Acidity-triggered zwitterionic prodrug nano-carriers with AIE properties and amplification of oxidative stress for mitochondria-targeted cancer theranostics

Junhuai Xua, Bin Yana, Xiaosheng Dua, Junjie Xiongb, Mi Zhoua, Haibo Wang*a and Zongliang Du*a
aTextile Institute, College of Light Industry, Textile and Food Engineering, Sichuan University, Chengdu, 610065, China. E-mail: whb6985@scu.edu.cn; dzl407@163.com
bDepartment of Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, China

Received 25th October 2018 , Accepted 11th January 2019

First published on 11th January 2019


Although biodegradable polymer–drug conjugate delivery systems exhibit great promise toward many types of cancers, several challenges still remain, associated with specific targeting of organelles as well as real-time tracking and observing. Herein, an innovative pH-responsive zwitterionic prodrug is prepared that can easily self-assemble into stable micelles with high drug loading. This new nanocarrier is composed of the following vital factors: (i) a zwitterionic outer shell to prolong the blood circulation and aggregate at the tumor site; (ii) surface-encoded triphenyl-phosphonium groups to enhance organelle targeting; (iii) ketal bonds of nanomicelles to realize the controlled release of therapeutic molecules; (iv) aggregation-induced emission (AIE) luminogen tetraphenylethene (TPE) to realize labelling and real-time monitoring of the mitochondria in living cells. This targeted pH-responsive polyprodrug can effectively release therapeutic molecules to activate the generation of the local ROS level and enable real-time monitoring of the status of mitochondria, which presents a practicable strategy for simultaneous cancer diagnostics and therapeutics.


Introduction

Apoptosis is a fundamental biological phenomenon of cells that plays a necessary role in the removal of unwanted or abnormal cells by multicellular organisms.1 It controls the evolution of organisms and the stability of the internal environment. Several pieces of evidence have shown that the defect of apoptosis could lead to an inflammatory environment which contributes to cancer initiation.2,3 Whereas apoptosis is frequently delayed in malignant tumor cells, activating apoptosis is a practical approach that could markedly retard their growth, and lead to an increase in the response to irradiation, cytotoxic chemotherapy, heating, and hormone ablation, sequentially to enable a better therapy. Therefore, many significant efforts to develop different methods to induce apoptosis in cancer cells have been explored.4–7

Reactive oxygen species (ROS) have been described to activate and modulate apoptosis.8–11 High intracellular ROS normally promotes apoptosis by stimulating pro-apoptotic signaling molecules. Zhang et al.12 demonstrated that capsaicin induced apoptosis is mediated by the activation of ROS generation which then significantly suppressed the growth of pancreatic tumor. According to previous reports, cinnamaldehyde (CA) and its analogs could also stimulate the increasing generation of ROS to induce cell death by apoptosis.13–17 However, their application as an efficient drug is at a standstill for various reasons including their poor water solubility, and lack of diseased tissue specific targeting ability, which are the typical drawbacks of small molecule drugs. Polymer–drug conjugates, one of the effective drug delivery systems, have shown great potential in clinical application in conquering these drawbacks in the treatment of cancer. In recent years, numerous studies18–22 confirmed the role of polymer–drug conjugates in enhancing the drug loading capability and controlling the drug release at the tumor site. For example, Lee et al.23 prepared a polymeric prodrug, which covalently incorporated CA in the backbone of polymers and allowed for a high percentage of deliverable drugs that are available as the polymer degrades.

Cellular ROS primarily arise from mitochondria. This organelle could decisively regulate the intrinsic pathway of apoptosis, which is regarded as the primary mode of cell death in cancer therapy. Therefore, a mitochondria-targeted prodrug can perturb ROS homeostasis and further induce cell apoptosis, which is beneficial to the cancer treatment.24,25 To date, specific mitochondria targeting drug delivery has been attempted by adopting various guiding agents such as peptides with arginine, triphenylphosphonium (TPP) cation salts, and pyridinium salts. Moreover, owing to abnormal mitochondria being closely related to the process of cell apoptosis, real-time detailed monitoring of morphological and functional changes of mitochondria is an urgent necessity, which has a significant effect on mitochondrial biology and associated diseases.26–29 Thus, many conventional organic dyes have been developed for mitochondrial imaging, but their poor photostability and the notorious aggregation-caused quenching (ACQ) effect limit their applications in living cell studies, leaving much to explore. Recently, AIE fluorescent probes have attracted increasing attention due to their excellent sensitivity and high photostability. Many kinds of AIE fluorescent probes have appeared as a substitute for live-cell imaging or real-time monitoring with strong photoluminescence and excellent photostability.30–37 Yuan and Peng's group epitomized the evolution of targetable fluorescent probes that allow subcellular imaging.38,39 More recently, Tang et al.28 and Yoon et al.40 developed the TPE luminogen modified with TPP groups, which can be employed for specific mitochondrial imaging and monitoring changes in mitochondrial membrane potentials and cell apoptosis. The combination of targetable amphiphilic polymers with AIE luminogens (AIEgens) will provide innovative approaches for bioimaging and theranostics.

To establish a better approach for mitochondria-targeting drug delivery and real-time monitoring, we herein synthesized a TPE-based polyurethane prodrug with a mitochondrial targeting group (TPP). In this contribution, the well-designed polymeric prodrug could self-assemble into nanomicelles. The zwitterionic shell of the nanomicelles is supposed to possess low cytotoxicity and excellent antifouling properties, and prolong blood circulation. Once these nanocarriers enter cells, the TPP groups would endow the nanomicelles with mitochondria-targeting ability. Then, the model drug (CA), conjugated to the polymer through the pH-responsive ketal bond, could release under the acidic conditions, and the generation of ROS by mitochondria would be triggered subsequently. Meanwhile, due to the strong fluorescence and photostability of TPE, the dynamic changes of mitochondria could be real-time monitored (Scheme 1).


image file: c8py01518j-s1.tif
Scheme 1 Illustration of the pH-responsive prodrug with mitochondrial-targeting to activate ROS generation for enhanced therapy and real-time monitoring in living cells.

Experiments

Materials

11-Hydroxyundecyltriphenylphosphonium bromide (TPP-OH), dihydroxy carboxybetaine (DHCB), dihydroxy TPE (DHTPE) and dihydroxy cinnamaldehyde (DHCA) were synthesized following published reports. All other solvents and reagents were of analytical purity and were purchased from commercial sources. Deionized water was used throughout the experiments.

Instrumentation

A Bruker AV III HD (400 MHz, Germany) was used to measure the 1H-NMR spectra with deuterated solvents. Molecular weights and polydispersities of the polymers were measured using a gel permeation chromatography (GPC) setup comprising PL gel 10 mm MIXED-B columns using polystyrene as standards for calibration. The eluent was DMF. An Analytic-Jena Specord S600 (Germany) spectrophotometer and a PerkinElmer LS-55 spectrofluorometer were used to collect the UV-visible absorption spectra and fluorescence spectra, respectively. Fourier transform infrared (FT-IR) spectra on KBr pellets were performed using a Nicolet 560 spectrum scanner (Thermo Scientific, USA) in diffuse reflectance mode. A Zetasizer Nano S90 (Malvern Instruments) was exploited to measure the size distribution and zeta potential of the micelles in water. To further certify the micelles, the morphology images were analysed on a transmission electron microscope (TEM).

Synthesis of PUs

The monomers DHCB, DHTPE, and HTPP were synthesized as in previous articles41,42 (1H NMR spectra shown as Fig. S7–9). DHCA was readily synthesized by mixing pentaerythritol and cinnamaldehyde in DMF, with p-toluene sulfonic acid (PTSA) as the catalyst, and then stirring at 85 °C for 24 hours. After that, the mixture was extracted with DCM and water 3 times and then dried using anhydrous sodium sulfate overnight. Afterwards, DCM was evaporated to obtain a white solid, and the crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate = 1[thin space (1/6-em)]:[thin space (1/6-em)]1) (1H NMR shown as Fig. S6).

TPE-CB PUs, TPE-CB-TPP PUs and TPE-CB-CA-TPP prodrug PUs were synthesized as the route of shown in Scheme 2 and Scheme S1. The difference between TPE-CB PUs and TPE-CB-TPP PUs was that TPE-CB-TPP PUs terminated with HTPP. As for TPE-CB-CA-TPP PUs, DHCB was placed in a vacuum drying oven at 105 °C for 2 hours to remove the trace of water. Then, briefly, 0.6 g DHCB and 0.964 g hexamethylene diisocyanate (HDI) were dissolved in a flask with 5 mL DMF, and after that 0.0002 g dibutyltin dilaurate was added sequentially. Then the temperature was increased to 85 °C. After polymerization for 4 hours, 0.2 g DHTPE and 0.3 g DHCA were added to the mixture to react for another 2 hours. Finally, 0.176 g HTPP was added to react with the remaining isocyanate group. 200 mL cold anhydrous ether was used to precipitate the reaction mixture. The fallow product (TPE-CB-CA-TPP PUs) was collected through filtration, and dried using a vacuum drying oven. Its chemical structure was confirmed using 1H NMR, UV, and FT-IR spectra.


image file: c8py01518j-s2.tif
Scheme 2 Detailed synthetic route for the TPE-CB-CA-TPP PUs.

Preparation of polyurethane prodrug micelles

The dialysis method was used to prepare polymer micelles. Briefly, products (10 mg) were dissolved in DMSO (1.5 mL). Then, 5 mL distilled water was added dropwise with vigorous stirring. After that, the mixture was fed into a dialysis membrane (MWCO = 3500 Da) and dialyzed against deionized water for 2 days to remove DMSO. Afterwards, the solution was filtered through a 0.45 μm pore-sized microporous membrane. Additionally, the morphology of TPE-CB-CA-TPP PU micelles was observed by TEM.

The stimulus-response behaviour and in vitro drug release

Micelles with a zwitterionic CB shell were expected to possess excellent physiological stability. Dimensions of micelles were determined using dynamic light scattering (Nano ZPS, Malvern instruments). The dialysis method was used to evaluate the pH-responsive drug release behavior in vitro. Briefly, 2 mL phosphate buffer (pH = 7.4) or acetate buffer (pH = 5.0) were used to suspend TPE-CB-CA-TPP PUs (50 mg), transferred into a dialysis bag (MWCO 3.5 kDa), and then dipped in 100 mL of PBS with continuous stirring at 37 °C. At the set breaks, 1 mL release medium of the sample was collected for measurement; meanwhile an equal volume of fresh PBS was replenished into the dialysis medium. UV spectroscopy was used to detect the accumulative amount of released CA at 365 nm. The experiments were performed three times, and the average was used in quantitative analysis.

Cell culture

HeLa cells were cultured in DMEM with 10% heat inactivated FBS, 1% streptomycin, and 1% penicillin. 37 °C and 5% CO2 atmosphere in the culture medium were maintained to culture the cells. In order to maintain the exponential growth of the cells, the culture medium was changed every 2 days.

In vitro cell labelling and imaging

6-Well microplates were used to seed HeLa cells for 24 h. Then, a specific concentration of TPE-CB-CA-TPP PU micelles (50 μg mL−1) was used to culture the cells for 5 h at 37 °C. Afterwards, the TPE-CB-CA-TPP PU micelles were removed by washing with PBS and then 2 mL DMEM with 100 nM Mito Tracker Deep Red was added. 30 min later, PBS solution was used to wash the cells twice and the cells were immediately observed by confocal laser scanning microscopy (CLSM). The confocal fluorescence images of cells were captured with 408 nm excitation for TPE and 600 nm excitation for Mito Tracker Deep Red.

Detecting ROS generation in vitro

6-Well microplates were used to seed HeLa cells with a density of 1 × 105 cells per dish and the cells were treated with TPE-CB-TPP PUs and TPE-CB-CA-TPP PUs (the concentrations were 100 μg mL−1). The blank culture medium was used for the control experiment. After 12 and 24 h incubation, the cells were washed with PBS three times to remove the culture medium, and then DCFH-DA was added to stain the cells for 20 min. A fluorescence microscope was exploited to collect the cell images.

Detection of the mitochondrial membrane potential (ΔΨm)

TPE-CB-CA-TPP PUs (100 μg mL−1) were added into the cells in DMEM culture medium for 4 and 24 h. Then, the tetramethylrhodamine methyl ester (TMRM) was delivered to incubate the cells for 20 min in darkness at 37 °C. Fluorescence images of TMRM-stained cells were captured using a fluorescence microscope.

Cell cytotoxicity assay

The cytotoxicity of TPE-CB-TPP PU and TPE-CB-CA-TPP PU micelles was evaluated using CCK-8 with HeLa cells, respectively. Cells were seeded in 96-well plates (the volume of each well was 100 μL). After 24 hours of cell attachment, the cells were cultured with micelle solutions at serial concentrations (10, 50, 100, 200, and 500 μg mL−1) for 48 hours. Then the free materials were removed by washing with PBS. CCK-8 solution (10 μL of CCK-8 in 100 μL of DMEM cell culture medium) was added to each well, and the cells were incubated for another 4 hours at 37 °C. Then a microplate reader (Thermo Multiskan GO) was utilized to analyze the plates. Three replicate experiments were conducted. Results are presented as the mean ± standard deviation (SD).

Results and discussion

The synthesis of PUs

As shown in Scheme S1 and Fig. S1, TPE-CB PUs were synthesized successfully and could be easily self-assembled into micelles. However, targeted delivery of drugs into specific organelles in cancer cells has been shown to be an effective way to evade overdosing. Herein, in this paper, TPP was used as the targeting moiety to link to the TPE-CB-CA PUs, which could help the drug delivery system selectively accumulate in mitochondria. Meanwhile, as the model medicine, CA could play a role in cancer therapy by increasing the cell membrane permeability and the concentration of ROS. Once the CA accumulated in the mitochondria, the concentration of ROS would increase to a certain high level, which could lead to a depletion of the proton gradient and then encourage cell apoptosis. TPE-CB PUs, TPE-CB-TPP PUs, and TPE-CB-CA-TPP PUs were synthesized successfully according to the following characterization. The synthesis route of TPE-CB-CA-TPP PUs is depicted in Scheme 2.

As shown in the route, the TPP group was introduced into the distal end of the polyurethane chain, and DHCA and TPE segments were conjugated in the backbone. The structure of the polymer was confirmed by 1H NMR, FT-IR, and UV-visible spectroscopy. The 1H NMR spectra of three polymers were compared and analyzed. The signal at 7.69–7.84 ppm (Fig. 1) can be assigned to the aromatic protons of the TPP group. The peaks at 6.11–6.20 ppm were attributed to the double bond of carbon. FT-IR spectra (Fig. 2A) further confirmed the structure of polymers, in which a typical signal at 1698 cm−1 (marked as a) reveals the appearance of –NH–COO–. Two peaks appearing at 1439 cm−1 and 1114 cm−1 (marked as c and d) in the spectra belonged to phenyl-phosphorus. The peak at 1650 cm−1 (marked as b) increasing from TPE-CB PUs to TPE-CB-CA-TPP PUs could be assigned to C[double bond, length as m-dash]C and the phenyl group on account of the conjugation of TPP and CA. As shown in Fig. 2B, the absorption peak at 263 nm was attributed to CA (marked as 1*), and the characteristic absorption band of TPP appeared at 286 nm (marked as 2*). In addition, there was a peak at 315 nm which could be considered as the characteristic absorption peak of TPE (marked as 3*). All the above results indicated the successful synthesis of TPE-CB-CA-TPP PUs. The number-average molecular weight of polyurethane was 6.5k with a narrow polydispersity index (Mw/Mn) of 1.5, which was characterized by GPC.


image file: c8py01518j-f1.tif
Fig. 1 1H NMR spectra of TPE-CB PUs, TPE-CB-TPP PUs, and TPE-CB-CA-TPP PUs.

image file: c8py01518j-f2.tif
Fig. 2 (A) FT-IR spectra of TPE-CB PUs, TPE-CB-TPP PUs, and TPE-CB-CA-TPP PUs; and (B) UV-visible absorption spectra of TPE-CB-CA-TPP PUs (λex = 365 nm).

Preparation and characterization of nanomicelles

To investigate the AIE properties of the nanocarrier, the fluorescence behavior of the prodrug polymer was studied in a DMSO/water system (Fig. 3A). It is easily observed that the FL curve is almost parallel to the baseline in DMSO solution; in other words, it is nearly nonemissive in the solution state. However, with the increasing concentration of water, the intense emission is observed. The FL intensity increases swiftly, as the water fraction is over 40%, demonstrating the formation of the aggregates. While the water fraction value is 90%, the FL intensity is 4200 times higher than that in pure DMSO. Evidently, this PU is AIE-active. To obtain the CMC of TPE-CB-CA-TPP PUs, FL spectra were recorded with different concentrations as presented in Fig. S2. The CMC value (11.4 μg mL−1) was calculated as shown in Fig. S3 via the FL intensity of TPE-CB-CA-TPP PUs with different concentrations. In order to verify the stability of the fluorescence intensity in different environments, the FL intensity of this nanocarrier was tested at pH 5 and 7.4. As shown in Fig. S10, the fluorescence intensity at pH 5 was slightly higher than that at pH 7.4, implying that the FL properties of the nanocarrier were little affected by the different pH conditions (pH: 5 or 7.4).
image file: c8py01518j-f3.tif
Fig. 3 (A) the FL intensity spectra of TPE-CB-CA-TPP PUs with the increase of the water fraction (DMSO/H2O) (λex = 350 nm); the left inset shows PU in DMSO, the right inset shows PU in mixture solution (DMSO[thin space (1/6-em)]:[thin space (1/6-em)]H2O = 1[thin space (1/6-em)]:[thin space (1/6-em)]9), under a UV lamp of 365 nm; (B) typical hydrodynamic size of TPE-CB-CA-TPP PU micelles at pH 7.4 as observed via DLS; the inset shows the TEM image of TPE-CB-CA-TPP PU micelles at pH 7.4.

Furthermore, the particle sizes of the prepared micelles at pH = 7.4 were tested by DLS as displayed in Fig. 3B. Functionalized TPE-CB-CA-TPP PUs exhibited a particle size of 78 nm, which is in accordance with the well-known optimum dimension. The morphology of micelles was further characterized by TEM. As shown in the inset of Fig. 3B, the micelles are almost spherical with an average size of 56 nm, which coincided with the results of DLS.

The stimulus-response behaviour of nanomicelles and in vitro drug release

CA, as a model drug, was linked to polyurethane by acid sensitive ketal bonds. Due to the special structure of the prodrug, the drug loading capability could be easily calculated based on the monomer ratio of the polymer, which was calculated to be 13.4% by weight. Furthermore, the results of the loading capability measured using a UV-vis spectrophotometer coincided with the calculated values. In order to investigate the release behaviour of the drug from the nanocarriers at different pH values, the micelles were kept at different pH values. The pH responsive behaviour could be observed in Fig. 4A via the change of the particle size at different pH values. As displayed in Fig. 4B, after incubation at pH = 7.4 for 50 h, almost 21% CA release was detected, while under acidic conditions at pH = 5, the release rate of CA was significantly accelerated more than 50% for 15 h. Ultimately, it reached the peak to 86% release for 50 h. The results showed that the resultant nanocarrier with the shielding of CB monomers could efficiently avoid the premature release of CA at the physiological pH value (such as cytoplasm or blood circulation). It turned out that the pH-responsive drug release behaviour of TPE-CB-CA-TPP PUs is beneficial for efficient intracellular drug release.
image file: c8py01518j-f4.tif
Fig. 4 (A) Typical hydrodynamic sizes of TPE-CB-CA-TPP PU micelles at pH 5 and 7.4 as observed via DLS; (B) the comparison of TPE-CB-CA-TPP PUs drug release curves.

In vitro cell labelling and imaging

To investigate the targeting ability of nanocarriers to mitochondria, the HeLa cells were incubated with TPE-CB-CA-TPP PU micelles for 5 hours, followed by staining the mitochondria with Mito Tracker Deep Red (100 nM) for 30 min. The typical effect of modifying with TPP is observed in mitochondrial localization. The results are presented in Fig. 5; the image with staining with TPP-modified TPE-CB-CA-TPP PUs is well overlapped with the image with labelling with Mito Tracker Deep Red, demonstrating that TPE-CB-CA-TPP PUs can target mitochondria with high specificity.
image file: c8py01518j-f5.tif
Fig. 5 The fluorescence images of live HeLa cells incubated with (A) Mito Tracker Deep Red and (B) TPE-CB-CA-TPP PUs; (C) merged image of (A) and (B).

The anti-photobleaching of TPE-CB-CA-TPP PUs was examined in living cells. As shown in Fig. S5, the intracellular fluorescence of this material changed slightly, even after continuous laser beam irradiation on the fluorescence microscope for 10 min. However, to conduct a comparison, Mito Tracker Deep Red was used for performing the same test. After irradiation by a laser in a fluorescence microscope, the fluorescence intensity of Mito Tracker Deep Red was attenuated sharply. Evidently, TPE possesses stronger anti-photobleaching properties. These results illustrated that the prepared TPE-CB-CA-TPP PUs are ideal fluorescent materials for targeting and real-time monitoring of mitochondria in living cells.

Detecting ROS generation in vitro

It is well known that CA could induce the production of intracellular ROS, and then the generated ROS would mediate the apoptosis of cancer cells. Therefore, the ability of prodrug micelles to generate ROS was assessed by the DCF assay. To conduct this assay, dichloro-dihydrofluorescein diacetate (DCFH-DA) was chosen as the ROS probe (DCFH-DA is non-fluorescent, but could be activated by ROS to emit green fluorescence). HeLa cells were incubated with TPE-CB-TPP PUs and TPE-CB-CA-TPP PUs for 12 h and 24 h, respectively. As a comparison, the cells were cultured in a blank medium.

The cells cultured in a blank medium were investigated for comparison. As shown in Fig. 6, the higher fluorescence intensity of the TPE-CB-CA-TPP PU group suggests a higher level of ROS generation than the TPE-CB-TPP PU and control groups, which fully testifies to the ROS generation ability of TPE-CB-CA-TPP PUs.


image file: c8py01518j-f6.tif
Fig. 6 ROS generation in HeLa cells treated with TPE-CB-TPP PUs and TPE-CB-CA-TPP PUs. ROS generation was detected with DCFH-DA. The fluorescence images of (A) untreated cells; (B) the cells incubated with TPE-CB-TPP PUs; and (C) the cells incubated with TPE-CB-CA-TPP PUs. The concentrations of both TPE-CB-TPP PUs and TPE-CB-CA-TPP PUs were maintained at 100 μg mL−1.

Detection of the mitochondrial membrane potential (ΔΨm)

Studies showed that the mitochondrial membrane potential is sensitive to ROS. As a result, CA is supposed to have an effect on the mitochondria. Therefore, we next explored the mitochondrial membrane potential of cells, which were treated with TPE-CB-CA-TPP PUs. HeLa cells were cultured with micelles of TPE-CB-CA-TPP PUs for 4 h and 24 h. The control comparison was performed in the blank medium. Then the TMRM was used to stain the cells for 20 min in darkness at 37 °C (TMRM is a cell-permeant dye which could accumulate in active mitochondria with membrane potentials and emit red fluorescence. Upon mitochondrial membrane potential loss, accumulation of TMRM ceases, and the red signal dims or disappears. As presented in Fig. 7, after incubation for 4 h, the red fluorescence intensity decreases a little compared with that of the control group, indicating that the ΔΨm has changed a little. Meanwhile, in the blue channel it could be observed that TPE-CB-CA-TPP PUs labelled the mitochondria owing to the membrane potential. At 24 h, the signal of red fluorescence dimmed which means that the ΔΨm of the TMRM group decreased sharply, while that of the control group barely changed. Simultaneously, it could be observed that TPE-CB-CA-TPP PUs aggregated into cell nucleus rather than labelling the mitochondria, and this mainly occurred out of the loss of mitochondrial ΔΨm. Associating anti-photobleaching and the above results, we can speculate that TPE-CB-CA-TPP PUs could realize the real-time monitoring of mitochondria.
image file: c8py01518j-f7.tif
Fig. 7 Mitochondrial membrane potential (ΔΨm) depolarization of HeLa cells was detected with TMRM. HeLa cells were observed at 4 and 24 h incubation with (B) TMRM and (C) TPE-CB-CA-TPP PUs. (A) The control experiment. The concentration of TMRM was 100 nm mL−1 and that of TPE-CB-CA-TPP PUs was 100 μg mL−1.

Toxicity of prodrug nanocarriers in vitro

To investigate the killing capacity of this polymer to cells, we explored the toxicity of TPE-CB-TPP PUs and TPE-CB-CA-TPP PUs. First, a series of concentration gradients was set for a standard CCK-8 assay in HeLa cells. As shown in Fig. 8, the results showed that the concentrations of TPE-CB-TPP PUs at 500 μg mL−1 for 24 hours resulted in relatively low cytotoxicity. However, while TPE-CB-CA-TPP PUs incubated with cells, they provoked significant cytotoxicity. The result could boil down to the release of CA in the acidic environment of the cell, which could efficiently generate ROS, and then lead to cell apoptosis.
image file: c8py01518j-f8.tif
Fig. 8 The cell cytotoxicity of TPE-CB-TPP PUs and TPE-CB-CA-TPP PUs.

Conclusion

In summary, novel mitochondria targeting prodrug nanocarriers with pH-responsive and AIE properties were successfully developed to utilize for real-time mitochondrial imaging of living cells and chemotherapy of cancer cells. Under the shielding of CB monomers and with the AIE feature of TPE, this nanocarrier is proved to exhibit plenty of advantages in cell imaging, such as low cytotoxicity and superior anti-photobleaching. Then, a colocalization study of PUs and mitochondria certified its appropriate mitochondrial targeting with the modification of TPP groups. Furthermore, the results of the in vitro experiment demonstrated that the released CA from micelles could induce the cells to generate a considerable amount of ROS; thereby the pH-stimulating TPE-CB-CA-TPP PU micelles exhibited promoted cytotoxicity in HeLa cells. On the whole, this multifunctional prodrug nanocarrier could realize high efficiency in killing cancer cells and real-time monitoring of the apoptosis process.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (No. 51773129 and 51503130), Support Plan of Science and Technology Department of Sichuan Province, China (2018SZ0174), International Science and Technology Cooperation Program of Chengdu (2017-GH02-00068-HZ), and Postdoctoral Research Foundation of Sichuan University (2018SCU12049) and supported by Graduate Student's Research and Innovation Fund of Sichuan University (2018YJSY084).

Notes and references

  1. J. F. Kerr, C. M. Winterford and B. V. Harmon, Cancer, 1994, 73, 2013–2026 CrossRef CAS.
  2. J. C. Reed, Cancer J. Sci. Am., 1998, 4(Suppl 1), S8–14 Search PubMed.
  3. S. W. Lowe and A. W. Lin, Carcinogenesis, 2000, 21, 485–495 CrossRef CAS.
  4. F. Bunz, P. M. Hwang, C. Torrance, T. Waldman, Y. Zhang, L. Dillehay, J. Williams, C. Lengauer, K. W. Kinzler and B. Vogelstein, J. Clin. Invest., 1999, 104, 263–269 CrossRef CAS.
  5. T. Hideshima, P. Richardson, D. Chauhan, V. J. Palombella, P. J. Elliott, J. Adams and K. C. Anderson, Cancer Res., 2001, 61, 3071–3076 CAS.
  6. K. Ahmed, Y. Tabuchi and T. Kondo, Apoptosis, 2015, 20, 1411–1419 CrossRef CAS PubMed.
  7. Y. Liu, W. Zhang, Y. Cao, Y. Liu, S. Bergmeier and X. Chen, Cancer Lett., 2010, 298, 176–185 CrossRef CAS.
  8. I. I. C. Chio and D. A. Tuveson, Trends Mol. Med., 2017, 23, 411–429 CrossRef CAS.
  9. H. U. Simon, A. Haj-Yehia and F. Levi-Schaffer, Apoptosis, 2000, 5, 415–418 CrossRef CAS.
  10. E. Panieri and M. M. Santoro, Cell Death Dis., 2016, 7, 2253 CrossRef.
  11. S. Galadari, A. Rahman, S. Pallichankandy and F. Thayyullathil, Free Radicals Biol. Med., 2017, 104, 144–164 CrossRef CAS.
  12. R. Zhang, I. Humphreys, R. P. Sahu, Y. Shi and S. K. Srivastava, Apoptosis, 2008, 13, 1465–1478 CrossRef CAS.
  13. K. R. Zodrow, J. D. Schiffman and M. Elimelech, Langmuir, 2012, 28, 13993–13999 CrossRef CAS PubMed.
  14. H. Ka, H.-J. Park, H.-J. Jung, J.-W. Choi, K.-S. Cho, J. Ha and K.-T. Lee, Cancer Lett., 2003, 196, 143–152 CrossRef CAS.
  15. L.-Y. Chuang, J.-Y. Guh, L. K. Chao, Y.-C. Lu, J.-Y. Hwang, Y.-L. Yang, T.-H. Cheng, W.-Y. Yang, Y.-J. Chien and J.-S. Huang, Food Chem., 2012, 133, 1603–1610 CrossRef CAS.
  16. G. Brackman, T. Defoirdt, C. Miyamoto, P. Bossier, S. Van Calenbergh, H. Nelis and T. Coenye, BMC Microbiol., 2008, 8, 149 CrossRef PubMed.
  17. H. Zhao, J. Yuan, Q. Yang, Y. Xie, W. Cao and S. Wang, J. Agric. Food Chem., 2015, 63, 6386–6392 CrossRef CAS PubMed.
  18. Z. Li, H. Wang, Y. Chen, Y. Wang, H. Li, H. Han, T. Chen, Q. Jin and J. Ji, Small, 2016, 12, 2731–2740 CrossRef CAS.
  19. M. Chang, F. Zhang, T. Wei, T. Zuo, Y. Guan, G. Lin and W. Shao, J. Drug Targeting, 2016, 24, 475–491 CrossRef CAS.
  20. N. Nishiyama and K. Kataoka, Pharmacol. Ther., 2006, 112, 630–648 CrossRef CAS PubMed.
  21. S. Wang, H. Wang, Z. Liu, L. Wang, X. Wang, L. Su and J. Chang, Nanoscale, 2014, 6, 7635–7642 RSC.
  22. P. Zhang, Y. Wang, J. Lian, Q. Shen, C. Wang, B. Ma, Y. Zhang, T. Xu, J. Li, Y. Shao, F. Xu and J. J. Zhu, Adv. Mater., 2017, 29, 1702311 CrossRef PubMed.
  23. B. Kim, E. Lee, Y. Kim, S. Park, G. Khang and D. Lee, Adv. Funct. Mater., 2013, 23, 5091–5097 CrossRef CAS.
  24. Y. Liu, X. Zhang, M. Zhou, X. Nan, X. Chen and X. Zhang, ACS Appl. Mater. Interfaces, 2017, 9, 43498–43507 CrossRef CAS.
  25. Y. Yamada and H. Harashima, Adv. Drug Delivery Rev., 2008, 60, 1439–1462 CrossRef CAS PubMed.
  26. M. Y. Wu, K. Li, Y. H. Liu, K. K. Yu, Y. M. Xie, X. D. Zhou and X. Q. Yu, Biomaterials, 2015, 53, 669–678 CrossRef CAS.
  27. S. A. Durazo and U. B. Kompella, Mitochondrion, 2012, 12, 190–201 CrossRef CAS PubMed.
  28. X. Gu, E. Zhao, J. W. Lam, Q. Peng, Y. Xie, Y. Zhang, K. S. Wong, H. H. Sung, I. D. Williams and B. Z. Tang, Adv. Mater., 2015, 27, 7093–7100 CrossRef CAS PubMed.
  29. W. Zhang, R. T. Kwok, Y. Chen, S. Chen, E. Zhao, C. Y. Yu, J. W. Lam, Q. Zheng and B. Z. Tang, Chem. Commun., 2015, 51, 9022–9025 RSC.
  30. X. Lu, M. Hou, Q. Xia, C. Yan, Y. Xu and R. Liu, Mater. Sci. Eng., C, 2017, 77, 129–135 CrossRef CAS PubMed.
  31. D. Ding, K. Li, B. Liu and B. Z. Tang, Acc. Chem. Res., 2013, 46, 2441–2453 CrossRef CAS.
  32. H. Shi, R. T. Kwok, J. Liu, B. Xing, B. Z. Tang and B. Liu, J. Am. Chem. Soc., 2012, 134, 17972–17981 CrossRef CAS.
  33. G. Feng, J. Liu, C. J. Zhang and B. Liu, ACS Appl. Mater. Interfaces, 2018, 10, 11546–11553 CrossRef CAS PubMed.
  34. G. Feng, D. Mao, J. Liu, C. C. Goh, L. G. Ng, D. Kong, B. Z. Tang and B. Liu, Nanoscale, 2018, 10, 5869–5874 RSC.
  35. G. X. Feng, R. T. K. Kwok, B. Z. Tang and B. Liu, Appl. Phys. Rev., 2017, 4, 21307 Search PubMed.
  36. M. Li, Y. Gao, Y. Yuan, Y. Wu, Z. Song, B. Z. Tang, B. Liu and Q. C. Zheng, ACS Nano, 2017, 11, 3922–3932 CrossRef CAS PubMed.
  37. W. Wu, D. Mao, F. Hu, S. Xu, C. Chen, C. J. Zhang, X. Cheng, Y. Yuan, D. Ding, D. Kong and B. Liu, Adv. Mater., 2017, 29, 1700548 CrossRef PubMed.
  38. H. Zhu, J. Fan, J. Du and X. Peng, Acc. Chem. Res., 2016, 49, 2115–2126 CrossRef CAS PubMed.
  39. W. Xu, Z. Zeng, J. H. Jiang, Y. T. Chang and L. Yuan, Angew. Chem., Int. Ed., 2016, 55, 13658–13699 CrossRef CAS PubMed.
  40. J. Li, N. Kwon, Y. Jeong, S. Lee, G. Kim and J. Yoon, ACS Appl. Mater. Interfaces, 2018, 10, 12150–12154 CrossRef CAS.
  41. J. Xu, R. Yan, H. Wang, Z. Du, J. Gu, X. Cheng and J. Xiong, RSC Adv., 2018, 8, 6798–6804 RSC.
  42. J. A. Jara, V. Castro-Castillo, J. Saavedra-Olavarria, L. Peredo, M. Pavanni, F. Jana, M. E. Letelier, E. Parra, M. I. Becker, A. Morello, U. Kemmerling, J. D. Maya and J. Ferreira, J. Med. Chem., 2014, 57, 2440–2454 CrossRef CAS PubMed.

Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c8py01518j

This journal is © The Royal Society of Chemistry 2019