Ti₃C₂T _x MXene Combined with Laser Photothermal for High-Performance Oocyte Cryopreservation

Abstract

Vitrification cryopreservation of oocytes represents a pivotal technique for the cryopreservation of female fertility. Nevertheless, conventional vitrification still faces the challenge of devitrification ice crystal damage stemming from an insufficient rewarming rate, which compromises the efficacy of oocyte cryopreservation. In this study, the two-dimensional nanomaterial Ti₃C₂T _x MXene was introduced into the oocyte cryoprotectants, and combined with 808 nm near-infrared (NIR) laser irradiation to achieve rapid photothermal rewarming. This nanophotothermal rewarming technology enables rapid temperature increase of cryopreserved oocytes, thereby inhibiting devitrification and mitigating ice crystal damage. The results revealed that the survival rate of oocytes subjected to nanophotothermal rewarming was 81.0 ± 3.2%, outperforming the traditional water bath rewarming group, and the cellular functionality post-thaw was also better preserved. Furthermore, no significant cytotoxic effects on oocytes were observed with Ti₃C₂T _x MXene. Overall, this study introduces a novel approach for optimizing the vitrification cryopreservation of oocytes, and offers fresh insights into the cryopreservation of fertility and the safeguarding of genetic resources in endangered species.

1. Introduction

Oocyte cryopreservation, a biomedical technique involving the storage of female gametes at ultra-low temperature for subsequent revival and application in assisted reproduction, constitutes a cornerstone strategy in the preservation of female fertility across both human clinical and veterinary contexts. − Despite its established utility, oocyte cryopreservation presents formidable technical challenges that distinguish it markedly from embryo cryopreservation. The unique biophysical properties of the oocyte, specifically its substantial cellular volume, elevated water content, and pronounced susceptibility to environmental stressors render it exceptionally vulnerable to cryoinjury throughout the freeze-thaw cycle. −

The commonly used cryopreservation protocols mainly include two principal methods, slow freezing and vitrification. The latter achieves solidification of the intracellular milieu into an amorphous glassy state through ultra-rapid cooling, effectively circumventing the lethal consequences of ice crystal nucleation and propagation. − Since the seminal work of Kuleshova et al., who documented the first live human birth derived from vitrified oocytes, vitrification has been progressively validated as the clinical gold standard for oocyte banking. − Nonetheless, the procedure remains constrained by intrinsic biophysical limitations. Although the deployment of highly concentrated cryoprotective agents (CPAs) facilitates partial intracellular water displacement and ice suppression, the inherently low surface-area-to-volume ratio of the oocyte impedes the kinetics of both water egress and CPAs permeation. Protracted handling during equilibration may engender substantial osmotic stress, while the phenomenon of devitrification, defined as the phase transition from a metastable glassy or supercooled liquid state to a crystalline lattice during rewarming beyond the glass transition temperature (T _g), poses a persistent threat to cellular ultrastructure. − As vitrified specimens traverse the thermal window exceeding their specific T _g (typically reported within the range of −120 °C to −130 °C for standard CPAs), they enter a thermodynamically precarious zone conducive to ice recrystallization. − Empirical evidence, such as the data presented by Sansinena delineating glass transition intervals between −126 °C and −121 °C, underscores the dependency of this critical window on solution formulation. This inherent thermal instability posits that the kinetics of the rewarming phase represent a principal rate-limiting determinant for efficacious cryopreservation outcomes, necessitating accelerated rewarming trajectories to mitigate the deleterious impact of devitrification on oocyte viability.

To address these physicochemical constraints, considerable research effort has been directed toward the engineering of novel vitrification carriers designed to augment rewarming rates and minimize ice-associated damage. These platforms are broadly categorized into closed systems (e.g., straws, glass capillaries) and open systems (e.g., electron microscopy grids). − While the strategic reduction of carrier volume has demonstrably enhanced thermal conductivity and rewarming velocity, the overall efficiency of oocyte survival post-thaw remains suboptimal. A major cause is the difficulty of preventing ice recrystallization and devitrification during rewarming. In recent years, the integration of nanomaterials and spatial heating of physical fields has emerged as a promising frontier for modulating ice behavior during vitrification. Specifically, nanomaterials with photothermal or magnetocaloric properties combined with near-infrared (NIR) lasers or alternating magnetic fields offer a compelling strategy to circumvent devitrification during the rewarming phase. , By leveraging nanoparticles with robust photothermal conversion efficiencies in conjunction with laser irradiation platforms, it is feasible to achieve not only ultra-rapid and uniform volumetric heating but also ancillary antimicrobial effects. ,, This paradigm shift was presaged by the foundational observations of Peter Mazur, who demonstrated that laser mediated rewarming substantially enhances post vitrification survival rates, thereby catalyzing extensive exploration of diverse photothermal nanoagents. Bischof et al. employed gold nanorods in combination with laser irradiation to enable successful rewarming of vitrified zebrafish embryos, underscoring the capacity of light-responsive nanoparticles to deliver the heating uniformity and speed required for millimeter-scale multicellular systems. Overall, nanoparticle driven rewarming technologies represent a pivotal advance in overcoming devitrification and improving post-thaw viability.

Although progress has been made in laser-assisted oocyte thawing, most previous studies have mainly focused on the thawing process itself, with insufficient attention devoted to the reproducibility and engineering robustness of the experimental apparatus. Furthermore, material innovation has been largely confined to conventional photothermal agents such as graphene oxide. In this context, the present study introduces Ti₃C₂T _x MXene, a two-dimensional transition metal carbide/nitride discovered in 2011. This material exhibits intrinsic metallic conductivity and pronounced laser-induced photothermal conversion capabilities. − For oocyte recovery, elevated rewarming rates are imperative to expedite transit through the devitrification prone thermal plateau, thereby ameliorating structural insult. Unlike traditional methods, this study synthesized Ti₃C₂T _x MXene nanomaterials (Figure A), combined them with 808 nm NIR laser, and worked in synergy with the water bath heating mode to facilitate the rewarming process. While this dual-source thermal strategy enables accelerated transition through the critical temperature zone, the inherent buoyancy of straws at the air-liquid interface compromises laser alignment and heating uniformity. To surmount this technical barrier, we have engineered a bespoke integrated laser rewarming platform comprising a laser source, a thermostatically controlled water bath, and a novel S-shaped straw immobilization apparatus (Figure B). This custom-designed retainer secures the straw within a recessed groove, thereby optimizing laser focus and maximizing photothermal transfer efficiency. Empirical validation confirms that this integrated system effectively resolves the challenges associated with straw stabilization during dual-mode rewarming, yielding superior rewarming performance. Subsequent biological characterization of post-thaw oocytes, including assessments of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, intracellular calcium (Ca²⁺) homeostasis, and viability (Figure C), revealed that the laser rewarmed cohort consistently outperformed the conventional control group across all quantitative metrics. These findings substantiate the reproducibility and feasibility of the developed platform, thereby providing a novel and highly efficient solution for the advancement of oocyte cryopreservation.

1.

Schematic of Ti₃C₂T_x MXene synthesis and photothermal rewarming platform. (A) Schematic of Ti₃C₂T _x MXene synthesis. (B) Schematic of the photothermal rewarming platform. (C) Schematic illustrating the advantages and efficacy of MXene-loaded laser-assisted rewarming for oocyte cryopreservation. (Some graphic elements were created with BioRender.com).

2. Materials and Methods

2.1. Preparation of Ti₃C₂T _x MXene

The preparation of Ti₃C₂T _x MXene follows the previous research. The specific process is as follows. 2.0 g of LiF was dissolved in 40 mL of 9 mol/L HCl. After 10 min of magnetic stirring, the mixture was transferred to a water bath maintained at 35 °C. 2.0 g of Ti₃AlC₂ powder was gradually added under continuous mechanical agitation for 24 h to ensure complete etching of the aluminum layer in the MAX phase. Upon completion of the reaction, centrifuge at 4000 rpm for 5 min, remove the supernatant, collect the precipitate, and wash repeatedly with deionized water until the filtrate pH stabilizes at around 6. Then, introduce 50 mL of deionized water to the neutral precipitate, sonicate under ice bath conditions (300 W, 40 kHz, 20 min), and thereafter centrifuge the dispersion at 4000 rpm for 2 h to get the Ti₃C₂T _x MXene dispersion.

2.2. Characterization of Ti₃C₂T _x MXene

The morphology of Ti₃C₂T _x MXene was characterized by transmission electron microscopy (TEM; Thermo Fisher Scientific, USA). When preparing samples, use deionized water as the dispersant. Place the MXene material in the dispersant, then use an ultrasonic cleaner to disperse the sample. Place a drop of the uniformly dispersed sample solution onto a copper mesh laid on filter paper and wait for 15 min. Atomic force microscopy (AFM; Bruker, Germany) was employed to measure the thickness and surface topography. Firstly, place 1–5 mg of MXene sample into 10 mL of deionized water and sonicate for 30 min. Then, place a drop of the dispersion onto a mica slide and let it stand for 10 min. Scanning electron microscopy (SEM; Zeiss, Germany) was used to examine the surface morphology. Since MXene samples are in powder form, they can be directly dispersed onto a conductive adhesive and secured to the SEM sample stage. The elemental composition of Ti₃C₂T _x MXene was analyzed by energy dispersive spectroscopy (EDS; Oxford Instruments, UK) using an X-MaxN 80 mm² silicon drift detector coupled to the SEM, with the detector engaged at a working distance of 10 mm, an accelerating voltage of 15 kV, and a live acquisition time of 60 s. The surface chemistry of Ti₃C₂T _x MXene was analyzed by X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific, USA). Using a spatula, transfer the MXene sample onto 3M double-sided tape affixed to a clean substrate; press gently and blow away any loose particles. Then trim the tape to fit the sample holder and mount it securely. Thermogravimetric analysis (TGA; TA Instruments, USA) was performed to evaluate the thermal stability. Before the experiment, set the temperature range to 20–800 °C, the heating rate to 10 °C/min, and purge with nitrogen. Ensure the sample is dry before placing it onto the sample pan. After confirming the settings, start the test to record the mass variation with temperature.

2.3. Photothermal Performance Test

The photothermal conversion efficiency of Ti₃C₂T _x MXene was evaluated under two independent variables: MXene concentration and laser power density. All irradiations employed an 808 nm NIR laser with an infrared camera for real-time temperature recording. Each experimental condition was replicated three times. Samples containing trehalose (0.855 g), propylene glycol (0.750 mL), ethylene glycol (0.750 mL), and M2 medium (to 5 mL final volume) were prepared with Ti₃C₂T _x MXene at 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL. Mixtures were ultrasonicated (30–40 min) for dispersion. Then, 20 μL of the sample was added to a straw and placed in liquid nitrogen (LN₂) for vitrification treatment. Subsequently, the samples were quickly taken out of the LN₂ and placed in a S-shaped holder to receive irradiation at a rate of 2 W/cm². The temperature distribution was recorded through thermal imaging. In addition, using a fixed MXene concentration of 0.6 mg/mL, samples were prepared identically. Irradiations were conducted at 1, 2, and 3 W/cm². Fresh 20 μL of the sample was used per trial, and temperature dynamics were recorded as described above.

2.4. Evaluation of Ice Inhibition and Thermal Properties of MXene Solutions

The Ti₃C₂T _x MXene supplemented PBS and CPAs solutions containing varying concentrations of Ti₃C₂T _x MXene were systematically characterized. This comprehensive assessment covers ice recrystallization inhibition (IRI) performance and thermal characteristics. The detailed experimental methodologies for these characterizations are provided in the Supporting Method S1.

2.5. Oocyte Retrieval

The mouse oocytes utilized in this research were sourced from 6- to 8-week-old female KM mice provided by Shiwens Experimental Supplies Sales Department (Hefei, China) and housed in the animal room. Administer 10 IU of pregnant mare serum gonadotropin (PMSG) (Nanjing Aibei Biotechnology Co., Ltd.) to induce superovulation in female mice. Two days later, a second intraperitoneal injection of 10 IU human chorionic gonadotropin (HCG) (Nanjing Aibei Biotechnology Co., Ltd.) was given. Oocytes were harvested 12–14 h following the second dose using cervical dislocation euthanasia. The ovaries were excised and positioned on a culture dish with PBS buffer. The ampullary area of the oviduct was incised with a 1 mL syringe needle under a stereomicroscope to extract the cumulus-oocyte complexes (COCs). The COCs were subsequently subjected to a 5 min treatment with hyaluronidase, followed by three washes in M2 medium to eliminate granulosa cells and leftover hyaluronidase. The cleaned oocytes were then transferred to M2 medium for further tests. All experimental procedures were conducted in accordance with the guidelines set by the Animal Ethics Committee of Anhui Medical University (Protocol No. LLSC20252757).

2.6. Oocyte Vitrification and Thawing

Oocyte vitrification and thawing follows the previous research. The oocytes, previously washed, were transferred via transfer pipette into a pre-prepared equilibration solution (ES) consisting of 7.5% (v/v) EG, 7.5% (v/v) PG, and M2, equilibrated at 4 °C for 5 min, and then swiftly transferred within 1 min into the vitrification solution (VS) comprising 15% (v/v) EG, 15% (v/v) PG, 0.5 M trehalose, and M2 within 1 min. The oocytes were then placed into the straws using a transfer pipette and were cryopreserved using the five-step method (CPAs-air-oocytes suspension-air-CPAs). Following exposure in LN₂ vapor for 3–5 s, the straws were immersed in LN₂ for vitrification.

Extracting cryopreserved straws from LN₂. For the experimental group with MXene doping, a S-shaped thawing platform was pre-assembled and an 808 nm NIR laser was calibrated to ensure that the laser beam was precisely focused at the center of the platform. After adjusting the laser output power to the target value, the straw was quickly removed from the LN₂ and placed on the platform. Then, the straw was exposed to laser irradiation for 5–7 s. Once it was confirmed that the internal crystals had completely dissolved, it was immediately removed. For traditional cryopreservation groups, swiftly immerse the cryopreserved straw in a water bath for 10–15 s until the crystals dissolve.

Following thawing, both groups underwent identical CPAs unloading and washing procedures. The contents were expelled from the straw using a transfer pipette, and the thawed oocytes were transferred into a pre-prepared thawing solution (TS; 1 M trehalose in M2) for 3 min. Subsequently, the oocytes were incubated consecutively in dilution solution I (DS-I, 0.5 M trehalose in M2) and dilution solution II (DS-II, 0.25 M trehalose in M2) for 3 min each at 37 °C. For the MXene-doped group, this standard multi-step solution transfer and dilution process effectively washed away extraneous nanomaterials, ensuring that no residual MXene adhered to the zona pellucida or persisted on the oocyte surface. Finally, the recovered oocytes were transferred to M2 medium, overlaid with mineral oil, and cultured in a 37 °C, 5% CO₂ incubator for further experimentation.

2.7. Fluorescence Staining and Quantitative Analysis of Post-Thaw Oocytes

JC-1 dye, reactive oxygen species (ROS) detection dye, and Fluo-4 AM were employed to assess MMP, ROS, and calcium ion concentrations in cryopreserved oocytes, respectively. Oocytes were treated with each dye for 20 to 30 min at 37 °C in a 5% CO₂ environment. After incubation, oocytes were rinsed three times with the appropriate buffer. Fluorescent pictures of oocytes were subsequently obtained using an inverted fluorescence microscope. All tests were conducted a minimum of three times. Image analysis was conducted using ImageJ software. The fluorescence intensity per oocyte was quantified to assess staining outcomes, with MMP represented as the ratio of red to green fluorescence intensity.

3. Results and Discussion

3.1. Characterization of Ti₃C₂T _x MXene

Ti₃C₂T _x MXene was produced using LiF-HCl etching, as seen in Figure A. The stratified structure of Ti₃C₂T _x MXene was examined by TEM and AFM. X-ray diffraction (XRD) was utilized to examine the evolution of its crystalline structure, while TGA assessed the thermal stability of the material. This thorough methodology facilitated accurate characterization of MXene’s multiple physicochemical attributes. Figure B illustrates the multilayer morphology of the material employed in this work. TEM characterization distinctly demonstrates that Ti₃C₂T _x MXene displays a characteristic ultrathin nanosheet shape (Figure C). Subsequent quantitative investigation utilizing AFM indicated a thickness ranging from 0.4 to 0.8 nm (Figure D). The ultrathin architecture of Ti₃C₂T _x MXene effectively inhibits devitrification during rewarming and mitigates thermal stress, thereby minimizing cellular injury. These findings align with earlier reports. ,

2.

Characterization of Ti₃C₂T_x MXene. (A) Schematic flowchart of Ti₃C₂T _x MXene preparation. (B) Schematic diagram of Ti₃C₂T _x MXene. (C) TEM image of Ti₃C₂T _x MXene. (D) AFM image of Ti₃C₂T _x MXene. (E) EDS distribution of Ti₃C₂T _x MXene. (F) Raman spectrum of Ti₃C₂T _x MXene. (G) XPS spectrum of Ti₃C₂T _x MXene. (H) C 1s XPS spectrum of Ti₃C₂T _x MXene. (I) O 1s XPS spectrum of Ti₃C₂T _x MXene. (J) FTIR spectrum of Ti₃C₂T _x MXene. (K) TGA curve of Ti₃C₂T _x MXene.

The integration of SEM with energy-dispersive X-ray spectroscopy (EDS) verifies that Ti₃C₂T _x MXene has uniformly distributed carbon, titanium, oxygen, and fluorine elements (Figure E). Furthermore, Raman spectroscopy analysis (Figure F) demonstrates characteristic peaks at 150 cm^–1 and 632 cm^–1, which are attributed to the out-of-plane stretching vibrations of Ti₂ and C. This result corroborates previous findings. , The subsequent XPS investigation of Ti₃C₂T _x MXene produced data depicted in Figure G. The deconvolution analysis of the high-resolution C 1s spectra (Figure H) identifies binding energy peaks at 284.8, 281.5, and 286.2 eV, which correspond to C–C bonds (sp²-hybridized carbon), C–Ti bonds, and C–O bonds, respectively. In the O 1s spectra (Figure I), peaks at 530.1 and 532.6 eV correspond to lattice oxygen (Ti–O) and surface-adsorbed oxygen (−OH/–O groups), respectively. This finding is consistent with previous reports. Fourier transform infrared (FTIR) and XPS analyses reveal that the surface chemistry of Ti₃C₂T _x MXene is primarily influenced by C–Ti bonds, Ti–O bonds, and terminal −OH/–F functional groups, establishing a structural foundation for its biocompatibility and interfacial reactivity.

The FTIR spectrum of Ti₃C₂T _x MXene displays a wide peak at 3429.09 cm^–1, which corresponds to O–H stretching vibrations, signifying a substantial presence of surface hydroxyl (−OH) functional groups (Figure J). A pronounced peak at 571.86 cm^–1 arises from the bending vibration of the Ti–O lattice, signifying the existence of (Ti–O) functional groups on the material’s surface. Faint absorption bands at 1623.82 cm^–1 and 2920.56 cm^–1 correspond to the bending vibration of adsorbed water (H–O–H) and the stretching vibration of C–H bonds in leftover organic matter, respectively. The distinctive peak at about 1115.06 cm^–1 indicates the vibrational properties of C–F bonds, hence affirming the successful incorporation of –F functional groups during the etching process. The multimodal vibrational characteristics suggest that the surface of Ti₃C₂T _x MXene creates a hydrophilic interface composed of −OH, –F, and adsorbed water molecules, establishing a chemical foundation for its use in biocompatible carriers.

TGA of Ti₃C₂T _x MXene in a nitrogen atmosphere reveals a total mass loss rate of merely 7.86%, signifying substantial thermal stability at elevated temperatures, with a weight loss rate remaining below 15% (Figure K). These thermal characteristics indicate that Ti₃C₂T _x MXene possesses exceptional stability across a wide temperature range, satisfying rigorous criteria for material structural integrity under demanding conditions such as high-temperature sterilization.

3.2. Investigation of Photothermal Conversion Characteristics of Ti₃C₂T _x MXene

Rapid and uniform warming is essential in the cryopreservation of biological materials, as the devitrification and recrystallization that occur during suboptimal thawing are primary threats to oocyte viability. − To assess the relevance of Ti₃C₂T _x MXene’s photothermal effect on oocyte vitrification reconstitution processes, the combined effects of laser power density (0–3 W/cm²) and Ti₃C₂T _x MXene concentration (0–1 mg/mL) were systematically measured by orthogonal experiments. Photothermal heating curves of Ti₃C₂T _x MXene suspensions were obtained under different power density gradients (808 nm NIR), concurrently monitoring the maximum equilibrium temperature (T _max) and the heating rate (dT/dt). The CPAs solution devoid of Ti₃C₂T _x MXene functioned as the blank control.

Initial experiments revealed that straws could not be consistently stabilized within the laser irradiation range during simultaneous water bath and laser rewarming. The brief photothermal rewarming duration utilized in this study considerably influenced the experimental results due to the time needed to identify the ideal laser spot. As a result, a laser rewarming platform was constructed, incorporating a laser source, a 37 °C water bath, an 808 nm NIR laser, and an S-shaped straw holder (Figure  A). This custom retainer facilitates the pre-adjustment of the appropriate laser spot position in relation to the S-shaped straw holder. During rewarming, straw can be swiftly positioned into the holder for effective infrared laser irradiation. Experiments indicate that this system efficiently contains straw within the laser’s operational range, hence mitigating the impact of surface water fluctuations on the straw. It also optimizes laser rewarming efficiency to attain superior rewarming outcomes. The infrared image demonstrates that the straw displays consistent heating accompanied by a significant temperature rise (Figure  B).

3.

Photothermal conversion properties of Ti₃C₂T _x MXene. (A) Modeling of photothermal rewarming device. (B) Temperature and infrared images of Ti₃C₂T _x MXene at different concentrations within 7 s. (C) Temperature curves for water bath heating and laser rewarming. (D) Temperature curves for laser rewarming at different Ti₃C₂T _x MXene concentrations. (E) Temperature curves for the same Ti₃C₂T _x MXene concentration under different laser powers. (F) Temperature curve slope plots for water bath heating versus laser rewarming. (G) Temperature curve slope plots for laser rewarming with different Ti₃C₂T _x MXene concentrations. (H) Temperature curve slope plots for the same Ti₃C₂T _x MXene concentration under different laser powers.

This study initially examined the rewarming effectiveness of laser compared to water bath. Figure  C illustrates that the laser rewarming group has markedly greater heating efficiency than the conventional water bath group. The study subsequently analyzed the effect of varying concentrations of Ti₃C₂T _x MXene on rewarming efficiency. Six concentrations were created with a 0.2 mg/mL gradient: 0 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1 mg/mL. Infrared images and temperatures at 7 s were compared under 808 nm NIR laser irradiation at 2 W/cm², as illustrated in Figure  B. The investigation indicated that the concentration of Ti₃C₂T _x MXene has a positive correlation with the rewarming rate within the 0–1 mg/mL range, as illustrated in Figure  D, in conjunction with the temperature curve. Taking into account photothermal efficiency and biocompatibility, a concentration of 0.6 mg/mL Ti₃C₂T _x MXene was identified as optimum. Moreover, the MXene suspension exhibited homogeneous heating under laser irradiation after a specific duration. Subsequently, a 0.6 mg/mL Ti₃C₂T _x MXene solution was employed with three laser power gradients (1 W/cm², 2 W/cm², 3 W/cm²) for experimentation. Data collection on temperature indicated that 2 W/cm² could elevate the straw temperature to 40 °C in 10 s with optimal control, as illustrated in Figure  E. The comparison of heating rates at varying MXene concentrations and laser strengths distinctly demonstrates that laser rewarming surpasses water bath (Figure  F). In MXene concentration trials, the rewarming rates for 0.6 and 0.8 mg/mL were almost comparable (Figure  G), which aligns with the previously stated finding. The rewarming rate charts for various laser powers indicate that the rewarming rates at 2 W/cm² and 3 W/cm² are comparable for the initial 10 s, with 2 W/cm² demonstrating the lowest energy usage and optimal controllability (Figure  H). In conclusion, according to photothermal data, the ideal group for photothermal conversion efficiency (2.0 W/cm² + 0.6 mg/mL Ti₃C₂T _x MXene) was chosen to facilitate targeted rewarming of cryopreserved biological specimens.

The ice recrystallization inhibition (IRI) performance and thermodynamic properties of the CPAs solution supplemented with MXene were further evaluated. As shown in Figure S1A, the mean largest grain size (MLGS) of ice crystals decreased significantly upon the addition of MXene compared to the control group, with the corresponding micrographs revealing markedly finer and more uniform ice structures. This indicates that MXene effectively inhibit ice growth during the rewarming process.

Furthermore, differential scanning calorimetry (DSC) was employed to investigate the thermal behavior of the CPAs solutions during melting. As shown in the DSC thermograms (Figure S1B), the incorporation of MXene influenced the phase transition characteristics of the CPA solutions. Specifically, the melting enthalpy showed an upward trend with increasing MXene concentrations (Figure S1C), whereas the crystallization temperatures remained relatively stable across different concentrations (Figure S1D). As noted in previous analyses, although MXene promotes heterogeneous nucleation, it does not substantially exacerbate the total amount of crystallization, and the simultaneous reduction in supercooling degree helps prevent the formation of sharp, dendritic ice morphologies.

3.3. Evaluation of Cytotoxicity of Ti₃C₂T _x MXene

Prior characterization studies of Ti₃C₂T _x MXene have revealed their remarkable photothermal conversion properties, suggesting potential utility in cryobiology. Building on this, the present work evaluates their potential for cryobiological applications using mouse oocytes, a crucial model system in reproductive biology. Figure  A depicts the experimental workflow. Following the multi-step solution transfer and dilution process, optical microscopy confirmed that the washing procedure completely removed residual nanomaterials from the zona pellucida surface (Figure S2), ensuring no adverse physical persistence prior to biological cultivation.

4.

Flowchart of biological experiments and cytotoxicity of Ti₃C₂T _x MXene. (A) Schematic of mouse oocyte cryopreservation and thawing experiments (Some graphic elements were created with BioRender.com). (B) Viability/necrosis plots of oocytes co-cultured with MXene at different durations. (C) Survival rate of co-cultured oocytes. All data were plotted as the mean ± s.d. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. n = 15–30 for three replicates. n: number of mouse oocytes used in each experiment.

To rigorously assess the biocompatibility of Ti₃C₂T _x MXene, the study incorporated a control group (CPAs solution devoid of MXene) and an experimental group (0.6 mg/mL MXene/CPAs composite solution). Mouse oocytes were incubated in each group at 37 °C with 5% CO₂ for durations of 0, 4, and 8 h. Oocyte membrane integrity was evaluated at each time point using acridine orange/ethidium bromide (AO/EB) dual-fluorescent staining (staining concentration: 100 μg/mL, incubated in the dark for 3–5 min). Figure  B displays fluorescent pictures obtained from an inverted fluorescence microscope, accompanied by a quantitative analysis of living oocytes (green fluorescence) and apoptotic/necrotic oocytes (red fluorescence). Figure  C illustrates that both the control and experimental groups demonstrated initial survival rates nearing 100% at 0 h. Survival rates remained high at 4 h but dropped to 0% after 8 h, respectively. This work involved the cultivation of oocytes using M2 solution. The combination of M2 solution with CO₂ in incubator results in acidity, causing the demise of oocytes in both the control and experimental groups after 8 h. Fluorescent images obtained before 4 h exhibited preserved oocytes shape in both groups, with no indications of damage, including membrane shrinkage or chromatin condensation. These results indicate that 0.6 mg/mL Ti₃C₂T _x MXene did not elicit substantial cytotoxicity relative to the control group after continuous treatment for up to 4 h, satisfying the requirements for oocyte processing.

3.4. Activity of Oocytes after Cryopreservation

This study utilized dual-fluorescent labeling to concurrently evaluate oocytes membrane integrity and metabolic activity (Figure  A), a traditional method for assessing post freeze/thaw oocytes viability. The comparative study indicates that the MXene group (0.6 mg/mL MXene/CPAs composite solution) attained oocyte survival rate of (81.0 ± 3.2)%, exhibiting a statistically significant difference (p < 0.01) relative to the Conventional Vitrification (CV) group of (72.1 ± 5.7)% (Figure  B). This visually substantiates that MXene laser-assisted thawing improves the effectiveness of oocyte cryopreservation.

5.

Viability of mouse oocytes post-cryopreservation. (A) AO/EB fluorescence image of oocytes after rewarming. (B) Survival rate of oocytes after rewarming. All data were plotted as the mean ± s.d. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. n = 10–30 for three replicates. n: number of mouse oocytes used in each experiment.

3.5. Functional Assessment of Oocytes after Cryopreservation

After confirming that Ti₃C₂T _x MXene treatment could enhance the survival rate of thawed oocytes, we further investigated the cryopreservation effects of Ti₃C₂T _x MXene loaded with different concentrations of CPAs (Figure S3). Finally, 15% CPAs (15% (v/v) EG, 15% (v/v) PG, 0.5 M trehalose) was selected for our next study to determine whether the surviving oocytes retained normal functional capabilities. To this end, a series of functional markers associated with oocyte viability and developmental potential were evaluated after vitrification and thawing. Given that oxidative stress is a major contributor to cryoinjury and can persist even in morphologically intact oocytes, we first assessed intracellular ROS levels (Figure  A). Because excessive ROS primarily originates from mitochondrial dysfunction during freeze-thaw cycles, monitoring this parameter serves as a key indicator of cellular stress. Figure  D illustrates that the ROS fluorescence intensity in the MXene group was diminished compared to the CV group and marginally elevated relative to the fresh group. This indicates that MXene combined with photothermal rewarming can optimize reactive oxygen species levels.

6.

Functional assessment of oocytes after cryopreservation. (A) ROS fluorescence image of oocytes after rewarming. (B) Ca²⁺ fluorescence image of oocytes after rewarming. (C) JC-1 fluorescence image of oocytes after rewarming. (D) ROS fluorescence intensity of oocytes after rewarming. (E) Ca²⁺ fluorescence intensity of oocytes after rewarming. (F) Relative JC-1 fluorescence intensity of oocytes after rewarming. All data were plotted as the mean ± s.d. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. n = 17–30 for three replicates. n: number of mouse oocytes used in each experiment.

In addition to oxidative stress, disrupted calcium homeostasis is another hallmark of cryoinjury that can impair oocyte function. To track these Ca²⁺ oscillations, we used the fluorescent probe Fluo-4 AM, whose sensing mechanism has been well established. , Intracellular Ca²⁺ concentrations act as an additional signal of mitochondrial activity. Mitochondria sequester Ca²⁺ to regulate calcium homeostasis in oocytes. Increased intracellular Ca²⁺ levels disrupt mitochondrial activity and induce premature exocytosis of cortical granules, resulting in zona pellucida hardness and diminished developmental potential of oocytes during cryopreservation. Intracellular Ca²⁺ concentrations were assessed using Fluo-4 AM labeling, with fluorescence pictures captured by inverted fluorescence microscope, the result is shown in Figure  B. ImageJ was utilized for quantitative analysis, with reduced fluorescence intensity signifying improved cellular outcomes. Figure  E demonstrates that the MXene group maintained significantly lower intracellular Ca²⁺ levels than the CV group, indicating better preservation of calcium homeostasis. MMP serves as a crucial measure of mitochondrial functionality. , The ratiometric response of JC-1 has been well characterized. , When the internal MMP (ΔΨ_m) is less than 100 mV, JC-1, a fluorescent probe for MMP, exists in its monomeric form and displays green fluorescence in the FITC channel, indicating low-polarized mitochondria. At ΔΨ_m > 140 mV, JC-1 forms J aggregates and produces red fluorescence, indicating highly polarized mitochondria. As illustrated in Figure  C, elevated mitochondrial polarization in oocytes was identified by assessing the relative intensities of red and green fluorescence emissions with an inverted fluorescence microscope. Quantitative analysis was conducted via ImageJ, with increased relative fluorescence intensity signifying superior oocyte quality. Figure  F illustrates that the cryopreservation efficacy of the MXene group was analogous to that of the fresh group and markedly superior to that of the CV group.

Experiments compared CPAs with and without Ti₃C₂T _x MXene by assessing four parameters: ROS levels, MMP, dual fluorescence staining (AO/EB), and intracellular Ca²⁺ levels. The MXene group outperformed the CV group across all assessed parameters and yielded results comparable to the Fresh group, demonstrating that Ti₃C₂T _x MXene enhances the cryopreservation efficacy of mouse oocytes. In the ROS assay, the MXene group demonstrated markedly reduced ROS levels compared to CV group, along with enhanced preservation of cellular structure and function. In the MMP test, the MXene group showed a phenomenon of predominantly red fluorescence and weakened green fluorescence, indicating an increase in MMP and intact structure, with no signs of cell apoptosis. In contrast, the CV group exhibited weakened red fluorescence and enhanced green fluorescence, suggesting a decrease in MMP and depolarization, which may imply the presence of oocyte apoptosis. Fluorescence microscopy during dual-fluorescence staining revealed that the proportion of damaged cells was markedly lower in the MXene group compared to the CV group, confirming enhanced survival. Measurements of Ca²⁺ levels indicated reduced intracellular calcium accumulation in the MXene group relative to the CV group. Moreover, laser rewarming exhibited significantly elevated warming rates and enhanced oocyte survival rates in comparison to traditional water bath rewarming.

This research successfully integrated Ti₃C₂T _x MXene with 808 nm NIR laser rewarming technology into an oocyte cryopreservation framework. The photothermal action facilitated rapid, homogeneous rewarming while markedly inhibiting ice devitrification. Furthermore, the intrinsic antibacterial properties of MXene diminished contamination hazards. Results indicate that this method significantly improves oocyte survival rates, sustains mitochondrial function, and preserves calcium homeostasis, presenting an innovative technical approach for reproductive medicine. This work examines how the concentration of Ti₃C₂T _x MXene, laser settings, and the photothermal conversion efficiency of Ti₃C₂T _x MXene affect cellular rewarming. The findings of the Ti₃C₂T _x MXene concentration variation test indicate that, within the 0–1.0 mg/mL range, the photothermal performance of Ti₃C₂T _x MXene exhibits a positive correlation with concentration. Nonetheless, the development of efficiency diminishes at elevated concentrations (>0.6 mg/mL). Consequently, a CPAs with 0.6 mg/mL Ti₃C₂T _x MXene was used for studies with varied power. The variable-power test findings for Ti₃C₂T _x MXene demonstrate that its photothermal performance enhances with increasing laser power. A laser strength of 3 W/cm² induces protein denaturation, resulting in diminished oocyte viability. A laser intensity of 2 W/cm² was chosen for the variable concentration studies.

Although the MXene-mediated photothermal heating has attracted attention in the field of cryopreservation such as stem cell preservation in recent years, successfully applying this strategy to mammalian oocytes faces significant biological and technical challenges. Unlike stem cells, mammalian oocytes have a large cell volume, high membrane sensitivity, and extremely high water content, making them prone to intracellular ice crystal formation and destructive desiccation during the conventional thawing process. To overcome these limitations, this study combines doped MXene CPAs with a specially designed S-shaped thawing platform. This platform can firmly fix the straws, ensuring precise laser focusing and uniform energy distribution, thereby achieving ultra-fast and repeatable rewarming. Therefore, this integrated design effectively enhances the heating efficiency and reduces devitrification, providing a reliable and oocytes protective strategy.

Although this method effectively improves the survival rate after thawing compared to conventional vitrification, there are still several challenges in its clinical practice. MXene are crucial for achieving rapid photothermal rewarming, their long-term biosafety still needs to be comprehensively evaluated to ensure that they do not have adverse effects on subsequent embryonic development or reproductive potential. In addition, the current integration of the S-shaped thawing platform and external laser component systems is limited, resulting in complex operations that urgently need further optimization to support future clinical applications.

4. Conclusions

This study investigated the photothermal conversion properties of Ti₃C₂T _x MXene and its application in oocyte cryopreservation. It was found that Ti₃C₂T _x MXene exhibited excellent photothermal response properties. Under NIR laser irradiation, it significantly increases the rewarming rate and suppresses ice crystal formation during thawing, thereby mitigating oocyte damage caused by devitrification. Survival rates and functional assessments of frozen-thawed oocytes further validated the efficacy and safety of this method. Furthermore, this work established a new platform for thawing cryopreserved samples, designed to stabilize the laser and straw, reduce positional interference during operation, and ensure more consistent and controllable temperature changes during thawing, thereby significantly improving experimental reproducibility. In summary, this study developed a new material and method for the vitrification cryopreservation of oocytes, and also provided a new approach and reference for the vitrification cryopreservation of biological samples such as embryos and organs.

Data will be made available on request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c07760.

• Ice recrystallization inhibition activity, DSC thermal behavior, MXene removal efficiency from the zona pellucida, optimal CPAs concentrations, and additional experimental methods (PDF)

The authors declare no competing financial interest.