Detoxification and decontamination using nanotechnology therapy
United States Patent: 7,563,613
Issued: July 21, 2009
Inventors: Dennis; Donn M.
(Gainesville, FL), Martin; Charles R. (Gainesville, FL), Morey; Timothy E.
(Gainesville, FL), Partch; Richard E. (Potsdam, NY), Shah; Dinesh O
(Gainesville, FL), Varshney; Manoj (Gainesville, FL)
Assignee: University of
Florida (Gainesville, FL)
Appl. No.: 11/195,046
Filed: August 1, 2005
A method of removing a toxic compound
comprising contacting the toxic compound with a particle having two
regions, the first region containing a detoxifying enzyme and the second
region containing a material selected to partition the toxic compound into
the second region. The particle may be a nanoparticle.
Description of the
A method for removal of at least one target chemical from a region
includes the steps of adding a nanoparticle size bioparticle to the region
and partitioning at least a portion of the target chemical into or onto
the bioparticle. The method results in reducing the active concentration
of the target chemical. The region can be a solution. Partitioning can
result from differences in physicochemical properties between the
bioparticle and the target chemical and/or from adsorption of the target
chemical on a surface of the bioparticle.
A method for removal of at least one target chemical from a region
includes the steps of adding a nanoparticle size bioparticle having at
least enzyme incorporated therein to the region and biotransforming at
least a portion of the target chemical into at least one substantially
inactive metabolite. The region can be a solution. The enzyme can
preferably be a genetically cloned enzyme.
A method for removal of at least one target chemical from a region
includes the steps of adding a bioparticle having at least one enzyme
incorporated therein and partitioning at least a portion of the at least
one target chemical into or onto the bioparticle. The method results in a
portion of the target chemicals being transformed into at least one
substantially inactive metabolite. The region can be a solution. The
bioparticle can be a nanoparticle. In one embodiment, the nanoparticle can
include a silica nanotube having an alkyl compound attached to the silica
nanotube. The bioparticle can have a size from approximately 1 to 100 nm
or preferably from approximately 1 to 5 nm. Enzymes can include
genetically cloned enzymes.
A method for treating a patient exposed to a toxic drug includes the steps
of providing a plurality of nanosized bioparticles capable of mitigating
the effects of the toxic drug through at least one mechanism selected from
the group consisting of partitioning the toxic drug into or onto the
bioparticle and transforming the toxic drug into at least one
substantially inactive metabolite, and introducing the plurality of
bioparticles and then introduced into the patient. This method can also be
applied to animals in the practice of veterinary medicine.
A composition for detoxification includes a plurality of nanoparticles.
The nanoparticles have at least one surface adapted for toxic drug
attachment. The nanoparticles are selected from the group consisting of
microemulsions with nanoscale oil cores having soft surface films,
hydrophobic cores having porous or soft shells and hard surfaces for
selective binding of toxins. The nanoparticles can include attached
enzymes for chemically degrading toxins. Preferably, the attached enzymes
include genetically cloned enzymes.
DETAILED DESCRIPTION OF THE INVENTION
Bioparticles have been discovered which provide effective and generally
complementary mechanisms for reducing the free concentration and
biological effects of a broad range of toxins including drugs. Recent
advances in particle science engineering combined with developing
molecular medicine have been found to provide highly effective therapeutic
strategies aimed at effectively treating a wide variety of toxic
poisonings, including drug poisonings. Previously, applications for
particle technology were limited by the size of available particles. For
example, particles available for use had been too large to be effectively
used with living organisms.
With a reduction in size of available particles to less than 100 nm, or
even as small as typical molecular dimensions (1-5 nm), the invention has
created bioparticles having nanoparticle sizes which are small enough to
effectively interact with living organisms. It is noted that molecules may
be less than 1 nm in diameter or larger than 5 nm in diameter. For
example, proteins which are polymers or macromolecules can be 5 nm or
larger in diameter. As used herein, the term nanoparticles refer to
particles having sizes less than approximately 100 nm. These bioparticles
can act to bind with targeted drugs and other toxic substances and/or to
quickly degrade these drugs and toxins into largely inactive reaction
products. The invention can have valuable applications for a wide variety
of uses such as drug detoxification (e.g., drug overdose), military (e.g.,
toxic warfare agents), industrial (e.g., manufacturing processes),
environmental (chemical spill clean up), as well as many other purposes.
Drug poisonings may be treated by bioparticles which can use any or all of
the following mechanisms:
1) partitioning a targeted drug onto the bioparticles by exploiting
differences in physicochemical properties and/or using molecular
templating to adsorb the drug onto functionalized surfaces of bioparticles;
2) biotransforming a targeted drug into an inactive metabolite(s). For
example, an enzyme, such as a human purified and genetically cloned high
activity enzyme, may be incorporated into a bioparticle to provide
biotransformation effects on a targeted drug or toxin, or
3) preferably, providing a bioparticle which combines both approaches (1)
These above methods are not limited to oral or intravenous use, but could
also be employed to remove toxins from biological surfaces such as skin or
non-biological surfaces such as metal, wood or plastic. Moreover, the
above methods can be used to detoxify a broad range of toxins.
The invention allows the synthesis of bioparticles that can directly
reduce the free drug concentration in the blood, either by exclusively
partitioning it inside the bioparticle, or more preferably, by
partitioning and biotransforming the drug into an inactive metabolite
within the bioparticle and ultimately promoting excretion from the body.
With this approach, the principles of lipid partitioning and/or adsorption
(via molecular templating) may act in a highly synergistic manner with
those of biotransformation to provide drug detoxification systems with
complementary detoxification mechanisms to provide added effectiveness.
Thus, by providing a very high local concentration of toxic drug
(substrate), partitioning and/or adsorption can dramatically increase the
rate of enzymatic degradation depending upon the KM of the enzyme and its
enzymatic efficiency in the bioparticle.
Nanoparticles (such as those shown in FIG. 1) have been created for
treating a broad range of toxins. For example, "soft particles" such as
microemulsions with nanoscale oil cores and "soft/hard" particles with
hydrophobic cores for lipophilic drugs and various shells each permit
relatively selective drug penetration. "Templated particles" allow for
toxins to be selectively fitted into pores in their surface generally with
1 or 2 point binding of the toxin to the nanoparticle. The above
nanoparticle types can preferably also include an attached enzyme for even
more rapid degradation of the target drug and/or toxin.
Soft/hard particles are ones consisting of a core composed of a liquid or
a (softer) polymeric organic material surrounded by or encapsulated in a
shell composed of an inorganic or (harder) polymeric organic material. The
example of a liquid encapsulated in a shell of metal oxide or metal
carbonate can be prepared by making a solution of the metal oxide
precursor in an oil and exposing droplets of the solution to water or
carbonate ion, respectively. This could be accomplished in aerosol or
microemulsion apparatus. The example of a liquid encapsulated in a shell
of organic polymer can be prepared by making a microemulsion stabilized by
surfactant having reactive vinyl functionality, followed by initiating
polymerization of the surfactant molecules around the surface of
individual oil droplets in the system. The example of a (soft) polymeric
organic core encapsulated in a (hard) polymeric shell can be prepared by
dispersing a polymer gel in a solution of organic monomer precursor to a
highly crosslinked or crystalline polymer, followed by initiation of
polymerization of the dissolved monomer.
Templated particles are ones consisting of material, either as the core or
shell component, that incorporates molecularly-sized and shaped pores into
its structure. These materials can be prepared by including in the
synthesis mixture suitably derivatized molecules or mimics (the templates)
of the chemicals (in this application the toxins) of interest to be
captured by the bioparticles. After the particles are formed and
collected, the templating species are removed from the particles by
dialysis or pyrolysis, leaving the desired pores as selective sites in
which toxic molecules to be removed can be trapped. Examples are various
metal oxides or solid organic polymers that have pores templated for
neural amines, aromatic compounds or terpenes.
A shell containing pores can provide significant improve the effectiveness
of nanoparticle-mediated drug detoxification. There are a number of
potential design benefits of encapsulating either a hydrophilic or a
hydrophobic environment with a solid shell containing nanopores. First, it
could act to stabilize a nanoemulsion injected into the blood to prevent a
dilutional effect on emulsion function. Second, it could act as a highly
selective molecular filter by allowing only molecules of certain physical
dimensions access to the bioparticle interior. Therefore, bioparticles
could be targeted against at toxic molecules with molecular weights (MWs)
at or below the selected cutoff points of molecular size. Third, by
"trapping" locally high concentrations of coenzymes and cofactors
important to CYP activity such as oxidoreductase (MW=75,000) and
cytochrome b5 (MW=17,500) within the bioparticle interior and preventing
their escape, a bioparticle with a shell incorporating nanopores would
potentially enhance (or at least help preserve) optimal P450 enzymatic
The reconstitution of P450 enzymes, especially the CYP 3A4 fraction, may
be technically challenging with regards to preserving its catalytic
activity, especially in blood. Inclusion of a shell can greatly aid this
process. Fourth, a bioparticle shell with nanopores could prevent
proteolytic degradation of CYP fractions while in the blood (i.e., armor
the enzymes). It will allow ingress of smaller sized toxic drug molecules
and easy egress of more water-soluble metabolites, but exclude those
molecules greater than the pore size cutoff. In contrast, CYP fractions
incorporated into soft bioparticles may not only be susceptible to
degradation in blood, but their local concentrations of cofactors and
coenzymes may not be preserved (see the second point given above). Fifth,
a solid shell with nanopores could be designed to provide a biodegradable
platform where the functional activity of the bioparticle is determined by
the blood half-life of biodegradable shell. That is, once the job of
biotransformation is complete, the biodegradable particle can slowly
disintegrate and releases the CYP fractions into the blood where they are
degraded. Sixth, design of the shell and its surface characteristics
(e.g., ionicity, lipophilicity) may be critical to prevent (or minimize)
uptake into RBCs or the reticuloendothelial system (RES), or eliminate the
risk of hemolysis. The goal of bioparticle design is to keep the
bioparticles within the vascular compartment. Seventh, if the shell of the
nanobioparticle is sized small enough to be directly excreted via the
kidney, it may be possible to directly sequester a highly lipophilic drug
within the bioparticle via partitioning and/or adsorption, or to attach it
to even smaller sized nanoparticles by adsorption onto its surface.
In a manner similar to what is observed clinically when the Fab antibody
Digibind.RTM. is used to treat digoxin toxicity, if the size of the "nanoparticulobody"
(a bioparticle acting functionally like an antibody) is less than a
50,000-60,000 MW molecule, it may be directly excreted from the body by
the kidneys, particularly if its surface has a neutral or positive charge.
Using molecular templating, highly selective adsorption of various
molecules (and members among their chemical class) can be achieved. In
this scenario, the P450 element of the bioparticle would not be needed. In
essence, the nanoparticle is accomplishing Phase I and II
biotransformation without the actual need for enzyme modification. That
is, it renders the toxic drug molecule more polar by complexing it to a
nanoparticle, which in turn allows excretion from the body. This type of
adsorption would be highly selective and would most likely be applicable
only to those molecules (or class of drugs sharing a common chemical
moiety) it was originally designed for. In contrast, if a lipid
partitioning system could be incorporated into a bioparticle, while
keeping its size less than 50-60 kDa, it would be useful for most
One approach for preparing such nanoparticles entails using the
template-synthesis method. In this method, the pores in a microporous
membrane or other solid are used as templates to prepare the nanoparticle.
The membranes employed contain cylindrical pores with monodisperse
diameters. The pore diameter can be controlled at will over a range from
iess than 1 nm to as large as tens of micrometers. A correspondingly large
range of nanoparticle sizes can be prepared. The template method is very
versatile. It has been used to prepare nanoparticles composed of metals,
semiconductors, other inorganic materials, carbons, etc. Nearly any method
used to prepare bulk materials can be adapted to allow for synthesis of
nanoparticles within the pores of a microporous template membrane.
Significantly, a hollow tubular nanostructures can be obtained.
Membranes that have been used to prepare nanoparticles via the template
method are shown in FIG. 3. These are microporous aluminas prepared
electrochemically from aluminum metal. The upper set of micrographs shows
an in-house prepared membrane of this type. In this case the pores are
approximately 60 nm in diameter. The lower micrograph in FIG. 3 shows a
commercially-available membrane of this type. In this case the pores are
200 nm in diameter. These micrographs illustrate the important point
discussed above that the pore diameter in such membranes (and
correspondingly the diameter of the nanoparticle obtained) can be
controlled at will. For these microporous alumina template membranes, the
pore diameter is varied by varying the potential used during the
electrochemical formation of the membrane.
Toxins can be adsorbed onto specialized nanoparticles to achieve drug
detoxification. Nanoemulsions have been synthesized for acutely reducing
the free concentration of potentially toxic molecules. FIG. 2 shows the
effect of Pluronic.RTM. L-44 Micelles and microemulsions in reducing the
concentration of the drug amitriptyline. A micelle may be defined as a
colloidal aggregate of amphipathic (surfactant) molecules, which occurs at
a well-defined concentration known as the critical micelle concentration.
The typical number of aggregated molecules in a micelle (aggregation
number) is from approximately 50 to 100.
Poloxamer, or Pluronic.RTM. gels are made from selected forms of
polyoxyethylene-polyoxypropylene copolymers in concentrations ranging from
15 to 50 weight %. Poloxamers generally are white, waxy, free-flowing
granules that are practically odorless and tasteless. Aqueous solutions of
poloxamers are stable in the presence of acids, alkalis and metal ions.
Commonly used poloxamers include the 124 (L-44 grade) used above as well
as 188 (F-68 grade), 237 (F-87 grade), 338 (F-108 grade) and 407 (F-127
grade) types, which are freely soluble in water. The trade name "Pluronic.RTM."
is used in the US by BASF Corp., of Mount Olive, N.J., for pharmaceutical
and industrial grade poloxamers.
In the example shown in FIG. 2, a nanoemulsion was produced using
Pluronic.RTM. L-44 (micelles) with or without addition of ethylbutyrate
ester. Ethylbutyrate ester was added at a low (ME-I) and high (ME-II)
concentration. As seen in FIG. 2, the emulsion effectively sequestered
significant quantities of amitriptyline, a tricyclic antidepressant agent
with potential cardiotoxic effects, when compared to the saline control
shown. The system labeled "Micelles" did not possess the oil core of
ethylbutyrate ester which was possessed by both ME-1 and ME-II. By
comparing the Micelles system to ME-1 and ME-2, it can be seen that the
oil core significantly increased the amount of amitripyline adsorbed, the
higher oil concentration (ME-II) adsorbing significantly more amitripyline
than the lower oil concentration (ME-I). Drugs that may be amenable to
this type of detoxification are not limited to tricyclic antidepressant
agents such as amitriptyline, but may include drugs and toxins from all
drug classes that have an affinity for, tend to combine with, or are
capable of dissolving in lipids (lipophilic drugs).
Many types of oils, surfactants, and cosurfactants may be used to produce
bioparticles based on nanoemulsion technology. The bioparticle composition
can be varied depending on the goal of the therapy and the specific
properties of the target toxin. For example, a particle system prepared
for intravenous use would, be preferably varied in composition compared to
a similar system prepared for skin or metal decontamination.
Another type of bioparticle that can be used to detoxify drugs can be
prepared using the template-synthesis method. Template-synthesized
nanoparticles can be either hollow nanotubules or solid nanoparticles and
they can remove the toxic substance by either adsorption on their surfaces
or extraction into the hollow part of the nanotubule. Such
template-synthesized nanoparticles can be composed of a wide variety of
materials including metals, polymers. semiconductors, other inorganic
materials, carbons, etc. The size of these nanoparticles (both the
diameter and the length) can be controlled at will from the nm regime to
the micrometer regime.
One embodiment of this technology is to use the template method to prepare
hollow cylindrical silica nanoparticles. These nanoparticles are
preferably prepared by using sol-gel template synthesis of silica within
the pores of a microporous alumina template membrane. The silica tubules
in this case were prepared using tetraethylorthosilicate as the starting
material; however, other precursors are available for preparing silica via
the sol-gel method. Sol-gel silica nanotubules of this type have been
prepared in the pores of various microporous alumina template of the type
shown in FIG. 3. It has been shown that both the inside and outside
diameters and the length of the silica nanostructures can be controlled by
varying the diameter of the pores and the thickness of the template used.
The tubular silica nanostructures prepared in this way can be derivatized
both on their outside and inside surfaces with chemical and/or biochemical
reagents. One approach for doing this is to use well-known silane
chemistry. Hundreds of silanes are available commercially, so this is a
very versatile route for chemically and biochemically derivatizing these
silica nanostructures. This is important because such derivatization
allows these nanotubules to extract or adsorb specific chemical reagents
and allows them to catalyze specific biochemical reactions. In addition
because the inside and outside of the tubules can be derivatized with
different reagents, the inside and outside chemistry/biochemistry can be
different. This is important because, for example, it might be desirable
to have the interior of the nanotubules hydrophobic so that they will
extract specific molecules but the outside hydrophilic so that the
nanotubules can be dispersed in an aqueous-based medium (e.g. blood).
Two embodiments of this concept are discussed herein. The first embodiment
derivatizes the inside surfaces of the silica nanocylinders with a
hydrophobic 18 carbon alkyl silane. In this case the outside surface is
left underivatized so that the outside surface retains the hydrophilic
character of silica. The second case entails derivatizing both the inside
and outside surfaces with trimethoxybutyl aldehyde to introduce the
aldehyde functionality to the surfaces. This aldehyde functionality can
then be reacted with terminal amino sites on a protein molecule to
covalently attach the protein to the nanotubules. Attachment of the
protein glucose oxidase (GOD) can be performed in this way. Many other
proteins have been attached using this general method. Accordingly, it is
a versatile way to attach proteins to the surfaces of these nanotubules.
Scanning electron micrographs of an alumina template membrane having a
plurality of open pores which can be filled with silica nanotubes are
shown in FIGS. 3(a)-3(c). FIG. 3(a) shows a surface image of an alumina
template membrane demonstrating a high packing density of pores, the pores
having diameters of approximately 60 nm. FIG. 3(b) shows a cross sectional
image of the template membrane shown in FIG. 3(a) while FIG. 3(c) shows a
perspective view of the template membrane. Although 60 nm nominal pore
diameters are shown, pore diameters of the template membrane can be
In an embodiment of the invention, a long chain alkyl carbon (such as
C.sub.18) is added to silica nanotubes positioned within a template
membrane for the purpose of adsorbing a target molecule. It is noted that
the field of protein and enzyme attachment to particles, otherwise
referred to prolifically in the literature as "enzyme immobilization", is
a mature science, and that the methodologies described in this portion of
the application are similar to ones described earlier but that the
selection of which enzymes used and the overall particle composition is
The 18-carbon alkyl (C.sub.18) silane was chosen because this renders the
insides of the nanotubules hydrophobic. The nanotubules with the C.sub.18
groups inside can then be used to extract hydrophobic molecules from a
contacting solution phase. Again, in this case the outsides of the
nanotubules remain hydrophilic silica and this allows these tubules to be
dispersed into solutions containing polar solvents. The most obvious
example is water, but the same principle applies for other polar solvents.
Obviously, the outside could also be derivatized with the hydrophobic
silane and such tubules could then be dispersed into solutions containing
nonpolar solvents. Other alkyl silanes could be used to tune the
extraction selectivity of the derivatized nanotubules. Examples include
using shorter chain (e.g. C.sub.8) alkyl silanes to make the tubules less
hydrophobic on the inside, using aromatic silanes, using silanes with
specific chemical functionalities (e.g., acidic or basic), etc.
The hydrophobic C.sub.18 silane-containing tubules were used to extract a
hydrophobic target molecule (7,8-benzoqunoline or BQ) from a dilute
aqueous solution. Extraction was accomplished in two ways. In the first
method the hydrophobic nanotubules were left embedded within the pores of
the template membrane, and a piece of the membrane was simply immersed
into and then removed from the solution of the target molecule. Removing
the membrane also accomplished the removal of the target molecule BQ
sequestered inside. In the second method, the nanotubuies were liberated
from the template membrane, by dissolving the membrane in phosphoric acid
solution. The liberated tubules were then collected by filtration. The
tubules were then dispersed into a solution of the target molecule. The
solution was then filtered to remove the tubules as well as the target
molecule BQ sequestered inside.
FIG. 4 (see Original Patent) shows an example of the second method,
dispersion of the liberated nanotubules. This figure shows first the UV
absorption spectrum of a solution that was 1.times.10.sup.-5 M 7,8
benzoquinoline solution (BQ). To this solution was first added silica
nanotubules that did not contain the hydrophobic C.sub.18 silane inside.
(10 mg of tubules added per 100 mL of solution.) The solution was then
filtered to remove these tubules and the solution spectrum was remeasured.
Note that there is essentially no change in the BQ absorbance. This
experiment showed that silica tubules that were hydrophilic on the inside
did not extract the hydrophobic BQ.
An identical quantity of the C.sub.18 derivatized tubules was then added
to the solution. The solution was then filtered to remove the tubules and
the spectrum was remeasured. As indicated by the lower absorbance, these
tubules extracted 82% of the BQ from the solution. FIG. 5 shows analogous
data after addition of a second 10 mg of tubules per 100 mL of solution.
After the second extraction 92% of the BQ was removed from the solution.
In another embodiment of the invention, reactive molecules such as enzymes
can be incorporated into nanoparticles to improve the drug detoxification
ability of the nanoparticles. Although enzymes are described herein,
incorporation of any molecule capable of generating a chemical reaction or
aiding in the rate of a chemical reaction with a target molecule can also
be incorporated in nanoparticles to produce enhanced detoxification
results. This approach detoxifies substances and surfaces by using
nanoparticles as a platform to incorporate molecules such as enzymes for
catalyzing the conversion of toxic substances into inactive substances (or
for generating chemical reactions with toxic substances).
Protein (i.e., enzyme) attachment to SiO.sub.2 nanotubules was achieved
using 2 steps. A tubule wall was functionalized with an aldehyde-terminated
silane. A protein was then coupled to the aldehyde through primary amino
sites on the protein. The enzyme used, glucose oxidase (GOD), was the
first protein tested. GOD effectively catalyzes the oxidation of glucose
to glucono-1,5-lactone. In the presence of glucono-1,5-lactone, electrons
shuttle to O.sub.2, creating hydrogen peroxide. In the presence of
peroxidase (POD), hydrogen peroxide oxidizes o-dianisidine from a
colorless to red form, which then can be assayed by monitoring its
absorbance (See FIG. 6).
For preliminary experiments performed, an intact alumina template membrane
with the SiO.sub.2/GOD nanotubules in the pores was utilized. The
absorbance of a solution of glucose, o-dianisidine and POD immersed in a
"blank membrane" (without nanotube incorporation of GOD) was determined to
establish a baseline absorbance. Al.sub.2O.sub.3/SiO.sub.2/GOD membranes
were then immersed into the glucose solution. The time dependent changes
in the absorbance (concentration) of glucose (assayed indirectly via
oxidation of o-dianisidine) were determined. The results are depicted in
FIG. 6. Based on the absorbance spectra shown, it is apparent that GOD
incorporated into pores of the nanotubules converted much of the glucose
in the solution to glucono-1,5-lactone upon immersion of the membrane (at
approximately 140 seconds). As shown in FIG. 6, at approximately 400
seconds the membrane was removed from the solution. No further oxidation
of glucose is observed because in removing the membrane the GOD
incorporated inside is removed.
The nanotubes are important because they are new morphologies of
particulate material. They are also important, being newly available, for
evaluation and use in biomedical applications either by themselves or
modified as described in this application. However, the nanotube is not
the only shape carrier/core particle that can be derivatized as is
described in this application. Many other shapes are useful, such as
derivatized polyhedral-shaped porous (templated or not) nanoparticles.
Based on this observation, it was concluded that enzymes incorporated into
nanoparticles can be used to degrade drugs. The linkage of enzymes to the
inner surface of pores, can be achieved without losing the enzyme's
reactivity. Although nanotubes having inner cavities for enzyme attachment
was used, any shaped particle, whether tubular or not, having pores
adapted for this purpose can have enzymes inside the pores. Thus, any
nanoparticles having pores, whether the pores are tubular or any other
shape may be used with the invention.
An identical approach (i.e., linking a cytochrome P-450 (CYP) enzyme
system) to the inner surface of a nanoparticle) can also be used to
efficiently reduce the free concentration of lipophilic agents in human
plasma and blood by a biotransformation dominated mechanism.
Analogous experiments have been done with nanotubules that were liberated
from the template membrane, and substantially identical results were
obtained. In this case the GOD was on both the inner and outer surfaces of
the membrane. This is an advantage of the hollow nanotubule approach.
Having available inner and outer surfaces increases the surface area
available for biocatalysis. As before, it might also be advantageous to
separately derivatize the inner and outer surfaces. For example, the inner
surface could be derivatized with a specific dye and the outer surface
could be derivatized with a specific enzyme or other protein. In a second
set of tubules, a second specific dye could be attached to the inside and
a second specific protein to the outside. This could be continued for n
tubules that contain specific dyes and specific proteins on the insides
and outsides. The dye could then be used to identify the tubules. For
example, green tubules could contain protein #1, blue tubules could
contain protein #2, etc. In this way, in a mixture of tubules one could
identify which tubules are catalyzing which biochemical process.
Hollow and solid nanoparticles can also be used for removal of lipophilic
toxins. For example, nanoparticles, whether hollow or solid, having
substantially polyhedral or spherical morphologies can be used for this
purpose. Studies were performed to establish whether the benzene ring
moiety of the prototypical amide local anesthetic, bupivacaine, possessed
sufficient electron enrichment to enable .pi.-.pi. electron bonding to an
electron deficient molecular moiety attached to a solid nanoparticle.
Specifically, when a mimic of bupivacaine was mixed with the
dinitrobenzamide moiety, a number of changes in spectral values occurred.
Specifically, the UV-VIS diffuse shoulder from 280-320 nm moved to a
diffuse shoulder from 340-400 nm.
Complexation of the pi-pi type between bupivacaine and its mimics, and
several electron deficient aromatics, including a dinitrobenzamide
(designed for convenient subsequent attachment to nanoparticles), has been
proven using proton NMR spectrometry. Tables 1 and 2 (see Original Patent)
show the chemical shift values observed for test systems relevant to this
This data is consistent with the ranges published (Dust, 1992) for other
.pi. complexed electron deficient and electron rich benzene rings. Thus,
the dinitrobenzamide moiety can be attached to carrier nanoparticles
during synthesis for use treating local anesthetic detoxification
primarily by .pi.-.pi. complexation which can take place on solid or
soft/hard nanoparticles, and with or without two-point binding or
templated cores or shells.
Further experiments were performed to determine the effectiveness of
various emulsions to reduce the free concentration and toxic effects of
other drugs. As shown in FIGS. 7-9, the cardiotoxic effects of bupivacaine,
a local anesthetic, were significantly reduced using a macroemulsion of
FIG. 7 (see Original Patent) shows a macroemulsion of Intralipid.
Intralipid attenuates bupivacaine-induced sodium current (I.sub.ns) in
guinea pig ventricular myocytes. Panel A shows examples of current traces
in response to depolarization to -20 mV from a holding potential of -100
mV during interventions shown near cach records. Panel B shows the change
in current peaking as a function of time. Horizontal bars above panel B
denote the duration of drug administration. Panel C summarizes the effect
of bupivacaine (5, 10 and 20 .mu.M) and Intralipid (1.5%) on I.sub.ns. All
data was normalized to a control current. Bars represent the mean.+-.SEM
of 5-7 myocytes.
FIG. 8 (see Original Patent)shows concentration-dependent in vitro
attenuation of bupivacaine (1 .mu.M). Bupivacaine induces QRS prolongation
in guinea pig isolated hearts, paced at 200 beats per minute (BPM) as
shown in FIG. 8. FIG. 8 shows the mean.+-.SEM of 4 experiments (P<0.05).
As shown in FIG. 8, increasing Intralipid concentations reduce QRS
prolongation caused by bupivacaine 1%.
FIG. 9 (see Original Patent) shows the attenuation of in-vivo cardiotoxic
effects of bupivacaine in isofluanc-anesthetized rates. Specifically, the
effect of an IV bolus of bupivacaine (8 mg/kg over 10 scc) on the QRS
interval is shown. Compared to time matched controls, Intralipid (3
ml/kg/min over 2 min) more rapidly attenuated bupivacaine induced
prolongation of the QRS interval. Two additional experiments were also
carried out, both yielding similar results.
Therefore, compared to available methods, bioparticles which may be
produced using the invention have numerous advantages over current methods
for treating drug toxicity. Advantages from the invention are enhanced
through use of complementary approaches including lipid partitioning,
adsorption and xenobiotic biotransformation.
Bioparticles using lipid partitioning and/or drug biotransformation
produced using the invention not only scavenge most toxic drugs that are
more lipophilic (active drug state normally) but also offer broader
substrate usage. For example, various soft bioparticles can effectively
reduce the free blood concentration of all virtually lipophilic drugs.
Moreover, appropriately chosen enzymes incorporated into bioparticles can
further improve the bioparticle's therapeutic performance and
applicability by adding metabolization effects applicable to a broad range
of drugs. If desired, the feature of chemical selectivity inherent in
immunotoxicotherapy can be incorporated into bioparticles by using the
processes of molecular templating and/or adsorption onto functionalized
Using bioparticles, large lipid-water partition coefficients for highly
lipid soluble substances such as amiodarone indicate that the free
concentration of this antiarrhythmic agent can be effectively reduced by
using a concentration of soft bioparticles in the bloodstream that should
not be detrimental to cell function (approximately 1.5% maximum). A
bioparticle having a large lipid-water partition coefficient (e.g.
10,000), where the lipid component of the bioparticle is either liquid
solid core, or lipophilic molecular entities attached to the surface of an
inorganic core can bind a large fraction of highly lipophilic drugs. Thus,
a drug's free blood concentration can be effectively reduced in a small
volume of soft bioparticles.
In a preferred embodiment of the invention, bioparticles containing P450
cytochrome components such as a CYP 3A4 fraction are used to not only
offer broad substrate detoxification, but also to produce rapid
elimination of toxins from the blood. CYP3A4 and CYP2D6 hepatic microsomal
fractions of the P450 system can biotransform approximately 55% and 25%,
respectively, of virtually all xenobiotics [Benet L Z, Kroetz D L and
Sheiner L B, Pharmacokinetics in Goodman and Gilman's, Pharmacological
Basis of Therapeutics (1996) eds Hardman JG and Limbird LE, 9th Edition,
McGraw Hill, pp 3-27.] Cytochrome results in an enhanced drug elimination
rate by either increasing the quantity or quality of enzyme (e.g.,
selecting high activity enzyme systems using genetic polymorphisms or
molecular cloning) and/or optimizing the environment of the enzyme
(substrate concentration, cofactor levels, hydrophilicity for optimal
Bioparticles can provide partitioning and a biotransformation to take
advantage of the potential synergistic actions which can result from
initially partitioning a drug at high local concentrations in an
environment containing high concentrations of an enzyme. This synergy can
dramatically increase the efficiency of the substrate degradation if the
toxic drug concentration occurs well below the KM value.
For example, partitioning a toxic drug from the bloodstream into the lipid
environment of a bioparticle containing a genetically engineered P450
enzyme designed for super high efficiency (e.g., supersomes) should not
only effectively and promptly reduce the free blood concentration of
xenobiotics in the blood but also concentrate the toxic drug in an area
adjacent to the active enzyme. This can promote extremely efficient
catalysis and degradation of target molecules.
The beneficial effects of bioparticles formed using the invention are
expected to be greatly enhanced by concentrating a large enzyme mass, such
as genetically engineering P450 fractions selected for super high activity
within the biocompatible particle. Preferably, the bioparticle should have
a large internal surface area whose efficiency of catalyzing its substrate
to its metabolite is marked augmented by the concentrating effect of the
soft bioparticle component (e.g., exposes the enzyme to a high
concentration of its substrate). In addition to its substrate
concentrating effect, another advantage gained by incorporating a lipid
matrix within the bioparticle is that this structure may dramatically
increase the intrinsic biocatalytic efficiency of an enzyme.
Salt-immobilized hydrolytic and co-factor requiring enzymes (lyophilizates
of enzyme in a salt matrix) in organic solvents have been shown to have
100-3000 times more activity than that observed in aqueous mediums (U.S.
Pat. No. 5,449,613 to Dordick, et al.). This technology applied to CYP
fractions located within bioparticles can produce extremely efficient
biocatalytical tools for drug detoxification, particularly when preferred
high efficiently molecular cloned supersomes are used.
Numerous products can be produced from the invention including those
composed of multiple types of nanoparticles for in-vivo detoxification of
drugs and toxins from humans or animals. For example, nanoparticles can be
synthesized for attenuating acute cardiotoxic effects of tricyclic
antidepressant drugs (e.g., amitriptyline). However, the invention is not
limited only to tricyclic antidepressants, but encompasses all drug
classes that may cause toxicity. In addition, biological toxins (e.g.,
snake and insect envenomation) may also be detoxified using the invention.
This can ensure human and animal safety and welfare. Furthermore,
endogenous toxins produced during organ dysfunction or failure (e.g.,
hepatic or renal failure) may also be removable using the invention to
create "circulating hepatocytes" or "circulation nephons."
Another potential product which can be produced from the invention is a
product for detoxification of poison warfare agents that are used for
military purposes (e.g., nerve gas). It is noted that warfare agents may
be solids, liquids or gases. Warfare agents can cause massive
intoxification of substances such as acetylcholine or tissue necrosis from
direct toxicity (e.g., mustard gases). Rapid and simultaneous removal of
both the warfare agent and molecules causing injury may prove to be
effective therapy to mitigate the dangers of these weapons of mass
destruction. For example, bioparticles produced using the invention could
be used intravenously to reduce the concentration of both the toxin (e.g.,
sarin) and acetylcholine. Alternatively, the invention can be used for
skin or metal decontamination for other types of toxic warfare agents such
as mustard gas.
Claim 1 of 13 Claims
1. A method comprising: contacting a
toxic compound with a particle comprising a hollow tube open at least at
one end, the hollow tube comprising a polymer or silica, an enzyme and a
hydrophobic compound that partitions the toxic compound to produce a high
local concentration of the toxic compound in contact with the enzyme,
wherein the hydrophobic compound and the enzyme are attached to a surface
of the hollow tube, whereby the enzyme transforms the toxic compound into
a substantially inactive compound.
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