CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro
<p>CD44-mediated endocytosis of HA@TPCS<sub>2a</sub>-NPs. (<b>a</b>) Uptake of HA@TPCS<sub>2a</sub>-NPs (50 μg/mL NPs) in MDA-MB-231 and MCF-7 cells after 2 h of incubation at 37 °C in medium with or without 10 mg/mL of free HA. (<b>b</b>) Concentration-dependent uptake of HA@TPCS<sub>2a</sub>-NPs in MDA-MB-231 and MCF-7 cells after 24 h of incubation at 37 °C. Data are expressed as means ± SD of at least three independent experiments, carried out in triplicate; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.001 (Student’s t test).</p> "> Figure 2
<p>Cytotoxicity of single and combined treatments in differentiated MCF-7 cells cultured as monolayers. (<b>a</b>) Dose-response curves of cells incubated for 24 h with single drugs or their combination loaded in HA-NPs and irradiated with 1 J/cm<sup>2</sup> of red light (600–800 nm) when PDT was part of the treatment. After additional 24 h in drug-free medium, cell viability was measured with the MTS assay. Total drug concentration is referred to DTX + TPCS<sub>2a</sub> concentration. Data are expressed as mean percentage ± SD of at least three independent experiments, carried out in triplicate. (<b>b</b>) Plots of combination index (CI) vs. fraction affected (Fa) relative to cells treated with HA@DTX/TPCS<sub>2a</sub>-NPs loaded with DTX and TPCS<sub>2a</sub> in the 1:35 (blue) or 1:5 (red) molar ratio.</p> "> Figure 3
<p>Mammosphere formation efficiency (MFE) after HA-NPs treatments. Percentage MFE measured in first generation mammospheres generated from MDA-MB-231 (<b>a</b>) and MCF-7 (<b>c</b>) monolayered cells incubated for 24 h with HA@DTX-NPs, HA@TPCS<sub>2a</sub>-NPs and HA@DTX/TPCS<sub>2a</sub>-NPs, irradiated with 1 J/cm<sup>2</sup>, and re-seeded in non-adherent conditions to allow formation of spheres (protocol 1). Percentage MFE measured in second generation mammospheres generated from first generation mammospheres of MDA-MB-231 (<b>b</b>) and MCF-7 (<b>d</b>) exposed to drug-loaded NPs for 24 h, irradiated with 1 J/cm<sup>2</sup>, and re-seeded in non-adherent conditions (protocol 2). MFE was evaluated after 7 and 4 days from re-seeding for MCF-7 and MDA-MB-231, respectively. Data are expressed as mean ± S.D. of at least two independent experiments, carried out in triplicate; * <span class="html-italic">p</span> < 0.05 (one-way ANOVA, Bonferroni’s correction).</p> "> Figure 4
<p>ALDEFLUOR assay in MCF-7 (<b>a</b>) and MDA-MB-231 (<b>b</b>) cells. Percentages of ALDH positive cells (<b>a</b>,<b>b</b>) and exemplificative flow cytometry plots (<b>c</b>) measured in first and second-generation mammospheres treated with drugs in HA-NPs. First generation mammospheres were incubated for 24 h with HA-NPs, irradiated with 1 J/cm<sup>2</sup> of red light and re-seeded in non-adherent conditions to allow formation of II-generation spheres. Seven (MCF-7) or 4 (MDA-MB-231) days after the reseed, the population of ALDH<sup>high</sup> cells was evaluated by gating the ALDH<sup>high</sup> cells, (green population and percentages) whose fluorescence in flow cytometry plots (DEAB−) exceeded that of the negative controls (DEAB+) stained with the ALDH inhibitor DEAB to control background fluorescence. Flow cytometry plots are referred to mammospheres treated with drug doses of 0.02 μg/mL DTX, 0.1 μg/mL TPCS<sub>2a</sub>, 0.12 μg/mL DTX + TPCS<sub>2a</sub>. Data are expressed as mean ± S.D. of at least two independent experiments; * <span class="html-italic">p</span> < 0.05 (one-way ANOVA, Bonferroni’s correction).</p> "> Figure 5
<p>Uptake of HA@TPCS<sub>2a</sub>-NPs in MDA-MB-231 and MCF-7 first generation mammospheres incubated for 2 or 24 h with 50 μg/mL HA@TPCS<sub>2a</sub>-NPs (TPCS<sub>2a</sub> dose 1.25 μg/mL). Column 1: Bright field images; Column 2: TPCS<sub>2a</sub> fluorescence at the equatorial plane of mammospheres; Column 3: Three-dimensional reconstruction of TPCS<sub>2a</sub> fluorescence distribution and intensity in the equatorial plane of the mammospheres; Column 4: Maximum projection obtained from the superimposition of 20 different acquired focal planes. Scale bar unit in the images is µm.</p> ">
Abstract
:1. Introduction
2. Results
2.1. NP Preparation and Characterization
2.2. CD44-Mediated Endocytosis Is Involved in the Cellular Uptake of NPs and Transported Drugs
2.3. Combination Therapy Using HA@DTX/TPCS2a-NPs Is Effective Toward Differentiated MCF-7 Cells
2.4. Combination Therapy Using HA@DTX/TPCS2a-NPs Is Effective in Reducing Stemness Capacity and Cancer Stem Cell Population in Mammospheres
2.5. CD44 Targeting Capability of HA@NPs in Mammospheres
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Preparation and Characterization of NPs
4.3. In Vitro Release of TPCS2a and DTX
4.4. Cell Lines
4.5. Uptake of HA@TPCS2a-NPs in Monolayered Cancer Cells
4.6. Cytotoxicity and Determination of Combination Index (CI)
4.7. Generation and Treatment of Mammospheres
4.8. Analysis of BCSC Population in Mammospheres
4.9. Aldehyde Dehydrogenase (ALDH) Activity Assay
4.10. Uptake of HA@TPCS2a-NPs in Mammospheres
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Formulation | Size a (nm ± SD) | PI b | ZP c (mV ± SD) | DTX Actual Loading (μg DTX/mg NPs) | DTX EE d (%) | TPCS2a Actual Loading (μg TPCS2a/mg NPs) | TPCS2a EE d (%) |
---|---|---|---|---|---|---|---|
HA@DTX | 163 ± 5 | 0.2 | −33 ± 2 | 5.2 | 98 ± 3 | - | - |
HA@TPCS2a | 208 ± 6 | 0.2 | −32 ± 7 | - | - | 25 | 97 ± 3 |
HA@DTX/ TPCS2a | 205 ± 3 | 0.2 | −37.2 ± 3 | 4.8 | 96 ± 4 | 24 | 95 ± 3 |
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Gaio, E.; Conte, C.; Esposito, D.; Reddi, E.; Quaglia, F.; Moret, F. CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro. Cancers 2020, 12, 278. https://doi.org/10.3390/cancers12020278
Gaio E, Conte C, Esposito D, Reddi E, Quaglia F, Moret F. CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro. Cancers. 2020; 12(2):278. https://doi.org/10.3390/cancers12020278
Chicago/Turabian StyleGaio, Elisa, Claudia Conte, Diletta Esposito, Elena Reddi, Fabiana Quaglia, and Francesca Moret. 2020. "CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro" Cancers 12, no. 2: 278. https://doi.org/10.3390/cancers12020278
APA StyleGaio, E., Conte, C., Esposito, D., Reddi, E., Quaglia, F., & Moret, F. (2020). CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro. Cancers, 12(2), 278. https://doi.org/10.3390/cancers12020278