(F-212) Cytotoxicity and Proliferative Effect of Nitric Oxide Encapsulated Liposome on the Human Fibroblast Cells

Introduction:: Introduction: Nitric oxide, a bioregulatory molecule produced by many cells has diverse effects on the human body, including peristalsis, hormone secretion, neurotransmission, smooth muscle vasodilation, and cell cycle based on its concentration. Due to its short half-life and high reactivity, the bioavailability of nitric oxide diminishes when directly introduced into the human body. Its complexation with a carrier before administration could address this challenge. One way is to load the nitric oxide into liposomes which are spherical vesicles of a lipid bilayer mainly consisting of phospholipids that can incorporate water-soluble drugs inside their hydrophilic core, or a lipid-soluble drug in their lipid bilayer with size ranges from 50 nanometers to several micrometers. At various concentrations of NO, this study aimed to understand the cytotoxic and proliferative effect of NO on fibroblast cells, the most common type of connective tissue cells, and how liposomes target these cells.

Materials and Methods:: Materials and Methods: Liposomes were prepared using Thin film hydration method. A total of 5.2 µmole (164.34 µL of chloroform/PL solution) phospholipids (PL) was dispensed into a 13x100mm glass tube using a 500 µL Hamilton syringe. It was then placed in a fume hood and connected to a spinning module and spun to dry overnight to remove the chloroform. A thorough mixture of DMHD/N2O2 (NO donor) and isopropyl alcohol (IPA) (2mL at 1mg/mL) with 1.73mL of 1X PBS was added to dried PL and allowed to wet for at least 1.5hours at room temperature. The mixture was vortexed to get a milky suspension and placed in a bath sonicator at constant temperature (35C) until suspension became clear or slightly hazy. An extrusion and filtration processes were then carried out to achieve PDI ≤ 0.3 and a previously determined encapsulation efficiency of 49%. The resulting liposome with NO solution was placed in a sample vial and stored at 4°C. Human Fibroblast cells (HFC) were passaged into a T-25 flask and later seeded in a 24 well plate with liposome/NO solution (LipoNO) at concentrations: 1000 µL cell solution (CS) set as the control, 900uL CS and 100uL of LipoNO, 500uL CS and 500uL of LipoNO, 500uL CS and 250uL of LipoNO and 400uL of cell solution and 100uL of LipoNO (Figure 1). This was repeated with smaller concentrations of LipoNO. Fluorescence activity was also studied in the liposome by placing carboxyfluoresceindiacetate (CFDA) probe in liposomes.

Results, Conclusions, and Discussions:: Results and Discussion: From the different concentrations of NO exposed to the cells, the cytotoxic effect of NO was evident at concentrations 250µL of LipoNO and 500µL of cell solution (1/2) after 48 hours, while at a higher concentration of lipoNO (500µL) and 500µL of cell solution (1/1), the cytotoxic effect is seen after 24 hours (Figure 1). Reducing the concentration of LipoNO, the cytotoxic effect on the cells was not observed until after 24 hours and lower concentration of lipoNO supported cell proliferation (not shown). The fluorescein probe indicated that the liposomes, when mixed into the cell media, cluster around cells (not shown).

Conclusion: The lipoNO construct’s interaction with human fibroblast cells and their dosing effect on cytotoxicity and proliferation supports a mechanism where the encapsulated NO is being delivered to the cells. The interaction between lipoNO and cells appears to be more stochastic in the absence of cell targeting ligands. While these results highlight the cytotoxic and proliferative effect of NO on HBCs, more studies need to be carried out to enhance targeted lipoNO interactions with various cells.

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