Cellular uptake of liposomes monitored by confocal microscopy and flow cytometry (original) (raw)
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Physica Medica, 2009
It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture. ª 2008 Published
International Journal of Nanomedicine
The uptake pathway of liposomes into cells is mainly via endocytosis or membrane fusion; however, the relationship between the uptake pathway and the intracellular pharmacokinetics of the liposome components remains unclear. This study aimed at revealing the relationship by using cationic liposomes having similar physical properties and different uptake pathways. Materials and Methods: We prepared cationic liposomes composed of amino acid-type lipids, K3C14 and K3C16, which have different uptake pathways by a hydration method, and fluorescently modified them by encapsulating FITC-dextran and surface conjugation with Alexa Fluor ® 488 (AF488). Then, we investigated their intracellular distribution in HeLa cells over time. Results: The liposomes had similar physical properties and did not cause significant cell mortality after treatment for 180 min. The delivery rate and efficiency of encapsulated FITCdextran with the fusogenic K3C16 liposomes were 3 and 1.6 times higher, respectively, than with the endocytic K3C14 liposomes. FITC-dextran molecules delivered with K3C16 liposomes were observed throughout the cytosolic space after 10 min, while those delivered with K3C14 liposomes were mainly observed as foci and took 60 min to diffuse into the cytosolic space. K3C14 lipids modified with AF488 were distributed mostly in the cytosolic space. In contrast, fluorescently labeled K3C16 lipids were colocalized with the plasma membrane of 50% of the HeLa cells after 10 min and were gradually internalized intracellularly. Conclusion: Fusogenic K3C16 liposomes internalized into HeLa cells faster than endocytic K3C14 liposomes, and their components differently distributed in the cells.
Liposomal Drug Delivery Systems: An Update Review
Current Drug Delivery, 2007
The discovery of liposome or lipid vesicle emerged from self forming enclosed lipid bi-layer upon hydration; liposome drug delivery systems have played a significant role in formulation of potent drug to improve therapeutics. Recently the liposome formulations are targeted to reduce toxicity and increase accumulation at the target site. There are several new methods of liposome preparation based on lipid drug interaction and liposome disposition mechanism including the inhibition of rapid clearance of liposome by controlling particle size, charge and surface hydration. Most clinical applications of liposomal drug delivery are targeting to tissue with or without expression of target recognition molecules on lipid membrane. The liposomes are characterized with respect to physical, chemical and biological parameters. The sizing of liposome is also critical parameter which helps characterize the liposome which is usually performed by sequential extrusion at relatively low pressure through polycarbonate membrane (PCM). This mode of drug delivery lends more safety and efficacy to administration of several classes of drugs like antiviral, antifungal, antimicrobial, vaccines, anti-tubercular drugs and gene therapeutics. Present applications of the liposomes are in the immunology, dermatology, vaccine adjuvant, eye disorders, brain targeting, infective disease and in tumour therapy. The new developments in this field are the specific binding properties of a drug-carrying liposome to a target cell such as a tumor cell and specific molecules in the body (antibodies, proteins, peptides etc.); stealth liposomes which are especially being used as carriers for hydrophilic (water soluble) anticancer drugs like doxorubicin, mitoxantrone; and bisphosphonate-liposome mediated depletion of macrophages. This review would be a help to the researchers working in the area of liposomal drug delivery.
Cancer research, 1982
Functional and ultrastructural studies of liposomes injected i.v. into inbred C57BL/6N mice were performed to determine whether free liposomes can traverse capillaries. In the liver and spleen, organs with discontinuous (sinusoidal) capillaries, ultrastructural and cell fractionation studies revealed that small (300- to 800-A diameter), sonicated, unilamellar liposomes were more efficient in penetrating liver sinusoids to interact with hepatocytes than were large (0.5- to 10-micrometers) multilamellar liposomes. Ultrastructural studies of the behavior of liposomes in the continuous capillaries of the lungs revealed that circulating phagocytic cells engulf the liposomes in the capillaries. Transcapillary migration of free liposomes was not observed. We conclude that free liposomes are unable to extravasate to reach the alveoli for subsequent engulfment by alveolar macrophages. Instead, liposomes in the lung capillaries are engulfed by circulating blood phagocytes which subsequently m...
Liposomes as Targetted Drug Delivery Systems Present and Future Prospectives: A Review
Journal of Drug Delivery and Therapeutics, 2013
Liposomes are the leading drug delivery systems have played a significant role in the formulation of potent drug to improve therapeutic effect. The mechanism giving rise to therapeutic advantages of liposomes such as the ability of long circulating liposomes to preferentially accumulate at disease sites such as tumours, site of infection, and site of inflammation. There are several new methods of liposomes preparation based on lipid drug interaction and liposomes disposition mechanism including the incubation of rapid clearance of liposomes by controlling particle size, and surface hydration. The liposomes are characterised with respect to physical, chemical and biological parameters. Present applications of liposomes are in field of immunology, dermatology, vaccine adjuvant, eye disorder, and brain targeting therapy. This review would be helpful to the researches working in the area of liposomal drug delivery and educates how this success is being built on to design, effective carriers for genetic drugs.
Archives of Biochemistry and Biophysics, 2000
The traditional mode of encapsulating drugs in liposomes poses risks to drug stability, especially when recognition agents are attached to the liposomal surface to obtain targeted liposomes. To reduce such risks, we devised a simple, novel method to entrap drugs in liposomes, consisting of (i) preparation and lyophilization of drug-free regular and surface-modified liposomes and (ii) drug encapsulation in the course of liposome reconstitution through rehydration in an aqueous solution of the drug. In this paper, we report physicochemical studies in which we compared regular and surface-modified liposomes made by this novel approach (denoted N-liposomes) to respective liposomes made by the traditional mode (denoted T-liposomes). The studies were performed with fluorescein, sucrose, histidine, mitomycin C (MMC), and chloramphenicol (CAM) encapsulated (each) in regular and in bioadhesive liposomes, the latter having hyaluronic acid as the surface-bound ligand. Our major findings are as follows: (1) The drug-specific encapsulation efficiencies spanning the range of 10 -90% were, excepting sucrose, either similar in the Nand T-liposomes or better in the N-than in the T-liposomes, for both regular and bioadhesive liposomes.
Assessment of liposome disruption to quantify drug delivery in vitro
Biochimica Et Biophysica Acta - Biomembranes, 2016
Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.
Liposomes as drug carriers and their characterization using different analytical methods
The physicochemical properties of liposomes are significantly affected by the composition of phospholipid bilayer; differences in composition allow the use of liposomes for analytical purposes and for therapeutic purposes. One of the most used components of phospholipid bilayer is cholesterol. Its concentration plays a significant role in the behaviour of liposomes. This study points to changes in the properties of liposomes and its influence on encapsulated doxorubicin according to the content of cholesterol in the phospholipid bilayer. The influence of SDS addition to liposomal variants was also evaluated. Three variants of liposomes differing in various concentrations of cholesterol were assessed. Firstly, the toxicity of all types of liposomal doxorubicin was evaluated and it was found that the content of cholesterol increases the IC50 values of encapsulated doxorubicin in liposome. The highest concentration of cholesterol in liposome increased the IC50 value even four times com...