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how we can control the cells' Oxygen in Nanoparticle Sensor Technology

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Answer 1

Photodynamic therapy (PDT) is a clinically approved anti-cancer treatment that involves the activation of an otherwise inactive sensitiser drug with light, which in the presence of molecular oxygen, generates cytotoxic reactive oxygen species (ROS). As oxygen is a key requirement for the generation of ROS in PDT and given the fact that hypoxia is a characteristic of most solid cancerous tumours, treating hypoxic tumours using PDT can be a challenge

CaO2 nanoparticle (NP) formulation coated with a pH-sensitive polymer to enable the controlled generation of molecular oxygen as a function of pH. The polymer coat is designed to protect the particles from decomposition while in circulation but enable their activation at lower pH values in hypoxic regions of solid tumours. The oxygen generating capability of the polymer coated NPs is in aqueous solution with minimal oxygen produced at pH7.4, whereas it increases significantly when the pH is reduced to 6.2. The polymer coated CaO2 NPs significantly increase tumour pO2 levels

MCF-7 cells incubated with CDM NPs for different periods of time were imaged by a confocal microscope to investigate the intracellular distribution. Both DOX and Ce6 fluorescence inside the cells significantly enhanced with the increase of incubation time. Interestingly, although Ce6 fluorescence was mostly located in the cytoplasm after incubation for 4 h, DOX preferentially accumulated inside the nuclei over time, indicating the gradual intracellular DOX release from CDM NPs. The quantitative cellular uptake of nanoparticles was also studied using Ce6 as the fluorescence probe. MCF-7 cells were incubated with free Ce6, Ce6 NPs and CM NPs with the same concentration of Ce6 for 4 h. the Ce6-loaded nanoparticles showed significantly enhanced uptake vs. free Ce6. Also, the comparable fluorescence intensities observed in cells treated with Ce6 NPs and CM NPs indicated that the introduction of MnO₂ did not interfere with the fluorescence of Ce

The generation of ROS such as O₂ during intracellular PDT. MCF-7 cells were treated with DCFH-DA, a non-fluorescent molecule that can passively diffuse into cells and be oxidized by ROS and O₂ to produce a bright green fluorescence compound DCF. The resulting oxidatively stressed cells showed green fluorescence indicating enhanced O₂ gained. cells treated with free Ce6, Ce6 NPs, or CM NPs without laser-irradiation presented a weak intracellular ROS signal .The control group not treated with any Ce6 formulation was also dim with/without laser-irradiation. However, in free Ce6, Ce6 NPs, and CM NPs groups under laser-irradiation, the bright green fluorescence signals of DCF were notably enhanced, suggesting that Ce6 could generate O₂ in the cells under irradiation. Among the laser-irradiated groups, weak fluorescence observed in the free Ce6 group was attributed to lower cellular uptake efficiency of Ce6. As expected, compared with the Ce6 NPs group, a significant increase in ¹O₂ production by CM NPs was observed due to the MnO₂-triggered generation of O₂ from H₂O₂ in TME. Flow cytometry analysis further revealed that the order of ROS level was CM NPs > Ce6 NPs > free Ce6, which was consistent with confocal microscopy. These results verified the better performance of CM NPs as the O₂ producer.

To elucidate the contribution of O₂-enhanced PDT , the cell apoptosis by flow cytometry. Annexin V-FITC and PI were introduced to distinguish viable cells from apoptotic and necrotic cells. The results in showed different cell populations (viable (Annexin V-FITC^-/PI^-), early apoptotic (Annexin V-FITC⁺/PI^-), and late-stage apoptotic or dead (Annexin V-FITC⁺/PI⁺)) were induced by different treatments. With 660 nm laser irradiation at a power density of 100 mW/cm² for 5 min, the CM NPs group was more effective at inducing apoptosis than free Ce6 and Ce6 NPs (54.1% vs. 36.04% and 35.40%) at 4 h post-irradiation. Given the ROS generation and apoptosis results above, the PDT efficiency was significantly improved using MnO₂-based photosensitizer-encapsulated nanoparticles relevant to the TME inside tumors.

The in vitro antitumor efficacy was evaluated by the MTT assay. First, the biocompatibilities of blank NPs, colloidal MnO_2, and MnO₂-loaded NPs . In striking contrast to the strong toxicity of positively charged colloidal MnO₂, after 24 h treatment with either blank NPs or MnO₂ NPs at the stated concentrations, the viabilities of both NIH 3T3 and MCF-7 cells were above 85%, indicating the good biocompatibility of the carriers. This result also indicated that colloidal MnO₂ was successfully encapsulated in nanoparticles to shield its positive charge. Furthermore, upon laser-irradiation (660 nm, 100 mW/cm²) for 5 min, the cells incubated with blank NPs showed no significant decrease in viability, suggesting the cytotoxicity was negligible at the studied power density. Next, the cell viability of MCF-7 cells was examined to determine the PDT efficiency. We observed no obvious dark cytotoxicity in cells treated with free Ce6, Ce6 NPs, or CM NPs for 24 h (Figure 7A). However, under laser-irradiation, the PDT efficiency of CM NPs was distinctly higher than that of free Ce6 or Ce6 NPs at different concentrations. The results were consistent with those of ROS generation and cell apoptosis. Subsequently, we compared the combined chemo-PDT efficacy with the single chemotherapy. As shown in Figure 7B, a dose-dependent inhibitory effect was observed in all groups. Compared to chemotherapy alone (free DOX, DOX NPs, or DM NPs), the combined treatment by CD NPs (+laser) without MnO₂ offered more effective cancer cell killing. As expected, the combination therapeutic efficacy could be further enhanced with O₂-generation (CDM NPs group with the laser) at all concentrations. The IC₅₀ values of free DOX, DOX NPs, DM NPs, CD NPs (+laser) and CDM NPs (+laser) against MCF-7 cells were 2.42, 3.30, 3.01, 1.98 and 0.85 μg/mL, respectively. Therefore, CDM NPs with MnO₂ producing O₂ from H₂O₂in situ appear to be an efficient agent to improve PDT, enhancing the chemotherapeutic efficacy of polymeric DOX formulations for combined therapy.