U.S. patent application number 17/165159 was filed with the patent office on 2021-07-15 for compositions and methods for inducing scarring by peri-tumoral cells.
The applicant listed for this patent is Duke University. Invention is credited to Ravi Bellamkonda, Tarun Saxena.
Application Number | 20210213141 17/165159 |
Document ID | / |
Family ID | 1000005482248 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210213141 |
Kind Code |
A1 |
Saxena; Tarun ; et
al. |
July 15, 2021 |
COMPOSITIONS AND METHODS FOR INDUCING SCARRING BY PERI-TUMORAL
CELLS
Abstract
Compositions are provided, the compositions comprising: (1) a
nanoparticle; (2) optionally, a linker and/or masking agent; and
(3) a ligand configured to activate peri-tumoral cells to induce
scarring by the peri-tumoral cells. In some aspects, administration
of the compositions to a subject may generate an environment
capable of walling-off and containing invasive tumors.
Inventors: |
Saxena; Tarun; (Durham,
NC) ; Bellamkonda; Ravi; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
1000005482248 |
Appl. No.: |
17/165159 |
Filed: |
February 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16696960 |
Nov 26, 2019 |
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17165159 |
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62771309 |
Nov 26, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 5/00 20130101; A61K
47/65 20170801; C07K 14/705 20130101; A61K 47/6929 20170801 |
International
Class: |
A61K 47/69 20060101
A61K047/69; C07K 14/705 20060101 C07K014/705; A61K 47/65 20060101
A61K047/65 |
Claims
1-12. (canceled)
13. A method for activating peri-tumoral cells to induce scarring
by the peri-tumoral cells, the method comprising administering an
effective amount of a composition to a subject in need thereof,
wherein the composition comprises a conjugate, the conjugate
comprising: (1) a nanoparticle; (2) optionally, a PEG, PEG
derivative, or hydrophilic polycarbon or polymer-based
polycarbonate-based linker; and (3) a ligand, wherein the ligand is
configured to target at least one of the following entities in a
peri-tumoral cell: (i) a TLR2 receptor; (ii) a TLR4 receptor; (iii)
a CSF-1 receptor; (iv) an IFN-gamma receptor 1; (v) an IFN-gmma
receptor 2; (vi) a xylosyltransferase; and (vii) a TNF-.alpha.
receptor, and thereby induce scarring by the peri-tumoral cell.
14. The method of claim 13, wherein the conjugate has an average
diameter sufficient to demonstrate an EPR effect.
15. The method of claim 13, wherein the nanoparticle comprises a
gold nanoparticle.
16. The method of claim 13, wherein the conjugate has an average
diameter of about 50 nm to about 200 nm.
17. The method of claim 13, wherein the ligand comprises one or
more of peptidoglycan, LPS, zymosan, Pam3CSK4, amyloid-beta
peptide, lipoteichoic acid, HMGB1, heat shock proteins, CSF-1R
inhibitors, LPS+M IFN-gamma, xyloside, IFN-gamma, TNF-alpha, IL-2,
lipocalin 2, and miRNA-155, a combination thereof, or a peptide
mixture extracted or derived from any one or combination of
them.
18. The method of claim 13, wherein the nanoparticle comprises a
gold nanoparticle and the ligand comprises a peptide mixture
extracted from zymosan.
19. The method of claim 19, wherein the entity is a TLR2 receptor
in a peri-tumoral stromal cell adjacent a GBM tumor.
20. The method of claim 19, wherein the administration stimulates
stromal CSPG expression.
21. The method of claim 18, wherein the zymosan extract comprises a
water-soluble mixture comprised of zymosan polypeptides.
22. The method of claim 21, wherein the water-soluble mixture
comprised of zymosan polypeptides is formed by a process
comprising: snap freezing zymosan; crushing the snap frozen
zymosan; mixing the crushed zymosan and an extraction buffer to
form an extraction mixture; centrifuging the extraction mixture and
collecting the extraction supernatant; applying the extraction
supernatant to a 10 kDa centrifugal spin column the to obtain a
water-soluble mixture comprised of zymosan polypeptides.
Description
RELATED U.S. APPLICATION DATA
[0001] This application is a divisional of U.S. application Ser.
No. 16/696,966, filed Nov. 26, 2019, which claims priority to U.S.
Provisional Patent Application No. 62/771,309, filed on Nov. 26,
2018, the entire contents of which are incorporated herein by
reference.
BACKGROUND
[0002] Tumor treatment and metastasis prevention remain a
challenge. This is particularly true where the tumors are difficult
to access by conventional means. As an example, primary brain
tumors are unique in that they rarely metastasize outside the
brain. Nevertheless, brain tumors are characterized by high
morbidity and mortality partly due to their localization and often
locally invasive growth. Gliomas, tumors arising from glial cells,
account for almost 30% of all primary brain tumors, and 80% of all
malignant ones, and are responsible for the majority of deaths from
primary brain tumors. WHO grade IV gliomas--glioblastoma multiforme
(GBM)--are the most malignant and frequently occurring gliomas. For
patients with newly diagnosed GBM, the therapeutic regimen is
maximally-safe and feasible resection of the tumor mass, followed
by concomitant and adjuvant temozolomide (TMZ) plus radiotherapy,
followed by TMZ alone. Despite these measures, survival rates of
GBM patients have remained dismal, with little improvement over the
last 50 years. An important reason for this is that GBMs are highly
invasive and can invade deep and into eloquent regions of the
brain, making resections highly risky. Accordingly, there is a need
for improved methods for treating brain tumors, and specifically
for treating gliomas.
[0003] A critical determinant of brain tumor invasion is its
extracellular matrix (ECM). In general, the ECM is a key component
in the pathophysiology of nervous system injury. The ECM of the
glial scar formed after traumatic brain or spinal cord injury is
inhibitory to axonal regeneration. Glial scar ECM is rich in
chondroitin sulfate proteoglycans (CSPGs), a diverse family of
covalently linked protein-chondroitin sulfate (CS)
glycosaminoglycan (GAG) polysaccharide complexes. The
growth-promoting or inhibitory/repulsive effects of CSPGs are
exerted predominantly by the various CS-GAGs that form the CSPGs.
CSPG-rich glial scarring around sites of traumatic brain or spinal
cord injury provides a critical barrier, quelling inflammation and
preventing wider spread of tissue damage by "walling-off" the
injury site.
[0004] With this in mind, what is needed are compositions and
methods for targeting and activating peri-tumoral cells via a
stimulus that recapitulates the sequelae of a traumatic CNS
(central nervous system) injury, to generate an environment capable
of walling-off and containing invasive tumors growth or spread by
the inducement of scarring by the peritumoral cells.
SUMMARY
[0005] This Summary is provided to introduce a selection of
concepts that are further described below in the Detailed
Description. This Summary is not intended to identify key or
essential features of the claimed subject matter; nor is this
Summary intended to be used as an aid in limiting the scope of the
claimed subject matter.
[0006] The present technology is directed to compositions and
methods for inducing scarring of peri-tumoral cells to slow the
growth of, contain, and/or substantially prevent the spread of
cancerous cells from the treatment locus. Several aspects of the
technology are directed to compositions and methods for inducing
scarring of peri-tumoral cells by targeting and activating
peri-tumoral cells with a stimulus that recapitulates the sequelae
of a traumatic CNS injury. Such activation of the peri-tumoral
cells creates a barrier or wall surrounding the existing tumor that
is at least partically impermeable to tumor cells, thereby impeding
invasive tumor growth or spread.
[0007] In one aspect, the present technology is directed to a
composition compromising: (1) a nanoparticle; (2) optionally, a
linker and/or a masking agent; and (3) a ligand configured to
activate peri-tumoral cells to induce scarring by the peri-tumoral
cells.
[0008] In one aspect, the present technology is directed to a
composition comprising a conjugate, an encapsulate, or both. The
conjugate, encapsulate, or both may compromise: (1) a nanoparticle;
(2) optionally, a linker and/or a masking agent configured to
enhance circulation time and prevent clearance of the nanoparticle
from the bloodstream; and (3) a ligand, wherein the ligand is
configured to target at least one of the following entities in a
peri-tumoral cell: (i) toll-like receptor (TLR) 2; (ii) TLR4
receptor; (iii) CSF-1 receptor; (iv) IFN-gamma receptor 1; (v)
IFN-gamma receptor 2; (vi) xylosyltransferase; (vii) tumor necrosis
factor alpha (TNF-.alpha.) receptor; and (viii) IL-2 receptor. In
some of the foregoing aspects, the linker and/or masking agent may
comprise polyethlene glycol (PEG), a PEG derivative, or a
hydrophilic polycarbonate. Additionally or alternatively, in some
of the foregoing aspects the conjugate, encapsulate, or both have
an average diameter of about 50 nm to about 200 nm.
[0009] In one aspect, a composition is provided, the composition
comprising: (1) a nanoparticle; and (2) a ligand, wherein the
ligand is configured to target at least one of the following
entities in peri-tumoral cells: (i) a TLR2 receptor; (ii) a TLR4
receptor; (iii) a CSF-1 receptor; (iv) an IFN-gamma receptor 1; (v)
an IFN-gamma receptor 2; (vi) xylosyltransferase; (vii) aTNF-alpha
receptor; and/or (viii) an IL-2 receptor, and wherein the
nanoparticle at least one of: (a) forms a conjugate with the
ligand; and (b) encapsulates the ligand, and wherein the conjugate
and/or encapsulate may have an average diameter sufficient to
demonstrate an enhanced permeability and retention (EPR) effect and
localize to peritumoral spaces when introduced intravenously. In
one aspect, the composition further comprises PEG, a PEG
derivative, a hydrophilic polycarbonate, or a derivative
thereof.
[0010] According to some aspects of the technology, including any
of the foregoing aspects, the nanoparticle may comprise a gold
nanoparticle having an average diameter between about 5 nm-200 nm.
In some aspects, the nanoparticle is a liposome having an average
diameter between about 50 nm-200 nm.
[0011] According to some aspects of the technology, including any
of the foregoing aspects, the ligand may comprise one or more of
peptidoglycan, lipopolysaccharide (LPS), zymosan, Pam3CSK4,
amyloid-beta peptide, lipoteichoic acid, high mobility group box 1
(HMGB1), heat shock proteins, CSF-1R inhibitors, LPS+IFN-gamma,
xyloside, IFN-gamma, TNF-alpha, IL-2, lipocalin 2, and miRNA-155, a
combination thereof, or a peptide or other mixture extracted or
derived from any one or a combination of them. In one aspect, the
ligand is a zymosan extract, referred to herein as ""Zpep.""
[0012] In one aspect, a composition comprising a conjugate is
provided, the conjugate comprising: (1) a gold nanoparticle; (2) a
linker and/or masking agent and (3) Zpep.
[0013] In another aspect, methods are provided for the preparation
of the conjugates and encapsulates provided herein.
[0014] According to some aspects of the present technology, a
method is provided for activating peri-tumoral cells to induce
scarring. In one aspect, this activation may be defined by a
process such as the production of ECM molecules that are inhibitory
to neural and glial migration, by the peri-tumoral cells. In one
aspect, the method comprises administering a nanoparticle
composition to target cells in close proximity to tumors in order
to activate the cells and stimulate scarring. In one aspect, the
method comprises administering gold nanoparticles coated with
polypeptides to target stromal cells in close proximity to GBM
tumors in order to activate the cells and stimulate stromal
chondroitin sulfate proteoglycans (CSPG) expression.
[0015] Several aspects of the present technology include methods
for slowing or substantially preventing tumor growth or spread of a
tumor by inducing scarring of peri-tumoral tissue. In some aspects,
inducing scarring includes administering a composition to a patient
that targets cells in close proximity to tumors in order to
activate the cells and stimulate scarring. In some aspects, the
compositions include one or more nanoparticles and one or more
ligands carried by the nanoparticles that are configured to
activate peri-tumoral cells and induce scarring thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0016] The description and claims may be more readily understood by
reference to the following figures.
[0017] FIG. 1 ((a)-(e)) shows the effects of CSPGs on repelling
tumor cells in an established in vitro model of glial scarring.
[0018] FIG. 2 ((a)-(f)) shows the effects of Zpep on glial cell
activation.
[0019] FIG. 3 ((a)-(c)) shows the effects of using a conjugate
comprising gold nanoparticles, PEG, and Zpep (the conjugate is
referred to herein as ""AuNP-Z"") to stimulate peri-tumoral CSPG
expression in vivo.
[0020] FIG. 4 ((a)-(b)) shows the effects of using AuNP-Z to
constrain tumors in vivo.
[0021] FIG. 5 ((a)-(c)) shows the effects of using AuNP-Z to
curtail tumor growth.
[0022] FIG. 6 ((a)-(c)) shows the results of a proteomic analysis
of in vivo response to AuNP-Z.
[0023] FIG. 7 shows the results of a gene set enrichment analysis
of AuNP-Z-treated animals relative to controls.
[0024] FIG. 8 shows the results of an overrepresentation analysis
of AuNP-Z-treated animals relative to controls.
[0025] FIG. 9 depicts astrocyte activation in AuNP, AuNP-PEG, and
AuNP-Z-treated animals.
DETAILED DESCRIPTION
[0026] The present technology is directed to compositions and
methods for inducing scarring of peri-tumoral cells to slow the
growth of, contain, and/or substantially prevent the spread of
cancerous cells from the treatment locus. Several aspects of the
technology are directed to compositions and methods for inducing
scarring of peri-tumoral cells by targeting and activating
peri-tumoral cells with a stimulus that recapitulates the sequelae
of a traumatic CNS injury. Such activation of the peri-tumoral
cells creates a local barrier or wall around the existing tumor
that is impermeable to tumor cells, thereby at least partially
restraining invasive tumor growth or spread.
[0027] Several aspects of the technology include a composition that
compromises (1) a nanoparticle; (2) optionally, a linker and/or a
masking agent; and (3) a ligand configured to activate peri-tumoral
cells to induce scarring by the peri-tumoral cells. In some
aspects, the composition does not include the linker and/or masking
agent and only comprises the nanoparticle and the ligand.
[0028] In some aspects, the composition may be configured to
activate production of chondroitin sulfate proteoglycans (CSPGs),
which is a diverse family of covalently linked protein-chondroitin
sulfate (CS) glycosaminoglycan (GAG) polysaccharide complexes.
Activating production of CSPG may be especially beneficial for
slowing or preventing the spread of brain cancer (such as GBM). A
critical determinant of brain tumor invasion is its extracellular
matrix (ECM). In general, the ECM is a key component in the
pathophysiology of nervous system injury. The ECM of the glial scar
formed after traumatic brain or spinal cord injury is inhibitory to
axonal regeneration. The growth-promoting or inhibitory/repulsive
effects of CSPGs are exerted predominantly by the various CS-GAGs
that form the CSPGs. CSPG-rich glial scarring around sites of
traumatic brain or spinal cord injury provides a critical barrier,
quelling inflammation and preventing wider spread of tissue damage
by ""walling-off"" the injury site. Additional details regarding
the compositions and methods of the present technology are
described below.
Nanoparticles
[0029] In some aspects, suitable nanoparticles will have an average
diameter between about 50 nm-200 nm. In some embodiments, suitable
nanoparticles will be configured to leverage the EPR effect to
target and accumulate in the tumor periphery of vascularized tumors
such as GBM.
[0030] In some aspects, the nanoparticles are gold nanoparticles.
Gold nanoparticles are known to optimize the biodistribution of
drugs to diseased organs, tissues, and cells, in order to improve
and target payload delivery. Nanoparticles are particularly useful
for difficult delivery sites (brain, retina, tumors, intracellular
organelles). The performance of the nanoparticles depends on the
size and surface functionality of the particles. Suitable gold
nanoparticles are well known, and include colloidal gold Product
Nos. 15701-1 through 15714-20 from Ted Pella, Inc. In a specific
aspect, a suitable gold nanoparticle may include product No. 15708
(60 nm diameter) purchased from Ted Pella, Inc. In other aspects,
any suitable iron, silica or poly(lactic-co-glycolic acid) (PLGA)
nanoparticle may be used in combination with or substituted for
gold nanoparticles. For example, suitable iron oxide nanoparticles
may include Product Nos. 747327, 747424, 747254, 747343, 7476319,
747300, 747408, 747416, 790508, 747335, 747432, 747459, 747467, and
747440 from Sigma Alrdrich, Inc., as well as poly(vinyl alcohol)
nanoparticles. Suitable silica microspheres may include, for
example, Product No. 24320-15, 24298-10, 24040-10, and 24041-10
from Poly sciences, Inc. Suitable PLEA nanoparticles may include,
for example, Degradex.RTM. Product Nos. 805092 and 805106 from
Sigma Aldrich, Inc.
[0031] In another aspect, the nanoparticle may comprise a liposome.
""Liposomes,"" as used herein, generally refer to spherical or
roughly spherical particles containing an internal cavity. The
walls of liposomes can include a bilayer of lipids. These lipids
can be phospholipids. Numerous lipids and/or phospholipids may be
used to make liposomes. One example are amphipathic lipids having
hydrophobic and polar head group moieties, which may form
spontaneously into bilayer vesicles in water, as exemplified by
phospholipids, or which may be stably incorporated into lipid
bilayers, with their hydrophobic moiety in contact with the
interior, hydrophobic region of the bilayer membrane, and their
polar head group moiety oriented toward the exterior, polar surface
of the membrane. In some aspects, suitable liposomes may include
those disclosed in U.S. Pat. No. 7,785,568, the entire contents of
which are incorporated herein by reference.
Linkers and Masking Agents
[0032] The linker may be any compound that will associate the
nanoparticle with the targeting ligand. In some aspects, the linker
may form a covalent bond to the nanoparticle, the ligand, or both.
In some aspects, interactions between the linker and either the
nanoparticle, ligand, or both are noncovalent (e.g., hydrogel
bonds, affinity, etc.). More generally, the linker may be a
molecule that has a functional group at each end with which the
linker binds the ligand to the body of the nanoparticle. The linker
may be cleavable or non-cleavable. The linker may also be a part of
the ligand; in that case, the ligand may be connected to the
nanoparticle directly.
[0033] In some aspects, the conjugate may comprise a masking agent
that protects the particles from being scavenged by the immune
system as well as associating with the nanoparticle and ligand. In
some aspects, the linker and/or masking agent may be a polymer. In
a specific aspect, the linker and/or masking agent may be PEG, a
PEG derivative, or a hydrophilic polycarbonate. The polymer can
have any of a variety of molecular weights. In one example, the
polymer comprises PEG and the PEG chain has a molecular weight
between about 1,000-10,000 Da.
Ligands
[0034] In some aspects, suitable ligands include any ligands that
will: (i) form a conjugate with, be encapsulated by, or otherwise
associate with the nanoparticle; and (ii) are configured to
activate peri-tumoral cells to induce scar deposition by the
peri-tumoral cells.
[0035] In one aspect, suitable ligands include those configured to
target at least one of the following entities in peri-tumoral
cells: (i) TLR2 receptor; (ii) TLR4 receptor; (iii) CSF-1 receptor;
(iv) IFN-gamma receptor 1; (v) IFN-gamma receptor 2; (vi)
xylosyltransferase; (vii) TNF-alpha receptor; and (viii) IL-2
receptor.
[0036] In yet another aspect, the ligand is selected from one or
more of peptidoglycan, LPS, zymosan, Pam3CSK4, amyloid-beta
peptide, lipoteichoic acid, HMGB1, heat shock proteins, CSF-1R
inhibitors, LPS+IFN-gamma, xyloside, IFN-gamma, TNF-alpha, IL-2,
lipocalin 2, and miRNA-155. The term ""ligand"" further includes
extracts thereof. For example, the ligand may comprise a zymosan
extract (Zpep) that is both water soluble and comprises a protein
fraction. Preferably, the extract comprises a protein fraction in
sufficient amounts to provide a concentration of about 25 .mu.g/mL
in an application. The protein fraction may comprise any proteins
routinely found in zymosan. The protein fraction may comprises
proteins, protein fragments, polypeptides, or a combination
thereof.
[0037] Zymosan, a yeast cell wall preparation, specifically
activates microglia and astrocytes when injected directly into the
brain, recapitulating the sequelae of a physical injury in its
absence. Microglial-conditioned medium, and not the direct addition
of Zpep, activated astrocytes, indicating that Zpep exerted its
inflammatory effects via macrophages and perhaps other myeloid
cells in vivo. Thus, the term ""induce"" may mean to directly
cause, and it may mean to activate endogenous processes that, in
turn, cause the desired effect.
[0038] By ""targeting molecule,"" what is meant is a compound that
serves to target or direct the ligand to a particular location or
cell type. In general, the targeting molecule specifically binds a
specific target epitope or receptor. ""Specifically binds"" means
that non-target cells either do not specifically interact with the
ligand or are only poorly recognized by the ligand. In some
aspects, the targeting molecule is all or a portion (e.g., a
binding portion) of a ligand for a cell surface receptor. Suitable
ligands include, but are not limited to, all or a functional
portion of the ligands that bind to a cell surface receptor.
[0039] By ""entities"" in a peri-tumoral cell, what is meant is any
moiety within or on the surface of a cell. The entity may be a cell
surface receptor or any molecule that interacts with the
appropriate ligand. Alternatively, the entities may be described as
on a peri-tumoral cell as well as embedded within the cellular
membrane of a peri-tumoral cell.
[0040] By ":peri-tumoral"" what is meant is any cell that is in the
near vicinity of a tumor. The cells may be immediately adjacent to
a tumor, may be in contact with the tumor, or may be near the
tumor.
[0041] By ""induce scarring by the peri-tumoral cell,"" or
iterations thereof, what is meant is that exposure of any
peri-tumoral cell to the compositions described herein begins a
process whereby a scarring process occurs. This process results in
a wall between a tumor and healthy tissue. The wall is generated by
the induced peri-tumoral cell and may include the peri-tumoral cell
and other components.
[0042] The enhanced permeability and retention (EPR) effect
describes the preferential accumulation of nanoparticles within
tumors owing to their leaky vasculature (enhanced permeation) and
poor lymphatic drainage (retention). As nanoparticles shielded from
the immune system traverse through the bloodstream, every time they
pass through the leaky tumor vasculature, they leak out of the
blood vessels into the tumor, and over time-with multiple passes,
they accumulate in the tumor. The particles are retained in the
tumor due to the poor lymphatic and vascular drainage of the tumor,
until they are phagocytosed by other phagocytotic cells, or
dissolved/disintegrated, or until mature vasculature or lymphatic
drainage in the tumor is established.
[0043] In another aspect, methods are provided for the preparation
of the conjugates and encapsulates provided herein.
[0044] In still another aspect, methods are provided for activating
peri-tumoral cells to induce scarring by the peri-tumoral cells. In
one aspect, the method comprises administering a nanoparticle
composition to target cells in close proximity to tumors in order
to activate the cells and stimulate scarring. In one aspect, the
method comprises administering gold nanoparticles coated with
polypeptides to target stromal cells in close proximity to GBM
tumors in order to activate the cells and stimulate stromal CSPG
expression.
[0045] In one aspect, compositions and methods are provided for
leveraging endogenous mechanisms of scar formation to moderate
tumor growth by modulating the behavior of cells other than those
of the tumor. In a specific aspect, compositions and methods are
provided for targeting of endogenous mechanisms for induced
inhibitory CSPG expression in the stromal space using the EPR
effect via gold nanoparticles.
[0046] In one aspect, methods are provided for tumor containment.
In a specific aspect, the method for tumor containment relies
partly on the activation of astrocytes, microglia, and/or the
production of CSPGs.
[0047] For the purposes of promoting an understanding of the
principles of the present disclosure, reference has been made to
specific, enabling aspects. For instance, the aspects described
herein are directed primarily to the administration of gold
nanoparticles coated with Zpep to target stromal cells in close
proximity to GBM tumors in order to activate the cells and
stimulate stromal CSPG expression. However, no such limitation of
the scope of the disclosure is intended. Rather, the inventors
contemplate the use of each of the nanoparticle-ligand combinations
disclosed herein for targeting to any peri-tumoral cells to induce
scarring and, thus, tumor containment.
[0048] Articles ""a"" and ""an"" are used to refer to one or to
more than one (i.e., at least one) of the grammatical object of the
article. By way of example, "an element" means at least one element
and can include more than one element.
[0049] ""About"" is used to provide flexibility to a numerical
range endpoint by providing that a given value may be ""slightly
above"" or ""slightly below"" the endpoint without affecting the
desired result.
[0050] The use herein of the terms ""including,"" ""comprising,""
or ""having,"" and variations thereof, is meant to encompass the
elements listed thereafter and equivalents thereof as well as
additional elements. Aspects recited as ""including,""
""comprising,"" or ""having"" certain elements are also
contemplated as "consisting essentially of and "consisting of"
those certain elements. As used herein, ""and/or"" refers to and
encompasses any and all possible combinations of one or more of the
associated listed items, as well as the lack of combinations where
interpreted in the alternative (""or"").
[0051] As used herein, the transitional phrase ""consisting
essentially of"" (and grammatical variants) is to be interpreted as
encompassing the recited materials or steps "and those that do not
materially affect the basic and novel characteristic(s)" of the
claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190
U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also
MPEP .sctn. 2111.03. Thus, the term "consisting essentially of"
should not be interpreted as equivalent to ""comprising.""
[0052] Moreover, the present disclosure contemplates that in some
aspects, any feature or combination of features can be excluded or
omitted. To illustrate, if the specification states that a complex
comprises components A, B, and C, it is specifically intended that
any of A, B, or C, or a combination thereof, can be omitted and
disclaimed singularly or in any combination.
[0053] Recitation of ranges of values are merely intended to serve
as a shorthand method of referring individually to each separate
value falling within the range, unless otherwise stated, and each
separate value is incorporated into the specification as if it were
individually recited. For example, if a concentration range is
stated as 1% to 50%, it is intended that values such as 2% to 40%,
10% to 30%, or 1% to 3%, etc., are expressly enumerated in this
specification. These are only examples of what is specifically
intended, and all possible combinations of numerical values between
and including the lowest value and the highest value enumerated are
to be considered to be expressly stated in this disclosure.
[0054] Unless otherwise defined, all technical terms have the same
meaning as commonly understood by one of ordinary skill in the art
to which this disclosure belongs.
[0055] As used herein, ""treatment,"" ""therapy,"" and ""therapy
regimen"" refer to the clinical intervention made in response to a
disease, disorder, or physiological condition manifested by a
patient or to which a patient may be susceptible. The aim of
treatment includes the alleviation or prevention of symptoms,
slowing or stopping the progression or worsening of a disease,
disorder, or condition and/or the remission of the disease,
disorder, or condition. The aim of the treatment may include
slowing the progression of cancerous tissue, increasing the
susceptibility of the cancerous tissue to more conventional
treatments or reducing the side effects of chemotherapy and/or
radiation therapy directed at the cancerous tissue. The term
"effective amount" or "therapeutically effective amount" refers to
an amount sufficient to effect beneficial or desirable biological
and/or clinical results.
[0056] The term ""disease"" as used herein includes, but is not
limited to, any abnormal condition and/or disorder of a structure
or a function that affects a part of an organism. It may be caused
by an external factor, such as an infectious disease, or by
internal dysfunctions, such as cancer, cancer metastasis, and the
like.
[0057] ""Administration"" as it applies to a human, primate,
mammal, mammalian subject, animal, veterinary subject, placebo
subject, research subject, experimental subject, cell, tissue,
organ, or biological fluid, refers without limitation to contact of
an exogenous ligand, reagent, placebo, small molecule,
pharmaceutical agent, therapeutic agent, diagnostic agent, or
composition (e.g., a gold nanoparticle:linker:ligand conjugate as
provided herein) to the subject, cell, tissue, organ, or biological
fluid, and the like.
[0058] As is known in the art, a cancer is generally considered as
uncontrolled cell growth. The methods of the present aspects can be
used to affect the peri-tumoral tissue surrounding any cancer, and
to impede or prevent any metastases/migration thereof. In some
aspects, the cancer comprises a solid tumor. Examples include, but
are not limited to, breast cancer, prostate cancer, colon cancer,
squamous cell cancer, small-cell lung cancer, non-small cell lung
cancer, ovarian cancer, cervical cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, liver cancer, bladder cancer,
hepatoma, colorectal cancer, uterine cervical cancer, endometrial
carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer,
vulval cancer, pancreatic cancer, thyroid cancer, hepatic
carcinoma, skin cancer, melanoma, uveal melanoma, brain cancer
(e.g., GBM), neuroblastoma, various types of head and neck cancer,
Ewing sarcoma, peripheral neuroepithelioma, adrenocortical cancer,
rectal cancer, esophageal cancer, thyroid cancer, stomach cancer,
mesothelioma, testicular cancer, and the like, including both
primary and metastatic.
[0059] As used herein, the term ""subject"" and ""patient"" are
used interchangeably herein and refer to both human and nonhuman
animals. The term ""nonhuman animals"" of the disclosure includes
all vertebrates, e.g., mammals and non-mammals, such as nonhuman
primates, sheep, dog, cat, horse, cow, chickens, amphibians,
reptiles, and the like.
[0060] The compositions described herein can be administered to a
subject, either alone or in combination with a pharmaceutically
acceptable excipient, in an amount sufficient to induce an
appropriate response. The response can comprise, without
limitation, specific immune response, non-specific immune response,
both specific and non-specific response, innate response, primary
immune response, adaptive immunity, secondary immune response,
memory immune response, immune cell activation, immune cell
proliferation, immune cell differentiation, and cytokine
expression. In some aspects, the response comprises the activation
of stromal cells to produce CSPGs.
[0061] In some aspects, the present disclosure provides a method
for providing a treatment in a subject by administering to the
subject an effective amount of proteoglycan. An ""effective
amount,"" as used herein means an amount that provides a
therapeutic or prophylactic benefit. Effective amounts of the
conjugates or encapsulates as provided herein can be determined by
a physician with consideration of individual differences in age,
weight, tumor size, extent of infection or metastasis, and
condition of the patient (subject). It can generally be stated that
a pharmaceutical composition comprising the conjugates or
encapsulates described herein may be administered at a dosage of 1
to 10.sup.11 particles/kg body weight, preferably 2 to 10.sup.10
particles/kg body weight, including all integer values within those
ranges. Conjugates or encapsulates may also be administered
multiple times at these dosages. The conjugates or encapsulates can
be administered by using techniques that are commonly known in the
art, including intravenous, intratumoral, subcutaneous, and
intraperitoneal administration. The optimal dosage and treatment
regime for a particular patient can readily be determined by one
skilled in the art of medicine by monitoring the patient for signs
of disease and adjusting the treatment accordingly.
[0062] An effective amount of the conjugates or encapsulates
described herein may be given in one dose, but is not restricted to
one dose. Thus, the administration can be two, three, four, five,
six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,
fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more
administrations of the composition. Where there is more than one
administration in the present methods, the administrations can be
spaced by time intervals of one minute, two minutes, three, four,
five, six, seven, eight, nine, ten, or more minutes, by intervals
of about one hour, two hours, three, four, five, six, seven, eight,
nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24
hours, and so on. In the context of hours, the term ""about"" means
plus or minus any time interval within 30 minutes. The
administrations can also be spaced by time intervals of one day,
two days, three days, four days, five days, six days, seven days,
eight days, nine days, ten days, 11 days, 12 days, 13 days, 14
days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21
days, and combinations thereof, including several weeks or even
months between administrations. The invention is not limited to
dosing intervals that are spaced equally in time, but encompass
doses at non-equal intervals, such as a priming schedule consisting
of administration at 1 day, 4 days, 7 days, and 25 days, just to
provide a non-limiting example.
[0063] A ""pharmaceutically acceptable excipient"" or
""diagnostically acceptable excipient"" includes but is not limited
to, sterile distilled water, saline, phosphate buffered solutions,
amino acid-based buffers, or bicarbonate buffered solutions. An
excipient selected and the amount of excipient used will depend
upon the mode of administration. Administration comprises an
injection, infusion, or a combination thereof.
[0064] An effective amount for a particular subject/patient may
vary depending on factors such as the condition being treated, the
overall health of the patient, the route and dose of
administration, and the severity of side effects. Guidance for
methods of treatment and diagnosis is available (see, e.g.,
Maynard, et al. (1996) A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good
Laboratory and Good Clinical Practice, Urch Publ., London, UK).
[0065] The conjugates and encapsulates of the present disclosure
can be administered in a dose, or dosages, where each dose
comprises at least 100 conjugates or encapsulates/kg body weight or
more; in certain aspects 1000 conjugates or encapsulates/kg body
weight or more; normally at least 10,000 conjugates or
encapsulates; more normally at least 100,000 conjugates or
encapsulates; most normally at least 1 million conjugates or
encapsulates; often at least 10 million conjugates or encapsulates;
more often at least 100 million conjugates or encapsulates;
typically at least 1 billion conjugates or encapsulates; usually at
least 10 billion conjugates or encapsulates; conventionally at
least 100 billion conjugates or encapsulates; and sometimes at
least 1 trillion conjugates or encapsulates/kg body weight.
[0066] A dosing schedule of, for example, once/week, twice/week,
three times/week, four times/week, five times/week, six times/week,
seven times/week, once every two weeks, once every three weeks,
once every four weeks, once every five weeks, and the like, is
available for the invention. The dosing schedules encompass dosing
for a total period of time of, for example, one week, two weeks,
three weeks, four weeks, five weeks, six weeks, two months, three
months, four months, five months, six months, seven months, eight
months, nine months, ten months, eleven months, and twelve
months.
[0067] Provided are cycles of the above dosing schedules. The cycle
can be repeated about, e.g., every seven days; every 14 days; every
21 days; every 28 days; every 35 days; 42 days; every 49 days;
every 56 days; every 63 days; every 70 days; and the like. An
interval of non-dosing can occur between a cycle, where the
interval can be about, e.g., seven days; 14 days; 21 days; 28 days;
35 days; 42 days; 49 days; 56 days; 63 days; 70 days; and the like.
In this context, the term "about" means plus or minus one day, plus
or minus two days, plus or minus three days, plus or minus four
days, plus or minus five days, plus or minus six days, or plus or
minus seven days.
[0068] The conjugates and encapsulates according to the present
disclosure may also be administered alongside one or more
therapeutic/anti-cancer agents/therapies. Methods for
co-administration with a therapeutic/anti-cancer agents/therapies
are well known in the art (Hardman, et al. (eds.) (2001) Goodman
and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.,
McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001)
Pharmacotherapeutics for Advanced Practice: A Practical Approach,
Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo
(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,
Williams & Wilkins, Phila., Pa.).
[0069] Co-administration need not refer to administration at the
same time in an individual, but rather may include administrations
that are spaced by hours or even days, weeks, or longer, as long as
the administration of multiple therapeutic agents is the result of
a single treatment plan. The co-administration may comprise
administering the conjugates and encapsulates of the present
disclosure before, after, or at the same time as the alternative
agents/therapies. In one treatment schedule, the conjugates and
encapsulates of the present disclosure may be given as an initial
dose in a multi-day protocol, with alternative agents/therapies
given on later administration days; or the alternative
agents/therapies given as an initial dose in a multi-day protocol,
with the conjugates or encapsulates of the present disclosure given
on later administration days. On another hand, alternative
agents/therapies and the conjugates and encapsulates of the present
disclosure may be administered on alternate days in a multi-day
protocol. This is not meant to be a limiting list of possible
administration protocols.
[0070] Yet another aspect of the present disclosure provides a
method of reducing and/or preventing the migration of a cancer in a
subject, the method comprising, consisting of, or consisting
essentially of administering to the subject a therapeutically
effective amount of a conjugate or encapsulate as provided herein
such that the migration of the cancer is reduced and/or
prevented.
[0071] In some aspect, the methods further comprise administering
to the subject an anti-cancer therapy. In some aspects, the
anti-cancer therapy is selected from the group consisting of
radiation, chemotherapy, immunotherapy, surgery, hormonal therapy,
target therapy, synthetic lethality, induced tumor migration,
immunotherapy, and combinations thereof.
[0072] In some aspects, such compositions and methods may
complement adjuvant interventions, such as radiation, chemotherapy,
surgery, hormonal therapy, target therapy, synthetic lethality,
designed tumor migration such as that described in U.S. application
Ser. Nos. 13/814,009 and 16/432,475, the entire contents of which
are incorporated herein by reference, immunotherapy, and
combinations thereof, for invasive tumors, such as invasive
GBM.
[0073] The following Examples are provided by way of illustration
and not by way of limitation.
EXAMPLES
[0074] One skilled in the art will appreciate that, for this and
other processes and methods disclosed herein, the functions
performed in the processes and methods may be implemented in
differing order. Furthermore, the outlined steps and operations are
only provided as examples, and some of the steps and operations may
be optional, combined into fewer steps and operations, or expanded
into additional steps and operations without detracting from the
essence of the disclosed aspects.
Methods
[0075] Cell Lines and Culture Procedures. U87 cells (human glioma,
HTB-14), F98 cells (Fischer rat glioma, CRL-2397), EOC cells (mouse
microglia, CRL-2469), LADMAC cells (mouse macrophages/monocytes,
CRL-2420), and C8D1A cells (mouse cerebellum astrocytes, CRL-2541)
were purchased from ATCC and maintained according to instructions
specific to each cell line. The CRL-2469 cell line was cultured in
Dulbecco's Modified Eagle Medium (DMEM; Corning) and 10% FBS with
either 30 ng ml-1 recombinant mouse CSF-1 (416-ML-010, R&D
Systems) or with conditioned medium from LADMAC cells. All cells
were grown at 37.degree. C. with 5% CO2, passaged with trypsin-EDTA
0.05% and maintained in respective complete cell culture medium
(ATCC recommended) and 1% penicillin-streptomycin (Gibco) unless
otherwise noted. As noted for some experiments, U87 cells were made
to stably express enhanced green fluorescent protein (eGFP), via
transfection with an eGFP expression plasmid using the Effectene
Transfection Reagent (Qiagen) and further selection of stable
transfectants with G418 Sulfate (Gemini).
[0076] Spot assays. Preparation of 14-mm glass-bottomed
Petri-dishes (In vitro Scientific, Sunnyvale, Calif.) was done
according to known methods with few modifications. Briefly,
surfaces were coated with poly-L-lysine (PLL, 1:10 dilution in
ultrapure water) (Sigma-Aldrich) and incubated overnight at
37.degree. C. The following day, the surfaces were rinsed thrice
with sterile water and allowed to dry completely. Various
concentrations of aggrecan, bovine serum albumin (BSA), and
fibronectin with Texas Red (Invitrogen) were spotted onto the
prepared surfaces in 2 .mu.L amounts and allowed to dry completely.
80,000 U87 mg cells (stably expressing eGFP) suspended in standard
growth medium were carefully added to the chambers. After 24 hours,
the cells were fixed with 4% paraformaldehyde and 0.4M sucrose
solution for 15 min and immunostained for CS-56 (Sigma C8035,
1:250). For determining the effect of GAG side chains, aggrecan
spots were treated extensively with cABC (1 U/mL; 3-5 h, 37.degree.
C.; Sigma). After aspirating the cABC and rinsing with PBS, 80,000
eGFP+U87 mg cells were plated as described above. After 24 h, cells
were fixed and immunostained for 2B6 (Seikagaku, 1:250) and CS-56
(1:200).
[0077] Zpep extraction. Zymosan (Sigma Aldrich, 250 mg) was snap
frozen in liquid nitrogen and crushed into a fine powder using a
mortar and pestle. The crushed zymosan was added to an extraction
buffer (10 mL) containing TRIS-HC (0.5 M), CHAPS (1%), DTT (1%),
and PMSF. The extraction process was allowed to go on overnight at
room temperature (rt) with gentle agitation. The next day, the
samples were centrifuged at 5000.times.g for 10 min and the
supernatant was collected, following which it was washed,
concentrated, and dialyzed (3.times.) using a 10 kDa centrifugal
spin column (EMD Millipore, Amicon Ultra-15) according to the
manufacturer's instructions. Protein concentrations were determined
using a NanoDrop device. Zpep aliquots were stored at -20.degree.
C. until further use.
[0078] In vitro experiments. Zpep was used at a concentration of 25
.mu.g/mL in vitro. Nitrite production was assessed at time points
(indicated in figure legends) using the Griess reagent system
(G2930, Promega). TLR2 blocking experiments were performed as known
in the art. TNF-.alpha. levels were assessed using anti-TNF-.alpha.
enzyme-linked immunosorbent assay (ELISA) kit as per the
manufacturer's instructions (BD Biosciences, RayBiotech).
Quantitative real-time PCR was performed on a multiplexed Fluidigm
system, and data were analyzed according to known methods. Primers
were purchased from Fluidigm Inc.
[0079] Gold nanoparticle conjugates (AuNP-Z). Gold nanoparticles
(AuNPs) were purchased from Ted Pella, Inc. (60 nm diameter,
15708-6). PEG-thiol (MPEG-SH-20K-1g) was purchased from Laysan Bio.
All reactions were performed in ultrapure DI water (18 M.OMEGA.
cm-1). The amount of AuNPs each animal received per dose was 57
microgram (=1 ml of 60 nm AuNPs, at a concentration of
2.6.times.10e.sup.10 particles/ml). PEG-SH concentration was
calculated such that each AuNP received 30,000 PEG-SH molecules.
Zpep and PEG-SH were conjugated to AuNPs as follows: PEG was
dissolved in ultrapure DDI water. Zpep was reconstituted at 1
mg/ml, such that each animal would receive 100 microgram final dose
(based on initial concentration of zymosan and assuming 100%
conjugation efficiency). Zpep was suspended in 3 mM Tris base
buffer. AuNPs were centrifuged (12000.times.g, 20 min) and
re-suspended in ultrapure DDI water. The AuNPs, PEG-SH, and Zpep
were combined and left on a rotating test-tube holder overnight at
rt. Particles were aliquoted into individual doses and washed twice
by centrifugation (12,000.times.g, 20 min) before being
re-suspended in sterile saline for in vivo tail vein injections, or
ultrapure DDI water for physical characterization. Dynamic light
scattering and UV-Vis spectroscopy were performed by known
techniques.
[0080] Tumor inoculation. All experiments were approved by the
Institutional Animal Care and Use Committee at the Georgia
Institute of Technology and Duke University. Rowett nude rats or
Fischer rats (175-200 g, male, Charles River Laboratories) were
inoculated with U87 mg (80,000 cells) or F98 tumor cells (10,000
cells), respectively. The animals were anesthetized using 5%
isoflurane and maintained with 2-3% isoflurane during surgical
procedures. The animals were placed in a stereotaxic device. A 1 cm
incision was made on the head. The periosteum was cut and removed
from the skull. A hole was made in the skull 2 mm lateral and 2 mm
posterior from bregma. A 26-gauge needle mounted on a 10 .mu.L
Hamilton syringe was inserted 2 mm deep from the surface of the
brain and retracted 0.5 mm. Tumor cells in 5 .mu.L of DMEM
(serum-free) were injected using an automated syringe pump at a
rate of 1 uL/minute. The needle was held in place an additional 2
min before removal and closing. Animals displaying symptoms of
distress from the glioblastoma were anesthetized with ketamine (1
mL/kg), xylazine (0.17 mL/kg), and acepromazine (0.37 mL/kg),
transcardially perfused with physiological PBS, followed by 4%
paraformaldehyde. The brains were dissected and incubated in 4%
paraformaldehyde overnight, and were stored in 30% sucrose
containing 0.01% sodium azide. For cohorts designated to proteomics
and histology, anesthetized animals were transcardially perfused
with physiological PBS followed by 10% formalin, and stored in 10%
formalin at rt (i.e. room temperature).
[0081] MRI Imaging. Rats were anaesthetized and placed in a Bruker
Pharmascan 7-T (Bruker BioSpin MRI) operating with the ParaVision
software with a 38-mm quadrature-detection volume coil as head
coil. The animals were anaesthetized using 2% isoflurane and placed
in a home-built cradle, allowing the easy placement of the animals'
heads within the MRI coil. The rapid acquisition of high quality T2
weighted images was achieved using the rapid imaging with refocused
echoes (RARE) sequence (RARE factor, 6; effective echo time, 36 ms;
repetition time [TR], 4,200 s; two averages per scan; total
acquisition time, 6 min). A slab of 40 transversal slices was
recorded using a field of view of 40 mm.times.40 mm with a
256.times.256 matrix and a slice thickness of 0.5 mm. This slab was
aligned to cover the injection site of the tumor cells using a
pilot scan, which was recorded immediately before the acquisition
of the RARE images. MR images were acquired roughly every week
following tumor implant to check for tumor growth or regression.
ImageJ software (FIJI, version 2.0) was used for further image
processing, and for tumor volume calculations. A region of interest
(ROI) following the tumor borders was drawn manually in the
T2-weighted images. The whole tumor volume was calculated by adding
up the voxel volumes within the ROIs of all image slices. In the
case that MRI was not possible on days of euthanasia, tumor volume
at time of death was extrapolated assuming a linear tumor growth
rate.
[0082] Proteomics at Duke proteomics and metabolomics shared
resource. U87 mg tumors that had previously been fixed in formalin
were macro-dissected from the surrounding normal tissue from the
brains of 9 rats: 3 different animals per group, 3 groups total.
The wet weight of each was noted. 1 mL of 50 mM ammonium
bicarbonate (AmBic) was added to each tumor, and heated at
80.degree. C. for 55 min while shaking at 750 rpm. The AmBic was
removed, and another 1 mL of AmBic was added for a second rinse.
The second rinse was then pipetted off, and the tumor was allowed
to cool completely at room temperature (<5 min). Each tumor was
transferred to a centrifuge tube, and 8 M urea in 50 mM AmBic was
added at 10 .mu.L per mg of wet weight. The tissue was then taken
through tissue tearing until no tissue pieces were visible, and the
samples were homogenized. The samples were then probe sonicated at
power level 3 for 5 s bursts, 3 bursts each while on ice. A
concentration was determined for each homogenate by Bradford assay.
50 .mu.g from each sample was taken out and concentration
normalized in 8 M urea in 50 mM AmBic. Then, enough AmBic was added
to each to get to 1.8 M urea for subsequent in-solution tryptic
digestion. The samples were reduced in 10 mM dithiothreitol (DTT)
at 32.degree. C. for 45 min, alkylated in 25 mM iodoacetamide (IAA)
at rt in the dark for 30 min, and trypsin was added to each at a
1:50 ratio of enzyme to protein for digestion at 32.degree. C.
overnight while shaking at 750 rpm. The following morning, the
samples were acidified with trifluoroacetic acid to give 0.5% TFA
final, and taken through a C18 SPE cleanup (Waters Sep-Pak Vac, 50
mg cartridges, Product #WAT054955). After some of the acetonitrile
was evaporated via Speed Vac, the remaining extracts were taken to
dryness by lyophilization overnight. The samples were then
reconstituted in 200 .mu.L of 1% TFA/2% ACN containing 25
fmol/.mu.L yeast alcohol dehydrogenase surrogate standard. A QC
pool was prepared by mixing equal volumes of all samples.
[0083] Quantitative Mass Spectrometry. Quantitative one-dimensional
liquid chromatography, tandem mass spectrometry (ID-LC-MS/MS) was
performed on the peptide digests per sample, with additional
analyses of conditioning runs and QC pools. Samples were analyzed
using a nanoACQUITY UPLC system (Waters) coupled to a QExactive
Plus high resolution accurate mass tandem mass spectrometer
(Thermo) via a nanoelectrospray ionization source. The sample was
trapped on a Symmetry C18 180 .mu.m.times.20 mm trapping column (5
.mu.L/min at 99.9/0.1 v/v H2O/MeCN), followed by an analytical
separation using a 1.7 .mu.m Acquity HSS T3 C18 75 .mu.m.times.250
mm column (Waters) with a 90 min gradient of 5 to 40% MeCN/H2O with
0.1% formic acid at a flow rate of 400 nL/min and column
temperature of 55.degree. C. Data collection on the QExactive Plus
MS was performed in data-dependent acquisition mode with a 70,000
resolution (@ m/z 200) full MS scan from m/z 375 to 1600 with a
target AGC value of 1e6 ions followed by 10 MS/MS scans at 17,500
resolution (@ m/z 200) at a target AGC value of 5e4 ions. A 20-s
dynamic exclusion was employed. The total analysis cycle time per
sample injection was approximately 2 h. Following 12 total
UPLC-MS/MS analyses (including 3 replicate QC injections), data
were imported into Rosetta Elucidator v 4.0 (Rosetta Biosoftware,
Inc.), and analyses were aligned based on the accurate mass and
retention time of detected ions (""features"") using PeakTeller
algorithm in Elucidator. Relative peptide abundance was calculated
based on area-under-the-curve (AUC) of the selected ion
chromatograms of the aligned features across all runs. Elucidator
was utilized to produce fragment ion spectra, and Mascot Server (v
2.5, Matrix Sciences) performed the database searches. The MS/MS
data were searched against two databases: a Swissprot database with
Homo sapiens taxonomy and a NCBI refseq database with Rattus
norvegicus taxonomy (both downloaded in August 2016) with
additional proteins commonly used as internal controls including
yeast ADH1, bovine serum albumin, and bovine alpha casein, as well
as an equal number of reversed-sequences (""decoys"") for false
discovery rate determination. Database search parameters included
fixed modification on Cys (carbamidomethyl) and variable
modifications on Asn and Gn (deamidation) and Met (oxidation), 2
missed cleavages, precursor tolerance of 5 ppm and product
tolerance at 0.2 Da, and trypsin as the enzyme specificity. After
individual peptide scoring using the PeptideProphet algorithm in
Elucidator, the data were annotated at a 0.9% peptide false
discovery rate.
[0084] Proteomic Differential Expression. For proteomics,
comparisons between fold changes of the protein-level intensities
(PLIs) were used to determine proteins that were differentially
expressed (DE) between the conditions (Z: AuNP-Z; P: AuNP-P; C:
control, no AuNP; n=3). The statistical comparison tool QPROT
(v1.3.3)72 (nburnin: 2,000; niters: 10,000; normalized: true) was
used to compute a z-statistic and FDR for each identified protein
compared pairwise between conditions. The p values were obtained
from the z-statistic using Python (Version 2.7.11, Anaconda 2.2.0;
https://www.python.org/). Proteins with a FDR<0.05 for a
particular fold change comparison between conditions were
considered statistically significant and treated as DE for that
condition-pair.
[0085] Pathway Overrepresentation Analysis. The DE proteins were
pre-filtered to exclude proteins with an absolute fold change of
less than 2-fold. For each condition-pair, gene ontology was
performed using g:profiler version: r1730_e88_eg35
(http://biit.cs.ut.ee/gprofiler/). Both Rattus norvegicus and Homo
sapiens datasets were used. Since parsimonious assignment of
species is unreliable for closely homologous proteins, each species
dataset was run for all proteins (species-indifferent), as well as
the subsets of proteins identified as either rat or human. The
search included Gene Ontology, KEGG, Reactome, and Regulatory Motif
databases and used the built-in g:SCS threshold for significance.
Default settings were used. QuickGO (https://www.ebi.ac.uk/QuickGO)
web service was used for obtaining additional gene ontology
information for visualization with Python.
[0086] Pathway Enrichment Analysis. Gene Set Enrichment Analysis
(GSEA) Release 3.0 (http://www.broadinstitute.org/gsea) was used to
perform pathway enrichment analysis. Pre-ranked analysis was
performed against the curated (c2.all.v6.0), and gene ontology
(c5.all.v6.0), datasets using the negative-log of the QPROT
p-value, signed according to fold-change direction, as the ranking
scheme for each condition-pair. The isoform with the lowest p-value
was selected in cases of gene symbol collision. Default settings
were used except the max and min pathway size exclusion criteria
were set to 1,000 and 5, respectively.
[0087] Immunohistochemistry, immunofluorescence, and microscopy.
All antibodies used for IHC and IF are listed in Figure Captions.
Fixed, frozen brain tissue was sectioned to 14-.mu.m thickness, and
prepared for immunofluorescence using known methods. Sections were
imaged on a Zeiss Axiovision inverted microscope. For IHC, tissues
were processed at the Emory Winship Pathology Core Lab, using known
methods. Tissues from paraffin-embedded blocks were sectioned at
5-.mu.m thickness. IHC was performed using DAB chromogenic kit
(Wako) following the manufacturer's protocol. Whole-slide scanning
was done using a Hamamatsu Nanozoomer 2.0 HT.
[0088] Graphing and Statistics. All graphs were made in Prism 7
(Graphpad Inc.), Python, or MATLAB (Version 9, Mathworks, Mass.).
Layout of figure panels was done with Illustrator (Adobe Inc.).
Outliers were omitted when indicated by a Grubb's test. Where
appropriate, Student's t-tests or one-way ANOVA (as described in
figure legends), followed by post-hoc tests were run in Prism 7.
Survival analysis was also performed in Prism 7, and significance
was assessed using the Mantel-Cox log rank test.
Example 1
[0089] Using CSPGs to repel GBM cells (FIG. 1(a)-(e)). To determine
the effects of CSPGs on repelling tumor cells, an established in
vitro model of glial scarring was used, namely, spot assay using a
prototypical inhibitory CSPG, aggrecan. Aggrecan was chosen because
it is a constituent of glial scars, and is sulfated with all of the
families of CS-GAGs. The results are shown in FIG. 1(a) (scale bar
is 50 .mu.m). U87 mg GBM cells expressing green fluorescent protein
(GFP) were used. ""DAPI"" indicates the nuclear stain
4',6-Diamidino-2-Phenylindole, and "CS56" is an antibody that
stains intact CSPGs. A spot of CS56+aggrecan (1 mg/ml, 2 ul spot)
repels the tumor cells: The dotted line (""Merge"") identifies the
zoomed-in region (scale bar is 200 .mu.m) on the far right,
confirming that tumor cells are at least partially prevented from
crossing the boundary posed by aggrecan.
[0090] Since CSPGs exert their inhibitory effects primarily via the
CS-GAG side chains, chondroitinase ABC (cABC) was used to
enzymatically digest and cleave the CS-GAG chains to determine
whether the boundary was sustained in the absence of CS-GAG side
chains. The results are shown in FIG. 1(b). 2B6 is an antibody that
stains the GAG stubs following enzymatic digestion of aggrecan by
chondroitinase ABC (chABC). Tumor cells were able to cross the spot
boundary posed by aggrecan upon chABC digestion, indicating that
CSPGs mediate their repulsive effects via CS-GAGs, and confirming
that CSPGs--the principal inhibitory components of the glial
scar--contributed a biochemical barrier to the invasion of GBM
cells in vitro.
[0091] To stimulate peri-tumoral expression of CSPGs, Fischer rats
were co-injected with highly motile, syngeneic F98 GBM cells and
zymosan. FIG. 1(c) shows the zymosan bead/stab injection sites and
the F98 tumor inoculation site. Animals were sacrificed 21 days
after tumor inoculation.
[0092] F98 tumor cells were excluded from regions of control stabs,
as shown in FIG. 1(d). Glial fibrillary acidic protein (GFAP) is an
immunofluorescent stain for reactive astrocytes. Stab wounds
(indicated by white arrow heads) partially repelled tumor growth,
but were unable to prevent tumor microsatellite migration
(indicated by red arrow heads). In contrast, zymosan beads caused
fulminant gliosis and cavitation (white arrow heads) and caused
tumors to remain as compact masses, as shown in FIG. 1(e)
(zoomed-in section scale bar is 200 .mu.m). In control animals,
tumors formed microsatellites away from stab sites, and in animals
treated with zymosan beads, tumor cells were constrained within the
boundaries of the bead injection sites. Zymosan injection caused
robust astroglial activation, indicating that astroglial scarring
largely contained and constrained tumor cell migration in vivo.
Example 2
[0093] Using Zpep to activate TLR2 (FIG. 2(a)-(f)). Direct
injection of zymosan into brain tumors is not practical for
multiple reasons, e.g., risk of injury due to intracranial
injections and incomplete coverage of tumor periphery.
Nanoparticles bearing zymosan are ideal to obviate these issues,
since nanoparticles leverage the EPR effect to target vascularized
tumors such as GBM, and accumulate in the tumor periphery--a
location ideal for constraining tumors. Thus, a water-soluble
mixture comprising zymosan polypeptides (Zpep) was extracted from
zymosan beads. Addition of Zpep to EOC mouse microglial cells
caused nitric oxide production (assessed by a nitrite assay), which
increased with time (Griess assay, FIG. 2(a)), and TNF-.alpha.
production (FIG. 2(b)). Since zymosan is a TLR2 stimulus, Zpep
activation of TLR2-related pathways was determined. Zymosan
recognition by mammalian cells is mediated by TLR2 and the
.beta.-glucan receptor dectin. EOC cells were treated with
laminarin (a soluble p-glucan that can block dectin-mediated
recognition) or TLR2-blocking antibody prior to exposure to Zpep.
The blocking of TLR2, but not dectin, led to a decrease in nitric
oxide production by EOC cells, indicating that Zpep retains the
TLR2-activating properties of zymosan (FIG. 2(c)), and that the
Zpep extract did not contain any water-insoluble .beta.-glucans.
Addition of Zpep to EOC cells caused robust upregulation of genes
associated with microglial activation, i.e., inflammatory gene
transcripts (FIG. 2(d)). Additionally, Zpep mimics the expected
response of zymosan on astrocytes--that is, zymosan cannot activate
astrocytes directly but through secretory factors of microglia
exposed to zymosan. These properties were confirmed, as direct
addition of Zpep was not able to classically activate C8D1A
astrocytes (FIG. 2(e), (f)), yet conditioned media from EOC cells
exposed to Zpep, induced robust astrocyte activation, as assessed
by nitric oxide production and inflammatory gene upregulation (FIG.
2(e), (f)) (""CM"" indicates EOC-conditioned medium). Taken
together, these data indicate that Zpep displays biological
properties similar to zymosan and causes robust inflammation in
glial cells.
Example 3
[0094] Using AuNPZ to stimulate peri-tumoral CSPG expression in
vivo (FIG. 3(a)-(c)). As described herein, the surface of AuNPs (60
nm diameter) were decorated with Zpep and 20 kDa PEG to create
inflammatory nanoparticles (AuNP-Z). Conjugation of polypeptides
onto AuNP surfaces was assessed by ultraviolet-visual spectroscopy.
As shown in FIG. 3(a), addition of PEG alone did not lead to a
red-shift (peak absorbance at 535 nm for naked AuNPs and AuNP-P).
However, addition of Zpep caused a small red-shift of 4-5 nm (peak
absorption at 540 nm for AuNP-Z). Dynamic light scattering of AuNPs
(60 nm diameter core) showed an increase in hydrodynamic radius of
AuNPs upon addition of PEG (100 nm diameter) and Zpep (90 nm
diameter).
[0095] To test the capacity of AuNP conjugates to cause
peri-tumoral CSPG expression in vivo, F98 glioma-bearing Fischer
rats were injected intravenously with Zpep-bearing AuNPs (100 .mu.g
of Zpep) at 6 days post tumor inoculation (DPI) and sacrificed at
20 DPI. As shown in FIG. 3(b), control tissue sections (b) from
animals bearing F98 tumors, but not receiving any treatment,
displayed low astrocyte activation (GFAP staining) and low CSPG
production (CS56 staining). Conversely, as shown in FIG. 3(c),
sections from Zpep-treated animals demonstrated robust astrocyte
activation and CSPG production. (Scale bar: 50 .mu.m.)
Example 4
[0096] Using AuNP-Z to constrain tumors in vivo (FIG. 4(a)-(b)).
F98 glioma-bearing Fischer rats were injected intravenously with
AuNPs at 6 days post tumor inoculation (DPI) and sacrificed at 20
DPI. As shown in FIG. 4(a), tissue sections from animals bearing
F98 tumors and receiving only PEG-bearing AuNPs display low
astrocyte activation (GFAP staining) and low CSPG production (CS56
staining). As shown in the whole-slide scans of FIG. 4(b), animals
receiving AuNP-Z had smaller and more constrained tumors. Tumors
are outlined with a red dotted circle. Constrainment is determined
as a qualitative assessment of how closely packed the tumor cells
are. (Scale bar is 100 .mu.m in FIG. 4(a) and 2 mm in FIG.
4(b).)
[0097] Thus, animals given AuNP-Z showed smaller, compact tumors
(FIG. 4(b)), similar to that observed with direct injection of
zymosan beads (FIG. 1(e)). Immunofluorescent staining of glial
fibrillary acidic protein (GFAP), a marker of reactive astrocytes,
and CSPGs (CS56 antibody) was weak in control and AuNP-P groups
(FIG. 3(b) & FIG. 4(a)). However, CS56 and GFAP
immunoreactivity was strong in the AuNP-Z group (FIG. 3(c)) with
high reactivity within and at the tumor periphery-a phenomenon
reminiscent of non-migratory gliomas. Taken together, these data
show that systemic injection of AuNP-Z particles induced elevated
CSPG and GFAP expression around brain tumors in vivo, and thus
constrained them.
Example 5
[0098] Using AuNP-Z to retard and contain tumor growth (FIG.
5(a)-(c)). Rowett nude (RNU) rats bearing xenogeneic U87 tumors
were injected intravenously with AuNP, AuNP-PEG, and AuNP-Z, single
dosed (SD) or double dosed (DD), starting at 9 DPI. As shown in
FIG. 5(a), tumor volume was plotted longitudinally for all five
experimental groups. Tumor volumes were calculated from MRI images
of animals imaged roughly weekly starting at 9 DPI until death. MRI
images were analyzed for growth by outlining the tumor on 0.5 mm
slices through tumor-bearing rat brains and calculating the volume
(mm3). The timeline of the dosing regime is indicated for the SD
and the DD groups. The dashed line indicates median volume at time
of death of animals in the Control group. Each dot on the growth
curve indicates an MRI session from which the tumor volume was
calculated. AuNP-Z administration led to slower growing tumors with
significantly smaller volumes in animals receiving AuNP-Z
(134.+-.100 mm3 in AuNP-Z SD vs. 311.+-.109 mm3 in Control group)
(FIG. 5(a)). A few days after AuNP administration, the tumors
seemed to pick up in growth rate (FIG. 5(a)); thus, the initial
dose was split equally into two halves and injected into the
animals at 9 DPI and 13 DPI. This dosing regimen led to even
smaller and slower growing tumors (86.+-.35 mm3 in AuNP-Z DD) in
comparison with the single-dose cohort (FIG. 5(b) & 5(c)). FIG.
5(b) shows box and whisker plots summarizing terminal volumes of
the five experimental groups in FIG. 5(a). AuNP-Z administration
curtailed tumor growth significantly compared to other groups. FIG.
5(c) shows representative T2-weighted MRI images of the
experimental groups at the tumor injection site. Images are shown
at 9, 16, and 23 DPI for the same animal. (* p<0.05; one-way
ANOVA (Kruskal-Wallis test), followed by the Uncorrected Dunn's
post-hoc test.)
Example 6
[0099] Proteomic analysis of in vivo response to AuNP-Z (FIG.
6(a)-(c)). Proteomic analyses of tumor biopsy showed that AuNP-Z
administration led to an increase in proteins involved in cell
adhesion, exocytosis, antigen processing, leukocyte-mediated
immunity, and extracellular cell structures (FIGS. 6, 7, & 8).
In stark contrast, administration of AuNP-PEG particles led to the
upregulation of proteins involved in the generation of metabolites,
energy derivation, cellular transport, and CNS growth indicating
continued tumor progression. Taken together, these data indicate
that AuNP-Z administration led to smaller malignant gliomas which
was in part mediated by regulation of pathways related to
inflammation and cell clustering.
[0100] FIG. 6(a) shows a volcano plot depicting differential
expression of proteins in AuNP-Z treated animals relative to
controls (significantly differentially-expressed proteins are
indicated by red dots). Each dot represents a uniquely identified
protein accession. Significance threshold was set to a false
discovery rate (FDR) of <0.05 and a fold change of greater than
2-fold. These proteins are involved in the pathways listed in FIG.
6(b) & (c). FIG. 6(b) is a select list of significantly
over-represented pathways in the AuNP-Z group relative to controls.
FIG. 6(c) is a list of all the significantly enriched pathways in
the AuNP-Z group from GSEA analysis. DE refers to differentially
expressed, ORA to over representation analysis, and NES to
normalized enrichment score.
Example 7
[0101] GSEA of proteomic data (FIGS. 7 & 8). GSEA of proteomic
data indicated enrichment of pathways related to cytostatic,
non-migratory behavior. FIG. 7 shows the results of a gene set
enrichment analysis of AuNP-Z-treated animals relative to controls.
Pathways and significantly enriched core genes are shown. FIG. 8
shows the results of an overrepresentation analysis of
AuNP-Z-treated animals relative to controls. Pathways and
significantly differentially expressed core genes are shown. For
example, the E-cadherin and MHC class pathways were up-regulated
upon AuNP-Z administration in vivo. AuNP-Z administration caused an
enrichment of proteins associated with E-cadherin expression.
AuNP-Z administration led to enrichment of pathways related to
upregulation of MHC class I and II, as well as those related to
complement signaling, and Fc-receptor-mediated phagocytosis.
Pathways related to cytotoxic and phagocytic functions of microglia
require microglia to up-regulated the expression of complement and
Fc-gamma receptors. In contrast, animals dosed with AUNP-PEG showed
enrichment of pathways related with neuronal cell communication and
synaptic transmission, reminiscent of a quiescent microenvironment
that allows for tumor growth. Overall, proteomic analysis points
toward a mode of action of Zpep that involves regulation of cell
adhesion, migration, and immune activation.
Example 8
[0102] Astrocyte activation in gold nanoparticle and AuNP-Z-treated
animals (FIG. 9). FIG. 9 depicts astrocyte activation in AuNP,
AuNP-PEG, and AuNP-Z-treated animals. In particular, FIG. 9 shows
astrocyte activation at the border between normal brain tissue and
the tumor mass. Individual color micrographs represent a unique
animal. The pseudo-colored brown channel image below each color
micrograph shows astrocyte activation as assessed by GFAP
reactivity. While activated astrocytes are present in all groups,
AuNP-Z treated animals show activation of astrocytes both within
and outside the tumor mass, with denser GFAP staining in comparison
to other groups. (Scale bar is 200 .mu.m.)
* * * * *
References