Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins

Article abstract of DOI:10.1002/smll.201803601

Current nanoparticle (NP) drug carriers mostly depend on the enhanced permeability and retention (EPR) effect for selective drug delivery to solid tumors. However, in the absence of a persistent EPR effect, the peritumoral endothelium can function as an a

Full text of DOI:10.1002/smll.201803601

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Polymeric Nanoparticles
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Quinic Acid-Conjugated Nanoparticles Enhance Drug
Delivery to Solid Tumors via Interactions with Endothelial
Selectins
Jun Xu, Steve Seung-Young Lee, Howon Seo, Liang Pang, Yearin Jun, Ruo-Yu Zhang,
Zhong-Yin Zhang, Pilhan Kim, Wooin Lee, Stephen J. Kron, and Yoon Yeo*
1. Introduction
Current nanoparticle (NP) drug carriers mostly depend on the enhanced per-
meability and retention (EPR) effect for selective drug delivery to solid tumors.
However, in the absence of a persistent EPR effect, the peritumoral endothelium
can function as an access barrier to tumors and negatively affect the effective-
ness of NPs. In recognition of the peritumoral endothelium as a potential
barrier in drug delivery to tumors, poly(lactic-co-glycolic acid) (PLGA) NPs are
modified with a quinic acid (QA) derivative, synthetic mimic of selectin ligands.
QA-decorated NPs (QA-NP) interact with human umbilical vein endothelial
cells expressing E-/P-selectins and induce transient increase in endothelial
permeability to translocate across the layer. QA-NP reach selectin-upregulated
tumors, achieving greater tumor accumulation and paclitaxel (PTX) delivery
than polyethylene glycol-decorated NPs (PEG-NP). PTX-loaded QA-NP show
greater anticancer efficacy than Taxol or PTX-loaded PEG-NP at the equivalent
PTX dose in different animal models and dosing regimens. Repeated dosing
of PTX-loaded QA-NP for two weeks results in complete tumor remission in
40–60% of MDA-MB-231 tumor-bearing mice, while those receiving control
treatments succumb to death. QA-NP can exploit the interaction with selectin-
expressing peritumoral endothelium and deliver anticancer drugs to tumors to
a greater extent than the level currently possible with the EPR effect.
Nanoparticles (NPs) have been considered
a promising carrier of chemotherapeutic
drugs.[1] The premise of NP-based chemo-
therapy is based on the notion that tumors
tend to develop hypervasculature and poor
lymphatic systems and, thus, provide
selective access for NPs.[2] This phenom-
enon, called the enhanced permeability
and retention (EPR) effect, has become
the governing principle of most NP-based
drug delivery. Nevertheless, the utility of
NP-based chemotherapy has recently been
challenged, due to the low tumor distri-
bution of NPs[3] and the lack of evidence
supporting the clinical benefits of NPs.[4]
One of the potential reasons that limit NP
delivery to tumors is the complex nature
of the disease and high intersubject and
intrasubject differences, which result in
variable efficiency of the EPR effect.[5] For
the clinical success of NP-based chemo-
therapy, there should be additional means
Dr. J. Xu, Prof. Y. Yeo
Dr. L. Pang
Department of Industrial and Physical Pharmacy
Purdue University
Department of Pharmaceutics
School of Pharmacy
575 Stadium Mall Drive, West Lafayette, IN 47907, USA
E-mail: yyeo@purdue.edu
Fudan University
Shanghai 201203, P. R. China
Dr. S. S.-Y. Lee, Prof. S. J. Kron
Y. Jun, Prof. W. Lee
Ludwig Center for Metastasis Research
The University of Chicago
College of Pharmacy and Research Institute of Pharmaceutical Sciences
Seoul National University
5758 South Maryland Avenue, MC 9006, USA
Seoul 08826, Republic of Korea
Dr. S. S.-Y. Lee, Prof. S. J. Kron
Dr. R.-Y. Zhang, Prof. Z.-Y. Zhang
Department of Molecular Genetics and Cellular Biology
The University of Chicago
Department of Medicinal Chemistry and Molecular Pharmacology
Purdue University
929 East 57th Street, GCIS W519, Chicago, IL 60637, USA
720 Clinic Drive, West Lafayette, IN 47907, USA
H. Seo, Prof. P. Kim
Prof. P. Kim
Graduate School of Nanoscience and Technology and KAIST Institute
for Health Science and Technology
Graduate School of Medical Science and Engineering
KAIST
Korea Advanced Institute of Science and Technology (KAIST)
291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea
291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea
Prof. Y. Yeo
Weldon School of Biomedical Engineering
Purdue University
West Lafayette, IN 47907, USA
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201803601.
DOI: 10.1002/smll.201803601
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to leverage the EPR effect and enhance the delivery efficiency of
NPs beyond the level currently possible.
Ligand-decorated NPs may be produced in various ways.
First, NPs can be incubated with functional ligands to allow
their physisorption on the NP surface.[24] However, the phys-
isorbed ligands have limited stability in blood, where multiple
molecular interactions can interfere with the NP–ligand inter-
actions. Alternatively, ligands may be covalently conjugated to
the surface via chemical reactions between functional groups of
ligands and NP surface.[25] However, this method requires the
presence of reactive functional groups on NPs, which are not
always available in a desirable quantity. This leads to an increas-
ingly complex adaptation of NP platforms, such as prefunc-
tionalization of constituent polymers or chemical activation of
the NP surface, imposing additional difficulties on the product
development. To address this challenge, we employed a simple
surface functionalization method based on dopamine polym-
erization, applicable to a variety of NP platforms irrespective
of their chemical reactivity.[26] The dopamine polymerization
method involves a simple incubation of NPs with dopamine in
an oxidizing condition, which allows dopamine to polymerize
and form a chemically reactive layer on the NP surface that
accommodates ligand molecules.[27] This method allows for
conjugation of various types of ligands, including a QA deriva-
tive, and flexible control of QA density on the NP surface. Such
versatility and flexibility enable us to investigate the optimal
conditions to conjugate QA on NPs for their transendothelial
transport and compare the QA-modified NPs (QA-NP) with
those containing typical stealth coating such as polyethylene
glycol (PEG).
We envision that the vascular endothelium—the first cell
layer circulating NPs encounter—serves as an access barrier to
the underlying tumors; therefore, NPs that can actively interact
with the endothelial barrier will have a greater chance to extrava-
sate into the tumors. In this regard, we note E- and P-selectins,
endothelial adhesion molecules expressed in response to local
inflammatory stimuli. In the vasculature surrounding inflamed
tissues, selectins mediate the interaction of leukocytes with the
endothelium and their trafficking into the underlying tissues.[6]
In addition to the well-known roles in inflammation, selectins
are also implicated in cancer as a mediator of cell adhesion in
metastasis and angiogenesis.[7] On the peritumoral endothelium,
E-selectin promotes angiogenesis[8] and tumor proliferation[9]
and serves as an adhesion point for circulating tumor cells and
endothelial progenitor cells to support metastasis.[10] P-selectin
is expressed constitutively in endothelial cells and platelets and
stored in Weibel–Pallade bodies and α-granules, respectively.[11]
Upon activation, P-selectin translocates from the intracellular
granules to the cell membrane to mediate the adhesion and acti-
vation of leukocytes and platelets.[12] In tumor, P-selectin medi-
ates endothelial interaction with circulating cancer cells as well
as their aggregates with platelets.[13] Consistent with their roles
in tumor progression, selectins are shown to be overexpressed
in various human cancers[14] including breast,[15] kidney,[16]
and lung cancers.[17] A recent study reports that among
420 clinical samples examined by immunohistochemistry, selec-
tive P-selectin expression is observed in multiple tumor types,
including lung (19%), ovarian (68%), lymphoma (78%), and
breast (49%) tumors.[14] In addition, selectin expression in tumor
can be artificially induced by local ionizing radiation,[12b,18]
which is a clinically used cancer treatment protocol. Although
the application for patients with other inflammatory diseases
may be limited, the prevalent selectin expression in various
types of tumor and the potential to induce local selectin expres-
sion in tumors suggest that NPs with affinity for selectins may
preferentially bind to peritumoral endothelium and afford an
increased chance to extravasate into tumors.
In this study, we demonstrate that QA mediates NP inter-
action with activated endothelial cells expressing selectins,
promotes the NP transport across the activated endothelial
layer in vitro, and brings more NPs to tumors than PEG-mod-
ified NPs (PEG-NP) in animal models. Consequently, pacli-
taxel (PTX) loaded in QA-NP (PTX@QA-NP) demonstrates
greater anticancer effects than Taxol or PTX loaded in PEG-NP
(PTX@PEG-NP) at the equivalent dose in multiple animal
models and dosing regimens.
One of the natural ligands for selectins is sialyl Lewis-
x (sLex), an oligosaccharide consisting of fucose, galactose,
N-acetyllactosamine, and N-Acetylneuraminic acid.[19] sLex and
its analogs have been widely explored as selectin antagonists for
the therapy of inflammatory diseases[20] and the prevention of
metastasis.[21] In particular, quinic acid (QA) and its derivatives
have been noted as promising sLex mimics due to the ease of
chemical modification.[22] These sLex mimics have been used
as targeting ligands of macromolecular and NP drug carriers
for the targeted delivery of chemotherapeutic drugs to the peri-
tumoral endothelium.[14,21b,22b,23] The affinity of QA derivatives
for selectins is not high, comparable to sLex, with a millimolar
range of the half maximal inhibitory concentration (IC50), in
the inhibition of P-selectin binding to its natural ligand.[20h] We
reason that the weak affinity ligand–receptor interactions may
be beneficial for NP delivery to tumors, as they will not prevent
the NP transport beyond the endothelial binding. Here, the
anticipated role of QA ligand on NP surface is to transiently
populate NPs on the peritumoral endothelium, permitting the
NPs to extravasate into the tumors.
2. Results and Discussion
2.1. Synthesis of QA-NH2, Synthetic Mimic of sLex
QA-NH2, a synthetic mimic of sLex, was synthesized according
to the previously reported scheme with slight modification
(Figure 1a).[23a] The overall yield was 62%. H NMR confirmed
1
the structure of the compound (Figure S1, Supporting Infor-
mation). Electron spray ionization mass spectrometry (ESI-
MS) found the mass to charge (m/z) ratio of the compound
to be 324.9 (negative ionization mode) as expected (Figure S2,
Supporting Information). QA-NH2 had no adverse effects on
human umbilical vein endothelial cells (HUVEC) proliferation
(Figure S3, Supporting Information).
2.2. Development and Characterization of QA-NP
QA-NP was prepared with the dopamine-mediated surface
modification method.[26] PLGA NPs (bare NP) were first
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Figure 1. a) Schematic of a quinic acid derivative, QA-NH2, a synthetic mimic of sLex. Overall yield: 62%. b) Transmission electron micrographs
of bare PLGA NPs (NP); pD-coated PLGA NPs (NP-pD); and QA-conjugated NP-pD (NP-pD-QA or QA-NP). Negative staining with 2% uranyl acetate.
Scale bar: 100 nm.
prepared by the single emulsion solvent evaporation method.
The bare NP was covered with a polymerized dopamine
(pD) layer, which accommodated QA-NH2 via Michael addi-
tion and/or Schiff base reactions,[27,28] to form QA-NP. The
ESI-MS analysis of QA-NP (Figure S4, Supporting Informa-
tion) provided qualitative evidence for successful conjuga-
tion of QA-NH2 on QA-NP. PLGA NPs simply incubated
with QA-NH2 without pD functionalization (QA/NP) had no
QA-NH2 signature peaks on ESI-MS, confirming the essential
role of pD coating as the mediator of QA-NH2 conjugation.
Transmission electron micrographs (TEM) (Figure 1b) identi-
fied a layer on the surface of the core NPs due to pD coating.
However, there was no visible difference between NP-pD and
NP-pD-QA (QA-NP) in the size and surface morphology, sim-
ilar to previous examples using other ligands.[26] The number
of QA-NH2 conjugated to the surface of QA-NP was indirectly
determined by subtracting the amount of QA-NH2 remaining
in the reaction medium from the original QA-NH2 feed. The
QA-NH2 conjugated to unit surface area of QA-NP increased
linearly with QA-NH2 feed (Figure S5, Supporting Informa-
tion) and did not saturate in the tested range. We attribute the
efficient conjugation to hydrogen bonding between multiple
hydroxyl groups of QA, which may allow for cooperative addi-
tion to the surface.
of aggregation in the buffer in which the DLS measurement was
performed. Prior to the biological evaluation of QA-NP, their
size distribution in 50% fetal bovine serum (FBS) was tested
at a NP concentration ranging from 0.05 to 0.2 mg mL−1. Irre-
spective of the concentration, QA-NP showed a consistent peak
at 120 nm (Figure S6a, Supporting Information), indicating the
resolution of NP aggregation. The two distinctive peaks at 10 and
80 nm were identified to be serum proteins and their aggregates
(Figure S6b, Supporting Information). Over 2 h in 50% FBS, no
aggregation or agglomeration of QA-NP occurred. QA-NP could
Table 1. Particle size, zeta potential, and polydispersity index (PDI)
of NPs. Data: mean s.d. (n = 3 independently and identically prepared
batches).
Name
Z-average [d, nm] Zeta potential [mV]
PDI
Bare NP
134 12
142 17
163 11
167 14
151 14
−10.4 9.3
−11.2 8.1
−10.7 6.2
−12.1 4.9
−11.8 4.9
−9.3 5.5
0.1 0.02
0.2 0.06
0.2 0.04
0.2 0.07
0.1 0.03
0.1 0.03
0.1 0.05
0.1 0.06
0.2 0.03
0.2 0.09
0.2 0.07
0.1 0.04
NP-pD
PEG-NP
PEG-NP (PLGA–DyLight 594)
QA-NP
QA-NP (PLGA–FITC)
QA-NP (PLGA–Alexa 555)
QA-NP (PLGA–ICG)
QA-NP (PLGA–DyLight 594)
Bare NP (PLGA–TPGS)
PTX@PEG-NP (PLGA–TPGS)
PTX@QA-NP (PLGA–TPGS)
149
7
147 11
154 20
158 14
171 33
184 41
179 26
−10.4 6.1
−10.8 5.7
−11.6 6.4
−12.3 6.6
−11.5 5.7
−10.8 6.4
QA-NP had a z-average of 151 nm in diameter and a zeta
potential of −11.8 mV according to dynamic light scattering
(DLS) (Table 1, based on n = 3 independent batches). The par-
ticle size was larger than that estimated by TEM (100–120 nm)
(Figure 1b). This difference is greater than the thickness of the
typically attributed hydration layer, which is no more than a few
nanometers, suggesting that the NPs underwent a mild degree
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be stored as lyophilized solid in 4 °C for up to one month without
HUVECs[31] deposited on the plate surface and interacted with
QA-NP. To quantify the NPs retained with the cells, fluores-
cence intensity of the cell suspension was measured by flow
cytometry. Activated HUVECs incubated with QA-NP showed
the greatest fluorescence intensity compared to control groups
(nonactivated HUVECs with QA-NP (or bare NP) or activated
HUVECs with bare NP), in agreement with confocal imaging.
These results suggest that QA-NP have the affinity for the acti-
vated HUVECs and thus the potential to interact with the peri-
tumoral endothelium.
To confirm the mediators of QA-NP and HUVEC interac-
tions, the incubation was performed after pretreatment with
free QA-NH2, anti-E-selectin, or anti-P-selectin antibodies.
QA-NP binding to the activated HUVECs was reduced by free
QA-NH2 (Figure 2d; Figure S8a, Supporting Information), anti-
E-selectin (Figure 2e; Figure S8b, Supporting Information), and
anti-P-selectin antibodies (Figure 2f; Figure S8c, Supporting
Information) in a dose-dependent manner, which indicates that
free QA-NH2 competes with QA-NP and that the blockade of
E-/P-selectin interferes with QA-NP binding to the activated
HUVECs, respectively. These results confirm that the binding
of QA-NP to the activated HUVECs was mediated by the QA
interaction with E-/P-selectin.
substantial changes in the physical properties (data not shown).
2.3. QA-NP Interact with Activated HUVECs
QA-NP interactions with endothelial cell layer were investi-
gated with HUVECs. The HUVEC model was chosen on the
basis of reported use as a cell model in the investigation of
tumor–endothelial cell interactions.[29] QA-NP–HUVEC inter-
action was examined with confocal microscopy and flow
cytometry (Figure 2a–c). Specifically, HUVECs were incu-
bated with fluorescein (FITC)-labeled QA-NP or bare NP with
or without tumor necrosis factor-alpha (TNF-α) treatment.
A common condition for endothelial expression of selectins
(10 ng mL−1 of TNF-α for 4 h) was followed.[30] Bare NPs
were also tested in the same manner. Figure 2a shows that
QA-NP were associated with the HUVECs with the majority
on the cell membrane, whereas bare NPs showed minimal
nonspecific binding. QA-NP were also observed in empty
space between cells. Given that QA-NP did not bind to the
plate without cells (Figure S7, Supporting Information), we
speculate that soluble E-selectin secreted by the activated
Figure 2. a) Confocal imaging of FITC-labeled NP and QA-NP incubated with resting or TNF-α-activated HUVECs. Green: NPs; blue: cell nuclei;
red: plasma membrane. The box in QA-NP/activated HUVEC image was magnified at the bottom in two channels. b) Representative flow cytometry
histogram. c) Quantitative measurement of HUVECs interacting with NP or QA-NP (n = 3 tests of a representative batch, mean s.d.). ****:
p < 0.0001 versus QA-NP with TNF-α activated HUVEC by Dunnett’s multiple comparisons test. Competitive inhibition of QA-NP binding to HUVECs
by d) free QA-NH2; e) anti-E-selectin antibody; and f) anti-P-selectin antibody. QA-NP to HUVEC binding was quantified by flow cytometry (n = 3 tests
of a representative batch, mean s.d.). ****: p < 0.0001 by Dunnett’s multiple comparisons test. The experiment was repeated with an independently
and identically prepared batch of QA-NP, and the results are presented in Figure S8 (Supporting Information).
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Figure 3. a) Schematic diagram of transendothelial transport of NPs. b) Representative TEER plot of HUVEC monolayer. The arrow indicates the
time of NP addition. c) The percentage of NP transport across the confluent HUVEC monolayer with and without TNF-α activation (n = 3 test
of a representative batch, mean s.d.). ****: p < 0.0001 versus NP with nonactivated HUVECs in each compartment by Holm–Sidak’s multiple
comparisons test. d) Representative TEER plot of HUVEC monolayer with values measured at shorter intervals from the time NP was added. The plot
in a dashed box was magnified on the right. e) Relationship between the ligand density and the transendothelial transport of QA-NP (n = 3 tests of a
representative batch, mean s.d.).
2.4. QA-NP Translocate Across the Activated HUVEC Layer
Inducing Transient Increase in Endothelial Permeaiblity
shorter intervals. With the addition of QA-NP (but not with
bare NP), the TEER value immediately started to decrease over
8 h (Figure 3d). Once QA-NP was removed, the TEER value
was gradually restored over the next 16 h. This result suggests
that the transendothelial transport of QA-NP may be mediated
by the transient increase in paracellular permeability of the
endothelium due to the QA-NP binding to E-/P-selectins.
To examine the relationship between ligand density and
the transendothelial transport, we measured the transport of
QA-NP across the activated HUVECs varying the ligand den-
sities (Figure 3e). QA-NP transport increased as the ligand
density of QA-NP increased from 8 to 36 QA-NH2 nm−2 but
rather decreased with further increase of the ligand density to
74 QA-NH2 nm−2. We speculate that excess QA-NH2 may have
formed intermolecular clustering due to hydrogen bonding
instead of interacting with selectins. Overall, the Transwell
experiment supports that QA-NH2 ligands with an optimal sur-
face density help transport QA-NP across the activated HUVECs.
We hypothesized that the interactions between QA-NP and the
activated HUVEC would increase the chance for NPs to travel
through the endothelial layer and reach the underlying tis-
sues. To estimate this potential in vitro, we assessed the QA-NP
transport across the endothelial cell layer grown on a Transwell
insert (Figure 3a). We placed QA-NP and control NPs on the
apical side of the Transwell containing a confluent HUVEC layer
indicated by the transendothelial electrical resistance (TEER)
values (Figure 3b), with and without TNF-α pretreatment, and
quantified the NPs remaining in the apical side and those recov-
ered from the basolateral side. As expected, the transport across
the endothelial layer was observed only with QA-NP and TNF-
α-activated (i.e., E-/P-selectin expressing) HUVECs (Figure 3c).
Literature suggests that the binding of antibodies or cancer
cells to E-selectin enhances the transendothelial permeability
and cell migration[32] by activating extracellular signal-regulated
kinase and p38 mitogen-activated protein kinase, which initiate
the disassembly of vascular endothelial–cadherin complexes.[33]
We suspect that the binding of QA-NP to activated HUVECs
may have enhanced transendothelial transport of QA-NP in a
similar manner. To test if QA-NP treatment increased transen-
dothelial permeability, we repeated the same experiment
measuring TEER values after QA-NP or bare NP treatment with
2.5. QA-NP Accumulate in Tumors with Forced Selectin
Expression by Focal Irradiation
To test the distribution of QA-NP in vivo, we first used an animal
model with forced E-/P-selectin expression via focal ionizing
radiation.[12b,18,34] For comparison with traditional PEG-modified
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NPs that depend mostly on the EPR effect, PEG-NP were pre-
pared in the same manner as QA-NP except for the replacement
of QA-NH2 with PEG-NH2 (Figure S9, Supporting Informa-
tion). Balb/c mice were inoculated with syngeneic CT26 colon
cancer cells on both hind limbs. One side was treated with 6 Gy
of X-irradiation, and the other was left untreated. One day after
intravenous (IV) injection of QA-NP or PEG-NP, tumors were
collected and optically cleared for confocal imaging with the
immunofluorescence staining of endothelial cells (anti-CD31),
P-selectin, E-selectin, and macrophages (anti-F4/80).[35] QA-NP
displayed greater accumulation in tumor than PEG-NP, even
in the absence of irradiation (Figure 4; Figure S10, Supporting
Information). The difference apparently increased in the irra-
diated tumors, which manifested stronger E- and P-selectin
stains, indicating that QA-NP accumulation in tumor increased
with selectin expression. The irradiated tumors also showed an
increased population of macrophages (a response to irradiation-
induced inflammation[36]); however, we exclude the possibility
of the tumor-associated macrophages mediating NP uptake[37]
for the following reasons: i) PEG-NP accumulation remained
negligible in the irradiated tumor despite the increased mac-
rophage population (Figure 4; Figure S10a, Supporting Infor-
mation), ii) there is little overlap between QA-NP and F4/80+
cells (Figure S10b, Supporting Information), and iii) our in vitro
study suggested that the surface QA suppresses the macrophage
uptake of QA-NP (Figure S11, Supporting Information), likely
due to the multiple hydroxyl groups increasing the hydro-
philicity of the surface. It is worth noting that, at the time of
observation, both E- and P-selectin signals were not colocalized
with CD31 but rather seen with macrophages. This may reflect
Figure 4. Confocal imaging of optically cleared, antibody-stained CT26 tumor sections from animals receiving QA-NP (top) and PEG-NP (bottom),
with or without 6 Gy X-irradiation ( ionizing radiation, IR). Tumors were sampled one day after injection. Different views of each section are shown
in Figure S10 (Supporting Information). n = 2 mice per NP, 2 sections per mouse.
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the transient nature of selectin expression on
endothelial surface[38,39] and the macrophage
uptake of the secreted selectins. To observe
QA-NP transport in cancer models with more
stable expression of endothelial selectins, we
next screened cancer cells that may induce
selectin expression in the endothelial cells.
2.6. Cancer Cells Induce Endothelial Selectin
Expression
Endothelial expression of selectins is
induced by cytokines from the neighboring
cells.[15a,40] To examine whether cancer cell
lines have such paracrine effects on endothe-
lial cells, we incubated endothelial cells in
the media conditioned with various cancer
cells of matching species and measured the
E-/P-selectin expression. All the tested human
cell lines induced the expression of selectins
in HUVECs (Figure S12a,b, Supporting Infor-
mation). MDA-MB-231-conditioned medium
induced E-selectin expression to the greatest
extent, followed by MCF-7-conditioned
medium. P-selectin expression was the
greatest with MCF-7-conditioned medium,
then with MDA-MB-231 and A2780-condi-
tioned media. Similarly, murine B16F10 and
4T1 cells induced significant expression of
E-/P-selectin in mouse hemangioendothe-
lioma (EOMA) cells (Figure S12c,d, Sup-
porting Information). These results support
broad applicability of E-/P-selectin targeting.
As the MDA-MB-231-conditioned medium
induced both E- and P-selectin expressions,
we chose a mouse model of MDA-MB-231
xenograft for subsequent in vivo evaluation
of QA-NP. Among murine cell lines, B16F10
cells were used to make a syngeneic model of
melanoma for additional evaluation of PTX@
Figure 5. a) Representative whole body imaging of animals over 24 h from the injection of each
treatment. Animals bearing subcutaneous MDA-MB-231 tumor xenografts were treated with
QA-NP* (top), PEG-NP* (middle), and free ICG (bottom) by tail vein injection. See Figure S16
(Supporting Information) for all animals receiving the treatments. b) Fluorescence intensity of
ex vivo images of major organs retrieved at the end of whole body imaging (24 h postinjection).
n = 5 mice per group. ****: p < 0.0001 by Bonferroni’s multiple comparisons test following
two-way ANOVA.
QA-NP. E-/P-selectin expression in MDA-MB-231 tumor was
confirmed by immunohistochemistry (Figure S13, Supporting
Information) as well as intravital imaging in live animals (Figure
S14, Supporting Information). In the intravital imaging, E-/P-
selectin expression was mainly observed in the blood vessel
unlike immunohistochemistry of MDA-MB-231 model or clinical
samples,[14] which showed broader selectin distribution. This dif-
ference may be explained by the way the selectins were stained.
While immunohistochemistry stains the sectioned tissues letting
the tumor tissues contact antibodies, intravital imaging provides
antibodies through circulation, where the antibodies are likely to
bind first to the endothelium without proceeding further.
endothelium via E-/P-selectin, translocate across the activated
endothelium, as it did with the activated HUVEC, and accu-
mulate in MDA-MB-231 tumors to a greater extent than PEG-
NP. To trace the NP distribution over time by whole body
fluorescence imaging, we labeled QA-NP with indocyanine
green (ICG), a near infrared fluorescence dye, by conjugating
the dye to PLGA via carbodiimide chemistry. NPs made with
the PLGA–ICG conjugate (ICG-NP) remained stable in 50%
FBS in contrast to those physically encapsulating ICG (ICG/
NP) (Figure S15, Supporting Information), indicating that the
fluorescence of the ICG-NPs would represent NPs in the whole
body imaging.
The ICG-labeled (indicated as *) QA-NP and PEG-NP or free
ICG with equivalent fluorescence intensity were injected via tail
vein and imaged over 24 h (Figure 5a; Figure S16, Supporting
Information). Free ICG spread throughout the body immedi-
ately following the administration and gradually disappeared by
hepatobiliary elimination.[41] On the other hand, QA-NP* and
2.7. In Vivo Fluorescence Imaging Shows QA-NP Distribution
On the basis of the above in vitro results, we hypothesized
that QA-NP might develop active interactions with peritumoral
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PEG-NP* showed up in the liver immediately after the injec-
tion. Significant tumor accumulation of QA-NP* was observed
starting at 2 h postinjection, whereas nearly no fluorescence
was detected in animals treated with PEG-NP*, let alone free
ICG. The QA-NP* signal in the tumor gradually decreased
over time but persisted throughout the 24 h experiment period
(Figure S17, Supporting Information). Animals receiving PEG-
NP* and free ICG did not show fluorescence in tumors at all
time points. It is curious that PEG-NP* was not observed at
all in the tumor. Although not as vascularized as highly angio-
genic LS174T xenografts[42] (Figure S18, Supporting Informa-
tion), MDA-MB-231 xenograft is still more vascularized than
muscle tissues, according to the extent of Evans blue accumula-
tion (LS174T/muscle = 1.34, MDA-MB-231/muscle = 1.04), in
accordance with the literature.[43] We speculate that the fluores-
cence detection threshold was set too high to capture weak fluo-
rescence intensity of tumors with PEG-NP*. Major organs were
excised and imaged ex vivo 24 h postinjection. Consistent with
live whole body imaging, animals receiving PEG-NP* showed
the greatest fluorescence signal in the liver, followed by the
lung and spleen (Figure 5b). QA-NP* showed less accumula-
tion in the reticuloendothelial system (RES) and significantly
higher tumor signals than PEG-NP*. This result was confirmed
in another set of animals (Figure S19, Supporting Information).
The ex vivo imaging obtained at 6 h postinjection showed a sim-
ilar trend as 24 h postinjection, except for the relative distribu-
tion of PEG-NP* in the RES organs. Although the contribution
of the EPR effect may not be excluded, the superior tumor accu-
mulation of QA-NP* relative to PEG-NP*, consistently shown
in Figures 4 and 5, indicates that the selectin-mediated trans-
port plays a dominant role in tumor distribution of QA-NP.
To investigate whether the interaction between QA-NP and
E-/P-selectin helped QA-NP to extravasate and enter tumors,
we used intravital microscopy to visualize QA-NP in mice with
GFP-expressing MDA-MB-231 tumors. QA-NP was observed
from 2 h postinjection near the blood vessel, and the signal
increased over time indicating gradual accumulation of QA-NP
in the tumor (Figure 6). This observation agrees with in vitro
Transwell result and provides an in vivo proof-of-concept that
QA-NP interaction with E-/P-selectin improved extravasation
and enhanced tumor accumulation. It is worth noting that
most QA-NP were nevertheless observed near the vasculature,
which indicates limited intratumoral transport of the NPs.
Given that efficient tumor penetration was reported with NPs
of 50 nm or smaller,[44] further reduction of NP size may be
necessary to improve intratumoral transport of QA-NP. Alter-
natively, conditionally disintegrated assemblies of ultrasmall
NPs[45] are also conceivable to afford both long-term circula-
tion (favoring relatively large size[44c]) and deep intratumoral
penetration (favoring small size[44c]).
superior anticancer efficacy. Typical PTX loading efficiency in
PLGA NPs is no more than 5 wt%.[46] The low drug loading
increases the NP dose requirement and thus the concentra-
tion and/or volume of injection, posing technical challenges
in administration. To increase the drug loading capacity, we
used a conjugate of PLGA and tocopheryl polyethylene glycol
1000 succinate (PLGA–TPGS) instead of PLGA following
the reported method.[47] PLGA–TPGS was synthesized by
ring opening polymerization and confirmed by 1H NMR
(Figure S20, Supporting Information) and matrix-assisted
laser desorption/ionization-MS (MALDI-MS). The molec-
ular weight (MW) ranged 4–8 kDa with an average of 5 kDa
according to MALDI-MS (Figure S21, Supporting Informa-
tion). NP made of PLGA–TPGS showed greater PTX loading
(13.0
ciency: 4.0
by TPGS (PLGA30k/TPGS, loading efficiency: 9.6
0.2%) than those made of PLGA150k (loading effi-
0.01%) or PLGA30k and physically stabilized
0.5%)
(Figure S22a, Supporting Information). Release kinetics per-
formed in phosphate-buffered saline (PBS) containing 0.2%
Tween 80 showed that in 48 h PTX@NP (PLGA–TPGS)
released 59.8 3.5% of the loaded PTX; PTX@NP (PLGA30k
TPGS) 79.1 9.8%; and PTX@NP (PLGA150k) 46.8 0.5%
/
(Figure S22b,c, Supporting Information). TEM images showed
2.8. PTX-Loaded QA-NP Demonstrates Greater In Vivo
Anticancer Efficacy than Taxol or PEGylated NPs
Figure 6. Intravital microscopy images of GFP-expressing MDA-MB-231
tumor in a dorsal skinfold chamber after 2, 6, and 24 h after IV injection
of Alexa Fluor 555-labeled QA-NP. Top: Overlays of blood vessels (blue),
QA-NP* (red), and MDA-MB-231-GFP tumor cells (green). Bottom:
Overlays of blood vessels and QA-NP only.
2.8.1. Production of PTX-Loaded QA-NP
We next examined whether QA-NP would deliver PTX, a
model anticancer drug, better than PEG-NP and achieve
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no significant morphological difference between NPs made
of PLGA and those of PLGA–TPGS (Figure 1 vs Figure S23,
Supporting Information). To confirm the functionality of QA-
conjugated PLGA–TPGS NPs, QA-NPs made of regular PLGA
or PLGA–TPGS (and bare NPs made of respective polymers as
negative controls) were used as competitive inhibitors for the
binding of FITC-labeled QA-NP to the activated HUVECs. The
binding was not affected by the added bare NPs but decreased
with the addition of QA-NPs in a dose-dependent manner, irre-
spective of the polymer type used for the core NPs (Figure S24,
Supporting Information). This result indicates that core NPs
did not affect the surface functionality and PLGA–TPGS NPs
might serve as a valid substitute for PLGA NPs as core parti-
cles. It also demonstrates the robustness and broad applica-
bility of QA conjugation process and suggests the potential for
modular design of QA-modified NPs. On the basis of high drug
loading and consistent surface functionality, we used PLGA–
TPGS in producing PTX-loaded QA-NP (PTX@QA-NP) for the
subsequent in vivo studies. Free PTX, a physical mixture of
PTX and blank QA-NP, and PTX@QA-NP showed similar half
maximal inhibitory concentration (IC50) values in MDA-MB-
231 cells, confirming that QA-NP itself had no additional tox-
icity (Figure S25, Supporting Information).
still induced superior tumor attenuation to Taxol at the same
dose.
Next, PTX@QA-NP was given every three days over 2 weeks
(total five times: q3d × 5) at 20 mg kg−1 per dose (Figure 7b).
PTX@QA-NP offered a significant survival benefit with com-
plete remission in 60% (3 out of 5) animals by 357 days (as of the
submission of this report), while only one surviving in the
Taxol group during the same period. Two animals treated with
Taxol reached the end point based on the tumor size, one with
deteriorating health condition with an ulcerated tumor, and the
other found dead with no apparent reason. These results collec-
tively demonstrate that PTX@QA-NP brings greater anticancer
efficacy than Taxol at the equivalent dose.
2.8.4. Anticancer Efficacy: PTX@QA-NP versus PTX@PEG-NP
PTX@QA-NP was compared with PTX@PEG-NP (both made
with PTX@PLGA–TPGS core particles) at the maximum
tolerated dose. Female nude mice with subcutaneous MDA-
MB-231 animals were treated with 10 injections of one of the
following treatments over the period of 2 weeks: i) saline; ii)
blank QA-NP; iii) blank PEG-NP; iv) PTX@PEG-NP at 30 mg
PTX kg−1 per dose; and v) PTX@QA-NP at 30 mg PTX kg−1 per
dose. Only the PTX@QA-NP group showed a survival benefit
compared to the saline group, with 40% complete remission
as of 367 days (as of the submission of this report) (Figure 8a).
All other groups succumbed to death with a median survival
time of 68 days (saline), 86 days (blank QA-NP), 80 days (blank
PEG-NP), and 70 days (PTX@PEG-NP). It was surprising that
the PTX@PEG-NP group was no better than the negative con-
trol groups and had no surviving animals. We reasoned that
PTX@PEG-NP might have induced the production of anti-PEG
antibodies. The accelerated blood clearance (ABC) phenom-
emon due to anti-PEG IgM is well documented with PEGylated
liposomes and shown to be responsible for rapid elimination of
repeatedly administered liposomes.[50] The extent of ABC may
vary with the extent of modification, and the length and density
of PEG, but since it has been reported with different types of NP
systems,[51] we infer that anti-PEG antibodies may have acceler-
ated the clearance of subsequently dosed PTX@PEG-NPs. To
investigate this possibility, we treated healthy nude mice with
saline, blank PEG-NP, or blank QA-NP, collected blood 5 days
later, and added the serum to a plate coated with mPEG or QA
to determine the presence of antibodies. Animals receiving
PEG-NP produced antibodies that bound to PEG-decorated
surface, while other treatments (saline, QA-NPs) did not
(Figure 8b). QA-NP injection did not induce the production
of anti-QA antibodies. These results demonstrate that QA-NP
delivered PTX to tumors more efficiently than PEG-NP by
repeated administration.
2.8.2. Design of Anticancer Efficacy Studies
The anticancer efficacy of PTX@QA-NP was tested in com-
parison with Taxol, a commercial surfactant-solubilized PTX
formulation, and PTX@PEG-NP, a control NP without QA
modification. The maximum tolerated dose of PTX@QA-NP
was first determined. Healthy female nude mice survived
10 injections of PTX@QA-NP at PTX 30 mg kg−1 per dose over 2
weeks without a significant weight loss (Figure S26, Supporting
Information). Since the reported maximum tolerated dose of
Taxol was relatively low (20 mg kg−1 PTX equivalent, qd × 5),[48]
the evaluation of QA-NP in vivo anticancer efficacy was per-
formed in two different settings: one compared with Taxol at
its known tolerated doses (20 mg kg−1 PTX equivalent, single
administration or q3d × 5 at 20 mg PTX kg−1 per dose over
2 weeks) and the other compared with PTX@PEG-NP at the
NP’s maximum tolerated dose (30 mg kg−1 per dose × 10 times
over 2 weeks).
2.8.3. Anticancer Efficacy: PTX@QA-NP versus Taxol
The anticancer efficacy of PTX@QA-NP was first tested with
a single dose (20 mg kg−1 PTX equivalent) in a female nude
mouse model of subcutaneous MDA-MB-231 tumor. Taxol was
used at the same dose as a reference. As shown in Figure 7a,
PTX@QA-NP attenuated the growth of MDA-MB-231 tumors
more efficiently than Taxol. The median survival time of ani-
mals receiving PTX@QA-NP was 62 days, significantly longer
than that of Taxol (44 days, p < 0.01, Log-rank test). The same
regimen was administered to male C57BL/6 mice with syn-
geneic B16F10 tumors (Figure S27, Supporting Information).
Reflecting the known aggressive nature,[49] the B16F10 tumors
grew much faster than MDA-MB-231 tumors, but PTX@QA-NP
2.8.5. Pharmacokinetics and Biodistribution of PTX Delivered
by QA-NP and PEG-NP
Pharmacokinetics (PK) and biodistribution of PTX were com-
pared following a single IV injection of PTX@QA-NP and PTX@
PEG-NP at a dose equivalent to PTX 20 mg kg−1. The two NPs
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Figure 7. Anticancer efficacy of PTX@QA-NP and Taxol in female nude mice with MDA-MB-231 xenografts. a) Treatments given as a single dose
equivalent to PTX 20 mg kg−1. Mice were sacrificed once the tumor volume (V) reached 2000 mm3 or showed any signs of morbidity defined in the
animal protocol. n = 5 mice per group. Tumor specific growth rate is defined as ∆logV/∆t (t: time in days). *: p < 0.05 by unpaired t-test. b) Treatments
given as five doses of 20 mg PTX equivalent per kg per dose over 2 weeks. n = 5 mice per group. *: p < 0.05 by Gehan–Breslow–Wilcoxon test. Inset
graphs show the tumor growth and survival over 150 days.
showed similar PK profiles and comparable plasma PK param-
eters upon noncompartmental analyses (Table 2 and Figure 8c).
However, PTX@QA-NP resulted in a significantly higher PTX
concentration in tumor than PTX@PEG-NP (7119 ng g−1 for
PTX@QA-NP and 145.5 ng g−1 for PTX@PEG-NP), when meas-
ured at 24 h after IV injection, whereas PTX concentration in
other major organs remained comparable (Figure 8d). This result
is consistent with the superior anticancer efficacy of PTX@
QA-NP relative to PTX@PEG-NP (Figure 8a). The PTX distribu-
tion did not exactly mirror the ex vivo imaging, which showed the
semiquantitative NP distribution at 24 h postinjection (Figure 5b).
This discrepancy may be explained by the preferential removal of
PTX from the RES organs, such as hepatic metabolism[52] and/or
PTX redistribution following phagocytic degradation.
HUVECs and translocated across the confluent HUVEC layer.
QA-NP showed greater accumulation and PTX delivery in
selectin-expressing tumors as compared to PEG-NP. PTX-
loaded QA-NP showed greater anticancer efficacy than Taxol
or PTX-loaded PEG-NP at equivalent PTX doses, leading to
complete tumor remission in 40–60% of MDA-MB-231 tumor-
bearing mice by repeated dosing over 2 weeks. The superior
anticancer efficacy of QA-NP is also partly due to the lack of
antibody formation. Our results support that QA-NP can exploit
the interaction with selectin-expressing peritumoral endothe-
lium and deliver anticancer drugs to tumors to a greater extent
than conventional PEG-NP.
4. Experimental Section
3. Conclusion
Materials: PLGA (LA:GA = 85:15, acid endcap, MW: 150 kDa), PLGA
(LA:GA = 50:50, acid endcap, MW: 30 kDa), FITC-conjugated PLGA
(LA:GA = 48:52, MW: 5 kDa), and PLGA-ethylenediamine (PLGA-NH2,
LA:GA = 57:43, MW: 5 kDa) were purchased from Akina, Inc. (West
Lafayette, IN, USA). Indocyanine green-N-succinimidyl ester (ICG-NHS)
was purchased from Intrace medical (Lausanne, Switzerland). Hoechst
QA was conjugated on the surface of PLGA NPs to pro-
mote the extravasation of the NPs via the interaction with
selectin-expressing peritumoral endothelium. The QA-
decorated NPs (QA-NP) interacted with selectin-expressing
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Figure 8. a) Anticancer efficacy of PTX@QA-NP, compared with those of saline, blank PEG-NP, blank QA-NP, and PTX@PEG-NP. Treatments were
given as 10 doses of 30 mg PTX equivalent per kg per dose over 2 weeks. n = 5 mice per treatment. *: p < 0.05 by Log-rank (Mantel-Cox) test. Inset
graphs show the tumor growth and survival over 150 days. b) Formation of antibodies to each ligand (PEG, QA), detected at 5 days after a single
injection of PEG-NP, QA-NP, or saline (as a negative control). n = 3 mice per group. c) Plasma PTX concentration profile after a single injection of
PTX@QA-NP or PTX@PEG-NP at a dose equivalent to 20 mg kg−1 PTX in MDA-MB-231 xenograft-bearing mice. n = 3 mice per time per group.
d) Biodistribution of PTX in major organs 24 h after a single injection of PTX@QA-NP or PTX@PEG-NP at a dose equivalent to 20 mg kg−1 PTX in
MDA-MB-231 xenograft-bearing mice. n = 3 mice per group. ****: p < 0.0001 by Sidak’s multiple comparisons test.
complete medium (EMEM), Gibco RPMI 1640 medium (RPMI), and
Gibco Iscove’s modified Dulbecco’s medium were purchased from
ThermoFisher Scientific (Waltham, MA, USA). Endothelial cell growth
medium EGM-2 BulletKit was purchased from Lonza, Inc. (Williamsport,
PA, USA). E-selectin antibody (P2H3) FITC, E-selectin antibody (UZ5)
FITC, P-selectin antibody (CTB201), and P-selectin antibody (CTB201)
FITC were purchased from Santa Cruz (Dallas, TX, USA). Transwell
polycarbonate membrane cell culture inserts (6.5 mm) with 3.0 µm pore
were purchased from Corning (Corning, NY, USA). PTX was a generous
gift from Samyang Biopharm (Seongnam, South Korea). All other
materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Synthesis of QA-NH2 (Scheme S1, Supporting Information)—(1S,3R,4S,
5R)-1,3,4,5-tetraacetoxycyclohexane-1-carboxylic acid (Compound 2): Quinic
acid (compound 1, 961 mg, 5 mmol) was dissolved in 12 mL of
acetic anhydride–pyridine 1:2 mixture. The solution was mixed with
4-dimethylaminopyridine (20 mg, 0.16 mmol) and stirred for 12 h at
5 °C. The reaction mixture was then added to ice water, acidified to pH
3 and extracted with dichloromethane (DCM). The extract was dried
with sodium sulfate and concentrated to give a white solid (1.75 g, 97%
Yield). ESI: (M+H)+: 361.
33342 and recombinant human TNF-α were purchased from Invitrogen
(Eugene, OR, USA). Methoxy-polyethylene glycol-amine (mPEG-NH2,
MW: 5 kDa) was purchased from JenKem Technology USA (Allen, TX,
USA). Collagen I rat-tail, Vacutainer PST Tubes with spray-coated lithium
heparin and a gel for plasma separation, Vacutainer Plus Plastic
Serum Tubes with spray-coated silica for serum separation, and
Calcein AM were purchased from BD Biosciences (San Jose, CA, USA).
Dopamine hydrochloride was purchased from Alfa Aesar (Ward Hill,
MA, USA). CellMask deep red membrane staining dye, Alexa Fluor
555 NHS ester (succinimidyl ester), DyLight 594-NHS ester, goat anti-
mouse IgM secondary antibody, HRP-conjugated, Gibco Dulbecco’s
modified eagle’s medium (DMEM), Gibco Eagle’s minimum essential
Table 2. Plasma PK parameters of PTX@PEG-NP and PTX@QA-NP.
PK parameters were obtained from noncompartmental analyses. The
numbers in parentheses denote the standard errors (SE) associated with
estimated parameters.
PK parameter
Formulation
PTX@QA-NP
PTX@PEG-NP
7.7
(1R,2S,3R,5S)-5-((3-amino-5-(methoxycarbonyl)phenyl)carbamoyl)
cyclohexane-1,2,3,5-tetrayl tetraacetate (Compound 3): Compound
2
t1/2 [h]
5.4
2.4 (0.05)
11.0 (0.1)
11.6
(958 mg, 2.7 mmol) was dissolved in 15 mL of dimethylformamide
(DMF). The solution was mixed with N-hydroxybenzotrizole (432 mg,
3.2 mmol) and stirred for 5 min at 0 °C. (1-ethyl-3-[3-dimethylaminopropyl]
carbodiimide (612 mg, 3.2 mmol) and triethyl amine (0.5 mL, 4.3 mmol)
were then added to the mixture and stirred for 1 h at 0 °C. Finally, a
solution of methyl 3,5-diaminobenzoate (1.7 g, 7.7 mmol) in DMF (5 mL)
was added to the mixture, which was warmed up to room temperature
and stirred for 72 h. The reaction mixture was then added to ice water
and extracted with DCM. The extract was dehydrated with sodium sulfate,
Cmax [µg mL−1
]
3.4 (0.5)
10.6 (0.4)
11.3
AUC0-last [h µg mL−1
]
AUC0-∞ [h µg mL−1
]
MRT [h]
5.8
6.3
CL [L h−1 kg−1
Vss [L kg−1
]
1.8
1.7
10.4
11.0
]
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filtered, and further evaporated. The residue was purified by column
chromatography on silica gel (hexane-ethyl acetate 3:7) to yield a brown
solid compound (1.3 g, 92% Yield). ESI: (M+H)+: 509.
functionalized NPs were designated as QA-NP (or NP-pD-QA) and
PEG-NP (or NP-pD-PEG), respectively.
Physical Characterization of NPs: NPs were dispersed in phosphate
buffer (1 × 10−3 m, pH 7.4), and their sizes and zeta potentials were
measured by a Malvern Zetasizer Nano ZS90 (Worcestershire, UK). NP
morphology was observed by Tecnai transmission electron microscopy
(FEI, Hillsboro, OR, USA) after negative staining with 2% uranyl acetate.
QA-NH2 surface decoration was confirmed by ESI-MS analysis of
QA-NP. PEG-NH2 surface decoration was confirmed by MALDI-TOF MS.
QA-NH2 Ligand Density on NPs: A known amount of NP-pD was
resuspended in Tris buffer (10 × 10−3 m, pH 8.5) containing a known
amount of QA-NH2 (QA-NH2 feed). After 30 min incubation, the NP was
collected via ultracentrifugation, and the supernatant was analyzed by an
Agilent 1100 high-pressure liquid chromatography (HPLC) system (Palo
Alto, CA, USA) equipped with Ascentis C18 column (25 cm × 4.6 mm,
particle size: 5 µm) to quantify unconjugated free QA-NH2. The mobile
phase was a 90:10 volume mixture of water and ACN and eluted at
0.5 mL min−1. QA-NH2 was detected by a UV detector at the wavelength
of 227 nm. The surface-conjugated QA-NH2 was calculated by subtracting
the unconjugated QA-NH2 from the QA-NH2 feed. The QA-NH2 ligand
density (QA-NH2 per nm2) was calculated as the number of surface-
conjugated QA-NH2 divided by the total surface area of NP-pD.
NP Stability in Serum: For evaluation of size stability, 5 mg of QA-NP
were suspended in 5 mL of 50% FBS and incubated for 2 h at room
temperature. Particle size distribution was measured by a Malvern
Zetasizer Nano ZS90. For evaluation of fluorescence stability, ICG-labeled
QA-NP was suspended in 50% FBS. NP pellet and supernatant were
separated by ultracentrifugation at prespecified time points and imaged
with IVIS Lumina to detect fluorescence intensity.
PTX Loading Efficiency: NPs with a premeasured mass were dissolved
in 50% ACN at a concentration of 50 µg mL−1 and filtered with 0.45 µm
polyvinylidene fluoride (PVDF) syringe filter. HPLC analysis was
performed with an Agilent 1100 HPLC system equipped with Ascentis
C18 column (25 cm × 4.6 mm, particle size: 5 µm). The mobile phase
was a 50:50 volume mixture of water and ACN and run at 1 mL min−1.
PTX was detected by a UV detector at a wavelength of 227 nm. Drug
loading efficiency was defined as the PTX amount divided by the
NP mass.
3-Amino-5-((1S,3R,4S,5R)-1,3,4,5-tetrahydroxycyclohexane-1-carboxa
mido)benzoic acid (Compound 4): A solution of compound 3 (102 mg,
0.2 mmol) in tetrahydrofuran (5 mL) was stirred with lithium hydroxide
monohydrate (63 mg, 1.5 mmol) for 48 h at room temperature. The
reaction mixture was then acidified to pH 5 using Amberlite acidic resin,
filtered, and purified by reverse column chromatography on C18-reversed
phase silica gel (water–acetonitrile (ACN) 1:2) to yield a white solid
compound (45 mg, 69% Yield). ESI: (M+H)+: 327.
Synthesis of ICG-Conjugated PLGA, Alexa Fluor 555-Conjugated
PLGA, and DyLight 594-Conjugated PLGA: 200 mg of PLGA-ethylene
diamine were dissolved in 2 mL dimethyl sulfoxide (DMSO) at room
temperature. 1.5 mg of ICG-NHS, Alexa Fluor 555-NHS, or DyLight 594-
NHS was dissolved in 1 mL DMSO at room temperature with rigorous
vortex mixing. 1 mL of N,N-diisopropylethylamine was added to PLGA
solution, followed by dropwise addition of ICG-NHS, Alexa Fluor 555-
NHS or DyLight 594-NHS solution. The mixture was constantly stirred
for 2 h, put in a regenerated cellulose dialysis bag with a MW cut off
3500 Da (Spectrum Labs, Rancho Dominguez, CA, USA) and dialyzed
against excess DMSO 3 times and DCM 3 times. The purified sample
was collected via rotary evaporation and stored at −20 °C.
Synthesis of TPGS-Conjugated PLGA (PLGA–TPGS): Glycolide 1 g,
lactide 1 g, D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)
400 mg were dissolved in toluene containing 0.5 wt% stannous octoate.
The reaction was carried out at 145 °C under oxygen- and moisture-free
environment for 16 h. The resulting product was dissolved in DCM
and then precipitated in excess cold methanol to separate unreacted
reagents. The final product, PLGA–TPGS, was collected by filtration,
washed twice with distilled water, and dried under vacuum at 60 °C for
72 h.
Preparation of Bare NP: Bare NP was produced by the single emulsion
solvent evaporation method. In brief, 100 mg of PLGA was dissolved
in 4 mL of DCM. The polymer solution was added to 12 mL of a 5%
polyvinyl alcohol (PVA) solution and emulsified using a Sonics Vibracell
probe sonicator (Sonics, Newtown, CT, USA) for 2 min. The emulsion was
added to 40 mL of deionized (DI) water and stirred for 4 h to evaporate
DCM. The NP was then collected by an Optima MAX-XP ultracentrifuge
(Beckman Coulter, Brea, CA, USA) at 34 000× g and washed with DI
water three times. For confocal microscopy and flow cytometry, NP was
prepared with FITC-conjugated PLGA. For imaging of NPs in focally
irradiated tumors, NP was prepared with DyLight 594-conjugated PLGA.
For optical in vivo imaging, NP was prepared with ICG-conjugated
PLGA. For intravital microscopy, NP was made with Alexa Fluor
555-conjugated PLGA. PTX-encapsulated NPs (PTX@NP) were prepared
with PLGA150k, PLGA30k, and PLGA–TPGS5k. For PTX@NP with PLGA150k
(PTX@NP (PLGA150k)), 20 mg of PLGA150k and 2 mg of PTX were dissolved
in 1 mL of DCM, homogenized in 4 mL of 5% PVA by probe sonication
(2 min, with 4 s on, 2 s off at 40% amplitude), and stirred in 20 mL of DI
water overnight. For PTX@NP with PLGA30k, 20 mg of PLGA30k and 2 mg
of PTX dissolved in 1 mL of DCM were homogenized in 4 mL aqueous
solution containing 0.5% PVA and 0.1% TPGS, and stirred in 20 mL of
0.5% PVA overnight. To indicate the use of TPGS as an emulsifier, we
called the NPs PTX@NP (PLGA30k/TPGS). PTX@NP with PLGA–TPGS5k
(PTX@NP (PLGA–TPGS5k)) was prepared by homogenizing 1 mL
DCM solution of 20 mg of PLGA–TPGS5k and 2 mg of PTX in 4 mL of
5% PVA and stirring in 20 mL of DI water. The NPs were collected via
ultracentrifugation, and washed three times prior to freeze drying.
NP Surface Modification: The core NPs were coated with pD by 3 h
incubation in 2 mL of dopamine hydrochloride solution in Tris buffer
(10 × 10−3 m, pH 8.5) at room temperature with rotation. The pD-coated
PLGA NPs (NP-pD) were collected by ultracentrifugation and washed
twice with DI water. For further surface functionalization, NP-pD
were resuspended in Tris buffer containing QA-NH2 or mPEG-NH2,
maintaining the weight ratio of NP-pD to the ligands at 1:2 unless
specified otherwise. After 30 min incubation, the NPs were collected
via ultracentrifugation and washed with DI water twice. The surface-
PTX Release Kinetics: NPs equivalent to 10 µg of PTX were suspended
in 1 mL of PBS containing 0.2% Tween 20 and incubated at 37 °C
under constant agitation. At timed intervals, NP suspension was
ultracentrifuged at 34 000× g for 15 min. 0.8 mL of supernatant was
sampled for HPLC analysis and replaced with 0.8 mL of fresh release
medium. NPs were resuspended in the medium by brief ultrasonication
and returned to incubation.
Cell Culture: HUVECs (ATCC, Manassas, VA, USA) at passage 4 were
grown in EGM-2 complete medium. Culture plates were precoated with
5 µg cm−2 of rat tail collagen type I. Human breast adenocarcinoma
cells (MDA-MB-231, ATCC; MCF-7, ATCC) were cultured in DMEM
supplemented with 10% FBS. Human colon adenocarcinoma cells
(LS174T, ATCC) were cultured in EMEM supplemented with 10% FBS.
Human ovarian cancer cells (A2780, ATCC) were cultured in RPMI
supplemented with 10% FBS. Human ovarian cancer cells (NCI/
ADR-RES) were cultured in DMEM supplemented with 10% FBS.
Mouse hemangioendothelioma cells (EOMA, ATCC) were cultured
in DMEM supplemented with 10% FBS. Mouse mammary carcinoma
cells (4T1, ATCC) were cultured in RPMI supplemented with 10%
FBS. Mouse melanoma cells (B16F10, ATCC) were cultured in RPMI
supplemented with 10% FBS. Mouse monocyte macrophages (J774A.1,
ATCC) were cultured in DMEM supplemented with 10% FBS. All
media were supplemented with 100 units mL−1 penicillin and 100 µg
mL−1 streptomycin. All cells were subcultured at a ratio of 1:7.5 when
they became 70–80% confluent. HUVECs was used between 4 and 7
passages, and all other cells were used in less than 20 passages.
Cytotoxicity of QA-NH2 on HUVECs: HUVECs were seeded in a 96 well
plate at a density of 10 000 cells per well coated with rat tail collagen
type I. A group of cells were incubated with 10 ng mL−1 of TNF-α for
4 h and then treated with QA-NH2 (1 × 10−9 m to 1 × 10−3 m) for 24 h.
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The cell viability was determined by the MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay. Cells were treated with 75 µg
of MTT and incubated for 4 h. The formazan crystals were dissolved in
stop/solubilization solution (50% DMF, 20% sodium dodecyl sulfate
(SDS), pH 5) and quantified by a SpectraMax M3 microplate reader
(Molecular Devices, CA, USA) at the wavelength of 562 nm. The
measured absorbance was normalized to the absorbance of control cells
that did not receive any treatment.
When HUVECs became 70–80% confluent, cells were exposed to
the culture media conditioned with MCF-7, MDA-MB-231, LS174T,
A2780, or NCI-ADR cells for 4 h. Respective cell-free media were used
as medium controls, and TNF-α (10 ng mL−1) was used as a positive
control. The HUVECs were trypsinized and collected via centrifugation.
The cell pellets were resuspended in PBS and stained with FITC-labeled
anti-human E-selectin or anti-human P-selectin antibody according
to the manufacturer’s protocol. The extent of E-selectin or P-selectin
expressions was quantified by flow cytometry.
Cytotoxicity of PTX@QA-NP on MDA-MB-231 Cells: MDA-MB-231 cells
were seeded in a 96 well plate at a density of 10 000 cells per well. After
overnight incubation, the culture medium was replaced with fresh DMEM,
to which PTX, PTX + QA-NP, and PTX@QA-NP were added to provide the
final PTX concentration ranging from 0.1 × 10−9 m to 100 × 10−6 m. After
72 h incubation, the cell viability was determined by the MTT assay.
Confocal Microscopy of In Vitro NP–Cell Interactions: HUVECs were
seeded in a 35 mm dish with a glass window (MatTek Corporation,
Ashland, MA, USA) coated with rat tail collagen type I and grown
in EGM-2 complete medium. When HUVECs were 80% confluent,
cells were treated with 10 ng mL−1 of TNF-α for 4 h. The medium
was replaced with EGM-2 containing 0.2 mg mL−1 of FITC-labeled NP
suspension and incubated for 2 h at 37 °C. The suspension was replaced
with fresh EGM-2 medium containing 0.5 × 10−6 m CellMask deep red
membrane staining dye. After 10 min, cells were gently rinsed with PBS
twice. Hoechst nuclear stain (10 µL at the concentration of 0.2 mg mL−1)
was added 10 min prior to the imaging. Imaging was performed with
a Nikon A1R confocal microscope (Melville, NT, USA) equipped with a
Spectra Physics 163C argon ion laser and a Coherent CUBE diode laser.
The NPs, cell nuclei, and cell membrane were excited using 495, 361,
and 649 nm laser, respectively, and detected at 519, 497, and 666 nm.
Flow Cytometry for Quantitative Analysis of NP–Cell Interactions: For
quantitative analysis of NP–cell interaction, HUVECs were treated with
FITC-labeled NPs in the same way as confocal microscopy and gently
washed with PBS twice to remove free or loosely bound NPs. The cells
were then trypsinized, collected by centrifugation at 930× g for 5 min,
resuspended in 0.2 mL of PBS at 4 °C, and analyzed by an Accuri C6 flow
cytometer (BD Biosciences, San Jose, CA, USA) equipped with an FL-1
detector (λex/λem = 488/525 nm).
For competition assay, free QA-NH2, E-selectin antibody, or P-selectin
antibody were preincubated with HUVECs for 30 min prior to the NP
treatment and flow cytometry analysis. In another competition assay,
known concentrations of NP, NP (PLGA–TPGS5k), QA-NP, and QA-NP
(PLGA–TPGS5k) were preincubated with the activated HUVECs for
30 min. The pretreated HUVECs were incubated with FITC-labeled
QA-NP for 2 h and analyzed by flow cytometry.
Macrophage interactions of bare NP and QA-NP were compared.
J774A.1 macrophages were seeded at a density of 100 000 cells cm−2
in a 6-well plate. When 70–80% confluent, the cells were incubated with
0.1 mg mL−1 of FITC-labeled QA-NP, NP, or NP-pD at an equivalent
fluorescence level in DMEM and incubated for 2 h at 37 °C. The cells
were washed with PBS twice and analyzed by flow cytometry.
Mouse Cell Lines: EOMAs at passage 2 were seeded in a 6-well plate at
a density of 10 000 cells cm−2. When EOMAs became 70–80% confluent,
cells were exposed to the culture media conditioned with 4T1 or B16F10
cells for 4 h. Respective cell-free media were used as medium controls,
and TNF-α (10 ng mL−1) and lipopolysaccharide (O111:B4 from E. coli,
10 ng mL−1) were used as positive controls. EOMAs were collected in
the same way as above, stained with FITC-labeled anti-mouse E-selectin
or anti-mouse P-selectin antibody, and analyzed by flow cytometry.
Immunohistochemistry of E-/P-Selectin Expression in MDA-MB-231
Xenograft Model: MDA-MB-231 xenograft tumors were fixed in formalin
and embedded in paraffin blocks. The tissue blocks were cut in 3 µm
thick sections, deparaffinized by xylene, and treated with 0.3% hydrogen
peroxide and the antigen retrieval buffer (10 × 10−3 m Tris, 1 × 10−3
m
ethylenediaminetetraacetic acid (EDTA), 0.03% Tween 20). The sections
were blocked with PBS containing 4% bovine serum albumin (BSA)
and 0.03% Tween 20. The sections were incubated with anti-E-selectin
antibody (1:800 dilution, AF575, R&D system) and anti-P-selectin
antibody (1:200 dilution, AF737, R&D system) for 1 h, followed by
corresponding secondary antibodies conjugated with horse peroxidase
(One-step or Two-step Polymer Detection Systems, Nichirei, Japan). The
selectin signals were visualized by incubation with 3, 3′-diaminobenzidine
(DAB) for 7 min. The sections were counterstained with hematoxylin and
imaged with an Eclipse 80i microscope (Nikon, Japan).
Confocal Imaging of QA-NP Accumulation in X-Irradiated Tumor: Balb/c
female mice (6–8 weeks old) were purchased from Envigo (Indianapolis,
IN, USA). All mice were maintained under pathogen-free conditions. The
study was approved by the Institutional Animal Care and Use Committee
of the University of Chicago, and all experiments conformed to the relevant
regulatory standards. CT26 cells (ATCC CRL-2638) were cultured in 5%
CO2 and maintained in DMEM supplemented with 10% heat-inactivated
fetal bovine serum, 2 × 10−3 m L-glutamine, and 100 units mL−1 penicillin.
To form subcutaneous tumors, 5 × 105 CT26 cells were injected into both
hind limbs of a mouse. Ten days after tumor implantation, CT26 tumor
on the right hind limb was X-irradiated with 6 Gy using an X-RAD 225
Cx image-guided radiotherapy system (Precision X-Ray) at 225 kV, 13 mV,
1.1 mm Cu half-value layer, and a dose rate of 2.5 Gy min−1. At 2 days
after radiation, DyLight 594-labeled QA-NP or PEG-NP (1 mg in 0.2 mL
PBS) solution was IV injected to the mouse. At 1 day postinjection of
NPs, CT26 tumors were harvested, washed with cold PBS, fixed with 2%
paraformaldehyde in PBS for 10 min at room temperature, and washed
with PBS. The washed tumors were cast in 2% agarose gel (dissolved
in distilled water, LE Quick Dissolve Agarose, GeneMate) in 24 well
plates. The gel plugs containing tumors were mounted on a vibrating
microtome (VT1200S, Leica) equipped with a buffer tray. Sections (in
0.4 mm thickness) were collected in order in cold PBS. The macrosections
were stained with DyLight 488-anti-F4/80 (BioXcell, clone CI:A3-1),
DyLight 550-anti-E-selectin (BioLegend, clone RME-1/CD62E), DyLight
633-anti-CD31 (BioLegend, clone MEC13.3), and DyLight 680-anti-P-
selectin (BioLegend, clone RMP-1) antibodies in staining buffer (SB,
RPMI 1640 media with 10 mg mL−1 BSA and 0.1% Triton X-100) at 4 °C
overnight. The macrosections were incubated in 10 mL of 80% D-fructose
solution for 1 h at 25 °C with gentle agitation. The stained sections were
imaged by a Leica TCS SP8 confocal laser scanning microscope, equipped
with a white light laser, a HC PL APO 40X/1.25 NA oil objective (0.24 mm
working distance), 488 nm excitation and 495–528 nm emission filter
for DyLight 488, 550 nm excitation and 563–579 nm emission filter for
DyLight 550, 594 nm excitation and 600–620 nm filters for DyLight 594,
633 nm excitation and 637–655 nm filter for DyLight 633, and 670 nm
excitation and 680–700 nm filter for DyLight 680.
Transendothelial NP Transport Across Confluent HUVECs: HUVECs were
seeded in a Corning Transwell insert coated with rat tail collagen type I
and grown in EGM-2 complete medium. The TEER across the HUVEC
layer in the Transwell insert was monitored daily by EVOM2 epithelial
voltohmmeter (World Precision Instruments, Sarasota, FL, USA). When
the TEER value reached a plateau (indicating confluency), the HUVEC
layer was treated with 10 ng mL−1 of TNF-α for 4 h, followed by incubation
with 0.1 mg mL−1 of FITC-labeled NP or QA-NP for 8 h. The fluorescence
intensity of total NPs prior to incubation and those of NPs in the apical
and basolateral sides of a Transwell insert after incubation period were
measured by FluoroMax-3 spectrofluorometer (Horiba Scientific, Edison,
NJ, USA). In a separate study, the confluent, TNF-α activated HUVECs
were treated with 0.1 mg mL−1 of NP or QA-NP for 8 h, followed by 16 h
incubation in NP-free medium. The TEER values were measured at 0, 4,
8, 12, and 24 h from the NP treatment.
Endothelial Expression of E-Selectin and P-Selectin by Tumor-Conditioned
Media—Human Cell Lines: HUVECs were seeded in a 6-well plate
coated with rat tail collagen type I at a density of 10 000 cells cm−2.
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Whole Body Imaging: Female athymic nude mice (Foxn1nu) at the age
of 6–7 weeks were purchased from Envigo (Indianapolis, IN, USA) and
acclimatized for 3 days prior to the procedure. All animal procedures
were approved by the Purdue Animal Care and Use Committee, in
compliance with the NIH guidelines for the care and use of laboratory
animals. Each mouse received 5 × 106 MDA-MB-231 cells in the flank
of hind leg by subcutaneous injection. When the average tumor volume
reached 200 mm3, each mouse received 6 mg of ICG-labeled QA-NP or
PEG-NP in 0.9% saline via tail vein injection. The animals were imaged
with an IVIS Lumina II system (Caliper Life Sciences, Hopkinton, MA,
USA) to detect near infrared fluorescence signal of ICG over 24 h. After
24 h, mice were sacrificed, and tumors and major organs were retrieved
and imaged with the IVIS Lumina system. The experiment was repeated
with another set of tumor-bearing animals receiving 6 mg of ICG-labeled
QA-NP or PEG-NP in 0.9% saline via tail vein injection. The image of ex
vivo organs was taken at 6 h postinjection using the IVIS Lumina system.
Intravital Microscopy: Female Balb/c nude mice at the age of 8 weeks
were purchased from OrientBio (Suwon, Korea). All animal experiments
were performed in accordance with the standard guidelines for the
care and use of laboratory animals as approved by the Institutional
Animal Care and Use Committee of KAIST (protocol no. KA2013-30). All
surgeries were performed under anesthesia. A dorsal skinfold chamber
was implanted into a mouse.[53] Immediately after the implantation, a
mixture of 3 × 106 MDA-MB-231-GFP cells and Corning Matrigel matrix
was injected to the center of a dorsal skinfold window chamber. After
1 week from the implantation, intravital imaging was performed. Two
milligrams of Alexa Fluor 555-labeled QA-NP was IV injected via tail
vein through a custom-built catheter composed of 30 gauge needle
tip and a polyethylene tube. At 2, 6, and 24 h after the NP injection,
tumor microenvironment was repeatedly visualized to locate the NPs. At
3 h before intravital imaging, 25 µg of anti-CD31 monoclonal antibody
(553 370, BD) conjugated to Alexa Fluor 647 (A20106, Invitrogen) was IV
injected to label the blood vessels. For P-selectin (CD62P) or E-selectin
(CD62E) labeling, 25 µg of anti-P-selectin monoclonal antibody (553742,
BD) or anti-E-selectin monoclonal antibody (553749, BD) conjugated to
Alexa Fluor 647 (A20106, Invitrogen) was IV injected at 3 h before the
intravital imaging. During intravital imaging, the body temperature of
the mouse was continuously monitored and maintained at 37 °C by the
homeothermic system (RightTemp, Kent Scientific).
growth rate of a tumor was calculated as ∆logV/∆t (t: time in days).[56]
Mice with ulcerated tumors or tumors greater than 2000 mm3 were
euthanized in a humane manner. Two other sets of experiments were
carried out to compare PTX@QA-NP with Taxol: One group received
a single dose at 20 mg kg−1 PTX equivalent, and the other received 5
doses of 20 mg kg−1 PTX equivalent every 3 days over 2 weeks.
Male C57BL/6 mice at the age of 5–6 weeks were purchased from
Envigo (Indianapolis, IN, USA) and acclimatized for 3 days prior to the
procedure. Each mouse received 5 × 106 B16F10 cells in the flank of
hind leg by subcutaneous injection. When the averaged tumor volume
reached 100 mm3, each mouse received a single dose of PTX@QA-NP
or Taxol at 20 mg kg−1 PTX equivalent and was monitored in the same
way as above.
Comparison of Tumor Vascularization: Female athymic nude mice
(Foxn1nu) at the age of 6–7 weeks received 5 × 106 MDA-MB-231 cells or
LS174T cells in the flank of hind leg by subcutaneous injection. When the
averaged tumor volume reached 200 mm3, 200 µL of 0.25% Evans blue
(Abcam, Cambridge, UK) in PBS was injected via tail vein. After 1 h, the
mice were sacrificed, and the tumors and muscle tissues were imaged.
Detection of Anti-PEG Antibody in NP-Treated Animals: Female
athymic nude mice (Foxn1nu) at the age of 6–7 weeks received tail-
vein injection of saline or PEG-NP or QA-NP (30 mg kg−1). Five
days later, blood was obtained by cardiac puncture and collected in
a BD Vacutainer Plus Plastic Serum Tube. Serum was separated by
centrifugation at 930× g for 15 min. In the meantime, 200 µL of 2 mg
mL−1 dopamine Tris solution was added to each well of a 96-well plate
for surface coating. After 4 h incubation at room temperature, the plate
was triple rinsed with DI water and incubated with 200 µL of 2 mg mL−1
mPEG-NH2 or QA-NH2 for each ligand decoration. After
1
h, the plate was triple rinsed with DI water and treated with
100 µL of 1% human serum albumin (HSA) blocking buffer (Tris
buffered saline + 1% HSA + 0.05% Tween 20) for 1 h, followed
by quintuple rinse. The blocked well was incubated with 100 µL
of diluted sera from the NP-treated animals (1:20 dilution) for 1
h and rinsed 5 times. Goat anti-mouse IgM IgG-HRP conjugate
(0.8 or 4 µg mL−1) was added to each well for 1 h incubation followed
by quintuple rinse. Another 100 µL of 1% HSA blocking buffer was
added to each well for 1 h incubation and rinsed 5 times. Finally, each
well was treated with 50 µL of 3,3′,5,5′-tetramethylbenzidine for 5 min
to develop a blue color corresponding to the amount of the bound
IgG-HRP, followed by quenching with 2 m sulfonic acid. The substrate
solution was transferred to another plate for absorbance measurement
at 450 nm.
A
custom-built video-rate laser-scanning confocal microscopy
system[54] was used for intravital imaging. Three laser modules with
output wavelengths at 488 nm (MLD488, Cobolt), 561 nm (Jive, Cobolt),
and 640 nm (MLD640, Cobolt) were used as excitation light sources.
Three photomultiplier tubes (R9110, Hamamatsu) with bandpass filters
(FF02-525/50, FF01-600/37, FF01-685/40, Semrock) were used for
multicolor confocal fluorescence imaging. To achieve 2D laser beam
scanning at video-rate of 30 Hz, a rapidly rotating polygonal mirror with
36 facets (MC-5, aluminum coated, Lincoln Laser) for x-axis scanning
and a galvanometer scanning mirror (6230H, Cambridge Technology)
for y-axis scanning were used. A commercial objective lens (CFI Plan
Apo lambda, 20X, NA 0.75, Nikon) was used to capture confocal
fluorescence images.
PK and Biodistribution of PTX: Female athymic nude mice (Foxn1nu) at
the age of 6–7 weeks were inoculated with 5 × 106 MDA-MB-231 cells
in the flank of hind leg by subcutaneous injection. When the average
tumor size reached 100 mm3, animals were administered with PTX@
PEG-NP, PTX@QA-NP, and Taxol at a PTX dose of 20 mg kg−1 by tail
vein injection. At predetermined time points, 3 mice were randomly
sacrificed for the collection of blood and major organs. Blood was
obtained by cardiac puncture and collected in a BD Vacutainer PST tube.
Plasma was separated from whole blood via centrifugation at 3000× g
for 15 min.
Plasma and tissue concentrations of PTX were quantified using
docetaxel (DTX) as the internal standard. Samples were prepared by
liquid–liquid extraction. Specifically, plasma samples were diluted with
water and extracted with methyl tertiary butyl ether. The samples were
mixed and spun at high speed. The organic layer was transferred to a tube,
evaporated to dryness, the tube was reconstituted with methanol, and a
small volume was injected into the HPLC-MS/MS. For tissues samples,
frozen tissue was weighed, phosphate buffer (100 × 10−3 m, pH 7.4) was
added, and the tissue was homogenized using a Qiagen TissueRuptor
with disposable probes. A volume of homogenate was removed,
internal standard added, and the tissue sample was extracted using
the same procedure that was used for plasma. Standards containing
known amounts of PTX were prepared to estimate the concentrations
in the samples. The standards ranged from 0.1–1000 ng mL−1
(n = 10 standards including a 0) for mouse plasma samples. The
Determination of Maximum Tolerated Dose of PTX@QA-NP: Female
athymic nude mice (Foxn1nu) at the age of 6–7 weeks were administered
with PTX@QA-NP varying the frequency of the dosing. Each time,
animals received the NPs at a PTX dose of 30 mg kg−1 through tail vein
injection and monitored over 15 days. The body weight was measured
every other day. Weight loss in excess of 20% was considered a humane
endpoint.
In Vivo Anticancer Efficacy: Female athymic nude mice (Foxn1nu) at the
age of 6–7 weeks received 5 × 106 MDA-MB-231 cells in the flank of
hind leg by subcutaneous injection. When the averaged tumor volume
reached 100 mm3, each mouse received saline, bare QA-NP, bare PEG-
NP, PTX@PEG-NP, or PTX@QA-NP at a dose equivalent to 30 mg kg−1
PTX 10 times over a 2-week period via tail vein injection. The tumor
volume was measured every other day. The length (L) and width (W) of
each tumor were measured by a digital caliper, and the volume (V) was
calculated by the modified ellipsoid formula: V = (L × W2)/2.[55] Specific
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standards ranged from 0.8 to 80 ng per sample (n = 6 including a
0) for tumor, spleen, lung, kidney, and heart tissues, and from 1.8 to
180 ng per sample (n = 6 including a 0) for liver tissue. The tissues from
mice receiving no treatment were used as the matrix in the standard
preparation for the respective tissues. The samples were analyzed
by HPLC-MS/MS (ABSciex 4000). The mass spectrometer utilized
an electrospray ionization probe and was run in positive mode. The
multiple reaction monitoring Q1/Q3 (m/z) transitions were 854.3/286.0
and 808.2/527.1, for PTX and DTX respectively.
The PK parameters were obtained via noncompartmental analyses
using the sparse data option of WinNonlin (version 7.0, Certara,
NJ, USA), allowing for the computation of the standard errors (SE)
associated with estimated parameters.
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Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
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This work was supported by the National Institutes of Health NIBIB
R01EB017791, the Lilly Endowment Gift Graduate Research Award (J.X.),
and NIBIB K99 EB022636 (S.S.-Y.L.). This work was also supported
by the Purdue Research Foundation and the Indiana Clinical and
Translational Sciences Institute, funded in part by grant #UL1 TR001108
from the National Institutes of Health, National Center for Advancing
Translational Sciences, Clinical and Translational Sciences Award.
HPLC-MS/MS analysis was performed by the Clinical Pharmacology
Analytical Core laboratory, a core laboratory of the Indiana University
Melvin and Bren Simon Cancer Center supported by the National Cancer
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Conflict of Interest
The authors declare no conflict of interest.
Keywords
drug delivery, polymeric nanoparticles, quinic acid, selectin, tumor
microenvironment
Received: September 3, 2018
Revised: October 11, 2018
Published online:
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