Ultrasound-powered MXene hydrogels for enhancing tumor inhibition and immune stimulation by the piezoelectric effect

Abstract

Cancer immunotherapy has attracted tremendous attention. To improve the response rate of immune checkpoint inhibitors and tumor antigens in immunosuppressive cancer, the induction of piezoelectric-triggered cancer cell death can increase antigenicity. Herein, we construct a piezoelectric poly(vinyl alcohol) (PVA)/polyvinylidene fluoride (PVDF)/MXene hydrogel loaded with a biomimetic cancer cell membrane (CCM) that incorporates TLR7/8a/anti-PD-L1. The CCM surface proteins act as tumor-specific antigens. Poly(lactic-co-glycolic acid) (PLGA) is used to enhance the stability and attachment of the MXene. After adding the MXene, the hydrogel exhibits a higher piezoelectric coefficient, greater electrical signal yield with superior stability, and excellent mechanical strength. Ultrasound (US) enhances the piezoelectric effect of the PVA/PVDF/MXene-CCM hydrogel. This is confirmed through in vitro reduction and oxidation catalysis reactions. The US-stimulated electrical signal inhibits cancer cells via apoptosis induction, endoplasmic stress, and mitochondrial membrane depolarization. It leads to the secretion of danger-associated molecular patterns into the cytoplasm, which promotes dendritic cell maturation and cytotoxic T-lymphocyte infiltration, thereby reversing the immunosuppressive tumor microenvironment. In vivo studies show that the hydrogel offers great therapeutic efficacy to control tumor growth due to the combined effects of the piezoelectric effect and immune checkpoint blockade (ICB) therapy. It improves dendritic cell maturation and increases cytotoxic T-cells. Therefore, our work presents a novel piezoelectric hydrogel and new therapeutic strategies with great potential and versatility for treating breast cancers.

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Introduction

Cancer immunotherapy is a promising approach employed with immune checkpoint inhibitors and Toll-like receptors, which was a standard strategy followed in the past decade. But, the response rate is poor, and it was reported that the tumor microenvironment impedes the immunotherapeutic effect.^1,2 Currently, it is known that the piezoelectric effect can activate the immune system to potentiate cancer immunotherapy. Several inorganic (barium titanate, zinc oxide, boron nitride, etc.) and organic nanomaterials (fluoropolymers, polyesters, polyureas, polyamides) have piezoelectric properties. An inorganic material exhibits good piezoelectric coefficients, but failed in vivo application and raises concerns about biocompatibility and degradability.^3,4 Conversely, piezoelectric polymers like polyvinylidene fluoride (PVDF) have high piezoelectric coefficients, are flexible, tunable, and biocompatible, and are widely used in biomedical applications. Several reports demonstrated that composite PVDF scaffolds stimulate the electrical signal to the cells during appropriate US stimuli. Also, the use of polar fillers like CNTs, graphene, MXenes, etc. in combination with PVDF has increased recently. The negative ions or polar groups on the filler are able to strongly interrelate with the PVDF molecule for effective polarization.^5–7 Bhunia et al. reported that the introduction of graphene in PVDF composites helped in enhancing the piezoelectric, ferroelectric, and triboelectric properties of the material. Here, the incorporation of MXenes into PVDF improves the composite's electrical conductivity and charge carrier mobility, increases its mechanical properties, and offers a highly electronegative surface. The MXene F group improves the interfacial compatibility and favors the uniform arrangement of PVDF molecular backbones.^8,9

In this context, a PVDF/MXene material with the piezoelectric effect delivers electric signals to cells non-invasively and remotely. Here, PVDF refers to polyvinylidene fluoride, a polymer known for its piezoelectric properties, while MXene is a type of two-dimensional nanomaterial. Mostly, nanomaterials activated through low-intensity pulsed ultrasound create mechanical stress that induces the direct piezoelectric effect, the generation of electrical charge in response to mechanical force.^10,11 This electric stimulation inhibits cancer cell proliferation, decreases invasion, induces necrosis, and generates reactive oxygen species such as hydroxyl radicals (–OH) and superoxide anions (–O₂⁻), which damage DNA, disrupt the endoplasmic reticulum, and affect the mitochondrial membrane potential. As a result, tumor-associated antigens and danger-associated molecular patterns (DAMPs), including high mobility group Box 1 (HMGB1), adenosine-5′-triphosphate (ATP), and calreticulin, are released. This also reverses the tumor microenvironment from immunosuppressive to immunoresponsive.^12–14 However, the inhibition of cancer cells by the piezoelectric effect alone does not produce a sustained immune response. To address this issue, combining this approach with interventions such as Toll-like receptor agonists or immune checkpoint inhibitors enhances immune stimulation. It has been reported that therapeutic outcomes are improved after combining with immune checkpoint inhibitors. The anti-PD-L1 antibody, a type of immune checkpoint inhibitor, activates tumor-specific effector T-cells by using a tumor antigen and helps maintain antitumor activity.^15–17

Biomimetic functionalized nanosystems were used to deliver therapeutic agents; currently, cell membranes from RBCs, NK, and cancer cells are used for engineering biomimetic nanoplatforms. The tumor cell membrane is able to retain the antigens and evoke the immune response and helps in homotypic adhesion for targeted delivery. Also, calreticulin on the cell membrane is a vital DAMP in immunogenic signal transmission, and it helps in the engulfment of tumor-associated antigens by antigen-presenting cells (APCs) to activate immune responses.^18–20 In this work, we constructed biomimetic nanoparticles composed of cancer cell membranes (CCMs) and poly(lactic-co-glycolic acid). The NPs are loaded with TLR, an agonist against toll-like receptor (TLR 7/8a), and encapsulated with the anti-PD-L1 antibody. CCM NPs were combined with an MXene, and they were introduced into the PVA/PVDF hydrogel formed with intermolecular interactions. Physiochemical characterization and in vitro analysis confirm the piezoelectric properties of the hydrogel. Also, under US stimulation, the PVA/PVDF/MXene-CCM hydrogel generates the piezoelectric effect to inhibit cancer cell proliferation and release DAMPs to activate the immune response against cancer cells. The release of adjuvants and the anti-PD-L1 antibody enhances the sensitivity of tumor cells, amplifying the immune response. The piezoelectric-induced cancer cell inhibition, DC maturation, and T-cell infiltration were confirmed through in vitro and in vivo studies. Therefore, the hydrogel's piezoelectric ability and biomimetic nanosystem-based ICB inhibit the cancer cells and induce immunotherapy.

Materials and methods

Materials

Poly(vinylidene difluoride) (PVDF), poly(vinyl alcohol) (PVA), poly(lactic-co-glycolic acid) (PLGA), titanium aluminium carbide MAX phase, TLR 7/8a (Resiquimod-SML0196), 7′-dichlorodihydrofluorescein diacetate (DCFH-DA), lipopolysaccharides (LPS), dimethyl sulfoxide (DMSO), 4,6-diamidino-2-phenylindole (DAPI), and hydrofluoric acid (HF) were procured from Sigma-Aldrich, India. Rhodamine B, 2,2,6,6-tetramethylpiperidine (DMPO), and a glutathione (GSH) assay kit were purchased from SRL, India. Anti-mouse-PD-L1 antibody (BE0101) was purchased from Thermo Fisher Scientific, and all other chemical reagents were analytically pure. MCF-7 and 4-T1 cells (breast cancer cells) were acquired from the National Centre for Cell Science (NCCS), Pune, India. Dulbecco's Modified Eagle's Medium (DMEM) and phosphate-buffered saline (PBS) were obtained from Gibco.

Isolation of the cell membrane

Breast cancer cells (MCF-7) were isolated using the Membrane Protein Extraction Kit. Briefly, the cells were collected and dispersed in membrane protein extraction buffer and incubated in an ice bath for 10–15 min. Then, the cells were freeze-thawed, and the mixture was centrifuged at 14 000g for 30 min. Then, the CCM was labelled with the red fluorescent lipophilic membrane stain Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo carbocyanine perchlorate) by mixing for 20 min. The unbound CCM was removed by centrifugation and washed with PBS.

Preparation of CCM-coated PLGA NPs

PLGA NPs were prepared by the oil-in-water emulsion method. The PLGA polymer was added to dichloromethane (10 mg mL⁻¹) and then mixed dropwise into 10 mL of deionized water. After evaporation of solvents, PLGA NPs were centrifuged at 5000 rpm for 20 min and washed with water. 0.5 mL of CCM was added to the PLGA NPs (2 mg mL⁻¹), and the mixture was extruded through a polycarbonate porous membrane for seven passes. Then, CCM-coated PLGA NPs were obtained by centrifugation and quantified by UV-vis spectroscopy.

Synthesis of the MXene

Following the preparation of PLGA NPs, the MXene was synthesized as follows. Briefly, Ti₃AlC₂ was added to HF (40 wt%), and the mixture was kept at 40 °C for 24 h. Then, the mixture was centrifuged at 3500 rpm for 5 min, followed by washing in deionized water until the pH < 6.0. Using a PVDF membrane, the Ti₃C₂T_x sediment was washed with 1000 mL of water using vacuum filtration. Finally, MXene powder was dried under vacuum for 24 h at 80 °C.

Fabrication of the CCM-MXene

The CCM-PLGA NPs were rehydrated in PBS and added to the MXene under vortexing. Then, the mixture was subjected to ultrasonication for 15 min under 4 °C conditions. The resulting mixture was centrifuged at 12 000 rpm for 10 min to remove the unbound components.

Preparation of the piezoelectric hydrogel

Initially, PVA/PVDF/MXene-CCM was added to DMSO, followed by freezing at −20 °C for 20 h (2 cycles). Then, the thawed mixture was poured into a mold, followed by the gels being soaked in distilled water for 2 days to remove the DMSO. The formed hydrogel was annealed at 40 °C for 12 h. Later, the hydrogel was immersed in water at 25 °C for 8 h to obtain the piezoelectric hydrogel.

Physiochemical characterization

FTIR analysis was performed on PLGA NPs, CCM-PLGA NPs, MXene, PVA/PVDF, and PVA/PVDF/MXene-CCM hydrogel to examine the functional groups, and FTIR spectra were recorded using a Jasco FTIR 460 plus, Japan. Specifically, the XRD patterns were obtained using a Philips Electronic Instruments Inc., (Mahwah, NJ) with Cu Kα radiation, operated at 40 kV and 40 mA. Scanning was performed over a 2θ range of 5°–80° at a rate of 2° min⁻¹. The hydrodynamic size and surface charge of the nanomaterial were determined using a Zetasizer Nano ZS90. The structural morphology of PLGA NPs, CCM-PLGA NPs, MXene, PVA/PVDF, and the PVA/PVDF/MXene-CCM hydrogel was observed by field-emission scanning electron microscopy (JSM 5900LV, JEOL Co., Ltd, Japan). Energy dispersive X-ray spectroscopy (EDS) measurements of the hydrogel were also performed using an energy dispersive X-ray spectrometer (INCA, PENTAFETX3, OXFRD). The tensile properties of materials were measured using the universal testing machine Instron 5566, USA. The dielectric properties of the hydrogel were measured using a broadband dielectric spectrometer (Novocontrol GmbH, Germany).

In vitro piezoelectric catalysis

The piezoelectric effect of the PVA/PVDF/MXene-CCM hydrogel was evaluated by spin resonance (ESR) spectroscopy to measure the production of –OH and superoxide anions under US irradiation. Singlet oxygen production was quantified by measuring the reduction in DPBF absorbance at 410 nm. Rhodamine B was used as a probe to assess the effectiveness of the hydrogel's piezoelectric effect in oxidizing H₂O to produce –OH radicals.

Cytotoxicity studies

5 × 10⁴ MCF-7 cells were seeded in a 24-well plate containing RPMI 1640 medium for 12 h. Subsequently, PVA/PVDF, the PVA/PVDF/MXene-CCM hydrogel, PVA/PVDF + US, and PVA/PVDF/MXene-CCM hydrogel + US of different concentrations (50–200 mg mL⁻¹) were added to the cells. Before addition, the hydrogels were equilibrated in the medium, and the swollen state of the hydrogel was used. To simulate physiological in vivo conditions, the samples were irradiated with ultrasound (1 MHz, 1 W cm⁻², 50% duty cycle) and subsequently incubated for 12 h. The in vitro cytotoxicity of the hydrogel was then assessed using the MTT assay.

DNA and mitochondrial membrane damage

Cancer cells were seeded in a 6-well plate with medium and PVA/PVDF, the PVA/PVDF/MXene-CCM hydrogel, PVA/PVDF + US, and PVA/PVDF/MXene-CCM hydrogel + US treatment and incubated for 12 h. The cells were stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), incubated for 15 min, washed with deionized water for 5 min, and visualized under a fluorescence microscope. Direct DNA damage and mitochondrial membrane damage were measured using the yH2AX marker and JC-1 dye.

Cellular uptake

BMDCs were obtained from the hind limbs of C57BL/6 mice, bones were left in 70% ethanol for 5 min for disinfection and washed with PBS. Then, ends were cut and the bone marrow was flushed with PBS using a syringe with a 0.45 mm diameter needle. For the DC uptake study, the FITC-loaded PVA/PVDF/MXene-CCM hydrogel (10 μg) was incubated with 2 × 10⁴ BMDCs for different time intervals. The cells were counterstained with DAPI for 10 min, washed, and visualized using a fluorescence microscope (Nikon Ti2F, Japan).

In vitro piezocatalytic immunotherapy effect

BMDCs were seeded into a 6-well plate and treated with the hydrogel for 16 h, and after incubation, the cells were collected. Then, extracellular levels of HMGB1 were measured using the mouse HMGB1 ELISA kit as per the manufacturer's instructions. The ATP level was measured using an adenosine 5′-triphosphate (ATP) bioluminescent assay kit using a microplate system, and DOX was used as a positive control. Then, the cells were rinsed with PBS, collected, and analyzed by flow cytometry.

DC activation

BMDCs (3 × 10⁵ cells) were treated with the hydrogel for 12 h; then, the cells suspended in PBS containing 1% FBS were incubated with the anti-mouse antibody and evaluated using a flow cytometer. For quantitative analysis of TNF-α and IL-12p40 release, the DC medium supernatants were subjected to ELISA.

Animal

All animal experiments were performed as per the protocols approved by the Institutional Animal Ethics Committee (AIEC) of J.J. College of Arts and Science, Pudukottai (JJ/BC/AH/014/2022). For the induction of a tumor in the C57BL/6 mouse model, 5 × 10⁴ 4T1 cells were subcutaneously injected into the right lower back of the mice. The tumor volume was measured, and after that the tumor volume reached about 50–75 mm³. The weight of the mice was tested throughout the study, and the tumor size was measured every day with a caliper. Tumor volumes were determined as follows: width × length × depth × 0.4.

Tumor combating studies

Mice were randomly divided into five groups treated with PBS, CCMPs, PVA/PVDF, and the PVA/PVDF/MXene-CCM hydrogel with and without US. They were intratumorally injected at an equivalent EPI dose of 3 mg kg⁻¹, and the treatment cycle was repeated three times every two days. The US was set at a 1 MHz frequency, 1 W cm⁻² intensity, 50% duty cycle, 5 min. During the experiment, the tumor volume and body weight were recorded every other day. At the end of the treatment, animals were sacrificed to collect the major organs and preserved in 4% paraformaldehyde for histological analysis.

In vivo activated immune cells

To examine potent immune responses of the piezoelectric hydrogel on a tumor-induced mouse model, the percentages of CD8⁺ T-cells and mature DCs were measured using a flow cytometer. The mice were euthanized two days after the last treatment, and the tumor, lymph nodes, and spleen were harvested. The tissue samples were cut into pieces and centrifuged to obtain the supernatant, which was subjected to flow cytometry analysis.

Statistical analysis

All the results are expressed as mean ± SD. The statistical significance was determined with Prism using ANOVA. P < 0.05 was considered statistically significant.

Results and discussion

The fabrication of PLGA NPs cloaked with the cancer cell membrane–MXene begins with the extraction of the MCF-7 cancer cell membrane. Then, the cancer cell membrane was coated on the surface of TLR/anti-PD-L1-containing PLGA NPs by extrusion (Fig. 1a).²¹ The Al layers were etched from the MAX phase precursor (Ti₃AlC₂) to obtain the MXene and CCM conjugated to the MXene with the help of intrinsic chemical groups, and the lateral size influences the aggregation of the CCM. A biocompatible polymer significantly improves the interfacial interaction between the CCM and the MXene with high binding efficiency.²² A piezoelectric PVA/PVDF/MXene-CCM hydrogel was fabricated by a freeze/thaw method, and stacking it with the MXene plays a vital role in the piezoelectric response of the hydrogel to produce a high piezoelectric coefficient and electrical signal output with excellent mechanical stability (Scheme 1).^23,24

Fig. 1 (a) Schematic illustration of the preparation of cancer cell membrane-coated TLR/anti-PD-L1-loaded PLGA NPs. (b and c) SEM analysis of TLR/anti-PD-L1-loaded PLGA NPs. (d) DLS analysis of CCM-cloaked TLR/anti-PD-L1-loaded PLGA NPs. (e–j) SEM analysis of CCM-cloaked TLR/anti-PD-L1-loaded PLGA NPs.

Scheme 1 Preparation of the PVA/PVDF/MXene-CCM hydrogel using TLR/anti-PD-L1; piezoelectric effect-induced effective tumor therapy via generation of oxygen and reactive oxygen species by external US irradiation. Further release of tumor antigen DAMPs strengthens the ICD, promotes the DC maturation, CD8⁺ T-cell infiltration and cytokine secretion to alter the immunosupportive microenvironment to enhance the immunostimulation effect of the anti-PD-L1 antibody.

Nanoparticle characterization

The structural morphology of the synthesized PLGA NPs presents a size distribution of 110 nm (Fig. S1a) and a positive zeta potential at 23.2 mV (Table S1). After loading, the size of the nanoparticles increased to 134 nm (Fig. S1b) and the zeta value changed to −18.2 mV. A monodisperse spherical morphology was observed for TLR/anti-PD-L1-loaded PLGA NPs by SEM (Fig. 1b and c). The PLGA NPs cloaked with cancer cell membrane fragments exhibited a spherical structure, the hydrodynamic size of the obtained CCM-PLGA NPs increased to 180.21 nm (Fig. 1d–j), and the charge potential decreased to −12.45 mV.

The preparation of the MXene is depicted in Fig. 2a; initially, in MXene synthesis, MAX phase Al atoms were etched using concentrated hydrogen fluoride. In the scanning electron microscopy images, the MXene shows interconnected ultrathin MXene sheet layers with a uniform surface with layer spacing (Fig. 2b–e). A single-layer MXene structure depicted a flat, layered structure (Fig. 2f–i). An HF-etched and completely removed Al layer was clearly observed, which was further confirmed using an energy dispersive spectrometer (Fig. S2a). It represents the compositional analysis of the MAX phase and MXene with their atomic ratios. It shows the purity of Ti₃AlC₂, which consists of only Ti, Al, and C. The EDX spectrum of the MXene implies the complete removal of Al from the MXene. The crystalline structures were investigated using X-ray diffraction, which is shown in Fig. S2c. MAX phase reflections between 5° and 20° disappeared and contamination peaks like AlTa₃ and Ta₂O were also removed, confirming the successful etching of Al. So, it presents a good crystalline structure, with a highly stacked order of Ti₃C₂. FTIR spectroscopy was performed to examine the surface conditions, interactions, surface functional groups, and hydrophilic properties of the MAX phase and MXene, as shown in Fig. S2b. MXene purity is verified through FTIR analysis, showing characteristic peaks at 1084 cm⁻¹, 1728 cm⁻¹, 1408 cm⁻¹, 2921 cm⁻¹, and a broad peak 3000–3500 cm⁻¹ correspond to CO, C–F, and OH bonds, respectively. The oxygen- and fluorine-containing peaks indicate a slight hydrophilic nature.²⁵

Fig. 2 (a) Fabrication of the MXene and MXene-CCM, and MXene with and without CCM labeled with Dil. FE-SEM analysis of (b–e) MXene and (f–i) single-layer MXene.

Cancer cell membrane-cloaked PLGA NPs

Cell membrane-cloaked PLGA NPs were coated over the MXene surface, and were visualized using a fluorescence microscope. These results indicate that the membrane proteins of the cancer cells were well preserved and successfully identified in the MXene sample, confirming effective coating of the cell membrane (Fig. S3). In the SEM image, the attachment of CCM-cloaked PLGA NPs on the MXene surface was directly visualized (Fig. S3). The presence of –F and –O functional groups helps in the attachment of particles, and they were completely wrapped over the MXene. They provide an excellent targeting ability and higher affinity recognition compared to the plain MXene. The functionalization of PLGA helps in diminishing the non-specific binding of PLGA NPs and serum protein, shielding them from aggregation, opsonization, and phagocytosis.²⁶

The chemical structures of PVA, PVDF, PVA/PVDF, and the PVA/PVDF/MXene-CCM hydrogel were analyzed by FTIR (Fig. S4). In the FTIR spectra of PVA/PVDF, the absorption peak represents the –OH stretching vibration of PVA, the weak peak at 768 cm⁻¹ was attributed to the nonpolar α-phase, and strong peaks at 515 cm⁻¹ and 830 cm⁻¹ represent the polar β-phase crystalline structure. The enhanced β-phase content in the PVA/PVDF/MXene-CCM hydrogel was confirmed by the characteristic peaks at 836 cm⁻¹ and 1272 cm⁻¹. The incorporation of the MXene increases the relative percentage of the electroactive β-phase and improves hydrogen bonding between PVA and PVDF, superior to PVA and PVDF alone.^27,28 The XRD crystallization properties of PVA, PVA/PVDF, and the PVA/PVDF/MXene-CCM hydrogel are shown in Fig. S4. The crystallinity and crystallite sizes were increased in the PVA/PVDF/MXene-CCM hydrogel compared to those of PVA and PVA/PVDF. This crystallization ability was increased due to the improved hydrogen bonding during the annealing steps. The hydrogen bonding between the PVA/PVDF and MXene -F groups improves the interfacial compatibility and uniform arrangement of the CCM of PVA/PVDF molecular backbones. It also transforms the α-phase into the electroactive β-phase of PVDF, easing the formation of a more crystalline structure.

The SEM images of the PVA/PVDF hydrogel are shown in Fig. 3a–c. PVA/PVDF shows a less porous structure with a uniform network, indicating the formation of intra- and inter-molecular hydrogen bonds between the polymeric networks. This confirms the formation of a more oriented crystalline structure, which is highly desirable for promoting the piezoelectric ability. In Fig. 3d–i, the PVA/PVDF/MXene-CCM hydrogel shows a more porous, stacked distribution of the MXene, and it acts as a connecting point to improve the cross-linking density and dense network. The oriented and regular stacked MXene shows a high shear rate and the confined microchannel helps in conducting and releasing electric charge.²⁹

Fig. 3 SEM images of (a–c) the PVA/PVDF hydrogel and (d–i) the PVA/PVDF/MXene-CCM hydrogel at different magnifications.

The distribution of the MXene and PVDF in the hydrogel was conducted using energy-dispersive spectroscopy (EDS) elemental mapping. The fluorine elemental concentration profiles of PVA/PVDF (Fig. 4a–c) and the PVA/PVDF/MXene-CCM hydrogel (Fig. 4d–g) show the uniform distribution of elements in different locations of the polymeric matrix. Compared to PVA/PVDF, the PVA/PVDF/MXene-CCM hydrogel shows high agglomeration; more difference in the fluorine concentration denotes the presence of the MXene and PVDF. The elemental distribution in the hydrogel influences the piezocatalytic effect, and the treatment with US directly increases the levels of O₂⁻ and –OH (Fig. 4h). In swelling analysis, PVA/PVDF and the PVA/PVDF/MXene-CCM hydrogel show similar swelling results of 52–53% in 8 h (Fig. S5).

Fig. 4 Energy-dispersive spectroscopy (EDS) elemental mapping of the PVA/PVDF hydrogel: (a) carbon, (b) oxygen and (c) fluorine; the PVA/PVDF/MXene-CCM hydrogel: (d) titanium, (e) fluorine, (f) oxygen and (g) carbon. (h) Piezoelectric activity of the PVA/PVDF/MXene-CCM hydrogel.

The tensile mechanical ability of PVA/PVDF and the PVA/PVDF/MXene-CCM hydrogel at different PVA/PVDF ratios is shown in Fig. 5a. After the addition of PVDF and the MXene to the hydrogel, there is an increase in the tensile strength and modulus. At PVA/PVDF ratios of 5 : 5 and 5 : 10, the tensile and modulus strength were 4.86, 5.46, 5.74, and 6.10 MPa, respectively. The increase in the MXene ratio of the PVA/PVDF hydrogel leads to an increase in the mechanical strength. At a 5 : 10 : 10 ratio, the tensile and modulus strength were 7.32 and 7.46 MPa, respectively, indicating the superior mechanical strength and toughness of the hydrogel.³⁰ In Fig. 5b, the electric conductivities at5 : 5 and 5 : 10 ratios of PVA/PVDF were 1.3 × 10⁻⁴ S cm⁻¹ and 1.6 × 10⁻⁴ S cm⁻¹ and they were increased to 2.4 × 10⁻⁴ S cm⁻¹ (5 : 10 : 5) and 3.8 × 10–4 S cm⁻¹ (5 : 10 : 10) for the MXene containing the PVA/PVDF hydrogel. This indicates the favorable electroactivity which was influenced by the MXene content. The output open-circuit voltage and short-circuit current signals of PVA/PVDF and the PVA/PVDF/MXene-CCM hydrogel are presented in Fig. 5c and d. The V_pp and I_sc values of the PVA/PVDF/MXene-CCM hydrogel were increased compared to those of PVA/PVDF. Also, the piezoelectric response properties at varying MXene concentration were compared. The V_pp and I_sc values were maximum for the PVA/PVDF/MXene-CCM hydrogel (5 : 10 : 10), and also the piezoelectric constant increased to 7.6 pC N⁻¹ (Table S2). The piezoelectric response was higher due to the increase in MXene content and the intermolecular hydrogen bonding.^31,32

Fig. 5 (a) Tensile properties of PVA/PVDF and the PVA/PVDF/MXene hydrogel with different ratios of PVDF and MXene. (b) Electrical conductivity of the hydrogel with various ratios of PVDF and MXene. (c and d) Output open-circuit voltage (V_pp) and short-circuit current (I_sc) signals of the hydrogel at different ratios of PVDF and MXene.

In vitro piezoelectric catalysis

The catalytic behaviors of the piezoelectric hydrogel under US irradiation were measured using electron spin resonance. A comprehensive study of hydrogel catalytic behaviors in the presence of US irradiation was conducted using DPBF and rhodamine B (Fig. 6a and b). It helps to measure the generation of O₂⁻, –OH levels, and the absorption level of DPBF. Rhodamine B was indirectly proportional to the O₂⁻ and –OH levels. Upon hydrogel treatment with DPBF without US irradiation, there is no change in the absorbance. Upon US irradiation, a gradual decrease in the absorption and further reduction occur with an increase in US treatment time. Similarly, a GSH assay kit was used to measure the hydrogel catalytic oxidation of GSH under US irradiation (Fig. 6c). Without US, no change in the GSH levels was observed, and after US treatment, GSH absorption was decreased, confirming the consumption of GSH by the hydrogel, which converts US stimuli into electrical signals to mimic endogenous electric fields.³³

Fig. 6 The generation of (a) O₂, (b) –OH and (c) oxidizing H₂O to produce –OH radicals by piezoelectric hydrogel under US irradiation identified using DPBF, rhodamine B, and GSH (d) Cytotoxicity of the piezoelectric hydrogel with and without US treatment to MCF-7 cells (n = 3) with statistical significance was determined using ANOVA followed by Tukey's multiple comparison test (**P < 0.0001, ***P < 0.0001).

Cell viability

The piezoelectric hydrogel exhibited a potential cytotoxicity effect to the cancer cells, as shown in Fig. 6d. PVA/PVDF, a piezoelectric hydrogel of concentration 25–200 mg mL⁻¹, was treated with/without US. After 5 min US irradiation, a 50 mg mL⁻¹ concentration shows a gradual decrease in cell viability. Similarly, at concentrations of 100, 150, and 200 mg mL⁻¹, the hydrogel shows an inherent increase in cytotoxicity, which highlights the piezoelectric effect-based tumor cell inhibition. A quantitative study supports that the complete DMSO removal was observed and shows residual below 50 ppm (Table S3). Furthermore, the piezoelectric cytotoxic effects of the hydrogel on cancer cells were measured using the ROS level using DCFH-DA and the results are presented in Fig. S5. Hydrogel-treated cancer cells without US irradiation showed no significant green fluorescence. An intense green fluorescence was observed for the hydrogel after treatment with US. The US-driven piezoelectric effect stimulates the higher ROS level in cancer cells, which leads to DNA damage.³⁴ It was elucidated from γH2AX, a DNA damage marker that measures the level of ROS that cause DNA damage. The PVA/PVDF/MXene-CCM hydrogel after treatment with US produces higher green fluorescence compared to the other groups and also indicates severe DNA damage.³⁵ Also, intracellular CO generation was measured using COP-1, as shown in Fig. S6. Noticeable green fluorescence was observed for both hydrogels during US treatment, and it was not observed for the hydrogel without US. This suggests the robust piezoelectric effect of the hydrogel, which is capable of converting CO₂ into CO under US treatment.³⁶ The excess amount of intracellular CO affects the mitochondrial membrane potential (MMP) in cancer cells, which can be monitored through the JC-1 dye (Fig. S7). Hydrogel alone-treated cells stained with JC-1 show a negligible change in MMP. After US treatment, there is a significant increase in green fluorescence, indicating mitochondrial depolarization (Fig. S8).³⁷

Cellular uptake

The cell penetration ability of the piezoelectric hydrogel was evaluated with BMDCs to identify the antigenicity and adjuvanticity of the hydrogel. In Fig. 7, the FITC-containing hydrogel presents green fluorescence determined using a fluorescent microscope. The cellular uptake efficiency of the hydrogel was increased based on the treatment time; in 5 min treatment, it shows a slight green fluorescence. After the treatment time, the green fluorescence intensity of the cells increases and there was clear visualization of the cellular structure (Fig. S9). The higher cell internalization of the hydrogel was due to the CCM and antigenic components, which also help in stimulating a stronger immune response^38,39

Fig. 7 Intracellular trafficking of the FITC-containing PVA/PVDF/MXene-CCM hydrogel at different time intervals (n = 3).

In vitro piezocatalytic immunotherapy effect

The potential of the piezoelectric hydrogel for tumor immune activation was assessed by measuring the HMGB1 and ATP release levels from cancer cells (Fig. S10). In PVA/PVDF and PVA/PVDF/MXene-CCM hydrogel treatment, the levels of HMGB1 release were 101 and 102 pg mL⁻¹, because it is located in the cell nucleus. After the US, the HMGB1 release was gradually increased and reached 270 and 296 pg mL⁻¹, and the HMGB1 release of 205 pg mL⁻¹ for DOX indicates the major HMGB1 found in the cytoplasm. The concentration of extracellular ATP is 1.0 and 1.1 pg mL⁻¹ for PVA/PVDF and the PVA/PVDF/MXene-CCM hydrogel, respectively. It shows that without US, immune stimulation was insignificant, and then cells treated with PVA/PVDF/MXene-CCM hydrogel + US show ATP release of 5.6 and 5.9 pg mL⁻¹. It was higher compared to the DOX treatment, which shows ATP release of 5.0 pg mL⁻¹. The amplified mitochondrial dysfunction, DNA damage, and ER stress strengthen the immune stimulation along with TLR/anti-PD-L1.^40,41

In vitro DC activation

Bone marrow-derived DCs were treated with the hydrogel, followed by assessing the DC maturation through flow cytometry (Fig. 8a). The BMDCs treated with PVA/PVDF/MXene-CCM hydrogel + US stimulate the immature dendritic cells, increasing the surface synapse formation. It shows a higher level of maturation compared to PVA/PVDF with/without US treatment and the activation is able to increase after combination with TLR/anti-PD-L1.^42,43 The ability of the piezoelectric hydrogel to stimulate CD8+ T-cells was measured by flow cytometry analysis and its hydrogel ability was compared with that of LPS (Fig. 8b). PVA/PVDF showed a lower percentage of CD8+ T-cells, and the PVA/PVDF/MXene-CCM hydrogel was found to have a higher percentage of CD8+ T-cells similar to the LPS. This indicates that the treatment suppresses Treg cell function within the tumor microenvironment and enhances effector T-cell activity. It was further analyzed to determine the secretion of cytokines like TNF-α and IL-12p40 by the ELISA assay (Fig. 8c and d). Compared to free PVA/PVDF, the hydrogel consisting of antigenic CCM and TLR/anti-PD-L1 shows increased cytokine secretion levels.⁴⁴ The higher DC stimulation by the piezoelectric hydrogel was due to the presence of antigenic CCM and TLR/anti-PD-L1 in the hydrogel, and it further helps in the activation of T-cells, inducing a subsequent immune response.^45,46

Fig. 8 Quantification percentages for the activation of (a) DCs and (b) CD8⁺ T-cells after treatment with piezoelectric hydrogel + US and secretion of (c) IL-12p40 and (d) TNF-α from BMDCs treated with piezoelectric hydrogel + US ((i) PBS, (ii) LPS, (iii) PVA/PVDF, (iv) PVA/PVDF/MXene-CCM, (v) PVA/PVDF + US, and (vi) PVA/PVDF/MXene-CCM + US) (n = 3); statistical significance was determined using ANOVA followed by Tukey's multiple comparison test (***P < 0.001 and ****P < 0.0001).

Tumor combating studies

The cell-killing effect and immune stimulation of the piezoelectric hydrogel were evaluated in a tumor-induced mouse model (Fig. 9a). During the experiment, animals were subjected to measurement for the body weight, as shown in Fig. 9b. The treatment does not affect the body weight of the mice, indicating biocompatibility, with no harmful effects of the hydrogel along with US treatment. The tumor inhibition rates of PBS and PVA/PVDF without US were only 2.32 and 2.21%. The PVA/PVDF/MXene-CCM hydrogel without the US group showed modest tumor regression efficacy. It was due to the antigenic cell membrane, TLR 7/8a, and the anti-PD-L1 nanoformulation, which stimulate the cancer-specific immunity.⁴⁷ After US treatment, PVA/PVDF showed tumor inhibition, and the PVA/PVDF/MXene-CCM hydrogel group exhibited a maximum inhibition rate of 89.25%. Additionally, the PVA/PVDF/MXene-CCM hydrogel + US treated mice had a longer lifespan compared to the other groups (Fig. 9c). Notably, 4 out of 6 mice were able to become tumor-free and survived more than 45 days of post-tumor challenge. These results demonstrate the remarkable tumor suppression based on the piezoelectric effect-induced antitumor immunity. The synergistic effect was feasible through the involvement of factors like the cancer cell membrane as a specific antigen, TLR adjuvant, and anti-PD-L1 as an APC recognition motif.^48,49

Fig. 9 Antitumor activities of the piezoelectric hydrogel. (a) The tumor volume of mice after pretreatment with the piezoelectric hydrogel with/without US. (b) Changes in the body weight of mice in different experimental groups. (c) Morbidity-free survival of mice in different groups. (d) The proportion level of CD8⁺ T-cells in the tumor tissue after hydrogel + US treatments ((i) PBS, (ii) LPS, (iii) PVA/PVDF, (iv) PVA/PVDF/MXene-CCM, (v) PVA/PVDF + US, and (vi) PVA/PVDF/MXene-CCM + US); (n = 6) statistical significance was determined using ANOVA followed by Tukey's multiple comparison test (***P < 0.001 and ****P < 0.0001).

In vivo immunological effect

The underlying mechanisms of piezoelectric hydrogel combination treatment in combating tumor progression, immune stimulation, and T-cell infiltration are illustrated in Fig. 9d and 10a and b. The percentage of CD8+ T-cells in the tumor, lymph node, and spleen was measured to determine the piezoelectric-based immunological effect. The levels of CD8+ T-cells were increased from 1.83% (control) to 33.0% for the PVA/PVDF/MXene-CCM hydrogel + US-treated group in the tumor region. There is also an increase in the level of CD8+ T-cells in the spleen and lymph region for PVA/PVDF/MXene-CCM hydrogel + US-treated group compared to the other group. This indicates that the dynamic piezocatalytic effect evokes cancer cell inhibition and generation of CRT, HMGB1, and ATP to stimulate the immune response. This confirms the M2 to M1 polarization of TAM, which is mediated through the ROS, CO, and anti-PD-LI.^50,51

Fig. 10 Quantification of CD8⁺ T-cells in the (a) spleen and (b) lymph nodes via flow cytometry analysis ((i) PBS, (ii) LPS, (iii) PVA/PVDF, (iv) PVA/PVDF/MXene-CCM, (v) PVA/PVDF + US, and (vi) PVA/PVDF/MXene-CCM + US). (c) Photographs of the lungs, spleen, and kidneys of mice after treatment with piezoelectric hydrogel + US (scale bar: 40 μm) (n = 6); statistical significance was determined using ANOVA followed by Tukey's multiple comparison test (***P < 0.001 and ****P < 0.0001).

Histopathology

Histology analysis of animals treated with the piezoelectric hydrogel with and without US is shown in Fig. 10c. All groups exhibited minimal inflammatory damage, as evidenced by the histological analysis of tissue samples (Table S4). In lung tissue histology, the sponge-like structure, clear alveoli, and interalveolar septa indicate the biosafe nature of PVA/PVDF/MXene-CCM + US. There is no significant change in spleen tissue; it formed a distorted lymphoid structure and lymphoid follicles. The kidney tissue sample after treatment with piezoelectric hydrogel + US shows good glomeruli and distinct tubules.⁵² Majorly, histological changes in the lung, spleen, and kidneys did not reach statistical significance, and they exhibited very similar results, which showed biocompatibility of the hydrogel.

Conclusions

In summary, we have successfully engineered a piezoelectric PVA/PVDF/MXene-CCM hydrogel consisting of anti-PD-L1, a TLR 7/8a agonist. The self-assembled hydrogel formed through hydrogen bonding, electroactive β-phase, and high cross-linking density provides superior mechanical strength and toughness to the hydrogel. Under US stimulation, the mechanical energy generated by physical activity converts into electrical energy. The piezoelectric stimulation to the tumor site regulates cancer cell inhibition, ER stress, and mitochondrial membrane depolarization through apoptosis pathways. Piezoelectric-induced cancer cell death releases the tumor antigens and DAMPs to enhance the immunogenicity along with TLR 7/8a and anti-PD-L1. The combined effect facilitates the binding and activation of antigen presenting cells, production of cytotoxic T-cells, and retention in the lymph node. Therefore, the developed piezoelectric hydrogel demonstrates substantial potential for cancer therapy by integrating piezocatalysis with immune checkpoint blockade (ICB), offering strong prospects for clinical translation.

Conflicts of interest

There are no conflicts of interest to declare.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of J.J. College of Arts and Science, Pudukottai and approved by the Institutional Animal Ethics Committee (AIEC).

Data availability

The author confirms that the data supporting the findings of this study are available within the article and its supplementary information (SI). Supplementary information: DLS and zeta potential analysis, FTIR, XRD, and EDAX analyses of the MXene, SEM analysis of MX/CCM, FTIR and XRD analyses of PVA/PVDF, dielectric constant of the PVA/PVDF/MXene, intracellular ROS generation, DNA damage, intracellular CO generation, intracellular mitochondrial membrane potential, relative fluorescence intensity, ATP secretion, and HMGB1 release. See DOI: https://doi.org/10.1039/d5bm01202c.

Acknowledgements

We gratefully acknowledge the support from MHRD-RUSA 2.0 and the University of Madras for providing financial support. We thank DST-FIST-funded X-Ray Diffractometer Facility, the National Centre for Nanoscience and Nanotechnology, the University of Madras.

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