A Simple Glutathione-Responsive Turn-On Theranostic Nanoparticle

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A Simple Glutathione-Responsive Turn-on Theranostic Nanoparticle for Dual-Modal Imaging and Chemo-Photothermal Combination Therapy Yihui Li, Yuxin Wu, Jitang Chen, Jiangling Wan, Chen Xiao, Jiankun Guan, Xianlin Song, Shiyou Li, Mengmeng Zhang, Huangchen Cui, Tiantian Li, Xiaoquan Yang, Zifu Li, and Xiangliang Yang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b02769 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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A

Simple

Glutathione-Responsive

Turn-on

Theranostic

Nanoparticle for Dual-Modal Imaging and Chemo-Photothermal Combination Therapy Yihui Lia,#, Yuxin Wua,#, Jitang Chena,#, Jiangling Wana,b,#, Chen Xiaoa, Jiankun Guana, Xianlin Songc, Shiyou Lia, Mengmeng Zhanga, Huangchen Cuia, Tiantian Lia, Xiaoquan Yangc, Zifu Lia,b,d,e,*, and Xiangliang Yanga,b,d,* a

Department of Nanomedicine and Biopharmaceuticals, College of Life Science and

Technology, Huazhong University of Science and Technology, Wuhan, 430074, China b

National Engineering Research Center for Nanomedicine, Huazhong University of

Science and Technology, Wuhan, 430074, China c

Key Laboratory of Biomedical Photonics (HUST), Ministry of Education, Huazhong

University of Science and Technology, Wuhan, 430074, China d

Hubei Key Laboratory of Bioinorganic Chemistry and Materia Medica, Huazhong

University of Science and Technology, Wuhan, 430074, China e

Wuhan Institute of Biotechnology, High Tech Road 666, East Lake high tech Zone,

Wuhan, 430040, China Author Contributions: #These four authors contributed equally to this work. * Corresponding

authors:

Professor Zifu Li Tel.: 86 27 87792234, Fax: 86 27 87792234, E-mail: [email protected] Professor Xiangliang Yang Tel.: 86 27 87792234, Fax: 86 27 87792234, E-mail: [email protected] 1

ACS Paragon Plus Environment

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Abstract Constructing tumor microenvironment stimuli activatable theranostic nanoparticle with simple components and preparation procedures for multimodality imaging and therapy remains a major challenge for current theranostic systems. Here we report a novel and simple glutathione (GSH) responsive turn-on theranostic nanoparticle for dual modal imaging and combination therapy. The theranostic nanoparticle, DHP, consisting of a disulfide bond linked hydroxyethyl starch paclitaxel conjugate (HES-SS-PTX) and a near infrared (NIR) cyanine fluorophore DiR, is prepared with a simple one-step dialysis method. As DiR is encapsulated within the hydrophobic core formed by HES-SS-PTX, the fluorescence of DiR is quenched by the aggregation caused quenching (ACQ) effect. Nonetheless, once DHP is internalized by cancer cells, the disulfide bond of HES-SS-PTX can be cleaved by intracellular GSH, leading to the synchronized release of conjugated PTX and loaded DiR. The released PTX could exert its therapeutic effect while DiR could adsorb onto nearby endosomes/lysosomes membranes and regain its fluorescence. Thus, DHP could monitor the release and therapeutic effect of PTX through the fluorescence recovery of DiR. Remarkably, DHP can also be used as an in vivo probe for both fluorescent and photoacoustic imaging and at the same time achieves potent antitumor efficacy through chemo-photothermal combination therapy. This study provides novel insights into designing clinically translatable turn-on theranostic systems. Keywords Turn-on theranostic nanoparticle, hydroxyethyl starch, polymer drug conjugate, glutathione responsive, dual-modal imaging, combination therapy

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Introduction Tumor microenvironment stimuli activatable theranostic nanoparticles are highly pursued for precise nanomedicine1. Compared with their turn-off counterparts, the turn-on theranostic systems have the advantages of higher signal-to-noise (S/N) ratio and lower background interference, providing a convenient means to simultaneously exert therapeutic effects and realize multimodal imaging for diagnosis2. Among various tumor pathological stimuli3, including slight acidity, hypoxia, reactive oxygen species (ROS), and enzymes, GSH responsive theranostic systems received tremendous attention due to the fact that the intracellular level of GSH inside cancer cells is several times higher than normal tissue cells and thousand-fold higher than that in blood4. Accordingly, numerous GSH triggered turn-on theranostic systems are designed5. For instance, a universal molecular structure6, composed of masked chemotherapeutic drug, disulfide bond, fluorescent reporter, and tumor targeting ligand, can selectively bind with cancer cells at high efficiency and release a cytotoxic agent and generate an evident fluorescence signal upon disulfide bond cleavage triggered by endogenous GSH. However, these disulfide bond linked fluorophore drug conjugates suffered from poor stability in blood post I.V. administration. Polyethylene glycol-polylactic acid nanoparticles were utilized to encapsulate such conjugates7 to prolong the circulation in blood and enhance tumor accumulation via the enhanced permeability and retention (EPR) effect8. Nonetheless, the multiple components complicate the preparation process and impede the clinical translation. Therefore, constructing GSH activatable theranostic nanoparticles with simple components and preparation procedures for multimodality imaging and therapy still

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remains a grand challenge for current theranostic systems. Alternatively, polymer drug conjugates might be an attractive component to prepare simple turn-on theranostic nanoparticles9. The polymer drug conjugates are usually composed

of

covalent-linked

hydrophilic

polymers

and

hydrophobic

active

chemotherapeutic agents and therefore intrinsically amphiphilic10. As a result, such conjugates could self-assemble and at the mean time encapsulate other components to obtain theranostic nanoparticles. Since the drug becomes part of the delivery vectors, they are “carrier-free” drug delivery system. Moreover, the drug release could be finely controlled by utilizing stimuli-responsive linker between polymer and drug9. To address the shortcomings of PTX, including poor aqueous solubility and serious side effects associated with Cremophor EL, a disulfide bond linked hydroxyethyl starch paclitaxel conjugate, HESSS-PTX, was prepared. Nanoparticles self-assembled from HES-SS-PTX, were not only α-amylase responsive but also sensitive to GSH, contributing to enhanced tumor penetration, prolonged blood circulation, and promoted antitumor efficacy11. In another case, a disulfide linked hydroxyethyl starch doxorubicin conjugate, HES-SS-DOX12, was crosslinked with indocyanine green (ICG) via noncovalent interactions to prepare NIR light dissociable theranostic nanoparticles for boosted tumor penetration13. To fabricate simple turn-on theranostic nanoplatforms14, the selection of fluorophore is also critical15. Among various fluorescent dyes, cyanine has become a promising candidate because of its favorable optical properties such as high absorption coefficient, high fluorescence quantum yield, and relatively long absorption and emission wavelength16. 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR)17, is a typical lipophilic

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probe and has strong NIR light absorption ability18. Since the two 18-carbon chains in DiR can insert into the hydrophobic domains in nanoparticles19, liposomes20, and cell membranes21, DiR is widely used for NIR fluorescent imaging to track the biodistribution of nanoparticles22, liposomes, and embryonic stem cells23. DiR is also attractive for applications in photoacoustic imaging and photothermal therapy (PTT), owing to the fact that DiR has a fluorescence emission peak at wavelength of 782 nm24. Nonetheless, to the best of our knowledge, leveraging DiR to fabricate a GSH-responsive turn-on theranostic nanoparticle has not been reported yet. Herein, based on DiR and HES-SS-PTX, we reported a novel and simple glutathioneresponsive turn-on theranostic nanoparticle, DHP, for dual-modal imaging and combination therapy, Scheme 1. DHP is prepared with a simple one-step dialysis method. Due to the high concentration in a limited space of hydrophobic core, the fluorescence of DiR was significantly quenched by the ACQ effect. Nonetheless, once DHP is internalized by cancer cells, the disulfide bond of HES-SS-PTX can be cleaved by endogenous GSH, leading to the simultaneous release of conjugated PTX and loaded DiR. The released PTX could

exert

its

therapeutic

effect

while

DiR

could

adsorb

onto

nearby

endosomes/lysosomes membranes and regain its fluorescence. Thus, DHP could monitor the release and therapeutic effect of PTX through the fluorescence recovery of DiR. DHP can also be used as an in vivo probe for both fluorescent and photoacoustic imaging and at the same time achieves potent antitumor efficacy through chemo-photothermal combination therapy.

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Scheme 1. Schematic illustration of DHP structure and the multiple theranostic applications.

Results and Discussion Based on HES-SS-PTX and DiR, DHP was prepared with a simple one-step dialysis method. HES-SS-PTX, as prepared by conjugating PTX with HES via a disulphide bond11, and DiR were first dissolved in DMSO, then the mixture was sealed in a dialysis bag and dialyzed against ultrapure water. As DMSO, which is a good solvent for both DiR and PTX, was gradually replaced by water, DiR aggregated and got entrapped in a hydrophobic core formed by HES-SS-PTX. Thus, DHP nanoparticle was obtained, with a DiR loading and encapsulation efficiency of 0.5±0.1% and 33.5±6.7%, respectively. Since the drug loading 6


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of PTX in HES-SS-PTX is 6.3±0.6%, the molar ratio of DiR to PTX is 1:15. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis show that DHP has uniform spherical morphology and hydrodynamic diameter of 160±7.8 nm, Figure 1A and 1B. DHP has a PDI below 0.2 and zeta potential around -0.25 mV. Figure 1A also reveals a hydrophobic core and hydrophilic shell structured nanoparticle is obtained from this simple one-step dialysis method. The core-shell structure of DHP is also confirmed with atomic force microscopy (AFM), Figure S1. On the basis of the results from TEM and AFM analysis, it is highly possible that DHP adopts a DiR/PTX hydrophobic core and HES hydrophilic shell structure, as outlined in Scheme 1, since PTX and DiR are hydrophobic while HES is hydrophilic. Figure S2 shows that there is no significant change in particle size during 20 days storage, suggesting that HES shell protected DHP could be dispersed well in PBS buffer. Moreover, the size does not change significantly during 10 days storage in 10% FBS, Figure S3. The slight decrease in diameter from 160 nm in PBS to 150 nm in 10% FBS can be ascribed to the presence of α-amylase in plasma, which degrades HES in the shell and lead to the decrease in size22. The slight decrease in size also corroborates hydrophobic core hydrophilic shell DHP is formed from the one-step dialysis method. Collectively, using a simple one-step dialysis method, we have successfully prepared a hydrophobic core hydrophilic shell nanoparticle DHP, which has uniform size, decent drug loading, and good colloidal stability. Since DiR is encapsulated within a hydrophobic core, DHP is further characterized with spectroscopy techniques to reveal the molecular interactions between DiR and DiR or PTX. Figure 1C shows that DHP has the maximum absorption at 760 nm, showing no

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significant difference compared with that of free DiR in DMSO. Besides, the absorbance of DHP at 760 nm is comparable to that of free DiR. But DHP exhibits superior stability than free DiR during one week storage, Figure S4. Interestingly, Figure 1D shows that the fluorescence of DHP is significantly quenched, suggesting that DiR molecules aggregate when they are encapsulated in the hydrophobic core. The inset images in Figure 1D show the macroscopic view in bright field and fluorescent imaging. It is worth noting that increasing PTX concentration does not affect DiR absorbance and fluorescence emission, suggesting that there is no significant interaction between PTX and DiR, Figure S5 and Figure S6. Nonetheless, increasing DiR concentration alone leads to fluorescence quenching of DiR, Figure S6C. Figure S6C and Figure 1D corroborate that DiR exhibits typical ACQ effect either in solution or tightly packed within a hydrophobic core25. In particular, with a considerable number of DiR molecules entrapped in a hydrophobic core, the π-π interaction between adjacent DiR is enhanced and the fluorescence of DiR is thus quenched26. Therefore, DHP is not fluorescent. Interestingly, the quenched fluorescence can be recovered in DHP by adding 10 mM GSH and the recovered fluorescence intensity is comparable to free DiR in DMSO, as revealed in Figure 1D. The GSH induced fluorescence enhancement in DHP is also observed by laser confocal microscopy, as shown in the inserted picture of Figure 1D. Along with the fluorescence recovery, Figure 1A and Figure 1B demonstrate that DHP dissociates into nanoparticles with diameter in the range of 20 to 30 nm. These smaller nanoparticles appear to be hydroxyethyl starch molecules12. Since DHP is composed of only two components, HES-SS-PTX and DiR, the responsiveness to GSH should originate

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from HES-SS-PTX. With the cleavage of disulfide bond by GSH, the hydrophobic core collapses, contributing to the dissociation of DHP and release of DiR. It should be noted in sample DHP+GSH the buffer solution contains not only 10 mM GSH but also 0.5% Tween80. As the aggregated DiR disintegrates along with collapse of hydrophobic core, the π-π stacking interaction between DiR molecules is significantly reduced15. Therefore, DiR switch from ACQ fluorescence “OFF” state to fluorescence “ON” state. GSH cleaves the disulfide bond in HES-SS-PTX, leading to the collapse of the hydrophobic core and simultaneous release of PTX and DiR. Nonetheless, the relationship between PTX release and DiR fluorescence recovery is not clear. To that end, the PTX release and DiR fluorescence recovery is examined in details. Strikingly, Figure 1E and Figure 1F demonstrate that the PTX release and DiR fluorescence recovery exhibit a similar trend. Without GSH, negligible fluorescence is recovered, Figure S7, and no evident PTX is released from DHP, Figure 1F, corroborating the DHP is highly stable. These results are highly consistent with Figure S2 and Figure S3. In stark contrast, both DiR fluorescence recovery and PTX release are time-dependent in PBS containing 10 mM GSH and 0.5% Tween-80. A quick fluorescence enhancement is observed during the first 30 min, followed by a gradual increase from 30 min to 240 min. Similarly, when incubated in the same reductive condition, the cumulative release of PTX from DHP is fast in the first half hour, then slows down gradually, and reaches a stable value in the fourth hour. Of particularly note, DiR fluorescence recovery and PTX cumulative release at the test endpoint are 92.5% and 54.1%, respectively, illustrating a more obvious change in DiR fluorescence recovery than that of released PTX, Figure 1G. This is probably attributed to (i) part of the released

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PTX is intercepted by the dialysis membrane in drug release test, (ii) the limited redox level (10 mM GSH) could not cleave all disulfide bonds of HES-SS-PTX. In the correlation analysis plot as displayed in Figure 1H, DiR fluorescence recovery shows outstanding correlation with cumulative PTX release (R2=0.9906). Therefore, detecting DiR fluorescence recovery can be used to monitor the cumulative release of PTX from DHP. This relationship is of great significance in subsequent in vitro and in vivo applications involving DHP. The fluorescence recovery of DiR provides a convenient route to monitor PTX release and possibly the therapeutic effect. Collectively, leveraging the ACQ effect of fluorescent dye, we have prepared a GSH responsive turn-on theranostic nanoparticle DHP based on two simple components, HESSS-PTX and DiR, with a simple one-step dialysis preparation method. Figure 1 demonstrates that the cleavage of disulfide bonds in HES-SS-PTX by GSH is responsible for PTX release and concomitant fluorescence recovery of DiR. Inspired by these results, we further constructed a similar nanoparticle by replacing DiR with coumarin 6 (C6) to obtain C6@HES-SS-PTX nanoparticle. Consistent with DHP, the fluorescence of C6 is significantly quenched via the ACQ effect. More importantly, the quenched fluorescence can be recovered by introducing 10 mM GSH whereas no significant fluorescence can be detected without GSH, Figure S8, suggesting the construction of GSH responsive turn-on theranostic nanoparticle based on a polymer drug conjugate and a fluorescent dye might be a universal and simple strategy25. Compared with existing theranostic systems, DHP has the advantages of simple elements and preparation procedures.

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Figure 1. Characterization of DHP. (A) TEM images of DHP with (right)/without (left) 10 mM GSH incubation. The scale bars are 200 nm. The inserted picture in (A) was digital photography of DHP solution. (B) Hydrodynamic diameter distribution of DHP with/without 10 mM GSH incubation, as determined by DLS. (C) UV-vis absorbance spectra of free DiR and DHP with/without 10 mM GSH incubation. (D) Fluorescence emission spectra of free

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DiR and DHP with/without 10 mM GSH incubation. Excitation wavelength = 750nm. The inserted picture was fluorescence images of DHP recorded by laser confocal microscope. (E) Fluorescence emission spectra of DHP with the incubation of 10 mM GSH for different time. Excitation wavelength = 750 nm. (F) Cumulative PTX release from DHP with the incubation of 10 mM GSH. (G) Time-dependent DiR fluorescence intensity and PTX release of DHP with the incubation of 10 mM GSH. (H) Correlation analysis between DiR fluorescence intensity and PTX release of DHP. The release medium was PBS (pH 7.4, 0.1 M, with 10 mM GSH and 0.5% Tween-80. Data in F and G represents the mean ± SEM, n = 3.

The above investigations show that the encapsulated DiR is quenched via the ACQ effect, while the released DiR can regain its fluorescence in solutions. It is not clear whether DHP still exhibits glutathione responsive turn-on behavior within cancer cells, a much more complex situation than buffer solution. To this end, murine breast cancer 4T1 cells were pre-treated with 10 mM GSH-OEt to enhance intracellular GSH concentration27, followed by DHP incubation, then the cellular fluorescence was evaluated with laser confocal microscope at pre-determined time points. Figure 2A shows that the DiR fluorescence signal appears within 1 hour, indicating cellular uptake and the dissociation of DHP within 4T1 cells. The fluorescent intensity increases over time, and the co-localization assay shows that most DiR molecules localize in endosomes/lysosomes within 4 hour. Reduction-responsive drug delivery systems are normally designed to disintegrate and release drugs in the cytoplasm28, which contains a high concentration of GSH29. The

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observation that most DiR was co-localized with endosomes/lysosomes in Figure 2A might owing to the fact that DiR could only regain its fluorescence in a relative hydrophobic microenvironment23. As depicted in Scheme 1, the disulfide bond of DHP is cleaved in cytoplasm after its release from endosomes/lysosomes, the released DiR adsorbs onto the nearby endosomes/lysosomes membranes and regains its fluorescence therein. The results from flow cytometry, Figure 2B and Figure 2C, further corroborate that the intracellular fluorescent intensity increases after GSH-OEt pre-treatment and DHP incubation. Figure 2C demonstrates that the fluorescent intensity at 4 hours is 17.6 times of the initial fluorescence intensity. Figure S9 shows that the fluorescent signal 1 hour post DHP

treatment

is

much

weaker

and

a

much

slower

co-localization

with

endosomes/lysosomes of DHP-treated cells than that of free DiR, thus proving that fluorescence turn-on of DHP is dependent on DiR dissociation and fluorescence recovery. In addition, unlike GSH-OEt pre-incubated cells, Figure S9B demonstrates that the nonpre-treated cells at a lower GSH level exhibits feeblish fluorescence after incubation of the same concentration of DHP, highlighting that the fluorescence turn-on of DHP is indeed GSH-dependent inside cancer cells. To further verify the GSH-mediated fluorescence turn-on behavior of DHP within cancer cells, a glutathione depletion agent dimethyl ester (DEM)30 or a glutathione supplement GSH-OEt27 were introduced to deplete or enhance the GSH concentration to various degrees. Cells pre-treated by different concentrations of DEM or GSH-OEt were then incubated with DHP, and the intracellular fluorescence intensity was quantified by flow cytometry. Figure 2D and 2E demonstrate that compared with non-pre-treated cells, the

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cellular fluorescence intensity of DEM pre-treated cells is significantly decreased, whereas the GSH-OEt pre-treated cells show evident stronger fluorescence signal. Moreover, the fluorescence enhancement and attenuation are both dependent on the dose of DEM and GSH-OEt. The cellular fluorescence intensity of 0.2 mM DEM, 2 mM DEM, 1 mM GSHOEt and 10 mM GSH-OEt pre-treated cells are 0.88, 0.63, 1.96, 3.97 times that of the nonpre-treated cells, respectively, suggesting that the fluorescence recovery of DHP is specifically dependent on intracellular GSH. The dissociation of DHP relies on the number of the cleaved disulfide bonds, while the disulfide bonds cleavage is dependent on intracellular GSH level in cytoplasm4. Therefore, once DHP ruptures under the reduction environment in 4T1 cancer cells, the quenched DiR aggregates dissociate gradually and regain fluorescence in a hydrophobic environment, for instance endosomes/lysosomes membranes. Via this mechanism, the fluorescence turn-on of DHP is sensitive to the reduction status of cancer cells. To study the impact of redox-status on therapeutic effect of PTX in DHP, 4T1 cancer cells were pre-incubated similarly as mentioned above, then were treated by DHP at various concentrations. Figure 2F and 2G show that the cytotoxicity of DHP against 4T1 cells is PTX dosage-dependent. Compared with non-intervened 4T1 cells, cells at relative lower GSH levels (pre-incubated with DEM) exhibit higher survival rate. In contrast, after pre-treated with GSH supplement, GSH-OEt, cells are more sensitive to DHP. Furthermore, the cell apoptosis analysis, which are determined by Annexin V-Propidium Iodide double staining, has a similar tendency in response to intracellular GSH level, Figure 2G. After 8 hours DHP treatment, the apoptosis of 0.2 mM DEM, 2 mM DEM, 1 mM GSH-OEt , 10 mM

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GSH-OEt pre-treated and non-pre-treated cells are 60.5%, 56.0%, 63.8%, 74.1% and 66.4%, respectively. No significant difference is observed between these groups. Nonetheless, the DEM-pre-treated cells at lower GSH level exhibit reduced apoptosis ratio, whereas GSH-OEt pre-treated cells at higher GSH level display higher apoptosis ratio when compared with cells of normal GSH concentration. On the basis of these results of Figure 2, we are able to conclude that while cancer cells are treated by DHP, both fluorescence intensity and cytotoxicity are dependent on intracellular GSH. Cancer cells, for instance those were pre-treated with GSH-OEt and subsequently incubated with DHP, consistently achieve a higher fluorescence intensity than the non-pre-treated cancer cells and at the meantime have a lower survival rate. Conversely, cancer cells, pre-treated with DEM, exhibit lower fluorescence intensity and higher survival rate than the non-pre-treated cancer cells. Therefore, DHP fluorescence status from ACQ to turn-on serves as a good indicator to report intracellular PTX release and therapeutic effects.

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Figure 2. Glutathione-mediated fluorescence recovery and cytotoxicity of DHP. (A) Cellular fluorescence variation after cells were pre-treated by 10 mM GSH-OEt and then were incubated with DHP for 0-4 hours, as determined by laser confocal microscope. The scale bar is 40 μm and 20 μm in the inset. Representative histogram (B) and cellular median fluorescence intensity (MFI) (C) of 10 mM GSH-OEt pre-treated cells incubated with DHP for 0-4 hours. Representative histogram (D) and cellular median fluorescence intensity (MFI) (E) of DEM/GSH-OEt pre-treated cells incubated with DHP for 2 hours. DEM concentration was 0.2 mM and 2 mM, respectively, the pre-incubation time was 1 hour. GSH-OEt concentration was 1 mM and 10 mM, respectively, the pre-incubation time was 2 hour. (F) Cell viability after DEM/GSH-OEt pre-treatment and 24 hours DHP incubation, as determined by MTT. DEM concentration was 0.1 mM and 1 mM, respectively, the pre16


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incubation time was 1 hour. GSH-OEt concentration was 1 mM and 10 mM, respectively, the pre-incubation time was 2 hours. (G) Cell apoptosis of DEM/GSH-OEt pre-treated cells incubated with DHP for 8 hours, as measured by Annexin V-PI staining. The percentages of Annexin V and PI-negative (lower-left, viable or no measurable apoptotic cells), Annexin V-positive (lower-right quadrant, early apoptotic cells), Annexin V/PI double-positive (upper-right quadrant, late apoptotic cells) and PI-positive (upper-left, death cells), were shown in the corresponding quadrants. DHP concentration was 2 mg/mL in all treated groups; DEM concentration was 0.2 mM and 2 mM, respectively, the pre-incubation time was 1 hour. GSH-OEt concentration was 1 mM and 10 mM, respectively, the preincubation time was 2 hour. Data in C, E, F and G represent the mean ± SEM, n = 3; *, p

A Simple Glutathione-Responsive Turn-On Theranostic Nanoparticle

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