Effective therapies targeting the molecular pathogenesis of liver diseases remain an unmet clinical need. Here, using acute and chronic liver pathology models, we demonstrate a dual therapeutic role for miR-200a encapsulated in poly(lactic acid) (PLA) nanoparticles (NPs) in both treating liver pathology and stimulating regeneration. This rationale is based on miR-200a's central role in regulating oxidative and endoplasmic reticulum (ER) stress, key drivers of liver diseases, enhancing liver regeneration and preventing disease development. Herein, we report the design and therapeutic validation of a nanomedicine platform comprising biocompatible PLA NPs for the hepatic delivery of miR-200a. The resulting PLA@miR-200a NPs (155 ± 39 nm in diameter) demonstrated successful cargo encapsulation and were internalized by hepatocytes in ex vivo liver slice models with minimal cytotoxicity. Pre-complexation of the miR with spermidine facilitated efficient encapsulation within the PLA matrix. Intracellular kinetic studies revealed a degradation-mediated, sustained release profile of the miR payload over 48 h. Crucially, the functional efficacy of this delivery mechanism was validated in vivo, where PLA@miR-200a successfully activated the Keap1/Nrf2 signaling pathway in target hepatocytes, achieving a reliable physiological effect. In a murine model of acute acetaminophen-induced liver injury, a single intraperitoneal administration of PLA@miR-200a profoundly mitigated hepatotoxicity, reduced dystrophic changes, normalized liver enzyme levels, and restored hepatocyte proliferation. Advanced metabolic FLIM imaging in combination with molecular analysis revealed revitalized of cellular biosynthetic activity and antioxidant capacity. Furthermore, in a chronic carbon tetrachloride-induced fibrosis model, the therapy demonstrated potent anti-fibrotic activity, reducing collagen deposition by two stages on the Metavir scale and promoting the resolution of fibrotic septa. In addition, the liver's regenerative capacity and metabolic state were normalized, as evidenced by restored hepatocyte proliferation and FLIM-based metabolic imaging. This work establishes that miR-200a-based nanotherapy has a high potential as a universal, dual-action strategy for not only treating a wide range of liver pathologies but also enhancing the organ's intrinsic regenerative potential, offering significant promise for clinical translation.

The most effective method of treating tumors localized in the liver remains resection. However, in the presence of concomitant pathology, the regenerative potential of the liver is significantly reduced. To date, there is insufficient fundamental data on the mechanisms responsible for the disruption of liver regeneration, and there is no effective method for assessing its regenerative potential. The most suitable model for these purposes is acute liver injury (ALI). Modern non-contrast methods of multiphoton microscopy with second harmonic generation and fluorescence lifetime imaging microscopy (FLIM) modes enable intravital evaluation of the metabolic status of the hepatocytes; therefore, this expands the possibilities for studying the processes occurring in cells during regeneration in the context of any pathologies.

The presence of molecular mutations in colorectal cancer (CRC) is a decisive factor in selecting the most effective first-line therapy. However, molecular analysis is routinely performed only in a limited number of patients with remote metastases. We propose to use tissue stiffness as a marker of the presence of molecular mutations in CRC samples. For this purpose, we applied compression optical coherence elastography (C-OCE) to calculate stiffness values in regions corresponding to specific CRC morphological patterns (n = 54). In parallel to estimating stiffness, molecular analysis from the same zones was performed to establish their relationships. As a result, a high correlation between the presence of KRAS/NRAS/BRAF driver mutations and high stiffness values was revealed regardless of CRC morphological pattern type. Further, we proposed threshold stiffness values for label-free targeted detection of molecular alterations in CRC tissues: for KRAS, NRAS, or BRAF driver mutation—above 803 kPa (sensitivity—91%; specificity—80%; diagnostic accuracy—85%), and only for KRAS driver mutation—above 850 kPa (sensitivity—90%; specificity—88%; diagnostic accuracy—89%). To conclude, C-OCE estimation of tissue stiffness can be used as a clinical diagnostic tool for preliminary screening of genetic burden in CRC tissues.

To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver's regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient.

A decrease in the regenerative potential of the liver during the development of non-alcoholic fatty liver disease (NAFLD), which is observed in the vast majority of patients with diabetes mellitus type 1, significantly increases the risk of postoperative liver failure. In this regard, it is necessary to develop new approaches for the rapid intraoperative assessment of the condition of liver tissue in the presence of concomitant liver pathology. A modern label-free approach based on multiphoton microscopy, second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) allow for the evaluation of the structure of liver tissue as well as the assessment of the metabolic state of hepatocytes, even at the cellular level. We obtained optical criteria and identified specific changes in the metabolic state of hepatocytes for a reduced liver regenerative potential in the presence of induced diabetes mellitus type 1. The obtained criteria will expand the possibilities for the express assessment of the structural and functional state of liver tissue in clinical practice.

Abuse with hepatotoxic agents is a major cause of acute liver failure. The search for new criteria indicating the acute or chronic pathological processes is still a challenging issue that requires the selection of effective tools and research models. Multiphoton microscopy with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are modern label-free methods of optical biomedical imaging for assessing the metabolic state of hepatocytes, therefore reflecting the functional state of the liver tissue. The aim of this work was to identify characteristic changes in the metabolic state of hepatocytes in precision-cut liver slices (PCLSs) under toxic damage by some of the most common toxins: ethanol, carbon tetrachloride (CCl4) and acetaminophen (APAP), commonly known as paracetamol. We have determined characteristic optical criteria for toxic liver damage, and these turn out to be specific for each toxic agent, reflecting the underlying pathological mechanisms of toxicity. The results obtained are consistent with standard methods of molecular and morphological analysis. Thus, our approach, based on optical biomedical imaging, is effective for intravital monitoring of the state of liver tissue in the case of toxic damage or even in cases of acute liver injury.

Liver regeneration has been studied for many decades, and the mechanisms underlying regeneration of normal liver following resection are well described. However, no less relevant is the study of mechanisms that disrupt the process of liver regeneration. First of all, a violation of liver regeneration can occur in the presence of concomitant hepatic pathology, which is a key factor reducing the liver's regenerative potential. Understanding these mechanisms could enable the rational targeting of specific therapies to either reduce the factors inhibiting regeneration or to directly stimulate liver regeneration. This review describes the known mechanisms of normal liver regeneration and factors that reduce its regenerative potential, primarily at the level of hepatocyte metabolism, in the presence of concomitant hepatic pathology. We also briefly discuss promising strategies for stimulating liver regeneration and those concerning methods for assessing the regenerative potential of the liver, especially intraoperatively.

BackgroundThere is an urgent clinical need for targeted strategies aimed at the treatment of bone defects resulting from fractures, infections or tumors. 3D scaffolds represent an alternative to allogeneic MSC transplantation, due to their mimicry of the cell niche and the preservation of tissue structure. The actual structure of the scaffold itself can affect both effective cell adhesion and its osteoinductive properties. Currently, the effects of the structural heterogeneity of scaffolds on the behavior of cells and tissues at the site of damage have not been extensively studied.MethodsBoth homogeneous and heterogeneous scaffolds were generated from poly(L-lactic acid) methacrylated in supercritical carbon dioxide medium and were fabricated by two-photon polymerization. The homogeneous scaffolds consist of three layers of cylinders of the same diameter, whereas the heterogeneous (gradient pore sizes) scaffolds contain the middle layer of cylinders of increased diameter, imitating the native structure of spongy bone. To evaluate the osteoinductive properties of both types of scaffold, we performed in vitro and in vivo experiments. Multiphoton microscopy with fluorescence lifetime imaging microscopy was used for determining the metabolic states of MSCs, as a sensitive marker of cell differentiation. The results obtained from this approach were verified using standard markers of osteogenic differentiation and based on data from morphological analysis.ResultsThe heterogeneous scaffolds showed improved osteoinductive properties, accelerated the metabolic rearrangements associated with osteogenic differentiation, and enhanced the efficiency of bone tissue recovery, thereby providing for both the development of appropriate morphology and mineralization.ConclusionsThe authors suggest that the heterogeneous tissue constructs are a promising tool for the restoration of bone defects. And, furthermore, that our results demonstrate that the use of label-free bioimaging methods can be considered as an effective approach for intravital assessment of the efficiency of differentiation of MSCs on scaffolds.