Fluorescent-Control-for-Clodronate

Introduction

Fluorescent liposomes are a new type of fluorescent marker by unilamellar liposomes encapsulated and surface-immobilized fluorophores to image flow profiles in microfabricated structures, which are popular in liposome-related experiments as a negative control marker.

Classification

Fluorescent liposomes are divided into five categories according to different fluorescent dyes:

• DiA
• DiD
• DiI
• DiO
• DiR

All five fluorescent liposomes incorporate different lipophilic dyes within their membranes. The five fluorescent dyes themselves are insoluble in water, but when these fluorescent liposomes pass through the cell membrane, the fluorescent dye in them spreads rapidly throughout the membrane, causing the cell to take on a specific fluorescence that can be easily detected.

Fig. 1 Macrophage uptake of fluorescent liposomes.

The excitation and emission wavelengths of these fluorescent dyes cover a wide range of wavelengths from 300s to 900s. One of them, DiA, has a broad emission spectrum and can detect green, orange and even red fluorescence if different filters are used. DiI, DiO, DiD and DiR, on the other hand, all belong to the family of dialkylcarbocyanine compounds, whose spectral properties are independent of the length of the alkyl chain and depend on the length of the heteroatoms and connecting bridges in the terminal ring system. They have very high extinction coefficients, moderate fluorescence quantum yields and short excited state lifetimes (~1 ns) in a lipid environment. The fluorescence excitation and emission maxima of DiI and DiO are separated by ~65 nm and can be used for two-color labeling. Depending on the fluorescence characteristics of the different dyes, fluorescent liposomes with trichromatic or even pentachromatic displays can also be developed.

Preparation

Fluorescently labeled liposomes are produced using phospholipids and cholesterol by extrusion through polycarbonate membranes. During preparation, hydroxyfluorescein was added to its water quality core and specific fluorescent dyes were added to the lipids, allowing the lipids in the bilayer to be labeled with fluorescein, resulting in fluorescently labeled liposomes with maximized fluorescence intensity. It is important to note that the formulation needs to be configured so that the final liposomes have a net negative charge to reduce self-aggregation and to be able to interact with the equally negatively charged channel glass surface to maintain the stability of the liposomes. The resulting liposomes are dispersed in solution as single molecules, are neutrally buoyant and hydrophilic and do not exhibit adsorption on the glass surface.

Fluorescent controls for clodronate liposomes are similar to the common negative controls, both are formulated and prepared identically to clodronate liposomes except that clodronate is not added to the liposomes, but the fluorescence controls have additional fluorescent dyes added to the liposomes and the normal negative controls do not.

Mechanism

When animals or cells are treated with clodronate liposomes, phagocytes recognize the liposomes as invading foreign particles and remove the liposomes from local tissues or serum through phagocytosis. The phagocytosed liposomes then release clodronate into the phagocyte, triggering the apoptotic program. In contrast, free clodronate cannot cross the cell membrane to initiate cell death.

Control liposomes are recognized and engulfed by the same mechanism as clodronate liposomes. Since control liposomes do not contain clodronate, phagocytes are not killed. However, phagocytes will also respond to the ingestion of control liposomes by secreting cytokines, temporarily suspending phagocytic activity and other responses described in the literature.