U.S. patent application number 11/993260 was filed with the patent office on 2010-07-01 for anti-inflammatory, radioprotective, and longevity enhancing capabilities of cerium oxide nanoparticles.
This patent application is currently assigned to EDWARD VIA VIRGINIA COLLEGE OF OSTEOPATHIC MEDICIN. Invention is credited to Ariane M. Clark, Beverly A. Rzigalinski.
Application Number | 20100166821 11/993260 |
Document ID | / |
Family ID | 37595977 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100166821 |
Kind Code |
A1 |
Rzigalinski; Beverly A. ; et
al. |
July 1, 2010 |
Anti-Inflammatory, Radioprotective, and Longevity Enhancing
Capabilities of Cerium Oxide Nanoparticles
Abstract
The present invention provides cerium oxide nanoparticles for
use both in therapeutic compositions in vivo and in research in
vitro. The cerium oxide nanoparticles are of a known range of sizes
having biological properties that are reproducible and beneficial.
Pharmaceutical and other compositions are provided, as are methods
of treatment.
Inventors: |
Rzigalinski; Beverly A.;
(Radford, VA) ; Clark; Ariane M.; (Bradenton,
FL) |
Correspondence
Address: |
LATIMER INTELLECTUAL PROPERTY LAW, LLP
P.O. BOX 711200
HERNDON
VA
20171
US
|
Assignee: |
EDWARD VIA VIRGINIA COLLEGE OF
OSTEOPATHIC MEDICIN
Blacksburg
VA
|
Family ID: |
37595977 |
Appl. No.: |
11/993260 |
Filed: |
June 27, 2006 |
PCT Filed: |
June 27, 2006 |
PCT NO: |
PCT/US2006/024963 |
371 Date: |
March 4, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60693930 |
Jun 27, 2005 |
|
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|
Current U.S.
Class: |
424/423 ;
424/489; 424/529; 424/617; 424/93.7; 428/402; 435/29; 435/375;
977/773 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
17/02 20180101; A61P 39/06 20180101; A61K 9/14 20130101; A61P 19/02
20180101; A61P 3/10 20180101; A61P 25/00 20180101; A61P 39/00
20180101; A61P 17/18 20180101; A61P 37/08 20180101; A61K 33/24
20130101; A61P 9/10 20180101; A61P 37/00 20180101; A61P 9/06
20180101; A61P 25/28 20180101; A61P 29/00 20180101; A61L 27/306
20130101; A61P 43/00 20180101; A61P 25/16 20180101; Y10T 428/2982
20150115 |
Class at
Publication: |
424/423 ;
428/402; 424/489; 424/617; 435/375; 424/93.7; 424/529; 435/29;
977/773 |
International
Class: |
A61F 2/30 20060101
A61F002/30; B32B 5/00 20060101 B32B005/00; A61K 9/14 20060101
A61K009/14; A61K 33/24 20060101 A61K033/24; C12N 5/00 20060101
C12N005/00; A61K 35/12 20060101 A61K035/12; A61K 35/14 20060101
A61K035/14; C12Q 1/02 20060101 C12Q001/02; A61P 29/00 20060101
A61P029/00; A61P 43/00 20060101 A61P043/00; A61P 39/00 20060101
A61P039/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0001] This invention was made partially with U.S. Government
support from the United States National Institutes of Health under
Contract No. NS40490 (National Institute of Neurological Disorders
& Stroke) and AG022617 (National Institute on Aging). The U.S.
Government has certain rights in the invention.
Claims
1-93. (canceled)
94. Cerium oxide nanoparticles having a size range of from 11 nm to
500 nm.
95. The cerium oxide nanoparticles of claim 94, wherein the
nanoparticles have been sonicated to avoid agglomeration.
96. A composition comprising the nanoparticles of claim 94, further
comprising one or more biologically tolerable substances.
97. The composition of claim 96, wherein the biologically tolerable
substance is one or more of water, a salt, phosphate buffered
saline, and a lipid.
98. Cerium oxide nanoparticles having a size range of less than 1
nm to about 500 nm, in an amount sufficient to provide a single
dose of particles to an animal to reduce or eliminate clinical
symptoms resulting from damage due to free radicals in cells of the
animal, or to prevent such clinical symptoms.
99. Cerium oxide nanoparticles having a size range of less than 1
nm to about 500 nm, in an amount sufficient to provide a single
dose of particles to an animal to reduce, eliminate, or prevent
clinical symptoms resulting from inflammatory responses in cells of
the immune system or to decrease the inflammatory capacity of cells
of the immune system.
100. A composition comprising a pharmaceutically suitable substance
and the cerium oxide nanoparticles of claim 98.
101. A composition comprising the cerium oxide nanoparticles of
claim 98, further comprising one or more substances having a
beneficial effect on the diet or nutrition of the animal.
102. The composition of claim 101, which is a nutritional
supplement or dietary supplement for the animal.
103. A kit comprising the cerium oxide nanoparticles of claim
98.
104. The kit of claim 103, wherein the cerium oxide nanoparticles
are of a size range between 11 nm and 500 nm.
105. A medical prosthesis made from joint replacements or joint
additives coated with or containing cerium oxide nanoparticles of a
size of at least about 1 nm to 500 nm in an amount sufficient to
reduce inflammation in the subject.
106. A method of increasing cell longevity, said method comprising:
contacting a cell to cerium oxide nanoparticles of claim 94 in an
amount sufficient to result in increased cell longevity.
107. The method of claim 106, wherein the amount of nanoparticles
is sufficient to reduce free radical damage within the cell.
108. The method of claim 106, wherein the cerium oxide
nanoparticles are produced from a process other than a sol-gel
method.
109. The method of claim 106, wherein the amount of nanoparticles
is sufficient to prevent aging of cells of a subject.
110. The method of claim 106, wherein the size of the cerium oxide
nanoparticles is from 11 nm to about 50 nm.
111. The method of claim 106, wherein the size of the cerium oxide
nanoparticles is from 40 nm to about 500 nm.
112. The method of claim 106, wherein the cerium oxide particles
have a size of about 20 nm.
113. The method of claim 106, wherein greater than 95% of the
cerium oxide particles have a size of between about 5 nm and about
40 nm.
114. The method of claim 106, which is a method of radioprotection
of cells exposed to radiation during a cancer treatment regimen or
a method of prevention or treatment of brain disease, a spinal cord
disease, neurological trauma, a neurodegenerative disorder,
Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis (ALS), multiple sclerosis,
toxin-mediated damage, stroke, arthritis, joint disease,
atherosclerosis, cardiovascular disease, diabetes, diseases of the
retina, an allergy, asthma, chronic obstructive pulmonary disease,
respiratory dysfunction, an autoimmune disease, inflammation, or a
wound.
115. The method of claim 106, which is an in vitro method.
116. The method of claim 106, which is an ex vivo method in which
at least one cell is removed from a multi-cellular organism,
treated with the cerium oxide nanoparticles, and then returned to
the organism.
117. The method of claim 116, in which the blood from an organism
is exposed to the cerium oxide nanoparticles, and then returned to
the organism.
118. An in vivo method of treating at least one cell with cerium
oxide nanoparticles comprising: contacting the cell with cerium
oxide nanoparticles of claim 98.
119. The method of claim 118, wherein the size of the cerium oxide
nanoparticles is from 11 nm to about 50 nm.
120. The method of claim 118, wherein the cerium oxide particles
have a size of about 20 nm.
121. The method of claim 118, wherein greater than 95% of the
cerium oxide particles have a size of between about 5 nm and about
40 nm.
122. The method of claim 118, which is a method of radioprotection
of cells exposed to radiation during a cancer treatment regimen or
a method of prevention or treatment of brain disease, a spinal cord
disease, neurological trauma, a neurodegenerative disorder,
Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis (ALS), multiple sclerosis,
toxin-mediated damage, stroke, arthritis, joint disease,
atherosclerosis, cardiovascular disease, diabetes, diseases of the
retina, an allergy, asthma, chronic obstructive pulmonary disease,
respiratory dysfunction, an autoimmune disease, inflammation, or a
wound.
123. A method of determining the effects of one or more
environmental stimuli on free radical production in a cell, said
method comprising: contacting at least one cell with cerium oxide
nanoparticles of a size of at least 11 nm to provide treated cells;
optionally providing identical cells, but which are not contacted
with cerium oxide nanoparticles to provide control cells; observing
the treated and, where provided, control cells for detectable
changes in free radical levels; and optionally, comparing the free
radical levels in the treated and control cells.
124. The method of claim 123, wherein the environmental stimuli is
one or more chemicals.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of medicine and
treatment of medically relevant diseases, disorders, and
complications of injury, inflammation, and aging. More
specifically, the invention relates to the use of nanoparticles to
treat subjects suffering from various diseases, disorders, and
complications due to injury, inflammation, radiation exposure, and
aging.
[0004] 2. Description of Related Art
[0005] Many approaches have been taken to treat, either
therapeutically or prophylactically, diseases, disorders, and other
medically important conditions that have, as a major component,
cell injury or death due to free radicals, such as oxygen radicals.
Among those approaches were the use of free radical scavengers,
such as Vitamin E and its related compounds, Vitamin C and its
related compounds, and melatonin, to name a few. While beneficial
effects of these compounds has been noted, researchers and
clinicians continue to search for compounds with higher activities
and half-lives.
[0006] In early experiments performed by the present inventors and
their colleagues, cerium oxide nanoparticles prepared by a sol-gel
process were utilized to enhance cell longevity. The cerium oxide
nanoparticles were proposed to act as free radical scavengers to
bring about the observed results. However, the sol-gel process
posed several difficulties. For example, particle size was not
well-controlled within the reported 2-10 nm range, making
variability between batches high. That is, the process, while
satisfactory for producing nanoparticles with free radical
scavenging activity, did not reproducibly produce particles of a
specific size range. Thus, each batch of particles needed to be
tested to confirm the size range and the suitability of the batch
for use. In addition, the process resulted in tailing of
surfactants used in the process into the final product. The
presence of these surfactants produced biological difficulties when
used, primarily due to the toxicity of the surfactants in the
product. Furthermore, the inability to control the amount of
surfactant tailing posed problems with agglomeration when
nanoparticles were placed in biological media. These difficulties
reduced particle efficacy and biological deliverability. Removal of
surfactant after sol-gel synthesis produced particles that appeared
prone to agglomeration in biological media, and had a lack of
biological effects. Further, difficulties were encountered with
changes in valence state of cerium associated with these particles,
causing alterations in the ratio of valence states of cerium
(+3/+4) that occurred over time, particularly when particles were
placed in biological media. It is possible that the +3/+4 ratio of
valence states in the nanoparticles might alter free radical
scavenging and cellular delivery, including delivery in vivo.
[0007] Thus, while the previous solution to use nanoparticles as
free radical scavengers was effective, it was highly variable from
batch to batch. Therefore, a need in the art still exists for
improved nanoparticles and methods of use of those particles to
treat various diseases and disorders involving production of oxygen
radicals and other radicals.
SUMMARY OF THE INVENTION
[0008] The present invention addresses this need in the art by
providing a method for the use of cerium oxide nanoparticles in
health. As a general matter, the method extends the life of a
living cell by exposing the cell to cerium oxide nanoparticles.
This exposure reduces or eliminates damage to the cell caused by
endogenous and exogenous free radicals. The cerium oxide
nanoparticles can be exposed to the cell before, during, or after
free radical damage.
[0009] Broadly speaking, the present invention provides a method of
treating at least one cell with cerium oxide particles. The method
generally comprises contacting at least one cell with an amount of
cerium oxide nanoparticles that reduces or eliminates damage caused
by free radicals. The method can be practiced in vivo as either a
therapeutic method of treating a disease or disorder involving free
radicals or as a prophylactic method to prevent free radical
damage. Likewise, the method can be practiced in vitro as a
research tool to study the effects of free radicals on cells or the
effects of combinations of nanoparticles with drugs on cells. In
preferred embodiments, the method is practiced with size-limited
cerium oxide nanoparticles made by a method other than a sol-gel
method. The method can also be practiced ex vivo or in vitro for
therapeutic or research purposes.
[0010] The present invention provides methods of treating
individuals suffering from, or suspected of suffering from, a
disease or disorder involving free radicals, such as oxygen
radicals. It likewise provides methods of treating individuals
suffering from, or suspected of suffering from a complication of an
injury that results from free radicals, such as oxygen radicals, or
results in the production of free radicals, such as oxygen
radicals. In general, the methods of the invention comprise
administering to an individual (used interchangeably herein with
"subject" and "patient") an amount of cerium oxide nanoparticles
sufficient to reduce or eliminate cell, tissue, or organ damage in
the individual that is caused by free radicals. Thus, the invention
encompasses the use of cerium oxide nanoparticles in enhancement of
cell and organism longevity, reduction of inflammation and
inflammatory disorders, reduction in tissue damage due to
inflammatory disorders, and reduction in radiation injury.
[0011] In a further aspect, cerium oxide nanoparticles and
compositions comprising cerium oxide nanoparticles are provided.
The cerium oxide nanoparticles are size-limited and provided in an
amount sufficient to provide one or more doses to a subject in need
of, or suspected of being in need of, treatment for a disease or
disorder involving free radicals. Compositions may comprise cerium
oxide particles of the invention along with one or more other
substances, which are typically substances that are biologically
tolerable in that they may be exposed to living cells without
killing the cells. In embodiments, the other substances are
pharmaceutically acceptable substances.
[0012] Certain aspects of the invention provide for the use of
cerium oxide nanoparticles in the treatment of diseases and
disorders associated with free radicals, such as oxygen free
radicals. The use is in particular for in vivo therapeutic or
prophylactic methods of protecting cells from free radical damage.
Certain other aspects of the invention provide for the use of
cerium oxide nanoparticles in the preparation of compositions for
medical use, such as pharmaceutical or therapeutic
compositions.
[0013] Another aspect of the invention provides a container
containing cerium oxide nanoparticles. In general, a container
according to the invention contains a sufficient amount of
size-limited cerium oxide nanoparticles made by a method other than
a sol-gel method to provide at least one dose of cerium oxide to a
subject suffering from, or at risk of suffering from, a disease or
disorder involving free radicals, such as oxygen radicals. In
certain embodiments, the container is provided in a package with
one or more other containers and/or with one or more articles of
manufacture or devices having use in delivery of substances to
subjects (e.g., syringes, needles, antiseptic swabs). In some
embodiments, kits comprising one or more containers are provided.
In some kits, single dose amounts of cerium oxide particles are
provided. In some embodiments, the single dose is 1 ng to 100 mg
per kg weight of subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and, together with the written
description, serve to explain principles of the invention.
[0015] FIG. 1 depicts the effects of cerium oxide nanoparticles on
the maximum lifespan of mixed neuronal cells in culture.
[0016] FIG. 2 depicts the effects of cerium oxide nanoparticles on
the lifespan of D. melanogaster flies.
[0017] FIG. 3 depicts the excitation spectra for intracellular
cerium oxide nanoparticles during a free radical scavenging
event.
[0018] FIG. 4 depicts a drug distribution graph of tissue cerium
content of BALB/c mice after injection with nanoparticles, as
assayed by inductively coupled plasma mass spectrometry.
[0019] FIG. 5 shows the response of brain cell cultures (neuronal
death) treated with nanoparticles, as assessed by propidium iodide
staining.
[0020] FIG. 6 shows the response of brain cell cultures (neuronal
death) treated with nanoparticles.
[0021] FIG. 7 demonstrates the response of brain cell cultures
treated with nanoparticles in terms of nitric oxide release.
[0022] FIG. 8 shows the morphological effect of cerium oxide
nanoparticles on brain microglia.
[0023] FIG. 9 shows the effect of pretreatment with cerium oxide
nanoparticles on exposure to UV radiation.
[0024] FIG. 10 demonstrates the effect of pretreatment with cerium
oxide nanoparticles on exposure to gamma-irradiation.
[0025] FIG. 11 shows the effect of pretreatment of a single dose of
cerium oxide nanoparticles against free radical mediated injury as
compared to a single dose of Vitamin E, n-Acetyl Cysteine, or
Melatonin.
[0026] FIG. 12 shows the effect of pretreatment of a single dose of
cerium oxide nanoparticles against free radical mediated injury as
compared to multiple doses of Vitamin E, n-Acetyl Cysteine, or
Melatonin.
[0027] FIG. 13 shows the change in female Drosophila life spans
when cerium oxide nanoparticles are given to the flies.
[0028] FIG. 14 demonstrates the change in male Drosophila life
spans when cerium oxide nanoparticles are given to the flies.
[0029] FIG. 15 shows the amount of neuron specific enolase (NSE) in
tissue culture medium.
[0030] FIG. 16 shows the effect of cerium oxide nanoparticles on
the longevity of tissue cultures.
[0031] FIG. 17 demonstrates the effect of paraquat on female
Drosophila fed 10 nM cerium oxide nanoparticles.
[0032] FIG. 18 demonstrates the effect of paraquat on female
Drosophila fed 1 uM cerium oxide nanoparticles.
[0033] FIG. 19 demonstrates the effect of paraquat on male
Drosophila fed 10 nM cerium oxide nanoparticles.
[0034] FIG. 20 demonstrates the effect of paraquat on male
Drosophila fed 1 uM cerium oxide nanoparticles.
[0035] FIG. 21 shows the effect of cerium oxide nanoparticles
against traumatic injury as compared to a single dose of other
antioxidants when given pre-trauma.
[0036] FIG. 22 shows the effect of cerium oxide nanoparticles
against traumatic injury as compared to a single dose of other
antioxidants when given post-trauma.
[0037] FIG. 23 demonstrates the release of NO by astrocytes in both
resting and injured states.
[0038] FIG. 24 shows the effect of cerium oxide nanoparticles on
the release of NO from microglia stimulated with medium conditioned
by injured astrocytes for 1 hour.
[0039] FIG. 25 shows the effect of cerium oxide nanoparticles on
the release of NO from microglia stimulated with medium conditioned
by injured astrocytes for 3 hours.
[0040] FIG. 26 shows the effect of cerium oxide nanoparticles on
the release of NO from LPS-stimulated microglia.
[0041] FIG. 27 demonstrates the morphology of microglia after
injury or exposure to LPS with and without cerium oxide
nanoparticles.
DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION
[0042] Reference will now be made in detail to various exemplary
embodiments of the invention, examples of which are illustrated in
the accompanying drawings. The following detailed description is
provided to give details on certain embodiments of the invention,
and should not be understood as a limitation on the full scope of
the invention.
[0043] A present inventor and her colleagues previously developed
cerium oxide nanoparticles for treatment of various diseases and
disorders, which was disclosed in U.S. provisional patent
application No. 60/408,275 and in a U.S. non-provisional patent
application filed on 4 Sep. 2003 under Attorney Docket Number
UCF-375, the entire disclosures of both of which are hereby
incorporated herein by reference. To address the shortcomings of
prior attempts to develop cerium oxide nanoparticles for use in
treating damage caused by free radicals, different methods of
synthesizing nanoparticles, and thus different nanoparticles, were
investigated. Efforts were directed toward examining the biological
efficacy of commercially available cerium oxide nanoparticles
prepared by existing manufacturing processes. These included cerium
oxide nanoparticles available from Nanophase Technologies
Corporation (Romeoville, Ill.), Advanced Powder Technology Pty Ltd.
(Welshpool, Western Australia), and NanoScale Materials Inc.
(Manhattan, Kans.). In summary, in a series of experiments, it was
found that cerium oxide nanoparticles produced by Nanophase
Technology Corporation, using specific, patented mechanisms of
synthesis, provided consistently reproducibly sized nanoparticles
that consistently showed high levels of biological activity. With
sizes of 20 nm and below, particles readily entered cells and
reduced free-radical mediated damage. Synthesis for these particles
has been described in the following patents, the disclosures of the
entireties of all of which are incorporated herein by reference:
U.S. Pat. No. 6,669,823, U.S. Pat. No. 5,460,701, U.S. Pat. No.
5,514,349, U.S. Pat. No. 5,874,684, Japanese Patents JP2980987 and
JP3383608, European Patent EP0711217B1, German Patent DE69426886,
French Patent FR94922757, Great Britain Patent GB94922757, and
Australian Patent AU068582882.
[0044] It was surprisingly found that the new source of cerium
oxide nanoparticles, as compared to those of the inventor's prior
invention, provided superior reproducibility of activity from batch
to batch, and showed lower toxicity to mammalian cells. It was
determined that the cerium oxide nanoparticles used in the present
invention were different from the prior nanoparticles in quality
and size distribution, factors that significantly contribute to
their improved characteristics in treating subjects according to
the methods of the invention. In developing the invention, it was
determined that, regardless of source, cerium oxide particles
having a small size, narrow size distribution, and low
agglomeration rate are most advantageous. Also, for delivery, the
nanoparticles are advantageously in a non-agglomerated form. To
accomplish this, stock solutions of about 10% by weight can be
sonicated in ultra-high purity water (16 megaohms) or in normal
saline prepared with ultra high purity water. These nanoparticles
are superior to previously developed cerium oxide nanoparticles for
treatment of and protection against, damage caused by free
radicals. This new and useful improvement allows cerium oxide
nanoparticles to be used in extending the life of a cell in vivo as
well as in vitro. In particular, it is shown herein the novel
finding that cerium oxide nanoparticles of a defined size range and
distribution and made by a method other than sol-gel synthesis
increase the lifespan of cells, such as cells of an organism in
vivo. Also shown is that cerium oxide nanoparticles enhance the
lifespan of mammalian cells in culture and in vivo, act as potent
free radical scavengers, and possess significant anti-inflammatory
and radioprotective properties in vivo.
[0045] While not wishing to be limited to any single method of
action, it is thought that cerium oxide nanoparticles have a unique
oxide lattice and valence structure that might confer them with the
ability to scavenge (detoxify) intracellular free radicals, and
might thus convey their anti-inflammatory, radioprotective, and
longevity-enhancing properties. Further, the data obtained by the
inventors, and provided herein, suggests that the valence and
oxygen lattice structure conveys the ability of cerium oxide
nanoparticles to regenerate a biologically active matrix after a
free radical scavenging event. This allows small, single doses of
nanoparticles to remain active within the cell for long periods of
time, conveying regenerative biological effects. In contrast, most
commonly available free radical scavengers, such as vitamin E,
nitrosone compounds, and vitamin C are inactivated by alteration of
their chemical structure after scavenging a single free radical.
This loss of structure limits their pharmacological efficacy and
requires high dosing regimens.
[0046] It appears that the regenerative activity of the cerium
oxide nanoparticles may be dependent on a well-known oscillating
chemical phenomenon, known as the Belousov-Zhabotinsky (B-Z)
reaction, in which cerium oxide serves to facilitate oscillation of
electrons (or free radicals) from one compound to another. Cerium
in the nanoparticles exists in two valence states, +3 and +4.
Adequate propagation of B-Z requires a specific ratio of Ce+3 to +4
in the nanoparticles. If the composition changes to have too much
+3 cerium, the reaction will not propagate. Research has shown that
as the cerium oxide nanoparticle size is reduced from 30 nm to 3
nm, lattice strain in the nanoparticles causes more cerium to be in
the +3 state. Although this mechanism has only been studied in
vitro up to now, this mechanism of action may also be true in vivo
and would provide a significant advantage to using larger sizes of
cerium oxide nanoparticles.
[0047] Broadly speaking, the present invention provides a method of
treating at least one cell with cerium oxide particles. The method
generally comprises contacting at least one cell with an amount of
cerium oxide nanoparticles that reduces or eliminates damage caused
by free radicals, which are unstable, highly reactive molecules
such as nitric oxide, superoxide, hydroxyl radicals, peroxynitrite,
and other unstable reactive compound formed from the above. They
cause aging and various diseases by taking electrons from other
molecules in the body, a process that causes cell or oxidative
damage. As used herein, cell or oxidative damage has the same
meaning as oxidative stress.
[0048] Contacting means any action that results in at least one
cerium oxide nanoparticle physically contacting at least one cell.
It thus may comprise exposing the cell(s) to cerium oxide
nanoparticles in an amount sufficient to result in contact of at
least one cerium oxide nanoparticle with at least one cell. The
method can be practiced in vivo, in which case contacting means
exposing at least one cell in a subject to at least one cerium
oxide nanoparticle. According to the invention, contacting thus may
comprise exposing at least one cell to at least one cerium oxide
particles, such as, for example by administering cerium oxide
particles to a subject via any suitable route. It also may comprise
exposing cells in vitro or ex vivo by introducing, and preferably
mixing, cerium oxide particles and cells in a controlled
environment, such as a culture dish or tube. Optionally, where
practiced in vitro or ex vivo, some or all of the cerium oxide
particles that are not taken up or adsorbed by cells are removed,
for example by washing the cells in suitable media, buffer, water,
etc. According to the invention, contacting may comprise
introducing, exposing, etc. the cerium oxide particles at a site
distant to the cells to be contacted, and allowing the bodily
functions of the subject, or natural (e.g., diffusion) or
man-induced (e.g., swirling) movements of fluids to result in
contact of the nanoparticle(s) and cell(s). Where practiced ex
vivo, the cells may also be re-introduced into a subject,
preferably the subject from which they were originally obtained. In
one embodiment, this includes putting the particles into a gel or
other packet that limits diffusion, followed by implanting it into
a body area such as a knee joint.
[0049] According to the method of the invention, the subject,
individual, or patient can be any organism to whom the cerium oxide
nanoparticles are administered. Thus, the subject may be a human or
a non-human animal, such as another mammal, including, but not
limited to a rodent (e.g., mouse, rat, rabbit), a canine (e.g., a
dog), a feline (e.g., a cat), an equine (e.g., a horse), an ovine
(e.g., a sheep), an orcine (e.g., a pig), or a bovine (e.g., a cow
or steer). The subject can be any other animal such as a bird,
reptile, amphibian, or any other companion or agricultural
animal.
[0050] The method can be practiced in vivo as either a therapeutic
method of treating a disease or disorder involving free radicals or
as a prophylactic method to prevent free radical damage. In
embodiments where the method is a method of treating (i.e., a
therapeutic method), the amount is an amount that is effective for
reducing or eliminating cell death or dysfunction or tissue or
organ damage due to free radicals that are being produce, or were
produced previously, in the subject. The subject, individual, or
patient may be one who is in immediate or apparent need of, or
suspected of being in need of, treatment for a disease or disorder
associated with free radicals, or it may be one who is in immediate
or apparent need of, or suspected of being in need of, treatment
for an injury or other trauma resulting from or known to result in
production of free radicals. In such situations, where a
pre-existing condition related to cell, tissue, or organ damage due
to free radicals is evident or suspected, the method is a
therapeutic method. For example, if a subject has had a stroke, it
may be beneficial to treat the subject with cerium oxide
nanoparticles to reduce the effects of the stroke.
[0051] In addition, according to the methods of the invention, the
subject, individual, or patient may be one who is not in or
suspected of being in need of treatment of a pre-existing disease,
disorder, or injury or trauma. In such situations, the method is a
prophylactic method. Prophylactic methods are useful in situations
where the subject is currently engaged in, or soon to be engaged
in, one or more activities that might result in an injury or
trauma. They are also useful in situations where the patient has a
likelihood of developing a disease or disorder associated with
cell, tissue, or organ damage due to free radicals. Thus, the
present methods are useful not only for treating patients with a
disease or disorder, but for treating patients who are suspected of
having a predisposition to a disease or disorder. For example, if
the family of a subject has been shown to be prone to a certain
neurodegenerative disease, the subject may be given cerium oxide
nanoparticles to avoid or reduce the effects of that disease.
Likewise, if a subject suspects he will be exposed to high levels
of radiation, such as a worker in the nuclear energy or weapons
industries, or a person about to go on a vacation in which he will
be exposed to high levels of sunlight and its UV component, may be
treated with the cerium oxide nanoparticles of the invention. In
another example, military uniforms, including clothes and helmets,
can be made containing cerium oxide nanoparticles to scavenge free
electrons and gamma irradiation for troops exposed to potential
radiation.
[0052] As another example to compare prophylactic and therapeutic
methods, in embodiments where the method is a prophylactic method,
the amount is an amount that is effective in reducing or blocking
cell death or dysfunction or tissue or organ damage due to free
radicals that might be produced in the subject in the future. For
example, in a therapeutic method, the cerium oxide nanoparticles
may be administered to a patient following a head injury to reduce
the amount of damage to the brain as a result of the injury. In
contrast, in a prophylactic method, the cerium oxide nanoparticles
may be administered to a subject prior to engaging in an activity
that has a likelihood of head injury, such as a car race or other
high-speed activity.
[0053] The act of administering cerium oxide nanoparticles can be
any act that provides the cerium oxide nanoparticles to a subject
such that the particles can function for their intended purpose.
For example, administering can be by injection or infusion. It can
thus be an intramuscular, intraparatoneal, subcutaneous, or
intrathecal injection, or a slow-drip or bolus infusion. Other
non-limiting examples of methods of administration include topical
administration, such as by way of lotions, salves, or bandages,
often on intact skin but also through open wounds, lesions, or
sores. Yet other non-limiting examples include administration
through mucous membranes, such as by way of intranasal
administration through inhalation of dry particles or a mist
comprising the particles, oral ingestion, sublingual absorption, by
subcutaneous means, and rectal or vaginal delivery. The vehicle of
delivery may be in any suitable form, such as the form of an oral
solution, gel, tablet, capsule, powder, suppository, infusible,
losenge, cream, lotion, salve, inhalant, or injection.
[0054] According to embodiments of the method, the method can
comprise repeating the act of contacting (e.g., administering) the
cerium oxide nanoparticles. In embodiments relating to
administering the cerium oxide to subjects, repeating the
administration can include one or more administrations in addition
to the original administration. The amount to be administered to
each subject will vary depending on usual factors taken into
consideration for dosing of pharmaceuticals, such as weight,
general health, and metabolic activities of the patient. Likewise,
the mode of administration (e.g., injection, oral administration)
will be taken into account when determining the proper amount of
nanoparticles to administer per dose.
[0055] In general, a dosing of about 0.01 ng to about 1 g, such as
about 0.05 ng, 0.1 ng, 0.5 ng, 1 ng, 10 ng, 50 ng, 100 ng, 500 ng,
1 ug, 5 ug, 10 ug, 50 ug, 100 ug, 500 ug, or 1 g per administration
or per kg body mass per administration should be effective in
providing the desired therapeutic or prophylactic result. Of
course, injection or infusion amounts will tend to be on the lower
end of the range while oral administration amounts will tend to be
on the upper end. Current results suggest that the optimal dose for
20 nm cerium oxide nanoparticles is 10 nM to 1 uM for blood and
intracellular fluid levels. However, the action of the particles is
highly dependent on other variables and so these amounts will vary
depending on the surface area, the species of the subject, the
reason for administration etc. Amounts may be higher when the
method is practiced in vitro or ex vivo because excess particles
may be easily removed at any time by washing, etc.
[0056] It should be noted that this method shows low toxicity in
mammalian cells, fruit flies, and mice, and thus is expected to
show low toxicity in other animal cells. This new and useful
improvement allows the method of the present invention to be used
in subjects with lower toxicity than in previous inventions. This
important feature of the present invention means that the cerium
oxide nanoparticles can be used in a broad range of applications.
In preferred embodiments, the cerium oxide nanoparticles do not
contain docusate sodium, which has been shown to produce toxicity
in tissue culture. Also, in preferred embodiments, there are less
than 1% (w/w or w/v) of any other contaminating ions, metals, or
other substances, which can also cause toxicity to cells.
[0057] Although the cerium oxide nanoparticles show very low
toxicity, in some instances it might be desirable to provide
multiple, low doses of particles to an individual. In such cases,
the method may comprise two or more administrations of less than
the total effective amount, where the amount ultimately
administered is an effective amount. Likewise, multiple
administrations of an effective dose may be desirable where the
second or subsequent administration is performed at a time well
separated from the first administration. That is, because the
cerium oxide nanoparticles are highly stable, even after being
administered, repeated administrations of effective doses are
envisioned as occurring at widely spaced intervals, such as months
or years apart.
[0058] Furthermore, where multiple administrations are performed,
different modes of administration may be used. For example, if two
doses are administered, one can be an injection whereas the other
can be oral. In addition, if three or more doses are administered,
two or more may be by the same mode, while the remaining may be
from one or more different mode, in any combination, number, and
order. Of course, where multiple administrations are used, each
administration may be by a different mode. The mode of
administration, the number of times it is repeated, and the
sequence of modes of administration may be selected by those of
skill in the art based on numerous considerations, and such
selection is well within the abilities of those of skill in the
art.
[0059] The method can also be practiced in vitro which means that
contacting at least one cell with at least one cerium oxide
nanoparticle can occur in a petri dish, a test tube, an IV tube, or
any other container applicable for contacting. When practiced in
vitro, it may be a method for identifying parameters that are
useful in in vivo treatment regimens. The method can be practiced
to study the effects of combinations of nanoparticles with drugs on
cells. For example, the cerium oxide nanoparticles can be combined
with other known antioxidants such as vitamin E, n-acetyl cysteine,
or melatonin. The cerium oxide nanoparticles could also be combined
with disease specific drugs. The in vitro methods can also comprise
using the cerium oxide nanoparticles as a research tool to observe
the effects of free radicals on cells or observe the cells for
changes in protein expression, cell morphology, or any other
characteristic of interest.
[0060] In preferred embodiments, the method is practiced with
size-limited cerium oxide nanoparticles made by a method other than
a sol-gel method. The nanoparticles useful in the present invention
have pre-defined sizes clustered tightly within a range. In
general, the particles have a size of about 1 nm or less to about
500 nm. In embodiments, the particles are 11 nm or more. In
embodiments where particles are taken into the interior of cells,
the preferable range of particles that are taken into the cell are
from about 11 nm to about 50 nm, such as about 20 nm. In
embodiments where particles exert their effects on cells from
outside of the cells, the preferable range of particles that are
extracellular are from about 11 nm to about 500 nm. In embodiments,
the particles are from about 40 nm to about 500 nm. In other
embodiments, the particles are from about 11 nm to about 40 nm,
such as from about 11 nm to about 20 nm, about 15 nm to about 20
nm, about 11 nm to about 15 nm, or about 30 nm to 40 nm. Of course,
any specific size range within these general sizes can be provided,
the size being selected by the practitioner based on any number of
parameters. According to the invention, the term "about" is used to
indicate a margin of error for a statistically significant portion
of the particles of 10%. Thus, particles of a size of 20 nm include
those in which a majority of the particles fall within the range of
18 nm to 22 nm. In embodiments, 95% of the cerium oxide
nanoparticles have a size of between about 15 nm and about 25 nm.
In embodiments, 95% of the cerium oxide nanoparticles are within 5%
of 20 nm. In other embodiments, 90% of the cerium oxide
nanoparticles have a size of between about 18 nm and about 22
nm.
[0061] The present invention provides methods of treating
individuals suffering from, or suspected of suffering from, a
disease or disorder involving free radicals, such as oxygen
radicals. It likewise provides methods of treating individuals
suffering from, or suspected of suffering from a complication of an
injury that results from free radicals, such as oxygen radicals, or
results in the production of free radicals, such as oxygen
radicals. In general, the methods of the invention comprise
administering to an individual (used interchangeably herein with
"subject" and "patient") an amount of cerium oxide nanoparticles
sufficient to reduce or eliminate cell, tissue, or organ damage in
the individual that is caused by free radicals. Thus, the invention
encompasses the use of cerium oxide nanoparticles in enhancement of
cell and organism longevity, reduction of inflammation and
inflammatory disorders, reduction in tissue damage due to
inflammatory disorders, and reduction in radiation injury.
[0062] While the above disclosure discusses administration in vivo,
it is important to recognize that the present invention also
encompasses administering ex vivo. Thus, a method according to the
invention can comprise removing at least one cell from an organism,
administering cerium oxide nanoparticles to that cell, then
returning the cell to its natural environment (e.g., into the body
of the patient). In such situations, the act of administering can
be simply exposing the nanoparticles to the cell, for example in a
culture dish or a tube. In one particular embodiment, the method of
ex vivo administration comprises obtaining blood from a patient,
exposing the blood to cerium oxide nanoparticles, and returning the
treated blood to the patient. The method can comprise separating
cerium oxide nanoparticles from the blood prior to returning the
blood to the patient.
[0063] In one embodiment of the method of the present invention,
the cerium oxide nanoparticles allow an increase in longevity of
prokaryotic cells. For example, adding the cerium oxide
nanoparticles to a large scale E. coli cell culture to allow longer
production of overexpressed protein may allow more efficient and
cost effective production. Relevant human proteins that could be
overexpressed include antibody fragments, single-domain antibodies,
and any other protein important in human health, including what are
presently known as "biologicals" in the pharmaceutical
industry.
[0064] In another embodiment, the cerium oxide nanoparticles allow
an increase in longevity of eukaryotic cells. In one example, the
nanoparticles could be used to increase the longevity of yeast cell
cultures that produce human proteins. Specifically, yeast cultures
that produce human proteins significant in human health, such as
Bacillus anthracis protective antigen, hepatitis vaccines, and
malaria antigens could be grown for longer periods of time.
Continuous fermentation using immobilized yeast cell bioreactor
systems to produce consumable and other products, such as beer,
could also benefit with increased longevity of the yeast cells
after addition of cerium oxide nanoparticles. The same effect of
the cerium oxide nanoparticles could be used in plant cell
cultures, such as cultures producing human vaccine antigens or
other human proteins. Also, mammalian cell cultures that produce
recombinant human antibodies and other important proteins for human
health could benefit from increased longevity due to the addition
of cerium oxide nanoparticles.
[0065] In another embodiment, the present invention is used to
affect, either prophylactically or therapeutically, cell longevity
in organisms. The methods treat or affect, either prophylactically
or therapeutically, diseases or disorders associated with free
radicals, or cell death or tissue or organ damage due to free
radicals. In general, the methods comprise administering to a
subject an amount of cerium oxide nanoparticles sufficient to
reduce, eliminate, or block cell, tissue, or organ damage caused by
free radicals in the subject.
[0066] In one embodiment, the cerium oxide nanoparticles can be
taken up by the cell. In this case, they can act to reduce or
eliminate free radicals within the cell. This method can be used
for the prevention or treatment of brain disease, spinal cord
disease, or other neurological trauma. This method can also be used
for the treatment or prevention of neurodegenerative disorders such
as Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis (ALS), multiple sclerosis,
toxin-mediated damage, or stroke. This method may be used in the
treatment or prevention of cardiovascular disease, diabetes,
diseases of the retina, asthma, respiratory dysfunctions, and
allergic or autoimmune diseases, such as chronic obstructive
pulmonary disease and lupus. It is to be understood that the
diseases stated above are only examples and are not to be
understood as limiting the invention in anyway.
[0067] In another embodiment, the cerium oxide nanoparticles are
not taken up in any significant amount by the cells, but go into
intravascular or interstitial spaces. In this embodiment, the
nanoparticles can act to reduce or eliminate free radicals outside
the cell. This can result in reduction of inflammation and
inflammatory disorders. The cerium oxide nanoparticles can reduce
inflammation systemically (throughout a subject's body) or locally
(at the site of the inflammatory cells). The nanoparticles can
reduce or eliminate inflammation that leads to preeclampsia or
inflammation caused by wounding. This can also reduce or eliminate
inflammation caused by the insertion of a medical prosthesis into
the subject. Nanoparticles may be retained at particular sites, at
least substantially retained for periods of time, by inclusion of
the nanoparticles into compositions, such as dissolvable or porous
matrices and the like.
[0068] The cerium oxide nanoparticles can also contact the surface
of the subject's skin and increase cell and organism longevity on
the surface of the skin. Skin aging and inflammation of the skin
are closely linked. In inflammation, there is an increase in
neutrophil activity that involves a change in the oxidation state
of the cell. Free radicals are generated which activate the
chemical mediators of inflammation. In skin aging, free radicals
are formed from normal metabolism, UV irradiation, and other
environmental factors. The use of cerium oxide nanoparticles on the
surface of the skin may prevent aging of the skin or reduce damage
already inflicted on the skin. This embodiment may be used in
makeup or anti-aging lotion. It may be in the form of a cream,
lotion, gel, solid stick, powder or any other acceptable
composition that is known in the art.
[0069] The cerium oxide nanoparticles can also be used in
protection against forms of radiation, such as IN irradiation. It
is known in the art that large cerium oxide molecules, as well as
other oxide molecules such as zinc oxide, have the ability to
protect a subject's skin from UV irradiation caused by the sun's
rays. However, it has not been shown until now that cerium oxide
nanoparticles, which enter a cell, have protective characteristics
against radiation intracellularly. The data presented here shows
that cerium oxide nanoparticles can function to protect against
forms of radiation such as UV and gamma radiation. The present
invention provides a method for protection against other forms of
radiation as well, such as beta and X-ray radiation. It is to be
noted that the mode of action of the cerium particles of the
present invention differs from the mode of action of larger
particles in that the larger particles known in the art act to
block, reflect, etc. UV light from entering cells, whereas the
nanoparticles of the present invention act at a biochemical level
to counteract the effects of the UV light within the cells.
[0070] Another embodiment of the invention is prophylactic
radioprotection of a subject. For example, if a subject requires
radiation treatment for cancer, some of the normal, healthy cells
surrounding the cancerous cells will be exposed to the radiation as
well. The present invention addresses this problem by providing a
method for protecting the normal, healthy cells by exposure to the
cerium oxide nanoparticles before radiation treatment. In other
examples, a subject can be exposed to cerium oxide nanoparticles
for radioprotection in work environments with high radiation
exposure or in military or bioterrorism uses.
[0071] In a further aspect, cerium oxide nanoparticles and
compositions comprising cerium oxide nanoparticles are provided.
The cerium oxide nanoparticles are size-limited and provided in an
amount sufficient to provide one or more doses to a subject in need
of, or suspected of being in need of, treatment for a disease or
disorder involving free radicals. Compositions may comprise cerium
oxide particles of the invention along with one or more other
substances, which are typically substances that are biologically
tolerable in that they may be exposed to living cells without
killing the cells. In embodiments, the other substances are
pharmaceutically acceptable substances. As used herein,
"pharmaceutically acceptable substance" is intended to include
solvents, coatings, antibacterial and antifungal agents, and any
other ingredient that is biologically tolerable. Examples of such
carriers include, but are not limited to, water, saline, dextrose
solution, human serum albumin, liposomes, and hydrogels. The use of
such media and agents for pharmaceutically active substances is
well known in the art, and thus further examples and methods of
incorporating each into compositions at effective levels need not
be discussed here.
[0072] Certain aspects of the invention provide for the use of
cerium oxide nanoparticles in the treatment of diseases and
disorders associated with free radicals, such as oxygen free
radicals. The use is in particular for in vivo therapeutic or
prophylactic methods of protecting cells from free radical damage.
Certain other aspects of the invention provide for the use of
cerium oxide nanoparticles in the preparation of compositions for
medical use, such as pharmaceutical or therapeutic compositions. In
general, use of the particles is in combining them with other
substances to make medicinal compositions.
[0073] Another aspect of the invention provides a container
containing cerium oxide nanoparticles. In general, a container
according to the invention contains a sufficient amount of
size-limited cerium oxide nanoparticles made by a method other than
a sol-gel method to provide at least one dose of cerium oxide to a
subject suffering from, or at risk of suffering from, a disease or
disorder involving free radicals, such as oxygen radicals. For
example, the container may contain sufficient cerium oxide
nanoparticles and, optionally, one or more other biologically
tolerable substance, for one dose to a human or non-human animal
subject. In certain embodiments, the container is provided in a
package with one or more other containers and/or with one or more
articles of manufacture or devices having use in delivery of
substances to subjects (e.g., syringes, needles, antiseptic swabs,
sterile saline solution). In some embodiments, kits comprising one
or more containers are provided.
[0074] Regardless of whether provided alone, as part of a
composition, or as part of a kit, the cerium oxide nanoparticles
may be provided in any suitable physical form. Thus, they may be
provided as dry particles or as part of a liquid composition. When
part of a liquid composition, the composition typically will
comprise water or an aqueous buffer, such as phosphate buffered
saline (PBS) or other salt buffers. In general, it is preferred
that the liquid composition be suitable for introduction into a
living organism or for contact with a living cell without causing
deleterious effects, such as cell toxicity. It is to be understood
that this general preference permits inclusion of toxic components
in the liquid composition as long as those components, when exposed
to a living cell upon exposure to the cell, are present in a
non-toxic form or at non-toxic levels. In embodiments where dry
nanoparticles are administered, the nanoparticles may be in a
purified state or may be in a composition comprising one or more
other component. It is preferred that the other component(s) be
non-toxic or, if toxic, present in an amount that, when
administered, is not toxic to the cell or subject as a whole.
Examples of non-toxic components include, but are not limited to,
salts (e.g., sodium salts such as sodium phosphate or sodium
chloride); sugars (e.g., glucose, sucrose); preservatives; and
antibiotics, anti-inflammatories, albumin, lipids, or other drugs.
The vehicle of delivery may be in the form of an oral solution,
gel, tablet, capsule, powder, suppository, infusible, losenge,
cream, salve, inhalant, or injection.
[0075] Typically, the particles or composition comprising the
particles will be sterile or will have been sterilized prior to
administration to a subject or other use. The particles may be
sterilized using any suitable technique known in the art,
including, but not limited to, heat sterilization, filtration, and
irradiation. Thus, in embodiments, the method of the invention
further comprises providing sterile or sterilized cerium oxide
nanoparticles, or further comprises sterilizing the nanoparticles
prior to administering them to a subject.
[0076] The invention provides compositions comprising cerium oxide
nanoparticles. The compositions can comprise a pharmaceutically
suitable carrier, a nutritional supplement, or a dietary
supplement. While not being so limited, typically the compositions
comprise one or more other substances other than the nanoparticles,
where the other substances are biologically tolerable (i.e.,
non-toxic or present in an amount that is non-toxic). Examples of
such substances are well known to those of skill in the art and
include, without limitation, sugars, salts, lipids, drugs,
excipients, carriers, flavorants, fillers, binders, gums,
colorants, water, buffers, detergents, biologically active
compounds, and the like.
[0077] The present invention also provides kits. In general, the
kits comprise cerium oxide nanoparticles in an amount sufficient to
treat at least one patient at least one time to reduce or eliminate
free radicals that can cause cell, tissue, or organ damage.
Typically, the nanoparticles of the kit will be supplied in one or
more container, each container containing a sufficient amount of
nanoparticles for at least one dosing of the patient. The kits can
comprise other components, such as some or all of the components
necessary to practice a method of the invention. For example, in
embodiments of the kit, albumin is included, either as a separate
component or as part of a composition comprising the nanoparticles.
The albumin is provided to lessen the amount or use of disruption
of the nanoparticles, for example by sonication at 5-20 Hz for 2
minutes, that can sometimes be needed to provide certain
formulations for delivery. The kits may contain a syringe for
administering a dose of the nanoparticles. The kits may also
comprise filters for sterilization of the particles prior to
delivery; however, it is preferred that the particles be sterilized
prior to packaging in the kits, or the entire kit be sterilized
after all components are packaged. It may likewise contain sterile
water or buffer for rehydration or reconstitution of dry
nanoparticles, prior to administration of the particles to a
patient. In embodiments, multiple doses of nanoparticles are
provided in the kit, either all in a single container (e.g., a
vial) or distributed among two or more containers. As the invention
contemplates administering or delivering (used synonymously herein)
of nanoparticles in liposomes, kits according to the invention may
comprise liposomes, particularly liposomes loaded with the
nanoparticles.
EXAMPLES
[0078] The invention will be further explained by the following
Examples, which are intended to be purely exemplary of the
invention, and should not be considered as limiting the invention
in any way.
Example 1
Extension of Cell and Organism Longevity
[0079] A single 10 nM dose of cerium oxide nanoparticles extended
the life span of cultured rat brain cells (neurons, astrocytes,
microglia) from 28 to 182 days (6 months). For delivery, the
nanoparticles were in a non-agglomerated form. To accomplish this,
stock solutions of about 10% by weight were sonicated in ultra-high
purity water (16 megaohms) or in normal saline prepared with ultra
high purity water. Stocks were sonicated with a probe sonicator for
3 minutes. Dilutions were made, beginning with 10 mM, down to 100
nM or lower. No phosphate or other ionic buffers were used because
these were found to increase agglomeration. All serial dilutions
were sonicated for 3 minutes prior to use or to further dilution.
Importantly, aged neurons and astrocytes were functionally
equivalent to their younger, untreated, counterparts.
Neurotransmission in response to glutamate, GABA, and acetylcholine
in cerium oxide nanoparticle-treated aged cultures was similar to
younger, cultured controls. Further, similar doses of cerium oxide
nanoparticles administered orally (in the food) extended the
lifespan of the fruit fly, Drosophila melanogaster.
[0080] FIG. 1 depicts the results of experiments to determine the
effect of nanoparticles on the maximum lifespan of organotypic
brain cells in culture. The mixed brain cell cultures from rat
cerebral cortex were treated with 10 nM cerium oxide nanoparticles
on day 10 in vitro. Controls received vehicle alone (normal
saline). The figure shows that the nanoparticles has a dramatic
effect on cell lifespan. DIV=Days In Vitro
[0081] FIG. 2 depicts the results of experiments to determine the
effect of nanoparticles on the lifespan of Drosophila melanogaster.
The results show that the lifespan of the flies is significantly
increased. Drosophila melanogaster (Oregon R strain) were fed from
enclosure with standard mix fly food with or without cerium oxide
nanoparticles at the indicated concentrations. Note that not only
is the maximum lifespan increased, but the time to 50% population
death in increased in nanoparticle-treated vs. controls (dotted
lines). Flies were fed food containing the indicated concentration
of cerium oxide nanoparticles, from enclosure throughout the
lifetime. Stock concentrations of cerium oxide nanoparticles were
prepared as described above (sonication methods) and added to the
fly food (Jazz Mix) during preparation (i.e., while the fly food
remained in liquid form). Food was sonicated 5 min after addition
of particles, to ensure non-agglomerated suspension of
nanoparticles in the food medium. Flies were growth under standard
conditions, in vials containing 5 ml food medium and 20 flies per
vial. Dead flies were counted every 1-2 days.
Example 2
Free Radical Scavenging Capacity of Cerium Oxide Nanoparticles
[0082] Given the structure of cerium oxide nanoparticles, we
hypothesized that cerium oxide nanoparticles promoted cell
longevity by acting as free radical scavengers. To test this
hypothesis, we exposed cultured brain cells to lethal and
sub-lethal doses of the free radical generating agents, hydrogen
peroxide, and UV light. Exposure to cerium oxide nanoparticles
afforded significant protection against both of these free radical
generating agents, and reduced cell death in excess of 60%.
Protection against UV and hydrogen peroxide-mediated injury was
preserved in 3 month old cultures that had been treated with cerium
oxide nanoparticles on day 10 in culture. Thus, the effects of
cerium oxide nanoparticles are long-lasting, following a single
dose.
[0083] Studies comparing the effects of cerium oxide nanoparticles
to the traditional free radical scavengers Vitamin E, melatonin,
and N-acetyl-cysteine demonstrated that only cerium oxide
nanoparticles were capable of enhancing longevity. Further, cerium
oxide nanoparticles provided superior protection to free radical
mediated injury, as compared to single and multiple doses of
traditional free radical scavengers.
[0084] To further confirm our hypothesis that cerium oxide
nanoparticles act via a free radical scavenging mechanism, we have
detected a novel shift in the excitation spectra of cerium oxide
nanoparticle solutions and in cells loaded with cerium oxide
nanoparticles, during free radical challenge. In cells and in
cerium oxide nanoparticle solutions, excitation scans reveal a peak
excitation of 451 for cerium oxide nanoparticles in the reduced
(+4) valence state. Upon free radical challenge, the excitation
maxima shifts to 356 nm, suggesting a change in cerium to the +3
valence state. After 5-20 minutes, the excitation spectra returns
to the normal resting state, with a peak maxima of 451 excitation,
suggesting regeneration of the original cerium oxide lattice
structure.
[0085] FIG. 3 depicts the excitation spectra for intracellular
cerium oxide nanoparticles, and shows that the spectra is altered
during a free radical scavenging event. For the experiments
depicted in the figure, astrocytes were treated with 10 nM cerium
oxide nanoparticles on day 10 in vitro, and examined
fluorimetrically on day 18. Cell cultures were washed, placed in
phosphate buffered saline, and subjected to excitation spectra scan
as shown. Emission was measured above 510 nm. Excitation scans were
collected every 0.01 msec using a high speed DeltaRam Scanner,
during the addition of 100 uM H.sub.2O.sub.2 as a free
radical-generating agent. Controls (untreated) cells revealed no
fluorescence emission in the range and magnitude shown. The shift
in excitation spectra of cerium indicates an electron shuffling
event in the oxide lattice or cerium atom, as shown in FIG. 3.
These results demonstrate that a similar shift in excitation
spectra occurs in cells containing cerium oxide nanoparticles,
which occurs during a reaction with a free radical, such as that
generated by H.sub.2O.sub.2. Importantly, the return to 456 nm
excitation maxima suggests that the cerium oxide nanoparticle can
regenerate its free radical scavenging capacity while in the
cell.
Example 3
Toxicity and Biodistribution
[0086] Using electron microscopy, microspectrophotometry, and
inductively coupled plasma mass spectrometry, we found that cerium
oxide nanoparticles of size less than 20 nm readily enter cultured
cells and cells of living organisms. Further, doses as high as
100-fold of that which extend cell culture lifespan exhibited no
overt toxicity in Drosophila. A single tail vein injection of 0.3-3
mM in the mouse produced no overt organ or behavioral
abnormalities. Cerium oxide nanoparticles were found to accumulate
preferentially in brain, heart, and lung with little excretion over
a 6 month time period. At the 0.3 mM dose, tissue cerium levels
approximately doubled (as compared to background), but remained in
the parts per billion range.
[0087] FIG. 4 depicts the results of tissue cerium measurements of
mice treated with nanoparticles. More specifically, Balb/c mice
were administered 5-10 ul tail vein injections each containing 300
nmoles cerium oxide nanoparticles. After 3 months, mice were
euthanized and organs were harvested. Tissue cerium was measured by
inductively coupled plasma mass spectrometry. It is interesting to
note that the highest increases in tissue cerium concentration
occurred in brain, heart, and lung, the most oxidative organs in
the body.
Example 4
Protection Against Trauma
[0088] Using an in vitro model representative of human head injury
that has been extensively published, we have demonstrated that
brain cell injury in response to trauma may be related, in part, to
generation of free radicals induced by injury. Brain cell cultures
treated with cerium oxide nanoparticles on day 10 in vitro showed a
60-70% reduction in cell injury when trauma was administered on
days 15-18 in vitro. Further, delivery of cerium oxide
nanoparticles up to 3 hrs post-injury reduced neuronal death by
40-50%, depending on the degree of injury. Thus, cerium oxide
nanoparticles represent a treatment for trauma and other forms of
neurodegeneration associated with free radical injury.
[0089] In brain trauma, neuronal dysfunction often manifests,
causing persistent neurological deficits. Here, we demonstrate this
correlates to human head injury with an in vitro model. We found
that pre- or post-injury delivery of nanoparticles significantly
reduced neuronal dysfunction, as measured by
neurotransmitter-stimulated calcium signaling, in both astrocytes
and neurons.
[0090] FIG. 5 shows the effect of nanoparticles on brain cells
subjected to trauma. Mixed organotypic brain cell cultures were
subjected to in vitro trauma as previously described (Zhang,
Rzigalinski, et al. Science 274: 1921-1923, 1997). Cerium oxide
nanoparticles (10 nM) were delivered to the cultures either on day
10 in vitro or 3 hours post injury and neuronal death was assessed
by propidium iodide staining at 24 hrs post injury. The positive
effects on cells is evident.
[0091] FIG. 6 further shows the effect of nanoparticles on brain
cells subjected to trauma. Mixed organotypic brain cells were
subjected to in vitro trauma as described above. Cerium oxide (10
nM) nanoparticles were delivered 3 hrs post injury and neuronal
intracellular free calcium ([Ca.sup.2+].sub.i) signaling was
determined at 24 hrs post injury using Fura-2
microspectrophotometry. Uninjured neurons (solid black line) showed
regular intracellular free calcium oscillations, indicative of
robust inter-neuronal signaling. Glutamate induced a rise in
[Ca.sup.2+].sub.i to 262 nM, followed by a return to basal. In
injured, untreated cultures (dashed line) [Ca.sup.2+].sub.i
signaling is perturbed. Neurons either had dramatically elevated
basal [Ca.sup.2+].sub.i with no response to glutamate, or a
dramatically enhanced response to glutamate, suggestive of
excitotoxicity. In injured cultures treated with cerium oxide
nanoparticles, normal basal [Ca.sup.2+].sub.i, oscillations and
glutamate signaling were preserved (lt gray line). Results shown
are representative of 12 separate experiments including over 90
neurons.
Example 5
Anti-Inflammatory Properties of Nanoparticles
[0092] Free radical production and the associated cell damage are
components of many inflammatory disorders, including arthritis,
Alzheimer's Disease, multiple sclerosis, atherosclerosis, ALS,
Parkinson's disease, autoimmune diseases, and allergic disorders.
We found cerium oxide nanoparticles to be potent inhibitors of
inflammation and inflammatory cell damage. Our studies indicate
that cerium oxide nanoparticles reduce the inflammatory response in
brain microglia (MG), reduce neuronal death induced by activated,
inflammatory brain MG, as well as reduce the release of interleukin
1-.beta. and inflammatory mediators of the arachidonic acid cascade
in brain MG. We also found that cerium oxide nanoparticles reduce
the inflammatory activation state of human neutrophil and
macrophage like cells lines, HL-60 and U937 and reduce the
inflammatory response initiated by histamine, bacterial
lipopolysaccharide (LPS), and fMLP (f-met-leu-phe, chemotactic
peptide) in human neutrophil and macrophage-like cell lines (HL-60
& U937). Therefore, cerium oxide nanoparticles represent a
novel treatment for inflammatory and immune disorders.
[0093] FIG. 7 shows that cerium oxide nanoparticles reduce the
inflammatory response initiated by lipopolysaccharide (LPS).
Experiments have shown that microglia (MG), as inflammatory cells,
respond to traumatic brain injury by up-regulation of inflammatory
functions, known as "activation". Once "activated", MG become
essential in the removal of damaged or malfunctioning neurons. MG
are hypothesized to exert a destructive force on healthy, bystander
neurons due to prolific release of free radicals, which damage
surrounding neurons. Our previous studies have shown that neuronal
death is reduced in traumatically injured organotypic brain cell
cultures by treatment with cerium oxide nanoparticles, a potent
free-radical scavenger. One of the free-radicals released by MG
when subjected to injury is Nitric Oxide (NO). MG were treated once
with 10 nM CeO.sub.2--NP for 24 hrs, to allow uptake of
nanoparticles. After washing and changing the media, MG were
treated with 100 ng/ml LPS to induce the inflammatory response.
Morphology and release of NO were examined. MG exposed to 100 ng/ml
LPS for 24 hours exhibited release of NO of 16.1 mM. When treated
with 10 nM CeO.sub.2--NP for 24 hours prior to exposure, NO release
decreased by 62.0%, demonstrating that CeO.sub.2--NP does decrease
release of inflammatory mediators that may enhance neuronal
death.
[0094] As shown in FIG. 8A, resting MG have compact cell bodies
with long, branched processes. In FIG. 8B, MG were stimulated with
LPS. Note the dramatic morphological changes as compared to the
resting state (8A). LPS-induced morphological changes are blocked
by CeO.sub.2--NP as shown in 8C.
Example 6
Radioprotective Effects of Nanoparticles
[0095] Radiation injury induces cell death by free radical-mediated
damage to cellular DNA, RNA, and proteins. Cerium oxide
nanoparticles reduced brain cell death associated with 1, 3, and 5
Gray by 78, 62, and 48%, respectively. In these experiments, a
single 10 nM dose of nanoparticles was administered on day 10 in
vitro, with irradiation of cultures on day 12-15. Further, a
reduction in injury was observed even when particles were
administered up to 3 hrs post irradiation. These results suggest
that cerium oxide nanoparticles have significant radioprotective
properties, and may be utilized in radiation protection for
military and anti-bioterrorism applications. Additionally,
nanoparticles have the potential for use in cancer therapy, by
protection of non-cancerous "bystander" cells from radiation
injury.
[0096] FIGS. 9 and 10 show the effect of pretreatment with cerium
oxide nanoparticles on exposure to radiation. Mixed organotypic rat
brain cells were obtained from neonatal rat pups and cultures as
previously described (Zhang et al., Science, 274, 1921-1923,
1996.). Cultures were treated +10 nM CeO.sub.2--NP on day 10 in
vitro, by delivery to the tissue culture medium for 24 hrs,
followed by regular medium replacements. After 14-16 DIV, free
radical damage was assessed by exposure to ultraviolet light for
increments of 5 minutes or 15 minutes, followed by measurement of
cell death with Propidium Iodide (PrI). For gamma-irradiation
studies, cells were exposed to 1.5 or 5 Gray radiation for 1
minute. Additionally, aged cultures (68 DIV) treated with
CeO.sub.2--NP were also exposed to UV and gamma-irradiation, to
determine whether the protective effects of CeO.sub.2--NP were
maintained in aged cultures.
[0097] For the experiments in FIG. 9, mixed brain cell cultures
were treated with CeO.sub.2--NP at 10 DIV, and exposed to UV light
at 16 or 68 DIV. Note that there are no 68 DIV untreated controls,
since untreated mixed brain cell cultures do not survive this long.
CeO.sub.2--NP treatment dramatically increased survival after 5 and
15 min UV exposure, which are known to induce cell death through
free radical production. Further, the protective effects of a
single 10 nM dose of CeO.sub.2--NP were maintained through the
extended lifespan of these cells.
[0098] For the experiments in FIG. 10, mixed brain cell cultures
were exposed to a second source of free radical generation,
gamma-irradiation. Cultures were treated with CeO.sub.2--NP and
exposed to irradiation as described above. A single 10 nM dose of
CeO.sub.2--NP delivered at 10 DIV provided significant protection
against gamma-irradiation, which was again maintained through the
extended lifespan of the cultures.
[0099] FIGS. 11 and 12 show that cerium oxide nanoparticles provide
greater protection against free radical mediated injury as compared
to single or multiple doses of Vitamin E, n-Acetyl Cysteine, or
Melatonin. In these experiments, cells were cultured in 6-well
plates. Three wells were used as controls while the other three
were treated with one of the following agents at 10-DIV: 10 nM
Cerium Oxide nanoparticles, 100 mM Vitamin E, 1 mM n-Acetyl
Cysteine, or 1 mM Melatonin. Drugs were delivered directly into the
tissue culture media and remained in the media for 24 his, followed
by media replacement. Nanoparticles were only delivered once, at 10
DIV. Other agents were delivered in single or multiple doses as
indicated. After 14-16 DIV, free radical damage was assessed by
exposure to ultraviolet light for increments of 5 minutes or 15
minutes, followed by measurement of cell death with Propidium
Iodide (PrI).
[0100] In the FIG. 11 experiments, cerium oxide nanoparticles or
other free radical scavengers were delivered to the tissue culture
medium on DIV 10. Medium was replaced 48 hrs later, followed by
regular medium changes every 2-3 days. UV exposure was performed on
DIV 14. Cerium Oxide nanoparticles reduced UV-light induced cell
death 24 hr after a 5 or 15 min. exposure, by 58%. MEL reduced cell
death associated with short term (5 min) UV exposure to a similar
extent, but was less effective after a long term (15 min) exposure.
Vitamin E afforded a modest degree of protection.
[0101] In the FIG. 12 experiments, a single 10 nM dose of cerium
oxide nanoparticles delivered on DIV 10 was compared to multiple
doses of other antioxidants. Vitamin E, n-Acetyl Cysteine, and
Melatonin were administered at DIV 10 and again on DIV 12. Cerium
Oxide nanoparticles were more efficient at decreasing UV-mediated
cell injury than multiple doses of Vitamin E, n-Acetyl Cysteine, or
Melatonin.
Example 7
Further Experiments on Extension of Cell and Organism Longevity
[0102] FIGS. 13 and 14 show that both male and female Drosophila
life spans are increased when cerium oxide nanoparticles are given
to the flies. These longevity studies were performed by adding 10
nM CeO.sub.2--NP directly to the fly food. To determine the effect
of CeO.sub.2--NP on survival after free radical challenge, male and
female flies were cultured continuously from the day of enclosure
on fly food containing 10 nM CeO2-NP. On day 35, flies were exposed
to filter paper saturated with 20 mM paraquat in 5% sucrose
solution for 24 hrs. Paraquat is a redox cycling pesticide known to
induce fly death via free radical production. Dead flies were
counted at regular intervals. Flies surviving in excess of 24 hrs
were placed back into vials containing control food or food treated
with the appropriate CeO.sub.2--NP concentrations. Surviving flies
continued to be monitored on a daily basis. Similar results were
obtained in vitro with cell cultures in which 1 nM, 10 nM, and 1 uM
cerium oxide nanoparticles protected the cells against death in the
presence of 0.1 mM, 0.5 mM and 1 mM paraquat (data not shown).
[0103] FIG. 15 shows the amount of neuron specific enolase (NSE) in
tissue culture. As neurons die off in a culture, they release a
characteristic enzyme, NSE. This experiment shows the amount of NSE
in the tissue culture medium, as a percentage of the total left in
the cultures. NSE release increases dramatically in the medium over
days 20-26, as the neurons die and lyse. At day 30, all the neurons
are dead. In the cerium oxide treated group (triangles), the NSE in
the medium does not rise, but stays at basal levels, denoting that
all the neurons are still alive.
[0104] FIG. 16 shows the percentage of tissue cultures surviving
with robust neurons and astrocytes. This experiment summarizes data
for over 75 control and cerium oxide-treated cultures. Each culture
was treated with a single dose of 10 nM cerium oxide nanoparticles
on day 10 in vitro. This experiment demonstrates that cerium oxide
nanoparticles increase the longevity of the cultures.
[0105] FIGS. 17 through 20 show that 10 nM cerium oxide
nanoparticles significantly extend the average and maximum lifespan
of male and female Drosophila when the fruit flies are introduced
to paraquat, an oxidative stress inducer. In this experiment we
tested the hypothesis that cerium oxide nanoparticles act as free
radical scavengers in Drosophila melanogaster. To induce oxidative
stress, we used paraquat (methyl viologen). The literature reports
that paraquat induces severe oxidative stress in the fruit fly, via
production of superoxide ions, with an LD50 of 10 mM. Hence,
paraquat is routinely used to test effects of various biochemical
agents on reduction of oxidative stress, via examining survival
after paraquat challenge. In this study, 100 male and female flies
were cultured continuously from the day of enclosure on fly food
containing 10 nM and 1 uM cerium oxide. On day 35, flies were
deprived of food for three hours, then exposed to filter paper
saturated with 20 mM paraquat in 5% sucrose solution for 24 hours.
Dead flies were counted at regular intervals. Flies surviving in
excess of 24 hrs were placed back into vials containing control
food or food treated with the appropriate nanoparticle
concentrations. Surviving flies continued to be monitored on a
daily basis.
Example 8
Further Experiments Showing the Protection Against Trauma
[0106] FIGS. 21 and 22 show that cerium oxide nanoparticles provide
enhanced protection against traumatic injury as compared to a
single dose of other antioxidants when given either pre-trauma
(FIG. 21) or post trauma (FIG. 22). Using an in vitro model for
traumatic brain injury (Ellis et al., J. Neurotrauma, 12, 325-339,
1995), we have previously shown that traumatic injury of mixed
brain cell cultures produces cell death, in part, via generation of
free radicals (Hoffman et al., Lamb, et al. J. Neurochem; 68,
1904-1910, 1997). Mixed brain cell cultures were injured at mild
(5.5 mm), moderate (6.5 mm), and severe (7.5 mm) levels, and cell
death was assessed with PrI, 24 hrs post injury.
[0107] FIGS. 23 to 26 show that cerium oxide nanoparticles decrease
the release of NO from brain microglia. Pure cultures of astrocytes
were injured using a well-characterized model for in vitro trauma.
We have previously shown that exposure to medium conditioned by
traumatically injured astrocytes induces microglial activation. MG
so activated induce neuronal death. In these experiments microglia
were activated by a 24 hour exposure to medium conditioned by mild,
moderate, or severely injured astrocytes. Controls consisted of
microglia exposed to medium conditioned by uninjured astrocytes. In
these experiments, LPS was utilized as positive control. LPS,
acting as an endotoxin, binds to receptors on microglia and
triggers the secretion of pro-inflammatory cytokines and promotes
the release of NO. Control or nano-treated microglia were exposed
to 100 ng/ml LPS for 24 hours followed by measurement of NO
released into the medium, as represented in FIG. 5. NO was measured
using kits provided by Oxis International and Calbiochem, via the
Griess reaction. Absorbance was read in a BioTek ELx800 automated
plate reader at 540 nm.
[0108] Because MG are activated by exposure to medium conditioned
by injured astrocytes, we first determined NO release from
astrocytes during the 1 and 3 hour post-injury period as shown in
FIG. 23. There is significant NO release from moderate and severely
injured astrocytes 1 hour after injury, suggesting that astrocytes
play an important role in oxidative stress in the brain.
[0109] In FIGS. 24 and 25, exposure of MG to medium conditioned by
astrocytes for 1 or 3 hours, regardless of injury, increased NO
release, suggesting that astrocytes regulate the inflammatory
potential of brain MG. Treatment of MG with 10 nM cerium oxide
nanoparticles reduced NO release in all cases. In MG activated by
exposure to medium conditioned by mild, moderate and severely
injured astrocytes for 1 hour, cerium oxide nanoparticles reduced
NO release by 29, 44, 70%, respectively. In MG activated by
exposure to medium conditioned by mild, moderate and severely
injured astrocytes for 3 hours, the decrease in NO release afforded
by cerium oxide nanoparticles was more modest.
[0110] In FIG. 26, MG exposed to 100 ng/ml LPS for 24 hours
exhibited release of NO of 16.1 mM. When treated with 10 nM Cerium
Oxide nanoparticles for 24 hours prior to exposure, NO release
decreased by 62.0%, demonstrating that Cerium Oxide nanoparticles
decrease release of inflammatory mediators that may enhance
neuronal death.
[0111] As shown in FIG. 27, resting MG have compact cell bodies
with long, branched processes. MG activated by exposure to medium
conditioned by severely injured astrocytes become more amoeboid in
shape, with refracted, short processes and highly granulated and
vacuolated cytoplasms. Pretreatment with Cerium Oxide nanoparticles
prevent some of the morphological changes observed in MG
activation. MG were also stimulated with LPS. Note the dramatic
morphological changes as compared to the resting state. LPS-induced
morphological changes are blocked by Cerium Oxide
nanoparticles.
[0112] It will be apparent to those skilled in the art that various
modifications and variations can be made in the practice of the
present invention without departing from the scope or spirit of the
invention. Other embodiments of the invention will be apparent to
those skilled in the art from consideration of the specification
and practice of the invention. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
* * * * *