Folic acid-functionalized human serum albumin nanocapsules for targeted drug delivery to chronically activated macrophages.

Activated synovial macrophages play a key role in Rheumatoid Arthritis (RA). Recent studies have shown that folate receptor beta (FRß) is specifically expressed by activated macrophages. Therefore a folate-based nanodevice would provide the possibility of delivering therapeutic agents to activated macrophages without affecting normal cells and tissues. This study shows for the first time the sonochemical preparation of HSA nanocapsules avoiding toxic cross linking chemicals and emulsifiers used in other methods. Production of HSA nanocapsules was optimized leading to a diameter of 443.5 ± 9.0 nm and a narrow size distribution indicated by a polydispersity index (PDI) of 0.066 ± 0.080. Nanocapsules were surface modified with folic acid (FA) and the FA content was determined to be 0.38 and 6.42 molecules FA per molecule HSA, depending on the surplus of FA employed. Dynamic light scattering was used to determine size, PDI and zetapotential of the produced nanocapsules before and after surface modification. FA distribution on the surface of HSA nanocapsules was localized three-dimensionally after fluorescence labeling using confocal laser scanning microscopy (CLSM). Furthermore, specific binding and internalization of HSA nanocapsules by FRß-positive and FRß-negative macrophages, obtained from human peripheral blood mononuclear cells, was demonstrated by flow cytometry. FRß-expressing macrophages showed an increased binding for FA-modified capsules compared with those without FA.

Heterogeneous liposome membranes with pH-triggered permeability enhance the in vitro antitumor activity of folate-receptor targeted liposomal doxorubicin.

The killing efficacy of doxorubicin from liposome-based delivery carriers has been shown to correlate strongly with its intracellular trafficking and, in particular, its fast and extensive release from the delivery carrier. However, previously explored pH-triggered mechanisms that were designed to become activated during liposome endocytosis have also been shown to interfere with the liposome stability in vivo. We have designed pH-triggered gel-phase liposomes with heterogeneous membranes for the delivery of doxorubicin. These liposomes are triggered to form "leaky" interfacial boundaries between gel-gel phase separated domains on the membrane bilayer with lowering pH. The pH-triggered mechanism does not compromise liposome stability in vivo and results in superior in vitro killing efficacy of delivered doxorubicin when liposomes are endocytosed by a clathrin-mediated pathway. In the present work, we evaluate the general applicability of these liposomes when targeted to the folate receptor (FR) of KB cancer cells in vitro and become endocytosed by a less acidic pathway: the caveolae pathway. FR-targeting liposomes exhibit almost 50% decrease in cell association for increase in liposome size from 120 to 280 nm in diameter after relatively short incubation times (up to 4 h). The fraction of internalized vesicles, however, is approximately 60% of the cell associated vesicles independent of their size. Our findings demonstrate that, for the same doxorubicin uptake per cancer cell, the killing effect of doxorubicin delivered by pH-triggered lipid vesicles is greater (IC(50) = 0.032 mM for a 6 h incubation) than when delivered by a conventional non-pH-responsive composition (IC(50) = 0.194 mM). These findings suggest higher bioexposure of cells to the therapeutic agent possibly via faster and more extensive release from the carrier. Animal studies of FR-targeting non-pH-responsive liposomal doxorubicin report stronger therapeutic potential for the targeted approach relative to nontargeted liposomes and to free doxorubicin. The findings of the present study suggest that the targeted pH-triggered liposomes could potentially further enhance the therapeutic outcomes of doxorubicin in vivo.

All-trans retinoic acid is capable of inducing folate receptor ß expression in KG-1 cells.

The high expression of folate receptor (FR) on cancer cells might be a potential target for cancer therapy. In this study, the FR-ß expression and the modulation effect of all-trans retinoic acid (ATRA) in a number of cancer cell lines were analyzed. The gateway of ATRA activity on FR-ß expression was further studied by a panel of retinoid activators and inhibitors. The results revealed that ATRA was capable of upregulating the expression of FR-ß protein in KG-1 cells in a dosage-dependent manner, not in KG-1a, NB4, HL60, 293, L1210, JAR, and Hela cells. FR-ß mRNA expression in KG-1 cells was higher when ATRA was present in culture medium at 10⁻6 mol/L for 5 days, and it went down to baseline when ATRA was removed from the medium, vice versa. The upregulation of FR-ß expression in KG-1 cells by ATRA was not associated with cell proliferation and differentiation. In addition, activators of retinoid acid receptor (RAR)α and RARγ, CD336, and CD2781 also induced FR-ß expression. The induction of FR-ß expression by CD336 could be inhibited by RARγ antagonist CD2665; RARß agonist CD-417 and CD-2314 as well as retinoid X receptor (RXR) agonist LG100364 could not induce FR-ß expression. These results indicate that ATRA within a certain range of concentration could reversibly induce the expression of FR-ß in a dosage- and cell type-dependent manner, and its action in KG-1 cells might be associated with the signal transduction of retinoid receptor RARα and RARγ, rather than RARß and RXRs.