The conjugation of rhodamine B enables carrier-free mitochondrial delivery of functional proteins

Article abstract of DOI:10.1039/d0ob01305f

The development of protein-based therapeutics faces many challenges, for example, carrier-dependence, safety concerns, endocytosis-dependence, and uncertain in vivo therapeutic outcomes. Small molecules are rarely used for intracellular organelle-targetin

Full text of DOI:10.1039/d0ob01305f

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ARTICLE
The conjugation of rhodamine B enables carrier-free
mitochondrial delivery of functional proteins
Jiayuan Shia, Dan Zhaoa, Xiang Lia, Feng Dinga, Xuemei Tanga, Nian liub, Hua Huanga, Changlin Liu1*a
Received 00th January 20xx,
Accepted 00th January 20xx
The development of protein-based therapeutics faces many challenges, for example, carrier-dependence, safety concerns,
endocytosis-dependence, and uncertain in vivo therapeutic outcomes. Small molecules are rarely used for intracellular
organelle-targeting and disease tissue-specific carrier-independent delivery of therapeutic proteins. Here, we report that
rhodamine B, after modification to protein, is able to guide carrier-free delivery into mitochondria and tissue-dependent
distributions of functional proteins through organic cation transporters (OCTs). The enrichment of the modified catalase in
the cancer tissue efficiently suppresses xenograft human lung tumor within mice. This carrier-free delivery platform of
proteins may emerge as a simple yet powerful approach for cancer treatment.
DOI: 10.1039/x0xx00000x
fluorescent biolabeling molecule, was unexpectedly found to be
capable of guiding direct entrance into a cell of proteins after the
specific modification to protein N-termini. By means of this
Introduction
The intravenously or subcutaneously administered proteins have
played an important role in improving human health and fighting off
diseases due to great advances in protein-based molecular medicine
over the past two decades1-3. Up to date, the administration of
protein drugs depends on the cellular delivery mainly mediated by
lipids4, various carriers5, cell-penetrating peptides (CPPs)6-8, cell-
modification, with a small molecule, we developed an efficient
carrier-free delivery platform of functional proteins, which provides
mitochondrion-targeting and carrier-free cellular entrance and
enables in vivo tissue-dependent enrichment of functional proteins.
Our mechanism study reveals that this small molecule-guided
carrier-free delivery into intracellular organelles of functional
proteins occurs via organic cation transporters (OCTs), rather than
relying on endocytosis. Furthermore, the RhB-modified catalase is
guided into the tumor tissue to efficiently suppress xenograft human
lung cancer in mice by elevating both the content and activity of cata-
lase in the tumor tissue. This direct and simple mitochondrion-
targeting carrier-free delivery and tissue-specific enrichment of
exogenous catalase may pave a new avenue for cancer treatment.
penetrating poly(disulfide)s (CPDs)9, supercharged proteins10,11
,
chemical modifications12,13, and iTOP14. Most of cellular protein
delivery is achieved in an endocytosis-dependent pathway15. The
major challenges in the protein delivery include carrier-dependence,
safety concerns, short-term blood circulation, lysosomal
degradation, tissue-nonspecificity, blood-brain barriers (BBBs).
Moreover, because many delivery systems stay at the test stages of
cultured cells, it remains uncertain whether the proteins delivered
can produce a desired in vivo therapeutic outcome16,17. In addition,
consider-able alterations in structures and activity of the proteins
cannot be ignored in many delivery cases15. Therefore, a powerful
and biocompatible carrier-free delivery platform of proteins is
urgently needed to address the challenges in protein administration
and to obtain positive in vivo therapeutic outcomes.
Results and discussion
N-terminal RhB modification and characterization of proteins
To establish an efficient organelle-targeting carrier-free delivery
platform of functional proteins, 10 wild-type (WT) proteins were
selected based on their types of structures, functions, molecular
weight, and medicinal uses (abbreviation: Cyt c, cytochrome c; HSA,
human serum albumin; Hb, hemoglobin; IgG, immunoglobulin G, Ins,
insulin; Mb, myoglobin; OVA, ovalbumin; RNase, ribonuclease A;
Among various reported delivery strategies of proteins, small
molecules are rarely employed to guide intracellular organelle-
targeting self-delivery and disease tissue-specific enrichment of
therapeutic proteins. In this study, rhodamine B (RhB), a well-known
CAT, catalase; SOD1, copper, zinc-superoxide dismutase; Table S1†).
RhB is one of the most used biolabeling fluorescent dyes with stable
red fluorescence, high quantum yields and low cytotoxicity18 and
targets mitochondria through OCTs19. This fluorescent molecule was
a. Key Laboratory of Pesticide and Chemical Biology of Ministry of Education,
International Joint Research Center for Intelligent Biosensing Technology and
Health, School of Chemistry, Central China Normal University, Wuhan, 430079
China, Email: liuchl@mail.ccnu.edu.cn.
specifically linked to N-termini of proteins by
a biomimetic
b. Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan
University, Wuhan, 430071 China.
transamination reaction20 (RhB and its linker together designated as
N-RhB, the modified proteins as protein-N-RhB, for example, SOD1-
N-RhB, Fig. 1a) that features mild reaction conditions, excellent site
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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Fig. 1 RhB modification and characterization of protein. (a) A reaction scheme of SOD1 N-terminal RhB modification. (b) MALDI-TOF-MS
determinations of SOD1-N-RhB peptide fragments provided by trypsin digestion. (c) Circular dichroism spectra of native and modified
SOD1. The conserved secondary structure of SOD1 was changed upon modification with RhB. (d) UV-Vis absorption and fluorescence
spectra of SOD1, SOD1-N-RhB (molar ratio of N-RhB to SOD1: 1, 5, 10, 50, 100), and N-RhB. (e) Activity of SOD1, SOD1-N-RhB, SOD1-
FITC, and SOD1-RhB. (f) The resistance of SOD1 and SOD1-N-RhB to proteolytic degradation monitored by enzyme activity
measurements, respectively, in the pH 7.4 trypsin solution and in the pH 2.0 pepsin solution at 37 oC.
specificity, promising scale-up potential and no inherent
resulted in significant dissociation of dimeric SOD1 (Fig. S2b†). In
requirement for genetic engineering20,21. For comparison, the amino
addition, SDS- and native PAGE showed respectively that this
groups on SOD1 were non-specifically modified with either RhB
modification occurred mainly at the N-terminus of IgG heavy chains,
and that no significant impact of the N-RhB attachment on IgG
(SOD1-RhB) or FITC (SOD1-FITC) (Fig. S1a†). The monitoring with UV-
vis absorption during purification indicated that the proteins were
successfully modified by either RhB or FITC, and unconjugated dyes
multimeric structures appeared (Fig. S2c†). The modification of the
other selected proteins was similarly characterized (Fig. S2d, e†).
were fully removed from the protein conjugates (Fig. S1b, c†).
Then, the impact of modification on structures of the WT proteins
was examined. The N-terminal RhB linkage altered the structure of
SOD1 only when the pair of SOD1 N-termini localized at its dimeric
interfaces was saturated by RhB (Fig. 1c), but the non-specific
modification did not alter the structure of SOD1 (Fig. S3a†). The
similar characterization was performed for the other nine modified
proteins, and no significant conformational changes were found in
their structures (Fig. S3b † ). In addition, the absorption and
fluorescent spectra showed that the proteins exhibited the elevated
absorbance and emission upon modification, and this modification
The specific and non-specific modification of the selected proteins
was first characterized by multiple assays following purification. For
example, MALDI-TOF-MS measurements verified the specific
attachment of RhB to the N-terminus of WT SOD1 (modified with 100
.eq N-RhB) (Fig. 1b), as indicated by the scheme (Fig. 1a). Native-
PAGE showed that the elevated RhB attachment to SOD1 N-termini
led to an increase in the transition of dimeric to monomeric forms
(Fig. S2a†). The non-specific modification with either RhB or FITC also
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did not significantly impact the spectral properties of dyes (Fig. 1d and to remain more than 75% of control even when its N-termini
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DOI: 10.1039/D0OB01305F
were all attached to RhB, whereas the non-specific RhB or FITC
and Fig. S4†).
Fig. 2 The carrier-free delivery of the modified proteins into multiple cell lines. (a) The carrier-free delivery of SOD1-N-RhB into A549,
RWPE-1, and DU145 cells observed under a confocal microscope (scale bars, 20 μm). (b) The carrier-free delivery of all the N-termini-
modified proteins with different molecular weights, structures and functions into A549 cells measured by a flow cytometry. (c) Detection
of SOD1-N-RhB within A549 cells via immunoprecipitation (IP). The endogenous and exogenous SOD1 immunoprecipitated by SOD1
antibody was captured by Protein A/G coupled to agarose beads through the bead surface-bound protein. (d) Western-blotting
observations of SOD1 within A549 cells treated with 10, 20 and 30 μM SOD1-N-RhB. (e) ELISA confirmation of the exogenous OVA within
A549 cells exposed to 1, 2 and 5 μM OVA-N-RhB. (f) Elevation in the total relative activity of intracellular SOD1 and CAT with
concentrations of SOD1-N-RhB and CAT-N-RhB. (g) Elevation or reduction of the intracellular H2O2 levels following the treatment with
SOD1-N-RhB or CAT-N-RhB. (h) RT-qPCR determinations of the SOD1 mRNA level within A549 cells treated with 2 and 5 μM SOD1-N-
RhB, respectively. (i) The cellular efflux of SOD1-N-RhB following replacement of culture media after 6 h incubation. (scale bars, 20 μm).
Error bars indicate ± SD from triplicates of independent experiments. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001
The activity of the modified proteins was then determined. The modification led to more than 40% reduction in activity relative to
SOD1 activity was observed to be slightly reduced with modification, WT SOD1 (Fig. 1e). Similar results were obtained for CAT (Fig. S3c†).
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incubation, and was partially excreted out of the cell after 6 h
DOI: 10.1039/D0OB01305F
incubation, as indicated by its partial flux out of the cell mediated by
These results indicated that the specific N-terminal modification
does not bring about significant loss of enzyme activity, although the
full modification can lead to the dissociation of multimeric enzymes
to a certain degree through interruption of their inter-subunit
interactions.
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exchanging the culture media (Fig. S6d †). Then, the carrier-free
delivery of SOD1-N-RhB into other cell lines RWPE-1, DU145,
HEK293, COS-7, HeLa, SPCA-1, and MRC-5 was also illustrated under
SOD1 modified with 50 .eq N-RhB indicates that one subunit in the
homo-dimeric wtSOD1 is conjugated to RhB and results in loss of <
20% native enzyme activity, so it was selected for the examination of
resistance to proteolytic degradation and follow-up experiments.
the same conditions (Fig. 2a and Fig. S7a, b†). Finally, the carrier-free
delivery of all other modified proteins into the cells was indicated to
be independent of their structures and molecular weight (Fig. 2b and
The data showed that the modified form of SOD1 maintained almost Fig. S7c†).
unchanged activity, but its WT form maintained only 70% of its initial
To verify intracellular maintenance of the modified proteins, their
activity after 3 h incubation in the pH 7.4 trypsin solution (Fig. 1f).
content and activity were determined inside A549 cells. Protein A/G
coupled agarose beads were used to specifically enrich the proteins
of interest via their antibodies. The results indicated that the protein
A/G-SOD1 antibody beads absorbed both SOD1-N-RhB in dilute
In fact, SDS-PAGE indicated that WT SOD1 was degraded in a
higher rate than SOD1-N-RhB by trypsin (Fig. S5a†). However, both
were found to be completely inactivated in the pH 2.0 pepsin
solution (Fig. 1f), because SOD1 was inactivated under less than pH solutions (Fig. S8a†), and the endogenous and exogenous SOD1 in
4.5 acidic conditions22,23
thoroughly, but SOD1-N-RhB appeared yet in a monomeric form in
. Moreover, WT SOD1 disappeared
A549 cell lysates through specific interactions between the bead
surface-bound protein and the SOD1 antibody, as confirmed by the
brilliant red RhB fluorescence (Fig. 2c). Heating led to release of the
the pH 2.0 pepsin solution (Fig. S5a†). These results demonstrated
that the RhB modification notably elevates resistance of WT SOD1 to modified SOD1 from the cellular SOD1-loading beads (Fig. S8b†).
proteolytic digestion in gastrointestinal milieus.
Moreover, western blotting directly demonstrated that the
intracellular SOD1-N-RhB content was elevated with its
concentration in culture media (Fig. 2d). Similar results were
obtained in quantification of the intracellular OVA-N-RhB with ELISA
assay (Fig. 2e). Moreover, the total cellular enzyme activity was
observed to be dramatically elevated in both cases of increased
SOD1-N-RhB and CAT-N-RhB delivery (Fig. 2f). In addition,
intracellular hydrogen peroxide content was increased or reduced
upon increased exposure of cells to SOD1-N-RhB or CAT-N-RhB (Fig.
2g). These results further revealed the successful carrier-free
delivery of modified proteins, because the expression of SOD1 was
not altered with their increased carrier-free delivery into cells (Fig.
2h). Overall, the carrier-free delivery of active modified proteins into
cells resulted in an elevation in both intracellular total content and
activity of these proteins.
The stability of protein-N-RhBs was further evaluated in cell
cultures, the intracellular environment, and the serum. HSA-N-RhB
was incubated first respectively with PBS, 1640 media, cell lysates
o
and serum for 5 days at 37 C. Then, a portion of the mixtures was
subject to SDS-PAGE, and the remaining was monitored using HPLC.
The results indicated that the protein-N-RhB remains intact during
treatment for 5 days (Fig. S5b, c†).
In brief, the successful N-terminal RhB modification does not
result in significant inactivation of the selected proteins, but elevates
their resistance to proteolytic degradation.
Cellular carrier-free delivery of modified proteins
The cellular delivery of SOD1-N-RhB under the carrier- and auxiliary
reagent-free conditions was first examined at 37 oC. Free small
molecule was first confirmed to be fully removed from protein-N-
RhB conjugates. To rule out the effect of the modified proteins bound
to cell surfaces, cells were washed three times with PBS containing
20 U/mL heparin (a general method used for removing the positively
charged cargos bound to cell membrane surface). The cellular
entrance of SOD1-N-RhB into A549 cells was supported by the RhB
fluorescence observed with confocal laser scanning microscope and
In addition, the maintenance of the modified proteins inside a cell
was monitored by dynamic RhB fluorescence. Following the
replacement of the culture medium with the protein-N-RhB-free
fresh one after 6 h incubation, the red RhB fluorescence was shown
to slightly reduce with incubation (Fig. 2i, also see Supporting movie
1), indicating that the RhB modification does not lead to significant
protein export out of cells with incubation.
Direct mitochondrial arrival of modified proteins
flow cytometry (Fig. S6a, b†). In addition, the cellular fluorescence
was enhanced with the concentration of SOD1-N-RhB (Fig. S6c†),
The movement of the RhB modified proteins toward their
intracellular destinations can be conveniently observed with the
fluorescent modification. First, the N-RhB moiety was observed to
enter into A549 cells (Fig. 3a) as free RhB19, and FITC modification
does not facilitate the delivery of the proteins under the same
conditions (Fig. 3a). These results suggested RhB plays a dominant
role in the cellular delivery of the proteins and might target its client
indicating it enters into a cell through carrier-free delivery in a
concentration-dependent manner. The intracellular modified SOD1
was found to reside in cytoplasm of the cell (Fig. 2a). Moreover, the
alteration of RhB fluorescence with incubation time showed that
SOD1-N-RhB reached its highest concentration in cytoplasm for 6 h
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Fig. 3 Mitochondrion-targeting carrier-free delivery of the modified SOD1. (a) The carrier-free delivery of SOD1-N-RhB, SOD1-RhB, and
N-RhB, but not SOD1-FITC, into A549 cells. (b) RhB fluorescence imaging of SOD1-N-RhB localization in mitochondria and lysosomes. (c)
RhB and FITC fluorescence imaging of intracellular FITC-SOD1-N-RhB. (d) Mitochondrial localization of FITC-SOD1-N-RhB indicated by the
merged imaging among Mito-Tracker Blue, RhB and FITC fluorescence. (e, f) Time-dependent RhB fluorescence imaging of SOD1-N-RhB
localization in mitochondria and lysosomes. (scale bars, 20 μm).
proteins into mitochondria. To prove this anticipation, nuclei, among Mito-Tracker Blue, RhB and FITC fluorescence (Fig. 3c, d), and
mitochondria and lysosomes were respectively labelled by the
fluorescent dyes blue Hoechest 33342 and either Mito-Tracker Green
by flow cytometric data (Fig. S6a†).
or Lyso-Tracker Green. Imaging showed that the localization of the
modified proteins overlapped well mitochondria, rather than
lysosomes in cytoplasm (Fig. 3b). FITC-SOD1-N-RhB prepared by
modifying SOD1-N-RhB with FITC provided the evidence to support
this direct mitochondrial localization of the modified proteins, not
the free dye stemmed from insufficient purification or degradation
of the protein-dye conjugates, as indicated by the merged image
To further confirm this RhB-guided mitochondrion-targeting
carrier-free delivery of the proteins, dynamic processes of both
entrance into an A549 cell and movement of SOD1-N-RhB toward a
mitochondrion were examined through the time-lapse imaging for
30 min. The dynamic imaging showed that the red RhB fluorescence
appeared only around a cell at the beginning and then moved
towards the interior of the cell upon incubation, suggesting that the
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Fig. 4 Cell membrane penetration of the modified SOD1 via OCTs and OCTNs. (a) RhB fluorescence images showed entrance of the RhB
fluorescence moiety, not SOD1-N-RhB, into A549 cells at 4 oC. (b) SOD1-N-RhB penetrates cells via a membrane-undamaged pathway.
Here, A549 cells were incubated, respectively, with SYTOX Green, SOD1-N-RhB, SYTOX Green + SOD1-N-RhB and SYTOX Green + 0.5%
Tweenat 37 oC. (c) The carrier-free delivery of SOD1-N-RhB was not impacted by the specific inhibitors of endocytic pathways. (d) The
pretreatment with inhibitors and substrates of OCTs/ OCTNs (for 1 h at 37 oC) reduced the entrance of SOD1-N-RhB into A549 cells, as
indicated by flow cytometry data of the intracellular fluorescence intensity. (e) Flow cytometry data of RhB fluorescence indicated that
the incubation in the culture medium without Na+ (for 6 h at 37 oC) reduced the entrance of SOD1-N-RhB into A549 cells. (f) Verapamil
inhibition of P-gps (for 1 h at 37 oC) elevated the content of SOD1-N-RhB within A549 cells (scale bars, 20 μm). (g) Schematic illustration
of the small molecule-guided carrier-free mitochondrial targeting of functional proteins.
modified SOD1 was absorbed first on the surface of a cell and then The carrier-free delivery suggests that the modified proteins
moved into the interior of the cell, and finally enriched in an penetrate cell membranes likely via an unique pathway. The red
organelle (Supporting movie 2). The dynamic fluorescence from co- fluorescence observed at 4 oC showed that the RhB moiety could
staining indicated that the organelle that enriched the modified enter into an A549 cell but its client proteins (e.g., SOD1-N-RhB) did
proteins was mitochondria (Fig. 3e, f). All together, these dynamic not (Fig. 4a), indicating that the delivery of the proteins into a cell did
o
o
and static results verified that the intracellular destination of protein- not occur at 4 C. Moreover, the observation at 37 C using SYTOX
N-RhBs is mitochondria.
Green, a green fluorescence dye that is permeable to damaged cell
membranes and displays green fluorescence once bound to nucleic
acids, indicated that the cell membranes remained their integrity
during the cellular entrance of the proteins (Fig. 4b). Obviously, these
results suggested that the modified proteins could penetrate
Cell membrane penetration of modified proteins via OCTs
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membranes through an energy- dependent manner without
disrupting the membrane, supported by the fact that the entrance
the proteins into A549 cells treated with sodium azide at 37 oC, a
general inhibitor of active transportation24, was inhibited (Fig. S9a†),
led to the reduced delivery of SOD1-N-RhB (Fig. 4e and Fig. S9e†).
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Evidently, these results revealed that the N-terminal RhB moiety
guides the cell membrane penetration of its client proteins through
OCTs and OCTNs, instead of through the endocytic pathway. In
addition, the intracellular accumulation and efflux of SOD1-N-RhB
were examined by specific inhibition of P-glycoprotein (P-gp). P-gp
can extrude drugs and organic cations including RhB out of cells29.
Incubation with Verapamil (an inhibitor of both P-gp) elevated
content of SOD1-N-RhB inside A549 cells through blocking its efflux
(Fig. 4f), demonstrating that P-gp contributes to the efflux of the
proteins.
owing to the NaN3–mediated energy deprivation.
To understand the nature of this energy-dependent pathway, we
first examined the endocytosis pathway. Endocytosis is a critical
pathway that a cell internalizes exogenous species and occurs mainly
through clathrin- and caveolin-mediated processes, as well as
dynamin-independent processes including micropinocytosis and
simple membrane recycling25. Following 1 h exposure of the cells to
the specific inhibitors of different endocytosis processes at 37 oC, the
The above results together revealed, for the first time, a new
results showed that the all inhibitors, i.e., chlorpromazine, nystatin, function of RhB: guiding the mitochondrial carrier-free delivery of its
nocodazole, chloroquine, cytochalasin D, and amiloride client proteins through OCTs and OCTNs (Fig. 4g). This cellular
hydrochloride, did not block the delivery of SOD1-N-RhB (Fig. 4c and carrier-free delivery ensures the intra-mitochondrial content and
activity of intact exogenous proteins by avoiding lysosomal
entrapment.
Fig. S9b†), revealing that the cellular delivery of SOD1-N-RhB did not
occur via any endocytosis process. Thus, the intracellular modified
proteins can avoid the lysosomal degradation during fusion of
endosomes with lysosomes. Then, we turned to consider the roles of
Toxicity of the modified proteins
the positive charge of the RhB moiety in the cellular delivery of the
modified proteins. It is well-documented that sulfated proteoglycans
on surfaces of mammalian cells greatly contribute to the anionic
feature of the cell surface26. Sodium chlorate, an inhibitor of
proteoglycan sulfation, was found to be capable of blocking the
delivery of positively supercharged proteins27. Our results showed
that the prior exposure to NaClO3 for 1 h at 37 oC did not reduce the
The toxicity of the modified proteins was examined with both
cultured cells and mice. First, their cytotoxicity was evaluated by MTT
with a cancer (A549) and a normal (RWPE-1) cell lines. The cell
viability data indicated that all of the unmodified proteins were non-
toxic, as they cannot enter into a cell. Although the modified forms
of HSA, Mb, Hb, Ins, IgG, OVA and Cyt c were non-toxic on both cell
lines in the tested range of concentrations (Fig. S10a), the modified
SOD1, CAT and RNase A were toxic on these two types of cells, but
much more toxic on the cancer cells than on the normal cells (Fig.
delivery of SOD1-N-RhB into an A549 cell (Fig. S9c†), suggesting that
the positive charge on RhB did not play a critical role in the
membrane penetration of the modified proteins.
S10b-d†). It is worthy that the RhB modification significantly elevated
cytotoxicity of CAT on the cancer cells (Fig. S10c†), well consistent
These results prompted us to explore the role of the proteins OCTs
and novel OCTs (OCTNs) in the membrane penetration of the
modified proteins, since OCTs and OCTNs were found to contribute
efficient transmembrane of RhB and its analogues via energy- and
Na+-dependent processes19. The transmembrane of the modified
proteins was first observed to increase and then to remain
approximately unchanged with their concentrations at 37 oC (Fig. 2d,
with our previous observations that the specific SOD1 inactivation-
mediated reduction of intracellular H2O2 content leads to the
decreased viability of cancer cells30. The cytotoxicity of these three
modified proteins could be attributed to their function, as the
fluorescent moiety did not reduce cell viability (Fig. S10e†). The
toxicity of the modified RNase A could stem from the exogenous
enzyme-mediated increase in degradation of intracellular RNAs. The
delivery of SOD1-N-RhB into A549 cells increased the mitochondrial
e and Fig. S6c†), indicating that the delivery is easily saturated by
protein-N-RhBs. Because RhB has been used to evaluate expression
and activity of OCTs and OCTNs as one of their substrates19, the
pretreatment with either their specific substrates or inhibitors could
reduce the delivery of protein-N-RhBs. The data showed that
incubation for 6 h resulted in the reduced delivery of SOD1-N-RhB
(67%, 80%, 59% and 80% of control, respectively) after the
pretreatment of A549 cells for 1 h at 37 oC, respectively, using L-
carnitine and mildronate (substrates and inhibitors of OCTN2),
quinidine (a substrate and inhibitor of OCTs) and free N-RhB (Fig. 4d),
agreeing well with the results observed by fluorescence imaging (Fig.
·−
H2O2 content (Fig. 2g) because SOD1 catalyzes the conversion of O2
into H2O231. The exposure to CAT-N-RhB decreased the mitochondrial
H2O2 content (Fig. 2g), because CAT functions as a catalyst to
promote decomposition of H2O232. These results suggested that (1)
cancer cells are more susceptible than normal cells to disruption of
the mitochondrial H2O2 homeostasis33, and (2) because an
appropriately high ROS level is essential for growth of cancer cells34,
increasing intracellular content and activity of the antioxidant
enzymes SOD1 or CAT results in the higher dying rate of cancer cells
than that of normal cells, implying that the direct mitochondrial
arrival of antioxidant enzymes is a new avenue to selectively kill
cancer cells35.
S9d†). OCTN2 is characterized by their ability to transport the L-
carnitine as well as other organic cations in a Na+-dependent
manner28. As expected, the removal of Na+ from the culture medium
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Then, the in vivo toxicity of modified CAT was examined with mice. OCTNs in liver and kidney (Fig. 5b)36,37. Although liver and kidney are
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15 Normal mice were randomly divided into three groups (5 mice key detoxifying and excretory organs, the fluorescent moiety and its
each group) and administered by intravenous injection of the client protein were yet enriched in these two organs even after
modified and WT CAT (doses: 10 mg/kg/2 days, 20 days). The data injection for 24 h, indicating high stability and long-term
showed that the modified and WT forms of CAT did not show maintenance of SOD1-N-RhB inside the living animal. Moreover,
observable impact on the growth of mice compared to the saline their distributions in the brain was not altered with long-term blood
group (Fig. 5a), indicating that CAT-N-RhB did not display in vivo circulation, demonstrating that the proteins can penetrate BBB,
toxicity in the normal animal.
because OCTN2 is highly expressed in brain capillary endothelial cells
that constitute BBB38. In addition, their enrichment in the lung was
ascribed to the limited expression of P-gp39. On the other hand, the
Tissue-dependent distributions of modified proteins
Fig. 5 In vivo toxicity and organ-dependent distributions of the modified proteins. (a) Body weight changes in three groups of mice (5
mice/group) during 20 days. (b) Organ-dependent distributions of N-RhB and SOD1-N-RhB in mice. Mice were administered by
intravenous injection of 15 nmol SOD1-N-RhB and its fluorescent moiety via tail veins. After injection for 2, 4, 6, 8, and 24 h, these mice
were dissected, and the major organs were collected and imaged by an Xtreme in vivo image system. (c) The relative activity of CAT in
tissues lysates from the three groups of mice that were used to evaluate the in vivo toxicity of the modified CAT. Error bars indicate ±
SD from triplicates of independent experiments. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
Because the carrier-free delivery of the proteins occurs through OCTs fluorescent moiety displayed a more time-dependent enrichment
and OCTNs, they could be enriched in the tissues that highly express than its client protein in lung, brain and spleen, but this enrichment
OCTs or OCTNs or both. Therefore, following intravenous injection was less than its client protein in kidney (Fig. 5b), suggesting that this
for 2, 4, 6, 8, 24 h via tail veins, the distributions of the RhB modified small molecule was removed more easily than the modified proteins
form of highly stable SOD1 and the fluorescent moiety in different from kidney. Overall, the distribution and enrichment of the proteins
organs of a mouse were observed under an Xtreme in vivo image and RhB were dependent on expression levels of OCTs and OCTNs in
system. On the one hand, co-imaging showed that both kinds of the tested organs.
fluorescence molecules entered rapidly into and enriched in
To prove this tissue-dependent enrichment of the modified
proteins, the total activity of CAT was assayed for the tissue lysates
from the organs of the above-tested three groups of mice that were
different organs. Their distributions in the liver and kidney were
much more noticeable than those in the other organs including
heart, lung, spleen and brain, owing to high expression of OCTs and
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used to evaluate the in vivo toxicity of the modified CAT (Fig. 5a). As
As expected, CAT-N-RhB was observed to efficiently suppress
View Article Online
DOI: 10.1039/D0OB01305F
expected, the total CAT activity in the liver, kidney and brain of CAT- growth of the malignant tumor inside mice. First, the average tumor
N-RhB mice was much higher than that in those of CAT and control volume of the CAT-N-RhB-treated group slightly increased with time
mice, but the activity in the heart, spleen and lung of CAT-N-RhB mice and was about one quarter of that of two control groups on day 22
did not display differences from that in CAT and control mice (Fig. 5c, (Fig. 6b). Moreover, the CAT-N-RhB-treated tumor mice were robust
see also Fig. 6e), revealing that the enrichment in the detoxifying and like those untreated mice. Then, tumor tissues and main organs were
excretory tissues of the protein contributes to their nontoxicity collected on day 22. Similar to the average volume, the average
towards normal mice (Fig. 5a).
tumor weight of the sample group was around one third of that of
two control groups following injection for 22 days (Fig. 6c).
Suppression of in vivo malignant tumor growth with CAT-N-RhB
Fig. 6 In Vivo anti-tumor effect of CAT-N-RhB. (a) Body weight changes of A549 tumor-bearing mice of three groups. (b) Tumor volume
changes of mice during 22 days. (c) Tumor weights obtained on day 22 and representative pictures of tumor tissues. (d) CAT-N-RhB
distributions in organ and tumor tissues on day 22. (e) The relative activity of CAT in organ and tumor lysates from the three groups. (f)
Histological observation of the tumor and kidney tissues. The tumor and kidney sections were stained with H&E. Error bars indicate ±
SD of three or five independent experiments. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
To probe the application of the RhB-guided carrier-free delivery of
Co-imaging showed that the tumor and kidney were the tissues
the proteins, CAT-N-RhB was selected for tests of whether a modified with the highest enrichment of CAT-N-RhB (Fig. 6d). In fact, the total
protein can suppress growth of a human malignant tumor within CAT activity in these two tissues from CAT-N-RhB-treated mice was
mice, because it exhibits the strongest cancer cell-killing ability much higher than that from control mice (Fig. 6e), well consistent
among the tested proteins, but is non-toxic towards normal mice with the organ-dependent distribution observed with normal mice
(Fig. S10c and Fig. 5a). 15 nude mice with human A549 tumor were (Fig. 5b). Slices of the tumor and kidney tissues from each group mice
randomly divided into three groups (5 mice per group) and were stained with hematoxylin and eosin (H&E) for imaging. The
intravenously injected every other day respectively with saline, CAT cross section imaging (Fig. 6f) showed that the kidney and tumor
(10 mg/kg, 1000 catalase units/mouse), and CAT-N-RhB (10 mg/kg, tissues from the saline and CAT groups and the kidney tissue from
1000 catalase units/mouse). The body weight and tumor sizes of the CAT-N-RhB group all were not damaged, indicating that the high
mice were measured every other day for 22 days. Body weight CAT-N-RhB enrichment is non-toxic to kidney. In great contrast, the
changes were found to be negligible for all three groups of tumor- tumor tissue from the sample group displayed seriously damaged
bearing mice (Fig. 6a), indicating that CAT-N-RhB was also non-toxic and necrotic tissues (Fig. 6f), revealing that the RhB-guided
within nude mice.
enrichment of CAT resulted in lesion and necrosis of tumor tissue. In
brief, these histological results validated the efficient and non-toxic
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suppression of malignant tumor growth mediated by the carrier-free targeting could prevent cells from carcinogenesis,ViebwecAartuicsleeOntlhinee
DOI: 10.1039/D0OB01305F
delivery and tissue-dependent enrichment of CAT. This anti-cancer appropriately high content of hydrogen peroxide originated from
effect of the exogenous H2O2-detoxifying enzyme can be attributed mitochondria is necessary for carcinogenesis46.
to mitochondrion targeting of CAT-N-RhB, the overexpression of
OCTs in tumors compared to normal tissues40, the enhanced
permeability and retention (EPR) effect41, low catalase levels and
high H2O2 concentrations within a cancer cell compared to normal
cells34,42. The low CAT level leads to the high mitochondrial content
of H2O2, because mitochondria are a main source of intracellular
H2O2, especially within vigorously metabolic cancer cells. The direct
arrival of active exogenous CAT can reduce the high mitochondrial
content of H2O2. Moreover, the overexpressed OCTs and EPR effect
ensure intracellular high levels of exogenous CAT, and the high CAT
activity can result in reduction in intracellular H2O2 content43.
Obviously, the low H2O2 content can inhibit cancer development, as
the appropriately high H2O2 content is essential for growth and
proliferation of cancer cells44.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work is supported by grants from National Natural Science
Foundation of China (21271079, and 21771073), and self-
determined research fund of CCNU from the colleges’ basis research
and operation of MOE. The support from 111 Program, B17019, and
Huabo project is also acknowledged. We thank Prof. Hongying
Zhong, Prof. Cuihong Wan for the help in the MALDI-TOF-MS
characterization. We thank Prof. Yao Sun, Prof. Pu Chen for their
support in animal experiments. We also thank Prof. Chunrong Liu and
Prof. Xiaopeng Li (University of South Florida, USF) for revising the
manuscript.
Conclusions
Our studies indicate that the specific protein N-terminal RhB
modification allows us to develop a safe and powerful mitochondrial
carrier-free delivery platform of functional proteins that is
independent of structures and sizes of proteins, and of cell types. The
N-terminal RhB linkage does not alter structures and activity of
proteins, but allows their long-term maintenance in blood and
elevated their resistance to proteolytic degradation. RhB guides the
intact and active proteins of different types to directly enter into cells
of multiple types, and to target mitochondria via OCTs. Therefore,
this protein entrance does not damage cell membranes, and survives
from both endosomal entrapment and lysosomal degradation. The
RhB modification elevates toxicity of the antioxidant enzymes on
cancer cells as the increased antioxidant content and activity can
reduce the intracellular high ROS levels essential for growth of cancer
cells.
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