Matrix metalloproteinase responsive, proximity-activated polymeric nanoparticles for siRNA delivery

Article abstract of DOI:10.1002/adfm.201202215

Small interfering RNA (siRNA) has significant potential to evolve into a new class of pharmaceutical inhibitors, but technologies that enable robust, tissue-specific intracellular delivery must be developed before effective clinical translation can be achieved. A pH-responsive, smart polymeric nanoparticle (SPN) with matrix metalloproteinase (MMP)-7-dependent proximity-activated targeting (PAT) is described here. The PAT-SPN is designed to trigger cellular uptake and cytosolic delivery of siRNA once activated by MMP-7, an enzyme whose overexpression is a hallmark of cancer initiation and progression. The PAT-SPN is composed of a corona-forming polyethylene glycol (PEG) block, an MMP-7-cleavable peptide, a cationic siRNA-condensing block, and a pH-responsive, endosomolytic terpolymer block that drives self-assembly and forms the PAT-SPN core. With this novel design, the PEG corona shields cellular interactions until it is cleaved in MMP-7-rich environments, shifting the SPN ζ-potential from +5.8 to +14.4 mV and triggering a 2.5 fold increase in carrier internalization. The PAT-SPN exhibits pH-dependent membrane disruptive behavior that enables siRNA escape from endo-lysosomal pathways. Intracellular siRNA delivery and knockdown of the model enzyme luciferase in R221A-Luc mammary tumor cells is significantly increased by MMP-7 pre-activation (p < 0.05). These combined data indicate that the PAT-SPN provides a promising new platform for tissue-specific, proximity-activated siRNA delivery to MMP-rich pathological environments. Copyright

Full text of DOI:10.1002/adfm.201202215

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Matrix Metalloproteinase Responsive, Proximity-Activated
Polymeric Nanoparticles for siRNA Delivery
Hongmei Li, Shann S. Yu, Martina Miteva, Christopher E. Nelson, Thomas Werfel,
Todd D. Giorgio,* and Craig L. Duvall*
therapies.[1,2] Ideally, cell- and tissue-spe-
Small interfering RNA (siRNA) has significant potential to evolve into a
new class of pharmaceutical inhibitors, but technologies that enable robust,
tissue-specific intracellular delivery must be developed before effective clinical
translation can be achieved. A pH-responsive, smart polymeric nanoparticle
cific siRNA delivery will be achieved in
tumor tissues in order to avoid off-target
effects likely to compromise therapeutic
[
3]
index and clinical applications. Cancer
targeting of nanoparticulate drug car-
riers typically involves optimizing carrier
biophysical properties for nonspecific
enhanced permeation and retention (EPR)
(SPN) with matrix metalloproteinase (MMP)-7-dependent proximity-activated
targeting (PAT) is described here. The PAT-SPN is designed to trigger cellular
uptake and cytosolic delivery of siRNA once activated by MMP-7, an enzyme
whose overexpression is a hallmark of cancer initiation and progression.
The PAT-SPN is composed of a corona-forming polyethylene glycol (PEG)
block, an MMP-7-cleavable peptide, a cationic siRNA-condensing block, and
a pH-responsive, endosomolytic terpolymer block that drives self-assembly
and forms the PAT-SPN core. With this novel design, the PEG corona shields
cellular interactions until it is cleaved in MMP-7-rich environments, shifting
the SPN ζ-potential from +5.8 to +14.4 mV and triggering a 2.5 fold increase
in carrier internalization. The PAT-SPN exhibits pH-dependent membrane dis-
ruptive behavior that enables siRNA escape from endo-lysosomal pathways.
Intracellular siRNA delivery and knockdown of the model enzyme luciferase
in R221A-Luc mammary tumor cells is significantly increased by MMP-7
pre-activation (p < 0.05). These combined data indicate that the PAT-SPN
provides a promising new platform for tissue-specific, proximity-activated
siRNA delivery to MMP-rich pathological environments.
[4]
tumor accumulation and/or through bio-
chemical targeting of cell surface receptors
[5–8]
that are overexpressed on cancer cells.
For example, in an exciting recent clinical
breakthrough, phase I human trial data
demonstrated safe siRNA-mediated gene
silencing in human melanoma patients
using nanoparticles molecularly targeted
to cancer cells that overexpress the trans-
[9]
ferrin receptor.
Alternatively, pathological environ-
mental signals and enzymatic activity can
be exploited to trigger changes in drug
carrier properties, exposing latent nano-
carrier functionalities that can increase
their internalization by target cells or
trigger drug payload release in pathologic
[
10,11]
sites.
environment is mildly acidic, which has
For example, the tumor micro-
[12,13]
been exploited by a variety of nanocarrier designs.
We and
1
. Introduction
others have also leveraged peptides that are cleaved by naturally
occurring proteases for “proximity activation” that uncovers
latent targeting ligands on inorganic nanoparticle image con-
Nanocarrier-mediated delivery of siRNA for silencing of genes
that inhibit apoptosis, promote oncogenesis, or induce multi-
drug resistance is an exciting new paradigm for anti-cancer
[
14–19]
trast agents.
In an analogous approach, Harishima and
Dr. H. Li, S. S. Yu, M. Miteva, C. E. Nelson, Prof. T. D. Giorgio,
Prof. C. L. Duvall
Department of Biomedical Engineering
Vanderbilt University
VU Station B, Box 351631, Nashville, TN, USA
E-mail: todd.d.giorgio@vanderbilt.edu; craig.duvall@vanderbilt.edu
T. Werfel
Vanderbilt Institute of Nanoscale Science
and Engineering REU program
Department of Engineering and Physics
Murray State University
Murray, KY, USA
Prof. T. D. Giorgio
Dr. H. Li, S. S. Yu, M. Miteva, C. E. Nelson, T. Werfel, Prof. T. D. Giorgio,
Prof. C. L. Duvall
Vanderbilt Institute of Nanoscale Science and Engineering
Vanderbilt University
Department of Cancer Biology
Department of Chemical and Biomolecular Engineering
Vanderbilt University
Nashville, TN, USA
Nashville, TN, USA
DOI: 10.1002/adfm.201202215
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co-authors have developed a multifunctional envelope-type
nanodevice (MEND) for siRNA delivery that incorporates a lipid
with a matrix metalloproteinase (MMP)-cleavable PEG layer that
2
. Results
2.1. PAT-SPN Synthesis and Characterization
[20,21]
blocks liposomal uptake until it is enzymatically removed.
The MMP-7-cleavable peptide (H-VPLSLYSGCG-OH)[45] was
made using solid phase peptide synthesis and purified by high
performance liquid chromatography (HPLC), as confirmed by
liquid chromatography-mass spectrometry (LC-MS) (Supporting
Information, Figure S1,S2). Proteolytic susceptibility of this
sequence to cleavage by human MMP-7 was verified using SDS-
PAGE (Supporting Information, Figure S3). Peptide PEGylation
was carried out by reacting a 5 kDa maleimide end-functional-
ized PEG with the MMP-7 cleavable peptide through its C-ter-
minal, cysteine residue as shown in Scheme 1. GPC analysis
confirmed the expected peak retention time shift between the
PEG precursor and the PEG-peptide conjugate (Supporting
Information, Figure S4). An N-hydroxysuccinimide-functional-
ized RAFT chain transfer agent (CTA), NHS-ECT, synthesized
MMPs are a family of zinc-dependent proteases that degrade
extracellular matrix[ and are known to be important in the
22]
establishment and growth of primary breast tumors and met-
astatic lesions.[
23,24]
MMP-7 is secreted even in the earliest
stages of breast tumor development. Small, benign “precan-
cerous” lesions, for example, secrete MMP-7 early in disease
progression, before any of the lesions would be detectable
clinically.[
24,25]
Furthermore, MMP-7 expression is predomi-
nantly localized to cells of epithelial origin, in contrast to the
expression of most MMP family members in cells of connective
tissue origin.[ These critical characteristics motivate the devel-
opment of “smart” breast cancer therapeutics that have latent
drug delivery functions triggered by MMP-7 activity.
26]
Here, a novel, pH-responsive micellar nanocarrier capable
of MMP-7 proximity-activated targeting for siRNA delivery is
reported. The base delivery system consists of a smart poly-
1
in-house ( H-NMR spectra in Supporting Information Figure S5
for ECT and Figure S6 for NHS-ECT) was coupled with the
peptide N-terminus. Successful coupling of NHS-ECT was con-
firmed using UV-vis spectroscopy based on the characteristic
spectrophotometric absorbance of the trithiocarbonate group in
the ECT at 320 nm (Supporting Information, Figure S7).
A 6 kDa pDMAEMA homopolymer was extended from the
PEG-peptide macroCTA via RAFT polymerization. Conversion
meric nanoparticle (SPN) that is capable of packaging and
delivering siRNA cargo.[
27–30]
This SPN consists of a positively
charged dimethylaminoethyl methacrylate (DMAEMA) corona
that functions as a siRNA-condensing block and a pH-respon-
sive, core-forming terpolymer block that mediates endosomal
[
27]
disruption and cytosolic delivery of the siRNA payload.
1
The DMAEMA block effectively mediates siRNA delivery,
but, like other polycationic delivery vehicles, it is internalized
indiscriminately by cells due to its interaction with negatively
charged elements of the cell membrane.[27,30,31] Furthermore,
polycationic carriers typically suffer from short circulation half-
lives and can cause undesirable interactions among cells and
was calculated from H-NMR spectroscopy analysis of the mono-
meric form of DMAEMA before and after polymerization relative
to the internal index trioxane. Retention of the trithiocarbonate
was confirmed in the resulting PEG-peptide-pDMAEMA (PEG-
pep-pD) by UV-vis spectrophotometric absorbance at 320 nm.
GPC analysis confirmed successful polymerization of
a
other blood constituents” to “and aggregation with cells and
21 kDa butyl methacrylate (BMA), propyl acrylic acid (PAA), and
DMAEMA copolymer block from the PEG-pep-pD macroCTA,
resulting in PEG-peptide-pDMAEMA-p(DMAEMA-co-PAA-
co-BMA) (PEG-pep-pD-pDPB) (Scheme 1). The full PEG-pep-
pD-pDPB polymer construct had a Mn of 33 kDa and a PDI
of 1.41. Using the GPC-determined Mn and 1H-NMR spec-
troscopy, the pDPB block was determined to be composed of
28.6 mol% DMAEMA, 21.7 mol% PAA, and 49.6 mol% BMA
serum proteins.[
32,33]
PEGylation can make cationic polymer-
based systems more stealthy in the systemic circulation by
shielding these carrier-cell interactions, reducing clearance
by the reticuloendothelial system (RES), and extending blood
circulation half-lives.[
34–37]
However, stable PEGylation can also
mask underlying functionalities that drive cell uptake and/or
endosomolytic activity.[
38,39]
1
The novel SPN design presented here incorporates surface
PEGylation that undergoes MMP-7-triggered shedding, or
PAT, of the outer PEG layer, which uncovers the underlying
cationic corona and triggers cellular uptake. This PAT-SPN was
designed to be sensitive to MMP-7 because, along with other
MMPs such as MMP-2 and MMP-9, high levels of MMP-7
activity have been correlated with invasive and metastatic
( H-NMR in Supporting Information, Figure S8).
The PEG-pep-pD-pDPB polymer spontaneously formed
micellar nanoparticles upon dropwise addition from ethanol
into PBS (pH 7.4). The resulting hydrodynamic particle diam-
eter of 46 nm, as determined by dynamic light scattering (DLS)
(Figure 1a), represents an optimal size for cellular uptake
while minimizing non-specific uptake by macrophages of the
[
19,27]
cancer phenotypes that yield poor outcomes in breast cancer
RES.
Transmission electron microscopy (TEM) imaging
patients.[
40–44]
Importantly, the current design is modular in
of the dehydrated micelles counter-stained with uranyl acetate
further confirmed the formation of the PAT-SPNs (Figure 1b).
Notably, sizing of dried micelles via TEM typically leads to
smaller measured diameters relative to sizing via DLS, due to
dehydration and partial collapse of the particle corona.
nature, and the sensitivity and specificity of MMP cleavage
can be tuned by altering the MMP-sensitive peptide used in
the PAT element.[ The ability to alter the proteolytically sen-
sitive peptide enables flexible selection of the most appropriate
sequence that can be selectively cleaved by the anticipated or
known MMP type(s) in the target tissue. This study focused
on proof-of-concept synthesis and characterization of MMP-
45]
2
.2. Assessment of Responsiveness of PAT-SPN Nanoparticles
to MMP Proteolysis
7
-sensitive PAT-SPNs, and the data reported indicate that this
delivery platform offers significant potential for environmen-
tally targeted tissue specific siRNA delivery.
GPC, ζ-potential, and DLS were used to confirm MMP-7 pro-
teolytic cleavage of self-assembled PAT-SPNs. PAT-SPNs were
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Scheme 1. Synthetic scheme of MMP-7-responsive PAT-SPN polymer construct.
treated with 50 nM MMP-7 in order to mimic the metastatic
tumor microenvironment.[ The resultant product was re-
isolated and analyzed by GPC with DMF as the mobile phase.
The small shift in the peak elution time of the PEG-pep-
pD-pDPB after treatment with MMP-7 is consistent with the
expected drop in molecular weight due to proteolytic removal
of the PEG segment. The sensitivity of the PAT-SPN to MMP-7
treatment was further supported by the appearance of a peak
eluting at 25 min (Figure 2), which aligns with the elution
time of the predicted 5 kDa PEG cleavage product. Untreated,
control PAT-SPN micelles had a ζ-potential of +5.8 mV.
Within 10 min of MMP-7 addition, the ζ-potential increased
from +5.8 to +7.2 mV, and it approached +13.8 mV after 3 h
based on the promiscuity of the VPLSLYSCG peptide, PAT-SPN
micelles were also activatable by MMP-2 or MMP-9, with con-
sequent increase in ζ-potential and decrease in hydrodynamic
diameter as observed for MMP-7 (Supporting Information,
46]
PEG-peptide (PAT component)
RAFT polymerization
PEG-pep-pD-pDPB (PAT-SPN)
MMP-7 cleavage
PEG + pD-pDPB (partial PAT + SPN)
(
Figure 3). Exposure to 1–5 nM concentrations of MMP-7
relevant to normal, healthy tissue generated a slower rate of
increase in ζ-potential following treatment. Over 3 h, with
the MMP-7-concentration dependent change in ζ-potential
was +0.26, +1.07, and +7.97 mV for 1, 5, and 50 nM MMP-7,
respectively (Supporting Information, Figure S9). As expected,
PEG
20
16
12
8
4
0
a
16
18
20
22
24
26
Retention Time (min)
Figure 2. GPC analysis of RAFT polymerization from PEG-peptide macro-
CTA and PAT-SPN construct responsiveness to MMP-7. PEG-pep-pD-pDPB
generated from PEG-peptide macro-CTA (dash-dot) after two successive
RAFT polymerizations was confirmed by the shift in GPC elution time
(dash-dot versus solid line traces). Elugrams of PEG-pep-pD-pDPB treated
with MMP-7 (dash) was consistent with cleavage of the distal PEG seg-
ment as indicated by the shift of the primary elution peak (solid versus
dashed traces at 23 min) and the appearance of a shoulder peak at 25 min
that corresponds to the PEG-peptide macro-CTA (dash-dot).
1
0
100
1000
Particle Size (d.nm)
Figure 1. Confirmation of PAT-SPN formation. a) DLS suggested that
PAT-SPNs exhibit an average hydrodynamic diameter of 46 nm. b) TEM
of dried PAT-SPN nanoparticles on TEM grids counterstained with 3%
uranyl acetate confirmed self-assembly of PEG-pep-pD-pDPB polymer
into micelles with average dehydrated diameter of 20 nm.
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1
1
1
4
2
0
8
6
4
2
[
+] layer
exposed
Figure 4. FRET-based demonstration of PAT-SPN/DNAFAM/DNACy5
FAM
Cy5
serum stability. PAT-SPN/DNA /DNA were incubated at 37 °C for
, 4, 8, and 24 h in the presence of 50% fetal bovine serum. FRET was
PEG-
shielded
2
measured with excitation at 485 ± 20 nm using FAM as a donor and
emission at 670 ± 25 nm using Cy5 as the acceptor and indicated that the
0
1
2
3
4
5
6
7
FAM
Cy5
PAT-SPN/DNA /DNA formulation was serum-stable over 24 h. Data
#
Cleavage Time (h)
presented as mean ± standard error with n = 3. p < 0.0001 compared
FAM
Cy5
with PAT-SPN/DNA /DNA in PBS (no serum).
Figure 3. MMP-7-dependent physicochemical switch in PAT-SPNs.
Nanocarrier ζ-potential increased from +5.8 to +14.4 mV over 6.5 h of
treatment with a physiologically relevant MMP-7 concentration (50 nM).
2
.4. PAT-SPN/siRNA Serum Stability
and siRNA Protection
Figure S10). Increased ζ-potential upon treatment with patho-
physiologic concentrations of MMP is consistent with detach-
ment of the peptide-tethered PEG chains from micelle surface,
unveiling the cationic pDMAEMA layer that forms the corona
of the underlying base SPN. These data support efficient PAT
properties of the siRNA nanocarrier.
The ability of the PAT-SPN/siRNA formulations to remain
stable under serum conditions and to protect siRNA from
degradation was demonstrated by a Förster (fluorescence) res-
onance energy transfer (FRET)-based assay (Figure 4) and aga-
rose gel electrophoresis (Supporting Information, Figure S12).
In both of these experiments, incubation of PAT-SPN/siRNA in
5
0% fetal bovine serum at 37 °C was performed to simulate in
2
.3. Particle Diameter and ζ-Potential Evaluations of PAT-SPN/
vivo conditions. For the serum particle stability assay, dsDNA
FAM
Cy5
siRNA Treated with MMP-7
molecules labeled with FAM (DNA
) or Cy5 (DNA ) were
co-loaded onto PAT-SPN/DNAFAM/DNACy5. These dsDNA were
21 base pairs and served as model siRNA molecules. The FAM/
Cy5 fluorophore pair have minimal emission overlap and make
a good donor/acceptor pair for use with FRET. When simply
mixed together in solution (with no PAT-SPN formulation),
the FRET-paired dsDNA did not produce a FRET signal. How-
ever, a robust FRET signal was observed when the FRET-paired
PAT-SPNs loaded with siRNA across a range of N(+)/P(-) charge
ratios exhibited an increase in ζ-potential and a decrease in
hydrodynamic particle diameter following treatment with
MMP-7, indicating that siRNA loading did not adversely affect
MMP-7 responsiveness (Table 1). Note that efficient PAT-SPN
siRNA loading occured across a range of N(+)/P(-) charge ratios
of 2 or greater (Supporting Information, Figure S11).
FAM
Cy5
dsDNA were co-loaded into PAT-SPN/DNA /DNA . Fur-
thermore, the FRET signal of the PAT-SPN/DNAFAM/DNACy5
formulations did not significantly change over a 24 h incubation
in 50% serum (student’s t-test, p > 0.05 all time points relative
to 0 h baseline sample, Figure 4). Because the FRET signal is
dependent on close packing of the two fluorophores, these data
suggest that siRNA loading onto PAT-SPNs is serum stable.
Agarose gel electrophoresis was utilized to confirm PAT-SPN
protection of siRNA from degradation. Free siRNA was signif-
icantly degraded within 15 min of incubation in 50% serum,
as evidenced by the disappearance of the bright band corre-
sponding to free siRNA (Supporting Information, Figure S12).
However, when SDS was utilized to release the siRNA from
PAT-SPN/siRNA following serum incubation, the siRNA was
found to remain relatively intact over the time course of the
experiment, though there was some visible decrease in stability
at 24 h (Supporting Information, Figure S12). These data indi-
cate that PAT-SPN loading improved the stability of siRNA in a
model in vivo environment.
T a b le 1 . MMP-7 dependent modulation of zeta potential and hydrody-
namic diameter of siRNA-loaded PAT-SPNs.
Formula
PAT-SPN/siRNA
Particle diameter Zeta
nm]
PAT-SPN/siRNA +MMP-7
Particle diameter
[nm]
Zeta
[
[mV]
[mV]
No siRNA
46 ± 6
45 ± 6
44 ± 5
44 ± 6
44 ± 5
42 ± 4
5.8 ± 0.0
7.5 ± 6.4
7.0 ± 5.8
6.6 ± 7.0
6.8 ± 6.4
6.2 ± 5.6
42 ± 6
42 ± 7
42 ± 7
40 ± 4
41 ± 6
37 ± 4
14.4 ± 0.0
14.5 ± 6.9
14.4 ± 7.6
15.1 ± 6.3
15.9 ± 5.5
12.0 ± 6.1
N(+)/P(−) = 12
N(+)/P(−) = 8
N(+)/P(−) = 6
N(+)/P(−) = 4
N(+)/P(−) = 2
All data are representative of repeated measures of two samples. MMP-7 treat-
ment significantly reduced particle diameter (p = 0.02) and significantly increased
ζ-potential (p < 0.0001) as determined by paired t-test.
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2
.5. Cytotoxicity Assessment
The high cytocompatibility of the PAT-SPNs loaded with siRNA
was in agreement with previous studies using the base SPN
[
27,30]
carrier.
Cell viability of R221A-Luc mammary tumor cells
treated with PAT-SPN/siRNA over a range of N(+)/P(-) ratios
indicated that significant cytotoxicity was apparent only at 12:1,
which was the highest N(+)/P(-) ratio tested (Figure 5). No statis-
tically significant cytotoxicity was measured at the other charge
ratios (2:1, 4:1, 6:1, or 8:1) examined. This data confirmed
that appropriately formulated PAT-SPNs can serve as cytocom-
patible siRNA nanocarriers.
Figure 5. PAT-SPN cytocompatibility across a range of N(+)/P(-) ratios
assessed on luciferase-expressing R221A-Luc tumor cells. Cell viability
was assessed by treating luciferase expressing R221A-Luc mammary
tumor cells with PAT-SPNs loaded with 50 nM scrambled siRNA at
N(+)/P(-) ratio of 2:1, 4:1, 6:1, 8:1, and 12:1 following pre-activation with
2.6. PAT-SPN Cell Uptake
Flow cytometry and confocal microscopy data demonstrated that
internalization of PAT-SPN/siRNA was significantly increased
in MDA-MB-231 breast cancer cells following MMP-7 treat-
ment. Cellular internalization of PAT-SPN siRNA nanocarriers
activated by MMP-7 was 2.5-fold greater than for unactivated
controls (Figure 6a, b; mean intracellular uptake increased
from 178 ± 9 relative fluorescence units before MMP-7
2
+
MMP (50 nM) and Zn (50 μM) (MMP+Zn). Cell viability was reported
relative to non-treated cells (NT). Significant cytotoxicity was observed
relative to NT at a charge ratio of 12:1 (58 ± 9% viability) but not at lower
charge ratios. Data presented as mean ± standard error with n = 3 from
#
three independent experiments. p < 0.05 compared with NT.
Figure 6. Enhanced PAT-SPN cell internalization following exposure to MMP-7. a,b) Flow cytometry analysis of DNAFAM delivered to MDA-MB-231
breast cancer cells using intact PAT-SPNs (PAT-SPN/FAM-dsDNA), MMP-7-pretreated PAT-SPNs (MMP7 pretreated PAT-SPN/FAM-dsDNA), or no
FAM
treatment (NT). Mean FAM fluorescence was significantly higher in cells treated with PAT-SPN/DNA
proximity activated by MMP-7 compared to
&
all other treatment groups. Data presented as mean ± standard error with n = 3. p < 0.05 compared with NT and #p < 0.05 compared with PAT-SPN/
FAM-DNA. c) Confocal microscopy images of MDA-MB-231 breast cancer cells treated with PAT-SPN/DNACy5 nanocarriers (at 50 and 100 nM DNA
concentration) with or without MMP-7 treatment correlated with quantitative flow cytometry data. PAT-SPN/DNA complexes of N(+)/P(−) = 6 were
used in all of these experiments.
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activation to 447 ± 41 relative fluorescence
units following MMP-7 activation). Confocal
microscopy images also supported the flow
cytometry findings that cell internalization
of the nanocarriers was enhanced following
MMP-7 proximity activation (Figure 6c). Fur-
thermore, uptake was found to remain low
at both 50 and 100 nM siRNA doses in PAT-
SPNs without MMP-7 activation, while the
level of uptake was dose-dependent for PAT-
SPNs that had been activated with MMP-7.
2
.7. PAT-SPN pH-Responsiveness
and Endosomal Escape
The pDPB block endows the PAT-SPNs with
the ability to undergo pH-dependent destabi-
[27,28,30]
lization of phospholipid membranes
,
and this behavior was confirmed for the PAT-
SPN formulations using the red blood cell
[
27,28]
hemolysis assay
hemolysis was measured at extracellular pH
7.4), and a sharp increase in hemolysis was
apparent in the endo-lysosomal pH range
≤6.2), suggesting that the PAT-SPNs can
(Figure 7a). Minimal
(
(
facilitate disruption and escape from endo-
somal vesicles without causing significant
cytotoxicity due to disruption of the outer
cell membrane. In agreement with the pH-
responsive hemolysis activity, MMP-7 pre-
activated PAT-SPNs facilitated enhanced
Cy5
escape of complexed DNA
from the
endo-lysosomal trafficking pathway, as visual-
ized by confocal microscopy (Figure 7b). In
this experiment, the presence of yellow signal
Cy5
(
co-localization of the DNA in red, and Lys-
oTracker in green) was used to indicate endo-
lysosomal entrapment. It was found that
Cy5
significant free DNA
(red fluorescence)
could be visualized intracellularly, indicating
that most of the DNA cargo had escaped the
endo-lysosomal pathway.
2
.8. PAT-SPN/siRNA Knockdown of
Luciferase Activity in R221A-Luc Mammary
Tumor Cells
Figure 7. pH-dependent membrane disruption and endo-lysosomal escape of PAT-SPNs.
a) pH-dependent hemolysis of erythrocytes was determined for intact PAT-SPN and MMP-7
To examine the ability of PAT-SPNs to facili-
pretreated PAT-SPN at 1, 5, 15, and 30 μg/mL relative to 1% Triton X-100 as a positive control tate functional, siRNA-mediated gene knock-
(100% lysis). Hemolysis was pH-dependent, with approximately 0% at extracellular pH 7.4 down, anti-luciferase siRNA was delivered
and more than 90% at pH 5.6. Hemolysis mediated by PAT-SPN was essentially unchanged
following MMP-7 proteolytic activation. Error bars indicate standard deviation of n = 4.
b) Confocal imaging of MDA-MB-231 cells treated with MMP-7 pretreated PAT-SPN/DNACy5
and stained with LysoTracker. Control cells only stained by LysoTracker are shown as a refer-
to R221A-Luc mammary tumor cells, which
constitutively express luciferase. siRNA
delivered by MMP-7-pretreated PAT-SPNs
facilitated significantly greater luciferase
Cy5
ence (upper panel). Intracellular DNA delivered by the MMP-7 pretreated PAT-SPNs was
predominantly distributed outside the lysosomes (red), and there was minimal colocalization knockdown, versus the control PAT-SPN/
with the LysoTracker signal (yellow).
siRNA group delivered without MMP-7
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pre-treatment (p = 0.0007; post-hoc two-way ANOVA). The
reduction in luciferase activity was significantly greater in the
MMP-7-activated PAT-SPNs for all charge ratios compared to
controls receiving no treatment (NT) (Figure 8a). When the
formulations were not pre-treated with MMP-7 for removal
of the PEG layer, delivery of 50 nM luciferase siRNA using
PAT-SPNs at N(+)/P(-) charge ratios of 2 and 4 did not pro-
duce any significant reduction of luciferase activity relative to
NT controls. However, at an N(+)/P(-) charge ratio of 6:1, sig-
nificant reduction in luciferase activity was observed without
MMP-7-pretreatment of PAT-SPNs. Importantly, MMP-7 treat-
ment of PAT-SPNs produced luciferase silencing to a level
that was not statistically different from that facilitated through
the commercial transfection reagent Lipofectamine. How-
ever, all formulations of PAT-SPNs that were not pre-treated
with MMP-7 produced statistically less luciferase knockdown
Figure 8. MMP-7 pre-treatment enhanced luciferase knockdown by PAT-SPNs. a) Protein level gene silencing readouts were acquired in R221A-Luc
mammary tumor cells that constitutively express firefly luciferase, following treatment with luciferase-targeted siRNA complexed into PAT-SPNs at
N(+)/P(-) charge ratios of 2, 4, and 6. Lipofectamine (Lipofec) was used as a positive control, and luciferase activity was reported relative to untreated
cells (NT). (Data presented as mean ± standard error with n = 3). &p < 0.05 compared with NT, p < 0.05 compared with Lipofec, and p < 0.001
between the intact and the MMP-7 pretreated PAT-SPN groups. b) Variance analysis by ANOVA (one way Tukey’s range test) on knockdown results
of PAT-SPN/siRNA versus no treatment (NT) (left) or versus Lipofectamine/siRNA (Lipofec) (right). Formulations with variance > 0 are significantly
different from NT (left) and to Lipofec (right).
#
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than Lipofectamine. An overview of the pairwise statistical
comparisons between treatment groups appears as Figure 8b.
On this plot, positive values represent significant differences,
while negative values represent the lack of statistically signifi-
cant differences. These data suggest that PAT-SPN mediated
siRNA delivery will induce significant gene silencing in MMP-
to an overexpressed receptor does not always enhance tumor
biodistribution following intravenous injection of nanoparti-
cles. For example, systemically injected transferrin-targeted
nanoparticles formed with cyclodextrin-containing polycations
and siRNA produced biodistribution and tumor accumulation
properties similar to nontargeted control particles; instead,
receptor targeting improved tumor cell internalization and bio-
activity of the nanoparticles. These interesting results sug-
gest that nanocarrier physicochemical properties should be
optimized for EPR-driven tumor accumulation and that addi-
tional targeting strategies may be best motivated by improving
tumor retention and cell uptake.
7
-rich environments and, depending on the precise formula-
[
50]
tion and exposure, have little or no effect in MMP-7-deficient
regions.
2
.9. PAT-SPN Activation by Endogenously Produced MMPs
The invasive breast cancer cell line MDA-MB-231, which has
been documented to overexpress several MMPs including MMP-
PEGylation is important for incorporating particle stealth,
increasing circulation time, and improving EPR-driven tumor
biodistribution, but conventional, permanent PEGylation can
inhibit interaction and uptake by cells in pathological tissues.
In order to increase carrier uptake and local retention following
EPR-driven distribution to tumor sites, the current PAT-SPN
was designed to be sensitive to MMP-7, an abundant constit-
[
47,48]
7
,
was used to confirm that cell secreted MMPs triggered
activation and enhanced cell uptake of the PAT-SPN/DNA. This
claim was supported by the confocal microsopy observation that
Cy5
decreased internalization of PAT-SPN/DNA
was observed
with MDA-MB-231 cell pre-conditioned media that was sup-
plemented with a broad-spectrum MMP inhibitor (Supporting
Information, Figure S13b). Cell-mediated and MMP-dependent
activation of the PAT mechanism was further confirmed by
temporal analyisis of PAT-SPN ζ-potential following exposure
to MDA-MB-231 conditioned media in the presence or absence
of MMP inhibitor. In the presence of the MMP inhibitor, the
ζ-potential was relatively stable over time, while exposure to
the conditioned media without the MMP inhibitor triggered a
[43,51–57]
uent of many solid tumors.
Here, MMP-7-dependent
cleavage of the PEG corona exposed the underlying, cationic
SPN and consequently triggered a significant increase in par-
ticle ζ-potential (Figure 3). Although pH-dependent PEG shed-
[
58]
ding from polymeric nanocarriers has been described, this
is, to our knowledge, the first report of a polymeric nanocarrier
utilizing tissue-specific enzymatic modulation of surface charge
as an approach for environmentally triggered siRNA uptake.
Reversible PEGylation removable by acidic conditions has been
shown to decrease cytotoxicity relative to non-PEGylated carriers
and to improve bioactivity relative to stable PEGylation for a
+6.6 mV increase after incubation for 18 h. Under these treat-
ment conditions, the ζ-potential value was comparable to that
of PAT-SPN/DNA treated with exogenous MMP-7 (Supporting
Information, Figure S13a). These results suggest that a signifi-
cant increase in PAT-SPN ζ-potential and cell internalization
can be triggered by physiologically relevant MMPs endog-
enously produced within breast tumor microenvironments.
[
58,59]
variety of applications using cationic polymers such as PEI.
The proteolytically meditated PEG deshielding demonstrated
here provides a potentially more broadly applicable platform
because it is not limited to local environments of reduced pH
and is able to accommodate essentially any short peptide that
can be enzymatically cleaved.
3
. Discussion
The modular nature of our design is derived from the syn-
thetic scheme that incorporates the proteolytically cleavable
PAT functionality onto the base SPNs through site-specific
orthogonal conjugation chemistries and by leveraging the
ability of reversible addition fragmentation chain transfer
(RAFT) polymerization to synthesize well-defined block copol-
ymer architectures. The presented strategy for pre-conjugation
of the CTA with the PAT components prior to conducting
RAFT polymerization of the SPN was used to achieve efficient
substitution of the PAT component onto the RAFT polymers
and a high density of PEG chains on the final PAT-SPN. This
“grafting-from” strategy for the RAFT polymerizations also
avoids inefficient reactions between large biomacromolecules
While siRNA-based therapies are promising, significant delivery
barriers have hindered the clinical translation of this new drug
class. The novel PAT-SPN disclosed herein was primarily moti-
vated by the desire to integrate a mechanism for tumor-specific
accumulation with other advanced polymer functionalities in
order to overcome each of the primary challenges to siRNA
delivery: siRNA protection, stealth in the circulation, site-spe-
cific biodistribution and cell uptake, and endosomal pathway
escape.
Proximity-activated targeting was chosen due to the poten-
tial shortcomings of cell surface receptor targeting. Cell sur-
face targeting of drugs is likely to achieve diminishing effects
upon repeat treatment and risks potentially selecting for a
population cancer cells that do not highly express the targeted
receptor. This could lead to incomplete tumor eradication and/
or recurrence. Also, because many of the commonly targeted
receptors that are overexpressed by cancer cells are also present
on some healthy cells (e.g., folate and transferrin), systemic
delivery of these molecularly targeted nanocarriers requires
finely tuned ligand presentation on the nanoparticle surface in
order to achieve sufficient selectivity and reduce the potential
for off-target side effects.[ Furthermore, molecular targeting
[
60,61]
and potentially cumbersome product purification.
Once this synthesis scheme was successfully performed, it
was important to verify that the resulting PAT-SPNs would be
selectively activated in tumor tissue in vivo. MMP activity levels
vary among tissues, and the elevated concentration of MMPs
[
21,62]
in tumors has been estimated to be as high as 1 mM.
In
our studies, we utilized 50 nM MMP, which falls at the lower
end of the range of MMP concentrations that other studies
typically use to assess the kinetics of cleavage of MMP-respon-
sive biomaterials under conditions that mimic the tumor
49]
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microenvironment.[14,16,21,46] We primarily assessed cleavage of
our construct in response to MMP-7, although we also studied
responsiveness to MMP-2 and MMP-9. The chosen substrate,
VPLSLYSG, can be cleaved by all three of these MMPs and
Activation of these nanocarriers by MMP-7 both increased intra-
cellular delivery and enabled RNA interference and protein-level
knockdown by siRNA. The combined results are consistent with
the design concept that MMP-7-dependent removal of an outer
PEG corona increased PAT-SPN ζ-potential, which consequently
increased rate of uptake into tumor cells. Once internalized, the
pH-responsive core of the PAT-SPN was activated and facili-
tated pH-dependent siRNA endosomal escape and bioactivity.
Overall, evidence suggests that this novel nanoscale vehicle
has important advantages in targeted and effective therapeutic
delivery to MMP-rich cancer tissues for gene-specific protein
silencing. It is anticipated that this approach will be useful for
the development of effective stand-alone therapies and/or as an
adjuvant used to sensitize cells to traditional chemotherapies.
The promising proof-of-concept data reported here inform our
ongoing work to optimize environmentally triggered targeting
of efficient siRNA nanocarriers for breast cancer applications.
has a mid-rate kcat/K for MMP-7 cleavage of 22 000 ± 3000
M
−1 −1 [45]
(
M s ), though it is anticipated that presence of the PEG
corona distal to the peptide sterically limits access of MMPs
to the peptide substrate and slows its cleavage. Treating the
PAT-SPN formulations with 1, 5, and 50 nM MMP-7 produced
a concentration-dependent rate of increase in ζ-potential, sug-
gesting that the PAT-SPN design is tuned to be activated under
pathophysiologically relevant MMP-7 concentrations (Sup-
porting Information, Figure S9). The rates of cleavage observed
were also comparable to results previously reported using the
RPLALWRS peptide in a PAT quantum dot system.[14]
While the VPLSLYSG sequence appears optimal for our
current breast cancer application, the modular nature of the
PAT-SPN design can be leveraged through substitution with
peptides possessing different proteolytic cleavage characteris-
tics and/or different PEG molecular lengths. The VPLSLYSG
peptide used here is cleavable by multiple MMPs in addition
to MMP-7, including MMP-2 (k /K = 61 000 ± 4000) and
5
. Experimental Section
cat
M
45]
[
Materials: Chemicals and materials were purchased by Sigma-Aldrich
MMP-9 (k /KM = 49 000 ± 3000). We have confirmed that
cat
or Fisher Scientific and used without purification unless otherwise
specified. DMAEMA monomers were purified by distillation under
vacuum prior to use in polymerizations and BMA was passed through
basic alumina prior to use. 2,2-Azobis(2-methylpropionitrile) (AIBN)
was recrystallized twice from methanol. siRNA was purchased from
Ambion and ssDNA from Sigma Aldrich. Two 21mer ssDNAs with
complementary sequences were dissolved in nuclease-free water, and a
stock concentration of 50 μM dsDNA was annealed by three cycles of
heating to 94 °C and cooling to 50 °C. PD-10 desalting columns were
purchased from from GE Healthcare, Lipofectamine high efficiency
transfection reagent was purchased from Invitrogen (Carlsbad, CA),
and active human MMPs (including MMP-7, MW = 19 kDa; MMP-2,
MW = 66 kDa; and MMP-9, MW = 83 kDa) were purchased from EMD
Chemicals. PAA monomer was synthesized following a protocol adapted
from previous literature.[
PAT-SPNs based on the VPLSLYSG peptide are also respon-
sive to MMP-2 and MMP-9 exposure, displaying simultaneous
increase in ζ-potential and decrease in hydrodynamic diameter
upon treatment with these MMPs (Supporting Information,
Figure S10). The tissue selectivity and efficiency of PAT-SPN
activity is predicted to be enhanced in environments simulta-
neously expressing multiple MMPs having proteolytic activity
against VPLSLYSG.
In addition to targeted delivery, key design criteria consid-
ered in development of the PAT-SPNs were stable packaging
and protection of the siRNA cargo, stealth in the circulation,
and endosomal pathway escape. As confirmed by gel electro-
phoresis and a FRET-based assay, the PAT-SPNs were able to
efficiently package siRNA into serum-stable particles that also
helped to decrease siRNA susceptibility to degradation (Sup-
porting Information, Figure S11,S12). Furthermore, PEGylation
of the particle surface is known to impart nanocarrier stealth
64]
Synthesis of 4-Cyano-4-(ethylsulfanylthiocarbonyl) Sulfanylpentanoic Acid
(
ECT): ECT was synthesized following protocols previously described
[27,65]
by Convertine et al. and Moad et al.
Briefly, ethanethiol (4.72 g,
7
6 mmol) was reacted with carbon disulfide (6.00 g, 79 mmol) in the
presence of sodium hydride (3.15 g, 79 mmol) in diethyl ether for 1 h.
The resulting sodium S-ethyl trithiocarbonate was further reacted with
iodine (6.3 g, 25 mmol) to obtain bis(ethylsulfanythiocarbonyl) disulfide,
which was then refluxed with 4,4-azobis(4-cyanopentanoic acid) in ethyl
acetate for 18 h to yield ECT. The crude ECT was purified by column
chromatography using silica gel as the stationary phase and ethyl
that reduces reticuloendothelial clearance and aggregation with
blood cells.[
34,35]
In combination with control of nanoparticle
size, PEGylation may also facilitate nanocarrier accumulation in
vascularized tumor tissues via the EPR effect. To this end, all of
the PAT-SPN formulations tested had diameters of 40–50 nm,
which is known be in the optimal size range for the EPR-driven
1
acetate:hexane (50:50) as the mobile phase. H NMR (400 MHz, CDCl3):
3.35 (q, 2H, S-CH -CH3), 2.69 (t, 2H, -CH -CH -COOH), 2.36-2.58 (m,
2
2
2
tumor accumulation, while minimizing RES-mediated clear-
2H, -CH -CH -CH ), 1.88 (s, 3H, CH -C-CN), 1.36 (t, 3H, S-CH -CH )
Synthesis of the N-Hydroxyl Succinimide-Functionalized ECT (NHS-ECT):
Purified ECT (1.25 g, 4.75 mmol) and N-hydroxylsuccinimide (0.55 g,
2 2 3 3 2 3
ance of the nanocarriers.[
19,27]
Furthermore, the pH-dependent
membrane-disruptive behavior of the terpolymer that forms the
4
.75 mmol) were added to a round-bottom flask and dissolved in
SPN core[
27,28]
was found to be retained in the PAT-modified
dichloromethane (DCM, 45 mL). The mixture was held at 4 °C and
SPN design, ensuring endosomolytic release of the siRNA into
the cytosol where it functions (Figure 7).
purged with N for 30 min. A solution of N,N’-dicyclohexylcarbodiimide
2
(DCC) (0.98 g, 4.75 mmol) in DCM (5 mL) was injected, and the mixture
was purged with N for 30 min. The reaction was kept at 4 °C for 2 h
2
and continued at room temperature for 22 h. The resulting solution was
filtered through a Buchner funnel, and the solvent was evaporated under
vacuum. The crude NHS-ECT was purified by column chromatography
on silica gel with methanol as mobile phase to rapidly elute unreacted
ECT, followed by ethyl acetate to elute purified NHS-ECT. The product
4
. Conclusions
A multifunctional, “smart” siRNA carrier (PAT-SPN) intended
for specific activation in vascularized tumors and metastases
was successfully constructed, characterized, and demonstrated.
1
was dried under vacuum to yield a solid pale yellow powder. H NMR
(400 MHz, CDCl ): 3.36 (q, 2H, -S-CH -CH ), 2.94 (m, 2H, -C-CH -CH -),
3
2
3
2
2
3
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2
1
.86 (s, 4H, -CO-CH -CH -CO-), 2.71-2.50 (m, 2H, -CH -CH -COOH),
from the resonances associated with the DMAEMA methylene, the BMA
methylene, the PAA methylene, and the PEG backbone.
2
2
2
2
.89 (s, 3H, CH -C-CN), 1.37 (t, 3H, -S-CH -CH ).
3
2
3
Synthesis of PEG-Peptide RAFT Macro Chain Transfer Agent: The MMP-
-cleavable peptide (H-VPLS↓LYSGCG-OH; arrow indicates expected
Molecular weight and polydispersity of the copolymers were
characterized by GPC using a mobile phase of N,N-dimethylformamide
(DMF) with 100 mM LiBr at 60 °C using a flow rate of 1.0 mL min
7
−1
MMP-7 cleavage site) was synthesized through standard Fmoc-based
solid phase techniques on a PS3 synthesizer (Protein Technologies,
Tucson, AZ). The peptide was purified using an HPLC (Waters Breeze
system) with a gradient mobile phase consisting of methanol and water,
both containing 0.05% formic acid, through a Phenomenex Luna semi-
prep 5U C18 (2) column (250 × 10 mm). Peptide sensitivity to MMP-7
was confirmed on a 16.5% tris-tricine polyacrylamide gel, containing
(columns: Tosoh Biosciences 1x TSKGel Alpha4000, 2x TSKGel Alpha3000
in series). Chromatograms were recorded with a Shimadzu SPD-10A
UV detector, a Shimadzu RID-10A refractive index detector (Shimadzu
Scientific Instruments, Columbia, MD), and a Wyatt miniDAWN Treos
multi-angle light scattering detector (MALS; Wyatt Technology, Santa
Barbara, CA). Absolute molecular weight was determined from first
principles via this GPC-MALS setup by measuring the polymer dn/dc
using known concentrations of the purified PEG-pep-pD-pDPB polymer
sample.
Nanoparticle Assembly and Size Measurement by DLS and TEM: PEG-
pep-pD-pDPB polymer was dissolved in ethanol at a concentration of 20
mg/mL. This freshly prepared stock solution in ethanol was then added
dropwise into a 20 fold volume excess of 1× phosphate buffer saline
(PBS) at pH 7.4 under stirring. This process triggered spontaneous self-
assembly of micellar PAT-SPNs at 1 mg/mL. PAT-SPNs were diluted with
PBS to 0.20 mg/mL for particle diameter measurement on DLS. Samples
were also prepared for transmission electron microscopy (TEM) imaging
using a Philips CM20 system. Carbon film-backed copper grids (Electron
Microscopy Sciences, Hatfield, PA) were sequentially dipped into ethanol
for 30 s, then 0.15 mg/mL nanoparticle suspension for 1 min, and finally
3% uranyl acetate in water. Following each dipping step, grids were
gently blotted dry, and after staining, they were further dried in a vacuum
desiccator for 6 h prior to imaging.
1
2% glycerol. Samples were separated in tris-tricine running buffer
under 150 V volt-constant current. Cleavage of the peptide (5 mg/mL)
by MMP-7 (1 μg/mL) in PBS buffer (pH 7.4) was tested before and after
the removal of the protecting group on the amino acid side chains. The
peptide with side chains that had not been deprotected was used as a
control since the tert-butyl protecting groups block MMP cleavage of the
peptide.
After validation of MMP-7 peptide cleavability, peptide (208 mg,
0
.21 mmol) and maleimide-PEG (mal-PEG 5 kDa, 520 mg, 0.11 mmol)
(
Laysan Bio, Inc.) were added in a 50 mL round-bottom flask and purged
with N2 for 30 min before dissolving into N2 pre-purged methanol.
Triethylamine (TEA) (24.2 mg, 0.24 mmol) was then added. The reaction
was stirred at room temperature for 12 h. PEG-peptide was purified from
unreacted peptide using PD-10 columns (GE Healthcare Life Sciences),
and the purified product was lyophilized. Purified PEG-peptide (270 mg,
.045 mmol) and NHS-ECT (162 mg, 0.45 mmol) were dissolved in a
co-solvent of 33 mL MeOH and 15 mL DMF and purged with N2 for
0 min. The reaction was stirred at 30 °C for 24 h and then dialyzed
against methanol (MWCO = 3000 Da, Fisher Scientific) to remove DMF.
Methanol was subsequently removed by rotary evaporation. Further
purification was completed by precipitating the PEG-peptide RAFT
macro-CTA two times from chloroform into ether.
Synthesis of pDMAEMA with PEG-Peptide RAFT Macro-CTA: The RAFT
polymerization of DMAEMA was conducted at 70° C under a nitrogen
atmosphere. DMAEMA (420 mg, 2.67 mmol), PEG-peptide macroCTA
0
3
pH-Dependent Membrane Disruption Hemolysis Measurement:
[66,67]
Hemolysis was measured according to a reported method
to
determine the membrane-disruptive activity of intact PAT-SPN and
MMP-7 pretreated PAT-SPN nanoparticles at pH values that mimic
intracellular trafficking. Following Vanderbilt University Institutional
Review Board approval of human subject protocols, whole human blood
was collected, centrifuged, plasma aspirated, and washed three times
in 150 mM NaCl to isolate the red blood cells (RBC). RBCs were then
resuspended in phosphate buffer (PB) in 96 well plates at pH 7.4, 6.8,
6.2 or 5.6 in the presence of the polymers at 1, 5, 15, 30 μg/mL and
incubated at 37 °C for 1 h. Samples were centrifuged to pellet intact
RBCs, and percent hemolysis relative to 1% Triton X-100 was determined
based on absorbance of the supernatants at 450 nm.
(
186 mg, containing 0.02 mmol of CTA functionality, as determined by
its UV-Vis spectroscopy extinction coefficient at 320nm), AIBN (0.42 mg,
.002 mmol), and trioxane (10 mg) were dissolved in 1.23 mL dioxane in
a 5 mL flask equipped with a magnetic stir bar. Initial monomer to CTA
[M] /[CTA] ) and CTA to initiator molar ([CTA] /[I] ) ratios were 133/1
0
(
0
0
0
0
and 10/1. Kinetic samples were removed periodically by nitrogen-purged
syringe to monitor monomer conversion by H NMR spectroscopy. The
Assessment of siRNA Loading by Gel Electrophoresis: Agarose gel
electrophoresis was used to determine siRNA loading capacity and
siRNA protection of the nanoparticles. The siRNA (21 base pairs, 0.04
nmol in each sample) was complexed with varied amounts of PAT-SPNs
to achieve N(+)/P(-) charge ratios of 1, 2, 4, and 8, and free siRNA and
the siRNA-loaded PAT-SPN formulations were run on a 2% agarose gel.
The N(+)/P(-) charge ratios were calculated using the positively charged
DMAEMA block, which is estimated to be 50% protonated at pH = 7.4
1
polymerization was quenched after 8.6 h (29% conversion) by opening
the vial to air and cooling the reaction in an ice bath. The resultant PEG-
peptide-pDMAEMA (PEG-pep-pD) product was isolated by precipitation
into cold 40/60 vol.% diethyl ether/pentane. The resultant polymer was
redissolved in deionized water, further purified using PD-10 desalting
columns, and lyophilized.
Synthesis of p(DMAEMA-co-PAA-co-BMA) Terpolymer Block from PEG-
pep-pD Macro-CTA: Stoichiometric quantities of DMAEMA, PAA, and
BMA (28/20/52 mol%), PEG-pep-pD macroCTA (200 mg, containing
+
(forming – NH-(CH ) ), divided by the negatively charged phosphate
2 2
27]
groups in the siRNA.[ The equation is expressed as:
0
.01 mmol of CTA functionality, as determined by its UV-Vis spectroscopy
Molpolymer ∗ RUDMAEMA ∗ 50%
N/P =
extinction coefficient at 320nm), and AIBN (0.27 mg, 0.0017 mmol)
were dissolved in 1.3 mL cosolvent of dioxane:DMF = 67:33 vol%. The
MolsiRNA ∗bp
∗2
siRNA
[
M] /[CTA] and [CTA] /[I] ratios were 285/1 and 5.6/1. The reaction
where Molpolymer is the number of mol of the complete PEG-pep-pD-
pDPB polymer, RUDMAEMA is the number of repeating units of DMEAMA
in the pDMAEMA block, MolsiRNA is the number of mol of siRNA, and
bpsiRNA is the base pair number in the siRNA. This equation was also
used for studies in which fluorophore-labeled dsDNA is used since the
charge per base pair is the same as for siRNA.
For the assessment of siRNA protection from degradation in serum,
formulations of N(+)/P(-) = 6 calculated from the above equation were
applied. PAT-SPN/siRNA were incubated in 50% FBS for 1, 4, 8, or 24 h.
Afterward, 1% SDS was added to each sample to disrupt the PAT-SPN/
siRNA and release the complexed siRNA. Free siRNA was incubated in
50% serum for 0.25, 0.5, 1, 2, and 4 h. Free siRNA and the siRNA-loaded
PAT-SPN formulations were run on 2% agarose gels.
0
0
0
0
mixture was purged with nitrogen for 20 min and allowed to react at
0 °C for 20 h. The resultant PEG-peptide-pDMAEMA-b-p(DMAEMA-
7
co-PAA-co-BMA) (PEG-pep-pD-pDPB) crude product was isolated by
precipitation into cold 50/50 diethyl ether/pentane twice. The resultant
polymer was resuspended in water, dialyzed against water overnight,
and then lyophilized.
Polymer Characterization: Polymer products were dissolved in
1
deuterated methanol (MeOD) and analyzed by H-NMR spectroscopy
using a 9.3T Oxford magnet equipped with a Bruker DRX console
operating at 400 MHz. The PEG-pep-pD composition was determined
from the resonance of DMAEMA methylene. Composition of the
completed PAT-SPN polymer (PEG-pep-pD-pDPB) was determined
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FAM
Nanoparticle Stability Study by FRET: Equal moles of DNA
have a linear relationship with cell number (Supporting information,
Figure S14). In gene silencing experiments, R221A-Luc were seeded at
Cy5
and DNA were mixed and incubated at room temperature for 1 h
before loading onto PAT-SPNs at room temperature for 1 h. PAT-SPN/
DNA /DNA formulations of N(+)/P(-) = 6 were incubated (125 nM
DNA /DNA in 200 μL medium) at 37 °C for 2, 4, 8, and 24 h in
the presence of 50% fetal bovine serum. FRET signal was determined
relative to PAT-SPN/DNA /DNA in PBS (lack of serum and without
incubation). Samples containing 50% serum were background-corrected
to account for any FRET signal resulting from 50% serum in PBS. FRET
signal was measured on an Infinite F500 TECAN plate reader with
excitation at 485 ± 20 nm using FAM as a donor and emission at 670 ±
4
2
a density of 5 × 10 cells/cm in 96 well plates and allowed to adhere
overnight. Different formulations of PAT-SPNs were made by complexing
with luciferase siRNA (Ambion) at N(+)/P(-) ratios of 2, 4, and 6. Some
PAT-SPNs were pre-activated with MMP-7 for 6 h as discussed for
uptake experiments. Lipofectamine dose (1 μL Lipofectamine in 200 μL
medium per 96 plate well) was chosen based on the manufacturer
instructions and used as a comparison with PAT-SPNs. Cells were
treated for 6 h with 50 nM siRNA concentration for all formulations.
Afterward, the cells were given fresh media and incubated for an
additional 18 h. To measure luciferase gene silencing, luminescence in
each sample was quantified on the IVIS-100 (Caliper Life Sciences) after
adding 0.15 mg/mL luciferin, and luciferase activity was normalized
to the total protein content measured from the cell lysate of each well
using the BCA assay.
For nanoparticle cytotoxicity assessment, a scrambled siRNA (proven
in other experiments to have no luciferase silencing activity) was
complexed with the PAT-SPNs at charge ratios of 2, 4, 6, 8, and 12. The
treatment protocol was the same as described above, and the relative
cell number was determined by quantification of luciferase activity in
each well. Each group was tested in triplicate and normalized to cells
receiving no treatment. Results from three independent experiments
were averaged to generate the final data reported here.
FAM
Cy5
FAM
Cy5
FAM
Cy5
2
5 nm using Cy5 as the acceptor.
Assessment of PAT-SPN MMP-7 Responsiveness: PAT-SPN proximity
activation by MMP-7 was tested in vitro by GPC and through
physicochemical assessments of particle size and surface charge.
PAT-SPNs in PBS (0.9 mg/mL) were treated with 1 μg/mL (50 nM)
active human MMP-7 (EMD chemicals) in the presence of 50 μM
2
+
Zn ions or PBS as a control. PAT-SPNs were incubated with MMP-7
at 50 nM at 37 °C, and, at various time points, samples were diluted
to 0.2 mg/mL with deionized water and used to measure size and
ζ-potential on a Malvern Zetasizer Nano-ZS. The change in ζ-potential
for PAT-SPNs incubated with MMP-7 at 1 and 5 nM at 37 °C was also
measured with the same method. An aliquot of the 1 mg/mL sample
after 6 h of MMP-7 exposure was dialyzed against deionized water for
one day to remove buffer salts, lyophilized, and analyzed by GPC.
Cell Culture: MDA-MB-231 breast cancer cells (ATCC, Manassas,
VA) and R221A-Luc mammary tumor cells that constitutively express
luciferase were cultured on standard tissue culture-treated polystyrene
surfaces in an incubator maintained at 37 °C/ 5% CO . MDA-MB-
31 cells were maintained in growth media consisting of Dulbecco’s
modified Eagle medium (Gibco) supplemented with 10% fetal bovine
serum (Gibco) and 50 μg/mL gentamicin (Mediatech). R221A-Luc cells
were grown in media consisting of Dulbecco’s modified Eagle medium
Confirmation of PAT-SPN Activation by Endogenously Produced MMPs:
The ability of cell-secreted MMPs to cleave the PAT component and
trigger cell uptake was assessed in confocal microscopy cell uptake
experiments using the MDA-MB-231 cell line. MDA-MB-231 cells were
4
2
seeded at a density of 5 × 10 cells/cm in 8-well chamber slides (Nunc/
Thermo-Fisher) and allowed to adhere for 6 h in DMEM with 10% FBS
before replacing the media with serum-free DMEM supplemented with
50 μg/mL gentamicin. The cells were then incubated for 12 h to allow
for cell secretion of MMPs. At that time, the existing media in a subset
of wells was supplemented with 25 μM broad-spectrum MMP inhibitor
GM 6001. Cells were then incubated for an additoinal 2 h, after which
2
2
(
Gibco) supplemented with 2 mM glutamine (Mediatech) and 5 μg/mL
puromicin dihydrochloride.
Flow Cytometry and Confocal Microscopy Cell Uptake Measurements:
the the conditioned media on all cells (with or without prior GM6001
Cy5
Flow cytometry was utilized to quantify intracellular delivery of PAT-
addition) was supplemented with PAT-SPN/DNA
N(+)/P(−) = 6
FAM
Cy5
SPNs. In these studies, DNA
was used to model siRNA and was pre-
(50 nM of DNA ). After 5 h of incubation, the samples were washed
twice with PBS, supplied fresh media, and the live cells were imaged on
a Zeiss LSM 510 confocal microscope.
complexed with PAT-SPN at N(+)/P(-) = 6 for 1 h. MDA-MB-231 breast
4
2
cancer cells were seeded at 5 × 10 cells/cm in 24 well plates (Costar)
and allowed to adhere overnight. PAT-SPNs were pre-treated with 50 nM
The MMP-dependent increase in PAT-SPN/DNA ζ-potential following
exposure to the conditioned media of breast cancer cells was also
characterized. MDA-MB-231 breast cancer cells were seeded at 2 ×
10 cells/cm2 in 12 well plates in 10% FBS, and after 6 h, the media
was replaced with serum-free and phenol red-free RPMI 1640. After 24 h,
the media was collected, centrifuged, and aliquoted into 50 μL samples.
GM 6001 was added at 25 μM into a subset of the samples, and all
conditioned media samples were incubated at 37 °C for 2 h. PAT-SPN/
DNA samples (0.5 mg/mL of polymer, N(+)/P(-) = 6) were then treated
with a 50% dilution of these conditioned media samples. To assess
PAT-SPN/DNA cleavage by the MMPs secreted into the conditioned
media, samples were taken after 0 h, 6 h, and 18 h of treatment, and the
ζ-potential was measured following sample dilution to 0.1 mg/mL with
deionized water. Conditioned media containing GM6001 and MMP-7
precleaved PAT-SPN/DNA nanocarriers were used as controls to confirm
MMP-dependency of ζ-potential changes and to benchmark ζ-potential
changes against exogenous MMP-7 treatment.
MMP-7 and 50 μM ZnSO , in the absence of serum, for 6 h prior to
4
use in cell experiments. PAT-SPN/FAM-labeled dsDNA complexes with
or without MMP-7 pre-treatment were then added to the cell wells with
5
5
0 nM FAM-labeled dsDNA in DMEM medium supplemented with 10%
FBS and 50 μg/mL gentamicin. After 6 h incubation, cells prepared for
flow cytometry were washed with PBS, trypsinized, and resuspended in
PBS (containing 0.04% trypan blue to quench extracellular fluorescence).
Cellular internalization of the PAT-SPNs was quantified by fluorescence
measurement of FAM label in the cell samples using a BD FACSCalibur
flow cytometer (San Jose, CA).
For confocal microscopy experiments, PAT-SPN/DNACy5 of N(+)/P(-) =
6
was prepared as for the flow cytometry studies. MDA-MB-231cells
4
2
were seeded at a density of 5 × 10 cells/cm in 8-well chamber slides
Cy5
(Nunc/ Thermo-Fisher). Cells were treated with PAT-SPN/DNA with or
without MMP-7 pre-activation (at 50 and 100 nM DNA concentrations),
or PBS in DMEM medium supplemented with 10% FBS and 50 μg/mL
gentamicin for 6 h. After washing twice with PBS and replaced with
fresh medium, the live cells were imaged on a Zeiss LSM 510 confocal
microscope. For LysoTracker co-localization imaging, some cells were
further incubated for 4 h, stained with 75 nM LysoTracker (Invitrogen)
for 30 min, washed twice with PBS, and imaged on a Zeiss LSM 510
confocal microscope.
Statistical Analysis: All data are reported as mean and standard error
of the mean (SEM). Analysis of Variance (ANOVA) was used to establish
statistical significance, and p < 0.05 was considered significant.
Assessment of PAT-SPN Cytocompatibility and siRNA Gene Knockdown:
PAT-SPNs were screened for cytocompatibility and gene silencing
bioactivity using R221A-Luc mammary cancer cells that constitutively
express luciferase. Luciferase activity in cell samples was confirmed to
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
3
050 wileyonlinelibrary.com
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Funct. Mater. 2013, 23, 3040–3052

www.afm-journal.de
www.MaterialsViews.com
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