for use in the treatment of a condition
resulting from the consumption of alcohol. Typical embodiments
multiple-enzyme nanocomplexes can comprise an alcohol oxidase
enzyme that generates hydrogen peroxide in a first enzymatic
reaction with alcohol in combination with a catalase enzyme that
converts the hydrogen peroxide into water in a second enzymatic
reaction. In this way, these multiple-enzyme nanocomplexes quickly
convert alcohol into water and 0.sub.2. Studies described in the
Examples below show how orally delivered multiple-enzyme
nanocomplexes can be used to accelerate of ethanol metabolism in
vivo.
[0010] In typical embodiments of the invention, the multiple-enzyme
nanocomplex system includes a polymeric network configured to form
a protective shell that encapsulates the alcohol oxidase and the
catalase. This shell is designed to form a polymeric matrix that
exhibits a permeability sufficient to allow the alcohol to diffuse
from an external environment outside of the shell to the alcohol
oxidase so that the hydrogen peroxide is generated as well as a
permeability sufficient to allow the hydrogen peroxide to diffuse
away from the alcohol oxidase and to the catalase so that the water
is generated. Typically in embodiments of the invention, the
alcohol oxidase is coupled to the catalase; and/or the polymeric
network is coupled to the alcohol oxidase or the catalase.
[0011] In certain embodiments of the invention, the multiple-enzyme
nanocomplex system is used in these methods to reduce blood alcohol
concentrations in vivo by at least 10%, 20% or 30% within 90
minutes following its administration. In some embodiments of the
invention, the method is used to treat a pathological condition
that results from the consumption of alcohol, for example, acute
alcohol intoxication. Optionally, in these embodiments, the
multiple-enzyme nanocomplex system is administered parenterally. In
other embodiments of the invention, the method is used to
ameliorate a condition that results from the consumption of
alcohol, for example, a hangover. Optionally, in these embodiments,
the multiple-enzyme nanocomplex system is administered orally.
[0012] Related embodiments of the invention include methods of
decreasing the concentration of ethanol in an individual, for
example a patient suffering from acute alcohol intoxication. These
involve administering a multiple-enzyme nanocomplex to the
individual. In these methods, the multiple-enzyme nanocomplex
comprises an alcohol oxidase enzyme that generates hydrogen
peroxide in a first enzymatic reaction with ethanol as well as a
catalase enzyme that converts the hydrogen peroxide into water in a
second enzymatic reaction. In this embodiment; a polymeric network
configured to form a shell that encapsulates the alcohol oxidase
and the catalase and, for example, protect these polypeptides from
proteolytic degradation (e.g. proteases present in in vivo
environments). In typical embodiments of the invention, the alcohol
oxidase is coupled to the catalase; or the polymeric network is
coupled to the alcohol oxidase or the catalase. In the methods of
using these multiple-enzyme nanocomplex systems, their
administration allows the alcohol oxidase and the catalase in the
multiple-enzyme nanocomplex to react with ethanol in the individual
and generate hydrogen peroxide and water, so that the concentration
of ethanol in the individual is decreased.
[0013] Embodiments of the invention are useful in a variety of
contexts. For example, in some embodiments of the invention, the
multiple-enzyme nanocomplex is administered prior to the
consumption of ethanol so as to avoid ethanol intoxication. In
other embodiments of the invention, the multiple-enzyme nanocomplex
is administered following the consumption of an ethanol by an
individual suffering from acute ethanol intoxication. In certain
embodiments, the multiple-enzyme nanocomplex is administered
orally. In other embodiments of the invention, the multiple-enzyme
nanocomplex is administered parenterally. Typically in these
methods, the multiple-enzyme nanocomplex reduces blood ethanol
concentrations in the individual by at least 25, 50, 75 or 100
mg/dL within 90 minutes following administration to the
individual.
[0014] Embodiments of the invention can include three or more
different enzymes encapsulated within the polymeric network, for
example enzymes selected for an ability to react with the products
of the other enzymes in the multiple-enzyme nanocomplex.
Optionally, for example, a third enzyme is an alcohol
dehydrogenase, an aldehyde dehydrogenase or an acetaldehyde
oxidase. In typical embodiments of the invention, one or more
enzymes are coupled to each other and/or to the polymer network. In
certain working embodiments of the invention, the first enzyme and
the second enzyme are coupled together by strands of complimentary
polynucleotides.
[0015] Yet another embodiment of the invention is a method of
decreasing a concentration of hydrogen peroxide in an aqueous
environment. Typically, such methods comprise the steps of
introducing a multiple-enzyme nanocomplex system into the aqueous
environment. In this embodiment of the invention, the
multiple-enzyme nanocomplex system comprises a first enzyme that
generates hydrogen peroxide in a first enzymatic reaction with a
first substrate present in the aqueous environment in combination
with a second enzyme that converts the hydrogen peroxide into water
in a second enzymatic reaction. The multiple-enzyme nanocomplex
system further includes a polymeric network configured to form a
shell that encapsulates the first enzyme and the second enzyme.
This polymeric network encapsulates the first enzyme and the second
enzyme in a manner that inhibits degradation of the first enzyme
and the second enzyme when the multiple-enzyme nanocomplex is
disposed in an in vivo environment. In addition, the polymeric
network is designed to exhibit a permeability sufficient to allow
the substrate to diffuse from an external environment outside of
the shell to the first enzyme so that the hydrogen peroxide is
generated, as well as a permeability sufficient to allow the
hydrogen peroxide to diffuse away from the first enzyme and to the
second enzyme so that the water is generated. In these methods, the
first enzyme and the second enzyme in the multiple-enzyme
nanocomplex system are allowed to react with the substrate and
generate hydrogen peroxide which is then converted to water by the
second enzymes, so that the concentration of hydrogen peroxide in
the aqueous environment is decreased. In one illustrative
embodiment, the first enzyme is an alcohol oxidase; and the second
enzyme is a catalase and the methods are preformed in an in vivo
environment. In another illustrative embodiment, the first enzyme
is a glucose oxidase; and the second enzyme is a horseradish
peroxidase and the methods are preformed in an in vitro
environment.
[0016] Other objects, features and advantages of the present
invention will become apparent to those skilled in the art from the
following detailed description. It is to be understood, however,
that the detailed description and specific examples, while
indicating some embodiments of the present invention are given by
way of illustration and not limitation. Many changes and
modifications within the scope of the present invention may be made
without departing from the spirit thereof, and the invention
includes all such modifications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Throughout the drawings and the application, enzyme
nanocomplexes (ENs) are denoted as n(Enzymes), where Enzymes within
the parentheses refer to the enzymes within the core of the
nanocomplexes. For instance, n(Cat) refers to an EN containing
catalase (Cat). n(HRP-GOx-Inv), in the same vein, refers to an EN
containing horseradish peroxidase (HRP), glucose oxidase (GOx) and
invertase (Inv).
[0018] Other abbreviations includes: AOx: alcohol oxidase; ssDNA:
single strand DNA; EDAC: 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide; NAS: N-hydroxysuccinimide ester; DLS: dynamic light
scattering; TEM: transmission electron microscopy.
[0019] FIG. 1 is a schematic illustration of synthesis of a
triple-enzyme nanocomplex by DNA-directed assembly and
nano-encapsulation. I. Spontaneous assembly of (A) invertase (Inv),
(B) glucose oxidase (GOx), and (C) horseradish peroxidase (HRP)
with an inhibitor-DNA scaffold containing their respective
competitive inhibitors: (a) lactobionic acid, (b) glucosamine, and
(c) 4-dimethylaminoantipyrine leading to the formation of a
triple-enzyme architecture. II. Stabilization of the triple-enzyme
architecture occurs by in-situ growth of a thin network polymer
which spatially constricts the enzyme nanocomplex; III. Removal of
the DNA scaffold leading to the formation of triple-enzyme
nanocomplexes with significantly enhanced stability and
close-proximity definition. Such a close-proximity architecture
enables active transport of their reaction intermediates among the
enzymes, leading to significantly enhanced reaction efficiency.
[0020] FIG. 2 includes images and charts to demonstrate the
structure and enhanced activity and stability of enzyme
nanocomplexes. (a) Transmission electron microscopic (TEM) image of
n(HRP-GOx) with uniform size. (b) TEM image of n(HRP-GOx) prepared
from HRP and GOx that were respectively labeled with a single gold
nanoparticle, confirming a dual-enzyme nanocomplex architecture.
(c) Fluorescence spectra of n(HRP-GOx) and a mixture of n(HRP) and
n(GOx) with the same protein content. The GOx and HRP were
pre-labeled with FITC and RhB, respectively. The spectrum was
recorded using Ex=450 nm. (d) Confocal microscopic images of (i)
n(FITC-labeled GOx) (Ex=488 nm, Em=510-530 nm), (ii) n(RhB-labeled
HRP) (Ex=532 nm, Em=570-600 nm), and n(HRP-GOx) synthesized with
the FITC-labeled GOx and RhB-labeled HRP (Ex=488 nm, Em=570-600
nm). (e) Comparison of enzyme stabilities of n(HRP-GOx),
n(HRP-GOx-Inv) and their native-enzyme-mixture counterparts at
65.degree. C. (1) Comparison of the turnover rates of n(HRP-GOx)
and n(HRP-GOx-Inv) (ENs) with their native-enzyme-mixture
counterparts (Free Enzymes), denoted as 2-enzyme system and
3-enzyme systems, respectively. The turnover rates were normalized
vs. those of the corresponding native enzyme mixtures. (g) Activity
comparison of n(HRP-GOx) and a mixture of native HRP and GOx with
same enzyme content with increasing poly(ethylene glycol) (PEG)
concentration in phosphate buffer (50 mM, pH=7.0). (h) Activity
comparison of n(HRP-GOx-Inv) and a mixture of native HRP, GOx, and
Inv with same enzyme content with increasing PEG concentration. The
relative activities were normalized by the activities of the
free-enzyme mixtures at the same enzyme content and PEG
concentration.
[0021] FIG. 3 presents the in vivo detoxifying capability of
Catalase-containing enzyme nanocomplexes. (a & b) Cell
viability assays after treated with n(GOx), mixture of n(GOx) and
n(Cat), and n(GOx-Cat). Cell proliferation rates were normalized
with those of the untreated cells. Cell viability assays were
performed by incubating the treated cells with CellTiter Blue for 3
h at 37.degree. C., where the viable cells reduced CellTiter Blue
exhibiting a highly fluorescent reddish color. (c&d) Photograph
of a mouse cutaneously injected with PBS, n(Cat), H.sub.2O.sub.2,
n(GOx), and n(GOx-Cat) at different sites and the corresponding
skin tissue slices obtained from each of the injecting sites: (i)
Hematoxylin and eosin (H&E) staining of skin tissue slices of
mice (original magnification 40.times.). (ii) Cell apoptosis in
mouse skin tissue slices. Apoptosis was determined by TUNEL
staining (green) identified with Cy3-conjugated monoclonal
.alpha.-smooth muscle actin antibody (red). DAPI was used for
nuclear staining (blue). The immunostained slices were evaluated
and photographed with fluorescence microscopy (original
magnification 200.times.).
[0022] FIG. 4 presents comparison charts to demonstrate the
efficacy of Oral n(AOx-Cat) Alcohol Antidote. (a) Blood alcohol
concentration (BAC) in mice after gavage with alcohol only, and
alcohol along with native AOx, n(Cat), n(AOx), a mixture of n(AOx)
and n(Cat), or n(AOx-Cat). BAC was determined with an alcohol test
kit. For mice treated with n(AOx-Cat), BAC decreases remarkably
within 3 hr (*P<0.05; **P<0.01) compared to mice fed alcohol
only (EtOH). Four mice were used in each group. (b) Corresponding
plasma alanine aminotransferase (ALT) of the mice. Mice in the
control group were fed with normal food, other groups of mice were
gavaged with a liquid diet containing ethanol along with PBS,
native AOx, n(Cat), n(AOx), a mixture of n(AOx) and n(Cat), or
n(AOx-Cat).
[0023] FIG. 5 shows the DNA-inhibitor scaffolds for syntheses of
n(HRP-GOx) (a) and n(HRP-GOx-Inv) (b).
[0024] FIG. 6 shows MALDI-TOF mass spectra of DNAs before (black)
and after (gray) 5'-modification with inhibitors. (a) ssDNA-1 and
4-Aminoantipyrine (4-AAP) modified ssDNA-1. (b) ssDNA-2 and
glucosamine modified ssDNA-2. (c) ssDNA-I and glucosamine modified
ssDNA-I. (d) ssDNA-II and 4-AAP modified ssDNA-II. (e) ssDNA-III
and lactobionic acid modified ssDNAIII. (0 ssDNA-IV.
[0025] FIG. 7 presents a size distribution chart of (a) n(HRP-GOx)
(gray) and n(HRP-GOx-Inv) (black).
[0026] FIG. 8 shows the viability of cells treated with EtOH and
n(AOx-Cat) in comparison with native AOx and mixture of n(AOx) and
n(Cat).
[0027] FIG. 9, similar to FIG. 4, shows that BAC reductions were
also obtained in animals via tail-vein injections. (a) BAC in mice
after injection of AOx containing nanocapsules (n(AOx), n(Cat),
n(AOx)+n(Cat), and n(AOx-Cat)) or native AOx. For mice treated with
n(AOx-Cat), BAC decreases dramatically before 3 hr (*P<0.05;
**P<0.01; compared to mice fed alcohol only (EtOH); n=4 mice in
EtOH-fed group). (b) ALT in mice treated by EtOH and the
nanocapsules. Mice in control group were fed with normal food,
other groups of mice were injected with different samples (PBS,
native AOx, n(Cat), n(AOx), n(AOx)+n(Cat) mixture, and n(AOx-Cat)),
followed by feeding a liquid diet containing EtOH.
[0028] FIG. 10. Structural characterization of dual-core enzyme
nanocapsule. a. TEM image of HRP-GOx dual-core nanocapsules. Both
HRP and GOx were single-labelled by 1.4 nm gold nanoparticles. b.
Fluorescence spectrum of HRPGOx dual-core nanocapsules including
pair of dyes. The spectrum was recorded at =450 nm (corresponding
the extinction wavelength of FITC) following =490-630 nm. The
emission at =585 nm (peak B), which corresponds to the fluorescence
of Rhodaomine B, is excited by the fluorescence of FTIC at =585 nm
(peak A). c. Confocal microscopy images of HRP-GOx dual-core
nanocapsules that include pair of dyes. HRP and GOx are labeled
with Rhodamine B and FITC, respectively. i. Fluorescence image of
GOx-FITC in dual-core nanocapsules. Image was recorded by the
extinction of FITC at =450 nm following the fluorescence of FITC at
510.about.530 nm. ii. Fluorescence image of HRP-RhB in dual-core
nanocapsules. Image was recorded by the extinction of RhB at =540
nm following the fluorescence of RhB at 570.about.600 nm. iii.
Fluorescence image of HRP-RhB (FRET) in dual-core nanocapsules.
Image was recorded by the extinction of FITC at =450 nm following
the fluorescence of RhB at 570.about.600 nm.
[0029] FIG. 11. Comparison of the activity and stability between
multi-core enzyme nanocapsule and free enzymes system. a. Relative
turnover rate of free enzymes (black) and multicore enzyme
nanocapsule (red). The turnover rates of each system are determined
with the oxidation rates of ODS by various concentration of
substrates (glucose for two enzymes system and sucrose for three
enzymes system) in the present of free enzymes (HRP/GOX for two
enzymes system, HRP/GOx/Inv for three enzymes system) or multicore
enzyme nanocapsules (HRP/GOx dual-core enzyme nanocapsules and
HRP/GOx/Inv triple-core enzyme nanocapsules, respectively).Both the
concentration and the ratio of enzymes in free enzymes system and
multi-core nanocapsules are exactly the same. All assays are
performed in 100 mM phosphate buffer (pH=7.0) b & c. Activity
comparison of two enzymes system b and three enzyme system c in
various concentration of PEG3000 phosphate buffer (50 mM, pH=7.0).
d. [MODEL]. e & f. Thermo-deactivation of free enzyme system
and multi-core enzyme nanocapsules (e. HRP/GOx, f. HRP/GOx/Inv) at
65.degree. C.
[0030] FIG. 12. a. Optical microscopy images of HeLa cells
incubating at 37.degree. C. for 2.5 h in present of uric acid (1.2
mg/mL, the black crystal in all pictures), PBS (10 .mu.L, for
control experiments, i), Uricase only (10 .mu.L, 0.0013 mg/mL, ii),
Uricase and Catalase (10 .mu.L in total, UOx 0.0013 mg/mL, Cat
0.0026 mg/mL, iii), and Uricase-Catalase dual-core nanocapsules (10
.mu.L, UOx 0.0013 mg/mL, Cat 0.0026 mg/mL, iv). b. Relative
turnover rates of uricase only, free uricase & catalase and
uricase-catalase dual-core nanocapsules. The turnover rates are
determined by the oxidation of uric acid. All concentrations of
uricase in each system are the same, and the concentration of
catalase in free UOx/Cat system and UOx/Cat dual-core nanocapsules
are also the same. c. Deactivation of free UOx/Cat system (black)
and UOx/Cat dual-core nanocapsules (red) by the proteinase
(trypsin). d. Cytotoxicity of UOx only system, free UOx/Cat system
and UOx/Cat dual-core nanocapsules.
[0031] FIGS. 13A-13C. In working embodiments of the invention,
multiple-enzyme nanocomplex systems were designed to deliver
functional enzymes orally in vivo. In these systems, an alcohol
oxidase (AOX) and a catalase enzyme were incorporated into small
polymer nanocapsules via an overall scheme is presented in FIG.
13A. In FIG. 13A, step I provides a schematic of the conjugation of
alcohol oxidase (AOX) and catalase (CAT) while step II provides a
schematic of the encapsulation of AOX-CAT through surface
modification and in-situ polymerization. In the first step, AOX was
oxidized with sodium periodate to produce reactive aldehyde
residues, to which CAT was added to produce a stabilized AOX-CAT
complex after conjugation by reductive animation using sodium
cyanoborohydride. This is summarized in FIG. 13B which provides a
schematic of the creation of AOX-CAT stabilized complexes. In step
I in FIG. 13B, AOX (a glycoprotein), is oxidized with sodium
periodate to produce reactive aldehyde residues. In step II FIG.
13B , the modified AOX was conjugated to CAT. In step III in FIG.
13B, conjugation was completed by reductive animation using sodium
cyanoborohydride. FIG. 13C then provides a schematic of the
Packaging of AOX-CAT complexes into nanocapsules. As shown in FIG.
13C, the AOX-CAT complex was first acryloxylated by reacting with
acrylic acid N-hydroxysuccinimide ester (NAS), followed by in situ
polymerization. In step I in FIG. 13C, the AOX-CAT conjugate
undergoes acryloxylation by reacting with acrylic acid
N-hydroxysuccinimide ester (NAS). As shown in step II in FIG. 13C,
this then allows in situ polymerization coating to produce
nanocapsules. The resulting multiple-enzyme nanocomplex system
contains functional AOX and CAT in complexes, allowing metabolism
of ethanol into aldehyde and then water and oxygen. The
nanocapsules are permeable to small molecule substrates (such as
ethanol), and can be designed to deliver enzymes into cells as
well. Such multiple-enzyme nanocomplex systems are particularly
suited for delivery to the liver due to the flow of blood from the
intestinal tract to the portal vein. In addition, these
nanocapsules are stable at normal body temperature as well as
protease resistant. The nanocapsules further protect the enzymes
from digestive processes, a feature which allows their uptake from
the gastrointestinal tract into blood and cells.
[0032] FIG. 14. Graphical data from experiments showing the
acceleration of ethanol metabolism in vivo through the use of
orally delivered multiple-enzyme nanocomplex systems in mice. In
these experiments, groups of three mice received ethanol and
either: Nothing (open circles), AOX enzyme unencapsulated (open
squares), or multiple-enzyme nanocapsules comprising AOX-CAT
complexes (closed triangles) through oral gavage. Blood alcohol
concentrations (mg/dL) are plotted over time.
DETAILED DESCRIPTION
[0033] Before the compositions and methods are described, it is to
be understood that the invention is not limited to the particular
methodologies, protocols, cell lines, assays, and reagents
described, as these may vary. It is also to be understood that the
terminology used herein is intended to describe particular
embodiments of the present invention, and is in no way intended to
limit the scope of the present invention as set forth in the
appended claims.
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All technical and patent publications cited herein are
incorporated herein by reference in their entirety. Nothing herein
is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0035] When a numerical designation is preceded by the term
"about", it varies by (+) or (-) 10%, 5% or 1%. When "about" is
used before an amount, for example, in mg, it indicates that the
weight value may vary (+) or (-) 10%, 5% or 1%.
Definitions
[0036] In accordance with the present invention and as used herein,
the following terms are defined with the following meanings, unless
explicitly stated otherwise.
[0037] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0038] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
not excluding others. "Consisting essentially of" when used to
define compositions and methods, shall mean excluding other
elements of any essential significance to the combination. For
example, a composition consisting essentially of the elements as
defined herein would not exclude other elements that do not
materially affect the basic and novel characteristic(s) of the
claimed invention. "Consisting of" shall mean excluding more than
trace amount of other ingredients and substantial method steps
recited. Embodiments defined by each of these transition terms are
within the scope of this invention.
[0039] As used herein, the term "nanocomplex" refers to a structure
sized between one and a few hundred nanometers in at least one
dimension. In some embodiments, it refers to a structure containing
a finite number of chemical and/or biological molecules optionally
connected with polymers.
[0040] As used herein, the term "spatially constrained" refers to
the location of a particle, such as an enzyme, being relatively
limited in space relative to another object, such as a polymer
network. In some embodiments, it refers to two or more particles
having substantially fixed locations relative to each other.
[0041] As used herein, the term "polymeric network" or
alternatively "polymeric mesh" refers to one or more polymers
interconnected within and/or between each other to form a net or
mesh. In some embodiments, the polymeric network is anchored to one
or more particles at one or more locations.
[0042] As used herein, the term "cooperatively" refers to two or
more enzymes functioning in a concerted manner. One example is that
the two or more enzymes form an enzyme complex, or alternatively
react with the same substrate simultaneously, to carry out their
activities. In another embodiment, one of the enzymes reacts with
the substrate first, producing a product, which in turn, is a
substrate that reacts with another of the enzymes.
[0043] As used herein, the term "metabolic pathway" refers to a
series of chemical reactions occurring within a cell. In each
pathway, a principal chemical is modified by a series of chemical
reactions. Lists of metabolic pathways are available publicly from,
e.g., the KEGG Pathway Database (available at
www.genome.jp/kegg/pathway.html).
Multiple-Enzyme Nanocomplexes
[0044] Enzymes are exquisite biocatalysts mediating every
biological process in living organisms. In eukaryote cells, most
enzymes are not freely diffused within the cytosols, but are
spatially defined within subcellular organelles or closely
co-localized as enzyme complexes along with other proteins (see,
e.g. Conrado et al., Curr Opin Biotech 19, 492-499, (2008); Yan et
al., J Proteomics 72, 4-11, (2009)). In consecutive reactions
catalyzed by multiple enzymes, such close confinement minimizes
diffusion of intermediates among the enzymes, enhancing overall
reaction efficiency and specificity (see, e.g. Yan et al., J
Proteomics 72, 4-11, (2009)).
[0045] Typically in vivo, toxic intermediates generated during a
metabolic process are promptly eliminated by the proximate enzymes
designed to degrade such toxins and which are co-localized within
the confined structures (see, e.g. Kristensen et al., Natl Acad Sci
USA 102, 1779-1784, (2005)). Peroxisomes, as an example, harbor a
variety of oxidases with important metabolic and catabolic
functions; toxic intermediates, such as hydrogen peroxide
(H.sub.2O.sub.2) (see, e.g. Wanders et al., Annu Rev Biochem 75,
295-332, (2006); and Schrader et al., Histochem Cell Biol 122,
383-393, (2004)). Interestingly, nature circumvents this dilemma by
incorporating catalase (Cat) within the peroxisomes. Catalase is
highly active and specific in decomposing H.sub.2O.sub.2; rapid
elimination of the as-generated H.sub.2O.sub.2prevents its
diffusion out from the peroxisomes and damage to other cellular
components (see, e.g. Fujiwara et al., J Biol Chem 275,
37271-37277, (2000); and Sheikh et al., P Natl Acad Sci USA 95,
2961-2966, (1998)).
[0046] Although multiple-enzyme architectures commonly exist in
nature, current enzyme-based applications, including enzyme
catalysis and therapeutics, are still limited to the use of single
enzyme or their mixtures (see, e.g. Leader et al., Nat Rev Drug
Discov 7, 21-39, (2008); Iso et al., J Microencapsul 6, 165-176,
(1989); Nouaimi-Bachmann et al., Biotechnol Bioeng 96, 623-630,
(2007); and Mateo et al., Enzyme Microb Tech 40, 1451-1463,
(2007)). Inorganic and polymeric materials incorporating multiple
enzymes have been extensively studied; however, the enzymes were
randomly immobilized within these materials in the forms of thin
films or large particles (.about.100 .mu.m in diameter), excluding
their therapeutic applications (see, e.g., Srere et al., P Natl
Acad Sci USA 70, 2534-2538, (1973); Mansson et al., P Natl Acad
Sci--Biol 80, 1487-1491, (1983); Kochschmidt et al., Eur J Biochem
81, 71-78, (1977) ; and Sheldon et al., Adv Synth Catal 349,
1289-1307, (2007)).
[0047] Fusion-protein techniques provide one strategy to construct
multiple-enzyme architectures (see, e.g. Riedel et al., Mol
Microbiol 28, 767-775, (1998); Shibuya et al., Biosci Biotech Bioch
56, 884-889, (1992); and Mao et al., J Biol Chem 270, 18295-18300,
(1995). Ideally, such architectures with designable functions may
be achieved by judiciously engineering the genomic inputs
transfected to the host cells; nonetheless, this approach often
leads to a loss or decreased enzyme specificity and activity (see,
e.g. Stempfer et al., Nat Biotechnol 14, 481-484, (1996); and Seo
et al., Appl Environ Microb 66, 2484-2490, (2000)). Moreover,
finding suitable host cells and designing suitable spacers further
place hurdles in the construction of fusion proteins (see, e.g.
Bulow et al., Bio-Technol 3, 821-823, (1985)). Recently,
post-translational assemblies were used to construct enzyme
complexes; however, such an assembling process solely relies on
specific bindings between the enzyme components and lacks general
applicability (see, e.g., Mingardon et al., Appl Environ Microb 73,
7138-7149, (2007); Fierobe et al., J Biol Chem 276, 21257-21261,
(2001) ; and Fierobe et al., J Biol Chem 277, 49621-49630,
(2002)).
[0048] The present disclosure provides multiple-enzyme
nanocomplexes, or enzyme nanocomplexes (ENs), that show enhanced
catalytic efficiency and increased stability, and at the same time
can reduce, when necessary, the release of intermediates that may
be toxic to the environment.
[0049] The invention disclosed herein has a number of embodiments.
Embodiments of the present invention include nanocomplexes
containing at least two different enzymes, and a polymer network.
Typically, the polymer network is anchored to at least one of the
enzymes. Such multiple-enzyme complexes enable the enzymes to carry
out their activities cooperatively. As demonstrated in the
experimental examples, these enzyme complexes (termed "Enzyme
Nanocomplexes (EN)") exhibit enhanced catalytic efficiencies as
well as an increased stability. Simultaneously, these enzyme
complexes can be designed to break down specific compounds, for
example those that can be toxic in in vivo environments (e.g.
hydrogen peroxide). In some embodiments, the activities of the
enzymes catalyze a cascade reaction. Further, the function of the
complexes is programmable through a simple chemical approach.
[0050] Embodiments of the invention include a multiple-enzyme
nanocomplex comprising at least two different enzymes and a
polymeric network. Typically, the polymer network is anchored to at
least one of the at least two different enzymes. In one embodiment,
the polymeric network is anchored to all of the at least two
different enzymes. In illustrative embodiments of the invention,
the at least two different enzymes are covalently or non-covalently
linked to one another. In one aspect, the covalent linkage is
degradable in vivo. In another aspect, the at least two different
enzymes are not linked to one another. In some embodiments,
different enzymes in the nanocomplex are anchored together, for
example via complementary polynucleotide strands. In some
embodiments of the invention, the polynucleotide strands are less
than 50, 45, 40, 35, 30, 25, 20 or 15 nucleotides in length. In
certain embodiments of the invention, the two different enzymes are
anchored in a manner that makes them a distance apart of less than
50, 40, 30, 20 or 10 nm.
[0051] Embodiments of the invention include a multiple-enzyme
nanocomplex comprising at least two different enzymes and a
permeable shell, the shell comprising a polymeric network which is
anchored to at least one of the at least two different enzymes. The
present disclosure further provides, in another embodiment, a
multiple-enzyme nanocomplex consisting essentially of a polymeric
network and at least two different enzymes, the polymeric network
is covalently anchored to at least one of the at least two
different enzymes and provides a permeable shell around the at
least two different enzymes. In one embodiment, the polymeric
network comprises a polymer composed of at least one monomeric
unit. In another embodiment, the polymeric network comprises a
polymer composed of at least two different monomeric units. In some
embodiments, the polymeric network further comprises a
crosslinker.
[0052] Yet another embodiment of the invention is multiple-enzyme
nanocomplex system that includes elements and architectures (e.g.
specific 3-dimensional constellations of elements) combined
together in a manner designed to facilitate certain enzymatic
reactions. Working embodiments of the systems disclosed herein
include a first enzyme that generates hydrogen peroxide in a first
enzymatic reaction with a first substrate, a second enzyme that
converts the hydrogen peroxide into water (H.sub.2O) and/or oxygen
(O.sub.2) in a second enzymatic reaction, and a polymeric network
configured to form a shell that encapsulates the first enzyme and
the second enzyme. In such system embodiments the polymeric network
exhibits a permeability sufficient to allow the first substrate to
diffuse from an external environment outside of the shell to the
first enzyme so that the hydrogen peroxide is generated. The
polymer network in this embodiment is designed to further exhibit a
permeability sufficient to allow the hydrogen peroxide to diffuse
away from the first enzyme and to the second enzyme so that the
water is generated. In certain embodiments of the invention, the
first enzyme is coupled to the second enzyme. In some embodiments
of the invention, the polymeric network is coupled to the first
polypeptide or the second polypeptide.
[0053] In typical systems of the invention, the polymeric network
encapsulates the first enzyme and the second enzyme in a manner
sufficient to inhibit degradation of the first enzyme or the second
enzyme when the multiple-enzyme nanocomplex is disposed in an in
vivo environment. In common embodiments, the first enzyme and the
second enzyme are coupled together, optionally for example, through
the use of a chemical linker or tether. In certain working
embodiments of the invention that are disclosed herein, the first
enzyme and the second enzyme are coupled together by strands of
complimentary polynucleotides.
[0054] A wide variety of enzymes can be used in various embodiments
of the invention. In one of the working embodiments disclosed
herein, the first enzyme is an alcohol oxidase; and the second
enzyme is a catalase. In another of the working embodiments
disclosed herein, the first enzyme is a glucose oxidase; and the
second enzyme is a horseradish peroxidase. Certain embodiments of
the invention include a third enzyme encapsulated within the
polymeric network (e.g. an aldehyde dehydrogenase, an alcohol
dehydrogenase or the like).
[0055] Embodiment of the invention include methods for using a
multiple-enzyme nanocomplex system that includes elements and
architectures (e.g. specific 3-dimensional constellations of
elements) designed to facilitate multiple enzymatic reactions.
Working embodiments of the systems disclosed herein include a first
enzyme that generates hydrogen peroxide in a first enzymatic
reaction with a first substrate, a second enzyme that converts the
hydrogen peroxide into water in a second enzymatic reaction, and a
polymeric network configured to form a shell that encapsulates the
first enzyme and the second enzyme. In such system embodiments, the
polymeric network exhibits a permeability sufficient to allow the
first substrate to diffuse from an external environment outside of
the shell to the first enzyme so that the hydrogen peroxide is
generated and further exhibits a permeability sufficient to allow
the hydrogen peroxide to diffuse away from the first enzyme and to
the second enzyme so that the water is generated. In certain
embodiments of the invention, the first enzyme is coupled to the
second enzyme. In some embodiments of the invention, the polymeric
network is coupled to the first polypeptide or the second
polypeptide.
[0056] Embodiments of the invention include methods of decreasing
the concentration of ethanol in an individual, for example a
patient suffering from acute alcohol intoxication. These methods
include the steps of administering a multiple-enzyme nanocomplex
system to the individual. In these methods, the multiple-enzyme
nanocomplex system comprises an alcohol oxidase enzyme that
generates hydrogen peroxide in a first enzymatic reaction with
ethanol as well as a catalase enzyme that converts the hydrogen
peroxide into water in a second enzymatic reaction. In this
embodiment; a polymeric network configured to form a shell that
encapsulates the alcohol oxidase and the catalase and, for example,
protect these polypeptides from proteolytic degradation (e.g.
proteases present in in vivo environments). Moreover, the polymeric
network exhibits a permeability sufficient to allow the ethanol to
diffuse from an external environment outside of the shell to the
alcohol oxidase so that the hydrogen peroxide is generated, as well
as a permeability sufficient to allow the hydrogen peroxide to
diffuse away from the alcohol oxidase and to the catalase so that
the water is generated. In addition, in typical embodiments of the
invention, the alcohol oxidase is coupled to the catalase; or the
polymeric network is coupled to the alcohol oxidase or the
catalase. In the methods of using these multiple-enzyme nanocomplex
systems, their administration allows the alcohol oxidase and the
catalase in the multiple-enzyme nanocomplex system to react with
ethanol in the individual and generate hydrogen peroxide and water,
so that the concentration of ethanol in the individual is
decreased.
[0057] Embodiments of the invention are useful in a variety of
contexts. For example, in some embodiments of the invention, the
multiple-enzyme nanocomplex system is administered prior to the
consumption of ethanol so as to avoid ethanol intoxication. In
certain embodiments, the multiple-enzyme nanocomplex system is
administered orally. In other embodiments of the invention, the
multiple-enzyme nanocomplex system is administered following the
consumption of an ethanol by an individual suffering from acute
ethanol intoxication. In certain embodiments of the invention, the
multiple-enzyme nanocomplex system is administered parenterally.
Typically in these methods, the multiple-enzyme nanocomplex system
reduces blood ethanol concentrations in the individual by at least
25, 50, 75 or 100 mg/dL within 90 minutes following administration
to the individual.
[0058] To confirm the power of working embodiments of the
invention, we tested nanocapsules comprising AOX-CAT in mice after
delivery by the gastric route. As shown for example in FIGS. 9 and
14, mice that received the treatment had considerably more rapid
clearance of ethanol compared to control mice. These data
demonstrate the feasibility of using nanocapsules to deliver
functional enzymes. Moreover, this approach can be adapted for use
with enzymes such as human alcohol dehydrogenase and aldehyde
dehydrogenase, as well as other key metabolic enzymes that are
lacking in diseases such as glycogen storage disorders (e.g.
glucose-6-phosphatase, acid maltase, glycogen branching enzyme) or
lipid metabolism disorders (e.g. coenzyme A dehydrogenases,
carnitine transporter). In addition, these nanocapsules can be
designed for hepatic uptake and intracellular delivery of
enzymes.
[0059] Embodiments of the invention a third enzyme encapsulated
within the polymeric network, for example one selected for an
ability to react with the products of the other enzymes in the
multiple-enzyme nanocomplex system. Optionally, for example, the
third enzyme is an alcohol dehydrogenase, an aldehyde dehydrogenase
or an acetaldehyde oxidase. In typical embodiments of the
invention, one or more enzymes are coupled to each other and/or to
the polymer network. In certain working embodiments of the
invention, the first enzyme and the second enzyme are coupled
together by strands of complimentary polynucleotides.
[0060] Embodiments of the invention also include a multiple-enzyme
nanocomplex system for use in a patient for the treatment of a
condition resulting from the consumption of alcohol. In such
embodiments, the multiple-enzyme nanocomplex system comprises an
alcohol oxidase enzyme that generates hydrogen peroxide in a first
enzymatic reaction with alcohol in combination with a catalase
enzyme that converts the hydrogen peroxide into water in a second
enzymatic reaction. In these embodiments, the multiple-enzyme
nanocomplex system includes a polymeric network configured to form
a protective shell that encapsulates the alcohol oxidase and the
catalase. This shell is designed to form a polymeric matrix that
exhibits a permeability sufficient to allow the alcohol to diffuse
from an external environment outside of the shell to the alcohol
oxidase so that the hydrogen peroxide is generated as well as a
permeability sufficient to allow the hydrogen peroxide to diffuse
away from the alcohol oxidase and to the catalase so that the water
is generated. Typically in embodiments of the invention, the
alcohol oxidase is coupled to the catalase; and/or the polymeric
network is coupled to the alcohol oxidase or the catalase.
[0061] In certain embodiments of the invention, the multiple-enzyme
nanocomplex system is used in these methods to reduce the blood
alcohol concentration of the patient by at least 10%, 20% or 30%
within 90 minutes following administration. In some embodiments of
the invention, the method used to treat a pathological condition
that results from the consumption of alcohol, for example, acute
alcohol intoxication. Optionally, in these embodiments, the
multiple-enzyme nanocomplex system is administered parenterally. In
other embodiments of the invention, the method is used to
ameliorate a condition that results from the consumption of
alcohol, for example, a hangover. Optionally, in these embodiments,
the multiple-enzyme nanocomplex system is administered orally. In
certain embodiments of the invention, the alcohol oxidase enzyme
and catalase enzyme are disposed within the polymeric network at a
distance from each other of less than 50, 40, 30, 20 or 10 nm.
[0062] Yet another embodiment of the invention is a method of
decreasing a concentration of hydrogen peroxide in an aqueous
environment. Typically, this method comprises the steps of
introducing a multiple-enzyme nanocomplex system into the aqueous
environment. In this embodiment of the invention, the
multiple-enzyme nanocomplex system comprises a first enzyme that
generates hydrogen peroxide in a first enzymatic reaction with a
first substrate present in the aqueous environment in combination
with a second enzyme that converts the hydrogen peroxide into water
in a second enzymatic reaction. The multiple-enzyme nanocomplex
system further includes a polymeric network configured to form a
shell that encapsulates the first enzyme and the second enzyme.
This polymeric network encapsulates the first enzyme and the second
enzyme in a manner that inhibits degradation of the first enzyme
and the second enzyme when the multiple-enzyme nanocomplex is
disposed in an in vivo environment. In addition, the polymeric
network is designed to exhibit a permeability sufficient to allow
the substrate to diffuse from an external environment outside of
the shell to the first enzyme so that the hydrogen peroxide is
generated, as well as a permeability sufficient to allow the
hydrogen peroxide to diffuse away from the first enzyme and to the
second enzyme so that the water is generated. In these methods, the
first enzyme and the second enzyme in the multiple-enzyme
nanocomplex system are allowed to react with the substrate and
generate hydrogen peroxide which is then converted to water by the
second enzymes, so that the concentration of hydrogen peroxide in
the aqueous environment is decreased. In one illustrative
embodiment, the first enzyme is an alcohol oxidase; and the second
enzyme is a catalase and the methods are preformed in an in vivo
environment. In another illustrative embodiment, the first enzyme
is a glucose oxidase; and the second enzyme is a horseradish
peroxidase and the methods are preformed in an in vitro
environment. In certain embodiments of the invention, an enzyme
encapsulated within the polymeric network is selected from the
group consisting of an alcohol oxidase, a catalase, a peroxidase,
an alcohol dehydrogenase, an aldehyde dehydrogenase or an
acetaldehyde oxidase.
[0063] Embodiments of the invention also include methods for
producing the multiple-enzyme nanocomplexes discussed above.
Optionally, these methods can comprise the steps of: coupling the
alcohol oxidase enzyme and the catalase enzyme to each other,
acryloxylating the coupled alcohol oxidase enzyme and/or catalase
enzyme, and then polymerizing the acryloxylated alcohol oxidase
enzyme and catalase enzyme in situ, using at least one monomeric
unit, and optionally a crosslinker, so that a multiple-enzyme
nanocomplex system is formed.
[0064] As noted above, a wide variety of enzymes can be used in
various embodiments of the invention. Certain embodiments of the
invention can comprise a catalase that can catalyze the
decomposition of hydrogen peroxide to water and/or oxygen.
Catalases can also catalyze the oxidation, by hydrogen peroxide, of
various metabolites and toxins, including formaldehyde, formic
acid, phenols, acetaldehyde and alcohols. Catalases typically do so
according to the following reaction:
H2O2+H2R.fwdarw.2H2O+R
A number of catalase polypeptides that can be used in embodiments
of the invention are well known in the art; for example, catalase
polypeptides derived from human erythrocytes (e.g. SIGMA-ALDRICH
product number C3556), bovine liver (e.g. SIGMA-ALDRICH product
number C1345), bison liver (e.g. SIGMA-ALDRICH product number
C9447), Aspergillus niger (e.g. SIGMA-ALDRICH product number
C3515), Corynebacterium glutamicum (e.g. SIGMA-ALDRICH product
number 02071), Micrococcus lysodeikticus (e.g. SIGMA-ALDRICH
product number 60634) etc.
[0065] Certain embodiments of the invention can comprise a
peroxidase. Peroxidases (EC number 1.11.1.x) are enzymes that
typically catalyze a reaction of the form:
ROOR'+electron donor (2e-)+2H+.fwdarw.ROH+R'OH
For many of these enzymes the optimal substrate is hydrogen
peroxide, but others are more active with organic hydroperoxides
such as lipid peroxides. Peroxidases can contain a heme cofactor in
their active sites, or alternately redox-active cysteine or
selenocysteine residues. A number of peroxidase polypeptides that
can be used in embodiments of the invention are known in the art;
for example, peroxidase polypeptides derived from horseradish,
(e.g. SIGMA-ALDRICH product number P8375), glutathione peroxidase
derived from human erythrocytes, (e.g. SIGMA-ALDRICH product number
G4013) etc.
[0066] Certain embodiments of the invention can comprise an alcohol
oxidase. Typical alcohol oxidases (EC 1.1.3.13) catalyze the
chemical reaction:
a primary alcohol+O.sub.2.fwdarw.an aldehyde+H.sub.2O.sub.2
In this illustration, the two substrates of this enzyme are primary
alcohol and O.sub.2, and its two products are aldehyde and
H.sub.2O.sub.2. A number of alcohol oxidase polypeptides that can
be used in embodiments of the invention are known in the art; for
example those derived from yeast (e.g. SIGMA-ALDRICH product number
A6941), Hansenula polymorpha (e.g. SIGMA-ALDRICH Catalog Number
A0438), Pichea pastoris (e.g. MP BIOMEDICALS Catalog Number:
190155) etc.
[0067] Certain embodiments of the invention can comprise an
aldehyde oxidase. Typical aldehyde oxidase enzymes generate
carboxylic acids from aldehydes, for example by catalyzing the
conversion of an aldehyde in the presence of oxygen and water to an
acid and hydrogen peroxide:
an aldehyde+H2O+O2<=>a carboxylate+H2O2+H+
A number of aldehyde oxidase polypeptides that can be used in
embodiments of the invention are known in the art; for example
human recombinant polypeptides (e.g. CYPEX product number CYP150,
LABOME product number TP319221) etc.
[0068] Certain embodiments of the invention can comprise a glucose
oxidase. Glucose oxidases are oxido-reductases that catalyze the
oxidation of glucose to hydrogen peroxide and
D-glucono-.delta.-lactone. A number of glucose oxidase polypeptides
that can be used in embodiments of the invention are known in the
art; for example those derived from Aspergillus niger (e.g.
SIGMA-ALDRICH product number G7141), Dactylium dendroides (e.g.
SIGMA-ALDRICH product number G7907) etc.
[0069] Certain embodiments of the invention can comprise an
invertase, for example to catalyze the hydrolysis of sucrose. A
number of invertase polypeptides that can be used in embodiments of
the invention are known in the art; for example, invertase
polypeptides purified from organisms such as yeast (e.g.
SIGMA-ALDRICH product number 14504) etc.
[0070] Certain embodiments of the invention can comprise a urate
oxidase. Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal
liver enzyme that catalyses the enzymatic oxidation of uric acid
into the more water-soluble allantoin. Urate oxidase is an
endogenous enzyme found in most mammals but not in humans. Urate
oxidase is used in humans for the control of increased serum uric
acid in patients with acute tumor lysis syndrome after receiving
chemotherapy. Rasburicase (SR 29142), a recombinant urate oxidase
expressed in Saccharomyces cerevisiae, has been demonstrated to
control of uric acid in a trial of patients at risk of acute tumour
lysis syndrome. In addition, urate oxidase is also used in the
treatment of gout. A number of urate oxidase polypeptides that can
be used in embodiments of the invention are known in the art; for
example recombinant forms (e.g. Rasburicase) as well as forms
derived from Aspergillus flavus (e.g. PROSPEC BIO product number
ENZ-312), yeast (e.g. WORTHINGTON BIOCHEMICAL product no. LS003857)
etc.
[0071] Certain embodiments of the invention can comprise an
aldehyde dehydrogenase such as a acetaldehyde dehydrogenase.
Acetaldehyde dehydrogenases (EC 1.2.1.10) are dehydrogenase enzymes
which catalyze the conversion of acetaldehyde into acetic acid. The
oxidation of acetaldehyde to acetate can be summarized as
follows:
CH3CHO+NAD++CoA.fwdarw.acetyl-CoA+NADH+H+
In humans, there are a number of genes which encode this enzymatic
activity including, ALDH1A1, ALDH2, and ALDH1B1 (also known as
ALDH5). These enzymes are members of the larger class of aldehyde
dehydrogenases. The CAS number for this type of the enzyme is
[9028-91-5]. Certain embodiments of the invention can comprise an
aldehyde dehydrogenase, for example to catalyze the oxidation
(dehydrogenation) of aldehydes. A number of aldehyde dehydrogenase
polypeptides that can be used in embodiments of the invention are
known in the art; for example, recombinant human aldehyde
dehydrogenase polypeptides (e.g. R & D SYSTEMS product number
5869-DH), as well as aldehyde dehydrogenase polypeptides purified
from organisms such as yeast (e.g. SIGMA-ALDRICH product number
A6338) etc.
[0072] Certain embodiments of the invention can comprise an alcohol
dehydrogenase, for example to facilitate the interconversion
between alcohols and aldehydes or ketones with the reduction of
nicotinamide adenine dinucleotide (NAD+ to NADH). A number of
alcohol dehydrogenase polypeptides that can be used in embodiments
of the invention are known in the art; for example, purified human
alcohol dehydrogenase polypeptides (e.g. ARP AMERICAN RESEARCH
PRODUCTS INC. product number 01-1746), recombinant human alcohol
dehydrogenase polypeptides (e.g. MYBIOSOURCE Catalog #MBS203604),
as well as alcohol dehydrogenase polypeptides derived from Equine
liver (e.g. SIGMA-ALDRICH product number 05646), Saccharomyces
cerevisiae (e.g. SIGMA-ALDRICH product number A7011), Parvibaculum
lavamentivorans (e.g. SIGMA-ALDRICH product number 75449) etc.
[0073] Certain embodiments of the invention can comprise a formate
dehydrogenase, for example to catalyze the oxidation of formate to
bicarbonate. A number of formate dehydrogenase polypeptides that
can be used in embodiments of the invention are known in the art;
for example, recombinant polypeptides (e.g. SIGMA-ALDRICH product
number 75274) as well as formate dehydrogenase polypeptides
purified from organisms such as yeast (e.g. SIGMA-ALDRICH product
number F8649) etc.
[0074] Certain embodiments of the invention can comprise a
glucose-6-phosphatase, for example to hydrolyze glucose-6-phosphate
in a manner that generates a phosphate group and free glucose. A
number of glucose-6-phosphatase polypeptides that can be used in
embodiments of the invention are known in the art; for example,
glucose-6-phosphatase polypeptides purified from rabbit liver (e.g.
SIGMA-ALDRICH product number G5758) etc.
[0075] Certain embodiments of the invention can comprise a
glucosidase, for example, one useful for the degradation of
glycogen to glucose (e.g. an acid maltase). A number of
.alpha.-glucosidase polypeptides that can be used in embodiments of
the invention are known in the art; for example, recombinant human
.alpha.-glucosidase .alpha. (e.g. NOVUS BIOLOGICALS product No.
H00023193-P01) as well as -glucosidase polypeptides purified from
organisms such as yeast (e.g. SIGMA-ALDRICH product number G0660)
etc.
[0076] Certain embodiments of the invention can comprise a glycogen
branching enzyme, for example one useful in converting glucose to
glycogen. A number of glycogen branching enzyme polypeptides that
can be used in embodiments of the invention are known in the art;
for example, recombinant human .alpha.-glucosidase .alpha. (e.g.
NOVUS BIOLOGICALS product No. H0002632-P01) as well as -glucosidase
polypeptides purified from organisms such as E. Coli (e.g. PROZOMIX
product number PRO-E0406) etc.
[0077] Certain embodiments of the invention can comprise a coenzyme
A dehydrogenase, for example, one useful to catalyze a step in each
cycle of fatty acid .beta.-oxidation. A number of coenzyme A
dehydrogenase polypeptides that can be used in embodiments of the
invention are known in the art; for example, recombinant human
coenzyme A dehydrogenase polypeptides (e.g. MYBIOSOURCE Catalog
#MBS142535) etc.
[0078] Certain embodiments of the invention can comprise a organic
cation/carnitine transporter, for example, one useful as an organic
cation transporter and/or as a sodium-dependent high affinity
carnitine transporter. A number of OCTN2 polypeptides that can be
used in embodiments of the invention are known in the art; for
example, recombinant human OCTN2 (e.g. NOVUS BIOLOGICALS SLC22A5
Recombinant Protein product no. H00006584-P01) etc.
[0079] In addition to the illustrative enzymes disclosed above, any
one of the large number of polypeptides encoded by known genes
(e.g. those disclosed herein and listed in GENBANK,
UniProtKB/Swiss-Prot and the like) can be readily made by
commercially available recombinant protein services such as those
provided by GENSCRIPT.
[0080] In some embodiments of the invention, a composition or
system embodiment of the invention is disposed in a kit comprising
a container and instructions for using the multiple-enzyme
nanocomplex system. Optionally in such kits, the system is combined
with one or more filling agents, binding agents or buffering agents
adapted for use in an orally administered formulation. In some
embodiments of the invention, the system is combined with one or
more filling agents, binding agents or buffering agents adapted for
use in an parenterally administered formulation.
[0081] Embodiments of the invention include articles of manufacture
and/or kits, for example those containing materials useful in
breaking down certain compounds in vivo such as hydrogen peroxide.
Optionally, the articles of manufacture and/or kits can contain
materials useful in treating a pathological condition such as acute
alcohol poisoning. In typical embodiments of the invention, the kit
comprises at least one container, typically with a label. Suitable
containers include, for example, blister packs, bottles, vials, and
test tubes. The containers may be formed from a variety of
materials such as metal (e.g. a metal foil), glass or plastic. In
some embodiments of the invention, the one or more containers holds
one or more compositions having an active agent which is effective
in imaging techniques. In certain embodiments of the invention, the
one or more containers holds one or more compositions having an
active agent which is effective in treating a pathological
condition such as acute alcohol poisoning. Typically, the label on
the one or more containers indicates that the one or more
compositions is used for in treating a pathological condition. Such
labels may also indicate directions for either in vivo or in vitro
use, such as those described herein. The kits of the invention can
also comprise the one or more containers described above and a
further container comprising a buffer. Kits may further include
other materials desirable from a commercial and user standpoint,
including other buffers, diluents, filters, needles, syringes, and
package inserts with instructions for use.
[0082] In certain embodiments of the invention, a composition or
enzyme nanocomplex system disclosed herein is formulated as an
agent designed to ameliorate or treat a pathological condition such
as alcohol toxicity. For example, in certain embodiments of the
invention, a composition or enzyme nanocomplex system disclosed
herein is formulated as an agent designed to prevent a pathological
condition such as alcohol toxicity. The agent may be administered
via a conventional route normally used to administer a medicament
including, but not limited to, oral and parenteral (including
subcutaneous, intradermal, intramuscular, intravenous,
intraarticular, and intramedullary) routes. Intravenous delivery
may take place via a bolus injection or via infusion; infusion may
be done over a period ranging from less than a minute to several
hours to continuously. In certain embodiments, a course of
treatment will involve administration by a combination of
routes.
[0083] The agent may be administered via a combination of
intravenous and oral routes. In one embodiment, a "loading" dose
may be administered IV in order to bring the concentration of drug
to the desired therapeutic level, followed by one or more
maintenance doses via the oral route to keep it there. The agent
may be administered as a pharmaceutical composition in a variety of
forms including, but not limited to, liquid, powder, suspensions,
tablets, pills, capsules, sprays and aerosols. The pharmaceutical
compositions may include various pharmaceutically acceptable
additives including, but not limited to, carriers, excipients,
binders, stabilizers, antimicrobial agents, antioxidants, diluents
and/or supports. Examples of suitable excipients and carriers are
described, for example, in "Remington's Pharmaceutical Sciences,"
Mack Pub. Co.; 17th edition New Jersey (2011). In some embodiments,
the agent may be administered via an IV infusion in an aqueous
sugar solution. The agent may also be associated with another
substance that facilitates agent delivery.
[0084] Formulations of the compounds disclosed herein suitable for
oral administration may be presented as discrete units such as
capsules, cachets or tablets each containing a predetermined amount
of the active ingredient; as a powder or granules; as a solution or
a suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The
active ingredient may also be presented as a bolus, electuary or
paste. Pharmaceutical preparations which can be used orally include
tablets, push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. Tablets may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with binders, inert diluents, or lubricating, surface active
or dispersing agents. Molded tablets may be made by molding in a
suitable machine a mixture of the powdered compound moistened with
an inert liquid diluent. The tablets may optionally be coated or
scored and may be formulated so as to provide slow or controlled
release of the active ingredient therein. All formulations for oral
administration should be in dosages suitable for such
administration. The push-fit capsules can contain the active
ingredients in admixture with filler such as lactose, binders such
as starches, and/or lubricants such as talc or magnesium stearate
and, optionally, stabilizers. In soft capsules, the active
compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition, stabilizers may be added. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses.
[0085] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. The formulations may be presented in
unit-dose or multi-dose containers, for example sealed ampoules and
vials, and may be stored in powder form or in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline or sterile pyrogen-free water,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described.
[0086] Formulations for parenteral administration include aqueous
and non-aqueous (oily) sterile injection solutions of the active
compounds which may contain antioxidants, buffers, bacteriostats
and solutes which render the formulation isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. Suitable lipophilic solvents or vehicles include fatty oils
such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate or triglycerides, or liposomes. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions.
[0087] Examples of fillers, or diluents, include, without
limitation, lactose, mannitol, xylitol, dextrose, sucrose,
sorbitol, compressible sugar, microcrystalline cellulose (MCC),
powdered cellulose, cornstarch, pregelatinized starch, dextrates,
dextran, dextrin, dextrose, maltodextrin, calcium carbonate,
dibasic calcium phosphate, tribasic calcium phosphate, calcium
sulfate, magnesium carbonate, magnesium oxide, poloxamers such as
polyethylene oxide, and hydroxypropyl methyl cellulose. Fillers may
have complexed solvent molecules, such as in the case where the
lactose used is lactose monohydrate. Disintegrants that can be
adapted for use with embodiments of the compositions herein
include, without limitation, sodium starch glycolate, sodium
carboxymethyl cellulose, calcium carboxymethyl cellulose,
croscarmellose sodium, povidone, crospovidone
(polyvinylpolypyrrolidone), methyl cellulose, microcrystalline
cellulose, powdered cellulose, low-substituted hydroxy propyl
cellulose, starch, pregelatinized starch, and sodium alginate.
Tablet binders to be used in the compositions herein include,
without limitation, acacia, alginic acid, carbomer, carboxymethyl
cellulose sodium, dextrin, ethylcellulose, gelatin, guar gum,
hydrogenated vegetable oil, hydroxyethylcellulose, hydroxypropyl
cellulose, hydroxypropylmethyl cellulose, copolyvidone, methyl
cellulose, liquid glucose, maltodextrin, polymethacrylates,
povidone, pregelatinized starch, sodium alginate, starch, sucrose,
tragacanth, and zein.
[0088] The addition of pH modifiers such as acids, bases, or
buffers may also be beneficial, retarding or enhancing the rate of
dissolution of the composition, or, alternatively, helping to
improve the chemical stability of the composition. In addition, the
formulations provided herein may include other agents conventional
in the art having regard to the type of formulation in question.
Proper formulation is dependent upon the route of administration
chosen. Any of the well-known techniques, carriers, and excipients
may be used as suitable and as understood in the art; e.g.,
Remington, supra. The pharmaceutical compositions may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or compression
processes.
[0089] As shown by the working embodiments of the invention, a
close-proximity architecture endows the ENs with significantly
enhanced catalytic efficiency. This was demonstrated by consecutive
reactions of sucrose and glucose mediated by invertase (Inv),
glucose oxidase (GOx) and horseradish peroxidase (HRP). Glucose
directly added in the reaction media or generated from the
Inv-mediated hydrolysis of sucrose is oxidized via the GOx-mediated
reaction, leading to the production of H.sub.2O.sub.2. The produced
H.sub.2O.sub.2 then further oxidizes o-dianisidine via the
HRP-mediated reaction. The architecture of this invention is unique
not only in that the multiple enzymes within the nanocomplexes are
able to be spatially confined with each other but that such spatial
confinement acts cooperatively (synergistically) rather than
antagonistically.
[0090] As shown in Example 1, the turnover rates (o-dianisidine
oxidation rates) of n(HRP-GOx) and n(HRP-GOx-Inv), compared to
those of their native enzyme mixtures with the same enzyme
contents, were 5 and 15 folds higher, respectively, confirming an
enhanced catalytic efficiency. As used throughout the disclosure,
enzyme nanocomplexes (ENs) are denoted as n(Enzymes), where Enzymes
within the parentheses refer to the enzymes within the core of the
nanocomplexes. For instance, n(Cat) refers to an EN containing
catalase (Cat) and n(HRP-GOx-Inv) refers to an EN containing
horseradish peroxidase (HRP), glucose oxidase (GOx) and invertase
(Inv).
[0091] Further, in the presence of 35 wt-% polyethylene glycol
(PEG, Mw .about.3000), a condition to simulate the viscous
environment within the cells or blood stream, n(HRP-GOx) achieved a
34 fold increase, as compared to the corresponding enzyme mixtures,
in turnover rates. Similarly, n(HRP-GOx-Inv) showed up to 24-fold
enhancement in relative turnover rates in PEG solutions.
[0092] Moreover, significantly enhanced stability of the enzymes in
the ENs have also been observed. As shown in FIG. 2e, n(HRP-GOx)
and n(HRP-GOx-Inv) retained 70% and 75% of the residual enzyme
activities, respectively, after incubation at 65.degree. C. for 60
minutes. Under the same condition, the free enzymes lose more than
98% of their activities.
[0093] Still further, the ENs release less intermediates than the
free enzyme mixtures. In the case of GOx and Cat, the intermediates
include H.sub.2O.sub.2, which is toxic and may cause damages to
organs and tissues. The in vitro data in Example 1 show that GOx
alone, when incubated with cells, led to a loss of 90% cell
viability owning to the H.sub.2O.sub.2 generated from GOx-mediated
glucose oxidation. When co-incubated with Cat, the loss went down
to 58%. In the presence of n(GOx-Cat), however, such loss went
down, dramatically, to 9%. Similar results, measured by skin
damages, were also observed in vivo.
[0094] Also shown in Example 1 is an enzyme nanocomplex,
n(AOx-Cat), containing alcohol oxidase (AOx) and catalase (Cat),
and the nanocomplex's potential as an antidote for alcohol. Both in
vitro and in vivo experiments demonstrated the significant effect
of n(AOx-Cat) in metabolizing alcohol and reducing alcohol-induced
liver damages, while minimizing the release of the toxic byproduct,
H.sub.2O.sub.2. Therefore, the experimental data clearly
demonstrate that the enzyme nanocomplexes (ENs) of the present
disclosure achieve enhanced catalytic efficiency and increased
stability, and at the same time can, when necessary, reduce the
release of intermediates that may be toxic, such as H.sub.2O.sub.2,
to the environment. Thus, one embodiment of the present disclosure
provides a nanocomplex comprising two or more enzymes including a
first enzyme and a second enzyme and a polymeric network anchored
to at least one, or alternatively at least two, or three, or four,
or five, or all of the enzymes, wherein the enzymes are spatially
constrained by the polymeric network and each enzyme cooperatively
reacts with a substrate.
[0095] Cooperatively reacting with a substrate, in one embodiment,
intends to mean that the enzymes, directly or indirectly, react
with the substrate in a concerted manner. In one aspect, the
enzymes react with the substrate(s) sequentially. In this respect,
the first enzyme may react with a substrate first, converting it
into one or more products, such as integrating a substrate with
another molecule and degrading it into more than one molecules. The
second enzyme then reacts with one or more of the products. In
other words, in one embodiment, the product of the first enzyme is
a substrate for the second enzyme.
[0096] Alternatively, the first and second enzyme can react with
the substrate simultaneously. It is well known in the art that
enzymes can form complexes and carry out their functions through
the complexes. In some instances, the enzymes can still function as
a group without forming a physical complex, such as enzymes and
their co-factors. Another example is that many enzymes interact
with a nucleic acid, such as DNA and RNA, simultaneously, to effect
replication, transcription or translation, without having direct
physical or functional relationship with each other. All these
combinations of enzymes are contemplated for the purpose of this
disclosure.
[0097] In any of the above disclosure, the nanocomplex further
includes a third enzyme, a fourth enzyme, and/or a fifth enzyme and
so forth. Each of the additional enzyme can carry out a serial
reaction with another enzyme in the nanocomplex, or concurrently
function with another enzyme.
[0098] In some embodiments, two or more of the enzymes in the
nanocomplex belong to a metabolic pathway. Metabolic pathways are
known in the art and further described herein. Non-limiting
examples of metabolic pathways include glycolysis/gluconeogenesis
pathway, citrate cycle (tca cycle) pathway, pentose phosphate
pathway, pentose and glucuronate interconversions pathway, fructose
and mannose metabolism pathway, galactose metabolism pathway,
ascorbate and aldarate metabolism pathway, starch and sucrose
metabolism pathway, amino sugar and nucleotide sugar metabolism
pathway, pyruvate metabolism pathway, glyoxylate and dicarboxylate
metabolism pathway, propanoate metabolism pathway, butanoate
metabolism pathway, c5-branched dibasic acid metabolism pathway,
inositol phosphate metabolism pathway, oxidative phosphorylation
pathway, photosynthesis pathway, photosynthesis-antenna proteins
pathway, carbon fixation in photosynthetic organisms pathway,
carbon fixation pathways in prokaryotes pathway, methane metabolism
pathway, nitrogen metabolism pathway, sulfur metabolism pathway,
fatty acid biosynthesis pathway, fatty acid elongation pathway,
fatty acid metabolism pathway, synthesis and degradation of ketone
bodies pathway, cutin, suberine and wax biosynthesis new! pathway,
steroid biosynthesis pathway, primary bile acid biosynthesis
pathway, secondary bile acid biosynthesis pathway, steroid hormone
biosynthesis pathway, glycerolipid metabolism pathway,
glycerophospholipid metabolism pathway, ether lipid metabolism
pathway, sphingolipid metabolism pathway, arachidonic acid
metabolism pathway, linoleic acid metabolism pathway,
alpha-linolenic acid metabolism pathway, and biosynthesis of
unsaturated fatty acids pathway.
[0099] Further non-limiting examples of metabolic pathways include
purine metabolism pathway, pyrimidine metabolism pathway, alanine,
aspartate and glutamate metabolism pathway, glycine, serine and
threonine metabolism pathway, cysteine and methionine metabolism
pathway, valine, leucine and isoleucine degradation pathway,
valine, leucine and isoleucine biosynthesis pathway, lysine
biosynthesis pathway, lysine degradation pathway, arginine and
proline metabolism pathway, histidine metabolism pathway, tyrosine
metabolism pathway, phenylalanine metabolism pathway, tryptophan
metabolism pathway, phenylalanine, tyrosine and tryptophan
biosynthesis pathway, beta-alanine metabolism pathway, taurine and
hypotaurine metabolism pathway, phosphonate and phosphinate
metabolism pathway, selenocompound metabolism pathway, cyanoamino
acid metabolism pathway, D-glutamine and d-glutamate metabolism
pathway, D-arginine and d-ornithine metabolism pathway, D-alanine
metabolism pathway, and glutathione metabolism pathway.
[0100] Further non-limiting examples of metabolic pathways include
n-glycan biosynthesis pathway, various types of n-glycan
biosynthesis pathway, mucin type o-glycan biosynthesis pathway,
other types of o-glycan biosynthesis pathway, glycosaminoglycan
biosynthesis-chondroitin sulfate pathway, glycosaminoglycan
biosynthesis-heparan sulfate pathway, glycosaminoglycan
biosynthesis-keratan sulfate pathway, glycosaminoglycan degradation
pathway, glycosylphosphatidylinositol (GPI)-anchor biosynthesis
pathway, glycosphingolipid biosynthesis-lacto and neolacto series
pathway, glycosphingolipid biosynthesis-globo series pathway,
glycosphingolipid biosynthesis-ganglio series pathway,
lipopolysaccharide biosynthesis pathway, peptidoglycan biosynthesis
pathway, other glycan degradation pathway, thiamine metabolism
pathway, riboflavin metabolism pathway, vitamin B6 metabolism
pathway, nicotinate and nicotinamide metabolism pathway,
pantothenate and CoA biosynthesis pathway, biotin metabolism
pathway, lipoic acid metabolism pathway, folate biosynthesis
pathway, one carbon pool by folate pathway, retinol metabolism
pathway, porphyrin and chlorophyll metabolism pathway, and
ubiquinone and other terpenoid-quinone biosynthesis pathway.
[0101] Still, further non-limiting examples of metabolic pathways
include terpenoid backbone biosynthesis pathway, monoterpenoid
biosynthesis pathway, sesquiterpenoid biosynthesis pathway,
diterpenoid biosynthesis pathway, carotenoid biosynthesis pathway,
brassinosteroid biosynthesis pathway, insect hormone biosynthesis
pathway, zeatin biosynthesis pathway, limonene and pinene
degradation pathway, geraniol degradation pathway, type I
polyketide structures pathway, biosynthesis of 12-, 14- and
16-membered macrolides pathway, biosynthesis of ansamycins pathway,
biosynthesis of type II polyketide backbone pathway, biosynthesis
of type II polyketide products pathway, tetracycline biosynthesis
pathway, polyketide sugar unit biosynthesis pathway, nonribosomal
peptide structures pathway, biosynthesis of siderophore group
nonribosomal peptides pathway, biosynthesis of vancomycin group
antibiotics pathway, phenylpropanoid biosynthesis pathway,
stilbenoid, diarylheptanoid and gingerol biosynthesis pathway,
flavonoid biosynthesis pathway, flavone and flavonol biosynthesis
pathway, anthocyanin biosynthesis pathway, isoflavonoid
biosynthesis pathway, indole alkaloid biosynthesis pathway,
isoquinoline alkaloid biosynthesis pathway, tropane, piperidine and
pyridine alkaloid biosynthesis pathway, acridone alkaloid
biosynthesis pathway, caffeine metabolism pathway, betalain
biosynthesis pathway, glucosinolate biosynthesis pathway,
benzoxazinoid biosynthesis pathway, penicillin and cephalosporin
biosynthesis pathway, beta-lactam resistance pathway, streptomycin
biosynthesis pathway, butirosin and neomycin biosynthesis pathway,
clavulanic acid biosynthesis pathway, puromycin biosynthesis
pathway, and novobiocin biosynthesis pathway.
[0102] Still, further non-limiting examples of metabolic pathways
include benzoate degradation pathway, aminobenzoate degradation
pathway, fluorobenzoate degradation pathway, chloroalkane and
chloroalkene degradation pathway, chlorocyclohexane and
chlorobenzene degradation pathway, toluene degradation pathway,
xylene degradation pathway, nitrotoluene degradation pathway,
ethylbenzene degradation pathway, styrene degradation pathway,
atrazine degradation pathway, caprolactam degradation pathway, DDT
degradation pathway, bisphenol degradation pathway, dioxin
degradation pathway, naphthalene degradation pathway, polycyclic
aromatic hydrocarbon degradation pathway, metabolism of xenobiotics
by cytochrome P450 pathway, and drug metabolism-cytochrome P450
pathway.
[0103] Metabolic pathways can also include MAPK signaling pathway,
MAPK signaling pathway-fly pathway, MAPK signaling pathway-yeast
pathway, ErbB signaling pathway, wnt signaling pathway, notch
signaling pathway, hedgehog signaling pathway, TGF-beta signaling
pathway, VEGF signaling pathway, Jak-STAT signaling pathway,
calcium signaling pathway, phosphatidylinositol signaling system
pathway, mTOR signaling pathway, plant hormone signal transduction
pathway, hematopoietic cell lineage pathway, complement and
coagulation cascades pathway, toll-like receptor signaling pathway,
NOD-like receptor signaling pathway, RIG-I-like receptor signaling
pathway, cytosolic DNA-sensing pathway, natural killer cell
mediated cytotoxicity pathway, antigen processing and presentation
pathway, T cell receptor signaling pathway, B cell receptor
signaling pathway, Fc epsilon RI signaling pathway, Fc gamma
R-mediated phagocytosis pathway, leukocyte transendothelial
migration pathway, intestinal immune network for IgA production
pathway, and chemokine signaling pathway,
[0104] In a particular embodiment, the two or more enzymes comprise
a horseradish peroxidase (HRP) and a glucose oxidase (GOx). In
another embodiment, the two or more enzymes comprise a horseradish
peroxidase (HRP), a glucose oxidase (GOx) and an invertase (Inv).
In another embodiment, the two or more enzymes comprise a glucose
oxidase (GOx) and a catalase (Cat). In yet another embodiment, the
two or more enzymes comprise an alcohol oxidase (AOx) and a
catalase (Cat).
[0105] In some embodiments, the nanocomplex is from about 5 nm to
about 2000 nm in length as measured along its longest axis. The
size may vary depending on the size and number of enzymes in the
nanocomplex and the characteristics of the polymer network. In some
embodiments, the length of the nanocomplex is at least about 10 nm,
20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 125
nm, 150 nm, 175 nm, 200 nm, 250 nm, 300 nm, 400 nm, or 500 nm. In
some embodiment, the length of the nanocomplex is no more than
about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 125 nm, 150 nm,
175 nm, 200 nm, 250 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800
nm, 900 nm, 1000 nm 1500 nm or 2000 nm.
[0106] The nanocomplex can be of any shape, depending on the size,
shape and number of the enzymes in the complex. In one embodiment,
the nanocomplex is substantially round. In another embodiment, the
nanocomplex is substantial oval, spherical, cylinder, or
pyramid-like.
[0107] In some embodiments, the polymer network is anchored to the
enzymes at, collectively, at least 1, 2, 3, 4, 5, 7, 10, 15, or 20
locations. In one embodiment, the polymer network is anchored to at
least two of the enzymes. In one embodiment, the polymer network is
anchored to each of the enzymes at least at 2, or 3, or 4, or 6
locations. It is contemplated that not every enzyme in the
nanocomplex needs to be anchored with the polymer network to
effectively constrain the enzyme in the nanocomplex. For instance,
for three or more enzymes, when two of them are anchored with the
polymer network, the others can be constrained by virtue of their
locations relative to the anchored enzymes. Such special constraint
can be reinforced by, but does not require, bonding between the
enzymes. It is also contemplated that the polymer network can be
anchored to just one of the enzymes in the nanocomplex and still
effects spatial constraint of the enzymes in the nanocomplex, by
virtue of the association of the other enzymes with the one
anchored to the polymer network. In one embodiment, the polymer
network is anchored to every enzyme in the nanocomplex.
[0108] The polymer network can be anchored to the enzymes through
any force, such as but not limited to covalent bond, hydrogen bond,
ionic force, hydrophobic bonding, and Van der Wall force. In
certain embodiments, multiple forces are involved in the anchoring.
In some embodiments, not all locations of the anchoring employ the
same type of force.
[0109] For the purpose of anchoring the polymer network, in one
embodiment, two or more enzymes in the nanocomplex have a plurality
of polymerizable groups. A polymerizable group is a chemical moiety
that polymerizes under certain chemical conditions. Examples of
polymerization conditions include photopolymerization, free radical
polymerization, and catalyst induced polymerization. In general,
the type of polymerizable group is not critical, so long as the
polymerizable group is capable of polymerization with a monomer
used to form the nanocomplex. Examples of polymerizable groups
include double-bond containing moieties which are polymerized by
photopolymerization or free radical polymerization. In some
embodiments, the polymerizable group is a vinyl group, acryl group,
alkylacryl group (i.e. acryl group having an alkyl substituent,
such as methacryl). As used herein, acryl (alkylacryl, methacryl,
etc) includes acryl t esters as well as acryl amides. In some
embodiments, the polymerizable group is an acryl group covalently
bonded to a lysine residue of the enzymes.
[0110] Enzymes can contain many different surface amino acids which
can readily be modified. For example, lysine, cysteine, threonine,
glutamic acid, aspartic acid, serine, histidine, arginine,
tyrosine, proline and tryptophan may be readily modified using
known processes and procedures. A reagent used to modify the
protein will have at least one polymerizable group, and at least
one reactive group that reacts with an amino acid side-chain on the
surface of the protein. Examples of reactive groups that are used
to react with amino acid side chains include activated esters (such
as acyl halides or N-hydroxysuccinimide esters) that react with
amine (such as lysine) and hydroxy 1 (such as threonine or serine)
containing residues; maleimides that react with thiol (such as
cysteine) containing residues; and amines which react with
carboxylic acid (such as glutamic acid and aspartic acid)
containing residues when activated with certain coupling reagents.
This way, the polymerizable group is covalently bonded to the
single-enzymes. The polymerizable group may be directly bonded, or
attached through a linker.
[0111] Ionic bonds can be used to anchor the polymer network on one
or more of the enzymes by virtue of the presence of positive or
negative charges of certain amino acids, such as arginine, aspartic
acid, glutamic acid, histidine and lysine, and amino acids that are
modified to have charges.
[0112] In some embodiments, a linker is present between the enzymes
and the polymerizable group. The linker is a chemical moiety
separating the polymerizable group, and the enzymes. A reagent used
as a linker will have at least one polymerizable group, and at
least one reactive group separated by a linker. The reactive group
reacts with the enzymes, usually by reaction with an amino acid
side chain on the surface of the protein, covalently bonding the
polymerizable group to the single-enzymes with the linker between
them.
[0113] In some embodiments, the linker may be degradable. A
degradable linker is a chemical moiety that is cleaved under
certain conditions. For example, a degradable linker may, for
example, hydrolyze at certain pH values (high pH or low pH), or may
be cleaved photolytically (i.e. when irradiated with light of
certain wavelengths), or under certain temperatures, under
reduction-oxidation conditions, or enzymatically (i.e. by
proteases). Any suitable degradable linker may be used, so long as
the linker has a reactive group to react with the enzymes, and a
polymerizable group to form the nanocomplex. The degradable linker
should also be stable during polymerization of the nanocomplex. The
type of linker may be selected based on the conditions under which
the linker will be cleaved. Numerous linkers are known, and will be
readily apparent to one of ordinary skill.
[0114] Enzymes in these nanocomplexes are more stable than the
enzymes in their native, non-complexed forms. Stability is
determined in the loss of enzyme activity over time or degradation
or denaturation of the enzyme. In some embodiments, the
nanocomplexes are resistant to degradation by proteases. The
polymer network reduces exposure of the enzymes to proteases. As a
result, the activity of the enzymes lasts longer in the presence of
proteases (for example, in vivo, in serum, or in cells) than
unprotected native enzymes. The polymer network stabilizes the
enzyme structure, and prevents aggregation of the enzymes. Thus,
the nanocomplexes are more resistant to changes in pH and
temperature. For example, nanocomplexes have a longer storage
lifetime at room temperature or decreased temperature (i.e.
refrigeration or freezing), than the native enzymes. Likewise,
nanocomplexes are less likely to lose activity after multiple
freeze/thaw cycles than native enzymes. Because the enzymes'
structures are stabilized by the polymer network, the nanocomplexes
are more resistant to organic solvents and surfactants.
[0115] The polymer network may also be adjusted to increase
solubility in organic solvents. Examples of organic solvents and
surfactants that may be used with the nanocomplexes include
methanol, ethanol, isopropyl alcohol, dimethyl sulfoxide,
tetrahydrofuran, 4-dichlorobenzene, para-dichlorobenzene,
1,4-dioxane, 1,4-dioxane PEG, polyethylene, polyethylene glycol,
polyoxyethylene, sodium laureth sulfate or oxynol, polysorbate 60,
2-bromo-2-nitropropane-1,3-diol, 2-butoxy-1-ethanol, alkyl phenoxy,
polyethoxy ethanol, among others widely used in cosmetics (i.e.
make-up) and pharmaceuticals. Other organic solvents will be
apparent to one of ordinary skill in the art. Because the polymer
network prevents aggregation, the nanocomplexes are more stable at
interfaces (i.e. gas/liquid or liquid/solid) where unprotected
enzymes tend to aggregate.
[0116] In some embodiments, the nanocomplex is two or more enzymes
co-polymerized with a monomer unit. A monomer unit is a chemical
moiety that polymerizes and forms a co-polymer with the
single-enzymes, forming the polymer network of the nanocomplex. In
some embodiments, when the enzymes bear polymerizable groups having
a double bond, such as a vinyl, acryl, alkylacryl or methacryl
group, the monomer unit also has a polymerizable group having
double bond, such as a vinyl, acryl, acrylamido, alkylacryl,
alkylacrylamido, methacryl or methacrylamido group. The
polymerizable group of the enzymes, and the polymerizable group of
the monomer unit may be the same or different, so long as they are
capable of forming a co-polymer under the conditions used to form
the nanocomplex. For example, vinyl and acryl groups may form
co-polymers under free-radical polymerization conditions.
[0117] In some embodiments, the nanocomplex is two or more enzymes
co-polymerized with two or more different monomer units. In
general, any number of different monomer units may be used to form
co-polymers with the enzymes, so long as the different monomer
units are all capable of forming a co-polymer under the conditions
used to form the nanocomplex. Monomer units with different
side-chains may be used to alter the surface features of the
nanocomplex. The surface features may be controlled by adjusting
the ratio between different monomer units. In some embodiments, the
monomers may be neutral, uncharged, hydrophilic, hydrophobic,
positively charged, or negatively charged. In some embodiments, the
polymer network as a whole is neutral, uncharged, hydrophilic,
hydrophobic, positively charged, or negatively charged. Solubility
of the nanocomplex may be adjusted, for example, by varying the
ratio between charged and uncharged, or hydrophilic or hydrophobic
monomer units. In some embodiments, the nanocomplex has a positive
or negative charge.
[0118] In some embodiments, at least one monomer unit has a
positive or negative charge at the physiological pH (.about.7.4).
By using monomer units having a charge at pH=7.4, the overall
charge of the nanocomplex may be varied and adjusted by changing
the ratio of the charged and uncharged monomer units. In some
embodiments, the monomer unit has a positive charge at pH=7.4.
Using positively charged monomer units enables the formation of
nanocomplexes having a positive charge. The charge may be adjusted
by changing the ratio of neutral and positively charged monomer
units.
[0119] In some embodiments, the polymer network further includes at
least a crosslinker. A crosslinker is a chemical compound having
two or more polymerizable groups. In general, any crosslinking
compound may be used, so long as the polymerizable groups on the
crosslinker are capable of forming a crosslinked co-polymer between
the enzymes and the at least one monomer unit under the conditions
used to form the nanocomplex. Examples of crosslinkers include
compounds having two vinyl, acryl, alkylacryl, or methacryl groups.
Examples of specific crosslinkers having two acryl groups include
N,N'-methylenebisacrylamide and glycerol dimethacrylate.
[0120] In some embodiments, the crosslinker is a degradable
crosslinker. A degradable crosslinker is cleaved under certain
conditions, resulting in decomposition or removal of at least a
portion of the polymer network of the nanocomplex. For example, a
degradable crosslinker may hydrolyze at certain pH (high or low),
may be cleaved by specific enzymes (such as esterases or
peptidases), may be photolytically cleaved upon exposure to certain
wavelengths, or be cleaved at certain temperatures. Examples of
crosslinkers which hydrolyze at reduced pH include glycerol
dimethacrylate, which is stable at physiological pH (about 7.4),
but hydrolyzes at lower pH (about 5.5). Other examples of
degradable crosslinkers include acetal crosslinkers described in
U.S. Pat. No. 7,056,901, which is incorporated by reference in its
entirety.
[0121] Further specific examples of degradable crosslinking groups
include N,N'-methylenebis(acrylamide), 1,4-bis(acryloyl)piperazine,
ethylene glycol diacrylate,
N,N'-(1,2-dihydroxy-ethylene)bisacrylamide, and poly(ethylene
glycol)diacrylate. Further examples of non-degradable crosslinking
groups include N,N'-bis(acryloyl)cystamine, glycerol
dimethacrylate, bis[2-(methacryloyloxy)ethyl]phosphate, and
bisacryloylated polypeptide.
[0122] In some embodiments, the polymer network is permeable. As
used herein, permeable means that molecules (e.g. ethanol) may pass
through the polymer network, either through pores or holes in the
polymer network, or by diffusion through the polymer matrix. For
example, substrates, co-factors and other chemical elements may
pass through the polymer network, allowing the nanocomplex to
retain the activity of the enzymes inside. In some embodiments of
the invention, the polymer matrix is formed from selected
constituents and/or under selected conditions designed to generate
a matrix having specific permselective properties that influence
the diffusion of compounds through the polymer matrix.
[0123] A degradable crosslinker can removes or reduces the density
of the polymer network and thus allow passing of various objects
such as co-factors and the substrates. For example, a degradable
crosslinker that decomposes at reduced pH may be used to remove or
reduce the polymer coating after the nanocomplexes are internalized
into cells by endocytosis. It is well known that serum and late
endosomes have pH values of -7.4 and -5.5, respectively. Thus, a
degradable crosslinker that is stable at pH -7.4, but degrades at
pH -5.5 will remove or reduce the polymer coating only after the
nanocomplex has entered the cell. In this way, the enzymes are
protected from proteases present in serum, but enzymes with large
substrates may still be effectively delivered to cells. After the
polymer coating is reduced, the activity of the enzymes is still
present. After the polymer coating is removed, however, the enzymes
becomes susceptible to degradation by intracellular proteases, but
this drawback is offset by the nanocomplex's increased stability in
serum, and resistance to serum proteases.
[0124] In certain embodiments, the nanocomplex further includes a
surface modification. Surface modifications are chemical moieties
which are added to the surface of the nanocomplex after formation
of the nanocomplex. Monomer units having reactive sidechains (or
protected reactive sidechains) may be used to form the nanocomplex
so long as the reactive sidechains do not interfere with formation
of the nanocomplex. The reactive sidechains may be (after
deprotection, if necessary) reacted with surface modifying agents
to covalently attach the surface modification to the nanocomplex. A
surface modifying agent may be a small molecule, polymer, peptide,
polypeptide, protein, oligonucleotide, polysaccharide, or antibody.
The surface modification may alter the solubility of the
nanocomplex, change the surface charge of the nanocomplex, or
impart an additional function to the nanocomplex, such as
light-emission, cell targeting or cell penetration. Surface
modifications that enhance cell targeting result in an increased
transduction of the nanocomplex into targeted cells, when compared
with non-targeted cells. Surface modifications that enhance cell
penetration result in increased transduction of nanocomplexes into
cells when compared with nanocomplexes lacking the cell penetration
enhancer. More than one surface modification may be present on the
nanocomplex. Examples of small molecule surface modifications
include light emitting compounds, such as fluorescein, or
rhodamine, or cell targeting compounds such as folic acid. Polymers
include polyethylene glycol to increase solubility. Peptides may be
used for cell targeting, such as antibodies to particular cell
surface features, cell signaling proteins, or growth hormones.
Other peptides may be used to increase cell penetration of the
nanoparticles (such as TAT or antennepedia homeodomain). In some
embodiments, the surface modification is an antibody.
Methods of Preparing Multiple-Enzyme Nanocomplexes
[0125] Multiple-enzyme nanocomplexes of the present disclosure can
be prepared by first forming a multiple-enzyme core and then
anchoring a polymer network about the core.
1. Preparation of Multiple-Enzyme Core
[0126] A method of preparation of a temporary multiple-enzyme core
is illustrated in FIG. 1 and described in Example 1. Further
details for such an exemplary procedure can be found in Example
2.
[0127] In the exemplary method, at a first step, a DNA-inhibitor
scaffold is synthesized. As illustrated in FIG. 1, competitive
inhibitors for each enzyme are respectively conjugated to a
single-strand DNA (ssDNA) with designed sequence; complementary
assembly of the DNA molecules forms a DNA-inhibitor scaffold linked
with the three inhibitors (see, e.g. Feldkamp et al., Angew Chem
Int Edit 45, 1856-1876, (2006)).
[0128] Conjugation of a ssDNA with an inhibitor can be achieved
with methods known in the art. For example, the conjugation can
occur at the 5' phosphate or 5' amineC6T group of the ssDNA (see, e
g., illustration in Example 1 section 3.1).
[0129] Once ssDNA-inhibitors are prepared, the DNA-inhibitor
scaffold can be self-assembled owning to the complementary
sequences on the ssDNA. FIG. 5a illustrates the assembly of two
ssDNA-inhibitor molecules. Therefore, the two ssDNA share a
complementary sequence with each other.
[0130] In FIG. 5b, on the other hand, the four ssDNA sequences can
assemble 3 or 4 inhibitors onto the scaffold. There, ssDNA-I has a
sequence that is complementary to a fragment of ssDNA-II, which in
turn has a sequence that is complementary to a fragment of
ssDNA-III, which in turn has a sequence that is complementary to a
fragment of ssDNA-IV, which in turn has a sequence that is
complementary to a fragment of ssDNA-I.
[0131] The design strategy illustrated in FIG. 5b can be readily
applied to form a 5, or 6, or more ssDNA-inhibitor scaffold by
including complementary sequences into ssDNA molecules.
[0132] Once the multiple-inhibitor scaffold is synthesized, the
enzymes can then be bound to the corresponding inhibitor to form an
enzyme-inhibitor-ssDNA core.
[0133] It is readily appreciated by the skilled artisan that the
multiple-enzyme core can be prepared with other methods known in
the art. For instance, one or more of the inhibitors can be
substituted with an enzyme substrate, ligands, or even an antibody
that recognizes the enzyme. The ssDNA, on the other hand, can be
replaced by a polymer that anchors the inhibitors, substrates or
antibodies.
[0134] Also provided, therefore, in one embodiment is a nanocomplex
comprising a polymeric scaffold anchored to a plurality of ligands,
the ligands recognized by a plurality of enzymes, the enzymes
cooperatively react with a substrate. A "ligand" as used herein,
broadly intends to cover any molecule that specifically recognizes,
or is recognized by, or binds to an enzyme. In one aspect, the
polymeric scaffold is a nucleic acid, such as ssDNA. Such polymeric
scaffold is useful in preparing multiple-enzyme nanocomplexes,
accordingly.
[0135] In a further embodiment, the polymeric scaffold further
includes the corresponding enzymes.
2. Preparation of Multiple-Enzyme Nanocomplexes
[0136] Once a multiple-enzyme core is prepared, a polymer network
can be then applied to the core. The polymer network can be
anchored covalently, or by other forces describe herein, to two or
more of the enzymes derivatizing the enzymes with at least one
polymerizable group; and then copolymerizing the derivatized
protein with a monomer unit.
[0137] An exemplary two-step procedure can be used to form a
polymer network about the enzymes. First, polymerizable groups are
linked to the enzyme surface. Then subsequent polymerization of
functional monomers and optionally, crosslinkers, in buffer
solution wraps two or more of the enzymes with a polymer skin.
[0138] Once the polymerizable groups are attached to the enzyme
surface, monomers can be used to form polymer coatings with tunable
composition, structure, surface property, and functionality. A room
temperature free-radical polymerization technique may be used to
ensure the retention of enzyme activity. Since these polymer
coatings serve as artificial membranes for the encapsulated
enzymes, they exhibit suitable mechanical strength to provide
structural integrity, possess effective transport pathways to allow
rapid substrate transport, and contain specific functionality to
provide substrate selectivity and moisture retention.
[0139] Nanocomplexes do not change the functions of the enzymes,
which, as noted above, may be obtained directly from commercial
sources or using other reported methods (e.g. recombinant
methodologies such as those offered by GENSCRIPT).
[0140] A reagent used to derivatize or modify the enzymes has the
following general structure where the polymerizable group is a
chemical moiety that forms a copolymer with one or more monomers
and/or crosslinkers under conditions used to form the nanocomplex.
In some embodiments, the polymerizable group is a double bond
containing group, such as vinyl, acryl, alkylacryl, and methacryl.
As discussed previously, acryl (and alkylacryl and methacryl)
includes both acryl esters and acrylamides.
[0141] A linking group is optional and may be present between the
polymerizable group and the protein. The linking group is a
chemical moiety that separates the reactive group from the
polymerizable group. The linking group is not limited to any
particular chemical structure, but should not interfere in the
polymerization reaction. In some embodiments, the linking group is
a degradable linking group, which is cleaved under certain
conditions. For example, acetals, ketals or esters may be
hydrolyzed at certain pHs. Linking groups having one or more of
these functional groups may decompose in response to changes in pH
(e.g. in endosomes).
[0142] A reactive group is a chemical moiety that reacts with an
amino acid side chain to covalently attach the polymerizable group
to the protein. Numerous reactive groups are known and are used to
react with different amino acid side chains. For example, acyl
halides, or activated esters (such as N-hydroxysuccinimide esters)
react with amines (e.g. on lysine) or hydroxyls (e.g. on serine or
threonine). Isocyanates react with amines. Epoxides react with
amines or thiols (e.g. cysteine). Maleimides react with thiols.
Other functional groups may react with amino acid side chains in
the presence of one or more coupling reagents (such as carbodiimide
reagents). For example amines may react with carboxylic acid amino
acid side chains (e.g. on glutamate or aspartate). Other reactive
groups will be readily apparent to one of ordinary skill in the
art.
[0143] During the protein modification mentioned above, to realize
the site-specific controlled modification, a genetic recombinant
technique can be used to introduce specific amino acids in
spatially defined locations. This technique allows precise control
of the site and density of the modification.
[0144] Examples of specific compounds used to derivatize proteins
are known the art, see for instance PCT Application Publication No.
WO 2010/104865, the contents of which are incorporated into the
present disclosure by reference. This PCT application, furthermore,
also discloses details methods of acrylating proteins during the
polymerization process, as well as generally preparing protein
nanocomplexes.
[0145] A monomer unit, in some embodiments, has a side chain and a
polymerizable group, where the polymerizable group is a chemical
moiety that forms a copolymer with the enzymes and optional
crosslinkers under conditions used to form the nanocomplex.
Polymerizable groups include all those discussed previously. The
polymerizable group of the enzyme, and the polymerizable group of
the monomer unit may be the same or different, so long as they are
capable of forming a co-polymer under the conditions used to form
the nanocomplex. For example, vinyl and acryl groups may form
co-polymers under free-radical polymerization conditions.
[0146] The side chain is a portion of the monomer unit that does
not participate in polymerization. In general, the side chain may
have any structure, and may be selected based on the desired
properties of the nanocomplex. The side chains of the monomer unit
affect the surface properties of the nanocomplex. In some
embodiments, the side chain may be neutral, neutral hydrophilic
(i.e. water soluble, but not charged), hydrophobic, positively
charged, or negatively charged. Neutral side chains include amides,
esters, ethers, hydroxyls some of which may be hydrophilic or
hydrophobic, depending on their structure. Positively charged side
chains include amines (including substituted amines, such as mono
and dialkyl amines, and tetrasubstituted ammonium compounds, and
cyclic variants thereof), guanidines, and heterocycles such as
pyridines and imidazoles. Negatively charged sidechains include
carboxylic acids. Hydrophobic side chains include alkyl groups
(including linear, branched or cyclic alkyl groups) and aryl
groups.
[0147] Examples of specific monomer units and their functions
include acrylamide (neutral, 1), 2-hydroxyethyl acrylate (neutral,
1), N-isopropylacrylamide (neutral, 2), sodium acrylate (negatively
charged, 3), 2-acryloylamido-2-methylpropanesulfonic sodium
(negatively charged, 3), allyl amine (positively charged, 4),
N-(3-aminopropyl)methacrylamide hydrochloride (positively charged,
4, 5), dimethylamino ethyl methacrylate (positively charged, 5),
(3-acrylamidopropyl)trimethylammonium hydrochloride (positively
charged, 5), methyl acrylate (hydrophobic, 6) and styrene
(hydrophobic 6). The numbers in the parentheses refer to functions:
1 to 5: hydrophilic surface and moisture retention; 2) temperature
responsive; 3) negatively charged surface; 4) reactive sidechain
for surface modification, 5) positive charge surface, 6)
hydrophobic surface.
[0148] Polymerization of the modified enzymes and monomer unit(s)
may use any method suitable for the polymerizable groups used on
the protein and monomer unit(s) and which does not destroy the
function of the protein during polymerization. Examples of
polymerization methods include photopolymerization and free-radical
polymerization of double bond containing polymerizable groups, such
as those described previously. In some embodiments, the
polymerization is a free radical polymerization.
[0149] In some embodiments, the polymerization is carried out at
room temperature, though the temperature may be increased or
decreased as desired, depending on the polymerization method, so
long as the function of the protein is not lost during
polymerization. Where degradable crosslinkers or linking groups are
used, the function of the nanoparticle may be measured after
degradation of the polymer coating. Reaction temperatures may be
increased where the polymerization reaction occurs too slowly, or
where elevated temperature is needed to initiate polymerization.
Temperatures may be decreased where polymerization reactions occur
too quickly.
[0150] In some embodiments, the polymerization reaction is
performed in water, or aqueous buffer. Other solvents may be used
as desired, so long as the solvent does not interfere with the
polymerization reaction, or degrade the protein. Mixtures of water
or aqueous buffer and organic co-solvents may also be used, if
necessary to dissolve reaction components, so long as the solvent
mixture does not interfere with the reaction, or damage the
enzymes. In some embodiments, the polymerization reaction is
performed in buffer.
[0151] In some embodiments, the copolymerization step comprises at
least two different monomer units. In general, any number of
different monomer units may be used to form copolymers with the
enzymes, so long as the different monomer units are all capable of
forming a co-polymer under the conditions used to form the
nanocomplex. Monomer units with different side-chains may be used
to alter the surface features of the nanocomplex. The surface
features may be controlled by adjusting the ratio between different
monomer units. In some embodiments, the monomer may be neutral,
neutral hydrophilic, hydrophobic, positively charged, or negatively
charged. Solubility of the nanocomplex may be adjusted by varying
the ratio between charged and uncharged, or hydrophilic or
hydrophobic monomer units.
[0152] In some embodiments, the copolymerization step further
includes a crosslinker. A crosslinker is a reagent having at least
two polymerizable groups, separated by a linking group. The
crosslinker may have more than two polymerizable groups. The
polymerizable groups on the crosslinker may be the same or
different, so long as all the polymerizable groups on the
crosslinker are able to form a copolymer with the monomer unit(s)
and enzymes under the conditions used to form the nanocomplex. A
crosslinker having two polymerizable groups has the general
structure where the polymerizable groups are the same as those
described previously. The linking group may have any structure so
long as it does not interfere with the polymerization reaction.
Examples of suitable linkers include alkyl groups (including
substituted alkyl groups), aryl groups (including substituted aryl
groups), ketones, amides, esters, ethers and combinations thereof.
Specific examples of crosslinkers include N,N'-methylene
bisacrylamide and glycerol dimethacrylate and others as described
above.
[0153] In some embodiments, the linking group is degradable. A
degradable linking group may be cleaved under certain conditions.
The structure of the degradable linking group determines the type
of conditions required to cleave the linking group. For example,
the linking group may be cleaved due to a change (i.e. increase or
decrease) in pH, exposure to certain wavelengths of light, or in
response to heat. An example of a degradable crosslinker is
glycerol dimethacrylate.
[0154] In some embodiments, the method of producing a nanocomplex
further includes a step of modifying the surface of the
nanocomplex. Sidechains of the monomer unit(s) are present on the
surface of the nanocomplex after polymerization. Monomer units
having a reactive sidechain (or protected reactive sidechain) may
be used to prepare the nanocomplex. The reactive sidechain does not
interfere with polymerization, but may undergo further chemical
modification after the nanocomplex is formed (i.e. after
polymerization is completed). A protected reactive sidechain may be
deprotected using standard chemical deprotection methods, then
reacted with a chemical modifying agent. A reactive sidechain is
treated with a chemical reagent to covalently attach the surface
modifying agent to the surface of the nanocomplex. The surface
modification may be a small molecule, polymer, peptide,
polypeptide, protein, oligonucleotide, polysaccharide, or antibody.
The surface modification may alter the solubility of the
nanocomplex (e.g. by adding polyethylene glycols or other
hydrophilic groups), change the surface charge of the nanocomplex
(e.g. by adding charged surface modifiers), or impart an additional
function to the nanocomplex, such as light-emission, cell targeting
or cell penetration. Examples of small molecule surface
modifications include light emitting compounds, such as
fluorescein, or rhodamine, or cell targeting compounds such as
folic acid. Polymers include polyethylene glycol to increase
solubility. Peptides and polypeptides may be used for cell
targeting, and may include antibodies selective to specific cell
surface features, cell signaling peptides, or hormones. Other
peptides may be used to increase cell penetration of the
nanoparticles (such as TAT or antennepedia homeodomain). In some
embodiments, the surface modification is an antibody. Because
nanocomplexes have an easily derivatizeded surface, specific
antibodies can be conjugated with nanocomplexes providing extra
ability of targeting delivery.
[0155] The size and surface features of the nanocomplexes may be
adjusted by varying the weight ratio of the different monomers
and/or crosslinkers used to form the nanocomplex. For example,
tuning the weight ratio of DMAEMA (positive charged) to AAm
(neutral) from 0:1, 1:3 and 1:1 allows the synthesis of BSA
nanocomplexes with adjustable zeta potentials from -12.8, 8.64 to
15.2 mV, respectively. Solubility may also be easily adjusted by
changing the types and ratios of monomer units and crosslinkers.
Increasing the amount of hydrophilic or charged monomers increases
water solubility. Increasing the amount of hydrophobic monomers
tends to increase nanocomplex solubility in organic solvents or
mixed organic/water solvent systems.
[0156] The permeability of the polymer network may be adjusted by
varying the ratio of the crosslinker in the reaction mixture used
to prepare the nanocomplex. In general, a lower amount of
crosslinker results in a higher permeability. Likewise, the
permeability of the polymer network may be varied by changing the
length of the linking group on the crosslinker. Generally, longer
linking groups lead to increased permeability.
[0157] After the multiple-enzyme core is anchored to a polymer
network, the ssDNA-inhibitor scaffold, or any other kind of
scaffold that serves the purpose of temporally maintaining the
core, can be removed. Removal of the scaffold can be done by a
condition or reagent that dissociates the inhibitors from the
enzymes, so long as such condition or reagent does not disrupt the
anchoring of the polymer network at the enzymes. Examples of such
conditions and reagents are provided in the examples and are
readily recognized by the skilled artisan.
Uses
[0158] The enzyme nanocomplexes described herein may be used to
carry out a series of enzymatic functions in a concerted manner
with increased efficiency, increased stability, and reduced
production of intermediates. In the event one or more the
intermediates are toxic, the enzyme nanocomplex also reduces or
eliminates the release of the toxic intermediates. The specific use
of the enzyme nanocomplex depends on what enzymes are enclosed in
the complex.
[0159] Further, the enzyme nanocomplexes can also be used to
deliver the enzymes to a cell in vitro or in vivo with improved
stability and/or long-term effect. Since the nanocomplexes are more
resistant to degradation by proteases, the protein activity has a
longer effect than when native or unprotected proteins are
administered. As a result, less enzymes (in the form of
nanocomplexes) is required for the same effect (when compared with
unprotected proteins), thereby improving efficiency.
[0160] Embodiments of the invention include a method of delivering
enzymes by exposing a cell to an effective concentration of
nanocomplexes described above. The cells may be in culture (i.e. in
vitro) or may be present in a living organism (i.e. in vivo).
[0161] In some embodiments, at least one of the enzymes is a
therapeutic enzyme. A therapeutic enzyme is one that can be used to
treat a disease or disorder in a subject. In some embodiments, the
subject is a mammal, or a human.
[0162] The nanocomplexes described herein may be used with enzymes
that may be used to treat a disease or disorder in a subject. For
example, nanocomplexes according to the invention may be used in
the treatment of a hyperproliferative disorder, cancer, or tumor.
In other embodiments, the nanocomplexes may be used in
cosmetics.
[0163] Furthermore, since such an enzyme nanocomplex is more
stable, than their isolated forms, in high temperature and acidic
and basic conditions, such an enzyme nanocomplex certainly shows
greatly improved use.
Pharmaceutical Compositions
[0164] The nanocomplexes discussed herein can be formulated into
various compositions, for use in diagnostic or therapeutic
treatment methods. The compositions (e.g. pharmaceutical
compositions) can be assembled as a kit. Generally, a
pharmaceutical composition of the invention comprises an effective
amount (e.g., a pharmaceutically effective amount) of a composition
of the invention.
[0165] A composition of the invention can be formulated as a
pharmaceutical composition, which comprises a composition of the
invention and pharmaceutically acceptable carrier. By a
"pharmaceutically acceptable carrier" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be administered to a subject without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the other components of the pharmaceutical composition in which
it is contained. The carrier would naturally be selected to
minimize any degradation of the active ingredient and to minimize
any adverse side effects in the subject, as would be well known to
one of skill in the art. For a discussion of pharmaceutically
acceptable carriers and other components of pharmaceutical
compositions, see, e.g., Remington's Pharmaceutical Sciences,
18.sup.th ed., Mack Publishing Company, 1990. Some suitable
pharmaceutical carriers will be evident to a skilled worker and
include, e.g., water (including sterile and/or deionized water),
suitable buffers (such as PBS), physiological saline, cell culture
medium (such as DMEM), artificial cerebral spinal fluid, or the
like. A pharmaceutical composition or kit of the invention can
contain other pharmaceuticals, in addition to the compositions of
the invention. The other agent(s) can be administered at any
suitable time during the treatment of the patient, either
concurrently or sequentially.
[0166] One skilled in the art will appreciate that the particular
formulation will depend, in part, upon the particular agent that is
employed, and the chosen route of administration. Accordingly,
there is a wide variety of suitable formulations of compositions of
the present invention.
[0167] One skilled in the art will appreciate that a suitable or
appropriate formulation can be selected, adapted or developed based
upon the particular application at hand. Dosages for compositions
of the invention can be in unit dosage form. The term "unit dosage
form" as used herein refers to physically discrete units suitable
as unitary dosages for animal (e.g. human) subjects, each unit
containing a predetermined quantity of an agent of the invention,
alone or in combination with other therapeutic agents, calculated
in an amount sufficient to produce the desired effect in
association with a pharmaceutically acceptable diluent, carrier, or
vehicle.
[0168] One skilled in the art can easily determine the appropriate
dose, schedule, and method of administration for the exact
formulation of the composition being used, in order to achieve the
desired effective amount or effective concentration of the agent in
the individual patient.
[0169] The dose of a composition of the invention, administered to
an animal, particularly a human, in the context of the present
invention should be sufficient to effect at least a detectable
amount of a diagnostic or therapeutic response in the individual
over a reasonable time frame. The dose used to achieve a desired
effect will be determined by a variety of factors, including the
potency of the particular agent being administered, the
pharmacodynamics associated with the agent in the host, the
severity of the disease state of infected individuals, other
medications being administered to the subject, etc. The size of the
dose also will be determined by the existence of any adverse side
effects that may accompany the particular agent, or composition
thereof, employed. It is generally desirable, whenever possible, to
keep adverse side effects to a minimum. The dose of the
biologically active material will vary; suitable amounts for each
particular agent will be evident to a skilled worker.
[0170] Another embodiment of the invention is a kit useful for any
of the methods disclosed herein, either in vitro or in vivo. Such a
kit can comprise one or more of the compositions of the invention.
Optionally, the kits comprise instructions for performing the
method. Optional elements of a kit of the invention include
suitable buffers, pharmaceutically acceptable carriers, or the
like, containers, or packaging materials. The reagents of the kit
may be in containers in which the reagents are stable, e.g., in
lyophilized form or stabilized liquids. The reagents may also be in
single use form, e.g., in single dosage form.
EXAMPLES
[0171] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0172] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0173] Terms listed in single tense also include multiple unless
the context indicates otherwise.
[0174] The examples disclosed below are provided to illustrate the
invention but not to limit its scope. Other variants of the
invention will be readily apparent to one of ordinary skill in the
art and are encompassed by the appended claims. All publications,
databases, and patents cited herein (e.g. U.S. Pat. No. 6,153,217;
U.S. Patent Application Publication No. 20090060894; International
Application Number PCT/US2012/33515 filed Apr. 13, 2012 for: REDOX
RESPONSIVE POLYMERIC NANOCAPSULES FOR PROTEIN DELIVERY) are hereby
incorporated by reference for all purposes.
[0175] Methods for preparing, characterizing and using the
nanocomplexes of this disclosures are illustrated in the following
Examples. Starting materials are made according to procedures known
in the art or as illustrated herein. The following examples are
provided so that the invention might be more fully understood.
These examples are illustrative only and should not be construed as
limiting the invention in any way.
Example 1
Design of Highly Robust Artificial Enzyme Nanocomplexes (ENs) with
Synergic Function
[0176] This example demonstrates a general design of highly robust
artificial enzyme nanocomplexes (ENs) with synergic function. This
is achieved by assembling or conjugating enzymes with synergic or
complementary functions, or enzymes that can cooperatively function
together, into a nanocomplex, followed by encapsulating the
nanocomplex within a network-polymer capsule.
Methods
[0177] Synthesis of Enzyme Nanocomplexes (ENs): The synthesis of
DNA-inhibitor scaffolds was achieved by conjugating the inhibitors
to single-strand DNA (ssDNA) with designed sequence followed by
their spontaneous assembly to form the scaffold. Briefly, 10 mg of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and 60 .mu.L
of 5'-phosphated ssDNA (5 nmol/.mu.L) were mixed, followed by
addition of 50 .mu.L of enzyme inhibitor (0.2 M) or substrate
derivatives (dissolved in 0.1M imidazole MES buffer). The mixtures
were then mixed thoroughly by vortexing. After centrifugation for 5
mM at 8000 rpm, the mixtures were kept at room temperature for 2 h
to allow a complete reaction. As-conjugated ssDNA was then purified
by gel filtration on desalting resin using EDTA-phosphate buffer
(10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH=7.4).
Complementary ssDNA-inhibitor molecules were then mixed, incubated
at 37.degree. C. for 10 min, and cooled to room temperature to form
the DNA-inhibitor scaffolds.
[0178] To synthesize n(HRP-GOx), GOx and HRP were anchored with
acryloyl groups by reacting with acrylic acid N-hydroxysuccinimide
ester (NAS) in 20 mM sodium carbonate buffer (pH=8.5) for 2 h.
After dialysis against phosphate buffer (20 mM, pH=7.0),
acryloxylated HRP, acryloxylated GOx, and DNA-inhibitor scaffold
(1.5:1.5:1, n/n/n) were mixed and kept at room temperature for 20
min to form the enzyme-scaffold complexes. The complexes were then
encapsulated by in-situ polymerization using acrylamide (4:1 weight
ratio to all enzymes) as the monomer, bis-methylacrylamide (1:6
weight ratio to acrylamide) as the cross-linker, and ammonium
persulfate/tetramethylethylenediamine as the initiator. The
encapsulated complexes were firstly purified by dialysis against
PBS, then heated to 37.degree. C. for 10 min and further purified
using size exclusion chromatography (Sepharose 6B). The synthesis
of n(HRP-GOx-Inv), n(GOx-Cat), n(AOx-Cat), n(Cat), n(AOx), n(GOx)
and other enzyme nanocomplexes were conducted using a similar
approach and the details are provided in Example 2. Detailed
characterization of the ENs, including dynamic light scattering
(DLS), transmission electron microscopy (TEM), fluorescent
microscopic images and spectra, cell viability assays, toxicity,
and other related information are also provided in Example 2.
[0179] In-vivo studies: In-vivo studies of alcohol antidotes were
achieved by monitoring the variation of blood alcohol concentration
(BAC) and the level of plasma alanine aminotransferase (ALT) of
mice after treating with ethanol, native enzyme, and enzyme
nanocapsules. Four groups of enzyme nanocapsules, including n(AOx),
n(Cat), mixture of n(AOx) and n(Cat), and n(AOx-Cat) with the same
enzyme content were used. Native AOx was used for comparison. Male
C57B6 mice of 8-10 week old from Jackson Laboratory (Bar Harbor,
Me.) were used for the studies. Mice were fasted for 8-12 hours,
followed by gavage with the enzyme nanocapsules and native AOx or
nanocapsules (dosed at 65 .mu.g AOx/animal) within the
alcohol-containing liquid diet (AIN76A, Dyets Inc., dosed at 6 g of
EtOH/kg body weight (w/w)). For the control group of mice,
isocaloric diet without EtOH were fed via gavage. Blood samples
were taken from tails at 45, 90, 180 and 360 min after the alcohol
gavage. BACs of the alcohol-fed animals were determined using an
ethanol assay kit (BioVision). Background BAC was deducted by using
control mice gavaged with 250 .mu.L of PBS buffer only. ALT values
in the plasma of mice were determined at six to eight hours after
the alcohol gavage by following methods described in Ji et al.,
Gastroenterology 124:1488-99 (2003). All animals were treated in
accordance with the Guide for Care and Use of Laboratory Animals
approved by local committee.
[0180] In-vivo detoxification capability of n(GOx-Cat) was
conducted using Male mice (C57B6). The mice were anesthetized using
ketaime (85 mg/kg) combined with xylazine (10 mg/kg), which were
intramuscularly injected into the posterior thighs. Hairs on the
back of animals were then removed using an animal shaver. n(GOx),
n(Cat) and n(GOx-Cat) were delivered into the skin via two
cutaneous injections, respectively, at 0 and 24 hours with a dosage
of 35 .mu.g nanocomplex dispersed in 50 .mu.l PBS buffer per group
per injection. Equal volumes of hydrogen peroxide (H.sub.2O.sub.2)
solution (3% w/v) and PBS buffer were injected at different spots
of the back of the same animals, respectively, as the positive and
negative controls. The animals were anesthetized at 24 hours
following the second injection; pieces of skin tissues were then
sampled, fixed and processed for H&E staining and for
fluorescent TUNEL staining using an in situ Cell Death Detection
Kit from Roche. H&E images were captured with a Nikon Eclipse
E600 microscope and confocal fluorescence images were obtained with
a Nikon PCM2000 confocal laser-canning microscope according to Ji
et al., Hepatology 40:442-51 (2004).
Results
[0181] As illustrated in FIG. 1, competitive inhibitors for each
enzyme are respectively conjugated to a single-strand DNA with
designed sequence; complementary assembly of the DNA molecules
forms a DNA-inhibitor scaffold linked with the three inhibitors;
specific bindings of the inhibitors and the enzymes construct the
triple-enzyme nanocomplexes (Step I).
[0182] Subsequent in-situ polymerization grows a thin layer of
network polymer around each nanocomplex, leading to the formation
of nanocomplexes containing a triple-enzyme core and a permeable
shell (Step II). Finally, removal of the DNA-inhibitor scaffolds
creates highly robust ENs denoted as n(Enzymes), where Enzymes
within the parentheses refer to the enzymes within the core of the
nanocomplexes (Step III). It is important to point out that
encapsulating the enzymes within the nanocomplexes effectively
stabilizes them against non-physiological environment and protease
attack, furthermore, as-formed nanocomplexes can be readily
functionalized to acquire desired surface properties and targeting
capability (see, e.g. Yan et al., Nat Nanotechnol 5, 48-53,
(2010)).
[0183] Successful construction of the ENs architecture was
demonstrated using horseradish peroxidase (HRP) and glucose oxidase
(GOx) as the model enzyme. FIG. 2a shows a Transmission electron
microscopic (TEM) image of n(HRP-GOx) with an average diameter of
30.+-.7 nm, which is confirmed by dynamic light scattering (DLS)
measurement (FIG. 6). To confirm the double-enzyme architecture,
each HRP and GOx molecule was labeled with a single 1.4-nm gold
nanoparticle prior to the ENs construction. As shown in FIG. 2b,
most gold-labeled ENs contain two gold nanoparticles, indicating
that each EN indeed contains one HRP and one GOx molecule
accounting specific bindings between the inhibitor and the
enzyme.
[0184] Such nanocomplex architecture spatially defines the
constituent enzymes within close proximity Using
Rhodamine-B-labeled HRP (HRP-RhB) and
fluorescein-isothiocyanatelabeled GOx (GOx-FITC) as an example,
FIG. 2c compares the fluorescence spectra of their mixture (equal
molar ratio) and the corresponding dual-enzyme ENs with the same
enzyme content. Since FITC and RhB possess excitation maximums at
495 and 540 nm, respectively, the mixture only exhibits the FITC
emission centered at 520 nm under 450 nm excitation. For
comparison, the ENs exhibit intense emissions from both FITC and
RhB (centered at 520 nm and 585 nm, respectively), indicating an
effective Foster resonance energy transfer (FRET) from GOx-FITC to
HRP-RhB. This observation clearly confirms that HRP and GOx are
closely associated within a short distance (<10 nm) (see, e.g.
Selvin et al., Nat Struct Biol 7, 730-734, (2000)). To further
confirm the close-proximity structure, FIG. 2d shows fluorescence
microscopic images of the ENs, exhibiting clear co-localization of
GOx-FITC and HRP-RhB. Upon excitation at 488 nm, FRET emission at
580 nm was also recorded at the same position, further validating
the close proximity architecture.
[0185] As observed, such close-proximity architecture endows the
ENs with significantly enhanced catalytic efficiency. This was
demonstrated by consecutive reactions of sucrose and glucose
mediated by invertase (Inv), GOx and HRP. Glucose directly added in
the reaction media or generated from the Inv-mediated hydrolysis of
sucrose is oxidized via the GOx-mediated reaction, leading to the
production of H.sub.2O.sub.2. As-produced H.sub.2O.sub.2 further
oxidizes o-dianisidine via the HRP-mediated reaction, which is used
to probe the overall enzymatic reaction efficiency.
[0186] FIG. 2f compares the turnover rates (o-dianisidine oxidation
rates) of n(HRP-GOx) and n(HRP-GOx-Inv) with those of their native
enzyme mixtures with the same enzyme contents. Compared with the
native enzyme mixtures, n(HRP-GOx) and n(HRP-GOx-Inv) show enhanced
turnover rate by 5 and 15 folds in buffer solution, respectively,
confirming an enhanced catalytic efficiency. To simulate the
viscous environment within the cells or blood stream 9 see, e.g.
Ellis et al., Trends Biochem Sci 26, 597-604, (2001)), polyethylene
glycol (PEG, Mw .about.3000) was added to the reaction media to
increase the viscosity (diffusion resistance of intermediates
within the media). As expected, n(HRP-GOx) systematically exhibits
increased relative turnover rates with increasing PEG
concentration; 34 fold increase in turnover rate was achieved in
the presence of 35 wt-% PEG. Similarly, n(HRP-GOx-Inv) shows up to
24-fold enhancement in relative turnover rates in PEG solutions.
The significantly improved rates of the chemical transformations
are attributed to the close-proximity architecture that effectively
minimizes diffusion of the intermediates. Besides the enhanced
catalytic efficiency, n(HRPGOx) and n(HRP-GOx-Inv) also exhibit
significantly enhanced stability.
[0187] FIG. 2e compares the residual enzyme activities of
n(HRP-GOx) and n(HRP-GOx-Inv) with their enzyme-mixture
counterparts. n(HRP-GOx) and n(HRP-GOx-Inv) retain 70% and 75% of
their original activities after incubation at 65.degree. C. for 60
minutes, whereas the mixtures lose more than 98% activities.
[0188] The capability of making ENs with significantly enhanced
activity and stability offers a novel class of enzyme machineries
for bio-catalysis, sensing, therapeutic and other applications.
Oxidases, for example, are being used or proposed for many
therapeutic applications such as the treatment of gouts (see, e.g.
Sherman et al., Adv Drug Deliver Rev 60, 59-68, (2008); Schelsinger
et al., Nat Rev Drug Discov 10, 17-18, (2011); Sundy et al.,
Arthritis Rheum 58, 2882-2891, (2008); and Kehrer et al., Crit Rev
Toxicol 23, 21-48, (1993)).
[0189] Nonetheless, oxidase-mediated reactions generally produce
toxic H.sub.2O.sub.2, which may cause damages to organs and tissues
(see, e.g. Kehrer et al., Crit Rev Toxicol 23, 21-48, (1993)).
Fortunately, this limitation can be effectively addressed using the
EN technology. Exemplified by n(GOx), its incubation with cells
leads to a loss of 90% cell viability owning to the H.sub.2O.sub.2
generated from GOx-mediated glucose oxidation (FIG. 3a). Adding
n(Cat) into the culture media effectively reduces the cell
viability loss (58%) due to catalase-mediated decomposition of the
H.sub.2O.sub.2 Amazingly, n(GOx-Cat) far outperforms the mixture of
n(GOx) and n(Cat) (with same enzyme content). Cells treated with
n(GOx-Cat) show similar viability to that of the untreated cells
(91%). FIG. 3b shows photographs of the cells cultured with n(GOx),
mixture of n(Cat) and n(GOx), and n(GOx-Cat) with the same enzyme
content after treating with CellTiter Blue for 3 h at 37.degree. C.
Consistently, the cells cultured with n(GOx-Cat) show a much
intense reddish fluorescent color than others, indicating a higher
cell viability due to more effective H.sub.2O.sub.2 elimination.
Without being bound by a specific scientific theory or mechanism of
action, this observation is consistent with an unexpected
phenomena, one where the close-proximity enzyme architecture not
only enhances the overall chemical-transformation efficiency, it
also furthers the effective removal of toxic intermediates so as to
avoid potential cell and tissue damages.
[0190] To further confirm the in-vivo detoxification capability of
ENs, n(GOx), n(Cat) and n(GOx-Cat) were injected cutaneously to a
mouse (C57BL/6) at different injection sites. As positive and
negative controls, equal volumes of H.sub.2O.sub.2 solution (3%
w/v) and PBS vehicle were injected to separate skin sites of the
same mouse, respectively. Skin lesions were observed in
H.sub.2O.sub.2-treated and n(GOx)-treated sites 48 hours after
injection.
[0191] In contrast, skin damages were not observed in the spots
injected with PBS, n(Cat), or n(GOx-Cat) (FIG. 3c). For further
toxicity studies, the mouse was sacrificed and skin tissues at the
injection sites were sectioned and stained with hematoxylin and
eosin (H&E) and TUNEL staining kit. Clearly, H.sub.2O.sub.2
administration caused tearing and ballooning in the dermis of the
skin; n(GOx) administration caused similar tissue ballooning and
neutrophil infiltration albeit to a smaller extent, indicating
pathophysiological response and injury due to the H.sub.2O.sub.2
generated (FIG. 3d). Cell apoptosis was evident in the skin tissues
treated with either H.sub.2O.sub.2 or n(GOx), whereas the cell
death in n(Cat) or n(GOx-Cat) treated skin tissues was minimal and
was comparable to that of PBS treated sample (FIG. 3d).
[0192] These observations clearly demonstrated that, by
encapsulating GOx and Cat in close proximity within the ENs, one
could effectively reduce the toxicity and tissue damage associated
with the administration of oxidases, which shines light on the
current oxidase-related therapeutics.
[0193] Further therapeutic significance of this technology was
demonstrated by ENs of catalase and alcohol oxidase, a potential
antidote for alcohol. Alcohol consumption is a millennium-old
component of human civilization; alcohol abuse, however, associates
with a series of organ injuries (e.g., liver damage) and social
problems (e.g., violence and driving under influence of alcohol
(DUI) see, e.g. Lee et al., Lancet 2, 759-761, (1979)).
Biologically, metabolic process of alcohol relies on consecutive
actions of alcohol dehydrogenase (conversion of ethanol to
acetaldehyde) and acetaldehyde dehydrogenases (conversion of
acetaldehyde to acetic acid) mediated by nicotinamide adenine
dinucleotide (NAD+) (see, e.g. Crabb et al., P Nutr Soc 63, 49-63,
(2004)).
[0194] However, low extracellular NAD+ concentration restricts the
use of such dehydrogenases as extracellular antidotes (see, e.g. Ji
et al., Hepatology 40,442-451, (2004)). To circumvent this
limitation, we designed a novel enzymatic machine based on
n(AOx-Cat), where AOx is alcohol oxidase that can effectively
mediate oxidization of alcohol to acetaldehyde using molecular
oxygen.
[0195] Similar to other oxidase, this oxidation process also
produces toxic byproduct H.sub.2O.sub.2; nevertheless, as-produced
H.sub.2O.sub.2 can be promptly eliminated by the catalase
proximately located within the ENs (see cell viabilities in FIG.
8). It is also important to point out that, both AOx and Cat
encapsulated within the n(AOx-Cat) are well protected by the
polymer shells from protease attack and non-physiological
condition, enabling their versatile administration orally and
systematically.
[0196] FIG. 4 demonstrates efficacy of oral alcohol antidotes based
on n(AOx-Cat). Mice were gavaged with a liquid diet containing
alcohol together with native AOX, n(AOX), n(Cat), an equal-ratio
mixture of n(AOx) and n(Cat), or n(AOx-Cat). All the animals were
under alcohol intoxication and slept within 20 minutes after the
alcohol administration; average blood alcohol concentration (BAC)
in mice with alcohol diet was 374.+-.25.8 mg/dL 30 min after the
administration. Compared with the mice administrated with alcohol
only, the BAC of the mice administrated with n(AOx-Cat) was reduced
by 10.1%, 31.8% and 36.8% at 45 min, 90 min and 3 hours after
injection, respectively (FIG. 4a). Only slight BAC reductions
during these periods were observed in those administrated with
n(AOx) (<8.5%) or the mixture of n(AOx) and n(Cat) (<10.6%).
No BAC reductions could be detected in those with n(Cat) or native
AOx; the ineffectiveness of native AOx in reducing BCA further
confirms the essential role of the polymer shells of ENs in
protecting the core enzyme complexes from inactivation. Six hours
after the alcohol gavage, the animals started to wake up; the
wakeup time for the group with n(AOx-Cat) was 1-2 hours earlier
than those of the other groups.
[0197] To probe liver injury associated with the alcohol feeding
and administrations of ENs, plasma alanine aminotransferase (ALT)
was analyzed 7 hours after the treatments. ALT was significantly
increased in all alcohol-fed animals. However, in comparison with
other groups, the mice with n(AOX-Cat) had a tendency of lower ALT
levels (FIG. 4b). Similar results of BAC reductions were also
obtained in animals via tail-vein injections (FIG. 9), confirming a
versatile administration of ENs.
[0198] Above studies confirm that it is feasible to develop alcohol
antidotes that effectively reduce BAC and liver injury. The
significantly higher effectiveness of n(AOX-Cat) than those of
n(AOx) and the mixture of n(AOx) and n(Cat) further confirms the
synergic effect of the AOx and Cat confined within the ENs. In
fact, oxidation of alcohol consumes molecular oxygen and releases
H.sub.2O.sub.2. Catalase does not only eliminate these toxic
H.sub.2O.sub.2 molecules to reduce liver damage; Catalase-mediated
H.sub.2O.sub.2 decomposition also regenerates molecular oxygen,
which can be rapidly fed back to the AOx owning to the
close-proximity architecture.
[0199] This synergic effect alleviates local depletion of oxygen,
leading to accelerated alcohol oxidation and more effective
reduction of BAC. Note that, besides the H.sub.2O.sub.2 production,
alcohol oxidation also generates acetaldehyde, another toxic
intermediate. More effective ALT reduction and complete liver
injury protection relies on effective removal of acetaldehyde. A
complete protection against alcohol-induced liver injury is
anticipated, when highly active ADOx becomes available for the
construction of n(AOx-ADOx-Cat) triple-enzyme nanocomplexes.
Nevertheless, to the best of our knowledge, this appears to be the
first alcohol antidote, clearly demonstrating great potentials of
using ENs for broad therapeutic applications.
[0200] In sum, this example demonstrates a design of robust enzyme
architectures by precisely assembling and stabilizing multiple
enzymes within a nano-space. Considering the vast library of
enzymes that are currently available or are being added, novel
classes of enzyme machineries could be built for broad applications
beyond therapeutic applications. More importantly, though
judiciously choosing building enzyme molecules and harvesting their
synergic and complementary effects, this example foresees creation
of novel families of enzyme machineries with programmable function
beyond evolution.
Example 2
Illustrative Methods and Materials for Making and Using Artificial
Enzyme Nanocomplexes (ENs) with Synergic Function
[0201] This example presents more details of the materials, methods
and results discussed in Example 1.
1. Materials
[0202] Reagents and solvents were purchased from Sigma-Aldrich (St.
Louis, Mo.) and used as received without further purification
unless otherwise noted. HeLa cells were purchased from American
Type Culture Collection (ATCC). The Dulbecco's Modified Eagle
Medium (DMEM) growth medium and Penicillin/streptomycin were
obtained from Invitrogen (Carlsbad, Calif.). Fetal Bovine Serum
(FBS) was obtained from Lonza Walkerrsville Inc (Walkerrsville,
Md.). CellTitra Blue cell viability kit was purchased from Promega
(Madison, Wis.). N-(3-Aminopropyl) ethacrylamide hydrochloride for
production of enzyme nanocomplexes was purchased from Polymer
Science, Inc. Mono-sulfo-N-hydroxy-succinimido Au-nanoparticles was
purchased from NanoProbe, NY. Fluorescence dyes including Rhodamine
B isothiocyanate and Fluorescein isothiocyanate were obtained from
Sigma-Aldrich (St. Louis, Mo.). Enzymes including horseradish
peroxidase, glucose oxidase, invertase and catalase were purchased
from Sigma-Aldrich (St. Louis, Mo.). Male C57B6 mice were purchased
from Jackson Laboratory (Bar Harbor, Me.). Alcohol liquid diet
AIN76A was obtained from Dyets, Inc. Ethanol Assay Kit was
purchased from BioVision (Mountain View, Calif.). H&E and
fluorescent TUNEL staining kits were purchased from Roche.
[0203] Single strand DNAs (ssDNAs) used for preparing enzyme
nanocomplexes were purchased from Sigma-Aldrich (St. Louis, Mo.).
Their sequences were provided in Table 1 below.
TABLE-US-00001 Name Sequence ssDNA-1 5'-[Phos]ATACGAATTCTAC-3' (SEQ
ID NO: 1) ssDNA-1' 5'-[ThiC6]ATACGAATTCTAC-3' (SEQ ID NO: 2)
ssDNA-2 5'-[Phos]AGTAGAATTCGTA-3' (SEQ ID NO: 3) ssDNA-2'
5'-[ThiC6]AGTAGAATTCGTA-3' (SEQ ID NO: 4) ssDNA-I
5'-[Phos]ATGGTTGAGGAAGTC-3' (SEQ ID NO: 5) ssDNA-II
5'-[Phos]AGACTTCCGCTATCG-3' (SEQ ID NO: 6) ssDNA-III
5'-[AmC6T]ACGATAGCATGTGTA-3' (SEQ ID NO: 7) ssDNA-IV
5'-ATACACATTCAACCA-3' (SEQ ID NO: 8) [Phos] stands for
phosphorylation, [ThiC6] stands for Thiol C6 S-S, and [AmC6T]
stands for Amine C6.
2. Instruments
[0204] UV-Visible spectra were acquired with a GeneSys 6
spectrometer (Thermo Scientific). Fluorescence spectra were
obtained with a QuantaMaster Spectrofluorimeter (Photon Technology
International). Dynamic light scattering studies of the enzyme
nanocomplexes was measured on Zetasizer Nano instrument (Malvern
Instruments Ltd., United Kingdom). Transmission electron microscope
(TEM) images were obtained on Philips CM120 electron microscope
operating with an acceleration voltage of 120 kV. Fluorescence
intensities were measured with a Fujifilm BAS-5000 plate reader.
Mass spectra were acquired with an Applied and photographed with a
Nikon TE2000S inverted fluorescent microscope.
3. Preparation of Enzyme Nanocomplexes (ENs)
3.1 Synthesis of DNA-Inhibitor Scaffolds
[0205] The syntheses of DNA-inhibitor scaffolds were achieved by
the combination use of conjugation and self-assembly technologies.
Briefly, single strand DNA was first conjugated with enzyme
reversible inhibitors (or their substrate derivatives) to get a
single-strand-DNA-derived enzyme inhibitor (ssDNA inhibitor). After
conjugation, different kinds of ssDNA inhibitors were mixed
together and heated to 37.degree. C. for 10 min. After cooling to
room temperature, ssDNA-inhibitors self-assembled with their
complementary strand(s) to form the DNA-inhibitor scaffolds.
Detailed synthesis schemes of the ssDNA inhibitors for horseradish
peroxidase (HRP), glucose oxidase (GOx) and Invertase (Inv) are
shown in the scheme below and in FIG. 5.
##STR00001## ##STR00002##
3.2 MALDI-TOF Characterizations of ssDNA-Inhibitors
[0206] Successful conjugation of ssDNA-inhibitors was demonstrated
by monitoring their molecular weight increase via MALDI-TOF
spectra. Ultrapure 3-hydroxypicolinic acid (3-HPA) was used as
matrix for the following measurements. Unconjugated ssDNAs were
used as controls for MALDI-TOF characterizations. Resulted spectra
of ssDNA before (black) and after (gray) conjugating with enzyme
inhibitors are shown in FIG. 6.
3.3 Synthesis of n(HRP-GOx-Inv)
[0207] n(HRP-GOx-Inv) was synthesized using a similar method as
that for the preparation of n(HRP-GOx) mentioned in the Methods
part. Briefly, HRP, GOx and Inv were first reacted with acrylic
acid N-hydroxysuccinimide (NAS), followed by purification by
dialyzing against phosphate buffer (20 mM, pH=7.2) using a dialysis
tubing membrane (MWCO=10 kDa). Acryloxylated HRP, GOx, Inv, and
HRP/GOx/Inv DNA inhibitor scaffold (1:1:1:5, n/n/n/n) were mixed
together to form HRP-GOx-Inv enzyme complexes, and then
encapsulated by in-situ polymerization on the surface of enzyme
complexes. After removal of the unreacted monomers by dialysis,
sample was heated to 37.degree. C. for 10 min, followed by passing
through a size exclusion chromatography (Sepharose-6B) to remove
DNA-inhibitor scaffolds.
3.4 Synthesis of Dual Fluorescence-Labeled n(HRP-RhB-GOx-FITC)
[0208] Rhodamine-B-labeled horseradish peroxidase (HRP-RhB) and
fluorescein isothiocyanate-labeled glucose oxidase (GOx-FITC) were
prepared by following the protocol provided by the manufacturer of
fluorescence dyes. Fluorescent dyes, RhB and FITC, were first
dissolved in anhydrous DMSO to get 10 mg/mL stock solution. Then 50
.mu.L of dye solutions were added gradually into 2-mg enzyme
solutions (pH=8.2, sodium carbonate, 100 mM) and reacting overnight
at 4.degree. C. Labeled enzymes were purified by gel permeation
chromatography (Sepharose-6B). The column was pre-equilibrated with
Phosphate-Buffered Saline (PBS) and protein was eluted with the
same buffer. The first color band was collected, condensed and
stored at 4.degree. C. for further use. The concentration and
dye/protein ratio (D/P) of HRP-RhB and GOx-FITC were determined by
the extinction coefficients of 102,000 M.sup.-1cm.sup.-1 at 403 nm
(HRP), 44,100 M.sup.-1cm.sup.-1 at 280 nm (GOx) and 108,000
M.sup.-1cm.sup.-1 at 555 nm (RhB), 81,000 M.sup.-1cm.sup.-1 at 495
nm (FITC).
[0209] n(HRP-RhB-GOx-FITC) enzyme nanocomplex was prepared by using
HRP-RhB, GOx-FITC as enzymes and HRP/GOx DNA-inhibitor scaffold to
form enzyme complex. Detailed procedure of its preparation is the
same as that of n(HRP-GOx) mentioned above. After preparation, dual
fluorescence-labeled ENs were further purified by gel filtration
(Superdex-75). The first band, which has a strong absorbance at 555
nm, was collected, condensed and stored at 4.degree. C. for
fluorescence characterizations.
3.5 Synthesis of Au-Nanoparticle Labeled ENs
[0210] Excess amounts of Au-nanoparticles
(mono-sulfo-N-hydroxy-succinimido Au nanoparticles) were reacted
with native HRP and GOx (3:1, n/n, Au/enzyme) in 1xPBS buffer for 1
hr, respectively. Excess amount of gold nanoparticles was removed
by gel filtration (Superdex-75). Concentrations of the
Au-nanoparticles, HRP and GOx were then determined by the UV/vis
spectra based on their molar extinction coefficients (nanogold,
155,000 M.sup.-1cm.sup.-1 at 420 nm, HRP, 102,000 M.sup.-1cm.sup.-1
at 403 nm, GOx, 11,200 M.sup.-1cm.sup.-1 at 453 nm). The resulted
Au-labeled HRP/GOx MCENs contain an AuNP/HRP ratio of 0.83 and
AuNP/GOx ratio of 0.95.
[0211] The Au-labeled HRP and GOx were then used to prepare dual
AuNP labeled ENs by using the same protocol for the preparation of
n(HRP-GOx). After preparation, the sample was purified by passing
through a size exclusion chromatography (Sepharose-6B), and
collecting the first colored band. Sample was then stored at
4.degree. C. for transmission electron microscopy (TEM)
observation.
3.6 Synthesis of ssDNA-Alcohol Oxidase (ssDNA AOx) and
ssDNA-Catalase (ssDNA-Cat) Conjugates
[0212] Alcohol Oxidase (AOx) and Catalase (Cat) were dissolved in
50 mM sodium phosphate buffer (pH=7.4, 0.15 M NaCl) with a
concentration of 2 mg/mL, respectively. N-succinimidyl
3-(2-pyridyldithio) propionate (SPDP) stock solution was first
prepared as a 2 mM DMSO solution, followed by reacting with enzymes
by gradual addition of 25 .mu.L of SPDP solution into 1 mL enzyme
solutions. Reaction was kept at 4.degree. C. under stirring for 4
h. After the reaction was finished, mixtures were purified by gel
filtration (Superdex-75) using 50 mM sodium phosphate, 0.15 M NaCl,
10 mM EDTA, pH 7.2 as eluting buffer. In the meanwhile, ssDNA-1'
and ssDNA-2' were reduced with dithiothreitol (DTT) and purified by
gel filtration (Superdex-75) immediately. After purification,
reduced ssDNA-1' and ssDNA-2' were then added into SPD-Pactivated
Cat and AOx solutions at a molar ratio of 1.2/1 (ssDNA to enzyme),
respectively. The mixture was kept reacting at 4.degree. C.
overnight for a complete conjugation, and then add NAS into the
mixture to anchor polymerizable sites to ssDNA-enzyme conjugates.
Samples were then purified by dialysis against 1xPBS with a
dialysis tubing membrane (MWCO.about.10 kDa) to remove unconjugated
ssDNAs.
3.7 Synthesis of n(GOx-Cat) and n(AOx-Cat)
[0213] ssDNA-2-glucosamine (ssDNA inhibitor for GOx) was first
mixed with equal amount of acryloxylated ssDNA-1' conjugated Cat
(1/1, n/n). The mixture was heated to 37.degree. C. for 10 min and
then cooled down with an ice bath. After recovering to room
temperature, in situ polymerization was carried out to encapsulate
a pair of GOx and Cat into an enzyme-nanocomplex.
[0214] After removal of unreacted monomers and initiators by
dialysis against 1xPBS, the sample was heated to 37.degree. C. for
10 min and immediately passed through a size exclusion
chromatography to remove ssDNA inhibitor conjugates for GOx. The
first band eluted was collected and condensed to get a stock
solution of n(GOx-Cat). The n(GOx-Cat) stock solution was kept at
4.degree. C. for further use.
[0215] The preparation of n(AOx-Cat) is similar to that of
n(GOx-Cat) mentioned above. Prior to polymerization, acryloxylated
ssDNA-1' conjugated Cat and ssDNA-2' conjugated AOx was mixed with
equal molar ratio. After polymerization, reaction mixture was
purified by passing through a size exclusion chromatography to
remove unreacted monomers and other by-products.
[0216] n(AOx-Cat) was then condensed and aliquoted to 65 .mu.g/tube
(100 .mu.L/tube) for in vitro and in vivo tests. Note that ENs of
AOx and Cat can also be prepared by directly conjugating the both
enzymes followed by a similar encapsulating technique.
4. Synthesis of Single Enzyme Nanocomplexes
[0217] Single enzyme nanocomplexes of GOx, AOx and Cat (denoted as
n(GOx), n(AOx) and n(Cat)) were synthesized using the method
reported in Yan et al., Nature Nanotechnology 5:48-53 (2010).
Briefly, the enzymes were first acryloxylated by reacting with NAS,
and then wrapped with a thin layer of polyacrylamine by in situ
polymerization. After removal of the unreacted monomers by dialysis
against 1xPBS, single enzyme nanocomplexes were then purified by
passing through a size exclusion chromography (Sepharose-6B).
Nanocomplexes were then collected, condensed and stored at
4.degree. C. for further use.
5. Activity Assays and Stability of ENs
5.1 Activity Assays
[0218] Activities of enzyme complexes, n(HRP-GOx) and
n(HRP-GOx-Inv), were assessed by monitoring the oxidation rate of
o-Dianisidine (ODS). Briefly, a phosphate buffer (0.1M, pH=7.0)
containing 0.2 mM o-Dianisidine, 500 .mu.M of ENs and a specific
amount of substrate (glucose for n(HRP-GOx) or sucrose for
n(HRP-GOx-Inv)), were incubated at 25.degree. C. for 5 min.
[0219] During the incubation, absorbance reading at 460 nm was
recorded continuously with a UV/vis spectrometer. The absorption
curve was plotted vs time and .DELTA.A.sub.460/min from the linear
potion of the curve was calculated. The activity assays were
repeated with a series of appropriate concentrations of substrate
to get a series of catalytic rate to obtain K.sub.M.sup.app and
K.sub.cut.sup.app by the Lineweaver-Burk plot. Similar assays were
also performed with free enzymes mixture (free HRP/GOx, and free
HRP/GOx/Inv) as the control experiments.
TABLE-US-00002 TABLE 2 Kinetic parameters of HRP/GOx and
HRP/GOx/Invertase free enzyme systems and their corresponding ENs
K.sub.cat.sup.app System K.sub.M.sup.app (mM) s.sup.-1 % Free
HRP/GOx 53.30 .+-. 0.44 244.6 .+-. 23.2 100 n(HRP-GOx) 361.85 .+-.
0.37 1262.0 .+-. 18.6 515.9 .+-. 7.6 Free HRP/GOx/Inv 53.30 .+-.
3.54 65.3 .+-. 2.5 100 HRP-GOx- 224.90 .+-. 13.64 1008.7 .+-. 61.2
1542.5 .+-. 93.5
[0220] Similar activity assays were also performed in polyethylene
glycol (PEG, Mw.about.3000) solutions of a series of concentrations
(0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, m/v) to simulate the viscous
environment within the cells or blood stream. Briefly, certain
amount of PEG was first added into phosphate buffer (0.1M, pH=7.0)
to get a stock PEG solution. Then enzyme nanocomplexes (or their
corresponding free enzymes mixtures) and ODS were added and mixed
thoroughly to get an assay solution with an enzyme concentration of
500 .mu.M, and ODS concentration of 0.2 mM. 145 .mu.L of
as-prepared assay solution was then passaged into a transparent
96-well plate. A series of substrates with different
concentrations, which were dissolved in 5 .mu.L of PEG solution,
were then added into each well of reaction solution. Plate was then
shaken for 30 s and further incubated for 5 min. Absorbance at 450
nm of each sample was then read out with a plate reader.
5.2 Thermal Stability
[0221] Solutions of n(HRP-GOx), n(HRP-GOx-Inv), free HRP/GOx
mixture, and free
[0222] HRP/GOx/Inv mixture were first incubated at 65.degree. C.
for certain periods (5-60 min), followed by quenching on ice bath
and recovering to room temperature. Activity assays were performed
with these samples to determine the residual activities of ENs and
their corresponding free enzymes mixtures.
6. Transmission Electron Microscopy (TEM) Studies of ENs
[0223] Morphology and structure of ENs were examined directly by
TEM. Studies were carried out on a Philips CM120 electron
microscope, operating at an acceleration voltage of 120 kV. TEM
samples were prepared by drop-coating of 2 .mu.L n(HRP-GOx)
solution onto the carbon-coated copper grids. Droplets of samples
were contacted with grids for 45 s, then excess amount of samples
were removed. The grid was then rinsed, followed by staining with
1% sodium phosphotungstate at pH 7.0.
[0224] For better imaging in TEM, silver enhancement of AuNPs was
performed prior to characterization. Briefly, AuNP-labeled
n(HRP-GOx) solution was first contacted with a TEM grid for 45 s.
After rinsing with deionized water, the grid was floated on a drop
of freshly prepared silver enhancement reagent (Nanoprobe, NY) for
1 min. The grid was then rinsed again, followed by staining with 1%
sodium phosphotungstate at pH 7.0. This process resulted in the
formation of negatively-stained n(HRP-GOx) with 3-4 nm
silver-coated AuNPs inside.
7. Dynamic Light Scattering (DLS) Studies of ENs
[0225] DLS experiments were performed with a Zetasizer Nano
instrument (Malvern Instruments Ltd., UK) equipped with a 10-mW
helium-neon laser (.lamda.=632.8 nm) and thermoelectric temperature
controller. Measurements were taken at 90.degree. scattering angle.
Results (FIG. 7) show that the centered hydrodynamic diameters of
ENs are 18.+-.5 nm for n(HRP-GOx), and 37.+-.8 nm for
n(HRP-GOx-Inv).
8. Cell and In-Vivo Tests
8.1 Cell Viability Assay
[0226] The detoxifying capability of ENs, enzyme nanocomplex
mixtures, and free enzyme mixtures was determined by cell viability
assay. This study takes n(GOx-Cat), n(GOx), and a mixture n(GOx) of
n(Cat) as an example. HeLa cells were seeded into a 96-well plate
(10.sup.4 cells/well, 100 .mu.L/well) and cultured in DMEM (normal
level of glucose in medium) for a day prior to exposure to the
nanoparticles. Same amounts (0.7 mg/mL, 2 .mu.L) of n(GOx), mixture
of n(GOx) and n(Cat), and n(GOx-Cat) were then added into the wells
and incubated at 37.degree. C. for 5 h. After incubation,
CellTiter-Blue (20 .mu.L) was added into each well and further
incubated for 3 h. The plate was then placed on a shaking table at
150 rpm for 5 min to thoroughly mix the solution. Viable cells
reduce CellTiter Blue and show fluorescent reddish color.
Quantification of the cell viability was achieved by measuring the
fluorescence intensities with a plate reader (Ex=535 nm, Em=585
nm).
8.2 Analysis of the Detoxification Capability of n(GOx-Cat) in
Mice
[0227] Hairs on the back of male mice (C57B6) (8.about.10 weeks
old) were removed by an animal shaver. n(GOx-Cat), n(GOx) or n(Cat)
were delivered into the skins of mice via cutaneous injection twice
at 0 and 24 hours with 35 .mu.g the enzyme (in 50 .mu.l of PBS
buffer) per injection. Equal volumes of hydrogen peroxide
(H.sub.2O.sub.2) solution (3% w/v) and PBS buffer were injected at
different spots on the back of the same animals as the positive and
negative controls.
[0228] Skin damage was directly observed and recorded with a
camera. The skin tissues were sectioned 48 hours after the first
injection. Tissues were processed for H&E staining as
previously described 2 in order to investigate the tissue damage.
Fluorescent TUNEL staining was also conducted with an in-situ Cell
Death Detection Kit from Roche to determine the apoptotic cell
death in the skin tissue.
8.3 Cytotoxicity of n(AOx-Cat)
[0229] The toxicity of n(AOx-Cat) was assessed by a similar
viability assay mentioned above, using native AOx, n(AOx), n(Cat),
mixture of n(AOx) and n(Cat) as control. HeLa cells (10.sup.4
cells/well, 100 .mu.L/well) were seeded on a 96-well plate the day
before exposure to the nanocapsules. Nanocapsules or native AOx (2
.mu.g) were first incubated with the cells for 4 hrs, removed from
the mixture, and incubated with fresh media. Equal amount of
ethanol (3 .mu.L) was added into each well and then incubated with
cells for another 5 h at 37.degree. C. After incubation, CellTiter
Blue (20 .mu.L) was added to each well and incubated for 3 h. The
plate was then placed on a shaking table, 150 rpm for 5 min to
thoroughly mix the solution. Cell viability was quantified by
measuring their fluorescence intensities with a plate reader
(Ex=535 nm, Em=585 nm).
8.4 In-Vivo Studies of Alcohol Antidotes and Hepatotoxicity
[0230] In-vivo studies of alcohol antidotes were achieved by
monitoring the variation of blood alcohol concentration (BAC) and
the level of plasma alanine aminotransferase (ALT) of mice after
treating with ethanol (EtOH) and the enzyme-based alcohol
antidotes. Alcohol antidote administrations were performed both
orally and systematically. For systematic administration, four
groups of nanocapsules were used, n(AOx), n(Cat), n(AOx)+n(Cat)
mixture, and n(AOx-Cat), as well as native AOx (without
nanocapsulation) used for comparison. Male C57B6 mice of 8-10 week
old from the Jackson Laboratory (Bar Harbor, Me.) were used for the
studies.
[0231] Mice were tail-vein injected with nanocapsules (65
.mu.g/animal) after being fasted for 8-12 hours. Mice were then
gavaged with an alcohol liquid diet (AIN76A, Dyets, Inc.) dosed at
6 g of EtOH/kg body weight (w/w) or isocaloric control at one hour
after injection.
[0232] BAC and ALT of mice were determined with the same methods
for mice administrated orally (detailed method are described in
Example 1). All animals were treated in accordance with the Guide
for Care and Use of Laboratory Animals approved by local committee.
As shown in FIG. 9, BAC and ALT in mice injected with n(AOx-Cat)
reduced signifcanly, which is consistent with the results obtained
with oral administration.
Example 3
Multi-Core Enzyme Nanocapsules (MCEN) for the Acceleration of
Cascade Enzyme Reactions
[0233] Metabolism, happening to maintain life for all living
organisms on earth, involves the millions of enzymatic cascade
chemical reactions. These complicate processes, in which enzymes
are dedicatedly confined in subcellular space and spatial position,
allow organisms to grow and reproduce, maintain their structures,
and respond to the environment. Although most of enzymes exhibiting
exquisite catalytic specificity and efficiency in metabolic pathway
have been well characterized and explored, successful
biotransformation demonstration through multiple enzyme cascade
reactions under mild conditions are still rare therefore highly
attractive to scientists in chemistry biology, material and
chemical engineering. An unresolved obstacle, however, is the huge
reduction in the activity and stability when multiple enzymes are
roughly mixed up through traditional immobilization or expressed
together by whole gene cluster cloning without carefully
considering their synergetic effects. The hints, shown in cases
like subcellular arrangement through cellular signal anchors,
compartmented polymersome, co-immobilization in mesoporous
nanostructure, proved that geometry-related neighborhood, dedicated
gyration control, precise ratio lock and stabilization are the key
principles to the success. However, all existing designs of
multiply enzyme systems, which lack homogeneity and quality
control, remains rudimentary and impractical as medicine,
microchip, tiny electrode and robot. We hypothesized that
single-protein-leveled control through confinement and face-to face
docking of several different enzymes in nanospace, the turnover
rate of multistep reactions will be dramatically increased while
the lifetime is fully extended.
[0234] Here we presented a highly active multi-core enzyme
nanocapsules (MCEN) (Scheme 1 as shown in FIG. 1), created by
integrating multiple catalytic enzymes into single small polymer
nanocapsule. First, inhibitor DNA scaffold was directly fabricated
by mixing four pre-designed short DNA sequences, of which three
short DNA are precisely conjugated with three enzyme inhibitors
(sucrose, glucosamine and 4-dimethylaminoantipyrine), respectively
(Scheme 1D). Then enzyme-inhibitor complexes are formed through
"key-lock" self-assembly by incubating invertase, glucose oxidase
(GOx) and horseradish peroxidase (HRP) (Scheme 1A,1B,1C) with their
corresponding inhibitor DNA scaffold. A very thin polymer network
was coated on enzyme-inhibitor complexes through a mild in-situ
polymerization to construct multi-core enzymes nanocapsule(MCEN)
(Scheme 1(II)). Finally, the three-enzyme nanocapsule is obtained
by removing inhibitor DNA scaffold (Scheme 1(III)). Polymer
coatings in MCEN serve as artificial membranes for the encapsulated
enzymes. Such artificial membranes therefore should exhibit
suitable mechanical modulus to provide structural integrity,
possess effective transport pathways to allow rapid substrate
transport, and contain specific functionality to provide substrate
selectivity. Our approach to make nanocapsules with dual- or
multi-core architectures, however, allow rapid transport of
reacting intermediates between the core proteins, which is of
particular interest for cascade reactions.
[0235] Negatively stained TEM image (FIG. 10a) showed that most of
HRP-GOx dual-core nanocapsules is round around 30 nm and have two
dark dots inside. As both of HRP and GOx are labeled with single
gold nanoparticle, respectively, we are able to speculate that most
of nanocapsules only have one set of HRP and GOx inside.
Fluorescence spectrum (FIG. 10b) and fluorescence confocal
microscopy (FIG. 10c) was used to investigate FRET(Fluorescence
Resonance Energy Transfer) effect of Rhodaomine B labeled HRP and
FITC labeled GOx in nanocapsule, which reflects the spatial
distance between two enzymes. There is only FITC emission peak at
520 nm (FIG. 10b) excited by blue light at 450 nm on the
fluorescence spectrum, indicating there is no FRET effect between
Rhodaomine B on HRP and FITC on GOx. On the contrary, there are two
fluorescence emission peaks (520 nm for FITC and 585 nm for
Rohamine B) excited only by blue light at 450 nm on the
fluorescence spectrum of dual-core enzyme nanocapsules solution,
indicating there is energy transfer from FITC to Rhodamine B. As
FRET effect only happens at really short distance (<10 nm), we
conclude that HRP and GOx are packaged in one nanocapsules. The
dual-core enzyme nanocapsule through confocal microscopy confirmed
similar conclusion as that from fluorescence spectrum and showed
yellow color (FIG. 11c-iii) excited at 450 nm, which actually is
mixed up by green color (FIG. 11c-i) and red color (FIG. 11c-ii).
Thus, those results suggested each dual-core nanocapsules has
single HRP and single GOx inside, which are really close to each
other.
[0236] We compared the activity and stability between multi-core
enzyme nanocapsule and free enzymes system. Here, HRP and GOx are
used as cascade reaction enzymes in dual-core enzyme nanocapsule
system while Invertase, GOx and HRP are enzymes used in triple-core
enzyme nanocapsule system. The turnover rates of each system are
determined with the oxidation rates of ODS by various substrates
(glucose for two enzymes system and sucrose for three enzymes
system) in the present of free enzymes combination or multicore
enzyme nanocapsules. Both the molar concentration and the ratio of
enzymes in free enzymes system and multi-core nanocapsules are
exactly the same. In aqueous buffer solution, enhancement of
turnover rate for dual-core and triple-core enzyme nanocapsules
over free enzyme system reached 460% and 1500%, respectively (FIG.
11a). Viscosity effect, which is simulated by different
concentration of PEG3000, to the turnover rate of multi-core enzyme
nanocapsule are also investigated, as most of cytoplasm is full of
high viscosity fluid. When PEG3000 content increases, turnover
rates of free multiply enzyme system and multi-core nanocapsules
decrease simultaneously (FIG. 11b,11c). But turnover rate for
dual-core enzyme nanocapsule compared with free two-enzyme system
is elevated from 4 fold to 34 fold when the activity assay is
conducted in the 0% -35% PEG buffer solution. Similar to the two
enzyme system case, triplecore enzyme nanocapsule system
(Invertase, GOx and HRP) has 9 fold to 24 fold enhancement over
free three enzyme system in turnover rate when PEG concentration
changed from 0% to 35%. Dual-core and triple-core enzyme
nanocapsules also presented a much higher stability against high
temperature compared with free two and three enzyme system. After
being incubated at 65.degree. C. for 60 minutes, residual activity
of dual-core and triple-core nanocapsules is 70% and 75%,
respectively. At the same condition, free two and three enzyme
system only have less than 2% activity left.
[0237] Uricase, a highly potential enzyme drug for gout therapy, is
able to use oxygen to oxidize uric acid and generate uricate and
hydrogen peroxide. Therefore, the main restraints for uricase are
the low clearance efficiency of uric acid at low oxygen area or
deep tissue and the heart-threaten product hydrogen peroxide. To
address these issues, we build up uricase and catalase dual-core
nanocapsule as a highly efficient and safe gout therapeutic drug
candidate. Compared with uricase only, uricase and catalase
dual-core nanocapsule achieve 400% enhancement in uric acid
degradation (FIG. 12c). MTT assay showed that uricase leads to more
than 75% reduction of cell viability and dualcore nanocapsule
elevates cell viability to 120% due to remove of uric acid from
cell culture medium (FIG. 12d). Dual-core nanocapsule still has
more than 75% activity left after incubation with trypsin at
37.degree. C. for 90 mins while activity of native uricase is less
than 1% after 20mins incubation with trypsin (FIG. 12e). Optical
microscopy images represented that HeLa cells (FIG. 12a) were
incubated at 37.degree. C. for 2.5 hrs in present of uric acid
crystal without uricase (FIG. 12a(i)), with uricase only (FIG.
12a(ii)), with uricase and catalase (FIG. 12a(iii)) and with
uricase and catalase dual-core nanocapsules (FIG. 12a(iv)). Uricase
alone was able to degrade the uric acid crystals but leads to cell
death, which is indicated by the round and shrinking of cell
morphology. Compared with uricase and catalase free system, uricase
and catalase dual-core nanocapsules leaded to full clearance of
uric acid crystal in the cell culture well without any negative
effects to the cell viability. All these investigations proved that
uricase and catalase dual-core nanocapsules are a highly potential
efficient and safe gout therapeutic drug candidate.
[0238] We have demonstrated a revolutionarily new method to
encapsulate multiply enzymes into one nanocapsules with
significantly enhanced activity and stability. Precise multi-core
architecture nanocapsules using DNA scaffold, allow rapid transport
of reacting intermediates between the core enzymes, which is of
particular importance for acceleration of cascade reactions.
Moreover, judicious choice of the core enzymes allows the synthesis
of nanocapsules with a wide range of cascade reactions. More
generally, this study will allow the awesome power of enzyme
catalysis to be harvested towards a wide range of nonphysiological
applications.
[0239] Scheme Multi-Core Enzymes Nanocapsule (MCEN).
I--Self-assembly of three enzymes (enzyme A--invertase; enzyme
B--glucose oxidase; enzyme C--horseradish peroxidase) and their
corresponding inhibitor DNA scaffold (inhibitor a--sucrose;
inhibitor b--glucosamine; inhibitor c--4-dimethylaminoantipyrine);
II--Multi-core Enzymes Nanocapsule(MCEN) formed through in-situ
polymerization; III--Inhibitor DNA scaffold removed from multi-core
enzymes nanogel.
[0240] It is to be understood that while the invention has been
described in conjunction with the above embodiments, that the
foregoing description and examples are intended to illustrate and
not limit the scope of the invention. Other aspects, advantages and
modifications within the scope of the invention will be apparent to
those skilled in the art to which the invention pertains.
Sequence CWU 1
1
8113DNAArtificial SequenceLinker 1atacgaattc tac 13213DNAArtificial
SequenceLinker 2atacgaattc tac 13313DNAArtificial SequenceLinker
3agtagaattc gta 13413DNAArtificial SequenceLinker 4agtagaattc gta
13515DNAArtificial SequenceLinker 5atggttgagg aagtc
15615DNAArtificial SequenceLinker 6agacttccgc tatcg
15715DNAArtificial SequenceLinker 7acgatagcat gtgta
15815DNAArtificial SequenceLinker 8atacacattc aacca 15
* * * * *
References