Intracellular Delivery of Functional Proteins and Native Drugs by Cell-Penetrating Poly(disulfide)s

Article abstract of DOI:10.1021/jacs.5b08130

The efficient delivery of bioactive compounds into cells is a major challenge in drug discovery. We report herein the development of novel methods for intracellular delivery of functional proteins (including antibodies) and native small-molecule drugs by

Full text of DOI:10.1021/jacs.5b08130

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Article
Intracellular Delivery of Functional Proteins and
Native Drugs by Cell-Penetrating Poly(disulfide)s
Jiaqi Fu, Changmin Yu, Lin Li, and Shao Q. Yao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b08130 • Publication Date (Web): 04 Sep 2015
Downloaded from http://pubs.acs.org on September 6, 2015
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Jiaqi Fu,† Changmin Yu,† Lin Li,†‡ and Shao Q. Yao*†
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†Department of Chemistry, National University of Singapore, Singapore 117543
‡Key Laboratory of Flexible Electronics & Institute of Advanced Materials, National Jiangsu Synergistic Innovation
Center for Advanced Materials, Nanjing Tech University, Nanjing, P. R. China 211816
ABSTRACT: The efficient delivery of bioactive compounds into cells is a major challenge in drug discovery. By making
use of cell-penetrating poly(disulfide)s, or CPDs, we report herein the development of novel methods for intracellular
delivery of functional proteins (including antibodies) and native small molecule drugs. CPDs were recently shown to be
rapidly taken up by mammalian cells in endocytosis-independent pathways, but their applications for delivery of proteins
and native small molecule drugs have not been demonstrated. With our newly developed, CPD-assisted approaches, rapid
and “bioorthogonal” loading of cargos was carried out with pre-synthesized CPDs, in two steps and in a matter of minutes
under aqueous conditions. The resulting CPD-cargo conjugates were used immediately for subsequent cell delivery stud-
ies. With the versatility and flexibility of these methods, we further showed that they could be used for immediate deliv-
ery of a variety of functional cargos with minimum chemical and genetic manipulations. The minimal cell cytotoxicity of
these CPDs and their cargo-loaded conjugates further highlights the unique advantage of this new cell-transduction
method over other existing strategies, and ensures our entire delivery protocol is compatible with subsequent live-cell
experiments and biological studies.
consuming and worse, often results in alteration of the
compound’s biological properties.3b Recent advances in
The efficient delivery of bioactive compounds, including
material chemistry have provided alternatives, where na-
nucleic acids, peptides/proteins and small molecules, into
cells is a major challenge in drug discovery.1 The difficulty
is more pronounced for large molecules such as proteins
and DNAs/RNAs,2 but due to the hydrophobicity and
poor water solubility of many small molecule drug candi-
dates, such compounds don’t enter cells readily either,
without proper formulation and/or delivery vehicles.3 To
achieve intracellular delivery of proteins, a variety of
methods have been developed in the last 20 years, includ-
ing the use of cell-penetrating peptides (CPPs), super-
charged proteins, liposomes, nanoparticles (NPs) and
polymers.4-7 Despite significant progress, these methods
have their share of shortcomings, including low/variable
delivery efficiency, the need for protein modification,
high cytotoxicity, and perhaps most importantly, ineffec-
tive endosomal/lysosomal escape.2a Take CPPs for exam-
ple, it is well-documented that, while CPP-conjugated
small- and medium-size cargos may be efficiently trans-
duced into cells via non-endocytic pathways, large cargos
such as proteins are mostly taken up by endocytosis, lead-
ing to subsequent endosomal trapping and lysosomal
degradation.8 To deliver hydrophobic small molecules
intracellularly, chemical modifications may be used to
make analogs that possess improved physicochemical and
pharmacokinetic profiles,9 but this is costly, time-
tive drugs are directly “loaded” into a suitable “container”
without the need of chemical modifications.3a,10 For ex-
ample, the use of small molecule-encapsulated mesopo-
rous silica nanoparticles (MSNs) for drug delivery is
worth-noting, in part due to the numerous desirable
properties that MSNs possess, including high loading ca-
pacity, biocompatibility and “zero premature release”.10b,c
In order to minimize unwanted leakage of the encapsu-
lated drug and improve cellular uptake of MSNs, the sur-
faces of these nanoparticles are “capped” with CPPs and
other forms of chemicals,11 which, in most cases, also
leads to severe endosome trapping and ineffective drug
release.11a,b
Herein, we focus on the development of novel methods
for intracellular delivery of functional proteins (including
antibodies) and native small molecule drugs by making
use of cell-penetrating poly(disulfide)s, or CPDs (Figure
1A).12 CPDs could be considered synthetic mimics of poly-
arginine CPPs, in which the polypeptide backbone was
replaced with poly(disulfide)s. Upon cellular uptake,
CPDs are rapidly degraded in the cytosol by glutathione
(GSH)-assisted depolymerization and show minimal cyto-
toxicity.12,13 Importantly, Matile et al. showed in a recent
study that, CPDs made of thiol-modified small fluoro-
phores (as initiators/cargos), a guanidinium-
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CPD
Cargo
Complexed Product
Formation via
Mw (KDa)
30
PDI
1.5
BiotinCPD
Ni-NTACPD
AvidinCy5
BRD-4Cy5
Casp-3
CPD-Avidin
CPD-BRD-4
CPD-Casp-3
CPD-BSA
Non-Covalent
Affinity Interaction
32
1.6
Protein
TCO-BSACy5
TCO-AbFC
MSN-Dox
Covalent
Bioorthogonal Ligation
TzCPD
22
30
2.3
1.5
CPD-Ab
BiotinCPD
CPD-MSN-Dox
Charge Interaction
Small Molecule
Figure 1. Overview of CPD-facilitated intracellular delivery of proteins (including antibodies) and native small molecule drugs.
(A) Newly developed initiators (I1/I2/I3), Monomer (M), terminator (T), the polymerization/depolymerization process of CPDs,
and the two-step approach for “conjugation” of protein cargos with CPDs. (B) Summary of non-covalent and covalent approach-
es for bioorthogonal attachment of CPDs to proteins. The highly efficient site-specific tetrazine-TCO ligation reaction is high-
lighted. (C) Schematic summary of intracellular delivery of native small molecules by drug-loaded MSNs capped with BiotinCPD.
(D) Table summary of all CPDs and cargos used in the current study.
propagating monomer (e.g. M in Figure 1A) and a termi-
nator (e.g. T), rapidly enter mammalian cells via thiol-
mediated pathways.12b The major issue of endosomal
trapping commonly associated with CPPs and other
means of delivery was thus minimized.14 Subsequently,
the authors proposed this substrate-initiated, cell-
penetrating poly-(disulfide)s (siCPDs) may be used for
intracellular delivery of other thiol-containing (or modi-
fied) cargos. This hypothesis was, however, never demon-
strated experimentally. Furthermore, during siCPD syn-
thesis, given the obligatory role of the thiol-containing
initiator (I), the need of millimolar concentrations of ini-
tiator/monomer/terminator as well as organic co-
solvents, it is not trivial how a protein could be directly
used as an initiator, nor is it practical to use thiol-
modified small molecule drugs, as few small molecule
drugs contain native thiols in their structures.
With the current work, we have confirmed that, for the
first time, proteins conjugated with CPDs, either cova-
lently (via bioorthogonal chemistry) or non-covalently
(via affinity interaction), could be rapidly and efficiently
delivered into the cytosol of different mammalian cells
without being trapped by endocytic vesicles. Similarly, by
making use of CPD-capped MSNs, we have successfully
achieved intracellular delivery of native small molecule
drugs (e.g. doxorubicin, or Dox). Our results indicate
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these novel protein and small molecule delivery methods polymerization. The monomer (M), as well as subsequent
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possess the following favorable properties when com-
pared to most existing strategies: (1) fast delivery, with
cell entry in less than 15 min, (2) flexible, enabling con-
venient delivery of different types of cargos, (3) less cyto-
toxic and applicable to different types of mammalian
cells, (4) efficient, facilitating cargo delivery at nanomolar
concentrations, and (5) immediately available upon cell
entry due to rapid CPD degradation, thus retaining the
biological activity of the delivered cargo.
polymerized products with the corresponding initiators
(and capped with T; Figure 1A), were synthesized accord-
ing to published protocols.12b Upon purification, the re-
sulting CPDs were further characterized by gel permea-
tion chromatography (GPC), giving an average molecular
weight (Mw) of 22-32 KDa with a polydispersity index
(PDI) of 1.5-2.3 (Figure 1D & S3A). The concentration of
TzCPD stock solution was determined by measurement
with UV-Vis spectroscopy at the tetrazine absorbance
(520 nm),18 and for the other two CPDs, they were similarly
estimated.
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Protein Attachment to CPDs. We next chose three
fluorescently labeled recombinant proteins having differ-
ent molecular weights, AvidinCy5 (~80 KDa in its tetram-
eric form), TCO-BSACy5 (~66 KDa) and BRD-4Cy5 (~15
KDa; an epigenetic reader protein19) as model cargos for
delivery by BiotinCPD, TzCPD and Ni-NTACPD, respectively.
Fluorescent labeling of these proteins was done by stand-
ard protein conjugation chemistry with commercially
available Cy5 dyes (Figure S1), thus allowing the entire
cargo delivery process to be monitored by fluorescence
microscopy and quantified by flow cytometry. Attach-
ment of CPDs, either non-covalently or covalently, to the
proteins was next done by simple “mix-and-go” protocols,
giving CPD-Avidin, CPD-BSA and CPD-BRD-4, respec-
tively (Figures 1D & S2). It should be highlighted that our
choices of highly bioorthogonal “conjugation” chemistries
between CPDs and proteins were critical, not only for
convenient sake and generality, but more importantly to
allow protein complexes to be directly used in subsequent
cell-based experiments. Furthermore, since most
CPD/protein-complexing reactions couldn’t be reliably
monitored by SDS-PAGE without addition of DTT, which
in turn caused CPD degradation (data not shown), we
needed such conjugation chemistries to be free of failure!
Nevertheless, we found that, for AvidinCy5 and CPD-
Avidin, they were well separated under modified DTT-
free SDS-PAGE conditions (presumably due to extremely
strong biotin/avidin interaction and avidin stability20),
and were thus chosen as the model system to monitor the
processes of CPD/protein complex formation and GSH-
assisted intracellular depolymerization/cargo release
(Figure S3B); upon incubation with a freshly prepared
Design and Synthesis of New CPDs. We were in-
trigued by the excellent properties of siCPDs (e.g. mini-
mal endosome trapping and cytotoxicity), as reported by
Matile et al.,12 and wondered whether robust, CPD-
mediated methods could be developed to facilitate intra-
cellular delivery of functional proteins, therapeutic anti-
bodies and native small molecule drugs (that is, without
any form of thiol modification). Instead of the substrate-
initiated CPD synthesis as originally proposed,12b we en-
visaged such cargos could be more conveniently append-
ed, in two steps, to pre-synthesized, functionally decorat-
ed CPDs by using suitable “conjugation” chemistries that
are already available for recombinant proteins/antibodies
and small molecules (Figure 1). Three types of thiol-
containing initiators, I1/I2/I3, were thus designed, con-
taining biotin, nitrilotriacetic acid (NTA) and tetrazine
(Tz), respectively. Upon polymerization, the correspond-
ing CPDs (BiotinCPD, Ni-NTACPD & TzCPD; Figure 1D),
could be obtained. Ni-NTACPD could be attached
bioorthogonally to readily available (His)6-tagged pro-
teins via non-covalent affinity interaction (Kd of Ni-
NTA/(His)6 < 10-7 M). BiotinCPD was designed to test
whether it could be used to deliver avidin via similar non-
covalent, but substantially stronger, interactions (Kd of
biotin/avidin < 1o-15 M). For a therapeutic antibody (Ab),
which might not possess a histag,15 we used the well-
known bis-sulfone chemistry that enables site-specific
introduction of a trans-cyclooctyne (TCO) into the native
disulfide present in the antibody (vide infra).16 Subsequent
bioorthogonal covalent attachment of TzCPD to the TCO-
modified antibody would result in quantitative formation
of CPD-Ab within minutes by the highly efficient te-
trazine-TCO ligation (Figure 1B).17 To “append” a native
small molecule drug to the CPD, we would use a positive-
ly charged CPD (BiotinCPD in this case) to cap the nega-
tively charged, drug-loaded MSNs (i.e. MSN-Dox) by
electrostatic interaction, giving CPD-MSN-Dox (Figure
1C-D); the capping of nanoparticles with CPDs is unprec-
edented and we were hopeful that, if CPDs could facilitate
endocytosis-independent cellular uptake of large, na-
nometer-size cargos such as MSNs, then a variety of hy-
drophobic drugs could potentially be delivered intracellu-
larly in their native form, in a controllable manner.10
0
HeLa lysate (1 mg/mL) for 1 h at 37 C, the higher-order
CPD-Avidin complex was found to have significantly
depolymerized, indicating our CPD-loaded protein cargos
would also be released readily in cytosolic environments.
Cellular Uptake. In order to unequivocally demon-
strate the cellular uptake of the CPD-conjugated proteins,
and their subcellular localization, we used confocal laser
scanning microscopy (CLSM) with live HeLa cells. It was
previously found that the use of fixed cells are not suita-
ble for such studies;8b upon fixation, cargos delivered by
CPPs and other means often artificially “escape” from
endocytic vesicles, thus resulting in misleading conclu-
sions.2a,21 Real-time imaging experiments together with
different fluorescent organelle trackers (CellMask™ mem-
brane tracker/LysoTracker™: pseudocolored in green;
Hoechst nuclear stain: pseudocolored in blue) were
All three initiators were conveniently synthesized (two
of which in their respective disulfide forms; Scheme S1),
and treated/reduced with TECP immediately prior to
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0o
90o
0 min
2 min
15 min
30 min
-90o
TCO-BSACy5
LysoTrackerTM
Hoechst
View
(A)
(C)
(B)
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Pro-JectTM
(D)
(H)
(I)
CPD (-)
CPD (+)
CPD
6
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ProteinCy5
CellMaskTM
Hoechst
120
160
FACS HCS
CPD-Avidin CPD-BSA CPD-BRD-4
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ProteinCy5
Pro-JectTM
CPD
+
-
+
+
+
-
(E)
(F)
-
(KDa)
20
-
+
CPD-Avidin
66
15
CPD-BSA
0
0
15
30
60
120
240
480 (min)
5
10
50
100 (nM)
CPD-BRD-4
180
(G)
(J)120
(K)
FACS HCS
CPD-Avidin
CPD-BSA
CPD-BRD-4
CPD-Avidin CPD-BSA CPD-BRD-4
CPD-Avidin
CPD-BSA
CPD-BRD-4
100
100
Temp (oC)
4
(KDa)
25 37
20
66
15
CPD-Avidin
CPD-BSA
CPD-BRD-4
0
0
0
0
TM
Pro-Ject
25
37 (oC)
TM
Blank
CPZ
w
MCD DTNB
CPD
CPD
Pro-Ject
4
Figure2. Cellular uptake of CPD-conjugated proteins. (A) Real-time CLSM of live HeLa cells treated with 50 nM of CPD-BSA
(red), LysoTracker™ (green) and Hoechst (blue) at indicated time intervals. (Inset) bright-field image of the corresponding fluo-
rescence channels. Scale bar = 20 µm. (B) 3D projections of z-stack images at different perspectives (step size, 0.186 µm) of HeLa
cells after 2-h CPD-BSA delivery. Pearson coefficient R = 0.35 (red/green). Scale bar = 13.2 µm. See Supporting Movies for details.
(C) CLSM images showing cellular uptake of CPD-BSA (50 nM) versus TCO-BSACy5 (50 nM) complexed to Pro-Ject™ reagent.
The images were taken 1 h after protein delivery. Cells with the delivered proteins (red) were co-stained with CellMask™ mem-
brane tracker (green) and Hoechst (blue). (Inset) bright-field images. Scale bar = 20 μm. (D) Same as (C), except CPD-Avidin
(top) or CPD-BRD-4 (bottom) was used, with the corresponding CPD-free AvidnCy5 or BRD-4Cy5 serving as negative controls.
(E) SDS-PAGE/in-gel fluorescence scanning of lysates from HeLa cells treated with CPD-Protein (50 nM each; 1 h incubation),
showing successful cellular uptake. Cells treated with the corresponding CPD-free protein (50 nM; 1 h incubation), either with or
with Pro-Ject™ reagents, were run concurrently as controls. (F) Quantitative analysis of CPD-assisted protein delivery to HeLa
cells by flow cytometry. Control experiments were done with Pro-Ject™ method on the same proteins. Surface-bound fluorescent
materials were removed by washing the cells with heparin-containing PBS. RFU = relative fluorescence units. Data from the Pro-
Ject™ results were normalized to each of the CPD-delivered experiments. (G) Cell viability measured with XTT assay for HeLa
cells treated with a protein (50 nM; 1 h incubation) delivered by either CPD or Pro-Ject™ method. Buffer-treated cells were used
for normalization (as 100% viability). (H) Concentration-dependent protein delivery to HeLa cells as determined by flow cytom-
etry analysis (FACS). Cells were treated with each protein (5, 10, 50, 100 nM) for 8 h before being quantified. The overall uptake
for each protein at different concentrations was normalized to data obtained at 100 nM. (I) Time-dependent protein uptake of
HeLa cells treated with CPD-BSA (50 nM). The protein uptake was quantified by both flow cytometry and high-content screen-
ing (HCS) of live cells. The overall uptake in each experiment was normalized to the data obtained at 8 h. (J) Temperature-
dependent protein uptake by HeLa cells (50 nM protein; 1 h treatment), as determined by flow cytometry. Data were normalized
to those obtained at 37 oC. (Inset) SDS-PAGE/in-gel fluorescence scanning of lysates from treated cells. (K) Flow cytometry/HCS
quantification of protein uptake (50 nM of CPD-BSA; 1 h incubation) by HeLa cells treated with different inhibitors, including
chlorpromazine (CPZ), wortmannin (w), methyl-β-cyclodextrin (MβCD) and 5,5’-dithioobis-2-nitrobenzoic acid (DTNB). Data
were normalized to those of HeLa cells treated with CPD-BSA only (Blank).
carried out. As shown in Figure 2A, 2 min upon addition
of 50 nM of CPD-BSA (pseudocolored in red) to the cul-
ture medium at 37 C, red fluorescence started to accu-
dation, presumably due to high protein concentration or
unfolding.2a We next directly compared the cellular up-
take efficiency of this CPD-assisted strategy with that of
the Pro-Ject™ reagent, a commercially available liposome-
based protein delivery system.22 In addition to CLSM
(Figure 2C-D), we analyzed the successfully delivered and
depolymerized proteins by SDS-PAGE/in-gel fluorescence
scanning of lysates from treated cells (Figure 2E). We fur-
ther quantified protein uptake by flow cytometry analysis
(Figure 2F). In order to minimize false readings of cells
derived from membrane-bound, but not internalized,
fluorescent proteins, cells were washed with heparin-
o
mulate around the cell membrane. After 15 min, a sub-
stantial amount of CPD-BSA was observed to have suc-
cessfully been transduced and evenly distributed
throughout the cytosolic space, with no evidence of endo-
some/lysosome trapping. This trend persisted for the next
2 h (Figures 2B, S4 & Supporting Movies). With prolonged
incubation (> 4 h), however, we started to observe some
merged green/red fluorescence signals, indicating some
delivered protein had been destined for lysosomal degra-
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containing PBS before analysis.12b In all cases, with all three
process completely (Figure 2J). The insensitivity of pro-
protein cargos (TCO-BSACy5, AvidinCy5 & BRD-4Cy5) hav-
ing different types of “conjugation” chemistries (covalent
and non-covalent), their resulting CPD-complexes (CPD-
BSA, CPD-Avidin & CPD-BRD-4) were delivered into
HeLa cells more efficiently than the Pro-Ject™ delivery
method. Successful intracellular uptake of a cargo was
found to be completely dependent on its complex for-
mation with the corresponding CPD, as none of the car-
gos alone (Figure 2D, left panels; Figure 2E, lanes 1) could
enter cells. We found that, unlike the liposome-based
Pro-Ject™ method which makes use of electrostat-
ic/hydrophobic interaction for complex formation with a
target protein (thus delivery efficiency varies significantly
with protein size/charge; see Figures 2E-F & S5),22 pro-
teins of different sizes and charges were efficiently deliv-
ered by the CPD-assisted method. Gratifyingly, even
(His)6-tagged proteins formed by comparatively moderate
non-covalent interaction with Ni-NTACPD (e.g. CPD-BRD-
4) could be delivered, thus setting the stage for more
widespread applications of this new protein transduction
technology in future. In addition, we found the CPD
method was significantly less cytotoxic than the Pro-Ject™
approach for protein delivery (Figure 2G). This is in good
agreement with previous cell-based experiments with
fluorophore-loaded siCPDs.12b In fact, the 10-20% cell
death observed in our experiments was likely caused by
trace amount of residual iodoacetamide from the
polymerization reaction, which could not be completely
removed with current purification method. Further opti-
mizations of CPD-Protein delivery were done by concen-
tration- and time-dependent experiments (Figures 2H-I &
S6). In addition to flow cytometry, we used imaging-
based, high-content screening (HCS) for comparative
studies. HCS could be used to simultaneously analyze
many live cells in the same experiment, without cell de-
tachment/fixation which might cause artifacts, thus was a
quantitative complement to our CLSM results. As ex-
pected, both longer incubation time and higher cargo
loading led to increases in the amount of delivered pro-
teins. With the issues of potential lysosomal degradation
and cytotoxicity in mind, we recommend the optimal
conditions for this CPD delivery method to be 25-50 nM
protein loading and 1-2 h incubation, which are still more
efficient than existing protein delivery strategies.4-7 The
CPD method was further tested on other mammalian cell
lines (NIH 3T3, MCF-7, A549 and PC3; Figure S7); in all
cases, CPD-BSA was successfully taken up intracellularly,
albeit at varying degrees of efficiency.
tein delivery with endocytosis-related inhibitors used
(chlorpromazine, wortmannin & methyl-β-cyclodextrin),
ruled out the endocytosis pathway. On the contrary,
blocking exofacial thiols on the cell surface with 5,5’-
dithiobis-2-nitrobenzoic acid (DTNB) significantly sup-
pressed protein uptake, further supporting thiol-
mediated cargo delivery mechanisms.12b In our hands,
flow cytometry gave less consistent results, presumably
due to non-specific, surface-bound CPD-Protein and the
need of cell detachment (Figure 2K). HCS on the other
hand, was more reliable, enabling direct quantification of
live cells having only intracellular fluorescence signals.
CPD-Assisted Transduction of Functionally Active
Caspase-3. Having confirmed the effect of this newly
developed CPD method for intracellular protein delivery
with minimal endosome trapping, we next investigated
whether it could deliver functional, therapeutic proteins.
Caspase-3 (a cysteine protease) is a promising therapeutic
protein owing to its key role in cell apoptosis.23 Intracellu-
lar delivery of active caspase-3 to tumor cells renders
them hypersensitive toward treatment by anticancer
drugs such as Dox.24 Functionally active, recombinant
(His)6-tagged caspase-3 was prepared as previously de-
scribed.25 Upon mixing with Ni-NTACPD, the resulting
CPD-Casp-3 was formed. Mindful of the trace amount of
iodoacetamide from CPD preparation, and that absence
of DTT might further reduce the enzymatic activity of
caspase-3,23 we carried out normalization of CPD-Casp-3
(Figure S9); results indicate that even without DTT, CPD-
Casp-3 was able to retain > 20% of the original caspase-3
activity. The activity of the complex was partially restored
upon DTT treatment, presumably due to either CPD de-
polymerization or/and DTT reduction of active-site cyste-
ine in the enzyme. Therefore for CPD-Casp-3, upon cell
entry, its enzymatic activity would also be restored under
the highly reduced cytosolic environment. HeLa cells
were incubated with 50 nM of CPD-Casp-3, and the re-
sulting cells were imaged for intracellular caspase-3 activ-
ity upon treatment with Ac-DEVD-AMC for 2 h (Figure
3A);26 significant fluorescence signals (from the liberated
AMC dye) throughout the cytosol of CPD-Casp-3-treated
cells, but not in control cells, were detected, indicating
successful cytosolic delivery of the functionally active
protein. These results were further confirmed by in vitro
enzymatic determination of caspase-3 activity, as well as
Western blotting (WB) analysis, of the corresponding cell
lysates (Figure 3B);11c,27 in all cases, the presence of intra-
cellular caspase-3 and its activity were unequivocally es-
tablished. Intracellular activation of caspase-3 is known to
promote subsequent translocation of the active enzyme
into nucleus, cleave PARP1, and finally cause cell death by
apoptosis.28 We observed similar phenomena in our “arti-
ficially” induced apoptotic cells as well (Figure 3B inset &
Figure 3C). These results further suggest that cytosolic
proteins delivered by this CPD system are not necessarily
confined to their original destination upon cell entry.
Instead, they may be further sorted/translocated in man-
ners similar to endogenous proteins.
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Our earlier CLSM results showed even cytosolic distri-
bution of intracellular fluorescence upon protein delivery
(Figure 2A-D), indicative of endocytosis-independent
pathways facilitated by these CPDs as previously pro-
posed.12b In order to further confirm this, we carried our
detailed uptake studies of CPD-Protein by HeLa cells at
different temperatures and in the presence of endocytosis
inhibitors (Figures 2J-K & S8); in general, cell uptake pro-
files observed with these CPD-conjugated proteins were
similar to what was previously reported with small fluor-
ophore-modified siCPDs.12b Reduced temperature de-
creased protein delivery efficiency but did not block the
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channel (Lanes 4), successful introduction of the TCO
moiety (giving the resulting TCO-AbFC) followed by liga-
tion with a tetrazine-containing tetraethylrhodamine
reporter (TER-Tz218) shifted the band to higher molecu-
lar-weight regions where they became detectable under
both FITC and TER channels (Lanes 2).
-
+
(A)
(B)
Casp-3
+
+
+
1
2
3
4
5
6
7
8
Ni-NTACPD
-
(1)
(2)
(3)
(A)
Ni-NTACPD
1
(C)
CPD-Casp-3
+
+
-
+
+
-
-
-
Casp-3
(KDa)
Ni-NTACPD
113
89
PARP1
Cleaved
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Casp-3
histag
(Mw 976)
17
55
β-tubulin
0
25
75
50
150
100
300
-
75
25
25
Casp-3 (nM)
-
-
Ni-NTACPD (nM) 75
(B)
(C)
-
CPD (+)
CPD (-)
Modified Non-modified
Figure 3. CPD-assisted delivery of functionally active caspa-
se-3 to HeLa cells. (A) CLSM images of HeLa cells treated
with CPD-Casp-3 (50 nM) for 2 h, followed by incubation
with Ac-DEVD-AMC (40 M; 2 h). Scale bar = 20 m. (Inset)
bright-field images. (B) RFU of in vitro enzymatic caspase-3
assay of treated HeLa lysates.11c,27 (Inset) WB results of deliv-
ered active caspase-3 and cleaved endogenous PARP1 in
HeLa lysates from the same cells. (C) Cell viability/apoptosis
caused by delivered active caspase-3 as measured by XTT
assay. Since it took some time for intracellular caspase-3 to
be translocated into the nucleus and cleave PARP1,28 for (B &
C), HeLa cells were treated with CPD-Casp-3 for 8 h prior to
analysis.
+
-
+
+
+
-
-
-
DTT
(KDa)
TER-Tz2
TER
250
250
FITC
Figure 4. (A) Labeling mechanism of antibodies by Thi-
oLinker-TCO™. (B) TCO-AbFC reacting with or without TER-
Tz2, before being analyzed by SDS-PAGE (with or without 10
mM DTT in the loading dye). The gel was visualized under
both FITC and TER channels. (C) HeLa cells treated with 50
o
nM of CPD-Ab (1 h at 37 C) before being imaged. CPD-Ab
(green); Hoechst (blue). Scale bar = 20 μm.
With TCO-AbFC being successfully prepared, we next
conjugated it to TzCPD. as earlier described, to generate
CPD-Ab which was subsequently used for confirmation
of successful intracellular antibody delivery by CLSM. As
shown in Figure 4C, live HeLa cells treated with 50 nM of
CPD-Ab for 1 h were shown to have taken up the fluores-
cently labeled antibody and distributed it throughout the
cytosol (right panel), whereas no fluorescence was detect-
ed in cells treated with the antibody alone (without con-
jugation to TzCPD; left panel). We thus concluded that
this CPD-assisted method could be used for intracellular
delivery of antibodies as well.
Small Molecule Drug Delivery by CPD-MSNs. Deliv-
ery of small molecule drugs to tumor sites often suffers
from low efficiency due to hydrophobicity and poor water
solubility. Approximately 40% of all current commercial
drugs and up to 75% of drug candidates are classified as
being poorly water-soluble.3b We reasoned the siCPD
method previously proposed by Matile et al. would have
been impractical, as it would require chemical modifica-
tion of small molecules with a thiol handle which is not
available in most native drugs.12b We therefore turned to
mesoporous silica nanoparticles (MSNs), which are wide-
ly used for intracellular delivery of native small molecule
drugs. Due to the large size of MSNs (> 100 nm in diame-
ter), they are often not efficiently taken up by cells unless
surface modification with CPPs or other chemicals is in-
troduced.10,11 In most cases, however, this leads to endocy-
tosis and poor cytosolic release of MSN-encapsulated
drugs. We wondered whether the unique endocytosis-
CPD-Assisted Antibody Delivery. Monoclonal anti-
bodies (mAbs) constitute one of the largest classes of
therapeutic proteins.29 There are currently more than 30
antibody-based, FDA-approved drugs, most of which tar-
get cancer. The number could have been much higher,
had an effective means for intracellular delivery of anti-
bodies been available.15 Common protein delivery meth-
ods are even more problematic for intracellular delivery
of antibodies, as most of them are large (> 150 KDa). Real-
izing the highly efficient, endosome-independent features
endowed by our newly developed CPD protein delivery
method, we anticipated it might be ideally suited for de-
livery of therapeutic antibodies, for which many robust
conjugation chemistries are already available in the forms
of antibody-drug conjugates (ADCs) and PEGylation
without compromising antibodies’ activity and stabil-
ity.15,16a,30,31 We used Alexa Fluor® 488 goat anti-rabbit IgG
(AbFC) as a model antibody and modified it with a com-
mercially available bis-sulfone reagent, ThioLinker-TCO™
(Figure 4A);16c upon TCEP reduction of inter-chain disul-
fides in the antibody, the TCO reagent underwent double
elimination-addition reactions with two cysteines from
the same reduced disulfide to form a three-carbon bridge,
thus successfully introducing TCO into the antibody
while leaving it structurally intake. The same approach
has been used for site-specific PEGylation and ADCs in
various therapeutic proteins/antibodies.31,32 As shown in
Figure 4B, while AbFC showed up on a DTT-free SDS-
PAGE gel as a 250-KDa fluorescent band under the FITC
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independent mechanisms of the CPD-assisted delivery CPD-capped MSNs was evident (Figure S14). In vitro
1
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3
4
5
6
7
8
could be successfully emulated in drug-loaded MSNs, e.g.
GSH-induced depolymerization/uncapping followed by
release of Dox was successfully observed (Figure S15).
To follow the entire process, from cellular uptake of
CPD-MSN-Dox in live HeLa cells and the subsequent
uncapping/depolymerization of CPD, to cytosolic release
of Dox, we directly added to cell medium 20 g/mL of the
drug-loaded MSNs and the resulting cells were imaged
over 24 h by CLSM, followed by WB and apoptosis analy-
sis (Figure 5). MSN-Dox, the drug-loaded nanoparticles
without capping with BiotinCPD, was used as a negative
control. Since Dox is an intrinsically fluorescent com-
pound, its intracellular distribution could be conveniently
monitored by CLSM. It is also one of the most effective
anti-cancer drugs. By intercalating to nuclear DNA of
target tumors, it is known to cause caspase-3 activation,
PARP1 cleavage and cell apoptosis.34 As shown in Figure
5A, we observed accumulation of most CPD-MSN-Dox
inside the cytosol of treated cells after only 3 h of incuba-
tion, at which point substantial release of the MSN-
encapsulated Dox (in red) was also observed (panels 1-3).
The slower cellular uptake of CPD-MSN-Dox, when
compared to that of CPD-Protein, was likely due to the
much larger size of MSNs, but was nevertheless still faster
than that of CPP-capped MSNs.11 Over the course of the
next 21 h, more Dox was released from CPD-MSN-Dox
and entered cell nucleus (panels 6, 10 & 14 in Figure 5A),
resulting in apoptosis in > 70% of cells (Figure 5B-C; 24-h
treatment). Successful activation of endogenous caspase-3
activity and cleavage of PARP1 were observed as well. For
cells treated with MSN-Dox (i.e no BiotinCPD capping; 12
and 24 h incubation), intracellular fluorescence signals
were detected in the red but not green channel, indicat-
ing cellular uptake of MSN-Dox was unsuccessful due to
a lack of the surface-bound BiotinCPD (Figure S16), but
leaked Dox from the nanoparticles was able to subse-
quently enter cells freely and cause cell death. Again, a
small percentage of cell death detected in cells treated
with CPD-MSN (i.e. no Dox; Figure 5C) was attributed to
the trace amount of iodoacetamide present in BiotinCPD.
CPD-MSN-Dox.
MSN
Dox
Merged
Hoechst
(A)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
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(9)
(10)
(14)
(11)
(15)
(12)
(16)
(13)
(C)
(B)
Dox
MSN
BiotinCPD
- -
-
+
+
-
+
+
+
-
+ +
1
(KDa)
- -
+
100
PARP1
Cleaved
113
89
β-tubulin
55
Casp-3
17
0
0
-
-
+
-
+
Dox
+
-
+
-
-
-
-
+
+
+
-
-
-
MSN +
BiotinCPD
+
-
+
+
+
-
+
-
+
+
+
Figure 5. (A) Confocal images of HeLa cells treated with
o
CPD-MSN-Dox (20 µg/mL) over 24 h at 37 C. Dox (red),
MSN (green) and Hoechst (blue) were colored accordingly.
Arrowed: apoptotic cells. Scale bar = 20 µm. (B) In vitro en-
zymatic assay of the cell lysates (after 24-h cell treatment).
RFU was recorded after 5 h-incubation with Ac-DEVD-AMC
(1 μM) with HeLa lysate (25 μg/reaction). (Inset) WB results
showing the successful activation of endogenous caspase-3
and cleavage of PARP1 of treated cells. (C) Cell viabil-
ity/apoptosis caused by release of intracellular Dox in (A &
B) as measured by XTT assay. Control experiments were con-
currently done with cells treated with MSNs alone, CPD-
MSN or MSN-Dox.
In this study, we have successfully designed and syn-
thesized several novel cell-penetrating poly(disulfide)s.
These CPDs (BiotinCPD, Ni-NTACPD & TzCPD), upon highly
efficient bioorthogonal “conjugation”, either non-
covalently or covalently, to readily available cargos in-
cluding recombinant proteins and suitably modified anti-
bodies, were able to rapidly and efficiently deliver these
cargos into different mammalian cells via endocytosis-
independent pathways. Rapid intracellular CPD depoly-
merization of the delivered cargos under highly reduced
cytosolic environments subsequently released the pro-
teins in their functionally active form, which may be fur-
ther translocated to their intended subcellular organelles
for additional biological processes. The successful intra-
cellular delivery of antibodies by TzCPD indicates this
method may be more broadly applicable in future for ef-
fective cellular delivery of many other therapeutic anti-
bodies, which at present could not be adequately
-
To make CPD-MSN-Dox, PO4 -modified MSNs were
first prepared according to published protocols.33 Such
MSNs, due to their negatively charged surface, were
known to be minimally taken up by mammalian cells. To
follow the cellular uptake of MSNs, they were first doped
with a small amount of fluorescein.11c We next loaded Dox
followed by capping the surface of the resulting drug-
loaded MSNs with positively charged BiotinCPD via
charge-charge interaction. The resulting MSNs were
shown to be highly monodisperse (mean diameter: ca. 155
nm) and possess well-defined pore sizes with high specif-
ic areas (Figure S10–S13). Reversal of Zeta potentials from
-
negatives in the PO4 -modified MSNs to positives in the
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achieved with other protein transduction methods.15 Un-
like the siCPDs approach recently developed by Matile et
al.,12b who suggested thiol-containing small molecule
drugs and probes may be directly “grown” onto CPDs dur-
ing polymerization, we have successfully developed CPD-
capped MSNs for encapsulation of native small molecule
drugs without the need of introducing thiol handles.
With doxorubicin as an example, we found CPD-MSN-
Dox entered mammalian cells rapidly and was able to
subsequently release free Dox into the cytosol. While
more studies are needed to investigate the utilities of
CPDs as novel “capping” agents for MSNs and other types
of nanoparticles, our preliminary finding herein indicates
that they may be more widely used in future for intracel-
lular delivery of otherwise difficult-to-deliver drugs in a
highly controllable manner.3,10
One of the key features of our two-step, CPD-assisted
approaches is their versatility and flexibility, enabling
immediate delivery of a variety of cargos (recombinant
proteins, antibodies and native small molecule drugs)
with minimum chemical and genetic manipulations. The
other feature is the rapid and “bioorthogonal” cargo-
loading process – with different types of pre-synthesized
CPDs in hand, the resulting CPD-cargo conjugates could
be prepared in a matter of minutes under aqueous condi-
tions, and used immediately for subsequent cell delivery
studies. The minimal cell cytotoxicity of these new CPDs
and their cargo-loaded conjugates further highlights the
unique advantage of this new cell-transduction method
over other existing strategies, and ensures our entire de-
livery protocol is compatible with live-cell experiments.
Future work will focus on the expansion of the types of
CPDs by using other conjugation chemistries, develop-
ment of better CPD purification protocols, and applica-
tion of these CPDs for cell type-specific delivery of other
therapeutically important drugs (including proteins, anti-
bodies and small molecules).
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Notes
The authors declare no competing financial interests.
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