U.S. patent application number 14/993769 was filed with the patent office on 2016-06-30 for system for screening particles.
The applicant listed for this patent is The Brigham and Women's Hospital, Inc., Massachusetts Institute of Technology. Invention is credited to Omid C. Farokhzad, Robert S. Langer, Aleksandar Filip Radovic-Moreno.
Application Number | 20160187323 14/993769 |
Document ID | / |
Family ID | 38163545 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160187323 |
Kind Code |
A1 |
Farokhzad; Omid C. ; et
al. |
June 30, 2016 |
SYSTEM FOR SCREENING PARTICLES
Abstract
Screening of a library of particles in vivo and/or in vitro
using Polyplex Iterative Combinatorial Optimization (PICO) allows
for the design of particles for targeting a specific organ, tissue
(e.g., cancer), or cell. Particles may, for example, include
different targeting agents (e.g., aptamers or plurality of
aptamers) on their surfaces, and the aptamer or aptamers may be
evolved to provide better targeting of the particles. Libraries of
particles are enriched in characteristics of particles that have
been found to migrate to a tissue of interest, be taken up by
cells, etc. The process may be repeated to engineer particles of a
desired specificity or biological function.
Inventors: |
Farokhzad; Omid C.;
(Chestnut Hill, MA) ; Radovic-Moreno; Aleksandar
Filip; (State College, PA) ; Langer; Robert S.;
(Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology
The Brigham and Women's Hospital, Inc. |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Family ID: |
38163545 |
Appl. No.: |
14/993769 |
Filed: |
January 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12097118 |
Feb 27, 2009 |
9267937 |
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PCT/US2006/047975 |
Dec 15, 2006 |
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14993769 |
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60750765 |
Dec 15, 2005 |
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60747240 |
May 15, 2006 |
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Current U.S.
Class: |
506/10 |
Current CPC
Class: |
A61K 47/66 20170801;
A61K 47/6891 20170801; A61K 47/555 20170801; G01N 33/5088 20130101;
B82Y 5/00 20130101; C40B 30/06 20130101; B82Y 15/00 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1-135. (canceled)
136. A high throughput, iterative, combinatorial screening method
of identifying particle properties providing enhanced delivery of
particles to targeted cells, the method comprising the steps of
providing a plurality of synthetic polymeric particle populations,
wherein the particles of each population have substantially the
same composition, size, density, surface chemistry, ligand or
density of ligand bound thereto, wherein the particles of a given
particle population differ from the particles of another given
particle population by at least one particle characteristic, and
wherein each particle of a given population has at least one
detectable label which is different from the detectable label of
the particles of another given particle population and is different
from the targeting agent; administering the plurality of particle
populations to an animal under conditions in which the particles
are delivered to targeted cells; and enriching the delivered
particles by repeating the process with different populations of
particles, each population having at least one different particle
characteristic, thereby determining the particle characteristics
which enhance delivery of the particles to the targeted cells.
137. The method of claim 1, wherein the particles have on the
surface thereof a ligand selected from the group consisting of an
oligonucleotide, a polysaccharide, an antibody, an antibody
fragment, a nucleic acid ligand, a lipoprotein, folate,
transferrin, an asialycoprotein, an enzymatic receptor ligand,
sialic acid, a glycoprotein, a lipid, a small molecule, metal,
metal complex, a bioactive agent, and an immunoreactive
fragment.
138. The method of claim 1, wherein the detectable label is
selected from the group consisting of a luminescent agent, a
chemiluminescent agent, a phosphorescent agent, a fluorescent
agent, a radionuclide, a small molecule, a mass spectroscopy tag, a
polynucleotide, a polypeptide, a semiconductor particle, a magnetic
material, an ultrasound contrast agent, an MM contrast agent, and
an x-ray contrast agent.
139. The method of claim 3, wherein the label is disposed on the
surface of the particle, in the interior of the particle, or
both.
140. The method of claim 1 further comprising the step of
recovering particles that have migrated to non-targeted cells.
141. The method of claim 1, wherein the particles are selected from
the group consisting of microparticles, nanoparticles, and
picoparticles.
142. The method of claim 1, wherein the polymer is selected from
the group consisting of polyesters, polyamides, polycarbonates,
polycarbamates, polyacrylates, polystyrene, polyureas, polyethers,
polyamines, polyanhydrides, poly(hydroxyacids), poly(lactic acid),
poly(glycolic acid), poly(orthoesters), polyphosphazene,
ethylene-vinyl acetate copolymer, polyurethanes, polyacrylates,
polymethacrylates, polyacrylonitriles, poly(amidoamine) dendrimers,
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester),
maleimide-poly(ethyleneglycol)-block-poly(D,L-lactic acid);
COOH-poly(ethyleneglycol)-block-poly(D,L-lactic acid);
methoxypoly(ethyleneglycol)-block-poly(D,L-lactic acid); proteins;
polysaccharides, PEGylated poly(hydroxy acids), PEGylated
poly(orthoesters), poly(caprolactone), PEGylated
poly(caprolactone), polylysine, PEGylated polylysine, poly(ethylene
imine), PEGylated poly(ethylene imine), and combinations
thereof.
143. The method of claim 142, wherein the particles are
poly(lactic-co-glycolic acid) (PLGA) particles.
144. The method of claim 1, wherein the particles comprise a
poly(hydroxy acid) polymer or copolymer or pegylated poly(hydroxy
acid) polymer or copolymer.
144. The method of claim 1, wherein the particles comprise at least
one targeting agent.
146. The method of claim 1, wherein the particles comprise a
plurality of targeting agents.
147. The method of claim 1 wherein the particles of each of the
given particle population have different ligand conjugated to their
surface.
148. The method of claim 1 wherein the particles comprise a
therapeutic or prophylactic agent for delivery to the targeted
cells.
Description
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. provisional patent applications, U.S. Ser. No.
60/750,765, filed Dec. 15, 2005, and U.S. Ser. No. 60/747,240,
filed May 15, 2006, which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to an in vitro and an in vivo system
for identifying particles with particular characteristics (e.g.,
targeting, cellular uptake, drug delivery) from a library of
particles.
BACKGROUND OF THE INVENTION
[0003] The development of targeted particles for the treatment and
detection of human diseases is expected to result in an explosion
of the market for this class of biomaterials. In some cases,
particles are functionalized with targeted molecules for the
specific delivery of particles to a subset of cells, tissues, or
organs. The delivery of particles is mediated by specific binding
of targeting molecules with distinct chemical moieties that are
present on the desired target. This approach has several
limitations. First, it requires unique chemical moieties on the
cells, tissue, or organ being targeted in order to achieve the
desired specificity. And it requires the availability of high
affinity targeting molecules that preferentially bind the unique
chemical moiety on the cell, tissue, or organ. One problem with
this approach is that there are a myriad of potentially useful
targets and targeting molecules that have not yet been isolated
and/or characterized.
[0004] In addition, other biophysiochemical characteristics of the
targeted particles frequently need to be optimized in order to make
them useful for in vitro or in vivo applications, for example,
composition of the particle, surface characteristics, surface
charge, and particle size. These many possibilities lead to a very
large number of possible particle formulations. Individual
evaluation of all these formulations or even a portion of them in
vivo would require an equal or larger number of animals.
Alternatively, particles could be initially screened using
cell-based systems, following which promising formulations could be
further evaluated in viva. However, the results of in vitro
evaluations are often not recapitulated by in vivo outcomes. For
example, rapid clearance of particles by the liver, spleen, lung,
lymphatic system, or bone marrow, the microenvironment of
inflammation, or the unique features of tumor microenvironment,
e.g., the tumor microvasculature, are not easily reproduced in in
vitro models.
[0005] Therefore, given these significant drawbacks to current
approaches for engineering particles, there remains a need for an
efficient system that can be used to identify particles with
particular characteristics, including targeting, cell uptake,
pharmacokinetics, clinical efficacy, and so forth.
SUMMARY OF THE INVENTION
[0006] The present invention provides a system for identifying
particles or particle compositions with desired characteristics.
The system is particularly useful for screening libraries of
particles for particular characteristics. The system includes both
in vitro and in vivo screening systems. In certain embodiments, the
system is performed in a high-throughput screening format. The
invention provides methods, compositions, and kits for carrying out
the inventive screening of particles.
[0007] In one aspect, the invention provides in vivo methods for
identifying particles in a library with desired characteristics
(e.g., targeting of a particular cell, tissue, or organ). The
method includes providing a library comprising a plurality of
particle populations that vary in at least one particle
characteristic (e.g., targeting moiety, surface charge, surface
hydrophilicity); administering the library to an animal under
conditions in which the particles can migrate to a tissue of
interest; and recovering a first plurality of particles that have
migrated to the cells, tissue, or organ of interest. The at least
one characteristic that may vary among particle populations may be
particle composition, particle size, surface chemistry, presence or
absence of a targeting agent at the surface of the particle, a
density of targeting agents conjugated to the surface, the
population of targeting agents attached to the particle, etc. The
targeting agent can be an oligonucleotide (e.g., aptamer), an
oligopeptide, a protein, a glycoprotein, a carbohydrate, a lipid, a
small molecule, a metal, or an organometallic complex. In certain
embodiments, the targeting agent is an aptamer. In certain
particular embodiments, the particles have attached to their
surface a plurality of different aptamers (e.g., 2, 3, 4, 5, 10,
20, 50, 100, or more different aptamers). The particles in the
library being screened may differ in the population of targeting
agents on their surfaces.
[0008] The method may further comprise characterizing the recovered
particles, for example, determining the identity of the targeting
agent(s) attached to the surface of the particles. Each particle
population may be characterized by an analytical signature provided
by at least one label. The label may include a luminescent agent, a
fluorophore, a radionuclide, a small molecule, a polynucleotide, a
polypeptide, a semiconductor particle, a magnetic material, a
polymer, an ultrasound contrast agent, an MRI contrast agent, or an
x-ray contrast agent. The at least one label may be disposed on the
surface of the particle, in the interior of the particle, or both.
The method may further include identifying at least one
characteristic of the first plurality of particles by
characterizing the analytical signature of at least a portion of
the first plurality of particles. Identifying may include
separating at least one label from its respective particle or
particles, and identifying the label. In certain embodiments, the
label is identified while attached to the particle. The label may
also function as a targeting agent (e.g., a nucleic acid
aptamer).
[0009] The method may further include recovering a second plurality
of particles that have migrated to a non-targeted cell, organ, or
tissue. Characterizing particles that have migrated to a
non-targeted cell, organ, or tissue allows for negative selection
of particles with these characteristics. The method may include
identifying at least one characteristic of the first plurality of
particles (which migrated to the cell, tissue, or organ of
interest), identifying at least one characteristic of the second
plurality of particles (which migrated to a non-targeted cell,
tissue, or organ), preparing an enriched population of particles
having many of the same characteristics of the particles that made
up the first plurality of particles and fewer or none of the
characteristics of the second plurality of particles, and
administering the enriched library and recovering particles from
the enriched population that migrated to the cells, organ, or
tissue of interest. Again, particles may also be recovered from a
non-targeted cell, organ, or tissue for negative selection
purposes.
[0010] The method optionally includes further enriching a library
of particles; administering the doubly enriched library; and
recovering particles. This iterative process provides for selecting
a particle with high specificity for targeting the tissue, cell, or
organ of interest. The characteristic that is selected for may be
the targeting agent(s) of the particles. In certain embodiments,
the aptamers on the surface of the particle are selected for ones
that target the tissue, cell, or organ of interest. The aptamers
may include a collection of different aptamers. The particles may
be microparticles, nanoparticles, or picoparticles. In certain
embodiments, the particles are polymeric microparticles,
nanoparticles, or picoparticles with an aptamer or plurality of
aptamers as the targeting agent. The resulting particles may target
a specific organ (e.g., heart, liver, brain, etc.), a specific
tissue (e.g., cancer, atherosclerotic plaque, etc.), or a specific
cell (e.g., endothelial cell, blood cell, epithelial cell,
etc.)
[0011] In another aspect, the invention provides in vivo methods
for identifying particles with a desired characteristic. The method
includes providing a library comprising a plurality of particle
populations that vary in at least one particle characteristic,
wherein each of the particle populations includes a targeting agent
or plurality of targeting agents conjugated to the surface of the
particles; administering the library of particles to an animal
under conditions in which the particles can migrate to a tissue of
interest; and recovering a first plurality of particles that have
migrated to the tissue of interest. The characteristic of the
particle may be particle composition, particle size, surface
chemistry, density of the targeting agents on the surface of the
particles, etc.
[0012] The method may further include identifying at least one
characteristic of the first plurality of particles. Each particle
population may be characterized by an analytical signature provided
by at least one label. The at least one label may be disposed on
the surface of the particle, in the interior portion of the
particle, or both. The method may further include identifying at
least one characteristic of the first plurality of particles by
characterizing the analytical signal of at least a portion of the
first plurality of particles. Identifying may include separating at
least one label from its respective particle or particles, and
identifying the label. In certain embodiments, the label is not
separated from the particle in order to be identified.
[0013] The method may further include recovering a second plurality
of particles that have migrated to a non-targeted tissue. Such
particles can provide for negative selection (i.e., characteristics
of these particles would be removed or lessened in any enriched
library of particles). The method may further include identifying
at least one characteristic of the first plurality of particles,
identifying at least one characteristic of the second plurality of
particles, preparing an enriched library of particles having
characteristics of the first plurality of particles and none or
fewer of the characteristics from the second plurality or
particles; administering the enriched library; and recovering a
plurality of the enriched particles from a tissue of interest. The
method may further include enriching the recovered plurality of
particles and administering a further enriched library and
recovering a plurality of the enriched particles from a targeted
cell, tissue, or organ.
[0014] In certain aspect, the in vitro and in vivo methods are
combined. For example, particles are designed first using the in
vitro method and then using the in vivo method.
[0015] In another aspect, the invention is a population of
particles having the characteristics of the first plurality of
particles identified by any of the above methods. In another
embodiment, the invention provides a population of enriched
particles prepared by any of the above methods.
[0016] In another aspect, the invention is an in vitro method of
screening for particles with a desired characteristic. The method
includes providing a library comprising a plurality of particle
populations that are each characterized by an analytical signature
provided by at least one label, wherein each population comprises a
plurality of particles having substantially the same analytical
signature; incubating a population of cells with the library for a
predetermined period of time under conditions where the cells can
take up the particles, wherein the particles include a substance
that is necessary to the survival or growth of the cells; and
recovering particles taken up by the living cells. The recovered
particles are then characterized. Characteristics of the recovered
particles that may be determined include particle composition,
particle size, surface chemistry, presence or absence of a
targeting agent, density of targeting agents on the surface, and
composition of targeting agents on the surface.
[0017] The method may further include characterizing the at least
one analytical signature present in the living cells and
correlating it with the corresponding particle population. The
method may further include enriching the corresponding particle
population; incubating the enriched particle with cells; and
recovering particles in living cells. The method may further
include recovering those particles that were not taken up by living
cells and identifying at least one characteristic of the recovered
particles by determining the analytical signature of at least a
portion of the recovered particles. Determining the analytical
signature may include separating at least one label from its
respective particle or particles, and identifying the label. The
method may further include determining the at least one analytical
signature present in the living cells and correlating it with the
corresponding particle population to identify a population of
positively correlated particles, recovering those particles that
were not taken up by living cells and identifying at least one
characteristic of the recovered particles by characterizing the
analytical signature of at least a portion of the recovered
particles to identify a population of negatively correlated
particles, preparing an enriched library of particles having more
of the same characteristics as at least a first predetermined
fraction of the positively correlated particles and none or fewer
of the characteristics of a second predetermined fraction of the
negatively correlated particles, and incubating the enriched
particles with cells.
[0018] In another aspect, the invention provides another in vivo
method of screening for particles with desired characteristics. The
method includes providing a first library comprising a plurality of
particle populations that vary in at least one particle
characteristic, wherein each population comprises a plurality of
particles having substantially the same characteristics, and
wherein each of the particle populations includes a targeting agent
conjugated to the surface of the particles; identifying the
targeting agent(s) conjugated to those particle populations from
the first library that preferentially accumulate in a predetermined
tissue or cell type; providing a second library comprising a
plurality of particle populations that vary in at least one
particle characteristic selected from composition, size, surface
chemistry, and density of a targeting agent on the surface of the
particle, wherein each population comprises a plurality of
particles having substantially the same characteristics, and
identifying the at least one particle characteristic of those
particle populations from the second library that preferentially
accumulate in a predetermined tissue or cell type.
[0019] Identifying the targeting agent or identifying the at least
one particle characteristic may include administering the library
of particles to an animal under conditions in which the particles
can migrate to a tissue of interest and recovering a first
plurality of particles that have migrated to the tissue of
interest. Identifying the targeting agent or identifying the at
least one particle characteristic may include incubating a
population of cells with the library for a predetermined period of
time under conditions where the cells can take up particles,
wherein the particles include a substance that is necessary for the
survival or growth of the cells and recovering particles from the
living cells. In another aspect, the invention is a population of
particles having the at least one particle characteristic
identified, wherein the identified targeting agent is conjugated to
the particles.
BRIEF DESCRIPTION OF THE DRAWING
[0020] FIG. 1 shows the complexity of an exemplary library of
nanoparticles that may be optimized using the inventive PICO
system.
[0021] FIG. 2 demonstrates the efficacy of nanoparticle (NP) ratio
prediction. Nanoparticles that were added to the wells were
expected to contain a certain quantity of DNA, the relative ratios
of which are indicated by the expected ratio. Observed ratios are
calculated relative to the lowest oligonucleotide content present,
so as to keep numbers greater than one for ease of
interpretation.
[0022] FIG. 3 demonstrates the efficacy of nanoparticle (NP) ratio
prediction. Nanoparticles that were added to the wells were
expected to contain a certain quantity of DNA, the relative ratios
of which are indicated by the expected ratio. Data was collected
similar similarly to the data presented in FIG. 2, only different
ratios of oligonucleotides are expected since different ratios of
particles were incubated together.
[0023] FIG. 4 demonstrates the efficacy of nanoparticle (NP) ratio
prediction. Nanoparticles that were added to the wells were
expected to contain a certain quantity of DNA, the relative ratios
of which are indicated by the expected ratio. Data was collected
similarly to the data presented in FIGS. 2-3, only different ratios
of oligonucleotides are expected since different ratios of
particles were incubated together.
[0024] FIG. 5 shows the uptake of nanoparticle in human prostate
cancer LNCaP cells. Whether nanoparticles are screened together as
a library or separately as individuals against LNCaP cells makes
little impace on the results of the screening assay. Results are
reported as percentages relative to maximum uptake observed, in
order to normalize for the effects of increased concentration of
particles in the "individual" experiments (the total concentration
of particles in both cases was the same).
DEFINITIONS
[0025] "Animal": As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to a human, at any stage of development. In some embodiments,
"animal" refers to a non-human animal, at any stage of development.
In some embodiments, animals include, but are not limited to,
mammals, birds, reptiles, amphibians, fish, and/or worms. In
certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, and/or a pig). In some embodiments, an animal
may be a transgenic animal, genetically-engineered animal, and/or
clone.
[0026] "Bioactive agents": As used herein, "bioactive agents" is
used to refer to compounds or entities that alter, inhibit,
activate, or otherwise affect biological or chemical events. For
example, bioactive agents may include, but are not limited to,
anti-AIDS substances, anti-cancer substances, antibiotics,
immunosuppressants, anti-viral substances, enzyme inhibitors,
including but not limited to protease and reverse transcriptase
inhibitors, fusion inhibitors, neurotoxins, opioids, hypnotics,
anti-histamines, lubricants, tranquilizers, anti-convulsants,
muscle relaxants and anti-Parkinson substances, anti-spasmodics and
muscle contractants including channel blockers, miotics and
anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or
anti-protozoal compounds, modulators of cell-extracellular matrix
interactions including cell growth inhibitors and anti-adhesion
molecules, vasodilating agents, inhibitors of DNA, RNA or protein
synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal
and non-steroidal anti-inflammatory agents, anti-angiogenic
factors, anti-secretory factors, anticoagulants and/or
antithrombotic agents, local anesthetics, ophthalmics,
prostaglandins, anti-depressants, anti-psychotic substances,
anti-emetics, and imaging agents. In a certain embodiments, the
bioactive agent is a drug.
[0027] A more complete listing of bioactive agents and specific
drugs suitable for use in the present invention may be found in
"Pharmaceutical Substances: Syntheses, Patents, Applications" by
Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999;
the "Merck Index: An Encyclopedia of Chemicals, Drugs, and
Biologicals", Edited by Susan Budavari et al., CRC Press, 1996, and
the United States Pharmacopeia-25/National Formulary-20, published
by the United States Pharmcopeial Convention, Inc., Rockville Md.,
2001, all of which are incorporated herein by reference.
[0028] "Biomolecules": The term "biomolecules", as used herein,
refers to molecules (e.g., proteins, amino acids, peptides,
polynucleotides, nucleotides, carbohydrates, sugars, lipids,
nucleoproteins, glycoproteins, lipoproteins, steroids, etc.)
whether naturally-occurring or artificially created (e.g., by
synthetic or recombinant techniques) that are commonly found in
nature (e.g., organisms, tissues, cells, or viruses). Specific
classes of biomolecules include, but are not limited to, enzymes,
receptors, neurotransmitters, hormones, cytokines, cell response
modifiers such as growth factors and chemotactic factors,
antibodies, vaccines, haptens, toxins, interferons, ribozymes,
anti-sense agents, plasmids, siRNA, DNA, and RNA.
[0029] "Biodegradable": As used herein, "biodegradable" polymers
are polymers that degrade (i.e., down to monomeric species or
oligomers that can be eliminated or processed by the body) under
physiological conditions. In preferred embodiments, the polymers
and polymer biodegradation byproducts are biocompatible.
Biodegradable polymers are not necessarily hydrolytically
degradable and may require enzymatic action to fully degrade. In
certain embodiments, the biodegradable polymer is degraded by the
endosome.
[0030] "Decomposition": As used herein, "decomposition" is the
process by which a material is broken down under physiological
conditions into components that may be metabolized or eliminated by
the body. For example, biodegradable polymers may be degraded to
oligomers or monomeric species. The oligomers or monomeric species
may then be eliminated by the body. In certain embodiments, the
polymer or its degradants are metabolized by the liver. In other
embodiments, the polymer or its degradants are eliminated by the
kidneys. In other embodiments, the polymer or its degradants are
eliminated by the digestive system.
[0031] "Endosomal conditions": The phrase "endosomal conditions",
as used herein, relates to the range of chemical (e.g., pH, ionic
strength) and biochemical (e.g., enzyme concentrations) conditions
likely to be encountered within endosomal vesicles. For most
endosomal vesicles, the endosomal pH ranges from about 5.0 to
6.5.
[0032] "Enrichment": As used herein, the term "enrichment" is used
to refer to creating a larger proportion of a material having the
same composition as a smaller sample of that material. For example,
a selected population of polynucleotides may be enriched to include
a large proportion of polynucleotides having substantially the same
sequences as the original selected population. It is not necessary
that the original population be a part of the enriched population,
e.g., the population need not be a template for the production of
the enriched population. However, in some instances, that may be
the case. For example, a population of particles may be enriched by
identifying or selecting a subset Of the population and
manufacturing a larger population having substantially the same
composition.
[0033] "Pharmaceutically active agent": As used herein, the term
"pharmaceutically active agent" refers collectively to
biomolecules, small molecules, and bioactive agents which exert a
biological effect upon administration to an animal.
[0034] "Physiological conditions": The phrase "physiological
conditions", as used herein, relates to the range of chemical
(e.g., pH, ionic strength) and biochemical (e.g., enzyme
concentrations) conditions likely to be encountered in the
intracellular and extracellular fluids of tissues. For most
tissues, the physiological pH ranges from about 7.0 to 7.4.
[0035] "Polynucleotide", "nucleic acid", or "oligonucleotide": The
terms "polynucleotide", "nucleic acid", or "oligonucleotide" refer
to a polymer of nucleotides. The terms "polynucleotide", "nucleic
acid", and "oligonucleotide", may be used interchangeably.
Typically, a polynucleotide comprises at least two nucleotides.
DNAs and RNAs are polynucleotides. The polymer may include natural
nucleosides (i.e., adenosine, thymidine, guanosine, cytidine,
uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and
deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine,
2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,
C5-propynylcytidine, C5-propynyluridine, C5-bromouridine,
C5-fluorouridine, C5-iodouridine, C5-methylcytidine,
7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,
biologically modified bases (e.g., methylated bases), intercalated
bases, modified sugars (e.g., 2'-fluororibose, 2'-methoxyribose,
2'-aminoribose, ribose, 2'-deoxyribose, arabinose, and hexose), or
modified phosphate groups (e.g., phosphorothioates and 5'-N
phosphoramidite linkages). Enantiomers of natural or modified
nucleosides may also be used. Nucleic acids also include nucleic
acid-based therapeutic agents, for example, nucleic acid ligands,
siRNA, short hairpin RNA, antisense oligonucleotides, ribozymes,
aptamers, and SPIEGELMERr, oligonucleotide ligands described in
Wlotzka, et al., Proc. Natl. Acad. Sci. USA, 2002, 99(13):8898, the
entire contents of which are incorporated herein by reference.
[0036] "Polypeptide", "peptide", or "protein": According to the
present invention, a "polypeptide", "peptide", or "protein"
comprises a string of at least three amino acids linked together by
peptide bonds. The terms "polypeptide", "peptide", and "protein",
may be used interchangeably. Peptide may refer to an individual
peptide or a collection of peptides. Inventive peptides preferably
contain only natural amino acids, although non natural amino acids
(i.e., compounds that do not occur in nature but that can be
incorporated into a polypeptide chain) and/or amino acid analogs as
are known in the art may alternatively be employed. Also, one or
more of the amino acids in a peptide may be modified, for example,
by the addition of a chemical entity such as a carbohydrate group,
a phosphate group, a famesyl group, an isofamesyl group, a fatty
acid group, a linker for conjugation, functionalization, or other
modification, etc. In one embodiment, the modifications of the
peptide lead to a more stable peptide (e.g., greater half-life in
vivo). These modifications may include cyclization of the peptide,
the incorporation of D-amino acids, etc. None of the modifications
should subtantially interfere with the desired biological activity
of the peptide.
[0037] "Polysaccharide", "carbohydrate" or "oligosaccharide": The
terms "polysaccharide", "carbohydrate", or "oligosaccharide" refer
to a polymer of sugars. The terms "polysaccharide", "carbohydrate",
and "oligosaccharide", may be used interchangeably. Typically, a
polysaccharide comprises at least two sugars. The polymer may
include natural sugars (e.g., glucose, fructose, galactose,
mannose, arabinose, ribose, and xylose) and/or modified sugars
(e.g., 2''-fluororibose, 2'-deoxyribose, and hexose).
[0038] "Small molecule": As used herein, the term "small molecule"
is used to refer to molecules, whether naturally-occurring or
artificially created (e.g., via chemical synthesis) that have a
relatively low molecular weight. Typically, a small molecule is an
organic compound (i.e., it contains carbon). The small molecule may
contain multiple carbon-carbon bonds, stereocenters, and other
functional groups (e.g., amines, hydroxyl, carbonyls, heterocyclic
rings, etc.). In some embodiments, small molecules are monomeric
and have a molecular weight of less than about 1500 g/mol. In
certain embodiments, the molecular weight of the small molecule is
less than about 1000 g/mol or less than about 500 g/mol. Preferred
small molecules are biologically active in that they produce a
biological effect in animals, preferably mammals, more preferably
humans. Small molecules include, but are not limited to,
radionuclides and imaging agents. In certain embodiments, the small
molecule is a drug. Preferably, though not necessarily, the drug is
one that has already been deemed safe and effective for use in
humans or animals by the appropriate governmental agency or
regulatory body. For example, drugs approved for human use are
listed by the FDA under 21 C.F.R. .sctn..sctn.330.5, 331 through
361, and 440 through 460, incorporated herein by reference; drugs
for veterinary use are listed by the FDA under 21 C.F.R.
.sctn..sctn.500 through 589, incorporated herein by reference. All
listed drugs are considered acceptable for use in accordance with
the present invention.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0039] The present invention provides a system for screening a
library of particles for particles with a desired characteristic.
The invention provides both in vivo and in vitro systems for
screening libraries of particles. The various screening
technologies may be used in combination to select a particle or a
population of particles for a specific use. In certain embodiments,
the particles are screened for the ability to target a specific
cell, tissue, or organ. In other embodiments, the particles are
screened for their ability to be taken up by a cell. In yet other
embodiments, the particles are screened for their ability to
deliver an agent to a cell, tissue, or organ. In other embodiments,
the particles are screened for their ability to rescue a cell. In
one embodiment, the inventive screening system is used to select
microparticles (having a diameter between 1 and 1000 microns),
nanoparticles (having a diameter between 1 and 1000 nm), or
picoparticles (having a diameter between 1 and 1000 pm) with
characteristics suitable for delivering an agent to a cell, tissue,
or organ of interest. The technology is referred to as Polyplex
Iterative Combinatorial Optimization, or PICO. A library of
particles is screened by first introducing a population of
different into a biological system in vivo or in vitro. Those
particles that are found in the cells, tissue, or organ of interest
are identified and a second population of particles is prepared by
enriching the particles in characteristics identified in the found
particles. The process is repeated until the characteristics of the
particles are sufficient for the desired preferential partition of
the particles in the cells, tissue, or organ of interest. Different
characteristics of the particles may be selected for using
different or simultaneous screening series. In another embodiment,
the library is screened by incubation with cells in vitro. For
example, the particles may deliver a necessary nutrient or
biologically active molecule to the cells, which require the
molecule for survival and/or growth, and the particles having
characteristics that facilitate targeted delivery are identified by
characterizing the surviving cells, which took up the particles, or
the particles taken up by the cells.
High Throughput Development of Targeted Particles
[0040] PICO may be used to optimize the biophysical characteristics
of particles for use in the targeted delivery of pharmaceutically
active agents, contrast agents, or other medically useful
materials. Parameters for optimization may include but are not
limited to any of size, polymer composition, surface
hydrophilicity, surface charge, and the presence, composition and
density of targeting agents on the particle surface. A library of
particles in which these or other parameters are varied may be
produced using combinatorial techniques. Combinatorial techniques
may also be used to provide a unique label for each particle or
population of particles.
Composition
[0041] Particles for use with PICO may be fabricated from a variety
of organic and inorganic materials. In one embodiment, particles
are fabricated from biodegradable polymers. In certain embodiments,
the particles are fabricated from biocompatible polymers. A variety
of biodegradable and/or biocompatible polymers are well known to
those skilled in the art. Exemplary synthetic polymers suitable for
use with the invention include but are not limited to
poly(lactide), poly(glycolide), poly(lactic co-glycolic acid),
poly(arylates), poly(anhydrides), poly(hydroxy acids), polyesters,
poly(ortho esters), polycarbonates, poly(propylene fumerates),
poly(caprolactones), polyamides, polyphosphazenes, polyamino acids,
polyethers, polyacetals, polylactides, polyhydroxyalkanoates,
polyglycolides, polyketals, polyesteramides, poly(dioxanones),
polyhydroxybutyrates, polyhydroxyvalyrates, polycarbonates,
polyorthocarbonates, poly(vinyl pyrrolidone), biodegradable
polycyanoacrylates, polyalkylene oxalates, polyalkylene succinates,
poly(malic acid), poly(methyl vinyl ether), poly(ethylene imine),
poly(acrylic acid), poly(maleic anhydride), biodegradable
polyurethanes and polysaccharides. In certain embodiment, the
particles include polyethylene glycol (PEG). In certain
embodiments, the polymer used to make the particles is PEGylated
(i.e., conjugated to a polyethylene glycol moiety). U.S. patents
that describe the use of polyanhydrides for controlled delivery of
substances include U.S. Pat. No. 4,857,311 to Domb and Langer, U.S.
Pat. No. 4,888,176 to Langer, et al., and U.S. Pat. No. 4,789,724
to Domb and Langer; each of which is incorporated herein by
reference.
[0042] Naturally-occurring polymers, such as polysaccharides and
proteins, may also be employed. Exemplary polysaccharides include
alginate, starches, dextrans, celluloses, chitin, chitosan,
hyaluronic acid and its derivatives; exemplary proteins include
collagen, albumin, and gelatin. Polysaccharides such as starches,
dextrans, and celluloses may be unmodified or may be modified
physically or chemically to affect one or more of their properties
such as their characteristics in the hydrated state, their
solubility, or their half-life in vivo. In certain embodiments, the
particles do not include protein.
[0043] In other embodiments, the polymer includes polyhydroxy acids
such as polylactic acid (PLA), polyglycolic acid (PGA), their
copolymers poly(lactic-co-glycolic acid) (PLGA), and mixtures of
any of these. In certain embodiments, the particles include
poly(lactic-co-glycolic acid) (PLGA). In certain embodiments, the
particles include poly(lactic acid). In certain other embodiments,
the particles include poly(glycolic acid). These polymers are among
the synthetic polymers approved for human clinical use as surgical
suture materials and in controlled release devices. They are
degraded by hydrolysis to products that can be metabolized and
excreted. Furthermore, copolymerization of PLA and PGA offers the
advantage of a large spectrum of degradation rates from a few days
to several years by simply varying the copolymer ratio of glycolic
acid to lactic acid, which is more hydrophobic and less crystalline
than PGA and degrades at a slower rate.
[0044] Non-biodegradable polymers may also be used to produce
particles. Exemplary non-biodegradable, yet biocompatible polymers
include polystyrene, polyesters, non-biodegradable polyurethanes,
polyureas, polyvinyl alcohol), polyamides,
poly(tetrafluoroethylene), poly(ethylene vinyl acetate),
polypropylene, polyacrylate, non-biodegradable polycyanoacrylates,
non-biodegradable polyurethanes, polymethacrylate, poly(methyl
methacrylate), polyethylene, polypyrrole, polyanilines,
polythiophene, and poly(ethylene oxide).
[0045] Any of the above polymers may be functionalized with a
poly(alkylene glycol), for example, poly(ethylene glycol) (PEG) or
poly(propyleneglycol) (PPG), or any other hydrophilic polymer
system. Alternatively or in addition, they may have a particular
terminal functional group, e.g., poly(lactic acid) modified to have
a terminal carboxyl group so that a poly(alkylene glycol) or other
material may be attached. Exemplary PEG-functionalized polymers
include but are not limited to PEG-functionalized poly(lactic
acid), PEG-functionalized poly(lactic-co-glycolic acid),
PEG-functionalized poly(caprolactone), PEG-functionalized
poly(ortho esters), PEG-functionalized polylysine, and
PEG-functionalized poly(ethylene imine). When used in formulations
for oral delivery, poly(alkylene glycols) are known to increase the
bioavailability of many pharmacologically useful compounds, partly
by increasing the gastrointestinal stability of derivatized
compounds. For parenterally administered pharmacologically useful
compounds, including particle delivery systems, poly(alkylene
glycols) are known to increase stability, partly by decreasing
opsinization of these compounds, thereby reducing immunogenic
clearance, and partly by decreasing non-specific clearance of these
compounds by immune cells whose function is to remove foreign
material from the body. Poly(alkylene glycols) are chains may be as
short as a few hundred Daltons or have a molecular weight of
several thousand or more.
[0046] Co-polymers, mixtures, and adducts of any of the above
modified and unmodified polymers may also be employed. For example,
amphiphilic block co-polymers having hydrophobic regions and
anionic or otherwise hydrophilic regions may be employed. Block
co-polymers having regions that engage in different types of
non-covalent or covalent interactions may also be employed.
Alternatively or in addition, polymers may be chemically modified
to have particular functional groups. For example, polymers may be
functionalized with hydroxyl, amine, carboxy, maleimide, thiol,
N-hydroxy-succinimide (NHS) esters, or azide groups. These groups
may be used to render the polymer hydrophilic or to achieve
particular interactions with materials that are used to modify the
surface as described below.
[0047] One skilled in the art will recognize that the molecular
weight and the degree of cross-linking may be adjusted to control
the decomposition rate of the polymer. Methods of controlling
molecular weight and cross-linking to adjust release rates are well
known to those skilled in the art.
[0048] Methods of producing polymer particles include emulsions,
for example, water/oil/water emulsions, oil/water emulsions,
spray-drying, freeze-drying, and other methods known to those
skilled in the art. Some exemplary methods are disclosed by U.S.
Patent Publications Nos. 20020131951 by Langer, 20040070093 by
Mathiowitz, 20050123596 by Kahane, 20020119203 by Wright,
20020142093 by Gibson, 20030082236 by Mathiowitz, and 20040086459
by Ottoboni; each of which is incorporated herein by reference.
Exemplary fluorescent particles are disclosed in U.S. Patent
Publication No. 20040225037 by Lam, incorporated herein by
reference.
[0049] Particles may also be produced from non-polymer materials,
e.g., metals, ceramics, and semiconductors. For example, where it
is desired to provide a contrast or imaging agent to a particular
tissue, it may not be necessary to combine a particulate agent with
a polymer carrier. Rather, a particulate contrast or imaging agent
may be conjugated to the selected targeting agent. For example, the
particles may be semiconductor particles or quantum dots. Exemplary
semiconductor particle compositions include but are not limited to
CdS, CdTe, CdSe, InGaP, GaN, PbSe, PbS, InN, InP, and ZnS.
Semiconductor nanoparticles are available from Quantum Dot
Corporation, Evident Technologies, and other sources known to those
skilled in the art. Exemplary methods of producing semiconductor
particles are disclosed in U.S. Pat. Nos. 6,576,291, 6,207,229,
6,319,426, 6,322,901, 6,426,513, 6,607,829, 5,505,928, 5,537,000,
6,225,198, 6,306,736, 6,440,213, 6,743,406, and 6,649,138; each of
which is incorporated herein by reference. Ceramic particles may
also be prepared by any technique known to those skilled in the
art, for example, precipitation, or using the techniques described
in U.S. Patent Publications No. 20040180096 by Prasad, 20050063898
by Ja Chisholm, 20020110517 by James, or 20050045031 by
Rajagopalan, each of which is incorporated herein by reference, may
also be employed. Metal particles may also be employed. Exemplary
methods of producing metal particles are known to those skilled in
the art. Exemplary methods may be found in U.S. Patent Publications
Nos. 20050274225 by Bocarsly, 20050235776 by He, 20050218540 by
Sastry, 20040099093 by Harutyunan, 20040009118 by Phillips, and
20030115986 by Pozarnsky, each of which is incorporated herein by
reference. Metal particles that can be detected by surface-enhanced
Raman spectroscopy are disclosed in U.S. Patent Publication No.
20050272160 to Natan, incorporated herein by reference.
[0050] The surface chemistry of the particles may be varied using
any technique known to the skilled artisan. Both the surface
hydrophilicity and the surface charge may be modified. Some methods
for modifying the surface chemistry of polymer particles are
discussed above. Silane or thiol molecules may be employed to
tether particular functional groups to the surface of polymer or
non-polymer particles. For example, hydrophilic (e.g., thiol,
hydroxyl, or amine) or hydrophobic (e.g., perfluoro, alkyl,
cycloalkyl, aryl, cycloaryl) groups may be tethered to the surface.
Acidic or basic groups may be tethered to the surface of the
particles to modify their surface charge. Exemplary acidic groups
include carboxylic acids, nitrogen-based acids, phosphorus based
acids, and sulfur based acids. Exemplary basic groups include
amines and other nitrogen containing groups. The pKa of these
groups may be controlled by adjusting the environment of the acidic
or basic group, for example, by including electron donating or
electron withdrawing groups adjacent to the acidic or basic group,
or by including the acidic or basic group in a conjugated or
non-conjugated ring. Alternatively, particles may be oxidized, for
example, using peroxides, permanganates, oxidizing acids, plasma
etching, or other oxidizing agents, to increase the density of
hydroxyl and other oxygenated groups at their surfaces.
Alternatively or in addition, borohydrides, thiosulfates, or other
reducing agents may be used to decrease the hydrophilicity of the
surface.
[0051] Particles may be any size between about 1 nm and about 1000
.mu.m, for example about 1 and about 50 nm, between about 50 and
about 100 nm, between about 100 and about 500 nm, between about 500
and about 1000 nm, between about 1 .mu.m and about 10 .mu.m,
between about 10 .mu.m and about 100 .mu.m or between about 100
.mu.m and about 1000 .mu.m.
Targeting Agents
[0052] Targeting agents may be employed to more precisely direct
the particles to a tissue of interest. One skilled in the art will
recognize that the tissue of interest need not be healthy tissue
but may be a tumor or particular form, of damage or disease tissue,
such as areas of arteriosclerosis or unstable antheroma plaque in
the vasculature. Targeting agents may target any part or component
of a tissue. For example, targeting agents may exhibit an affinity
for an epitope or antigen on a tumor or other tissue cell, an
integrin or other cell-attachment agent, an enzyme receptor, an
extracellular matrix material, or a peptide sequence in a
particular tissue. Targeting agents may include but are not limited
to antibodies and antibody fragments, nucleic acid ligands (e.g.,
aptamers), oligonucleotides, oligopeptides, polysaccharides,
low-density lipoproteins (LDLs), folate, transferrin,
asialycoproteins, gp120 envelope protein of the human
immunodeficiency virus (111V), carbohydrates, polysaccharides,
enzymatic receptor ligands, sialic acid, glycoprotein, lipid, small
molecule, bioactive agent, biomolecule, immunoreactive fragments
such as the Fab, Fab', or F(ab').sub.2 fragments, etc. A variety of
targeting agents that direct pharmaceutical compositions to
particular cells are known in the art (see, for example, Cotton, et
al., Methods Enzym. 217:618; 1993; incorporated herein by
reference). Targeting agents may include any small molecule,
bioactive agent, or biomolecule, natural or synthetic, that binds
specifically to a cell surface receptor, protein or glycoprotein
found at the surface of cells. In one embodiment, the targeting
agent is an oligonucleotide sequence including 10.sup.10-10.sup.20
nucleotides. In certain embodiments, the aptamer includes 5-50
nucleotides, preferably 10-40 nucleotides. In another embodiment,
the targeting agent is a naturally occurring carbohydrate molecule
or one selected from a library of carbohydrates. Libraries of
peptides, carbohydrates, or polynucleotides for use as potential
targeting agents may be synthesized using techniques known to those
skilled in the art. Various macromolecule libraries may also be
purchased from companies such as Invitrogen and Cambridge
Peptide.
[0053] The targeting agent may be conjugated to the particle by
covalent interactions. For example, a polymeric particle may be
modified with a carboxylate group, following which an aminated
targeting agent, or one that is modified to be aminated, is coupled
to the polymer using a coupling reagent such as EDC or DCC.
Alternatively, polymers may be modified to have an activated NHS
ester which can then be reacted with an amine group on the
targeting agent. Other reactive groups that may be employed to
couple targeting agents to particles include but are not limited to
hydroxyl, amine, carboxyl, maleimide, thiol, NHS ester, azide, and
alkyne. Standard coupling reactions may then be used to couple the
modified material to a second material having a complementary group
(e.g., a carboxyl modified targeting agent coupled to an aminated
polymer). Particles fabricated from inorganic materials may be
modified to carry any of these groups using self-assembled
monolayer forming materials to tether the desired functional group
to the surface.
[0054] Alternatively, the targeting agents can be attached to the
particles directly or indirectly via non-covalent interactions.
Non-covalent interactions include but are not limited to the
following:
[0055] 1) Electrostatic Interactions: For example, the particle may
have a cationic surface or may be reacted with a cationic polymer,
such as poly(lysine) or poly(ethylene imine), to provide a cationic
surface. The particle surface can then bind via charge interactions
with a negatively charged targeting agent. One end of the targeting
agent may be attached to a negatively charged polymer (e.g., a
poly(carboxylic acid)) or other negatively charged material or
molecule that can interact with the cationic polymer surface
without disrupting the binding affinity of the targeting agent.
[0056] 2) Affinity Interactions: For example, biotin may be
attached to the surface of the particle and streptavidin may be
attached to the targeting agent, or vice versa. The biotin group
and streptavidin may be attached to the particle or to the
targeting agent via a linker, such as an alkylene linker or a
polyether linker. Biotin and streptavidin bind via affinity
interactions, thereby retaining the targeting agent on the
particle.
[0057] 3) Metal Coordination: For example, a polyhistidine may be
attached to the targeting agent material, and a nitrilotriacetic
acid can be attached to the surface of the particle, or vice versa.
A metal, such as Ni.sup.+2, will chelate the polyhistidine and the
nitrilotriacetic acid, thereby binding the targeting agent to the
particle.
[0058] 4) Physical Adsorption: For example, a hydrophobic tail,
such as polymethacrylate or an alkyl group having at least about 10
carbons, may be attached to the targeting agent. The hydrophobic
tail will adsorb onto the surface of a hydrophobic particle or a
hydrophobic coating on a particle, for example, a polyorthoester,
polysebacic anhydride, unmodified poly(lactic acid), or
polycaprolactone.
[0059] 5) Host-Guest Interactions: For example, a macrocyclic host,
such as cucurbituril or cyclodextrin, may be attached to the
particle or the targeting agent, and a guest group, such as an
alkyl group, a polyethylene glycol, or a diaminoalkyl group, may be
attached to the other. In one embodiment, the host and/or the guest
molecule may be attached to the particle or the targeting agent via
a linker, such as an alkylene linker or a polyether linker. Where
the particle is fabricated from a polymeric material, the host or
guest group may be incorporated into the polymer.
[0060] 6) Hydrogen Bonding Interactions: For example, an
oligonucleotide having a particular sequence may be attached to the
surface of the particle, and an essentially complementary sequence
may be attached to the targeting agent. The targeting agent will
then bind to the particle core via complementary base pairing with
the oligonucleotide attached to the particle. Two oligonucleotides
are essentially complimentary if about 80% of the nucleic acid
bases on one oligonucleotide form hydrogen bonds via an
oligonucleotide base pairing system, such as Watson-Crick base
pairing, reverse Watson-Crick base pairing, Hoogsten base pairing,
etc., with nucleic acid bases on the second oligonucleotide. In
some embodiments, it is desirable for an oligonucleotide sequence
attached to the particle to form at least about 6 complementary
base pairs with a complementary oligonucleotide attached to the
targeting agent. For example, a poly(cytosine) tag may be attached
to the particle and a poly(guanine) tag may be attached to the
targeting agent. Where the particle is fabricated form a polymer,
the entire polymer may be so modified. Some of the poly-C tags will
end up on the surface of the particle, and others will remain in
the interior portions of the particle. In another embodiment,
polysaccharides may be used as a targeting agent. The hydroxyl
groups on sugar residues such as glucose and galactose will
hydrogen bond with polar moieties on polymers such as poly(vinyl
alcohol).
Labels
[0061] In one embodiment, each particle in the library has a unique
analytical signature, e.g., a molecular bar code defined by an
oligonucleotide, provided by one or more labels. The label may
include a pattern of luminescence or radioactive emission, a small
molecule, a polynucleotide, a polypeptide, or some combination of
these. In one embodiment, the label is a short oligonucleotide,
e.g., 10-100 bases, that may be incorporated inside or on the
surface of the particle. Quantum dots may also be exploited as
labels. For example, the techniques described in U.S. Pat. No.
6,602,671, which is incorporated herein by reference, for using
quantum dots for inventory control may be employed. Of course, a
quantum dot or other semiconductor particle may serve as its own
label. Radioisotopes may also be employed. Exemplary radionuclides
may include gamma-emitters, positron-emitters, X-ray emitters, beta
emitters, and alpha-emitters and include but are not limited to
.sup.123I, .sup.125I, .sup.130I, .sup.131I, .sup.133I, .sup.135I,
.sup.47Sc, .sup.72As, .sup.72Se, .sup.90Y, .sup.88Y, .sup.97Ru,
.sup.100Pd, .sup.101mRh, .sup.119Sb, .sup.128Ba, .sup.197Hg,
.sup.211At, .sup.212Bi, .sup.212Pb, .sup.109Pd, .sup.111In,
.sup.67Ga, .sup.68Ga, .sup.67Cu, .sup.75Br, .sup.77Br, .sup.99mTc,
.sup.14C, .sup.13N, .sup.15O, .sup.32P, .sup.33P, and .sup.15F. A
pattern of luminescence may include one or more of a wavelength, an
emission time, and an emission polarization, for example, as
discussed in U.S. Pat. No. 6,696,299, incorporated herein by
reference. The emission time of various luminescent moieties varies
with their decay or relaxation mechanism; indirect decay mechanisms
may result in extended phosphorescence of the moiety. Luminescent
agents may include materials commonly used as clearing agents, such
as gadolinium and europium chelates with DTPA, DTPA-BMA, DOTA and
HP-DO3A, which are reviewed in Aime, et al., Chemical Society
Reviews (1998), 27:19-29. These materials and iron oxide particles,
among others, are also used as contrast agents for MRI, which may
be used as labels for the particles in certain embodiments. Other
ceramic or metal agents that can be used to enhance contrast in
x-ray, ultrasound, MRI, or other diagnostic techniques may also be
employed
[0062] The signature may include one or more of these labels.
Indeed, different kinds of labels (e.g., luminescent and
oligonucleotide labels) may be combined to increase the number of
unique signatures, or different types of the same labels (e.g., two
luminescent labels with different excitation or emission
wavelengths or emission times) may be combined.
[0063] Labels may be combined with particles according to any
method known to those skilled in the art. In one embodiment,
labeling agents are conjugated to the particles in the same manner
as described for the conjugation of targeting agents, using silane
or thiol tethers, or using other techniques known to those of skill
in the art. Reactive chemical groups at the surface of polymer
particles may be used to covalently link labels to the surface, and
any of the non-covalent interactions described above may be
employed as well. Alternatively or in addition, combinatorial
methods may be employed. Exemplary combinatorial methods for
conjugating materials to microparticles are disclosed in U.S.
Provisional Applications Nos. 60/750,711 and 60/652,881, the
contents of which are incorporated herein by reference.
[0064] Alternatively or in addition, contrast or imaging agents may
be encapsulated in polymer particles. A variety of methods of
making particles in which active agents are encapsulated are well
known to those skilled in the art. For example, a double emulsion
technique may be used to combine a polymer and label in particles.
Alternatively, particles may be prepared by spray-drying. For
example, gadolinium or europium complexes such as those described
above or diagnostic contrast agents may be encapsulated in polymer
particles.
[0065] Any analytical technique known to those skilled in the art
may be employed to identify the signatures of recovered particles.
Where an oligonucleotide is used, quantitative PCR may be employed
to determine the amount of each nucleotide present. High throughput
multi-plex ELISA systems such as the Bioplex (Bio-Rad, U.S.A.) may
also be used to quantitively determine the molecular signature
concentrations. Where luminescence or a radioactive emission is
used as the label, Fourier transform techniques may be used to
identify the various emitters present in a particular sample.
Depending on the number of labels, it may be desirable to use
several analytical techniques to completely identify all the
particles. For example, it is not necessary that all the labels be
detectable using one technique, e.g., luminescence or quantitative
PCR. Alternatively or in addition, particles may encapsulate one or
more labels, and the label may be released from the particle either
during screening or after recovery of the particle from a sample
and identified separately from the particle.
High-Throughput Optimization of Particles
[0066] A library of particles in which specified parameters are
varied may be produced using combinatorial chemistry techniques.
The combinatorial techniques are also used to provide a unique
label for each particle. One or more parameters such as
composition, size, surface chemistry (including surface charge and
hydrophilicity), the presence of a targeting agent, a density of
one or more targeting agents, and the identity of one ore more
targeting agents, may be varied. The particles may be screened
using either in vivo or in vitro techniques.
[0067] In some embodiments, PICO is performed several times to
optimize various properties of the particles. For example,
substantially identical particles may be used to screen targeting
agents. The particle-targeting agent conjugates are administered to
an animal or a population of cells. The conjugates recovered from
the tissue of interest, or the living cells are enriched. The
process is repeated with the enriched conjugates. After several
repetitions, e.g., 2-20 iterative rounds, this process provides a
population of particle-targeting agent conjugates that are
selective for a particular tissue or that are taken up by cells
without actually purifying targets or ligands for the tissue in
advance. Optimal selection is marked by a plateau in enrichment.
The population may include more than one targeting agent. As used
herein, the term "selective targeting agents" refers to the
targeting agent or agents identified by this embodiment of the PICO
process. Other characteristics of the particles, e.g., a density of
selective targeting agents on the surface, composition, size,
and/or surface chemistry, may then be optimized by further rounds
of optimization using the PICO method.
In Vivo Screening
[0068] For in vivo screening, the library of particles is
administered into a biological system, e.g., dog, rodent, mouse,
human, or other animal model. Any class of animal may be used. In
one embodiment, the animal is a mammal. In some embodiments, the
animal model may be engineered or treated to produce a tumor or
other defect. In addition, the choice of animal may be dictated by
a variety of factors, such as cost and the suitability of the
animal as a model for a particular tumor, disease, or tissue. The
composition may be administered by intravenous injection.
Alternatively, the composition may be administered by other routes,
e.g., intra-arterial, inhalational, intradermal, subcutaneous,
oral, nasal, bronchial, ophthalmic, transdermal (topical),
transmucosal, peritoneal, rectal, and vaginal routes. In some
embodiments, the particles are not only optimized to reach a
particular tissue site but for a particular delivery route.
[0069] After a defined period of time, the tissue of interest is
excised and the particles that are present are identified and/or
enriched. In one embodiment, the particles are dissolved to release
an encapsulated label, which is used to identify the particles that
were present in the tissue. The amount of each particle may also be
quantitated. Alternatively or in addition, samples from
non-targeted organs (e.g., liver, spleen, lung, bone marrow,
lymphatic system) are collected, and the particles are identified.
Those particles with undesirable biophysiochemical properties, such
as non-specific tissue targeting, may be identified and eliminated
from subsequent rounds of enrichment.
[0070] Those formulations that demonstrate effective targeting of
the desired organ (e.g., a high level of signal from particles
recovered from a tumor) while optionally demonstrating a low level
of uptake by non-targeted organs may be enriched. Where the only
variable among the particles is the identity of a polynucleotide
targeting agent (that is, the targeting agent also serves as the
label), the particles may be enriched using PCR without first
identifying the particles, or quantitative PCR may be used to
identify the particles. The screening may be repeated several
times, for example, to improve the resolution of the assay. In
addition, the strength of the screen may be modified by requiring
higher or lower levels of signal from a particular label in order
to select the corresponding particle for enrichment.
In Vitro Screening
[0071] In in vitro methods, the population of particles is
administered to a stable cell line in vitro. For example, the cell
line may be stable except for requiring an external nutrient or
other material, for example, an antibiotic. The particles may
encapsulate the required material, and those cells that are able to
take up the particles will survive. As for the in vivo methods,
those particles that were taken up may be enriched and the
procedure repeated. In some embodiments, it may be desirable to
perform a negative selection by removing the living cells and
assaying the particles in the vicinity of the dead cells. This may
identify those particles that have a low rate of cellular
uptake.
[0072] In some embodiments, a single particle type is produced with
a variety of targeting agents. A single particle may be produced
with a combination of several targeting agents to screen the
targeting agent combinations. In these embodiments, it is not
necessary to identify the targeting agents between rounds of
screening. Rather, those particles that were prefeientially taken
up by the cells may simply be amplified. After screening is
complete, the targeting agents may be identified by PCR or other
suitable techniques. Thus, the targeting agents serve as the label.
In some embodiments, particles are produced with common targeting
agents, but the properties of the particles themselves (e.g.,
composition, surface chemistry and charge, etc,) are varied. The
surviving cells may be assayed for the particles' label to identify
the particular particle composition and surface characteristics.
For example, where an oligonucleotide "molecular bar code" is
employed, PCR or some other nucleic acid assay may be used to
identify the label. This allows the particle characteristics to be
optimized for preferential targeting. By employing both these
techniques, both the biophysiochemical characteristics of the
particles and the particular targeting agents employed with the
particles may be optimized to maximize preferential binding and
reduce non-specific uptake of the particles.
[0073] These and other aspects of the present invention will be
further appreciated upon consideration of the following Example,
which is intended to illustrate certain particular embodiments of
the invention but are not intended to limit its scope, as defined
by the claims.
Example
"DNA Barcodes" in High Throughput Screening of Nanoparticles for
Desirable Characteristics
[0074] A procedure was developed to screen different nanoparticle
formulations for desirable characteristics in parallel using
cultured cells in vitro. This procedure is based on tagging
distinct nanoparticle formulations with unique segments of DNA
(i.e., "DNA barcodes"), thereby allowing one to quantitatively
trace the amount of each particle type present in a cell or tissue,
for example. Nanoparticles were incubated at chosen conditions,
purified, dissolved by incubation in a mildly basic (0.01 N NaOH)
solution (12 hr, room temperature), and assayed directly for DNA
content by multiplex assay using Luminex beads (published U.S.
Patent Application 2006/0177850, published Aug. 10, 2006; which is
incorporated herein by reference) conjugated with the appropriate
complementary DNA sequences, all using a Bioplex platform
(published U.S. Patent Application 2005/0123455, published Jun. 9,
2005; which is incorporated herein by reference). This system
allows for scale-up of nanoparticle optimization for high
throughput screening protocols.
[0075] We have previously developed methods of creating complex
libraries of particle formulations by varying the identity of the
polymers used to create them, starting from
poly(D.L-lactic-co-glycolic acid) (PLGA), poly(D,L-lactic acid),
and poly(ethylene glycol) (PEG) precursors (FIG. 1) together with
cell or tissue targeting agents, including, but not limited to
antibodies, aptamers, antibody fragments, peptides, carbohydrates,
vitamins, small molecules, or magnetic materials. See U.S.
Provisional patent application, U.S. Ser. No. 60/747,240, filed May
15, 2006; incorporated herein by reference. As part of a
proof-of-concept experiment, we have chosen to screen four distinct
formulations of particles, each encapsulating trace amounts of
biotinylated DNA oligonucleotides (8.37, 6.76, 4.49, 6.10 parts per
million (wt/wt)) which were unique for each particle type. First,
to establish the validity of the method, we demonstrated that the
relative ratios of particles to one another in a sample could be
calculated by quantifying the relative ratios of each type of DNA
present (FIGS. 2-4). Nanoparticles were added to wells not
containing any cells, dissolved in 0.01 N NaOH solution, and then
assayed for the resulting free DNA content. Ratios were arbitrarily
normalized to the quantity of the lowest nanoparticle added (based
on the expected ratios), for ease of interpretation. By simple
inspection, one can see that the observed and expected ratios are
generally in agreement.
[0076] The method was then applied to screen the four nanoparticle
preparations (diameters: 238.7.+-.8.6 nm, 291.3.+-.8.3 nm,
230.3.+-.8.3 nm, 278.2.+-.1.1 nm, zeta potentials: -4.10.+-.1.47
mV, -2.95.+-.0.52 mV, 1.17.+-.5.06 mV, -2.63.+-.1.54 mV, from left
to right in FIGS. 2-4, respectively) for their ability to penetrate
human prostate cancer (LNCaP) cells by incubating them together
(37.degree. C., 5% CO.sub.2, 1 total NP concentration, 6 well plate
format, 20 hr) in two formats: (1) each particle formulation was
incubated with cells individually (i.e., one nanoparticle
formulation per well in a 6 well plate of cells); (2) a mixture of
the four distinct particle types was incubated with the cells
together (i.e., four nanoparticle formulations per well in a 6 well
plate of cells). After incubation, cells were washed twice with
isotonic PBS, treated with trypsin, collected, centrifuged,
resuspended in PBS, and then lysed by several rounds of flash
freezing in liquid nitrogen and thawing in lukewarm water.
Nanoparticles released from the intracellular milieu were dissolved
in 0.01 N NaOH, and DNA content was assayed directly. The results
show that the two methods are comparable (Figure S), suggesting
that screening particles as a library in parallel is a viable
method to greatly reduce the number of individual experiments that
are required for optimization of particles (at least by the size of
the library, which in the present embodiment is by a factor of
four). By decreasing the number of individual experiments that must
be run, the present invention facilitates the high throughput
optimization of particles with the desired characteristics.
EQUIVALENTS AND SCOPE
[0077] The foregoing has been a description of certain non-limiting
preferred embodiments of the invention. Those skilled in the art
will recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the invention described herein. Those of ordinary skill in the art
will appreciate that various changes and modifications to this
description may be made without departing from the spirit or scope
of the present invention, as defined in the following claims.
[0078] In the claims articles such as "a,", "an" and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a grdup are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention also includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process. Furthermore, it is to be understood that the invention
encompasses all variations, combinations, and permutations in which
one or more limitations, elements, clauses, descriptive terms,
etc., from one or more of the claims or from relevant portions of
the description is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Furthermore, where the claims recite a
composition, it is to be understood that methods of using the
composition for any of the purposes disclosed herein are included,
and methods of making the composition according to any of the
methods of making disclosed herein or other methods known in the
art are included, unless otherwise indicated or unless it would be
evident to one of ordinary skill in the art that a contradiction or
inconsistency would arise. For example, it is to be understood that
any of the compositions of the invention can be used for inhibiting
the formation, progression, and/or recurrence of adhesions at any
of the locations, and/or due to any of the causes discussed herein
or known in the art. It is also to be understood that any of the
compositions made according to the methods for preparing
compositions disclosed herein can be used for inhibiting the
formation, progression, and/or recurrence of adhesions at any of
the locations, and/or due to any of the causes discussed herein or
known in the art. In addition, the invention encompasses
compositions made according to any of the methods for preparing
compositions disclosed herein.
[0079] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that each subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It is also noted that the term "comprising" is intended
to be open and permits the inclusion of additional elements or
steps. It should be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, steps, etc., certain
embodiments of the invention or aspects of the invention consist,
or consist essentially of, such elements, features, steps, etc. For
purposes of simplicity those embodiments have not been specifically
set forth in haec verba herein. Thus for each embodiment of the
invention that comprises one or more elements, features, steps,
etc., the invention also provides embodiments that consist or
consist essentially of those elements, features, steps, etc.
[0080] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and/or the understanding of one of
ordinary skill in the art, values that are expressed as ranges can
assume any specific value within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates otherwise.
It is also to be understood that unless otherwise indicated or
otherwise evident from the context and/or the understanding of one
of ordinary skill in the art, values expressed as ranges can assume
any subrange within the given range, wherein the endpoints of the
subrange are expressed to the same degree of accuracy as the tenth
of the unit of the lower limit of the range.
[0081] In addition, it is to be understood that any particular
embodiment of the present invention may be explicitly excluded from
any one or more of the claims. Any embodiment, element, feature,
application, or aspect of the compositions and/or methods of the
invention (e.g., any derivative of zolpidem, any release-retarding
ingredient, any buffering agent, any carbohydrate, any fatty acid,
any formulation of zolpidem, any dissolution characteristic, any
method of producing a formulation, any dosage regimen, any route or
location of administration, any method of use, any purpose for
which a composition is administered, etc.), can be excluded from
any one or more claims. For example, in certain embodiments of the
invention the biologically active agent is not an
anti-proliferative agent. For purposes of brevity, all of the
embodiments in which one or more elements, features, purposes, or
aspects is excluded are not set forth explicitly herein.
* * * * *