A Nd–Yb ratiometric luminescent nanothermometer for assessing thermal resistance discrepancies between A549 and BEAS-2B cells to achieve selective hyperthermia

Abstract

Temperature is a crucial physical parameter in living organisms, directly associated with cellular activities. Elevated temperatures induce cell death, thereby establishing hyperthermia as a viable modality for cancer therapy. The demand for determining appropriate cancer types for hyperthermia lies in identifying cancer cells that exhibit poorer heat tolerance compared to normal cells. Herein, we have designed NaNdF₄:4%Yb@NaYF₄ with bright luminescence in the near-infrared region for the purpose of achieving in situ cellular temperature detection. The Nd–Yb nanothermometer provides temperature feedback based on a ratiometric luminescence intensity signal. By employing a universal cytobiology method to assess the heat resistance differences between cancer cells and normal cells across various organs, it has been observed that lung epithelial cells exhibit superior heat resistance compared to lung cancer cells. Once the Nd–Yb nanothermometer incubates within lung cells, the temperature differences between live and dead cells can be detected. The absolute temperature differences between live and dead lung cancer cells (0.1 °C) and lung epithelial cells (1.4 °C) under identical thermal stimulation (50 °C) are detected by the Nd–Yb co-doped nanothermometer, confirming that the heat resistance of normal lung cells is significantly superior to that of lung cancer cells. The differential heat resistance of lung cells enables selective hyperthermia for killing A549 cells while maximally protecting BEAS-2B cells. This research may establish rare earth nanothermometry as a valuable protocol for assessing cellular heat resistance, thereby guiding selective hyperthermia for precise lung cancer treatment.

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1 Introduction

Temperature is a critical factor influencing the life cycle, which also plays a pivotal role in regulating cellular functions.¹ Temperature variations could affect multiple cellular processes such as gene expression,² protein interaction,³ enzyme-catalyzed reactions,⁴ and cell death.⁵ In turn, the biochemical reactions within cells would also dynamically affect the endogenous cellular temperature.⁶ Cells constitute the fundamental structural units of organisms and serve as essential components in biological processes.⁷ Based on the differential expression of heat shock proteins and apoptosis-associated mRNA in normal and cancer cells,⁸ these two cell types exhibited distinct heat resistance properties, allowing for the possible selective elimination of cancer cells by hyperthermia.⁹ Hyperthermia is a cancer treatment that exposes cells to higher temperatures to induce cell death, which is more effective for cancer cells with lower heat resistance. Hyperthermia offers distinct advantages in terms of eliminating dependence on exogenous agents, reducing side effects and establishing clinical use for broader oncological applications.¹⁰ Recently, research found that when the external temperature rose, cells activated their self-regulatory mechanisms to protect themselves from overheating, resulting in a decrease in actual cellular temperature.¹¹ The self-regulatory function of cells in response to elevated external temperatures may serve as a typical characteristic to evaluate cellular heat resistance. Therefore, measuring actual cellular temperature variations using thermometers could be employed to assess cellular heat resistance properties, thereby identifying the optimal cancer types suitable for hyperthermia.

In the context of biological and medical research, precise temperature detection for organisms is essential for accurately characterizing the corresponding biological activity patterns. The temperature detection methods for organisms include calorimetry,¹² ultrasonic thermometry,¹³ and luminescence nanothermometry.¹⁴ Luminescence nanothermometry acquires the temperature information based on the temperature-responsive luminescence signal of luminescent probes.¹⁵ Luminescent probes for temperature detection mainly include fluorescent dyes,¹⁶ polymer nanoparticles,¹⁷ quantum dots,¹⁸ and rare earth nanoparticles.¹⁹ Among the above luminescent thermometers, rare earth luminescent nanoparticles²⁰ show excellent optical stability,²¹ low cellular toxicity,²² bright luminescence signals,²³ and high thermal sensitivity,²⁴ fulfilling the stringent requirements for accuracy and real-time performance in cellular temperature detection.

Rare earth nanothermometers acquire temperature information mainly by detecting the temperature-sensitive signal which includes luminescence intensity²⁵ and luminescence lifetime.²⁶ The signal acquisition time of luminescence lifetime was relatively long (seconds to minutes), and the detection instrument was complex.²⁷ Luminescence intensity as a temperature detection signal showed the advantage of short signal acquisition time (microseconds to milliseconds).²⁸ As the cellular physiology changed rapidly, the luminescence intensity signal was preferred over the luminescence lifetime signal for cellular temperature detection.²⁹ The possible signal fluctuation induced by the non-uniform concentration distribution of probes can be avoided by utilizing the ratiometric detection method with two emission peaks.³⁰ The ratio of the two luminescence peaks can be correlated with temperature, which remains constant across different probe concentrations.²⁸ A Nd–Yb ratiometric luminescent nanothermometer is a commonly used type of rare earth luminescent ratiometric nanothermometer. The temperature-sensitive process primarily relies on the back energy transfer from Yb³⁺ to Nd³⁺ ions.³¹ As the temperature increases, the energy transfer rate from Yb³⁺ to Nd³⁺ would significantly enhance while the efficiency of energy transfer from Nd³⁺ to Yb³⁺ would almost remain unchanged.^32,33 When the temperature elevates, the luminescence intensity of Yb³⁺ significantly decreases, whereas the luminescence intensity of Nd³⁺ remains constant. As a result, the ratiometric luminescence intensity from Yb³⁺ to Nd³⁺ decreases with increasing temperature, therefore, the temperature variation can be detected due to luminescence ratiometric measurement. Once the temperature results are detected, the corresponding thermal resistance of the cells can be inferred.

Herein, we investigated the differences in heat tolerance between normal and cancer cells originating from the same organ by the traditional cellular biological method (CCK-8).³⁴ Consequently, we developed a core@shell nanothermometer (NaNdF₄:Yb@NaYF₄) to measure cellular temperature based on the ratiometric luminescence intensity signal. The differences in cellular temperature response measured by rare earth nanothermometers could be utilized as a faster testing tool to validate the variations in cellular thermal resistance determined through cellular biological methods. As the considered cancer cells exhibit significantly lower heat resistance compared to the corresponding normal cells, selected killing of cancer cells at a suitable hyperthermia temperature could be achieved.

2 Experimental

2.1 Materials

Nd₂O₃ (>99.99%), Yb₂O₃ (>99.99%), Y₂O₃ (>99.99%), oleic acid (OA, >90%), 1-octadecene (ODE, >90%) and Na(CF₃COO) were purchased from Sigma-Aldrich Ltd. RECl₃ (RE³⁺ = Nd³⁺ and Yb³⁺) and RE(CF₃COO)₃ (RE³⁺ = Y³⁺) solid powders were all synthesized from their corresponding rare earth oxides with excessive hydrochloric acid and trifluoroacetic acid (TFA), respectively. Methanol, absolute ethanol, cyclohexane, dichloromethane (CH₂Cl₂), hydrochloric acid and trifluoroacetic acid (TFA) were purchased from Sinopharm Chemical Reagent Co., China. Nitrosonium tetrafluoroborate (NOBF₄) was bought from Alfa Aesar. Saline (0.9% NaCl) was purchased from Beyotime. Cell counting kit-8 (CCK-8) was purchased from Vazyme. A calcein-AM/PI double stain kit was purchased from Yeasen Biotechnologies Co., Ltd. All experiments used deionized water.

2.2 Cells

Lung cancer cells (A549), colon cancer cells (LOVO), cervical cancer cells (HeLa) and human renal proximal tubular cells (HK-2) were purchased from the National Collection of Authenticated Cell Cultures. Normal lung epithelial cells (BEAS-2B), normal colon mucosal epithelial cells (NCM460), oophoron cancer cells (A2780), normal oophoron epithelial cells (HOSEpiC), esophagus cancer cells (ECA109), normal esophagus epithelial cells (Het-1A), stomach cancer cells (AGS) and normal stomach epithelial cells (GES-1) were purchased from Fenghuishengwu Co., China. Pancreatic cancer cells (MIA PACA-2), prostate cancer cells (DU145), breast cancer cells (MCF-7), normal breast epithelial cells (MCF-10A) and adrenocortical carcinoma cells (NCI-H295R) were purchased from Procell Co., China. Normal pancreatic duct epithelial cells (HPDE6-C7) were purchased from Shanghai Yaji Biological Co., China. Normal cervical epithelial cells (HUCEC) were purchased from Otwo Biotech Co., China. Normal prostatic stromal myofibroblast cells (WPMY-1) were purchased from Taize Biotechnology Co., China.

2.3 Characterization

High-resolution TEM images, HAADF-STEM images, SAED images and EDS mapping of the disparate proportions of β-NaNdF₄:x%Yb@NaYF₄ (Nd–Yb nanothermometer) were obtained using a Tecnai G2 F20 S-Twin (FEI, America) instrument operated at an accelerating voltage of 200 kV. Hydrated particle size was measured using a nanoparticle size-Zeta potential analyzer ZS-90. X-ray powder diffraction (XRD) results were obtained on a Bruker AXS D8 advance diffractometer at a scanning rate of 11.4° min⁻¹ (λ = 1.5418 nm, Cu Kα radiation) with a 2-theta range of 10–60°. The FT-IR spectrum was recorded using a Nicolet 6700 Fourier transform infrared spectrometer. Luminescence emission spectra were recorded using an optical fiber spectrometer with a temperature control system. The luminescence images of lung cells (A549 and BEAS-2B) were acquired using a homemade microscopic imaging system. The cell viability test results were acquired with the absorbance values at 450 nm of different cells incubated with CCK-8 with Biotek Synergy H1. Live and dead fluorescence images of A549 and BEAS-2B cells without hyperthermia as well as A549 mixed with BEAS-2B cells before and after hyperthermia were obtained using a confocal microscope FV1000 (IX81, Olympus) with a 60× lens.

2.4 Synthesis of the hexagonal (β) NaNdF₄:x%Yb@NaYF₄ nanoparticles

The hexagonal (β) phase NaNdF₄:x%Yb nanoparticles were prepared using a modified solvothermal method based on reported work.³⁵ Firstly, 1 mmol RECl₃ (RE = (100-x)%Nd, x%Yb, x = 4, 7, 10, 17, 25, 32 and 40), 10 mmol oleic acid (OA) and 20 mmol octadecene (ODE) were added to a 100 ml three-necked flask. Then, the mixture was heated to 115 °C under vacuum and continuous stirring to become transparent. After the mixture was allowed to cool to room temperature, NaOH (2.5 mmol) and NH₄F (4 mmol) dissolved in methanol were added slowly into the mixture under a nitrogen atmosphere. The above mixed solution was heated to 115 °C after stirring for 10 min and then heated to 300 °C for 1 h with protection of nitrogen. Subsequently, the solution was cooled to room temperature, and the nanoparticles were washed and centrifuged with absolute ethanol and then eventually dispersed in cyclohexane.

The thermal decomposition method was used to synthesize the core@shell NaNdF₄:x%Yb@NaYF₄ structure.³⁶ 0.25 mmol β-NaNdF₄:x%Yb (Core), 0.5 mmol Na(CF₃COO), 0.5 mmol Y(CF₃COO)₃, 10 mmol OA and 10 mmol ODE were added to a 100 mL three-necked flask and heated to 110 °C under a nitrogen atmosphere. Then, the reaction system was vacuumized and refilled with nitrogen. After that, the solution was quickly heated to 300 °C for 45 min. Ultimately, the solution was washed with absolute ethanol and then dispersed in cyclohexane solution to acquire β-NaNdF₄:x%Yb@NaYF₄.

2.5 Surface modification of β-NaNdF₄:x%Yb@NaYF₄ nanoparticles

In order to achieve cellular imaging, it is necessary to obtain hydrophilic nanoparticles. 0.05 mmol NaNdF₄:x%Yb@NaYF₄ nanoparticles were added into a solution of NOBF₄ with CH₂Cl₂.³⁷ After centrifugation at 15 000 rpm for 10 min, ligand-free nanoparticles were acquired. Then, ethanol was used for washing the ligand-free nanoparticles. Eventually, the ligand-free nanoparticles were dispersed in saline so that aqueous β-NaNdF₄:x%Yb@NaYF₄ nanoparticles were acquired.

2.6 Temperature measurement in vitro

In order to obtain the temperature of lung cancer cells and lung epithelial cells, the aqueous NaNdF₄:4%Yb@NaYF₄ nanoparticles were incubated into the above cells. Initially, the frozen A549 and BEAS-2B cells were revived and transferred to cell culture flasks. Once the cells adhered and reached a density of 80–90%, they were digested by trypsin-EDTA (0.25%). Following centrifugation at 800 rpm for 5 minutes, the digested cells were distributed into a six-well plate, with approximately 10 000 cells per well. The six-well plate was placed in an incubator (37 °C, 5% CO₂) for a duration of 24 h. After washing each well twice with saline, 1900 μL of saline was added. Subsequently, the aqueous Nd–Yb co-doped nanothermometer (400 μg mL⁻¹, 100 μL) was added into each well. The six-well plate was placed back in the incubator for an incubation duration of two hours before proceeding to digestion. The digested cells were then transferred into a quartz cuvette containing 2 mL of saline for temperature detection. The temperature detection was based on the variation of the ratio of the peak area of the Yb³⁺ (²F_5/2 → ²F_7/2) emission to that of the Nd³⁺ (⁴F_3/2 → ⁴I_11/2) emission.

2.7 Calculation of relative thermal sensitivity

We use eqn (1) to calculate the relative thermal sensitivity of β-NaNdF₄:x%Yb@NaYF₄:where is the derivative of the logarithm of the peak area ratio of Yb³⁺ (²F_5/2 → ²F_7/2) to Nd³⁺ (⁴F_3/2 → ⁴I_11/2) with respect to temperature (T) and r is the concrete value of ln(I_Yb/I_Nd) in the temperature calibration curve. The calculated sensitivities are used to evaluate the temperature sensing abilities of the probes with different compositions and at different temperatures here. However, it cannot be used directly to compare the performance of the probe with that of the others reported in the literature, which, due to the calculation method, is different.

2.8 Cellular thermal resistance experiment

In order to attain the heat resistance of cancer cells and normal cells of the same organ, we seeded about 7000 cells into 96-well plates and separately placed two plates into a water bath for 30 min. We chose 10 organs to determine the heat resistance difference between cancer cells and normal cells. For the choice of water bath temperature, we chose 44.7 °C as a mild temperature and 48.7 °C as a higher temperature. After water bath heating for 30 min, the 96-well plates were placed back into the incubator (37 °C, 5% CO₂) to continue the cellular culture. As for the post-incubation time, we chose a relatively short time (4 h) and a relatively long time (24 h). Then, the blank culture medium was used to wash the 96-well plates and fill the wells (100 μL of the blank culture medium). The control 96-well plates were not heated by the water bath where the other conditions were the same as the corresponding temperature and incubation of the cancer cells and normal cells. We used the CCK-8 (cell counting kit-8) method to determine the cell viability of the cancer cells and normal cells of the same organ with different temperatures and incubation times. Each test group contained six 96-well plates: control group (cancer cells), mild temperature group (cancer cells with 44.7 °C and 4 h), high temperature group (cancer cells with 48.7 °C and 4 h), control group (normal cells), mild temperature group (normal cells with 44.7 °C and 4 h) and high temperature group (normal cells with 48.7 °C and 4 h). For another test group, the post-incubation time was just changed to 24 h. To ensure the cell viability of the cells under different heat conditions, 10 μL of CCK-8 solution was added into each well of the 96-well plates with 100 μL of culture medium and then put back into the incubator for 3 h. Finally, the 96-well plates were all put into a microplate reader to test the absorbance at 450 nm. The cell viability could be calculated using the formula:³⁸

2.9 Microscopic cellular imaging

The cell culture procedure is similar to section 2.6 while the container chosen for incubating the Nd–Yb co-doped nanothermometer is a confocal dish. When a continuous excitation laser (808 nm) was applied on the lung cells incubated with aqueous NaNdF₄:4%Yb@NaYF₄ nanoparticles, the emission light would transmit to the sCMOS camera through the objective, filter and reflector. The luminescence emission from 970 nm to 990 nm was collected both in A549 and BEAS-2B cells.

2.10 Cellular temperature measurement in vitro

Firstly, the luminescence emission spectrum of the aqueous NaNdF₄:4%Yb@NaYF₄ solution was detected from 20 °C to 50 °C with an interval of 5 °C by the excitation of continuous 808 nm to acquire the temperature standardized working curve (ln(I_Yb/I_Nd) to T, I_Yb represented the integral area of the Yb peak (929–1032 nm) and I_Nd represented the integral area of the Nd peak (1032–1076 nm)). Then, the live and dead cells of A549 and BEAS-2B cells which were incubated with the aqueous NaNdF₄:4%Yb@NaYF₄ were detected to acquire the luminescence spectrum, respectively. By calculating the tested ln(I_Yb/I_Nd) of the live and dead cells of the lung cells and taking into account the standardized working curve, the detected temperature curve of the live and dead cells of A549 and BEAS-2B cells could be acquired.

2.11 Cytotoxicity test of the Nd–Yb nanothermometer

The CCK-8 (cell counting kit-8) method was used to evaluate the toxicity of the aqueous β-NaNdF₄:4%Yb@NaYF₄ nanoparticles of the A549 and BEAS-2B cells. The A549 and BEAS-2B cells were all seeded in 96-well plates with about 7000 cells per well. Cells were incubated for 24 h in a cell incubator (37 °C, 5% CO₂). After washing with blank DMEM, the six wells in each column were added to the blank DMEM containing a series of aqueous β-NaNdF₄:4%Yb@NaYF₄ nanoparticles (0, 0.2, 0.4, 0.6, 0.8, 1, 1.4, 1.7 and 2 mg mL⁻¹). We chose four incubation times (4 h, 12 h, 24 h and 48 h) to ensure the toxicity of β-NaNdF₄:4%Yb@NaYF₄ in both A549 and BEAS-2B cells. As the incubation time was up, the cells were gently washed three times with blank DMEM and added to 100 μL of blank DMEM with 10 μL of CCK-8 solution per well. After again incubating for 3 h, the absorbance at 450 nm of each 96-well plate was measured using a Biotek multifunction microplate reader (SYNERGY H1). The cell viability could be calculated using eqn (2).

2.12 Selective hyperthermia for A549 mixed with BEAS-2B cells

Live A549 cells (30 000) and live BEAS-2B cells (30 000) were mixed in the same confocal dish. The above confocal dish was placed in a water bath at 48.7 °C for 30 min to achieve selective hyperthermia.

2.13 Calcein-AM and PI double staining

The Calcein-AM and PI double staining method was used for verifying the hyperthermia therapeutic effect. The A549 and BEAS-2B cells without hyperthermia as well as A549 mixed with BEAS-2B cells before and after hyperthermia were directly double stained with Calcein-AM (2 μM) and PI (5 μM) for 15 min. The cells were washed with saline three times after staining. The live and dead status of cells was observed using a confocal microscope. The live status of cells was determined by Calcein-AM staining with the excitation of 488 nm and the emission from 500 nm to 530 nm. The dead status of cells was determined by PI staining with the excitation of 543 nm and the emission from 570 nm to 670 nm.

3 Results and discussion

3.1 Synthesis and characterization of β-NaNdF₄:x%Yb@NaYF₄ nanothermometers

The core@shell nanoparticles (β-NaNdF₄:x%Yb@NaYF₄) were developed as a near-infrared ratiometric nanothermometer to obtain real-time cellular temperature information. The core (β-NaNdF₄:x%Yb) was used for temperature measurement, while the shell (NaYF₄) served to increase the emission intensity and protect the functional core from external chemical environmental factors. Given that the energy transfer between Nd³⁺ ions and Yb³⁺ ions was temperature-sensitive,³³ the NaNdF₄:x%Yb@NaYF₄ nanoparticles could function as a nanothermometer for cellular temperature detection (Scheme 1).

Scheme 1 Schematic diagram of the Nd–Yb co-doped nanothermometer for cellular temperature detection in normal and cancer cells.

To obtain a nanothermometer with optimized temperature detection performance, we regulated the doping ratio of Nd³⁺ ions and Yb³⁺ ions in hexagonal NaNdF₄:x%Yb@NaYF₄ nanoparticles (Fig. 1a). Seven distinct proportions of β-NaNdF₄:x%Yb@NaYF₄ were synthesized where the proportions of Yb³⁺ ions in the nanoparticles were 4%, 7%, 10%, 17%, 25%, 32% and 40%, respectively (Fig. 1b–h). Initially, the seven compositions of cores (Fig. S1†) were synthesized separately using a modified solvothermal method. The corresponding average sizes of the cores were 10.2 nm (NaNdF₄:4%Yb), 10.3 nm (NaNdF₄:7%Yb), 7.9 nm (NaNdF₄:10%Yb), 12.7 nm (NaNdF₄:17%Yb), 14.5 nm (NaNdF₄:25%Yb), 16.9 nm (NaNdF₄:32%Yb) and 17.9 nm (NaNdF₄:40%Yb) (Fig. S2†). The selected area electron diffraction (SAED) images indicated that all the cores were hexagonal nanoparticles (Fig. S3†). The inert shell (NaYF₄) was epitaxially grown using the thermal decomposition method, which was used for isolating the external physicochemical environment. As the inert shell was grown, the average particle sizes of the core@shell nanoparticles were measured at 18.7 nm (NaNdF₄:4%Yb@NaYF₄), 18.9 nm (NaNdF₄:7%Yb@NaYF₄), 17 nm (NaNdF₄:10%Yb@NaYF₄), 25 nm (NaNdF₄:17%Yb@NaYF₄), 24.9 nm (NaNdF₄:25%Yb@NaYF₄), 32.5 nm (NaNdF₄:32%Yb@NaYF₄) and 34.4 nm (NaNdF₄:40%Yb@NaYF₄) (Fig. S4†). The scanning transmission electron microscopy high-angle annular dark-field (STEM-HAADF) images clearly revealed the core@shell structure of NaNdF₄:x%Yb@NaYF₄ nanoparticles (Fig. 1i and Fig. S5†). The high-resolution transmission electron microscopy (HR-TEM) images (Fig. 1j and Fig. S6†), selected area electron diffraction (SAED) patterns (Fig. S7†) and X-ray diffraction (XRD) results (Fig. S8†) all corresponded well to the hexagonal phase of β-NaYF₄ (JCPDS No. 16-0334). STEM-HAADF images (Fig. 1k and Fig. S9†) and energy dispersive X-ray spectroscopy (EDS) analysis (Fig. S10 and Table S1†) of the seven β-NaNdF₄:x%Yb@NaYF₄ nanoparticles confirmed the presence of Nd, Yb, Y, Na and F elements within the nanoparticles. The aforementioned experimental data proved that the seven disparate dopants of β-NaNdF₄:x%Yb@NaYF₄ nanoparticles were successfully synthesized.

Fig. 1 The morphology of disparate proportions of β-NaNdF₄:x%Yb@NaYF₄. (a) Core@shell structure scheme of β-NaNdF₄:x%Yb@NaYF₄. TEM images of β-NaNdF₄:4%Yb@NaYF₄ (b), β-NaNdF₄:7%Yb@NaYF₄ (c), β-NaNdF₄:10%Yb@NaYF₄ (d), β-NaNdF₄:17%Yb@NaYF₄ (e), β-NaNdF₄:25%Yb@NaYF₄ (f), β-NaNdF₄:32%Yb@NaYF₄ (g) and β-NaNdF₄:40%Yb@NaYF₄ (h). (i) STEM-HAADF image of β-NaNdF₄:4%Yb@NaYF₄. (j) HR-TEM image of β-NaNdF₄:4%Yb@NaYF₄. (k) EDS image of β-NaNdF₄:4%Yb@NaYF₄. (l) TEM image of aqueous β-NaNdF₄:4%Yb@NaYF₄.

Since the β-NaNdF₄:x%Yb@NaYF₄ nanoparticles were subsequently used for cellular temperature detection, it is essential to convert the original hydrophobic nanoparticles into hydrophilic nanoparticles. By removing the oleic acid (OA) ligand from the hydrophobic NaNdF₄:x%Yb@NaYF₄ nanoparticles using NOBF₄, the aqueous NaNdF₄:x%Yb@NaYF₄ nanoparticles were obtained.³⁹ The morphologies of the aqueous NaNdF₄:x%Yb@NaYF₄ nanoparticles are shown in Fig. 1l and Fig. S11† which revealed no significant differences in morphology and particle size compared to the hydrophobic nanoparticles. The Fourier-transform infrared (FT-IR) spectra (Fig. S12†) demonstrated the successful surface modification of the aqueous NaNdF₄:x%Yb@NaYF₄ nanoparticles. The absorption at 1562 cm⁻¹ was attributed to the C=C bond, while the band at 1720 cm⁻¹ corresponded to the C=O group of OA. In contrast, the hydrophilic nanoparticles exhibited a peak at 1381 cm⁻¹ corresponding to the –OH bond due to the ethanol used for washing the aqueous NaNdF₄:x%Yb@NaYF₄ nanoparticles. These results indicated that the hydrophobic nanoparticles were successfully converted into hydrophilic nanoparticles, which could be further utilized for cellular temperature detection.

3.2 Choosing the Nd–Yb co-doped nanothermometer with optimal temperature detection performance

Upon excitation of the NaNdF₄:x%Yb nanoparticles with a continuous 808 nm laser, the excited Nd³⁺ ions could transfer the energy to Yb³⁺ ions which resulted in the emission at 976 nm (²F_5/2 → ²F_7/2). Subsequently, the excited Yb³⁺ ions could transfer the energy back to Nd³⁺ ions through a phonon-assisted process, thereby generating emissions at 864 nm, 895 nm (⁴F_3/2 → ⁴I_9/2), 1058 nm (⁴F_3/2 → ⁴I_11/2) and 1350 nm (⁴F_3/2 → ⁴I_13/2). As for the NaNdF₄:x%Yb@NaYF₄ nanoparticles with the shell of NaYF₄, the wavelengths of emission peaks were identical to the NaNdF₄:x%Yb nanoparticles while the luminescence intensity showed a difference (Fig. S13†). It was indicated that the coating of an inert shell would not alter the energy transfer pathway while effectively mitigating luminescence quenching induced by external environmental factors. As the hydrophobic nanoparticles (Fig. S14a†) were converted to the hydrophilic nanoparticles (Fig. 2a), the corresponding energy transfer pathway remained unchanged while the luminescence intensity at 980 nm decreased significantly (Fig. S14b–h†). The luminescence quenching of the hydrophilic nanoparticles was attributed to the stretching vibrations of the hydroxyl (–OH) oscillators, which were well-matched with the luminescence emission peak of NaNdF₄:x%Yb@NaYF₄ nanoparticles at around 980 nm.⁴⁰ For the various compositions of NaNdF₄:x%Yb@NaYF₄ nanoparticles, an increase in the doping ratio of Yb³⁺ ions resulted in a higher luminescence intensity ratio of 976 nm to 1058 nm, which indicated that a greater number of Yb³⁺ ions as the luminescence centers could accept more energy from Nd³⁺ions (Fig. S14a† and Fig. 2b).

Fig. 2 Luminescence spectra and temperature-sensitive properties of aqueous NaNdF₄:x%Yb@NaYF₄ nanothermometers with disparate proportions. (a) The luminescence emission spectra of aqueous NaNdF₄:x%Yb@NaYF₄ nanothermometers under 808 nm excitation. (b) The luminescence intensities at 976 nm (Yb) and 1058 nm (Nd) for aqueous NaNdF₄:x%Yb@NaYF₄ nanothermometers. (c) The plot of ln(I_Yb/I_Nd) versus T indicates the temperature response characteristics of aqueous NaNdF₄:x%Yb@NaYF₄ nanothermometers. (d) The relative thermal sensitivity of aqueous NaNdF₄:x%Yb@NaYF₄ nanothermometers. (e) The logarithm of the ratio between the areas of the Yb peak (929–1035 nm) and the Nd peak (1035–1076 nm) for aqueous NaNdF₄:x%Yb@NaYF₄ nanothermometers at 45 °C. (f) The relative thermal sensitivity of aqueous NaNdF₄:x%Yb@NaYF₄ nanothermometers at 45 °C.

The as-synthesized NaNdF₄:x%Yb@NaYF₄ nanoparticles with different compositions all showed temperature-responsive properties based on the temperature-sensitive energy transfer path between Nd³⁺ and Yb³⁺ ions. As the temperature increased, the luminescence intensity from Yb³⁺ ions (²F_5/2 → ²F_7/2) decreased, while the luminescence intensity from Nd³⁺ ions (⁴F_3/2 → ⁴I_11/2) remained nearly constant (Fig. S15†). The aforementioned phenomenon was observed in the temperature-dependent spectra of NaNdF₄:x%Yb@NaYF₄. In other words, in the NaNdF₄:x%Yb@NaYF₄ nanothermometers, the luminescence intensity of Yb³⁺ ions (²F_5/2 → ²F_7/2) played a pivotal role in temperature response, while the luminescence intensity of Nd³⁺ ions (⁴F_3/2 → ⁴I_11/2) served as a reference for temperature detection. For both hydrophobic (Fig. S16a†) and hydrophilic nanoparticles (Fig. 2c), the area ratio of luminescence emission peaks from Yb³⁺ ions to Nd³⁺ ions decreased with increasing temperature. The various particle sizes among the different compositions of NaNdF₄:x%Yb@NaYF₄ nanothermometers significantly affected the luminescence intensity of both hydrophobic (Fig. S14a†) and hydrophilic nanothermometers (Fig. 2b), thereby, the luminescence intensity was inadequate for evaluating the temperature detection efficiency. The luminescence ratiometric measurement remained consistent when the concentrations of Yb³⁺ and Nd³⁺ were maintained at fixed levels, making the relative thermal sensitivity less influenced by particle size.

The relative thermal sensitivity was preferably used to evaluate the performance of the as-prepared nanoparticles (Fig. S16b† and Fig. 2d). By calculating the temperature detection sensitivities of various NaNdF₄:x%Yb@NaYF₄ nanothermometers using eqn (1), NaNdF₄:4%Yb@NaYF₄ exhibited the highest relative thermal sensitivity which is 2.3% K⁻¹ at 20 °C, 2.6% K⁻¹ at 25 °C, 3.1% K⁻¹ at 30 °C, 3.6% K⁻¹ at 35 °C, 4.4% K⁻¹ at 40 °C, 5.5% K⁻¹ at 45 °C and 7.3% K⁻¹ at 50 °C (Fig. 2d). As the relationship between ln(I_Yb/I_Nd) and T was fitted by linear fitting, the derivative of ln(I_Yb/I_Nd) with respect to T in eqn (1) would be a constant. The luminescence ratio between Yb³⁺ and Nd³⁺ would be inversely proportional to the relative thermal sensitivity. Therefore, NaNdF₄:4%Yb@NaYF₄ nanoparticles with the lowest luminescence ratio of Yb³⁺ to Nd³⁺ exhibited the highest relative thermal sensitivity. Although NaNdF₄:4%Yb@NaYF₄ showed the weakest luminescence intensity among the whole NaNdF₄:x%Yb@NaYF₄ nanothermometers, the detected luminescence signal was sufficiently high for cellular temperature detection. Given that the typical hyperthermia therapy temperature was approximately 45 °C,⁴¹ NaNdF₄:4%Yb@NaYF₄ exhibited the lowest luminescence ratio of Yb³⁺ ions to Nd³⁺ ions (Fig. 2e) and the highest relative thermal sensitivity (Fig. 2f). Based on the above evaluation, aqueous NaNdF₄:4%Yb@NaYF₄ (named Nd–Yb co-doped nanothermometer) was selected for subsequent cellular temperature detection. Dynamic light scattering (DLS) results revealed that the hydrated particle size of the selected nanothermometer was 32.3 nm at 15 °C and 32.4 nm at 40 °C, demonstrating the thermal stability of the nanothermometer for subsequent cellular temperature detection (Fig. S17†).

3.3 Investigation of the thermal resistance discrepancies between cancer cells and normal cells derived from disparate organs

Given that different cancer cells exhibited varying thermotoxicities to temperature, the thermotolerance of both normal and cancer cells should be specifically investigated within the same organ. Detecting the thermal resistance discrepancies between cancer cells and normal cells within the same organ could enhance the identification of specific cancers that are more susceptible to hyperthermia, ideally killing cancer cells with lower heat resistance while preserving normal cells with higher heat resistance. Cells from ten common organs were selected to investigate the differences in heat resistance, including lung, pancreatic, colon, cervical, oophoron, prostate, breast, kidney, esophagus and stomach cells. These cells were initially heated at different water bath temperatures (mild temperature: 44.7 °C, higher temperature: 48.7 °C). After heating in the water bath, the cells were returned to the incubator for post-incubation at different durations (short duration: 4 h, long duration: 24 h) (Fig. 3a). The CCK-8 method showed superior detection accuracy in cell viability assessment which was preferred for directly detecting cell viability under thermal stimulation conditions. The cell viability of the different cell types was subsequently assessed to evaluate the cellular heat resistance, as the cell viability was positively correlated with cellular heat resistance. By determining the cell survival rates under varying water bath temperatures and post-incubation durations, the differences in heat resistance between cancer cells and normal cells could be comprehensively confirmed.

Fig. 3 The experiment protocol and results regarding the differences in heat resistance between cancer cells and normal cells from various organs. (a) The scheme of acquiring the heat resistance differences between cancer cells and normal cells with different heating temperatures (44.7 °C and 48.7 °C) and different post-incubation durations (4 h and 24 h). (b) Differences in cell viability between cancer cells and normal cells from ten organs with different heating temperatures and post-incubation durations. (c) The cell viability of lung cells (cancer cell: A549, normal cell: BEAS-2B) after a post-incubation duration of 4 h. (d) The cell viability of lung cells (cancer cells: A549, normal cells: BEAS-2B) after a post-incubation duration of 24 h.

Among the above cell types (Fig. 3b), only the normal lung cells (BEAS-2B) exhibited significantly better heat resistance compared to lung cancer cells (A549), regardless of the water bath temperatures or post-incubation durations (Fig. 3c and d). Regarding the cells from the other nine organs, the differences in heat resistance between cancer cells and normal cells were not uniform. The normal pancreatic duct epithelial cells (HPDE6-C7) showed superior cellular heat resistance compared to pancreatic cancer cells (MIA PACA-2) at higher temperatures (Fig. 4a and b), whereas the prostate cells (Fig. 4c and d), esophagus cells (Fig. 4e and f) and stomach cells (Fig. 4g and h) showed the same differences in heat resistance as pancreatic cells. The normal colon mucosal epithelial cells (NCM460) demonstrated better cellular heat resistance compared to colon cancer cells (LOVO) at lower temperatures (Fig. 4i and j) whereas the cervical cells (Fig. 4k and l) and oophoron cells (Fig. 4m and n) showed the same differences in heat resistance as colon cells. As for the kidney cells, only under conditions of higher temperature and short-term culture did the heat resistance of immortalized proximal tubule epithelial cells (HK-2) surpass that of adrenocortical carcinoma cells (NCI-H295R) (Fig. 4o and p). Furthermore, normal breast epithelial cells (MCF-10A) showed better heat resistance than breast cancer cells (MCF-7) under the conditions of higher temperature and long-term culture (Fig. 4q and r).

Fig. 4 The cell viability of pancreatic cells (cancer cells: MIA PACA-2, normal cells: HPDE6-C7) with post-incubation durations of 4 h (a) and 24 h (b). The cell viability of prostate cells (cancer cells: DU145, normal cells: WPMY-1) with post-incubation durations of 4 h (c) and 24 h (d). The cell viability of esophagus cells (cancer cells: ECA109, normal cells: Het-1A) with post-incubation durations of 4 h (e) and 24 h (f). The cell viability of stomach cells (cancer cells: AGS, normal cells: GES-1) with post-incubation durations of 4 h (g) and 24 h (h). The cell viability of colon cells (cancer cells: LOVO, normal cells: NCM460) with post-incubation durations of 4 h (i) and 24 h (j). The cell viability of cervical cells (cancer cells: HeLa, normal cells: HUCEC) with post-incubation durations of 4 h (k) and 24 h (l). The cell viability of oophoron cells (cancer cells: A2780, normal cells: HOSEpiC) with post-incubation durations of 4 h (m) and 24 h (n). The cell viability of kidney cells (cancer cells: NCI-H295R, normal cells: HK-2) with post-incubation durations of 4 h (o) and 24 h (p). The cell viability of breast cells (cancer cells: MCF-7, normal cells: MCF-10A) with post-incubation durations of 4 h (q) and 24 h (r).

Concerning the cells that revealed significant differences in heat resistance, normal lung epithelial cells (BEAS-2B) demonstrated superior heat resistance to the lung cancer cells (A549) whether at moderate or higher temperatures and short-term or long-term culture.

3.4 Cellular imaging of lung cancer cells and lung epithelial cells incubated with the Nd–Yb co-doped nanothermometer

By introducing the rare earth nanothermometer, the intrinsic temperature and heat resistance of various cell types could also be detected. The nanothermometer was incubated within lung cells to enable in situ cellular temperature detection. A549 cells (Fig. S18†) and BEAS-2B cells (Fig. S19†) with the incubation of the Nd–Yb co-doped nanothermometer exhibited high cell viability, which indicated that the nanothermometer possessed significant biosafety. A custom-built microscopic imaging system (Fig. 5a) was used for acquiring the luminescence images of the lung cancer cells (A549) and normal lung epithelial cells (BEAS-2B) to confirm the successful incubation of the Nd–Yb co-doped nanothermometer. The excitation light (808 nm) was directly applied above the lung cells incubated with the Nd–Yb co-doped nanothermometer. Upon excitation of the nanothermometer, both the excitation light and the emission light were collected simultaneously through the objective. A designated filter was incorporated into the imaging system to filter out the excitation light and capture the corresponding emission signals. After passing through two reflectors, the emission light signal was collected by a sCMOS camera to obtain the cellular luminescence image.

Fig. 5 Microscopic imaging system and luminescence images of lung cancer cells and lung epithelial cells incubated with the Nd–Yb co-doped nanothermometer. (a) Schematic representation of the optical path in the microscopic imaging system. (b) Luminescence image of A549 cells incubated with the Nd–Yb co-doped nanothermometer, exhibiting emission wavelengths ranging from 970 nm to 990 nm. (c) Luminescence image of BEAS-2B cells incubated with the Nd–Yb co-doped nanothermometer, exhibiting emission wavelengths ranging from 970 nm to 990 nm.

After the Nd–Yb co-doped nanothermometer was incubated in A549 and BEAS-2B cells, cellular imaging could be performed using the above imaging system. Upon excitation with a continuous 808 nm laser, luminescence images were acquired from 970 nm to 990 nm in both A549 and BEAS-2B cells. The luminescence images of A549 (Fig. 5b) and BEAS-2B (Fig. 5c) revealed NIR emission signals from the Nd–Yb co-doped nanothermometer within the cells, indicating successful incubation of the nanothermometer into lung cells.

3.5 Different thermal resistances between A549 and BEAS-2B cells acquired by the Nd–Yb nanothermometer

The spectral detection system (Fig. 6a) was used to measure the temperature of lung cells based on the luminescence peak area ratio between Yb³⁺ and Nd³⁺. Firstly, by measuring the temperature response of the aqueous Nd–Yb co-doped nanothermometer dispersed in saline, a calibration curve for temperature detection was established (Fig. 6b). Since the temperature-sensitive property of the rare earth nanothermometer with inert shell protection was nearly unaffected by the surrounding environment,¹⁴ the calibration curve demonstrated stability in temperature detection. Subsequently, the nanothermometer was incubated into A549 and BEAS-2B cells to obtain the real-time temperature response of the cells (Fig. 6c and d). The temperature of the live cells was inferred from the obtained ratio and the calibration curve (red symbols in Fig. 6e and f). The live cells were then heated to 70 °C for 1 h to obtain the corresponding dead cells (Fig. S20†). Similarly, the measured temperature of the dead cells (black symbols in Fig. 6e and f) was derived from the luminescence peak area ratio and the calibration curve. The measured temperatures of lung cells in various survival states all demonstrated differences compared to the specified external temperature (Fig. S21 and Table S2†).

Fig. 6 The thermal response differences between lung cancer cells (A549) and lung epithelial cells (BEAS-2B) upon incubation with the Nd–Yb co-doped nanothermometer. (a) The thermal sensing detection scheme by utilizing a spectral detection system. (b) A calibration curve for temperature detection using the Nd–Yb co-doped nanothermometer. (c) The temperature detection curve of live and dead A549 cells. (d) The temperature detection curve of live and dead BEAS-2B cells. (e) The temperature measurements of live and dead A549 cells obtained using the Nd–Yb co-doped nanothermometer. (f) The temperature measurements of live and dead BEAS-2B cells using the Nd–Yb co-doped nanothermometer.

The nanothermometer inside the cells can determine the temperature discrepancies between live and dead cells, thereby facilitating the assessment of heat resistance differences among various cell types. Under identical external temperature stress conditions, the cells would regulate the expression of HSP proteins to enhance cellular thermal resistance and prevent intracellular temperature elevation.⁴² Cell viability remained uncompromised under elevated external temperatures owing to enhanced cellular thermal resistance, which was attributed to a simultaneous reduction in intracellular temperature. The greater the difference between intracellular and external temperatures, the higher the thermal resistance and viability of the cells. In comparison with the absolute temperature difference observed in live and dead BEAS-2B cells (1.4 °C at 50 °C), the A549 cells (0.1 °C at 50 °C) exhibited a negligible thermal difference between live and dead states. When exposed to a higher temperature environment, normal living lung cells exhibited significantly greater temperature downregulation compared to lung cancer cells, which demonstrated that the normal living lung cells (BEAS-2B) showed superior heat resistance to external hyperthermia (Fig. S22 and Table S3†). The Nd–Yb co-doped nanothermometer could obtain cellular temperature in situ, allowing for the assessment of the difference between cellular intrinsic temperature and external temperature. The greater the differences between the cellular temperature measured by the Nd–Yb co-doped nanothermometer and the elevated external temperature, the higher the thermoregulatory capacity of the cells. The above results confirmed that rare earth nanothermometry can provide an alternative detection approach for assessing cellular thermal resistance.

3.6 Selective hyperthermia for lung cancer cells

The Nd–Yb ratiometric nanothermometer has validated that BEAS-2B cells exhibit superior thermoregulatory capacity to A549 cells. The cytobiology method synchronously verified that BEAS-2B cells showed higher survival discrepancies than A549 cells under the conditions of a water bath at 48.7 °C for 30 min. Therefore, the optimal hyperthermia conditions for selective cancer therapy were heating A549 mixed with BEAS-2B cells at 48.7 °C for 30 min. Before hyperthermia treatment, A549 cells (Fig. 7a–d), BEAS-2B cells (Fig. 7e–h), and A549 mixed BEAS-2B cells (Fig. 7i–l) only displayed the signal of Calcein-AM in the Calcein-AM/PI double staining results which confirmed the live status of cells. In the bright field images, A549 cells (Fig. 7a) exhibited more separated distribution with prominent leading edges⁴³ while BEAS-2B cells (Fig. 7e) exhibited more aggregated distribution and less defined leading edges. These morphological differences can be utilized to effectively differentiate A549 from BEAS-2B cells in the mixture.

Fig. 7 The live and dead status of A549 and BEAS-2B cells before and after hyperthermia. Confocal fluorescence images of A549 cells without hyperthermia: bright field image (a), Calcein AM (b) and PI (c) double-staining images, and overlay image (d). Confocal fluorescence images of BEAS-2B cells without hyperthermia: bright field image (e), Calcein AM (f) and PI (g) double-staining images, and overlay image (h). Confocal fluorescence images of A549 cells mixed with BEAS-2B cells before hyperthermia: bright field image (i), Calcein AM (j) and PI (k) double-staining images, and overlay image (l). Confocal fluorescence images of A549 cells mixed with BEAS-2B cells after hyperthermia (48.7 °C for 30 min): bright field image (m), Calcein AM (n) and PI (o) double-staining images, and overlay image (p) with the survival status label of A549 and BEAS-2B cells (green for live, red for dead).

The mixture of A549 and BEAS-2B cells (Fig. 7m) was then simultaneously heated under the optimal hyperthermia conditions. The Calcein-AM/PI double staining results after hyperthermia showed fluorescence emissions of both Calcein-AM (Fig. 7n) and PI (Fig. 7o). The aggregated cells represented BEAS-2B cells and possessed more Calcein-AM emission signal. The separate cells corresponded to A549 cells and possessed more Calcein-AM emission signal than the PI signal. Therefore, the status of BEAS-2B cells (Fig. S23a†) was nearly all live (∼93%) while the status of A549 cells (Fig. S23a and b†) was more dead (∼59%) than live (∼41%). This result indicated that the specific lung cancer cells could be selectively killed while the corresponding normal lung cells showed a better survival rate.

4 Conclusions

In summary, various proportions of nanothermometers were synthesized to identify the nanothermometer with optimal relative thermal sensitivity where NaNdF₄:4%Yb@NaYF₄ showed the best thermal sensing performance. The heat resistance discrepancies between cancer cells and normal cells of ten distinct organs were initially detected using a universal cytobiology method, revealing that the heat resistance of normal lung cells was consistently superior to that of lung cancer cells. Following the verification of lung cells exhibiting a greater difference in heat tolerance between normal and cancer cells, the Nd–Yb co-doped nanothermometer was utilized for in situ cellular temperature detection. The temperature differences between live and dead cells of lung cancer cells and normal lung cells under different temperature conditions were successfully detected. Rare earth nanothermometry provided a real-time methodology for the direct detection of intrinsic temperature and evaluation of heat resistance performance in diverse cell types. The temperature response differences between live and dead cells confirmed that the considered normal lung cells exhibited superior temperature regulation performance and better cellular heat resistance. Under optimal hyperthermia, the A549 cells were precisely killed while keeping the live status of BEAS-2B cells almost unaffected. These results demonstrate that rare earth nanothermometry offers a methodology to investigate the heat resistance differences between cancer cells and normal cells, enabling the effective identification of the most suitable lung cancer for precision hyperthermia.

Author contributions

Yishuo Sun: data curation, formal analysis, investigation, visualization and writing – original draft. Qingbing Wang: conceptualization, methodology and investigation. Na Wu: methodology and investigation. Mengya Kong: investigation. Yuyang Gu: investigation. Wei Feng: conceptualization, supervision, funding acquisition and writing – review & editing.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

Financial support from the National Key R&D Program of China (2023YFC3605704), the National Natural Science Foundation of China (22171054), the Shanghai Sci. Tech. Comm. (22XD1420300), and the State Key Laboratory for Modification of Chemical Fibers and Polymer Materials (KF2206) is gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4bm01729c

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