ABSTRACT
Drug delivery to corneal epithelial cells is challenging due to the intrinsic mechanisms that protect the eye. Here, we report a novel liposomal formulation to encapsulate and deliver a short sequence peptide into human corneal epithelial cells (hTCEpi). Using a mixture of Phosphatidylcholine/Caproylamine/Dioleoylphosphatidylethanolamine (PC/CAP/DOPE), we encapsulated a fluorescent peptide, resulting in anionic liposomes with an average size of 138.8 ± 34 nm and a charge of -18.2 ± 1.3 mV. After 2 h incubation with the peptide-encapsulated liposomes, 66% of corneal epithelial (hTCEpi) cells internalised the FITC-labelled peptide, demonstrating the ability of this formulation to effectively deliver peptide to hTCEpi cells. Additionally, lipoplexes (liposomes complexed with plasmid DNA) were also able to transfect hTCEpi cells, albeit at a modest level (8% of the cells). Here, we describe this novel anionic liposomal formulation intended to enhance the delivery of small cargo molecules in situ.
Subject(s)
DNA, Complementary , Epithelium, Corneal/metabolism , Peptides , Transfection/methods , Cells, Cultured , DNA, Complementary/chemistry , DNA, Complementary/pharmacology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelium, Corneal/cytology , Humans , Liposomes , Peptides/chemistry , Peptides/pharmacology , Plasmids/chemistry , Plasmids/pharmacologyABSTRACT
This paper focuses on the targeting of single-walled carbon nanotubes (SWNTs) for the treatment of breast cancer with minimal side effects using photothermal therapy. The human protein annexin V (AV) binds specifically to anionic phospholipids expressed externally on the surface of tumour cells and endothelial cells that line the tumour vasculature. A 2 h incubation of the SWNT-AV conjugate with proliferating endothelial cells followed by washing and near-infrared (NIR) irradiation at a wavelength of 980 nm was enough to induce significant cell death; there was no significant cell death with irradiation or the conjugate alone. Administration of the same conjugate i.v. in BALB/c female mice with implanted 4T1 murine mammary at a dose of 0.8 mg SWNT kg(-1) and followed one day later by NIR irradiation of the tumour at a wavelength of 980 nm led to complete disappearance of implanted 4T1 mouse mammary tumours for the majority of the animals by 11 days since the irradiation. The combination of the photothermal therapy with the immunoadjuvant cyclophosphamide resulted in increased survival. The in vivo results suggest the SWNT-AV/NIR treatment is a promising approach to treat breast cancer.
Subject(s)
Hyperthermia, Induced , Mammary Neoplasms, Animal/therapy , Nanotubes, Carbon/chemistry , Phototherapy , Animals , Annexin A5/isolation & purification , Annexin A5/metabolism , Biotinylation , Cell Line, Tumor , Cell Proliferation , Cell Survival , Endothelial Cells/cytology , Endothelial Cells/metabolism , Female , Fluorescein-5-isothiocyanate/metabolism , Humans , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Maleimides/chemistry , Mammary Neoplasms, Animal/pathology , Mice , Mice, Inbred BALB C , Microscopy, Atomic Force , Microscopy, Fluorescence , Phosphatidylethanolamines/chemistry , Polyethylene Glycols/chemistry , Recombinant Proteins/isolation & purification , Spectroscopy, Near-Infrared , Staining and Labeling , SuspensionsABSTRACT
A new approach for enzyme prodrug therapy for cancer was tested using human endothelial cells and two breast cancer cell lines in vitro. The concept is to use the human annexin V protein to selectively target the enzyme L-methioninase to the tumor vasculature. The major finding was that enzyme prodrug treatment using the L-methioninase-annexin V fusion protein and selenomethionine as the prodrug over 3 days was shown to be lethal to the endothelial cells and the cancer cells, while having little or no effect with the prodrug but with no fusion protein present. Thus, this new approach appears promising.
Subject(s)
Endothelial Cells/drug effects , Prodrugs/pharmacology , Recombinant Fusion Proteins/pharmacology , Selenomethionine/pharmacology , Annexin A5/genetics , Annexin A5/metabolism , Breast Neoplasms/blood supply , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Carbon-Sulfur Lyases/genetics , Carbon-Sulfur Lyases/metabolism , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Endothelial Cells/metabolism , Female , Humans , Kinetics , Methanol/analogs & derivatives , Methanol/metabolism , Methanol/pharmacology , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/prevention & control , Organoselenium Compounds/metabolism , Organoselenium Compounds/pharmacology , Prodrugs/metabolism , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Selenomethionine/metabolismABSTRACT
A method for the non-covalent attachment of proteins to single-walled carbon nanotubes (SWNTs) is described. In this method, the protein is adsorbed to SWNTs that are suspended using sodium cholate, a surfactant and bile salt. The sodium cholate is then removed by dialysis with retention of the protein on the SWNTs. This method has resulted in good protein loadings and good retention of protein activity.
Subject(s)
Nanotubes, Carbon/chemistry , Proteins/chemistry , Adsorption , Bile Acids and Salts/chemistry , Sodium Cholate/chemistry , Surface Properties , Surface-Active Agents/chemistryABSTRACT
In this study, we describe the use of a sodium cholate suspension-dialysis method to adsorb the redox enzyme glucose oxidase (GOX) onto single-walled carbon nanotubes (SWNT). By this method, solutions of dispersed and debundled SWNTs were prepared that remained stable for 30 days and which retained 75% of the native enzymatic activity. We also demonstrate that GOX-SWNT conjugates can be assembled into amperometric biosensors with a poly[(vinylpyridine)Os(bipyridyl)2Cl(2+/3+)] redox polymer (PVP-Os) through a layer-by-layer (LBL) self-assembly process. Incorporation of SWNT-enzyme conjugates into the LBL films resulted in current densities as high as 440 microA/cm2, which were a 2-fold increase over the response of films without SWNTs. We also demonstrate that the adsorption pH of the redox polymer solution and the dispersion quality of SWNTs were important parameters in controlling the electrochemical and enzymatic properties of the LBL films.
