title: Folate receptor targeted three-layered micelles and hydrogels for gene delivery to activated macrophages.
abstract: New folic acid (FA) coupled three layered micelles (3LM) were designed to encapsulate DNA, and their application as delivery system that specifically targets activated macrophages was investigated for new treatment options in rheumatoid arthritis (RA). FA coupled poly(l-lactide)-b-poly(ethylene glycol) (FA-PEG-PLLA) was synthesized via the NHS-ester activated/amine coupling method. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor β in LPS-activated and resting macrophages. FA coupled 3LM were formulated in a two-step procedure and characterized regarding hydrodynamic diameters and zeta potentials. The presence of the targeting ligand was shown not to increase the size of the 3LM compared to their non-targeted counterparts. Targeted and non-targeted 3LM were used in vitro to optimize uptake conditions in the RAW 264.7 macrophage cell line. The amount of FA coupled polymer in the final formulation was found to be optimal at 75% FA-PEG-PLLA and 25% PLLA-PEG-PLLA. Subsequently, transgene expression in vitro in RAW 264.7 cells and ex vivo in primary activated and resting mouse macrophages was determined as a function of FR-mediated internalization of 3LM encapsulating GFP expressing plasmid. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher GFP expression in vitro and ex vivo than resting macrophages with only a basal level of FR expression. Lastly, injectable hydrogels as depot formulation were formed by stereocomplexation, and their degradation, DNA release profiles, and dissociation into intact 3LM were found to be beneficial for potential in vivo application. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and that their hydrogels release intact 3LM for efficient transfection of primary macrophages. Therefore, FA-3LM could become a promising delivery system for receptor-mediated drug or gene delivery and novel therapy for rheumatoid arthritis in an in situ forming gel formulation.
authors: Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA; Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilian University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany.
authors: Mariam
authors: M
authors: Mohammadi
authors: Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA.
authors: Ying
authors: Y
authors: Li
authors: Department of Chemistry, The University of Memphis, Memphis, TN 38152, USA.
authors: Daniel G
authors: DG
authors: Abebe
authors: Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA.
authors: Yuran
authors: Y
authors: Xie
authors: Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA; Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilian University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany.
authors: Rima
authors: R
authors: Kandil
authors: Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA; Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilian University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany.
authors: Teresa
authors: T
authors: Kraus
authors: Department of Obstetrics and Gynecology & Immunology and Microbiology, School of Medicine, Wayne State University, CS Mott Center for Human Growth and Development, 275 E. Hancock, Detroit, MI 48201, USA; Perinatology Research Branch, NICHD/NIH/DHHS, Detroit, MI, USA.
authors: Nardhy
authors: N
authors: Gomez-Lopez
authors: Department of Chemistry, The University of Memphis, Memphis, TN 38152, USA.
authors: Tomoko
authors: T
authors: Fujiwara
authors: Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA; Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilian University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany; Department of Oncology, Molecular Therapeutics Program, Wayne State University, 4100 John R St, Detroit, MI 48201, USA. Electronic address: Olivia.merkel@lmu.de.
authors: Olivia M
authors: OM
authors: Merkel
mesh_terms: D000818:Animals; D004247:DNA / Q000008:administration & dosage / Q000737:chemistry; D005260:Female; D058976:Folate Receptor 2 / Q000378:metabolism*; D005492:Folic Acid / Q000008:administration & dosage / Q000737:chemistry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D020100:Hydrogels / Q000008:administration & dosage* / Q000737:chemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008823:Micelles; D011092:Polyethylene Glycols / Q000008:administration & dosage / Q000737:chemistry; D000067996:RAW 264.7 Cells
chemical_list: D058976:Folate Receptor 2; D020100:Hydrogels; D008070:Lipopolysaccharides; D008823:Micelles; C542623:monomethoxypolyethyleneglycol-polylactide block copolymer; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D004247:DNA; D005492:Folic Acid
keywords: Folate receptor; Macrophages; Nanotechnology; Rheumatoid arthritis, hydrogel, depot, degradation; Three layered micelles