A nitroreductase and glutathione responsive nanoplatform for integration of gene delivery and near-infrared fluorescence imaging

Article abstract of DOI:10.1039/c9cc10071g

A novel platform rationally integrating indocyanine green analogues and an arginine-rich dendritic peptide with both nitroreductase (NTR) and glutathione (GSH) reduction responsive linkers was developed. This multifunctional platform can enable selective

Full text of DOI:10.1039/c9cc10071g

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Nitroreductase and glutathione responsive nanoplatform for
integration of gene delivery and near-infrared fluorescence
imaging
Received 00th January 20xx,
Accepted 00th January 20xx
Hong Lianga, †, Qunjie Bi a, †, Ao Hu a, Xiaobing Chen a, Rongrong Jin a, Xu Songa,b,*, Bowen Ke a,
Matthias Barzc and Yu Nie a, *
DOI: 10.1039/x0xx00000x
“main” portion has been designed as drug carrier,20 and PEGylated
A novel platform rationally integrated indocyanine green analogues
and arginine-rich dendritic peptide with both nitroreductase (NTR)
and glutathione (GSH) reduction responsive linker was developed.
This mutifunctional platform can enable selective and efficient
gene delivery and specific turn-on fluorescence imaging in tumor.
platinated-BODIPY molecules have been developed to combine
chemical, photodynamic and photothermal therapy together for
tumor ablation.21 Our previous work have proved arginine-rich
amphiphilic lipopeptides owned excellent gene transfection
activity,22-24 and microenvironment-responsive modification could
strongly promote both specificity and efficiency of gene
expression.25,26 While our recent investigations further confirm that
dual or multi-responsive triggers in carrier design showed deeper
biological understanding of tumor micro-environment, and resulted
in high-specific, high-efficient and low-toxic therapy.27,28 The high
GSH concentration (at least 4 fold higher) is one of important
characteristics that distinguish tumor tissue/cells from normal
tissue/cells. 29-30 Therefore, we intend to utilize the feature of high-
concentrated NTR and GSH around tumor and intra-tumor cells
Tumor cells, tumor associated cells, cellular and extracellular
matrix components constitute an orchestrated “tumor
microenvironment”, promoting the growth, invasion and metastasis
of tumors.1 A hallmark of solid tumor microenvironment is hypoxia,
originating from the insufficient oxygen supply of blood from
disorganized vasculature.2 Hence, the biomarkers of hypoxia, such as
the overexpressed reductive enzymes (including glutathione,
nitroreductase, azoreductase and DT-diaphrose) ,3-5 could be useful
as triggers for tumor diagnosis and antitumor treatment.6,7 Among
them, NTR switchable fluorophores have received lots of attention
due to their exceptional sensitivity.8-11 The combining of
fluorescence recovery together with chemotherapy, photodynamic
therapy and radiotherapy12,13 have achieved obvious development in
theranostic platform. However, tumor hypoxia is also known as the
“Achilles’ heel” of traditional photodynamic therapy (PDT), because
severe tumor hypoxia hampers therapeutic outcomes of oxygen-
dependent PDT and PDT potentiates hypoxia.
Gene therapy is a promising treatment through silencing
abnormally overexpressed gene or compensating defective gene,14,
15 which has no conflict to the hypoxia condition. It might be a good
choice for the NTR triggered theranostic therapy. Especially, visual
tracing gene gives intuitive and quantitative evaluation of the
dynamic delivery processes at both cellular and tissue levels in a real-
time fashion.16,17 It is generally known that directly labeling genes
could influence their biological effect, the probes are usually
physically encapsulated or chemically bonded to periphery of the
nano-carriers. However, these loading modes for fluorescent
nanomaterials often suffer from premature leakage.18,19 Thus,
integrated fluorescent probe boron-dipyrromethene (BODIPY) as the
simultaneously.
Accordingly,
NTR-sensitive
near-infrared
fluorescence molecule was designed as the hydrophobic segment,
which would be quenched due to photoinduced electron transfer.31-
33
We herein developed an NTR and GSH-induced “turn-on”
assemblies (RNNS) for gene delivery and targeted imaging with real-
time visualization of carrier metabolism (Scheme 1). The carriers
possess arginine-rich hydrophilic moiety for efficient gene
condensation and cytomembrane penetration, a derivative of
cyanine dye in the skeleton structure of amphiphilic molecule as
hydrophobic moiety for NTR-sensitive fluorescence imaging, and a
disulfide bond as trigger for GSH-responsive cargo release in tumor
sites.
aNational Engineering Research Center for Biomaterials, Sichuan University, No.
29, Wangjiang Road, Chengdu 610064, P. R. China.
b Institute of Regulatory Science for Medical Devices, National Engineering
Research Center for Biomaterials, Sichuan University, No. 29, Wangjiang Road,
Chengdu 610064, P. R. China.
c Johannes Gutenberg-University Mainz, Organic Chemistry, Duesbergweg 10-14,
MainZ, DE 55099.
* Corresponding authors: E-mail: nie_yu@scu.edu.cn, xusong2016@scu.edu.cn
†The authors contributed equally to this article.
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x.
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Scheme 1. NTR and GSH responsive nanoplatform for gene delivery
and fluorescence imaging.
In order to evaluate the selective disassembly response of
DOI: 10.1039/C9CC10071G
assemblies, RLS, RNNF, RNNS-1, 2, 3 and 4 were incubated with GSH,
View Article Online
The synthetic route of targeted molecule RNNF and details
were presented in Fig. S1, and the structure characterization of each
molecule has been performed through nuclear magnetic resonance
spectrum (NMR) and MS (Fig. S2-S13). The amphiphilic molecule
RNNF could self-assemble into nano-sized assemblies with Z-Average
size of 340 nm and zeta potential of +29 mV. It could be also mixed
with another amphiphilic molecule (RLS) synthesized in our previous
work23 with different molar ratios (1/1, 1/2 and 1/3) to obtain smaller
assemblies (RNNS, size from 130-185 nm) and better size
distribution. The RNNS-3 assemblies (RNNF/RLS, molar ratio= 1/3)
showed the minimal size of 136 nm with positive charge (+22 mV)
(Table S1). The larger diameter might be owing to both strong
hydrophobicity and rigidity of fluorescence molecule (RNNF) during
self-assembly.34-36 While addition of flexible molecule RLS might be
conducive to tight integration between molecules, inducing smaller
size in process of assembly.23 The size of all assemblies increased
after DNA condensation, and the zeta potential reduced slightly. As
a representative, the morphology of RNNS-3 assemblies and RNNS-
3/DNA complexes were observed through the transmission electron
microscopy (TEM). RNNS-3 assemblies showed a uniform discoid
shape, and RNNS-3/DNA complexes revealed a uniform ellipsoidal
shape (Fig. 1A and B). The fluorescence spectral characteristic of
various molecule and assemblies in organic or aqueous solutions
were investigated to detect the on-demand “OFF” and “ON” signals,
respectively (Fig. 1C). RNNF had no fluorescence signal in organic
solution, even after incubation with GSH. While the spectral peak
significantly enhanced in the presence of both NTR and NADH. It
indicated specific response of RNNF molecules to NTR with
outstanding turn-on effect on fluorescence recovery, which might be
beneficial for reducing the background interference.
NTR or their mixture, respectively. The changes of size and zeta
potential were shown in Fig. 2A and S14. RLS and RNNF displayed the
most rapid increase on size when incubated with GSH and NTR,
respectively. While RNNF/RLS assemblies showed relatively slow
responses in the conditions only with GSH or NTR. It might be RNNF
partly prevented the degradation of RLS by GSH, and the RLS also
disturb the interaction between RNNF and NTR. What’s more,
noticeable changes of size and zeta potential in all assemblies were
shown on condition with both GSH and NTR, revealing the successful
disassembly.
Fig. 2. The selective disassembly response and corresponding gene
release of various assemblies in different conditions. (A) The changes
of size for various assemblies (RNNF, RLS and RNNS) after incubation
with 10 mM GSH, 5 μg/mL NTR and 1 mM NADH, or mixture of GSH
and NTR, respectively. Date are presented as means ± SD (n = 5). (B)
The gene release ability of different assemblies/DNA complexes in
the presence of 10 mM GSH, 5 μg/mL NTR with 1 mM NADH, or
mixed GSH and NTR, respectively.
Some research found assemblies with rigid structure could
conduce to building tight interaction with cargos and improve the
ability of gene condensation.35,36 Thus, we evaluated gene
compaction status in various assemblies with different N/P ratios
through gel electrophoresis (Fig. S15) and RNNS-3 was chosen as a
representative for the mixed assemblies. It was clear that pDNA
could be completely dragged in the loading position by RLS at N/P
ratio of 60, by RNNF at N/P ratio of 20 and by RNNS-3 at N/P ratio of
30, respectively. RNNF with relatively rigid structure really showed
benefit for the condensation, and addition of it could contribute for
gene compaction with lower N/P in the mixed assemblies.
Followingly, gene release ability of these assemblies was investigated
in the presence of GSH, NTR or their mixture, respectively (Fig. 2B).
The images showed that in the presence of either GSH or NTR alone,
the release of gene in RNNF, RLS and RNNS assemblies was
incomplete. This revealed that fluorescence could recovery in NTR
solution but with no gene release (Fig. 1). However, all the
Fig. 1. Characterization of RNNS-3 assemblies and RNNS-3/DNA
complexes by TEM and fluorescence spectra of RNNF in various
solutions. TEM image of RNNS-3 assemblies (A) and RNNS-3/DNA
complexes (B), respectively. (C) Fluorescence spectra of RNNF in
various solutions with maximal excitation wavelength (dotted line,
Em 870 nm) and emission wavelength (solid line, Ex 690 nm),
respectively. Black lines: RNNF in methanol, red lines: RNNF in mixed
solution (methanol/water, 1/1, v/v) with 10 mM GSH for 2 h, blue
lines: RNNF in mixed solution (methanol/water, 1/1, v/v) with 5
μg/mL NTR and 1 mM NADH for 2 h.
2 | J. Name., 2012, 00, 1-3
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assemblies could completely release gene in the combined action of
GSH and NTR.
In vitro gene transfection activity of these assemblies was also
studied on HeLa, and pEGFP plasmid was aDctOeId: 1a0s.10th3e9/Cm9oCdCe1l0g0e7n1Ge
View Article Online
Although it has been reported that NTR has relatively high (Fig. 3B and S18). More green fluorescent spots were observed from
content in tumor cells due to the hypoxia environment,37 seldom
relevant data could support this statement.8-11 Therefore, the
content of NTR in tumor cells (HeLa) and non-tumor cells (Human
umbilical vein endothelial cells, HUVEC) were detected by Human
NTR ELISA Kit, showing certainly higher in Hela cells than that in
RNNS-3 group compared with PEI or lipofectamine 2000, either in the
presence or absence of 10% FBS. On the culture medium with 10 %
FBS, the gene transfection activity of these mixed assemblies has
decreased in a certain extent, but still far surpassed that in the
control groups (Fig. 3B and S18). RNNS-3 assemblies showed the best
gene transfection effect at N/P = 30, reaching up to 30-fold higher
than commercial reagents (Lipofectamine 2000 and PEI). Flow
cytometry data also confirmed RNNS-3 (N/P=30) retained a high
gene expression on HeLa cells with 10% serum (Fig. S19). The gene
transfection efficiency would be gradually improved with the
increase ratio of RNNF. But excessive rigidity would obstruct the
interaction between nanocarriers and cell membrane, even hindered
the endocytosis and gene release.39 Meanwhile, the cytotoxicity was
another factor that influence the gene transfection effects, which
might lead to the poor gene transfection efficiency of RNNS-1 (Fig.
S20). The order of cytotoxicity for these assemblies was RNNS-1 >
RNNS-2 > RNNS-3 > RNNS-4. Almost no toxicity was observed in
RNNS-3 groups on the concentration below 60 μg/mL, which was
suitable for in vitro and in vivo application. According to our previous
works, RLS showed very low cytotoxicity.22-24 We speculated that the
cytotoxicity was mainly caused by RNNF. The addition of RLS has
significant neutralizing effect on the rigidity of RNNF, thus reducing
the cytotoxicity of mixed assemblies. As a result, the RNNS-3
assemblies with part rigid structure and low cytotoxicity have the
best performance on gene transfection whether in the condition of
culture medium with or without FBS.
HUVEC (Fig. S16). In addition, GSH concentration has also been
22,38
confirmed highly expressed in Hela.
Followingly, fluorescence
recovery of RNNS-3 gene complexes was studied on HeLa with
HUVEC as control (Fig. 3A). It was obviously that the fluorescent
signal in HeLa was gradually enhanced with the incubation time.
While only slight changes were observed in HUVEC. The semi-
quantitative evaluation also confirmed these results, showing almost
5-fold difference in intensity (Fig. S17). We could speculate that the
fluorescence of RNNS-3 could recovery in the interaction with NTR
and GSH in Hela.
In the animal experiment, RNNS-3/pEGFP showed turn-on
effect in tumor site (Fig. S21). The near-infrared (NIR) fluorescence
could maintained strong until 6 h post-injection, as shown in living
mice imaging (Fig. S21A) and ex vivo imaging of the isolated tissues
(Fig. S21B). Images also reflected that the indocyanine green (ICG)
analogs degraded from RNNS-3 could be metabolized by liver and
excreted by kidney.40 This result confirmed that RNNS-3 could be
degraded or metabolize and safe for humans. In vivo gene
transfection was studied at 48 h post-injection (Fig. 4). Compared
with PEI/pEGFP group, the frozen sections of isolated tumors in
RNNS-3/pEGFP group showed that considerable green fluorescence
corresponded well to the result of NIR channel, indicating that NIR
provide the accurate location for gene expression in tumor site.
Fig. 3. Fluorescence recovery and in vitro gene transfection efficiency
of RNNS-3. (A) Fluorescence recovery image of RNNS-3 observed
through laser scanning confocal microscope (CLSM). The plasmid
DNA were condensed by RNNS-3 assemblies at N/P = 30. HeLa (tumor
cells) and HUVEC (non-tumor cells) were incubated with the RNNS-
3/DNA complexes for different time (1, 2, 4 and 6 h). The fluorescent
signals (red) were observed with 663 nm laser-excitation and 700 ~
800 nm emission signals. Scale bar= 100 μm. (B) Fluorescence
microscopy images of Hela with EGFP transfection for 48 h in the
culture medium without or with 10% FBS. pEGFP plasmid DNA were
condensed by RNNS-3 assemblies at different N/P ratios (N/P = 20,
30, 40 and 60). Lipofectamine 2000 and PEI (MW = 25000) were
acted as control groups. The N/P ratio of PEI/DNA complexes were
10.22-24 Liposome 2000/DNA complex was 0.2 µL/ 100ng according to
the protocol. Scale bar= 100 μm.
Fig. 4. In vivo green fluorescent protein expression and fluorescent
signals in tumors at 48 h after intratumoral injection of RNNS-
3/pEGFP (N/P = 30) and PEI/ pEGFP (N/P = 10).
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J. Name., 2013, 00, 1-3 | 3
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In summary, we designed a kind of amphiphilic lipopeptide
molecule RNNF which could be used as theranostic nanoplatform for
tumor disease through self-assembly. The mixed assemblies RNNS-3
(RNNF/RLS = 1/3) have minimal size at 136 nm and their fluorescence
showed NTR-responsiveness with the maximal emission wavelength
at 870 nm. Although RNNF and RLS assemblies have different
sensitive responsiveness to NTR and GSH respectively, RNNS-3
assemblies showed good responsiveness to both and good
performance on gene condensation and release in the condition of
NTR or GSH. Moreover, with low cytotoxicity, RNNS-3 presented the
excellent performance on fluorescence recovery and efficient gene
transfection both in vitro and in vivo. Their gene transfection activity
was even much better than commercial reagents such as
Lipofectamine 2000 and PEI (MW = 25000).
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This work was supported by the National Key Research and
Development Plan of China (2017YFC1104601), the National Natural
Science Foundation of China (NSFC, No. 81873921 and 51903174),
Sino-German cooperation group project (GZ1512) and Sichuan
Science and Technology Program (2019JDJQ0027).
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