Conjugated Polymer for

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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 37−41

Soft Particles of Gemini Surfactant/Conjugated Polymer for Enhanced Anticancer Activity of Chemotherapeutics Hua Wang,†,§ Weiwei Zhao,†,§ Lingyun Zhou,‡,§ Jianwu Wang,‡,§ Libing Liu,‡ Shu Wang,*,‡,§ and Yilin Wang*,†,§ †

Key Laboratory of Colloid, Interface and Chemical Thermodynamics, and ‡Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China § University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: In this work, we developed a kind of novel nontoxic soft particle self-assembled by gemini surfactant (SDHC) and conjugated polymer (PMNT). The soft particle shows strong ability in incorporating into cell membrane, and alters the membrane permeability, especially under light irradiation. The anticancer activities of doxorubicin (DOX) were enhanced 6−9 times after cancer cells were treated with the soft particles under light irradiation. The cell viabilities of three kinds of cancer cells testify that this effect of the soft particles on chemotherapy is universal. This work provides a new strategy to enhance the anticancer activities of drugs.

KEYWORDS: gemini surfactant, conjugated polymer, light irradiation, cell membrane permeability, anticancer activity

C

This light irradiation technique displays potential applications in cancer therapy because of its noninvasiveness to normal tissues and high spatiotemporal accuracy.25−28 Therefore, the strategy of combining surfactants, photosensitizers, and light irradiation is desired to significantly enhance anticancer activity of drugs in the present work. Herein, we utilized carboxylate gemini surfactant (sodium 2,6-didodecyl-4-hydroxy-2,6-diaza-1,7-heptanedicarboxylate, SDHC) and conjugated polymer (polythiophene, PMNT) to prepare soft particle through self-assembling, and found that the SDHC/PMNT soft particle can significantly enhance the anticancer activity of drugs upon light irradiation. The proposed mechanism of the soft particle for enhanced anticancer activity and three steps for this strategy are illustrated in Figure 1. SDHC exhibits strong self-assembly ability, and forms vesicles at very low concentration, which makes SDHC easy to incorporate into the lipid bilayer and disrupt the integrity of cell membrane. PMNT acts as a photosensitizer to generate singlet oxygen and other reactive oxygen species (ROS) under light so as to oxidize the unsaturated phospholipid molecules and increase the permeability of cell membrane. So the SDHC vesicles are used to disperse the PMNT aggregates, and assemble into soft

hemotherapy of cancers is an important method to combat this devastating disease. Up to the present, numerous natural products and synthetic compounds have been developed as anticancer drugs, such as camptothecin, paclitaxel, doxorubicin, and platinum compounds.1−4 However, anticancer drugs suffer from their severe side effects to healthy tissues and result in toxicity to the patients, which is the major obstacle in chemotherapy. Therefore, it is very important to reduce the dosage of drugs by enhancing the anticancer activity of drugs during cancer therapy. Surfactants with excellent self-assembly ability have been widely utilized in nanomedicine through enhanced permeability and retention (EPR) effect.5−8 Surfactants can be used to prepare nanoparticles with target agents and anticancer drugs by ultrasonication and evaporation.9−11 Moreover, micelles or vesicles formed by surfactants can deliver both hydrophobic and hydrophilic drugs into cancer cells.12−15 Besides, surfactants can anchor to cell surfaces and cause the disintegration of cell lipid bilayers.16−18 All these factors are expected to improve the solubility of drugs, protect drugs from in vivo condition, reduce nonspecific side-effects and toxicity of drugs, and enhance their anticancer efficacy. Photosensitizers show the ability to generate singlet oxygen under light irradiation,19 which can oxidize the lipid or other unsaturated molecules20 and then release the drugs encapsulated in these carriers.21 Photosensitizers are also used to kill cancer cells in photodynamic therapy (PDT) because of this property.22−24 © 2017 American Chemical Society

Received: October 28, 2017 Accepted: December 20, 2017 Published: December 20, 2017 37

DOI: 10.1021/acsami.7b16396 ACS Appl. Mater. Interfaces 2018, 10, 37−41

Letter

ACS Applied Materials & Interfaces

employed to evaluate the role of SDHC in the soft particles. The MCF-7 cells were seeded and cultured for 12 h, and then the culture medium was changed by fresh medium with 10 μM SDHC, PMNT, or SDHC/PMNT. After being incubated for 1 h at 37 °C, the MCF-7 cells were further incubated with DOX in dark for 1 h. Confocal laser scanning microscopy (CLSM) images (Figure 2c) demonstrate that a small amount of DOX enters into the cytoplasm of MCF-7 cells in control and PMNT groups, while more DOX distributes in and around the cell nucleus in SDHC and SDHC/PMNT groups. This means that the anticancer drug DOX easily accumulates in the MCF-7 cells treated with SDHC and SDHC/PMNT, while PMNT does not affect the uptake of DOX in dark. In order to determine the enhanced anticancer activity by SDHC, the MCF-7 cell viability was monitored. Figure 2d displays that the lowest half maximal inhibitory concentrations (IC50) of MCF7 cells are 3.9, 2.8, 3.6, and 2.5 μM for the control, SDHC, PMNT, and SDHC/PMNT group, respectively. Obviously, the anticancer activity of DOX increases with the addition of SDHC. In final, the effect of light irradiation on anticancer activity was studied. As verified above, DOX and SDHC cannot sensitize oxygen to ROS, so the MCF-7 cells treated with PMNT and SDHC/PMNT for 1 h were further incubated with DOX under white light irradiation (a fluence rate of 5 mW/cm2) for 1 h. The CLSM images (Figure 2c) show that DOX extensively distributes in cytoplasm and nucleus, especially in the SDHC/PMNT group. The cytotoxicity coincides with this result. The results show that IC50 are further reduced to 2.3 and 0.6 μM in the PMNT and SDHC/ PMNT groups, respectively (Figure 2d). That is to say, after light irradiation, IC50 of MCF-7 cells declines from 3.9 to 0.6 μM, which indicates that the anticancer activity of DOX in the SDHC/PMNT group increases almost 7-fold in comparison with the control group in dark. It is evident that the surfactant, PMNT and light irradiation synergistically act with DOX in killing cancer cells. Therefore, it is sufficient to prove that the SDHC/PMNT soft particle is very effective in improving the anticancer activity of the drug under light irradiation. Two other cancer cells (HeLa cells and A549 cells) were also chosen to verify the universality of the above strategy. The cell viability against the PMNT concentration (0−40 μM) at 10 μM SDHC in dark and under white light irradiation (Figure S4) indicates that the same soft particles with 10 μM SDHC and 10 mM PMNT is also suitable for these two cells. Figure S5 shows that the IC50 values of HeLa cells and A549 cells are 13.4 and 12.2 μM for the control group in dark and 1.5 and 2.3 μM in the SDHC/PMNT group under light irradiation. The anticancer activity of DOX is enhanced 9-fold and 6-fold, proving that the effect of the soft particle is universal for other cancer cells. To further understand why the SDHC/PMNT soft particle can significantly enhance the anticancer activity of drug, we have studied the interaction between the soft particle and cancer cells as follows. A new fluorescence marker SDHC-Cy5 was synthesized to track the location of SDHC.29 The MCF-7 cells were incubated in DMEM containing 10% FBS with SDHC (10 μM), PMNT (10 μM) and SDHC-Cy5 (1 μM) for 1 h before the cell imaging assay. The CLSM images (Figure 3a) prove that both SDHC and PMNT accumulate on the cell membrane, suggesting that the interaction occurs between the soft particles and the cell membrane. The SDHC/PMNT location in MCF-7 cells against uptake time (1−12 h) was observed with CLSM (Figure S6), which exhibited the uptake

Figure 1. Preparation of the soft particle by gemini surfactant (SDHC) and conjugated polymer (PMNT), and the proposed mechanism of the soft particle for enhanced anticancer activity. Three steps for this anticancer strategy: (1) SDHC/PMNT treatment: the cells were incubated with 10 μM SDHC/PMNT for 1 h at 37 °C. (2) DOX treatment: after SDHC/PMNT treatment, the culture medium was changed for fresh medium with DOX quickly. (3) Light treatment: after DOX treatment, the cells were immediately incubated under the white light at a fluence rate of 5 mW/cm2 for 1 h.

particles. Upon light irradiation, the soft particles are expected to efficiently damage the cell membrane while approaching to cancer cells, and afterward the preadded DOX is proposed to be accelerated into cancer cells. Finally, the growth of cancer cells would be inhibited, and thus the anticancer activity would be enhanced. The following section will indicate that the results we obtained have confirmed this designed strategy and the related mechanism. Three typically cancer cell lines, breast cancer cells (MCF-7 cells), cervical carcinoma cells (HeLa cells) and human alveolar basal epithelial cancer cells (A549 cells), are selected in the following study. At first, soft particles are constructed by gemini surfactant (SDHC) and conjugated polymer (PMNT) through selfassembling. As shown in Figure S1 and S2 and Table S1, SDHC itself forms anionic vesicles with diameters of 100 nm, whereas PMNT itself associates into very large aggregates at the micrometer scale because of the hydrophobic effect and favorable π−π stacking between the conjugated backbones. The prepared SDHC/PMNT soft particles keep the SDHC vesicles while dispersing the PMNT aggregates by electrostatic and hydrophobic interaction. MCF-7 cells as a representative were used to evaluate the effect of the SDHC/PMNT soft particles on anticancer activity. The results confirm that the singlet oxygen and other ROS are sensitized by the SDHC/ PMNT soft particles rather than DOX under white light irradiation (400−800 nm) at a fluence rate of 5 mW/cm2. Figure 2a shows the variation of the fluorescence intensity of DCF at 525 nm with the irradiation time in the presence of SDHC, PMNT, SDHC/PMNT or DOX. The fluorescence intensities of PMNT and SDHC/PMNT are much higher than those of SDHC, DOX and control group. These results prove that the ROS originated from DOX is negligible compared with that from the SDHC/PMNT soft particles. SDHC was previously testified to be nontoxic to mammalian cells.29 Here, the cell viability against the PMNT concentration (0−40 μM) with 10 μM SDHC in dark and under white light irradiation (400−800 nm) shows that the SDHC/PMNT soft particles also show less toxic to mammalian cells (Figure 2b). Then the soft particles consisting of 10 μM SDHC and 10 μM PMNT were selected for further experiments. The dark condition was 38

DOI: 10.1021/acsami.7b16396 ACS Appl. Mater. Interfaces 2018, 10, 37−41

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ACS Applied Materials & Interfaces

Figure 2. (a) Fluorescence intensity of DCFH against light irradiation time (5 mW/cm2) in the presence of SDHC, PMNT, SDHC/PMNT and DOX with an excitation of 488 nm. [SDHC] = 10 μM, [PMNT] = 10 μM, [DOX] = 10 μM. The background emission was deducted. (b) Cell viabilities of MCF-7 cells as a function of PMNT concentrations with 10 μM SDHC in dark or under light. (c) The CLSM images of DOX (excitation: 559 nm; collection: 570 to 670 nm) and PMNT (excitation: 488 nm; collection: 500 to 545 nm) in MCF-7 cells after different treatments. The false colors of DOX and PMNT are red and green, respectively. [SDHC] = 10 μM, [PMNT] = 10 μM, [DOX] = 5 μM. (d) Cell viabilities of MCF-7 cells as a function of DOX concentrations after different treatments.

altering the membrane permeability and solubilizing lipid vesicles.17,18,30 As PMNT is titrated into the MCF-7 cell solution, the ΔHobs shows less exothermic than that of titrating PMNT into HBSS, and the entropy change (TΔS) is positive, suggesting the PMNT aggregates bind to the cells accompanied by the disaggregation process. With regard to the SDHC/PMNT soft particles, the ITC curve for the particles being titrated into MCF-7 cells displays the same tendency as that of SDHC, but he exothermic ΔHobs becomes smaller, because the aggregation behavior of the SDHC/PMNT soft particles is similar to SDHC, and PMNT molecules weaken the interaction between soft particles and cells. ESI-MS technique was used to confirm whether ROS produced from PMNT could oxidize the unsaturated phospholipid on cell membrane (Figure S7). The unsaturated phospholipid DOPC was used to simulate the phospholipid molecules in cell membrane. The peaks at 808.5 and 485.4 in the ESI-MS spectra correspond to DOPC and PMNT, respectively. The peak from the SDHC

process of SDHC/PMNT from the cell membrane into cytoplasm. When incubation time was less than 1−2 h, SDHC/PMNT was mainly distributed on the cell membranes, while SDHC/PMNT was delivered to lysosome upon more than 2 h incubation.29 Small changes in the zeta potential values of MCF-7 cells with the different treatments (Figure 3b) suggest that the SDHC and PMNT molecules may insert into cell membrane or bind on the surface of cell membrane. Then, isothermal titration calorimetric (ITC) was employed to investigate the thermodynamic changes in the binding process of MCF-7 cells with SDHC, PMNT or SDHC/PMNT (Figure 3c, and Table S2). Only when SDHC is titrated into the Hank’s Balanced Salt Solution (HBSS) of MCF-7 cells, the observed enthalpy (ΔHobs) value undergoes a marked change from larger exothermic to less exothermic, indicating that SDHC vesicles are fused into the cell membrane through hydrophobic interaction. Previous works proved that surfactants show strong ability in incorporating into lipid bilayer, 39

DOI: 10.1021/acsami.7b16396 ACS Appl. Mater. Interfaces 2018, 10, 37−41

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ACS Applied Materials & Interfaces

Figure 3. (a) CLSM images of SDHC-Cy5 (excitation: 559 nm; collection: 570 to 670 nm) and PMNT (excitation: 488 nm; collection: 500 to 545 nm) in MCF-7 cells after incubation for 1 h. The false colors of SDHC-Cy5 and PMNT are red and green, respectively. The scale bar is 10 μm. (b) Zeta potentials of MCF-7 cells in HBSS after different treatments. (c) Observed enthalpy changes ΔHobs against the final concentration of SDHC, PMNT and SDHC/PMNT by titrating 30 μM solution into HBSS or MCF-7 cells (160 000/mL).

molecule cannot be found in the ESI-MS spectra under positive charge state, whereas the alkyl segments of SDHC produces the peaks at 101.0 and 179.0. The mixture of DOPC and SDHC/PMNT under dark shows no difference in ESI-MS. After the mixture was irradiated by light for 1 h, the peak at 808.5 decreases, whereas the new peaks at 153.0 and 530.2 appeared. The new peaks may come from the DOPC segments. The C−C double bonds on the alkyl chain and the spacer tend to be broken when DOPC is oxidized. So the above ESI-MS spectra prove that PMNT could generate ROS to oxidize the unsaturated phospholipid on cell membrane. In brief, the SDHC/PMNT soft particles can interact with the cancer cell membrane and alter the membrane permeability. In conclusion, we developed a new strategy to enhance the anticancer activity of chemotherapeutics. The anticancer activity of DOX has improved 6 to 9 times after the cancer cells are treated by the SDHC/PMNT soft particles and by exerting light irradiation. The effect of the soft particles is universal as testified by three kinds of cancer cells. In this strategy, the soft particles interact strongly with the cell membrane, and disintegrate the membrane permeability in two ways: (I) the gemini surfactant molecules are distributed into the lipid bilayer to disrupt the integrity of cell membrane; (II) the conjugated polymer acts as a photosensitizer to generate ROS under light, also increasing the permeability of cell membrane. The strategy provides a new perspective to enhance the anticancer activity of drugs through treating cancer cells with the soft particles of surfactant and conjugated polymer.

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of HeLa cells and A549 cells for the sublethal doses and anticancer activities; time imaging of SDHC/PMNT in MCF-7 cells; thermodynamic parameters of SDHC, PMNT and SDHC/PMNT with MCF-7 cells derived from ITC curves; ESI-MS spectra of DOPC, SDHC/ PMNT and SDHC/PMNT/DOPC (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.W.). *E-mail: [email protected] (Y.W.). ORCID

Lingyun Zhou: 0000-0002-8506-856X Libing Liu: 0000-0003-4827-6009 Shu Wang: 0000-0001-8781-2535 Yilin Wang: 0000-0002-8455-390X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21633002, 21327003).

ASSOCIATED CONTENT

REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b16396. Experimental section; SEM and Cryo-TEM images of SDHC, PMNT, and SDHC/PMNT; size and ζpotential values of SDHC, PMNT, and SDHC/ PMNT; surface tension curve of SDHC/PMNT; UV− vis spectra of PMNT and SDHC/PMNT; cell viabilities 40

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DOI: 10.1021/acsami.7b16396 ACS Appl. Mater. Interfaces 2018, 10, 37−41