Triphenyl Phosphine-Functionalized Chitosan Nanoparticles Enhanced Antitumor Efficiency Through Targeted Delivery of Doxorubicin to Mitochondria
© The Author(s). 2017
Received: 18 December 2016
Accepted: 18 February 2017
Published: 28 February 2017
Mitochondria as an important organ in eukaryotic cells produced energy through oxidative phosphorylation and also played an important role in regulating the apoptotic signal transduction process. Importantly, mitochondria like nuclei also contained the functional DNA and were very sensitive to anticancer drugs which could effectively inhibit the synthesis of nucleic acid, especially the production of DNA. In this work, we designed novel triphenyl phosphine (TPP)-conjugated chitosan (CS) nanoparticles (NPs) for efficient drug delivery to cell mitochondria. The results showed that compared with free doxorubicin (Dox), Dox-loaded TPP-NPs were specifically distributed in mitochondria of tumor cells and interfered with the function of mitochondria, thus resulted in the higher cytotoxicity and induced the significant cell apoptosis effect. Taken together, triphenyl phosphine-conjugated chitosan nanoparticles may become a promising mitochondria-targeting nanocarrier candidate for enhancing antitumor effects.
Doxorubicin (Dox) was an anthracycline glycoside antibiotic drug derived by chemical semisynthesis from a bacterial species and widely clinically used in the hydrochloride form. It was commonly used in the treatment of a wide range of cancers, including hematological malignancies (blood cancers, like leukemia and lymphoma), many types of carcinoma (solid tumors), and soft tissue sarcomas [1–6]. Although Dox effectively killed tumor cells and showed the excellent clinical effect in the treatment of cancer, significant systemic adverse reactions were also emerged such as bone marrow suppression, congestive heart failure, and typhlitis [7–11]. Especially, after a time period of drug exposure, the treatment response to chemotherapy in some patients was highly declined due to an effect known as acquired resistance [12, 13]. Therefore, it was necessary to find an efficient treatment strategy on the application of Dox for enhancing cytotoxicity to tumor tissues and overcoming drug resistance. In recent years, liposomal Dox (liposome doxorubicin) and Dox-loaded nanoparticles enhanced the intracellular accumulation of Dox or changed the cellular distribution of drug, thus strengthening the cytotoxicity of anticancer drugs and limiting the cardiotoxicity of Dox [14–20]. Mitochondria as the main provision of cellular power played a vital role on cell survival and death, and it not only produced most ATP which the cell metabolism required but also regulated metabolism and programmed cell death. Some anticancer drugs directly interfered with mitochondrion respiratory chains and eventually led to tumor cell death . Therefore, dysfunction of mitochondria would be a preferential target of many antineoplastic agents [22–25]. It was reported that the anticancer drug Dox-loaded nanocarrier delivered Dox to mitochondria in a targeted manner to interact with mtDNA, leading to the direct dysfunction of mitochondria in cancer cells. Accumulation of Dox in mitochondria generated excessive reactive oxygen species and caused damage to the mitochondrial respiratory chain, further resulting in mitochondrial lipid oxidation and increasing release of cytochrome c . Chitosan (CS) was a natural cationic polymer with the excellent biocompatibility, low toxicity, and especially low cost compared to other nano carriers. CS had the positive charges and easily combined with the negatively charged membrane, facilitating its internalization. As membrane potential of mitochondria was much higher than other organelles because of ATP synthesis and ion transport, therefore, triphenyl phosphine (TPP) as a kind of lipophilic cationic compound could be effectively targeted in mitochondria. Combination of CS and TPP could smartly target the loaded drug to mitochondria, thus triggering programmed cell death process, leading to cell stress response, cytochrome c release, and then the damage and cytotoxicity of the tumor cells.
Herein, we designed novel triphenyl phosphine (TPP)-conjugated chitosan nanoparticles (TPP-CS NPs) for the targeted mitochondrial delivery of Dox to enhance antitumor efficiency. Triphenyl phosphine was grafted onto the surface of (as-prepared) chitosan nanoparticles for targeting the mitochondria, and Dox was encapsulated into NPs for investigating the drug loading and in vitro release process. Furthermore, the cellular distribution, in vitro cytotoxicity, and cellular apoptosis were also investigated to evaluate cancer treatment efficacy by the targeted mitochondrial delivery of Dox via triphenyl phosphine-conjugated chitosan nanoparticles.
Chitosan (CS) of medium molecular weight (deacetylation degree, 80%; molecular weight, 400,000) was purchased from Haixin Biological Product Co., Ltd (China); Dox hydrochloride was purchased from Beijing Huafeng Lianbo Technology Co., Ltd. (China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), triphenyl phosphine (TPP), and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich Co. (St Louis, MO, USA); A549 cell lines and Hela cells were purchased from the Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai, People’s Republic of China). All other chemicals purchased were of analytical grade and were obtained from a variety of vendors.
Preparation and Characterization of Dox-Loaded TPP-CS NPs
According to the previous reported literature , Dox-loaded triphenyl phosphine-conjugated chitosan nanoparticles were prepared by ionic crosslinking method. Briefly, 10 mL stock solution of Dox at a concentration of 1 mg/mL was pre-mixed with CS solution followed by the addition of sodium tripolyphosphate reserve liquid, and drug-loaded NPs with the mass ratio of CS and sodium tripolyphosphate at 2:1 were formed until the mixture appeared opalescence. TPP was activated by reacting with EDC and NHS  to form semi-stable amine-reactive HNS-ester at constant vibration for 1 h and was further conjugated to the surface of NPs by the formation of stable amide bond. The structure of TPP to CS NPs was investigated by using affinity-1 infrared spectroscopy (Shimadzu, Kyoto, Japan). Nanoparticles were dispersed in distilled water to obtain the homogenous suspension, and the shapes, sizes, and zeta potentials of the particles were determined by a transmission electron microscope (JEM-1200EX; Jeol, Tokyo, Japan) and laser particle analysis (Nano ZS90; Malvern Instruments, Malvern, UK). The encapsulation efficiency of Dox in NPs and drug release characterization in medium with different pH were also evaluated by a previously reported method .
MTT assay was used to detect antitumor activity of Dox-loaded TPP-NPs in vitro. Briefly, predetermined amount of Dox-loaded NPs were added into distilled PBS buffer at the different volume to obtain a homogenous suspension. Cells were exposed to suspension of NPs at different amounts. Hela cells and A549 cells were seeded in 96-well plates (seeding density was 5 × 104 cells/well) and cultured to logarithmic growth phase. Next, culture medium was removed and re-added serum-free medium respectively containing free Dox and Dox-loaded NPs with different concentrations of Dox into cells for continuous incubation for 24 h. Twenty microliters of MTT (5 mg/mL) was placed into each well to incubate for 4 h followed by the addition of 150 μL of DMSO in each well for dissolving crystal violet precipitation, and the absorbance at 490 nm was detected in a microplate reader.
Intracellular Distribution and Quantitative Analysis of free Dox and Dox-Loaded TPP-NPs in Cells
In order to investigate the intracellular distribution and uptake intensity of Dox, free Dox and Dox-loaded TPP-NPs were chosen to incubate with A549 cells and Hela cells for observing their internalizing process and quantifying the uptake rate. Briefly, A549 cells and Hela cells at logarithmic growth phase were treated with 0.25% trypsin, and after centrifugation, cell suspension was obtained and seeded in six-well plates at a density of 5 × 104/mL for incubation at 37 °C for 24 h. Then, cells were treated with free Dox and Dox-loaded TPP-NPs containing the same amount of Dox for a certain time. The nucleuses were stained with Hoechst (blue) for 15 min at 37 °C, and the mitochondria were stained by Mitotracker Green FM. Their locations were tracked in cells by confocal laser scanning microscopy (FluoView FV10i; Olympus Corporation, Tokyo, Japan) at given time intervals. Free Dox and Dox-loaded TPP-NPs at the same amount of Dox were treated with both cells, and the intensity of fluorescence from Dox which was excited at 479 nm and emitted at 587 nm was quantified using a microplate reader. The relative uptake ratios of free Dox and Dox-loaded TPP-NPs were determined by calculating the ratio of intracellular fluorescent intensity from internalized free Dox and Dox-loaded TPP-NPs to the initial fluorescent intensity from the total added free Dox and Dox-loaded TPP-NPs at the different time interval.
Mitochondrial Membrane Potential Change
Cells were digested and seeded into the culture plate to reach a density at cell coverage of 50–70% followed by the separate addition of free Dox and Dox-loaded TPP-NPs. After 24 h, fresh culture medium containing JC-1 at 1 mg/mL was placed into wells and incubated for 30 min at 37 °C under dark conditions. A laser scanning confocal microscope (FluoView FV10i, Olympus, Japan) was used to observe the image change of mitochondrial membrane. JC-1 dye was excited using the 488 nm, and emission light collection was set to 515–545 nm (green) and 570–600 nm (red).
Intracellular Reactive Oxygen Species (ROS) Measurement
Free Dox and Dox-loaded TPP-NPs were incubated with cells for 12, 24, and 48 h followed by the continuous incubation with 10 μM 2,7-dichlorofluorescein diacetate (DCFH-DA, Sigma, MO, USA) for about 30 min. After washing with PBS, the intracellular DCF fluorescence intensity which was excited at 485 nm and emitted at 530 nm was detected using a microplate reader (Synery-2, Biotek, USA) to investigate the extent of oxidative stress.
Cell Apoptosis Evaluation by Western Blot
According to the protocol of our previous study , free Dox and Dox-loaded TPP-NPs were incubated with cells for 48 h, and then protein supernatant was lysed in RIPA buffer and separated by SDS-PAGE electrophoresis. The protein was transferred from the gel to the PVDF membrane and blocked with 1% BSA at 4 °C overnight. The PVDF membrane was incubated with the primary antibodies (caspase-3, caspase-9, Bax, cytochrome c) at 4 °C overnight, followed by the continuous incubation with secondary antibody and being stained with ECL. The levels of the targeted apoptosis proteins (caspase-3, caspase-9, Bax) and cytochrome c were analyzed by UVP gel analysis system.
The Preparation and Characteristics of Various Kinds of NPs
Cellular Viability Study
In Vitro Cellular Uptake
Mitochondrial Membrane Potential Analysis
Western Blot Analysis
Here, we designed a new functional nanoparticle system for enhancing drug accumulation in the mitochondria and leading to the significant apoptosis of cancer cells. The cytotoxicity of Dox caused by the targeted accumulation of Dox mediated by TPP-NPs was enhanced by activating the mitochondria signaling pathway in cancer cells, demonstrating increased activity and the rapid release of cytochrome c, caspase-9, and caspase-3. In order to achieve targeted drug delivery to mitochondria, the cationic lipophilic compound triphenyl phosphine (TPP) had been conjugated to nanoparticles. The results showed that Dox-loaded TPP-NPs were spherical in shape and dispersed homogenously with lower polydispersity index. Dox-loaded TPP-NPs showed sustained release of Dox from nanoparticles and with the decrease of pH in medium; the releasing rate of Dox was increased and the cumulative release of drug was significantly enhanced. At the same time, compared with targeted location of free Dox in nucleus, when cells were incubated with Dox-loaded TPP-NPs, red spots representing Dox were homogenously distributed in mitochondria instead of location at nucleus. The mitochondrial membrane potential was decreased in cells and the mitochondria in the cells were almost completely damaged. After treated with free Dox and Dox-loaded TPP-NPs, the generation of ROS in A549 cells and Hela cells was significantly enhanced, suggesting induction of ROS for triggering the cell apoptosis. Taken together, Dox-loaded TPP-NPs increased the mitochondrial delivery of Dox, therefore increasing cytotoxicity of drug and inducing the significant apoptosis effect.
We had constructed a mitochondrial-targeted functional nanoparticle delivery system for enhancing antitumor efficiency. The cationic lipophilic compound triphenyl phosphine (TPP) with high mitochondrial binding specificity was introduced, and broad-spectrum anticancer drug Dox was entrapped in the cavity of nanoparticles for achieving the better physiological functions. It showed that Dox-loaded TPP-NPs were internalized into the cytoplasm and further quickly located at the mitochondria to release Dox with the mediation of TPP-NPs. Dox in mitochondria activated the mitochondrial apoptotic pathway, reduced mitochondrial membrane potential, and opened mitochondrial membrane hole, thus releasing cytochrome c and promoting expression of Bax, caspase-3, and caspase-9. Taken together, triphenyl phosphine-conjugated chitosan nanoparticles provided preferential mitochondrial delivery and accumulation of Dox, thus improving drug efficacy in tumor cells.
This work was supported by Liao’ning Educational Committee (No. L2014339), Natural Science Foundation of Liaoning Province (No. 2015020692 and No. 201602337).
JH, XY, and LZ designed the study and conducted the experiments. YS, YS, CS, and LZ performed treatment of experimental data and calculations. JH, XY, YS, YS, CS, and LZ participated in the discussion of the results. JH and XY prepared the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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