The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum

Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the “protein corona”. To simplify studies of protein–NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography–mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.     

Formation of a protein corona influences the biological identity of nanomaterials

The development and testing of nanomaterials is an area of interest due to promising diagnostic and therapeutic applications in the treatment of diseases like cancer or cardiovascular disease. While extensive studies of the physicochemical properties of nanoparticles (NPs) are available, the investigation of the protein corona (PC) that is formed on NPs in biofluids is a relatively new area of research. The fact that few NPs are in clinical use indicates that the biological identity of NPs, which is in large part due to the PC formed in blood or other bodily fluids, may be altered in ways yet to be fully understood. Herein, we review the recent advances in PC research with the intent to highlight the current state of the field. We discuss the dynamic processes that control the formation of the PC on NPs, which involve the transient soft corona and more stable hard corona. Critical factors, like the environment and disease-state that affect the composition and stability of the PC are presented, with the intent of showcasing promising applications for utilizing the PC for disease diagnosis and the identification of disease-related biomarkers. This review summarizes the unique challenges presented by the nanoparticle corona and indicates future directions for investigation.     

An integrative method for evaluating the biological effects of nanoparticle-protein corona

BackgroundNanoplastics in the environment can enter the human body through gastrointestinal intake, dermal contact, and pulmonary inhalation, posing a threat to human health. Protein molecules in body fluids will quickly adsorb on the surfaces of the nanoplastics, forming a protein corona, which has implications for the interaction of the nanoplastics with cells and the metabolic pathways of the nanoplastic within cells. For years, practical tools such as dynamic light scattering, transmission electron microscopy, and liquid chromatography have been developed to understand the protein corona of nanoparticles (NPs), either in vitro or in cellular or molecular level. However, an integrated approach to understand the nanoparticles-protein corona is still lacking.MethodsUsing the most frequently observed environmental nanoplastics, polystyrene nanoplastics (PS), as a standard, we established an integrative structural characterization platform, a biophysical and biochemical evaluation method to investigate the effect of surface charge on protein corona composition. The cellular and molecular mechanisms were also explored through in vitro cellular experiments.ResultsThe first integrative method for characterizing biological properties of NPs-protein corona has been established. This method comprehensively covers the critical aspects to understand NPs-protein corona interactions, from structure to function.ConclusionsThe integrative method for nanoplastics microstructure characterization can be applied to the structural characterization of nanoparticles in nanoscale, which is of universal significance from in vitro characterization to cellular experiments and then to molecular mechanism studies.General significanceThis strategy has high reliability and repeatability and can be applied both in environment and nanomedicine safety assessment.     

A health concern regarding the protein corona,aggregation and disaggregation

Nanoparticle (NP)–protein complexes exhibit the “correct identity” of NP in biological media. Therefore, protein–NP interactions should be closely explored to understand and modulate the nature of NPs in medical implementations. This review focuses mainly on the physicochemical parameters such as dimension, surface chemistry, morphology of NPs, and influence of pH on the formation of protein corona and conformational changes of adsorbed proteins by different kinds of techniques. Also, the impact of protein corona on the colloidal stability of NPs is discussed. Uncontrolled protein attachment on NPs may bring unwanted impacts such as protein denaturation and aggregation. In contrast, controlled protein adsorption by optimal concentration, size, pH, and surface modification of NPs may result in potential implementation of NPs as therapeutic agents especially for disaggregation of amyloid fibrils. Also, the effect of NPs-protein corona on reducing the cytotoxicity and clinical implications such as drug delivery, cancer therapy, imaging and diagnosis will be discussed. Validated correlative physicochemical parameters for NP–protein corona formation frequently derived from protein corona fingerprints of NPs which are more valid than the parameters obtained only on the base of NP features. This review may provide useful information regarding the potency as well as the adverse effects of NPs to predict their behavior in vivo.     

In-vitro in-vivo correlation (IVIVC) in nanomedicine: Is protein corona the missing link?

One of the unmet challenges in nanotechnology is to understand and establish the relationship between physicochemical properties of nanoparticles (NPs) and its biological interactions (bio-nano interactions). However, we are still far from assessing the biofate of NPs in a clear and unquestionable manner. Recent developments in the area of bio-nano interface and the understanding of protein corona (PC) has brought new insight in predicting biological interactions of NPs. PC refers to the spontaneous formation of an adsorbed layer of biomolecules on the surface of NPs in a biological environment. PC formation involves the spatiotemporal interplay of an intricate network of biological, environmental and particle characteristics. NPs with its PC can be viewed as a biological entity, which interacts with cells and barriers in a biological system. Recent studies on the bio-nano interface have revealed biological signatures that participate in cellular and physiological bioprocesses and control the biofate and toxicity of NPs. The ability of in-vitro derived parameters to forecast in-vivo consequences by developing a mathematical model forms the basis of in-vitro in-vivo correlation (IVIVC). Understanding the effect of bio-nano interactions on the biological consequences of NPs at the cellular and physiological level can have a direct impact on the translation of future nanomedicines and can lead to the ultimate goal of developing a mathematical IVIVC model. The review summarizes the emerging paradigms in the field of bio-nano-interface which clearly suggests an urgent need to revisit existing protocols in nanotechnology for defining the physicochemical correlates of bio-nano interactions.     

Personalized liposome–protein corona in the blood of breast,gastric and pancreatic cancer patients

When nanoparticles (NPs) are dispersed in a biofluid, they are covered by a protein corona the composition of which strongly depends on the protein source. Recent studies demonstrated that the type of disease has a crucial role in the protein composition of the NP corona with relevant implications on personalized medicine. Proteomic variations frequently occur in cancer with the consequence that the bio-identity of NPs in the blood of cancer patients may differ from that acquired after administration to healthy volunteers. In this study we investigated the correlation between alterations of plasma proteins in breast, gastric and pancreatic cancer and the biological identity of clinically approved AmBisome-like liposomes as determined by a combination of dynamic light scattering, zeta potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) and semi-quantitative densitometry. While size of liposome–protein complexes was not significantly different between cancer groups, the hard corona from pancreatic cancer patients was significantly less negatively charged. Of note, the hard corona from pancreatic cancer patients was more enriched than those of other cancer types this enrichment being most likely due to IgA and IgG with possible correlations with the autoantibodies productions in cancer. Given the strict relationship between tumor antigen-specific autoantibodies and early cancer detection, our results could be the basis for the development of novel nanoparticle-corona-based screening tests of cancer.     

Toward a molecular understanding of nanoparticle–protein interactions

Wherever nanoparticles (NPs) come in contact with a living organism, physical and chemical interactions take place between the surfaces of the NPs and biomatter, in particular proteins. When NP are exposed to biological fluids, an adsorption layer of proteins, a “protein corona” forms around the NPs. Consequently, living systems interact with the protein-coated NP rather than with a bare NP. To anticipate biological responses to NPs, we thus require comprehensive knowledge of the interactions at the bio–nano interface. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP–protein interactions. In this brief review, we present the latest findings regarding the composition of the protein corona as it forms on NPs in the blood stream. We also discuss molecular aspects of this adsorption layer and its time evolution. The current state of knowledge is summarized, and issues that still need to be addressed to further advance our understanding of NP–protein interactions are identified.     

Identifying Metal Nanoparticle Size Effect on Sensing Common Human Plasma Protein by Counting the Sensitivity of Optical Absorption Spectra Damping

Colloidal nanoparticles (NPs) interact with biological fluids such as human plasma to form a protein coating (corona) on the surface of NPs (NP-protein complex). However, the impact of size and type of NPs on binding of the hard corona to the surface of NPs as well as damping of their optical spectra has not been systematically explored. To elucidate the interaction between biological environment (human plasma) and NPs, a photophysical measurement was conducted to quantify the interaction of two different types of NPs (gold (Au) and silver (Ag)) with common human plasma proteins. The colloidal AuNPs and AgNPs were electrostatically stabilized and varied in diameter from 10 to 80 nm in the presence of common human plasma. The sizes of the NPs were determined using transmission electron microscopy (TEM). Optical absorption spectra were obtained for the complexes. Dynamic light scattering (DLS) measurement and zeta potential were used to characterize the sizes, hydrodynamic diameters, and surface charges of the protein-NPs complexes. Protein separation was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to isolate and identify the protein bands. The absorption of proteins to the NPs was found to be strongly dependent on the size and type of NPs. The distance between surface of NPs by absorbed protein bound to the NPs gradually increased with size of NPs, particularly for AgNPs with primary diameter of < 50 nm. The chi-square test proved that AgNPs are a good candidate in sensing the protein complex in human plasma compared with AuNPs mainly for the AgNPs with diameter sized 50 nm.

     

Physicochemical properties and in vitro intestinal permeability properties and intestinal cell toxicity of silica particles,performed in simulated gastrointestinal fluids

Background

Amorphous silica particles with the primary dimensions of a few tens of nm, have been widely applied as additives in various fields including medicine and food. Especially, they have been widely applied in powders for making tablets and to coat tablets. However, their behavior and biological effects in the gastrointestinal tracts associated with oral administration remains unknown.

Methods

Amorphous silica particles with diameters of 50, 100, and 200 nm were incubated in the fasted-state and fed-state simulated gastric and intestinal fluids. The sizes, intracellular transport into Caco-2 cells (model cells for intestinal absorption), the Caco-2 monolayer membrane permeability, and the cytotoxicity against Caco-2 cells were then evaluated for the silica particles.

Results

Silica particles agglomerated in fed-state simultaneous intestinal fluids. The agglomeration and increased particles size inhibited the particles' absorption into the Caco-2 cells or particles' transport through the Caco-2 cells. The in vitro cytotoxicity of silica particles was not observed when the average size was larger than 100 nm, independent of the fluid and the concentration.

Conclusion

Our study indicated the effect of diet on the agglomeration of silica particles. The sizes of silica particles affected the particles' absorption into or transport through the Caco-2 cells, and cytotoxicity in vitro, depending on the various biological fluids.

General significance

The findings obtained from our study may offer valuable information to evaluate the behavior of silica particles in the gastrointestinal tracts or safety of medicines or foods containing these materials as additives.     

Transferrin coated nanoparticles: study of the bionano interface in human plasma

It is now well established that the surface of nanoparticles (NPs) in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC) and after washing (hard corona, HC) in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.     

Biosynthesized silver nanoparticles for inhibition of antibacterial resistance and biofilm formation of methicillin-resistant coagulase negative Staphylococci

The ability of a natural stabilizing and reducing agent on the synthesis of silver nanoparticles (Ag NPs) was explored using a rapid and single-pot biological reduction method using Nocardiopsis sp. GRG1 (KT235640) biomass. The UV–visible spectral analysis of Ag NPs was found to show a maximum absorption peak located at a wavelength position of ∼422 nm for initial conformation. The major peaks in the XRD pattern were found to be in excellent agreement with the standard values of metallic Ag NPs. No other peaks of impurity phases were observed. The morphology of Ag NPs was confirmed through TEM observation, demonstrating that the particle size distribution of Ag NPs entrenched in spherical particles is in a range between 20 and 50 nm. AFM analysis further supported the nanosized morphology of the synthesized Ag NPs and allowed quantifying the Ag NPs surface roughness. The synthesized Ag NPs showed significant antibacterial and antibiofilm activity against biofilm positive methicillin-resistant coagulase negative Staphylococci (MR-CoNS), which were isolated from urinary tract infection as determined by spectroscopic methods in the concentration range of 5–60 µg/ml. The inhibition of biofilm formation with coloring stain was morphologically imaged by confocal laser scanning microscopy (CLSM). Morphological alteration of treated bacteria was observed by SEM analysis. The results clearly indicate that these biologically synthesized Ag NPs could provide a safer alternative to conventional antibiofilm agents against uropathogen of MR-CoNS.     

Toxicity evaluation of manufactured CeO2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells

Background

Engineered nanomaterials may release nanosized residues, by degradation, throughout their life cycle. These residues may be a threat for living organisms. They may be ingested by humans through food and water. Although the toxicity of pristine CeO₂ nanoparticles (NPs) has been documented, there is a lack of studies on manufactured nanoparticles, which are often surface modified. Here, we investigated the potential adverse effects of CeO₂ Nanobyk 3810™ NPs, used in wood care, and their residues, altered by light or acid.

Results

Human intestinal Caco-2 cells were exposed to residues degraded by daylight or in a medium simulating gastric acidity. Size and zeta potential were determined by dynamic light scattering. The surface structure and redox state of cerium were analyzed by transmission electronic microscopy (TEM) and X-ray absorption spectroscopy, respectively. Viability tests were performed in Caco-2 cells exposed to NPs. Cell morphology was imaged with scanning electronic microscopy. Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs. All CeO₂ NPs suspended in the culture medium became microsized. Cytotoxicity tests showed no toxicity after Caco-2 cell exposure to these various NPs up to 170 μg/mL (24 h and 72 h). Nevertheless, a more-sensitive whole-gene-expression study, based on a pathway-driven analysis, highlighted a modification of metabolic activity, especially mitochondrial function, by altered Nanobyk 3810™. The down-regulation of key genes of this pathway was validated by qRT-PCR. Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.

Conclusion

The degraded nanoparticles were more toxic than their coated counterparts. Desorption of the outside layer was the most likely cause of this discrepancy in toxicity. It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-700) contains supplementary material, which is available to authorized users.     

Study of the Adsorption of Human Hemoglobin to Silver (Ag) Nanoparticle Surface for the Detection of the Unfolding of Hemoglobin

In the present report, we focused on the detail study of the optical properties and structural characterization of the Ag NPs for the nanobioconjugate analysis and detection of the conformational structural change of the Hb. The detail optical and structural analysis of Ag NPs has been studied from UV–Vis absorption, emission spectrum, XRD, and HRTEM study. The proteins/Hb are attached immediately onto Ag NPs surface when NPs touch the biological fluids, forming protein corona (PC), which gives their biological identity. The NPs-PC bioconjugate is, more specifically, the true identity of NPs in the physiological world. The adsorption of Hb with Ag NP surfaces has been studied by monitoring the soret band and tryptophan band of Hb. The dynamics of the Hb adsorption on the Ag NPs showed the time constant of surface binding t₁?=?5.79 min and 10.23 min and surface reorganization t₂?=?500 min and 251.75 min with the use of small and large concentrations of Ag NPs, respectively. The absorption peak shape and size around the wavelength, λ ≈ 406.2 nm of the bioconjugate has been examined by Gaussian and Lorentz curve fitting analysis. The bioconjugate along with the PC formation has been analyzed by HRTEM images and DLS observations. The tertiary deformation of Hb and energy transfer efficiency connecting Ag NPs and Hb are discussed from the emission-quenching phenomenon. The change of the secondary structural elements (α-helix, β-sheets, intermolecular aggregates, intramolecular aggregates) of the bioconjugate has been analyzed from FTIR spectrum.

     

Stability and absorption mechanism of typical plant miRNAs in an in vitro gastrointestinal environment: basis for their cross-kingdom nutritional effects

Plant miRNAs, a group of 19–24 nt noncoding RNAs from plant foods, were recently found to have immunomodulatory and nutritional effects on mammalian and human bodies. However, how the miRNAs survive gastrointestinal (GI) environment and how the stable miRNAs are absorbed, which serve the basis for their biological functions, were not unraveled. Here, we investigated the stabilities of six typical plant miRNAs in simulated gastric and intestinal environments, and the absorption mechanisms by Caco-2 cells. The results showed that the miRNAs can survive the environment with certain concentrations. The mixture of food ingredients enhanced the stabilities of the plant miRNAs in the gastric conditions, while 2′-O-methyl modification protects the miRNAs in intestinal juice. The stabilities of the miRNAs vary significantly in the environment and are related to their secondary structures. The stable plant miRNAs can be absorbed by Caco-2 cells via clathrin- and caveolin-mediated endocytosis. Uptake of the miRNAs was sequence dependent, facilitated by NACh and TLR9, two typical receptors on cell membrane. The results suggest that some of plant miRNAs are stable in the mimic GI environment and can be absorbed by Caco-2 cells, underlying the potential of their cross-kingdom regulation effects.     

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO₂ NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO₂ NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.     

A hard-sphere model of protein corona formation on spherical and cylindrical nanoparticles

A nanoparticle (NP) immersed in biological media rapidly forms a corona of adsorbed proteins, which later controls the eventual fate of the particle and the route through which adverse outcomes may occur. The composition and timescale for the formation of this corona are both highly dependent on both the NP and its environment. The deposition of proteins on the surface of the NP can be imitated by a process of random sequential adsorption, and, based on this model, we develop a rate-equation treatment for the formation of a corona represented by hard spheres on spherical and cylindrical NPs. We find that the geometry of the NP significantly alters the composition of the corona through a process independent of the rate constants assumed for adsorption and desorption of proteins, with the radius and shape of the NP both influencing the corona. We further investigate the roles of protein mobility on the surface of the NP and changes in the concentration of proteins.     

Modeling the Time Evolution of the Nanoparticle-Protein Corona in a Body Fluid

Background

Nanoparticles in contact with biological fluids interact with proteins and other biomolecules, thus forming a dynamic corona whose composition varies over time due to continuous protein association and dissociation events. Eventually equilibrium is reached, at which point the continued exchange will not affect the composition of the corona.

Results

We developed a simple and effective dynamic model of the nanoparticle protein corona in a body fluid, namely human plasma. The model predicts the time evolution and equilibrium composition of the corona based on affinities, stoichiometries and rate constants. An application to the interaction of human serum albumin, high density lipoprotein (HDL) and fibrinogen with 70 nm N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles is presented, including novel experimental data for HDL.

Conclusions

The simple model presented here can easily be modified to mimic the interaction of the nanoparticle protein corona with a novel biological fluid or compartment once new data will be available, thus opening novel applications in nanotoxicity and nanomedicine.     

Evaluation of Lactobacillus strains for selected probiotic properties

Eleven strains of Lactobacillus collected in the Culture Collection of Dairy Microorganisms (CCDM) were evaluated for selected probiotic properties such as survival in gastrointestinal fluids, antimicrobial activity, and competition with non-toxigenic Escherichia coli O157:H7 for adhesion on Caco-2 cells. The viable count of lactobacilli was reduced during 3-h incubation in gastric fluid followed by 3-h incubation in intestinal fluid. All strains showed antimicrobial activity and the three most effective strains inhibited the growth of at least 16 indicator strains. Antimicrobial metabolites of seven strains active against Lactobacillus and Clostridium indicator strains were found to be sensitive to proteinase K and trypsin, which indicates their proteinaceous nature. The degree of competitive inhibition of non-toxigenic E. coli O157:H7 adhesion on the surface of Caco-2 cells was strain-dependent. A significant decrease (P?<?0.05) in the number of non-toxigenic E. coli O157:H7 adhering to Caco-2 cells was observed with all lactobacilli. Three strains were selected for additional studies of antimicrobial activity, i.e., Lactobacillus gasseri CCDM 215, Lactobacillus acidophilus CCDM 149, and Lactobacillus helveticus CCDM 82.     

Toxicity of commercially available engineered nanoparticles to Caco-2 and SW480 human intestinal epithelial cells

The effects of ingestion of engineered nanoparticles (NPs), especially via drinking water, are unknown. Using NPs spiked into synthetic water and cell culture media, we investigated cell death, oxidative stress, and inflammatory effects of silver (Ag), titanium dioxide (TiO₂), and zinc oxide (ZnO) NPs on human intestinal Caco-2 and SW480 cells. ZnO NPs were cytotoxic to both cell lines, while Ag and TiO₂ NPs were toxic only at 100 mg/L to Caco-2 and SW480, respectively. ZnO NPs led to significant cell death in synthetic freshwaters with 1 % phosphate-buffered saline in both cell lines, while Ag and TiO₂ NPs in buffered water led to cell death in SW480 cells. NP exposures did not yield significant increased reactive oxygen species generation but all NP exposures led to increased IL-8 cytokine generation in both cell lines. These results indicate cell stress and cell death from NP exposures, with a varied response based on NP composition.     

Stiffness of the extracellular matrix affects apoptosis of nucleus pulposus cells by regulating the cytoskeleton and activating the TRPV2 channel protein

It is known that nucleus pulposus cells (NPs) play an important role in intervertebral disc degeneration (IVDD), and a previous study indicated that the stiffness of NP tissue changes during the degeneration process. However, the mechanism underlying the cellular response to ECM stiffness is still unclear. To analyze the effects of extracellular matrix (ECM) with different degrees of stiffness on NPs, we prepared polyacrylamide (PA) gels with different elastic moduli, and cells grown under different stiffness conditions were obtained and analyzed. The results showed that the spreading morphology of NPs changed significantly under increased ECM elastic modulus conditions and that TRPV2 and the PI3K / AKT signaling pathway were activated by stiffer ECM. At the same time, mitochondria released cytochrome c (Cyt c) and activated caspase proteins to promote the apoptosis of NPs. After TRPV2 was specifically knocked out, the activation of the PI3K / AKT signaling pathway decreased, and the release of Cyt c and NP apoptosis were reduced. These results indicate that TRPV2 is closely linked to the detection of extracellular mechanical signals, and that conversion of mechanical and biological signals plays an important role in regulating the biological behavior of cells. This study offers a new perspective on the cellular and biochemical events underlying IVDD which could result in novel treatments.