Intracellular Delivery of Native Proteins Facilitated by Cell-Penetrating Poly(disulfide)s

Article abstract of DOI:10.1002/anie.201711651

Intracellular delivery of therapeutic proteins is highly challenging and in most cases requires chemical or genetic modifications. Herein, two complementary approaches for endocytosis-independent delivery of proteins to live mammalian cells are reported. By using either a “glycan” tag naturally derived from glycosylated proteins or a “traceless” tag that could reversibly label native lysines on non-glycosylated proteins, followed by bioorthogonal conjugation with cell-penetrating poly(disulfide)s (CPDs), we achieved intracellular delivery of proteins (including antibodies and enzymes) which, upon spontaneous degradation of CPDs, led to successful release of their “native” functional forms with immediate bioavailability.

Full text of DOI:10.1002/anie.201711651

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Intracellular Delivery of Native Proteins Facilitated by Cell-
Penetrating Poly(disulfide)s
Authors: Shao Q. Yao, Linghui Qian, Jiaqi Fu, Peiyan Yuan, Shubo Du,
Wei Huang, and Lin Li
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711651
Angew. Chem. 10.1002/ange.201711651
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Intracellular Delivery of Native Proteins Facilitated by Cell-
Penetrating Poly(disulfide)s
Linghui Qian,† Jiaqi Fu,† Peiyan Yuan, Shubo Du, Wei Huang, Lin Li* and Shao Q. Yao*
Abstract: Intracellular delivery of therapeutic proteins is highly
challenging and in most cases requires chemical or genetic
modifications. Herein, two complementary approaches for
endocytosis-independent delivery of proteins to live mammalian cells
are reported. By using either a “glycan” tag naturally derived from
glycosylated proteins or a “traceless” tag that could reversibly label
native lysines on non-glycosylated proteins, followed by bioorthogonal
conjugation with cell-penetrating poly(disulfide)s, we achieved
intracellular delivery of proteins (including antibodies and enzymes)
which, upon spontaneous degradation of CPDs, led to successful
release of their “native” functional forms with immediate bioavailability.
Cell-penetrating poly(disulfide)s (CPDs) are synthetic mimics
of poly-arginines in which the polypeptide backbone was replaced
with poly(disulfide)s (Figure 1A), and have been shown to
facilitate endocytosis-independent cell uptake of various
cargos.[14-18] The positively charged guanidinium groups in CPDs
help cargo accumulation near the negatively charged mammalian
cell surface and their disulfide backbone helps intracellular cargo
delivery via disulfide-exchange mechanisms with no apparent
endosomal trapping.[19] In the cytoplasm, CPDs undergo rapid
depolymerization (< 5 min), a process catalyzed by endogenous
glutathione (GSH). At present, both non-covalent and covalent
conjugations are available to append CPDs to proteins modified
either genetically or chemically with suitable “tags” (i.e. histidine
tag). Such “artificial” modifications might affect protein activities
and conformations. Alternatively, CPD-coated biodegradable
nanocapsules were used as carriers to encapsulate native
antibodies leading to successful intracellular delivery and on-
demand release.[20] This material chemistry-based delivery
method is however not applicable to many native proteins which
are much more fragile than antibodies and could be readily
denatured by the harsh conditions used during nanocapsule
formation.[7] More general and practical approaches for
endocytosis-independent intracellular delivery of “native”
functional proteins (including antibodies) are urgently needed.
Herein, by using protein chemistries,[21] we report two
complementary CPD-facilitated approaches for endocytosis-
independent delivery of “native” proteins (Figure 1). In the first
strategy (named PTM-based tagging), we took advantage that
glycosylation (a process in post-translation modification, or PTM)
occurs in > 50% of mammalian proteins including all antibodies
(i.e. IgG).[22] As shown in Figure 1B (top), different from our
previously reported method that relies on genetic engineering to
introduce artificial tags into recombinant proteins (Table S1),[15]
the glycan naturally present in a glycoprotein was converted into
a bioorthogonal tag (Step 1), either chemically (by NaIO4
Seven of the top-ten selling drugs are protein-based biologics,
and one of the largest classes of therapeutic proteins is that of
antibodies because of their ability to bind to targets with high
affinity and specificity.[1,2] Despite obvious benefits of protein-
based therapy, most proteins do not enter mammalian cells
directly, which severely limits the number of disease-relevant
targets they can target/modulate.[3] In fact, of all existing
therapeutic antibodies, almost all of them target cell-surface
antigens overexpressed in malignant cells in order to bypass the
need for intracellular delivery.[2,4] Therefore, intracellular delivery
of functional, native proteins (including antibodies) remains a key
challenge in protein-based therapy. Although various approaches
for intracellular protein delivery have thus far been reported,[5-9]
most rely on endocytosis-based cell uptake mechanisms and
necessitate significant genetic or chemical modifications to the
protein. Futaki et al reported an endosomolytic peptide (i.e., L17E)
that forms noncovalent complexes with various proteins, leading
to cell entry via endocytosis followed by successful cytosolic
protein liberation.[10] Although the delivered cargos were
unmodified, their slow endosome escape was not ideal for
immediate bioavailability. The noncovalent L17E/cargo
formulation also limits the type of proteins that could be used and
delivery efficiency. Of few reports that showed successful delivery
of proteins directly into the cytoplasm of mammalian cells, all of
them require genetic and/or chemical modifications to the
cargos,[11-13] and almost none allows intracellular release of
“native” proteins to enable full restoration of their activities.
oxidation to aldehyde[23]
)
or chemoenzymatically (by -
galactosidase/GalNAz to N3[24]), followed by bioorthogonal
conjugation to the corresponding CPD (oxime-containing AOCPD
or tetrazine-modified TzCPD/TCO-PEG12-DBCO, respectively;
Step 2). Upon successful intracellular delivery via CPD-assisted
endocytosis-independent pathways,[19] followed by spontaneous
GSH-catalyzed depolymerization inside mammalian cells,[14,15]
the resulting CPD-free cargo was essential “native” barring
minimal changes to the glycan, which was not expected to alter
the protein in any noticeable way. In the second strategy (named
traceless tagging; Figure 1B, bottom), in order to label non-
glycosylated proteins with CPD, two cleavable linkers (NBL and
TCOL) were developed to reversibly label lysines in a protein
(Step 1). These two linkers contain p-nitrophenylcarbonate which
could readily react with primary amines (e.g. lysines), and a
disulfide-bridged norbornene and trans-cyclooctene (TCO),
respectively, that could be conjugated with TZCPD (Step 2).[15]
[*]
Dr. L. Qian, Dr. J. Fu, Dr. P. Yuan, S. Du, Prof. Dr. S. Q. Yao,
Department of Chemistry, National University of Singapore,
3 Science Drive 3, Singapore 117543 (Singapore)
E-mail: chmyaosq@nus.edu.sg
Prof. Dr. W. Huang, Prof. Dr. L. Li
Key Laboratory of Flexible Electronics (KLOFE) & Institute of
Advanced Materials (IAM), Nanjing Tech University (NanjingTech),
30 South Puzhu Road, Nanjing 211800, China
E-mail: iamlli@njtech.edu.cn
[†]
These authors contributed equally to this work
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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Figure 1. Overview of CPD-facilitated intracellular delivery of native proteins. (A) Structures of AO-SH and Tz-SH used to synthesize AOCPD and TzCPD. (B) Scheme
showing the two CPD-facilitated protein delivery approaches: (I) PTM-based tagging, and (II) traceless tagging.
Following intracellular delivery (Step 3), the resulting protein
complex could undergo GSH-catalyzed disulfide cleavage
followed by spontaneous cleavage of the self-immolative linker,[25]
leading to successful release of the “native” protein. It should be
noted that while our manuscript is in preparation, Raines et al
reported diazo compounds derived from (p-methylphenyl)glycine
for reversible esterification of carboxyl residues in proteins
leading to subsequent intracellular delivery.[13] Our traceless
approach thus provides an alternative for protein delivery through
reversible tagging of lysines instead.
All compounds were synthesized and characterized (Figure
S1 and Schemes S1/S2). Subsequently, the two tagging
strategies were examined by using model cargos (Figure 2). As
antibodies are amongst the most important therapeutic
glycoproteins,[2] human IgG, which has a single N-linked glycan
present in the Fc region and numerous surface-exposed lysines,
was chosen as a model to test both strategies. In order to image
cellular uptake, IgG was first fluorescently labeled (giving IgGCy3
or IgGCy5). Bovine serum albumin (BSA) was chosen as a model
for non-glycosylated proteins (Figure 2).
sulfone reagent, ThioLinker-TCO™ (Scheme S3),[27] we found
both the oxime and chemoenzymatic strategies were able to
modify the antibody with comparable efficiency (Figure S2). With
both oxidized IgGCy3 and IgGCy3-N3 in hand, we next carried out
CPD conjugation by mixing them with AOCPD and TzCPD,
respectively. Both conjugations occurred rapidly within 1 h in a
“plug-and-play” manner without additional reagents and the
resulting CPD conjugates were directly used for intracellular
delivery. Live-cell imaging with confocal laser scanning
microscope (CLSM) was carried out in HeLa cells co-stained with
an endolysosomal tracker and Hoechst nuclear stain (Figure
S2B); similar to CPD-conjugated IgGCy3 modified by ThioLinker-
TCO™ as previously reported,[15] both oxidized IgGCy3 and IgGCy3
-
N3 were able to successfully enter cells upon CPD conjugation,
and the uniformly distributed fluorescent patterns of delivered
cargos throughout the cytosol with few overlaps with the
endolysosomal tracker indicated successful endocytosis-
independent uptake mechanisms.[14-19] More quantitative
fluorescence-activated cell sorting (FACS) analysis was carried
out (Figure 2A); consistent with live-cell imaging, the PTM-based
tagging with oxime conjugation showed the highest intracellular
IgGCy3 delivery efficiency upon CPD conjugation, followed by that
of chemoenzymatic conjugation and the previously reported
ThioLinker approach.[15] Without CPD conjugation, however, none
of the strategies was effective for intracellular delivery.
Consequently, oxime conjugation was chosen as the method-of-
choice for PTM-based tagging in subsequent experiments.
We next investigated whether the traceless tagging could be
used for CPD conjugation of a model non-glycosylated protein
(e.g. BSA) followed by successful cellular delivery and
spontaneous intracellular release of the native cargo. As shown
in Figure 2B (and Figure S3), following a concentration-
dependent labeling of BSA by both NBL and TCOL, the resulting
linker-modified, as well as the subsequent GSH-catalyzed linker
cleavage, BSA was visualized by in-gel fluorescence scanning
following click chemistry with TER-Tz1 and SDS-PAGE
In PTM-based tagging with oxime conjugation (Figure 1B,
top), IgGCy3 was first oxidized with 10 mM NaIO4 for 0.5 h on ice
by following published protocols.[23] The aldehyde formation was
successfully monitored by incubating the oxidized product with
FITC-ONH2. Alternatively, IgGCy3 was chemoenzymatically
modified by following a previously published protocol with some
optimizations (Scheme S3);[24] upon treatment with β-
galactosidase to trim off the sugar chains, the exposed GlcNAc
residue on IgGCy3 was enzymatically incorporated with GalNAz
(an azide-containing GalNAc analog) by using a mutant β-1,4-
galactosyltransferase, providing the N3-labeled IgGCy3 (i.e. IgGCy3
-
N3). Following bioorthogonal strain-promoted alkyne-azide
cycloaddition (SPAAC) with TCO-PEG12-DBCO,[26] the resulting
TCO-modified IgGCy3 was ready for subsequent CPD conjugation
and intracellular delivery. By comparing with a previously reported
antibody conjugation strategy using a commercially available bis-
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Figure 2. (A) FACS quantification of IgGCy3 uptake in live HeLa cells with “PTM-based tagging“ in comparison with ThioLinker conjugation,[15] before/after adding
the corresponding CPDs. Relative fluorescence (RFU) in each experiment was normalized to that of cells treated by oxime conjugation (set as 1). (B) GSH-
dependent cleavage (0 to 10 mM) of BSA labeled by “traceless tagging“. Native BSA was labeled by NBL/TCOL prior to being clicked with TER-Tz1, followed by
GSH treatment (0 to 10 mM; 2 h at 37 oC) and SDS-PAGE before in-gel fluorescent scanning (FL, indicating only labeled BSA) and silver staining (SS, indicating
total BSA). (C) MALDI-TOF spectra of native BSA (bottom) and TCOL-labeled BSA (top) after GSH cleavage (5 mM, 2 h at 37 oC). (D) Time-dependent GSH
cleavage between NBL- and NBC-labeled BSACy5. BSACy5 was first tagged by NBL or NBC, clicked with TER-Tz1, then treated with GSH (5 mM at 37 oC) for 0 to
2 h, before SDS-PAGE/in-gel fluorescence scanning. (Cy3): TER-Tz1/linker-labeled BSA; (Cy5): total BSACy5 intensity. (E) 3D projections of z-stack images showing
intracellular delivery of IgGCy5 conjugated to CPDs by using different approaches (oxime/NBL). Controls via L17E/ProJect strategies were run. HeLa cells were
incubated with IgGCy5 (50 nM for 1.5 h; colored in red) following each delivery formula (Supporting Information), then co-stained with Hoechst (blue) and LysoTracker
(green). Cells treated with unmodified IgGCy5 (panel 7) or “tagged” IgGCy5 but without CPD conjugation (panels 2 & 4) were imaged as negative controls. Scale bar
= 20 μm. (F) FACS quantification of IgGCy5 uptake in HeLa cells treated with different delivery methods described in (E). The RFU in each sample was normalized
to that from cells treated with unmodified IgGCy5 (set as 1), and indicated on top of each bar. (Inset): SDS-PAGE/in-gel fluorescence scanning of lysates from the
corresponding treated cells. (G) FACS of CPD-conjugated IgGCy5 uptake in HeLa cells after oxime or NBL tagging, in the presence of endocytosis inhibitors (left) or
at different incubation temperatures (right). Inhibitors include chlorpromazine (CPZ), wortmannin (w), methyl-β-cyclodextrin (MβCD), and 5,5′-dithiobis-2-nitrobenzoic
acid (DTNB). Data were normalized against cells treated with no inhibitors (Blank; set as 1; left graph), or cells incubated at 37 °C (set as 1; right graph).
separation. We found both NBL- and TCOL-labeled BSA were
efficiently cleaved by 5 mM GSH in as little as 15-30 min (Figure
S3). With 1 mM GSH, however, a minimum of 2-h incubation was
needed to afford complete linker cleavage (Figure 2B). Since
GSH is present at 1-10 mM in most mammalian cells, we
reckoned these linkers would be intracellularly cleaved in a matter
of 1-2 h. The “traceless“ tagging/release of BSA was further
verified by MALDI-TOF mass spectrometry with TCOL-labeled
BSA as a representative (Figures 2C & S4); virtually identical
molecular weights of native BSA and GSH-cleaved BSA following
TCOL labeling were detected, indicating a complete linker
removal from the labeled BSA. No GSH-catalyzed linker release
was observed with BSA labeled by a non-cleavable control linker,
NBC (Figures 1B/2D & S5). Nonetheless following TzCPD
conjugation, even NBC-labeled BSA was successfully delivered
to the cytosol of mammalian cells (Figure S5C), underscoring the
essential role of CPD in facilitating intracellular cargo delivery.
We next used IgG to compare the delivery efficiency of the
newly developed approaches. We were surprised to notice that
IgG labeling by TCOL was substantially weaker than NBL, even
though no obvious difference was observed in similar labeling
reactions with BSA (Figure S6A). This indicates the efficiency of
our traceless tagging approach was protein-dependent. For
simplicity, NBL was chosen as the method-of-choice for traceless
tagging in subsequent experiments. When oxime and NBL
conjugations were compared side-by-side with the same cargo
(IgGCy5; Figure S6B), the former appeared to label IgGCy5 much
more strongly than the latter, which might have been caused by
competitive Cy5-NHS pre-labeling on IgG, thus resulting in less
available free lysines for subsequent NBL labeling. Importantly,
upon CPD conjugation, both methods enabled intracellular
delivery of IgGCy5 (Figure 2E/F); compared to L17E and ProJect™
(a commercial protein transfection kit),[10,15] both CLSM and
quantitative FACS showed our newly developed approaches
were more effective (oxime > NBL > L17E ≈ ProJect). The
endocytosis-independent advantage of the methods was further
verified to be similar with previously reported systems (Figure
2G);[14,15] while common endocytosis inhibitors didn’t noticeably
affect protein delivery, both DTNB and reduced temperature
(4 °C) led to significant inhibition in cellular uptake, indicating thiol-
mediated uptake mechanisms as previously reported.[14-19]
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Oxime + AOCPD
+NaIO4 + AOCPD
Oxime- AOCPD
+NaIO4 - AOCPD
NBL + TzCPD
+NBL + TzCPD
NBL - TzCPD
+NBL - TzCPD
+ L17E
+L17E
+ ProJect
+ProJect
HRP only
(A)
(B)
HRP only
(7)
(1)
(2)
(3)
(4)
(5)
(6)
(D)
(C)
6000
4. RNase+GSH
5. RNase+NBL+GSH
6. RNase+NBC+GSH
1. RNase
2. RNase+NBL
3. RNase+NBC
15
12
1. RNase + NBL
2. RNase + NBC
3. RNase + NBL+CPD
4. RNase + NBC+CPD
5. RNase + L17E
6. L17E only
7. RNase + ProJect
8. ProJect only
9. Blank
13.5
100
RNase A
3 4 5 6
2.6
2.0
1.0
kDa 1
2
2.2
1.3
17
-
FL
SS
2
1
0
10
17
10
-
-
-
1.0
0
0
(1) (2) (3) (4) (5) (6) (7)
(min)
60
0
20
40
1
2
3
4
5
6
7
8
9
Figure 3. Intracellular delivery of native proteins for immediate bioavailability. (A) HRP delivery to live HeLa cells followed by measurement of enzymatic activities.
Cells were treated with HRP (50 nM, 1.5 h) following each delivery formula, before being washed, fixed and incubated with ECL reagents (20 min at RT) then
imaged. Scale bar = 20 μm. (B) Relative chemiluminescence intensity (CHL) extracted from different images in (A). The signals were normalized from cells treated
with HRP only (panel 7; set as 1), and indicated on top of each bar. (C) In vitro RNase A activity assays towards (1) native RNase A, (2) NBL-labeled RNase A and
(3) NBC-labeled RNase A. Upon GSH treatment (5 mM, 37 oC, 15 min), samples (4, 5, 6) were measured again. (Inset): in-gel fluorescence scanning (FL) of the
six samples upon click chemistry with TER-Tz1 and SDS-PAGE. (D) XTT cell viability assay of HeLa cells upon intracellular delivery of RNase A (0.52 μg/well; 24
h) via different methods. Error bars were obtained from triplicate.
Having shown both the PTM-based and traceless tagging
coupled with CPD conjugation were effective for endocytosis-
independent intracellular protein delivery, we next investigated
their ability to deliver native functional proteins with immediate
bioavailability. We turned to enzymes as the cargos. Horseradish
activity. Upon subsequent GSH-triggered “traceless” cleavage of
NBL to release native RNase A, the enzyme’s activity should be
fully restored. NBC-tagging on RNase A, on the other hand,
should not be capable of restored activity due to the linker’s non-
cleavable nature (Figures 3C & S5A). Indeed, when compared to
native RNase A, tagging of RNase A with NBL and NBC
significantly attenuated its enzymatic activity (compare line 1 vs
2/3 in Figure 3C). Upon successful GSH-catalyzed linker
cleavage, the enzymatic activity of NBL- but not NBC-labeled,
RNase A was fully restored (compare lane 4 vs 5/6). Following
CPD conjugation and intracellular delivery of NBL-tagged RNase
A to live HeLa cells for 24 h, we detected significant cell death
(Figure 3D, in red); even positive cells treated with L17E/Project™
methods showed significantly less cell death. This indicates our
traceless tagging/delivery strategy was more effective than other
existing approaches for intracellular delivery and release of native
proteins with immediate bioavailability. Consistent with the in vitro
assays, NBC-labeled intracellularly delivered RNase A showed
very weaker cytotoxicity even after 48-h incubation (Figure S6E).
To sum up, we have developed novel conjugation
chemistries that enabled convenient tagging with cell-penetrating
poly(disulfide)s, endocytosis-independent cellular delivery and
intracellular release of “native“ functional proteins (including
antibodies) with immediate bioavailability. In future, other types of
peroxidase (HPR),
a
glycoprotein whose endogenous
accumulation in mitochondria leads to neurodegenerative
diseases and cancer,[28] is widely used in biology as a sensor to
image endogenous H2O2. By catalyzing oxidization of a suitable
substrate in the presence of H2O2, detectable chemiluminescent
or fluorescent signals could be generated. Previous reports
indicate chemical modifications on any of the eight glycosylation
sites in HRP had negligible effects on its enzymatic activity and
our in vitro enzymatic assay also supported this (Figure S6C).[29]
Upon successful conjugation with either PMT-based or traceless
tagging (Figure S6D), followed by bioorthogonal CPD attachment,
the resulting conjugates were used for intracellular delivery
(Figure 3A/B); by imaging (and quantifying) the enzymatic activity
of intracellularly delivered HRP from various delivery methods,[30]
we detected significant chemiluminescent signals in HeLa cells
treated with both CPD conjugates ( e.g. panels 1 & 3) as well as
L17E and ProJect™ methods (panels 5 & 6), with the oxime
conjugation again showing significantly more superior delivery
efficiency (see Figure 3B; compare 1 vs others). This is similar to
what was observed with IgG in our earlier experiments (Figure 2F). post-translational modifications may be similarly exploited for
In the absence of CPD, however, neither tagging produced any
noticeable endogenous HRP activity (panels 2 & 4). In another
example for intracellular delivery of native, functionally active
RNase A (a non-glycosylated protein), we further studied the
traceless tagging with NBL by comparing it with the non-cleavable
NBC. It was previously reported that lysine residues in RNase A,
a therapeutic protein which has recently entered clinical trials for
cancer treatments, are essential to its activity,[31] Upon successful
cell uptake, this enzyme causes strong cytotoxicity. We
hypothesized that RNase A conjugation with NBL should block its
CPD-facilitated intracellular delivery of other native proteins.
Acknowledgements
Funding was provided by National Medical Research Council
(CBRG/0038/2013), the GSK-EDB Trust Fund (R-143-000-688-
592) and MOE-T1 (R-143-000-694-114) of Singapore, and the
National Natural Science Foundation of China (81672508,
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10.1002/anie.201711651
Angewandte Chemie International Edition
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Intracellular Delivery of Native
Proteins Facilitated by Cell-
Penetrating Poly(disulfide)s
Delivery free of “tag”: By making use of either a glycan (for glycoproteins) or
lysines (for a a non-glycosylated protein), cell-penetrating poly(disulfide)s were
conjugated to the cargo, resulting in endocytosis-independent cellular uptake and
spontaneous release of the “native” functional protein in the cytosol.
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