Interfacial cationization to quicken redox-responsive drug release

Article abstract of DOI:10.1039/d1cc00156f

Interfacial cationization could greatly increase the redox-responsiveness of disulfide bond-linked lipid-drug nanoassemblies (NAs) due to the generation and concentration of ionized thiols at the alkalized NAs’ cationic interface. This strategy could be used to prepare redox-ultrasensitive nanocarriers for efficient intracellular drug delivery.

Full text of DOI:10.1039/d1cc00156f

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Interfacial cationization to quicken
redox-responsive drug release†
Cite this: Chem. Commun., 2021,
57, 2943
a
b
Yaxin Zheng,
Jie Lei,a Qi Li,a Xuan Jin,a Qingyuan Lia and Yang Li
*
Received 10th January 2021,
Accepted 10th February 2021
DOI: 10.1039/d1cc00156f
rsc.li/chemcomm
Interfacial cationization could greatly increase the redox-responsiveness chemical optimizations of the position of SS in the linkage
of disulfide bond-linked lipid-drug nanoassemblies (NAs) due to the and the type of adjacent linkers (e.g. ester, amide, carbamate
generation and concentration of ionized thiols at the alkalized NAs’ and carbonate). For example, combining SS with carbonate is
cationic interface. This strategy could be used to prepare redox- reported to be more effective to trigger drug release due to its
ultrasensitive nanocarriers for efficient intracellular drug delivery.
more effective self-immolation mechanism upon reduction.14
The position of SS in the carbon chain linkage also exerts
The disulfide bond (SS)-containing drug delivery systems significant effects on their redox responsiveness, which can
(DDSs) have been widely used for the intracellular delivery of further influence the drug release rate and in vivo antitumor
various drugs by responding to the high level of glutathione efficacy of SS-containing DDS.15 On the other hand, the steric
(B2–10 mM) in the cytosol, of which the concentration is hindrance and electrostatic microenvironment around SS
around 2 to 3 orders higher than that in the extracellular fluids linkers can significantly influence the redox-responsiveness of
(approximately 2–20 mM).1–4 Glutathione is readily able to SS-containing linkers.16,17 These chemical methods are effec-
cleave the SS via a thiol–SS exchange reaction, thus accelerating tive to increase the redox-responsiveness of SS linkers, but may
drug release due to the direct nanocarrier destruction or the also change their chemical stability to induce the premature
rapid hydrolysis of adjacent esters linked to the parent drugs. drug release.16 To address this, we developed a novel strategy to
The introduction of SS in the DDSs is always associated with increase the redox-responsiveness of SS-containing DDSs, with-
improved antitumor activity, which is mainly ascribed to the out the need to change the chemical structure of the linkers.
selective release of active drugs at the tumor site.5–7 Therefore, This strategy was associated with the interfacial cationization of
the design of sensitive redox-responsive DDSs has received nanocarriers, which could modulate redox-responsiveness as
great interest in the past few decades.
high as 3 orders of magnitude due to greatly the changed pH at
A variety of redox-activatable DDSs with SS linkages have the interfaces.
been developed with promising antitumor efficiency.8–11 Once
As a proof of concept, self-assembling redox-responsive
internalized into cells, the active drugs are expected to be nanoassemblies (NAs) formed by SS-linked lipophilic prodrugs
released as soon as possible to exert therapeutic effects. There- were used as model DDSs. For this purpose, camptothecin
fore, a great many efforts have been made to enhance the redox- (CPT) and curcumin (CUR) were chosen as model drugs, which
responsiveness of DDSs.12,13 The strategies to increase the were then conjugated with oleic acid (OA) via the disulfanyl-
redox responsiveness of DDSs are mainly based on the ethyl carbonate (ETCSS) linker to obtain the corresponding
1
chemical modification of the SS-containing linker, such as CPT-SS-2OA and CUR-SS-2OA. Their chemical structures, H-NMR
and mass spectra (MS) are shown in Fig. 1A and Fig. S1 (ESI†),
respectively. The PEGylated NAs consisting of CPT-SS-2OA and
CUR-SS-2OA (1/2, mole/mole) (CPT/CUR-NAs) were prepared by
a School of Pharmacy, Key Laboratory of Sichuan Province for Specific Structure of
Small Molecule Drugs, Chengdu Medical College, Chengdu, China
b Department of Pharmaceutics, College of Pharmacy, Chongqing Medical
University, Chongqing 400016, P. R. China. E-mail: beyond20061330@163.com
† Electronic supplementary information (ESI) available: Experimental section,
1H-NMR and MS of CPT-2S-2OA, CUR-2S-2OA, DON-PEG2000 and DON-COOH,
cellular uptake, size distributions and fluorescence response of CPT/CUR-NAs
and +CPT/CUR-NAs in plasma and NaCl solutions, combination index of +CPT-
NA and CUR-NAs, selective cytotoxicity on CT26 and 3T3 cells, hemolytic and RBC
aggregation assay. See DOI: 10.1039/d1cc00156f
dispersing their ethanol solutions (containing 10% DON-PEG2000
as a neutral stabilizing agent, H-NMR and MS shown in Fig. S2,
,
1
ESI†) in water under vigorous stirring. To prepare the cationic CPT/
CUR-NAs (+CPT/CUR-NAs), the cationic lipid of DOTAP (10%, by
weight) was co-assembled with lipophilic prodrugs using the same
preparation procedure. Dynamic light scattering revealed that
both unmodified and cationic CPT/CUR-NAs had similar average
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probably ascribed to the high interfacial pH of NAs (Fig. 1C). As
supporting evidence for this explanation, the fluorescence
response of CPT/CUR-NAs was also highly dependent on the
pH in bulk solutions (Fig. S6, ESI†). It is worth mentioning that
there is nearly no fluorescence increase for +CPT/CUR-NAs after
24 h incubation in 10 mM phosphate buffer (pH 7.4) at 37 1C
(Fig. S7, ESI†), indicating that +CPT/CUR-NAs were stable in the
absence of reductive agents, despite the high pH at NA-water
interfaces.
Considering that the low pH in lysosomal organelles can
make the thiol–SS exchange reactions less efficient,16 the redox-
responsiveness of +CPT/CUR-NAs at a lower pH was investigated. As
can be seen in Fig. S5B (ESI†), the fluorescence change of CPT/
CUR-NAs was greatly decreased at pH of 6.8 and 5.5. By contrast,
+CPT/CUR-NAs still displayed an obvious fluorescence response at
the low pH of 5.5, which was even 4 folds faster than that of CPT/
CUR-NAs at pH 7.4. On the other hand, the cationic NAs were readily
able to absorb various anionic ions and proteins in the physiological
media, which might potentially interfere with the redox-
responsiveness of +CPT/CUR-NAs. To address this concern, the
DTT-induced fluorescence change of +CPT/CUR-NAs in plasma
and various concentrations of NaCl was also investigated. As shown
in Fig. S8 (ESI†), NaCl at the physiologically-relevant concentration
Fig. 1 Schematic illustration of co-assembling FRET CPT/CUR-NAs to monitor
CPT release by detecting the recovery of CPT fluorescence (A). Comparative
properties of unmodified and cationic CPT/CUR-NAs: zeta potentials
(B), appearance (C, CUR solutions at various pH were used as a control), kinetic
change of fluorescence and emission spectra in 10 mM DTT (D).
diameters in the range of 124.8–159.1 nm (Fig. S3, ESI†), but (150 mM) did not influence the fluorescence response of +CPT/
showed different zeta potentials (À18.4 mV vs. +28.5 mV, CUR-NAs. After 30 min incubation in plasma, the positive zeta
Fig. 1B). Interestingly, the unmodified CPT/CUR-NAs show a yellow potential of +CPT/CUR-NAs was inversed from +41.8 mV to
appearance, whereas +CPT/CUR-NAs had a brownish red appear- À16.4 mV, and the particle size increased from 124.8 nm
ance (Fig. 1C). This result indicated that cationic NAs had an to 147.7 nm, indicating the extensive adsorption of anionic
alkalized interface, because CUR itself was a pH colorimetric proteins. Despite this, +CPT/CUR-NAs still exhibited an around
indicator with a strong redness-shift under alkaline conditions.18 15 fold higher redox-responsivity than that of CPT/CUR-NAs
Such high pH at the cationic surface might be attributed to the (Fig. S9, ESI†). These results indicated that the interfacial
electrostatic adsorption of OHÀ ions at the NAs’ interface.19,20
cationization was a robust strategy to increase the redox-
Due to fluorescence resonance energy transfer (FRET) effects responsiveness of CPT/CUR-NAs, which could be even valid at
between CPT and CUR, the co-assembly of CPT-SS-2OA and a lower pH or in physiologically relevant media.
CUR-SS-2OA could result in the complete quenching of CPT
We next investigated the effects of proportion of cationic or
fluorescence. The recovery of CPT fluorescence at 426 nm could anionic lipids on the redox-responsiveness of CPT/CUR-NAs
reflect CPT release (Fig. 1A). This was demonstrated by HPLC (Fig. 2A). It is shown that the particle sizes of +CPT/CUR-NAs
analysis (Fig. S4, ESI†), indicating that the cleavage of ETCSS were less dependent on the DOTAP proportions (Fig. S10, ESI†),
was directly coupled with CPT or CUR release. Therefore, the but the zeta potentials of +CPT/CUR-NAs highly depended
redox-responsiveness of CPT/CUR-NAs could be readily evaluated on the fraction of DOTAP (Fig. 2B). The appearance of CPT/
by simply detecting the kinetic change of CPT fluorescence, which CUR-NAs was changed from yellow into red as the DOTAP
made them an ideal platform to investigate the effects of various increased, indicating the gradual alkalization at the NAs’ inter-
factors on the redox-responsiveness of NAs.21
face. Accordingly, the increasing rate of fluorescence was
Based on such FRET NAs, we firstly investigated the effect of positively correlated with the DOTAP proportions, with the
interfacial cationization on the redox-responsiveness of CPT/ maximum fluorescence response at 5% DOTAP (Fig. 2C). Similarly,
CUR-NAs. The dithiothreitol (DTT) was utilized as the model the redox responsiveness of NAs could also be adjusted by incor-
reductive stimulus agent. As shown in Fig. 1D, +CPT/CUR-NAs porating anionic lipids. It is shown that the zeta potentials of CPT/
displayed a rapid CPT fluorescence increase with the maximum CUR-NAs became more negative with the increase of anionic lipid
level reached after 1 h incubation, which was much faster than of DON-COOH (Fig. 2D,1H-NMR and MS shown in Fig. S11, ESI†).
that of unmodified CPT/CUR-NAs. Based on the change of CPT The increase of CPT fluorescence was accordingly lowered, and
fluorescence intensity at 426 nm, +CPT/CUR-NAs exhibited an nearly stopped for NAs containing 20% DON-COOH (Fig. 2E).
around 53 fold higher redox-sensitivity than that of unmodified Based on the fluorescence change before and after 1 h incubation
CPT/CUR-NAs. Furthermore, +CPT/CUR-NAs at 0.1 mM DTT in 10 mM DTT, the maximum difference in the redox-
could still provide a high fluorescence response, which was responsiveness of CPT/CUR-NAs can be up to B1000 folds. These
comparable to that of unmodified CPT/CUR-NAs at 10 mM DTT results indicated that the interfacial microenvironment of NAs
(Fig. S5A, ESI†). Such high redox-sensitivity of cationic NAs was can modulate their redox-responsiveness by nearly 3 orders of
2944 | Chem. Commun., 2021, 57, 2943À2946
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Fig. 3 Real-time visualization of the CPT release in CT26 cells by
monitoring the fluorogenic process of CPT/CUR-NAs (B). MTT assay (B)
and apoptotic analysis (C) of CT26 cells after the treatment of unmodified
and cationic CPT/CUR-NAs.
Fig. 2 Schematic illustration of the preparation of cationic and anionic
CPT/CUR-NAs (A). Zeta potentials (B and D) and fluorescence response
(C and E) of cationic or anionic CPT/CUR-NAs with different weight
percentages of DOTAP or DON-COOH.
probably attributed to the faster intracellular drug release from
cationic NAs in CT26 cells (Fig. S14C, ESI†), despite the lower
cellular uptake of cationic NAs in CT26 cells as compared with that
in 3T3 cells (Fig. S14D, ESI†). These results indicated that the
increased redox-responsivity of +CPT/CUR-NAs could not only
magnitude in a predictable and finely tunable manner, which increase in vitro cytotoxicity but also enhance the selective
provided an opportunity to induce a selective cytotoxicity against cytotoxicity against tumor cells.
tumor cells by utilizing different concentrations of glutathione in
cancer cells and normal cells.
The hemolysis and erythrocyte aggregation of CPT/CUR-NAs
and +CPT/CUR-NAs were tested to predict the in vivo safety. It is
The cellular uptake of unmodified and cationic CPT/CUR-NAs shown that both NAs only caused slight hemolysis at high
was next comparatively investigated in colorectal cancer cells concentrations, but the cationic NAs resulted in remarkable
(CT26) using a confocal laser scanning microscope. As shown in erythrocyte aggregation as compared to the unmodified NAs
Fig. S12 (ESI†), there was nearly no CPT fluorescence (blue) (Fig. S15, ESI†). This result indicated that cationic NAs could
observed for the cells after 0.5 h of incubation with unmodified not be directly intravenously injected, and a ‘‘charge reversal’’
CPT/CUR-NAs. By contrast, +CPT/CUR-NAs treated cells exhibited strategy might be required for intravenous injection.22 How-
a bright CPT fluorescence, which was mainly co-localized with the ever, such redox-ultrasensitive cationic NAs could be directly
lysosomal signal (in red). This result indicated that +CPT/ used for localized cancer therapies, such as in situ antitumor
CUR-NAs could be rapidly internalized in cells and mainly loca- vaccine and intravesical therapy of bladder diseases.
lized in acid lysosomes. As the CPT release can be self-reported via
Fig. 4 shows the proposed mechanism of the increased
the FRET signal, the recovering of CPT fluorescence suggested that redox-responsivity of +CPT/CUR-NAs. Firstly, the directional
the CPT could be released from cationic NAs. The intracellular CPT arrangement of positive charges at the cationic nanoparticle’s
release was further detected in a real-time manner by capturing the surface resulted in a high positive surface potential, which
kinetic change of the fluorescence images. It is shown that the blue further led to the high interfacial pH due to the extensive
CPT fluorescence rapidly appeared within the cationic NA-treated adsorption of OHÀ ions (Fig. 1C). Therefore, thiol species
cells within several minutes, indicating a faster CPT release from (RSH) approaching the cationic nanoparticle’s surface were
+CPT/CUR-NAs as compared with that from CPT/CUR-NAs readily converted into ionized thiolate (RSÀ). As the chemical
(Fig. 3A). Such faster CPT release from +CPT/CUR-NAs further reactions of thiol mainly involved the nucleophilic attack of the
resulted in a more potent in vitro cytotoxicity, with a much lower RSÀ,23 the generation of RSÀ would facilitate the thiol–SS
IC50 (CPT equivalent concentration, 0.9 versus 3.2 mg mlÀ1, Fig. 3B) exchange reactions, thus enhancing the sensitivity of redox-
and higher apoptosis percentage (31.5% versus 7.4%, Fig. 3C) than responsive drug release. Furthermore, RSÀ was also hypothe-
that of the unmodified NAs. It is worth mentioning that the sized to be electrostatically adsorbed at NAs’ surface, which also
combination of CPT and CUR in the +CPT/CUR-NAs displayed contributed to greatly increased redox-responsivity. As supporting
a synergistic antitumor effect with a low combination index of evidence for this explanation, the interfacial cationization was
0.24–0.4 at the CPT/CUR ratio of 1/2 (Fig. S13, ESI†). On the other more effective to increase the redox-responsivity of NAs as com-
hand, a cytotoxicity study was also performed on a normal cell line pared with that by enhancing the bulk pH of solution (Fig. 1D and
(mouse fibroblast, 3T3 cells) to evaluate the selective cytotoxicity of Fig. S6, ESI†). Although the pH in the vicinity of the nanoparticle
NAs between tumor cells and normal cells. As shown in Fig. S14A surface has been found to be quite different from the bulk,19,20,24
(ESI†), +CPT/CUR-NAs also induced a higher apoptosis in 3T3 cells, to our knowledge, this is the first report that the redox-
but it also achieved around 2-fold higher selectivity index against responsivity of nanocarriers could be adjusted by their interfacial
CT26 cells than that of CPT/CUR-NAs (Fig. S14B, ESI†). This was change properties.
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This research was supported by the Foundation for New Teacher
Training Program of Chongqing Medical University (YXY20
20XSZ02), and the National Undergraduate Training Program for
Innovation and Entrepreneurship (S202013705008, S20201370
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Conflicts of interest
There are no conflicts to declare.
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