Changes In HeLa Cells Research Articles

H-Aggregation of Squaraine Dye as Generic Colorimetric Molecules to Detect Cu2.

An infrared squaraine dye was utilized to detect Cu2+ in solvents based on H-aggregates of squaraine dye. H-aggregates are a type of aggregation with enhanced photophysical properties compared to monomers. In the presence of a Ca2+ solution, F-Cl offers exceptional H-aggregators that can be transformed into monomers by adding Cu2+. Furthermore, this mode successfully demonstrated fluorescence changes in HeLa cells cultured in vitro after the addition of Ca2+ or Cu2+. A highly specific detection of Cu2+ was achieved using this transformation mode.

Fluorescence lifetime imaging of dynamics of mitochondrial and nucleolar microenvironment during stimuli response in living cells

Nucleolus and mitochondria play an important role in maintaining cell balance, and studying their physiological processes is helpful in understanding the biological functions. In this work, a red fluorescent pyrene rhodamine probe is used to target and label cell mitochondria and nucleolus under different conditions, and the binding mode of probe and RNA is also clarified by bio-computational simulation results. Confocal laser scanning microscopy is used to analyze the morphological changes of apoptosis in HeLa cells under the action of laser light, paclitaxel and colchicine, and the changes of micro-environmental viscosity between mitochondria and nucleolus are quantitatively analyzed by fluorescence lifetime imaging phase map. It is determined that the average fluorescence lifetime of mitochondria labeled by probes in steady-state HeLa cells is about 3.65 ns. The mitochondrial viscosity is about 66×10<sup>–3</sup> Pa·s. After laser irradiation, mitochondrial fracture and fusion occur, the fluorescence lifetime of the probe increases to 3.82 ns and the mitochondrial viscosity increases to about 131×10<sup>–3</sup> Pa·s. The mean fluorescence lifetime of the nucleolus of HeLa cells increases from 4.23 ns to 4.32 ns, indicating that the changes of the nucleolus and mitochondrial microenvironment are induced by prolonging laser irradiation. Apoptosis is induced by paclitaxel and colchicine, and the nucleolus moves out of the nucleus and into the cytoplasm. Meanwhile, the fluorescence lifetime of nucleolus and mitochondria labeled by the probe first increase and then decrease. The treatment time of paclitaxel increases from 0.5 h to 4 h, and the average lifetime of nucleolus of HeLa cells labeled by the probe increases from 4.19 ns to 4.47 ns, and finally decreases to 4.42 ns, reflecting the differences in nucleolar microenvironment of HeLa cells induced by different treatment times of paclitaxel. Comparing with the blank HeLa cell, the average lifetime of the probe increases from 4.10 ns to 4.34 ns after 1 h treatment with colchicine at low concentration (10 nM), and continuously increases to 4.47 ns after 1 h treatment with high concentration (100 nmol/L) colchicine. The microenvironments of nucleolus and mitochondria induced by apoptosis induced by colchicine at different concentrations are shown. The above three ways of inducing apoptosis, i.e. by laser light, paclitaxel and colchicine, prove that the changes of nucleolar and mitochondrial microenvironment and functional changes of HeLa cells under the condition of cell instability provide a new method of studying the dynamic process of apoptosis induced by different pathways and the diseases related to nucleolar and mitochondrial dysfunction as well.

Rational Design of MMP-Independent Near-Infrared Fluorescent Probes for Accurately Monitoring Mitochondrial Viscosity.

Mitochondrial viscosity affects metabolite diffusion and mitochondrial metabolism and is associated with many diseases. However, the accuracy of mitochondria-targeting fluorescent probes in measuring viscosity is unsatisfactory because these probes can diffuse from mitochondria during mitophagy with a decreased mitochondrial membrane potential (MMP). To avoid this problem, by incorporating different alkyl side chains into dihydroxanthene fluorophores (denoted as DHX), we developed six near-infrared (NIR) probes for the accurate detection of mitochondrial viscosity, and the sensitivity to viscosity and the mitochondrial targeting and anchoring capability of these probes increased by increasing the alkyl chain length. Among them, DHX-V-C12 had a highly selective response to viscosity variations with minimum interference from polarity, pH, and other biologically relevant species. Furthermore, DHX-V-C12 was used to monitor the mitochondrial viscosity changes of HeLa cells treated by ionophores (nystatin, monensin) or under starvation conditions. We hope that this mitochondrial targeting and anchoring strategy based on increasing the alkyl chain length will be a general strategy for the accurate detection of mitochondrial analytes, enabling the accurate study of mitochondrial functions.

A coumarin/cyanoacetamide-derived lysosome-targeting probe for detecting bisulfite and viscosity changes in living cells and zebrafish

A coumarin/cyanoacetamide-based probe was produced by a simple condensation reaction. The probe exhibits high selectivity towards bisulfite (HSO3−) and viscosity changes based on either fluorescence or UV–Vis spectra in almost pure aqueous media. The addition of HSO3− induced a significant blue shift in the emission maximum of the free probe (from 575 nm to 485 nm) with a short response time (5 min), a broad function pH range (3.0–11.0), and a low detection limit (74 nM), via the recognition mechanism of bisulfite-involved Michael addition reaction. In parallel, the fluorescence intensity at 575 nm of the probe increased significantly along the increasing viscosity of the test media owing to the prohibited rotation of CC bridged rotors in probe 1. Notably, the probe could monitor the level of lysosomal HSO3− and viscosity changes in HeLa cells and zebrafish.

Infrared spectroscopy and flow cytometry studies on the apoptotic effect of nano-chrysin in HeLa cells

Mapping the structural changes in membrane lipids, proteins, polysaccharides and nucleic acids has opened new channels for understanding the mode of action of anticancer natural products. Earlier, we synthesized chrysin nanoparticles (NChr) with good bioavailability, and characterized its size, surface charge, entrapment efficiency, and drug release pattern using PLGA polymer. NChr induced concentration dependent cytotoxicity in HeLa cells with an IC50 of 61.54 ± 1.2 µM in comparison with free chrysin with IC50 of 86.51 ± 2.9 µM. Since nanoparticles interact dynamically with cell membranes, organelles, proteins and DNA, it is necessary to understand the interplay of nanodrug induced macromolecular changes in cancer cells. In this work, we obtained signatures of NChr-induced biochemical changes in HeLa cells by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy technique coupled with flow cytometry. NChr induced cell membrane disruption, G1 phase cell cycle arrest, and increased externalization of phosphatidylserine leading to apoptosis indicating the biochemical perturbations in membrane lipids and DNA of HeLa cells in comparison with untreated cells. The 1300–1000 cm−1 spectral region indicated NChr interaction with the ribose sugar backbone and DNA denaturation. Spectral range 1800–1400 cm−1 indicated a concentration dependent decrease in α helical and β sheet structures which may lead to protein degradation during apoptosis. The spectral range 3000–2800 cm−1 indicated the lipid peroxidation in response to NChr treatment. This is the first study describing the bio-macromolecular changes induced by a nano encapsulated drug and can pave the way to investigate unconventional modes of action for bioactive formulations.

A mitochondrial suitable ratiometric fluorescent probe for tracking pH change during mitophagy

Intracellular pH is one of the important parameters of cellular metabolism. The function of mitochondria is highly dependent on pH. Developing ratiometric fluorescent probes with suitable alkaline pKa for the detection of mitochondrial pH is urgently demanded. Herein, a mitochondria-targeted ratiometric pH fluorescent probe SNRF was designed based on 1,6-dihydroxynaphthalene and 4-diethylaminoketo acid. SNRF contains a hydroxyl electron donor and a nitrogen positron acceptor with excellent molecular charge transfer (ICT) performance. The hydroxyl group is a pH-sensitive group, which is prone to be protonated under acidic conditions and deprotonated under alkaline conditions. The ratiometric performance of SNRF can avoid the interference from instrumental factors, concentration of probe and microenvironment around the probe. SNRF exhibits a suitable pKa (8.30) with a linear variation between pH 7.4–9.0, making it ideal for monitoring mitochondrial pH changes. And, SNRF can be used to detect pH changes in HeLa cells and zebrafish without interference from other biologically active molecules. Furthermore, SNRF showed excellent mitochondrial targeting and was successfully used to track pH changes during rapamycin-induced mitophagy.

In vitro anticancer activity studies of Aspergillus terreus on uterine cervix Cancer cells

The exploration of new anticancer agents is in high demand owing to limitations of conventional therapies. This study was designed to evaluate the cytotoxic potential of Aspergillus terreus ethyl acetate extract on uterine cervix cancer cells. Secondary metabolites were extracted from broth cultures and total phenolic content was determined using the Folin-Ciocaltiu method. Antioxidant activity was evaluated using DPPH and ABTS free radical scavenging assays. The cytotoxic activity of the fungal extract was assessed using Methyl ThaizolylTetazolium bromide (MTT) assay on HeLa cells. Morphological changes in HeLa cells upon treatment with the extract were determined. The effect of the fungal extract on cell cycle arrest was evaluated using flow cytometry. The extract showed a significant free radical scavenging activity. The total phenolic content was found to be 16.76 mg of gallic acid equivalents (GAE) per gram dry weight of the extract. The extract exhibited a cytotoxic effect on the HeLa cells with an IC50 value of 92.68 μg/mL. Cell cycle analysis revealed that A. terreus induced G0/G1 arrest confirming its anticancer activity. Results of the present investigation revealed that crude extract of the A. terreus possesses potential anticancer properties.

Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy.

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells’ nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.

Proteomic analysis of the influence of CO2 pneumoperitoneum in cervical cancer cells.

The effect of CO2 pneumoperitoneum (CDP) on the oncology outcomes of patients undergoing laparoscopic radical hysterectomy for cervical cancer remains unclear. In this study, we investigated the effects of CDP on the proliferation of cervical cancer cells and examined the molecular mechanism. We established an in vitro CDP model to study the effects of CDP on the proliferation of cervical cancer cells by Cell Counting Kit-8 (CCK-8) assay, xenografted tumor assay. Tandem mass tag-based quantitative proteomics were used to study the proteomic changes in HeLa cells after CDP treatment. Western blot assay was used to detect the expressions of PI3K/Akt signaling pathway proteins. CDP increased cell proliferation after a short period of inhibition in vitro and promoted tumorigenesis in vivo. Proteomic analysis showed that the expression levels of 177 and 309 proteins were changed significantly 24 and 48 h after CDP treatment, respectively. The acidification caused by CO2 inhibited the proliferation of cervical cancer cells by inhibiting the phosphorylation of PI3K and Akt. CDP promoted the proliferation of human cervical cancer cells after a short time of inhibition. The mechanism of which is related to the inhibition of phosphorylation of the PI3K/Akt signaling pathway.

Physcion Induces Potential Anticancer Effects in Cervical Cancer Cells.

Background: The extent of morphological and ultrastructural changes in HeLa cells was assessed by optical, fluorescence and electron microscopy after exposure to various concentrations of physcion, taking into account the biological properties of the test compound. Methods: Cell viability was assessed by MTT assay, while the cell cycle, LC3 expression, apoptosis, change of mitochondrial potential, Bcl-2 protein expression level and the level of reactive oxygen species were analyzed by flow cytometry. Results: As a result of physcion encumbrance, concentration-dependent inhibition of HeLa cell viability and the G0/G1 phase of the cell cycle was observed. Activation of the lysosomal system was also revealed, which was expressed by an increased number of lysosomes, autophage vacuoles and increased expression of the LC3 protein, a marker of the autophagy process. Transmission electron microscopy and fluorescence microscopy showed that physcion induced clear changes in cervical cancer cells, especially in the structure of the nucleus and mitochondria, which correlated with the production of reactive oxygen species by the test compound and indicated the induction of the oxidative process. At the same time, the pro-apoptotic effect of physcion was demonstrated, and this mechanism was dependent on the activation of caspases 3/7 and the reduction in Bcl-2 protein expression. Conclusion: The obtained results indicate an antitumor mechanism of action of physcion, based on the induction of oxidative stress, autophagy and apoptosis.

A new biocompatible silver/polypyrrole composite with in vitro antitumor activity

We used an in situ chemical oxidation method to prepare a new composite of silver nanoparticles (AgNPs) with polypyrrole (PPy), whose properties were optimized through a 23-factorial design of the synthesis conditions. The successful formation of the AgNPs/PPy composite was confirmed by UV–Visible and FTIR spectroscopies. Transmission electron microscopy revealed the presence of AgNPs smaller than 100 nm, dispersed into the PPy matrix. This hybrid composite exhibits a blue fluorescence emission after excitation in the ultraviolet region. In MTT assays, the AgNPs/PPy composite exhibited low cytotoxicity toward non-tumoral cell lines (fibroblast, Vero, and macrophages) and selectively inhibited the viability of HeLa cells. The AgNPs/PPy composite induces ultrastructural changes in HeLa cells that are consistent with the noticeable selectivity exhibited toward them when compared to its action against non-tumoral cell lineages. Also, the AgNPs/PPy exhibited a hemolytic activity below 14% for all blood groups tested, at concentrations up to 125 μg/mL. These results suggest that the AgNPs/PPy composite has a promising potential for use as an antitumoral agent.

Colorimetric and fluorescent turn-on detection of chloride ions with ionophore and BODIPY: Evaluation with nanospheres and cellulose paper

Here, we present the optical detection of chloride ions with co-extraction based anion-selective optodes containing a BODIPY derivative (BDP-OH) and the ionophore In(OEP)Cl. Spectroscopic studies suggested that BDP-OH and In(OEP)Cl formed an adduct BDP-O-In(OEP), which was converted to BDP-OH and In(OEP)Cl upon increasing sample Cl− concentration, and induced signal changes in both fluorescence and absorbance modes. The method was evaluated in polystyrene-graft-poly(ethylene oxide) (PS-PEO) nanospheres (ca. 40 nm in diameter) and on cellulose paper. In contrast to Cl− probes based on fluorescence quenching, the nanospheres exhibited turn-on fluorescence and ratiometric absorbance responses to a tunable Cl− concentration range (10 μM–1 M). Through fluorescence microscopy, the nanospheres were able to respond to Cl− concentration changes in HeLa cells. Cellulose paper-based Cl− optodes with colorimetric response were successfully used to measure Cl− in artificial sweat, providing a potential analytical tool for clinical diagnosis of cystic fibrosis.

APOPTOSIS INDUCTION OF ETHANOLIC EXTRACT OF AMOMUM SUBULATUM ROXB. IN HELA CELL LINES

Deficiency in apoptosis is one of the key factors that plays a pivotal role in cancer cell growth and proliferation. A procedure used in the treatment of cancer is the triggering of apoptosis in cancer cells. The current study aims to investigate the anticancer property of ethanolic extract of Amomum subulatum Roxb. against HeLa cell line. The MTT 3-(4, 5-di-methylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay is the quantitative cytotoxicity assay used, maximal inhibitory concentration (IC50) value was selected as the cytotoxicity parameter. The IC50 value of A. subulatum Roxb. extract is 62.5 μg/ mL and for 5-fluorouracil it is 7.8 μg/mL which indicates anticarcinogenic properties against HeLa cells. The apoptotic morphological changes in HeLa cells were observed using an inverted microscope and changes in HeLa cells were noticed after treatment with 62.5 mg/mL of ethanolic extracts, followed by acridine orange and ethidium bromide staining. The induction of apoptosis by extract of A. subulatum Roxb. was determined using the DNA fragmentation study. The results of the DNA fragmentation study, which exhibits prototypical intrinsic apoptotic characterization, also included fragmentation of nuclear DNA. We also found that the expression of Bcl2 and p53 mRNA were measured using RT-PCR. Overall, the current study results suggest that the

A simple dual-response fluorescent probe for imaging of viscosity and ONOO− through different fluorescence signals in living cells and zebrafish

Cellular viscosity is a prominent micro-environmental parameter and peroxynitrite is an essential reactive oxygen special, both of which are involved in various pathological and physiological processes. When the intracellular viscosity is abnormal or the ONOO− concentration is irregular, the normal function of cells will be disturbed. Herein, we rationally designed and synthesized a novel multichannel fluorescent probe (probe 1) for multichannel imaging of viscosity and peroxynitrite. Probe 1 displayed about 108-fold enhancement as the viscosity increased from 1.005 cP to 1090 cP. Moreover, the fluorescence intensity at 540 nm was quickly increased after adding ONOO−. It should be noted that probe 1 has high sensitivity, selectivity and low cytotoxicity, which can be successfully employed for the visualization of exogenous and endogenous ONOO− and imaging viscosity changes in HeLa cells by different fluorescent signals. Furthermore, probe 1 could monitor the change of ONOO− induced by LPS (lipopolysaccharide) and IFN-γ (interferon-γ) in zebrafish. This result reveals that probe 1 may inspire more diagnostic and therapeutic programs for viscosity-peroxynitrite related diseases shortly.

Preparation of Porous Hydroxyapatite Using Cetyl Trimethyl Ammonium Bromide as Surfactant for the Removal of Lead Ions from Aquatic Solutions.

In the present study, a new low-cost bioceramic nanocomposite based on porous hydroxyapatite (HAp) and cetyl trimethyl ammonium bromide (CTAB) as surfactant was successfully obtained by a simple chemical co-precipitation. The composition and structure of the HAp-CTAB were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), scanning electron microscope (SEM) equipped with an energy dispersive X-ray (EDX) spectrometer, and N2 adsorption/desorption analysis. The capacity of HAp-CTAB nanocomposites to remove the lead ions from aqueous solutions was studied by adsorption batch experiments and proved by Langmuir and Freundlich models. The Pb2+ removal efficiency of HAp-CTAB biocomposite was also confirmed by non-destructive ultrasound studies. The cytotoxicity assays showed that the HAp-CTAB nanocomposites did not induce any significant morphological changes of HeLa cells after 24 h of incubation or other toxic effects. Taken together, our results suggests that the obtained porous HAp-CTAB powder could be used for the decontamination of water polluted with heavy metals, such as Pb2+.

Studies on apoptosis induced by B-norcholesteryl benzimidazole compounds in HeLa cells

Certain B-norcholesteryl benzimidazole compounds were found to mediate marked anti-tumor proliferative effects in vitro in our earlier study. Here, the mechanism of action of these anti-tumor effects was evaluated using HeLa human cervical cancer cells. Methods for detecting cell invasion and migration, Annexin V-PI double staining, cell cycle status, and mitochondrial membrane potential Δψm were employed. These compounds were confirmed to significantly inhibit the proliferation of HeLa cells in vitro. Compound 1 induced apoptosis in S phase, compound 2induced apoptosis in the G0/G1 phase and compound 3 induced late apoptosis in the G2/M phase. These compounds induced HeLa cell apoptosis through depolarization of mitochondrial membrane potential Δψm in a dose-dependent manner. B-norcholesteryl benzimidazole compounds induced morphological changes in HeLa cells and inhibited proliferation, invasion and metastasis. Apoptosis was promoted by mechanisms involving p21 and p53 in this cervical cancer cell line.

SR-FTIR spectro-microscopic interaction study of biochemical changes in HeLa cells induced by Levan-C60, Pullulan-C60, and their cholesterol-derivatives

Objects of the present study are improved fullerene C60 drug carrier properties trough encapsulation by microbial polysaccharides, levan (LEV), pullulan (PUL), and their hydrophobized cholesterol-derivatives (CHL and CHP), that show better interaction with cancer cells. The zeta potential, polydispersity index, and the diameter of particles were determined, and their cytotoxicity against three cancer cell lines were tested. Biochemical changes in HeLa cells are analyzed by synchrotron radiation (SR) FTIR spectro-microscopy combined with the principal component analysis (PCA). The most significant changes occur in HeLa cells treated with LEV-C60 and correspond to the changes in the protein region, i.e. Amide I band, and the changes in the structure of lipid bodies and membrane fluidity are evident. The highest cytotoxicity was also induced by LEV-C60. In HeLa cells, cytotoxicity could not be strictly associated with biochemical changes in lipids, proteins and nucleic acids, but these findings are significant contribution to the study of the mechanism of interaction of C60-based nanoparticles with cellular biomolecules. In conclusion, LEV, PUL, CHL, and CHP enhanced fullerene C60 potential to be used as target drug delivery system with the ability to induce specific intracellular changes in HeLa cancer cells.

Investigation of potential antibiofilm properties of Antimicrobial Peptide (AMP) from Linckia laevigata against Candida albicans: An in vitro and in vivo study

Biofilm-associated Candida albicans infection remains to be challenging, though there are several investigations and developments in the medical therapy. Antimicrobial peptides are promising candidates due to their efficacy to target pathogens and cancer cells. In the present study, LLpepII identified from Linckia laevigata (star fish), has been predicted as ɑ-helical, amphipathic antimicrobial peptide. LLpepII was investigated for their potency to inhibit C. albicans biofilm formation using crystal violet. LLpepII shows antibiofilm activity which inhibits only the biofilm formation and without inhibiting the growth of the yeast cells. This biofilm specific activity of LLpepII was confirmed through SEM and XTT assay. Cell viability assay revealed that LLpepII significantly inhibited the proliferation of human cervical (HeLa) cell lines. Morphological changes in HeLa cells, apoptosis and cell death was studied using AO/EB. It is also confirmed that LLpepII can prevent infection and it is non toxic using zebrafish egg infection model. These results suggest that amphipathic LLpepII as promising candidate with dual property can be explored further as antibiofilm and antiproliferate candidate for therapeutic applications.

Single-/Dual-Responsive pH Fluorescent Probes Based on the Hybridization of Unconventional Fluorescence and Fluorophore for Imaging Lysosomal pH Changes in HeLa Cells.

As a weak acidic organelle, lysosomes maintain acidic pH ranging from 4.5 to 5.5 and abnormal lysosomal pH levels can result in functional deficiency of lysosomes. Tracking the pH changes in lysosomes will help us to understand lysosome-related biological processes and diseases. However, pH-stimuli-responsive polymer-based lysosomal pH fluorescent probe with good water solubility and biocompatibility have rarely been reported. In this work, on the basis of naphthalimide chromophore and poly(N,N-dimethylaminoethyl methacrylate), we designed lysosome-targeted fluorescent probe NapBr-PDM or NapMl-PDM with single- or dual-responsive sites for monitoring pH changes in lysosomes. Both NapBr-PDM and NapMl-PDM exhibited a narrow polydispersity index, excellent fluorescence properties, water solubility, and high photostability. The synthesized probe NapBr-PDM, with a single-responsive site, showed fast response to pH changes and well-stained lysosomes, which could monitor lysosomal pH changes in cells after incubation with chloroquine. NapMl-PDM had good lysosomes-targeting property and highly sensitive response to pH, which could track lysosomal pH changes. Moreover, NapMl-PDM achieved tracking of lysosomal pH changes in cells during lysosomal storage disorder induced by high-concentration sucrose solution for the first time. Therefore, this work provided useful tools for monitoring intracellular pH changes as well as studying the relationship between lysosomal pH and its related diseases.

Two-photon semiconducting polymer nanoparticles as a new platform for imaging of intracellular pH variation

Intracellular pH (pHi) plays a crucial role in cell physiological and pathological processes. We herein report an efficient pH-sensitive sensor based on two-photon excitable semiconducting polymer nanoparticles (PFV/PSMA-DA NPs) for pHi sensing. PFV/PSMA NPs were functionalized with redox-active dopamine (DA) and the obtained PFV/PSMA-DA NPs showed sensitive and reversible pH response over the pH range of 5.0–9.0. Owning to the high biocompatibility and pH-responsive DA, PFV/PSMA-DA NPs show low cytotoxicity and the quantification and imaging of intracellular pH changes of HeLa cells were successfully realized. Moreover, the detection of intracellular pH fluctuation induced by redox species such as NAC (N-acetylcysteine) and H2O2 was also achieved by both one- and two-photon excitation of the PFV/PSMA-DA NPs probe. This work clearly shows that nanoprobe based on two-photon PFV/PSMA-DA NPs could serve as a promising platform for quantitatively monitoring the intracellular pH fluctuations.