Osmosis, as a force effect, plays a crucial role in facilitating the flow of water and regulating water–electrolyte metabolism. It maintains vital physiological functions in the human body, including cell volume and structural alterations. Furthermore, osmosis significantly influences the occurrence and progression of certain diseases. Cytotoxic astrocyte swelling is responsible for the pathogenesis of brain edema in trauma, ischemia, hemorrhage, inflammation, and hepatic encephalopathy ^[1][2](Zhang et al. 2019, Zheng et al. 2025). Osmotic gradients across the cell membrane upregulate the permeability of pulmonary endothelial and alveolar barriers, which is associated with acute respiratory distress syndrome and pulmonary edema (Qian et al. ^[3]2022). Moreover, an increase in the intracellular osmotic effect destroys the blood–brain barrier function, resulting in the development of degenerative neurological disorders, including stroke, Alzheimer’s disease, Parkinson’s disease, and glioblastoma ^[4](Li et al. 2021). In addition, the electrolyte injection of sodium ions may affect cytoplasmic PNs and regulate transmembrane osmotic gradient by changing the concentrations of albumin and various ions in hypertensive human umbilical vein endothelial cells, which eventually damage vascular endothelial cells ^[5](Song et al. 2023). Furthermore, Osmotic effect also exerts a pivotal role in the progression of cancer, in which osmotic tension asymmetry orchestrates tumor cell migration (Zhu et ^[6]al. 2025). Thus, osmosis investigations into physiological and pathological activities are crucial for the development of modern medicine and for providing reasonable interpretations of osmotic effects in regulating water metabolism, cell swelling, and pathological morphogenesis.

Van't Hoff pioneered osmotic pressure (OP) research using semipermeable membranes and showed that OP depends solely on the number of solute particles, regardless of their chemical identity or size (Lu et ^[7]al., 2012). This principle led to the invention of the osmometer ^[8](Larkins et al., 2025), which essentially measures total solute concentration rather than the mechanical effect of osmotic gradient.

However, physiological and cellular osmotic behavior diverges from the traditional view that OP and cell volume are determined only by total solute concentration. For instance, isotonic urea solutions, unlike saline or glucose, can induce sequential shrinkage followed by hypertonic rupture in red blood cells, as well as hypotonic shrinkage and even rupture in nerve cells, which indicates that the osmotic effect depends on solute type rather than total concentration in live cell. Furthermore, under Van't Hoff theory, colloid osmotic pressure (COP) from plasma proteins like albumin, contributes only marginally (e.g., 42 mg/mL albumin yields ~25 mmHg or 1.3 mOsm/kg) to total OP. Clinically, albumin-driven COP plays a critical role in fluid homeostasis ^[9][10](Campos Munoz et al., 2025; Moman et al., 2025), suggesting more complex regulatory mechanisms beyond classical OP theory.

Notably, aside from red blood cells and neurons, many cell types exhibit a regulatory volume decrease (RVD) in response to hypotonic stress and a regulatory volume increase (RVI) under hypertonic conditions, behaviors that are inconsistent with the classical view that cell volume changes are solely determined by the total solute concentration ^[11](Bortner & Cidlowski, 2020;). Furthermore, astudies have clearly shown that intracellular and extracellular ion concentrations are not always in equilibrium. A prime example is found in early as the middle of the last century, study has clearly pointed out that the total amount of ions inside and outside cells is not equal (Conway et al., 1955), especially, renal medullary cells, which can tolerate urea concentrationsa hypertonic urea environment up to four times the physiological levels (around concentration (1200 mOsm/kg) while maintaining their urea filtration and urine-concentrating functions ^[13](Sands & Layton, 2014).

Experimental results suggest that the Van't Hoff theory fails to adequately explain regulation of osmotic effect in live cells. In the context of the widespread use of osmometers, the view of relying on "the total amount of ions and particles" to evaluate the osmotic effect of cells is likely to lead to misunderstandings about the role of the cell osmosis. Therefore, it is essential to reconsider the mechanisms underlying cellular osmotic effect and explore a more comprehensive theoretical framework to accurately determine its role in physiological and pathological processes.

To overcome these limitations, we developed a novel bio-osmotic force detection system based on fluorescence resonance energy transfer (FRET)-labeled intermediate filament (IF) tension probes and tension vector analysis tools (Guo et al., 2014; Zhang et al., ^[1][14]2019). The system employs cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) as the acceptor. In the absence of a mechanical force, CFP and YFP remain closely aligned, yielding high FRET efficiency. Upon application of mechanical tension (such as that from osmotic force changes), the conformation of the cpstFRET module is altered, reducing FRET efficiency in a quantifiable manner. This detection system utilizes mechanical effects as the indicator, enabling the analysis of the dynamic change of the transmembrane osmotic gradient of human living cells. It thereby breaks the dependence on semi-permeable membrane assumptions and total solute concentrations, providing a more physiologically relevant and mechanically precise view of bio-osmotic force. This bio-optomechanical approach offers a powerful new direction to explore the interplay between osmotic force and bioelectric activity in live-cell systems ^{[2][3][15][16]}(Qian et al., 2022; Zheng et al., 2025; Zheng et al., 2023; Zheng, Wang, et al., 2022).

Free protein nanoparticles (PNs), formed through cytoskeletal depolymerization and inflammasome activation, was found to play a key role in modulating the bio-osmotic force, exerting ion-specific regulatory effects that vary with ionic species ^[3](Qian et al., 2022). Based on this, we propose the concept of the bio‑osmotic force ^[17][18](Wang & Guo, 2025; Wang et al., 2023; Zheng, Hu, et al., 2022), a physiologically relevant osmotic phenomenon dependent on the selectivity and permeability of membrane channels. Unlike the classical osmotic theories, the bio‑osmotic force theory reflects a force-electricity coupling mechanism wherein cellular mechanical responses are tightly coordinated with PN levels, ionic composition, and membrane potential dynamics. By establishing an osmotic imbalance, cells gain the ability to autonomously regulate shape deformation, volume changes, and migratory behaviors. This theory provides a reasonable explanation for how increased the PN levels can induce both hypotonic-like shrinkage and hypertonic-like expansion in living cells, depending on specific ionic and membrane conditions. Furthermore, it also reveals the mechanisms underlying membrane potential transitions (hyperpolarization and depolarization), the differential activation of sodium and potassium ion channels and the selective diffusion of various ions.

Cytoplasmic PNs play a pivotal role in regulating the osmotic force of live cells by modulating membrane potential dynamics through synergistic interactions with calcium ions and water molecules. Inhibition of PNs formation has been shown to restore membrane polarization, suppress the activation of voltage-gated channels (Sur1-TRPM4, TMEM16A), and alleviate edema. PNs and Ca²⁺ exhibit antagonistic effects on membrane potential transitions, contributing to the regulation of hyperpolarization and depolarization (Zheng ^[2]et al., 2025).

Moreover, study has proven that mercury ions can modulate cellular ion diffusion independently of water flux ^[2](Zheng et al., 2025), indicating that the movement of ions and water are two independent events in cells. Ion influx mediated by channels is not necessarily associated with osmotic force regulation. Osmotic force regulation of human cells does not follow the characteristics of a semi‑permeable membrane.

Additionally, the activity of non-selective ion channels (such as TRPM4 and TMEM16A) is chemically regulated. For instance, in glutamate receptor-induced cell swelling, intracellular signaling molecules, such as calmodulin (CaM) and protein kinase C-α (PKCα), significantly increase the sensitivity of these channels (Zheng ^[15]et al., 2023), leading to enhanced ion influx and water retention. These findings underscore the indispensable role of intracellular signaling pathways in bio-osmotic force regulation.

Importantly, it is the differential permeability of voltage-dependent ion channels, rather than the total ion concentration, that governs water movement and modulates the mechanical effects of biological osmotic forces. The activation of calcium-induced voltage-dependent non-selective ion channels (e.g., TRPM4 and TMEM16A) is crucial for cellular edema formation, primarily through a mechanism involving massive influx of sodium and chloride ions, coupled with limited potassium efflux, resulting in an intracellular hypertonic state ^[15](Zheng et al., 2023). Conversely, the activation of hyperpolarization-activated potassium channels promotes potassium efflux, driving cell shrinkage, a phenomenon known as the Gardos effect ^[6](Zhu et al., 2025). Together, these findings indicate that the generation of an intracellular hypotonic or hypertonic condition depends on the differential ion permeability of various ion channels. Thus, transmembrane osmotic gradient is a product of differential ion channel regulation, reflecting an active and dynamic process that cannot be fully explained by traditional passive diffusion models. Therefore, based on the above research and experimental evidence, we propose that the cell does not exist in a state of osmotic equilibrium, but rather in a state of an opposing balance between different ion-specific osmotic effects. During cell swelling, the selective influx of sodium and chloride ions diminishes outheir inward osmotic effect of water. However, this does not directly promote corresponding water entry, which is a consequence of the inverse relationship between ion diffusion and water osmosis. Instead, this swelling is primarily driven by the activation of non‑selective ion channels, including TRPM4 and TMEM16A, which is accompanied by the reverse diffusion of intracellular potassium and other anions. This process enhances the inoutward osmotic force, drawing water into the cell and leading to cellular swelling ^[17][18](Wang & Guo, 2025; Zheng, Hu, et al., 2022). A similar mechanism underlies cell shrinkage, in which the large efflux of K⁺ reduces their inoutward osmotic effect, but does not induce a hypotonic state, because the efflux cannot directly promote water exit. Rather, the reverse influx of sodium ions during this process increases the outinward osmosis, thereby driving water out of the cell and resulting in cell shrinkage (Wang & Guo, ^[17][18]2025; Zheng, Hu, et al., 2022). If intracellular and extracellular environments were strictly isotonic, this form of reverse energy conversion would not occur, highlighting the dynamic and directional nature of osmotic regulation in live cells.

The bio-osmotic force theory thus offers a conceptual framework that is fundamentally distinct the classical definition of osmosis, describing a unique osmotic force regulation mechanism. The theory proposes that autonomous volume regulation (swelling and shrinkage) and cell movement are mediated through the coordinated activity of membrane potential shifts and ion channel dynamics. It also provides a mechanistic basis for PNs-dependent hypertonic expansion and hypotonic shrinkage, which are induced by various physicochemical stimuli, including extracellular hypertonic and hypotonic treatments, and regulated by PN-mediated modulation of membrane excitability and selective ion transport.

In summary, we propose a novel theory of biological osmotic force based on the electromechanical synergy of live cells, offering a conceptual breakthrough beyond traditional osmotic pressure models, such as Fick's law, Van't Hoff’s theory, the Donnan effect, and the electrical double-layer theory. By identifying PNs as key regulators of membrane potential and ion channel activity, this framework redefines osmotic force of live cell as an actively modulated, dynamic biophysical process, rather than a passive diffusion phenomenon. The development of a FRET-based intermediate filament tension probe system enables, for the first time, real-time mechanical monitoring of transmembrane osmotic force dynamics in live cells, providing a methodological innovation that overcomes the inherent limitations of conventional osmometry.

More importantly, this theory bridges the gap between physical chemistry and cell physiology, revealing how bio-osmotic force integrates with electrical signaling, cytoskeletal remodeling, and biochemical pathways to maintain cellular homeostasis. It not only challenges the prevailing understanding of osmotic balance, but also opens new avenues to decipher the mechanobiological basis of complex diseases, such as cerebral edema, inflammation-induced swelling, and cancer metastasis.

Ultimately, the bio-osmotic force theory is not merely an update of classical models—it represents a paradigm shift in how we understand cell-environment interactions. Its integration into future physiological, pathological, and clinical frameworks holds the potential to reshape foundational knowledge in cell biology and inspire the development of novel diagnostic and therapeutic strategies.

References

Bortner CD, Cidlowski JA (2020) Ions, the Movement of Water and the Apoptotic Volume Decrease. Front Cell Dev Biol 8:611211. https://doi.org/10.3389/fcell.2020.611211

Campos Munoz A, Jain NK, Gupta M (2025) Albumin Colloid. StatPearls Publishing, Treasure Island (FL)

Conway, E. J., Geoghegan, H., & McCormack, J. I. (1955). Autolytic changes at zero centigrade in ground mammalian tissues. The Journal of Physiology, 130(2), 427-437. https://doi.org/10.1113/jphysiol.1955.sp005416

Guo J, Wang Y, Sachs F, Meng F (2014) Actin stress in cell reprogramming. Proc Natl Acad Sci U S A 111:E5252-5261. https://doi.org/10.1073/pnas.1411683111

Larkins, M. C., Zubair, M., & Thombare, A. (2025). Osmometer. In StatPearls. StatPearls Publishing.

Li C, Chen L, Wang Y, Wang T, Di D, Zhang H, Zhao H, Shen X, Guo J (2021) Protein Nanoparticle-Related Osmotic Pressure Modifies Nonselective Permeability of the Blood-Brain Barrier by Increasing Membrane Fluidity. Int J Nanomedicine 16:1663–1680. https://doi.org/10.2147/IJN.S291286

Lu Y, Wang L, Chen D, Wang G (2012) Determination of the concentration and the average number of gold atoms in a gold nanoparticle by osmotic pressure. Langmuir 28:9282–9287. https://doi.org/10.1021/la300893e

Moman RN, Gupta N, Varacallo MA (2025) Physiology, Albumin. StatPearls, Treasure Island (FL)

Qian Z, Wang Q, Qiu Z, Li D, Zhang C, Xiong X, Zheng Z, Ruan Q, Guo Y, Guo J (2022) Protein nanoparticle-induced osmotic pressure gradients modify pulmonary edema through hyperpermeability in acute respiratory distress syndrome. J Nanobiotechnology 20:314. https://doi.org/10.1186/s12951-022-01519-1

Sands, J. M., & Layton, H. E. (2014). Advances in Understanding the Urine-Concentrating Mechanism. Annual Review of Physiology, 76(1), 387-409. https://doi.org/10.1146/annurev-physiol-021113-170350

Song X, Li D, Gan L, Xiong X, Nie A, Zhao H, Hu Y, Li G, Guo J (2023) Intravenous Injection of Na Ions Aggravates Ang II-Induced Hypertension-Related Vascular Endothelial Injury by Increasing Transmembrane Osmotic Pressure. Int J Nanomedicine 18:7505–7521. https://doi.org/10.2147/IJN.S435144

Wang Y, Zhou J, Guo J (2023) Protein Nanoparticles and Electromechanical Balance in live cells. Chinese Bull Life Sci 35:1322-1327. doi:10.13376/j.cbls/2023144

Zhang J, Wang Y, Zheng Z, Sun X, Chen T, Li C, Zhang X, Guo J (2019) Intracellular ion and protein nanoparticle-induced osmotic pressure modify astrocyte swelling and brain edema in response to glutamate stimuli. Redox Biol 21:101112. https://doi.org/10.1016/j.redox.2019.101112

Zheng Z, Hu Y, Guo J (2022a) Protein nanoparticles and bioosmotic pressure. Chemistry of life 42:2066-2070. doi:10.13488/j.smhx.20220455

Zheng Z, Qiu Z, Xiong X, Nie A, Zhou W, Qiu H, Zhao H, Wu H, Guo J (2023) Co-activation of NMDAR and mGluRs controls protein nanoparticle-induced osmotic pressure in neurotoxic edema. Biomed Pharmacother 169:115917. https://doi.org/10.1016/j.biopha.2023.115917

Zheng Z, Wang Y, Li M, Li D, Nie A, Chen M, Ruan Q, Guo Y, Guo J (2022b) Albumins as Extracellular Protein Nanoparticles Collaborate with Plasma Ions to Control Biological Osmotic Pressure. Int J Nanomedicine 17:4743–4756. https://doi.org/10.2147/IJN.S383530

Zheng Z, Nie A, Wu X, Chen S, Zhang L, Yang D, Shi Y, Xiong X, Guo J (2025) Electromechanical Regulation Underlying Protein Nanoparticle-Induced Osmotic Pressure in Neurotoxic Edema. Int J Nanomedicine 20:4145-4163. https://doi.org/10.2147/IJN.S503181

Zhu L, Zheng ZH, Li W, Shou CY, Zhao Y, Zhang LJ, Shi XL, Hu YF, Zhao HH, Wu HW, Guo J. Osmotic Tension Asymmetry Drives Electrotactic Migration via PDLIM7-Polarized Microfilament Coordination in Breast Cancer Cells. Adv Sci. 2025 Dec 22; e15246. doi: 10.1002/advs.202515246