Self-Motivated Supramolecular Combination Chemotherapy for Overcoming Drug Resistance Based on Acid-Activated Competition of Host–Guest Interactions

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Aug 2021Self-Motivated Supramolecular Combination Chemotherapy for Overcoming Drug Resistance Based on Acid-Activated Competition of Host–Guest Interactions Hua Wang, Han Wu, Yu Yi, Ke-Fei Xue, Jiang-Fei Xu, Hao Wang, Yuliang Zhao and Xi Zhang Hua Wang Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Google Scholar More articles by this author , Han Wu Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Google Scholar More articles by this author , Yu Yi *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 Google Scholar More articles by this author , Ke-Fei Xue Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 Google Scholar More articles by this author , Jiang-Fei Xu Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Google Scholar More articles by this author , Hao Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 Google Scholar More articles by this author , Yuliang Zhao CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 Google Scholar More articles by this author and Xi Zhang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202100964 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail In this work, we propose a nanoparticle-based strategy of self-motivated supramolecular combination chemotherapy that carries drugs stably and releases them actively in the acidic tumor microenvironment to overcome the drug resistance of cancer cells. This self-motivated nanoparticle consists of a cucurbit[7]uril-containing polymer (PCB)-based core, in which the oxaliplatin and a mitochondria-targeting cytotoxic peptide with an N-terminal phenylalanine are stably loaded through host–guest interactions, and a polymeric protection shell containing acid-activated competitors for releasing drugs. In the acidic tumor environment, the protected aminomethyl phenylalanine moieties on the polymer shell were recovered with a remarkable increase in the binding constant toward cucurbit[7]uril, which competitively replaced and released the combination drugs in PCB. With the synergistic effect of released mitochondria-targeting cytotoxic peptides that effectively inhibited the energy-dependent drug efflux, this self-motivated nanoparticle augmented the anticancer activity of oxaliplatin against drug-resistant cells. This strategy of self-motivated supramolecular combination chemotherapy provides a new method for efficient combination of chemical drugs and bioactive peptides with on-demand drug release, and opens a great prospect for supramolecular chemotherapy toward overcoming the drug resistance of cancers. Download figure Download PowerPoint Introduction Drug resistance is a major hurdle to effective and safe cancer chemotherapy.1–4 With long-term use, most anticancer drugs can cause cancer cells to develop drug resistance. Therefore, how to overcome drug resistance is an urgent problem in cancer chemotherapy.5–10 Among complicated mechanisms, the overexpression of adenosine triphosphate (ATP)-binding cassette efflux transporters, for example, multidrug resistance P-glycoprotein, is a common characteristic of drug resistance, which pumps out multiple drugs from cancer cells by consuming the energy of ATP hydrolysis.11–14 Combination chemotherapy has proven to be effective.15–22 The initial strategy of combination chemotherapy involves the use of different anticancer agents with nonoverlapping mechanisms of action. This approach works well in clinical treatment and has led to a series of gradually more complex strategies, such as combination chemotherapy with peptides, transporter protein inhibitors, and monoclonal antibody immunity. However, how to achieve efficient combinations of different types of drugs like chemical drugs and bioactive peptides or proteins still remains a big challenge. Along with the development of nanotechnology, nanoparticle-based drug delivery systems have increasingly attracted interest.23–30 On the one hand, nanocarriers can stably transport drugs to cells, which improve the chemical stability of anticancer drugs, increase the cellular accumulation, and enhance the length of circulation stability. On the other hand, on-demand drug release is also an issue of concern in nanomedicines. Controlled drug release can be realized by responding to both endogenous and exogenous stimuli. Particularly, the tumor microenvironment, including acidity, redox potential, and overexpressed biomarkers, can be sensed by stimuli-responsive nanocarriers, leading to more precise intracellular drug release.31–34 Among the stimuli-responsive strategies based on microenvironments, the “prodrug” strategy is an ingenious method that introduces a cleavable group on drugs to block their efficacy, and can be converted to an active drug within a biological system by cutting off the protective group.35–37 Here, we propose a “self-motivated” strategy derived from the “prodrug” strategy that can be applied to the controlled release of drugs from nanocarriers. In this strategy, nanocarriers not only possess drug containers, but inactive competitors that facilitate drug release. The drugs can be stably loaded by shielding the competitors in the delivery process, and the competitors can be released by stimulus response at the target, achieving the controlled drug release. Compared with the “prodrug” strategy, the “self-motivated” strategy may reduce the unpredictable influence of direct modification on drug efficacy. Considering that the polyethylene glycol (PEG)-based poly-cucurbit[7]uril (PCB) is effective in delivering platinum-based drugs and N-terminal phenylalanine (N-Phe)-containing therapeutic peptides, it should be an ideal carrier to achieve stable and safe combination chemotherapy.38–44 Notably, the oxaliplatin (OxPt) and N-Phe-containing peptide are comparable in binding constants upon cucurbit[7]uril (CB[7]) (ca. 2 × 106 M−1), indicating the possibility of precise control of the dosage ratio between different drugs. To further achieve both stable loading and on-demand release of combination drugs for improved anticancer activity against drug-resistant cancer cells, herein a strategy of self-motivated supramolecular combination chemotherapy is presented. To this end, as shown in Scheme 1, a clinical anticancer drug OxPt and a positively charged mitochondria-targeting cytotoxic peptide with an N-Phe (N-Phe-KLAK) were simultaneously carried in PCB through host–guest interactions, resulting in multipositively charged nanoparticles (PCB-NPs). Afterward, a negatively charged block copolymer mPEG-PLL(AF-CAA), which consisted of a backbone of α-methoxy PEG (mPEG)-block-poly(l-lysine) with lysine side chains modified with acid-cleavable cis-aconitic acid (CAA)-linked 4-(aminomethyl)-d-phenylalanine,45 was wrapped around PCB-NPs by electrostatic interaction to construct PEGylated nanoparticles [supramolecular combination chemotherapy nanoparticles (SCC-NPs)]. Under the acidic microenvironment in drug-resistant cancer cells, CAA protection groups of aminomethyl phenylalanine moieties in mPEG-PLL(AF-CAA) were removed, with a charge reversal of the polymer from negative to positive charges, resulting in the disintegration of SCC-NPs. The recovered aminomethyl phenylalanine moieties, whose binding affinity with PCB exhibited an order of magnitude higher than drugs, led to the competitive replacement of the drugs from PCB-NPs. This supramolecular combination chemotherapy was expected to have the following four advantages: (1) This strategy would achieve stable loading for small anticancer drugs as well as biological peptide drugs and their on-demand drug release. (2) These different types of drugs would combine in a tuned and defined dose ratio. (3) Nearly complete release of drugs would be achieved through the so-called “self-motivated drug release” based on the acid-activate competition of host–guest interaction. (4) This supramolecular combination chemotherapy would show enhanced anticancer activity against OxPt-resistant colon cancer cells, with reduced side effects for normal intestinal cells. Scheme 1 | Schematic representation of the self-motivated supramolecular combination therapy to overcome drug resistance. (a) The preparation of self-motivated nanoparticles (SCC-NPs). The OxPt and positively charged N-Phe-KLAK are first encapsulated in PCB simultaneously though host–guest interactions, followed by coverage of PEGylated polyions containing acid-activated competitors via electrostatic interactions. (b) Proposed mechanism of supramolecular combination chemotherapy for overcoming drug resistance. Under the acidic tumor microenvironment, OxPt and N-Phe-KLAK are released out from SCC-NPs through acid-activated competition of host–guest interactions. The released N-Phe-KLAK destroy the mitochondria and reduce the production of ATP in cancer cells, probably resulting in an inhibition of the drug efflux mediated by the P-glycoprotein, which augments the chemotherapy of OxPt toward drug-resistant cancer cells. Download figure Download PowerPoint Experimental Methods Size and zeta-potential The size and their distribution of nanoparticle solutions with different pH were measured by a dynamic light scattering (DLS) analyzer (Zetasizer Nano ZS90, Malvern Instruments, Malvern, UK) with an incident He–Ne laser (λ = 633 nm) and a detection angle of 90° at 25 °C. Zeta-potential values were determined by the same instrument with a capillary cell (DTS1070, Malvern Instruments) at 25 °C. For drug release, zeta-potential values of SCC-NPs or Ctr-NPs solutions at pH 6.5 or 7.4 were measured every 2 h after the solutions were freshly prepared. Isothermal titration calorimetry measurement Isothermal titration calorimetry (ITC) experiments were carried out on a MicroCal VP-ITC apparatus (Malvern Instruments) at 37.0 °C. To measure the binding constants, 1.0 mM OxPt, N-Phe-KLAK, mPEG-PLL(AF-CCA), or acridine orange (AO) solution was titrated into 0.10 mM PCB solution. The sample cell was initially loaded with 1433 μL titrated solution, and then titrating solution was injected consecutively into the stirred sample cell in portions of 10 μL. The sample was stirred at 70 rpm with a propeller, and the interval between two injections was long enough to reach equilibrium. The heat of dilution for the titrating solution was corrected in separate experiments. The ITC data were analyzed using ORIGIN software (OriginLab, Northampton, MA). Cellular uptake The human normal colonic epithelial (NCM460), colorectal cancer (HT29R), and OxPt-resistant colorectal cancer (HCT116/OxPt) cells were seeded in a 15 mm confocal microscope dish with a density of 6 × 104 cells/well, respectively. After 12 h incubation, the culture media were replaced with new culture media containing free AO or AO-labeled SCC-NPs with the AO concentration of 5 μM, followed by incubation for 4 h. Then, the cells were sequentially washed by the cold phosphate-buffered saline (PBS) three times, and observed by using a ZEISS LSM710 confocal microscope (Carl Zeiss, Jena, Thuringia, Germany) with a 40× objective lens. For flow cytometry, the cells were dispersed by using trypsin after incubation with free AO or AO-labeled SCC-NPs for 4 h, and measured by a BD Calibur flow cytometry (BD FACSCalibur, Franklin Lakes, NJ) by counting 1 × 104 cells per test (n = 3). Evaluation of cell cytotoxicity NCM460, HT29R, and HCT116/OxPt cells were seeded in a 96-well plate (8000 cells/well), respectively. After 12 h incubation, the cells were cultured for 48 h in fresh culture media containing OxPt, PCB, PCB-NPs, SCC-NPs, or Ctr-NPs with different concentrations, respectively. Then, the cells were incubated with new media containing 10% CCK-8 solution for 1 h. The absorbance at 450 nm of the bioreduced soluble formazan product was measured by using an EnVision Multimode Plate Reader (EnVision, Perkin, Beaconsfield, UK). The cell viability was calculated by manually deducting the blank value from each value then normalizing them against the control values (n = 6). The 50% inhibitory concentration (IC50) values were calculated by GraphPad Prism software (GraphPad, San Diego, CA) using a Variable slope model (four parameters). In vitro drug release NCM460, HT29R, and HCT116/OxPt cells cultured in a 15 mm confocal microscope dish (6 × 104 cells) were incubated with AO-labeled SCC-NPs or Ctr-NPs with the AO concentration of 5 μM for 4 or 12 h. Then, the cells were washed by cold PBS three times and incubated with fresh media containing LysoTracker Red (75 nM) for 30 min. After being washed by PBS three times, the cells were observed by using a ZEISS LSM710 confocal microscope (Carl Zeiss) with a 40× objective lens. The plot profiles for colocalizations of LysoTracker Red and AO-labeled nanoparticles in the obtained photo were analyzed by ZEN 3.0 software (blue edition; Carl Zeiss, Jena, Thuringia, Germany). The Pearson’s correlation coefficients were analyzed from 10 individual cells for each sample. Cell apoptosis HCT116/OxPt cells were seeded in a six-well plate with a density of 1 × 105 cells/well. After 12 h incubation, cells were incubated with 50 μM OxPt, PCB-NPs, SCC-NPs, and Ctr-NPs (with 8.8 μM N-Phe-KLAK in nanoparticles) for 48 h. Then, the cells were sequentially washed by cold PBS three times, dispersed by using trypsin, washed by a fresh medium, and suspended in cold PBS. Cells were stained with propidium iodide (PI) and Annexin V using an Annexin V-FITC apoptosis detection kit. Finally, the apoptosis of cells was measured by a BD Calibur flow cytometry (BD FACSCalibur) by counting 1 × 104 cells per test (n = 3). Nontreated cells were used as the control. The data were analyzed with FlowJo software (BD, Franklin Lakes, NJ). Mitochondria-regulated apoptosis HCT116/OxPt cells were cultured in a 15 mm confocal microscope dish (6 × 104 cells). After 12 h incubation, the cells were exposed to new culture media containing SCC-NPs or Ctr-NPs with different concentrations, followed by incubation for 48 h. Similarly, the positive control (carbonyl cyanide 3-chlorophenylhydrazone, CCCP, 10 μM) dispersed in the culture media were added into cells grown in a confocal microscope dish for 20 min. After the cells were washed with PBS three times, the mitochondria were stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) working solution in culture medium for another 20 min. After removing the medium and washing it with PBS three times, cells were imaged using a Zeiss LSM710 confocal laser scanning microscopy (CLSM) with a 40× objective lens. ATP level HCT116/OxPt cells were seeded in six-well plates (1 × 105 cell) for 12 h. The cells were exposed to new culture media containing SCC-NPs or Ctr-NPs with different concentrations, followed by incubation for 48 h. After being washed three times with PBS, the cells were lysed and centrifuged at 130,00 rpm for 10 min to get the supernatants. The supernatants were further incubated with 100 μL ATP Assay Kit (S0026, Beyotime, Shanghai, China) for 5 min. The luminescence was measured by using an EnVision Multimode Plate Reader (EnVision, Perkin, UK). The ATP level of each sample was proportional to the luminescence intensity of each sample (n = 6). Statistical analysis The experimental data were analyzed by Student’s t-test in Microsoft Excel (Microsoft Corp., Redmond, WA). The p < 0.05 was considered statistically significant. Results and Discussion Preparation and characterization of SCC-NPs To fabricate the acid-responsive self-motivated drug delivery system, PCB and mPEG-PLL(AF-CAA) were first synthesized based on published methods ( Supporting Information Scheme S1 and Figures S1–S6). Subsequently, the anticancer drug OxPt and the mitochondria-targeting cytotoxic peptide N-Phe-KLAK with a sequence of FGGD(KALKALK)2 were encapsulated in PCB with a molar ratio of OxPt:N-Phe-KLAK:CB[7] as 85:15:100 using a vortex mixer at a stirring speed of 2500 rpm for 1 min at the ambient temperature. As shown in Figures 1a and 1b, the host–guest interaction between OxPt or N-Phe-KLAK and PCB was explored by ITC measurement, and the apparent binding constant (Ka) was determined to be 6.94 × 105 M−1 and 2.64 × 105 M−1, respectively, indicating the stable encapsulation of the two drugs in PCB. Besides the synergistic treatment, N-Phe-KLAK also endowed positive charges to the PCB-NPs surface. Then, the drugs-loaded nanoparticles SCC-NPs were constructed through electrostatic interaction with positive PCB-NPs trapped in the core and negative mPEG-PLL(AF-CAA) wrapped as the shell (Scheme 1a). As a control, Ctr-NPs were fabricated by the same method by a nonresponsive polymer, mPEG-PLL(AF-CCA) ( Supporting Information). The average hydrodynamic diameter of the nanoparticles was determined to be approximately 130 nm by using DLS (Figure 2a). The zeta-potential value was about −35 mV ( Supporting Information Table S1), which was expected to favor longer blood circulation for improved tumoral accumulation. The spherical morphology of SCC-NPs with the diameter of 130 nm was confirmed by the cryogenic transmission electron microscopy (Cryo-TEM) images (Figure 2b), in line with the data from DLS. To verify the competitive replacement of the drugs, as shown in Figure 1c, ITC measurements were performed to investigate the host–guest interaction between mPEG-PLL(AF) and PCB. The apparent binding constant was determined to be 5.32 × 106 M−1 at pH 6.5, which was an order of magnitude higher than those between drugs and PCB. The competitive replacement rate from CB[7] by aminomethyl phenylalanine was calculated to be 95.7% ( Supporting Information). These results suggest that the drugs-loaded nanoparticles SCC-NPs were successfully constructed and the mPEG-PLL(AF-CAA) was effective in competitively replacing OxPt and N-Phe-KLAK encapsulated in PCB-NPs under acidic condition. Figure 1 | Characterization of the host–guest interactions between (a) OxPt and PCB, (b) N-Phe-KLAK and PCB, and (c) mPEG-PLL(AF) and PCB using ITC measurement (PBS, pH 6.5, 25 °C). Download figure Download PowerPoint The zeta-potential measurement and fluorescence spectra were carried out to reveal the drug release behaviors of SCC-NPs. As illustrated in Figure 2c, under the acidic condition, the acid-cleavable CAA protection groups on the mPEG-PLL(AF-CAA) were removed, and the aminomethyl phenylalanine was exposed, accompanied by a reverse in polymer charge from negative to positive. Then, the aminomethyl phenylalanine could competitively replace the drugs in the PCB-NPs and release them effectively. As shown in Figure 2d, the freshly prepared solution of both SCC-NPs and Ctr-NPs were negatively charged with zeta potentials of about −35 and −27 mV, respectively. After 12 h at pH 6.5, the SCC-NPs gradually became positively charged, suggesting the recovery of aminomethyl phenylalanine moieties for sustained release of the drugs.45–47 While SCC-NPs at pH 7.4 and Ctr-NPs at pH 6.5 and 7.4 remained negatively charged. The stability of SCC-NPs at pH 7.4 would effectively restrain the drug release in normal tissues. Notably, after 12 h under acidic condition, the average hydrodynamic diameter and zeta potential value of SCC-NPs turned into 110 nm and 20.6 mV, respectively, which were consistent with those of the PCB/mPEG-PLL(AF) complexes ( Supporting Information Table S1). To further evaluate the competitive drug release, AO was used as a model compound to track the drug release process. The binding constant of AO and PCB was determined to be 3.43 × 105 M−1 ( Supporting Information Figure S7), which was similar to those of OxPt and PCB. Through time-dependent fluorescence spectra, the release rate after 12 h at pH 6.5 and 7.4 was calculated as 93% and 24%, respectively (Figure 2e and Supporting Information Figure S8). The above results indicate that SCC-NPs can stably carry the drugs under neutral conditions and release the drugs under acidic conditions. Figure 2 | Characterization of SCC-NPs. (a) Size distribution of freshly prepared SCC-NPs in PBS buffer (pH 7.4) with a concentration of 100 μM at 25 °C. (b) Cryo-TEM images of SCC-NPs with a concentration of 100 μM. (c) The proposed competition of host–guest interactions under acidic condition. (d) The acid responsiveness profiles of SCC-NPs or Ctr-NPs by detecting variations of zeta-potential values at pH 6.5 or 7.4. (e) The drug release profile of SCC-NPs and Ctr-NPs at pH 6.5 or 7.4. Download figure Download PowerPoint Cellular uptake and drug release in vitro Cellular uptake and drug release behavior of SCC-NPs were investigated in human colorectal normal epithelial cells, cancer cells, and drug-resistant cancer cells. Cultured HT29R, HCT116/OxPt, and NCM460 cells were incubated with either free AO or AO-labeled SCC-NPs for 4 h, respectively, followed by observation using CLSM. As shown in Figure 3, all three types of cells presented green fluorescence with similar intensities, indicating the efficient uptake of SCC-NPs. Free AO was distributed in the cytoplasm and tended to accumulate in the nucleus of all three colorectal cells, whereas AO-labeled SCC-NPs appeared as bright dots located in the organelles. Meanwhile, the cell uptake of free AO and AO-labeled SCC-NPs was further confirmed by quantitative analysis using flow cytometry ( Supporting Information Figure S9). In three cells, the AO signal intensities were around 40, while the AO-labeled SCC-NPs signal intensities were around 35, possibly due to the PEG protection and negatively charged surface. Figure 3 | Cellular uptake behavior of SCC-NPs. The images of NCM460, HT29R, and HCT116/OxPt cells with AO or AO-labeled SCC-NPs 5 μM) for 4 h. The of AO is 10 Download figure Download PowerPoint To the drug release behavior of SCC-NPs in HCT116/OxPt cells, a was used for analysis by using CLSM. As illustrated in Figure after incubation with SCC-NPs or Ctr-NPs for 4 h and stained with LysoTracker Red for 20 the green fluorescence from SCC-NPs or Ctr-NPs the cells was well with fluorescence from LysoTracker with Pearson’s coefficients of and respectively. These results that SCC-NPs and Ctr-NPs into the cells by and in the and After further incubation for 12 h green fluorescence from SCC-NPs longer with the and the intracellular distribution was similar to that of free The Pearson’s was to only indicating that the encapsulated AO was released from SCC-NPs and from the and to the cytoplasm and the green fluorescence from Ctr-NPs well with fluorescence with a Pearson’s of suggesting their stable of drugs and of These results reveal that SCC-NPs can be effectively by cells and accumulate in and on-demand drug release can be realized by acid-activated competition of host–guest interactions. Figure 4 | drug release behavior of SCC-NPs. The images show the cellular of AO-labeled SCC-NPs or Ctr-NPs with LysoTracker Red after 4 and 12 h against cultured HCT116/OxPt cells 5 respectively. The green the free AO or AO-labeled SCC-NPs and Meanwhile, the stained by LysoTracker The plot profile the of fluorescence intensities derived from AO and LysoTracker Red the of the in each 10 Download figure Download PowerPoint activity in vitro To the drug resistance of cancer cells could be overcome through self-motivated supramolecular combination the cytotoxicity of cultured cells after with different for 48 h was by a CCK-8 the values were calculated by using a Variable slope model (four in GraphPad Prism software (GraphPad, San Diego, Based on different pH values in normal and tumoral and HCT116/OxPt cells were cultured at pH 6.5, while NCM460 cells were cultured at pH respectively. of the polymer including PCB, mPEG-PLL(AF-CAA), and mPEG-PLL(AF-CCA), remarkable cytotoxicity with the concentration to μM, great ( Supporting Information Notably, as shown in Figure in to OxPt that was to NCM460 cells SCC-NPs with cell in the concentration of μM, suggesting reduced side effects for normal cells. This of reduced side effects was to the stable encapsulation by which a host–guest interaction with As to cells, as shown in Figure the anticancer activity of SCC-NPs was with an value of μM, which was similar to that of OxPt In Ctr-NPs an anticancer activity against cells, with an value higher than μM. These results indicate that the exposed by an acidic environment, can effectively release the OxPt encapsulated in PCB and the cytotoxicity of the improved anticancer activity and reduced normal cell of SCC-NPs an therapeutic Figure 5 | of anticancer for SCC-NPs and other groups with against (a) NCM460 cells, (b) cells, and (c) HCT116/OxPt cells. The cultured cells are incubated with different drugs for 48 h, followed by of the viability using CCK-8 Results are as and (n = 6). < Download figure Download PowerPoint To further investigate the anticancer activity of SCC-NPs for overcoming the drug resistance of cancer cells, we their anticancer activity against OxPt-resistant HCT116/OxPt cells as As shown in

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