Formation and Investigation of Cell-Derived Nanovesicles as Potential Therapeutics Against Chronic Liver Disease

Abstract

This study aims to offer a new therapeutic approach using cell-derived nanovesicles (cdNVs) to overcome the lack of effective treatments for liver fibrosis, a reversible chronic liver disease. To achieve this goal we established the formation and purification of cdNVs from untreated, quiescent-like, or activated LX-2 cells, an immortalized human hepatic stellate cell (HSC) line with key features of transdifferentiated HSCs. Analysis of the genotype and phenotype of naïve and transdifferentiated LX-2 cells activated through transforming growth factor beta 1 (TGF-β₁), following treatment with cdNVs, revealed a concentration-dependent fibrosis regression. The beneficial fibrosis-resolving effects of cdNVs are linked to their biomolecular corona.

Liposomes generated using lipids extracted from cdNVs exhibit a reduced anti-fibrotic response in perpetuated LX-2 cells and show a reduced cellular uptake. However, incubation with soluble factors collected during purification results in a new corona, thereby restoring fibrosis regression activity. Overall, cdNVs display encouraging therapeutic properties, making them a promising candidate for the development of liver fibrosis resolving therapeutics.

Introduction

The liver is involved in various vital functions, such as vitamin storage,[1] clearance of damaged erythrocytes,[2] elimination of xenobiotics,[3] synthesis of plasma proteins,[4] lipid management,[5] immune response,[6] and more. Injuries to this organ, caused e.g., by viral infections, fatty diets and/or alcohol abuse[7] lead to the formation of extracellular matrix (ECM) proteins,[8] which are an essential part of the wound-healing response. However, if the cause of injury is not resolved, such as in chronic liver diseases, fibrosis formation will be promoted leading, eventually, to switching from a reversible[7] condition to irreversible liver cirrhosis, liver cancer and liver failure, due to non-resolved excessive ECM protein production with consequent scar formation and loss of organ function.[9] In such cases only a transplant can save the patient, making the liver the second most transplanted solid organ worldwide.[10] The development of liver fibrosis involves different cell types, cytokines, and response cascades.[7, 11] Hepatic stellate cells (HSCs) have been described to be the major driver of fibrosis progression in the liver.[12] In physiologically healthy conditions, HSCs are in a quiescent (inactive) state in which they store vitamin A (retinol) in cytoplasmic lipid droplets.[13] Upon activation, HSCs undergo a trans-differentiation into a highly proliferative myofibroblast-like status [12, 14] In this activated state, HSCs proliferate, rapidly lose their ability to store lipid droplets, and start producing a lot of ECM components, such as alpha-smooth muscle actin (αSMA), fibronectin, type I and III collagens, as well as the profibrogenic cytokine transforming growth factor beta 1 (TGF-β1) which further activates other HSCs.[9, 12] The Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD 2017) estimated that 1.5 billion people were suffering from chronic liver diseases worldwide,[15] 2 million annual deaths[16] with the tendency increasing. Considering worldwide rising obesity and diabetes rates[17] and the fact that only 11 high-income countries are projected to be able to eliminate all hepatitis virus C infections by 2030,[18] new effective therapies are needed to fight the already present increase in patients, in order to reduce the need of lifesaving transplantations.

Extracellular vesicles (EVs) have been extensively studied in the past two decades and show great promise for both diagnostic and therapeutic purposes in a variety of diseases.[19] Because of their physicochemical properties, EVs can pass through biological barriers, elicit a low immune response, and thus have a longer circulation time in vivo.[20] Proteins, peptides, nucleic acids, metabolites, and lipids can be carried by EVs and functionally transferred to target cells.[20] Expression of cell-specific epitopes on the EV surface enhanced targeted administration in vivo[21-23]. As a result, EVs released from various cell sources, such as hepatocytes,[24] endothelial cells,[25] fibrocytes,[24] macrophages,[24] mesenchymal stem cells (MSCs)[24, 26], induced pluripotent stem cells[11, 24] and HSCs[24, 27] have been used to treat liver fibrosis. Depending on the cell state and harvesting point, various outcomes can be obtained. For example, EVs from palmitic acid (fibrotic)-treated hepatocytes,[28] as well as EVs from injured hepatocytes,[24, 29] led to the activation and progression of HSCs. Consequently, it is crucial to understand the source of the EVs. Yet, the use of EVs is not without its challenges and limitations, which pose obstacles to their translation into clinical trials and their viability as a biotherapeutic Extracellular vesicles (EVs) have been extensively studied in the past two decades and show great promise for both diagnostic and therapeutic purposes in a variety of diseases.[19] Because of their physicochemical properties, EVs can pass through biological barriers, elicit a low immune response, and thus have a longer circulation time in vivo.[20] Proteins, peptides, nucleic acids, metabolites, and lipids can be carried by EVs and functionally transferred to target cells.[20] Expression of cell-specific epitopes on the EV surface enhanced targeted administration in vivo[21-23] . As a result, EVs released from various cell sources, such as hepatocytes,[24] endothelial cells,[25] fibrocytes,[24] macrophages,[24] mesenchymal stem cells (MSCs)[24, 26], induced pluripotent stem cells[11, 24] and HSCs[24, 27] have been used to treat liver fibrosis. Depending on the cell state and harvesting point, various outcomes can be obtained. For example, EVs from palmitic acid (fibrotic)-treated hepatocytes,[28] as well as EVs from injured hepatocytes,[24, 29] led to the activation and progression of HSCs. Consequently, it is crucial to understand the source of the EVs. Yet, the use of EVs is not without its challenges and limitations, which pose obstacles to their translation into clinical trials and their viability as a biotherapeutic.

To induce quiescence in LX-2 cells, two established approaches were applied: treatment with retinol and palmitic acid, recognized as the gold standard for in vitro deactivation of HSCs,[41] and treatment with the polyenylphosphatidylcholine-rich (>75%) S80 liposomes, which our group demonstrated to exhibit anti-fibrotic effects on LX-2 cells[42]. To generate perpetuated, highly fibrogenic LX-2 cells, TGF-β1 was employed, as previously described.[35, 43] Afterwards, cdNVs were produced by subjecting differently pre-treated LX-2 cells (antifibrotic/fibrotic) to serial extrusion. cdNVs were then purified using a combination of density-gradient ultracentrifugation steps, followed by standard ultracentrifugation. Characterization of the formed cdNVs included assessment of their size, particle concentration, protein and lipid content, and their biological activity on both naïve LX 2 cells and TGF-β1-perpetuated LX-2 cells.

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Formation and Investigation of Cell-Derived Nanovesicles as Potential Therapeutics Against Chronic Liver Disease
Aymar Abel Ganguin, Ivo Skorup, Sebastian Streb, Alaa Othman, Paola Luciani
First published: 05 September 2023 https://doi.org/10.1002/adhm.202300811

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