ABSTRACT
The immunosuppressive factors in tumor microenvironment enhance tumor growth and suppress anti-tumor immune responses. Adenosine is an important immunosuppressive factor which can be secreted by both tumor and immune cells trough action of two cell surface ecto-nucleotidase molecules CD39 and CD73. Blocking the adenosine generating molecules has emerged as an effective immunotherapeutic approach for treatment of cancer. In this study, CD73-siRNA encapsulated into chitosan-lactate (ChLa) nanoparticles (NPs) was employed to suppress the expression of CD73 molecule on 4T1 breast tumor cells, in vitro. ChLa NPs were generated through ionic gelation of ChLa by tripolyphosphate (TPP). Small interfering RNA (SiRNA)-loaded NPs had about 100 nm size with a polydispersive index below 0.3 and a zeta potential about 13. Our results showed that ChLa NPs with Ch 50 kDa exhibit the best physicochemical features with the high siRNA encapsulation capacity. Synthesized NPs were able to fully bind with siRNA, protect them against serum and heparin degradation, and promote the transfection process. While the NPs exhibited low toxicity during 72 h cell culture, the transfection of Ch-plasmid expressing green fluorescent protein (pEGFP) NPs was efficient in 4T1 cells with a transfection rate of 53.6 % as detected by flow cytometry. In addition, CD73-siRNA-loaded ChLa NPs could efficiently suppress the expression of CD73 as assayed by real-time polymerase chain reaction and flow cytometry. As a conclusion, CD73-siRNA-loaded ChLa NPs may be considered as a promising therapeutic tool for cancer therapy; however, further in vivo investigations are necessary.
Subject(s)
5'-Nucleotidase/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Nanoparticles/administration & dosage , RNA, Small Interfering/genetics , Animals , Cell Line, Tumor , Cell Survival , Chitosan , Down-Regulation , Female , Flow Cytometry , Gene Expression , Lactic Acid , Mice , Real-Time Polymerase Chain ReactionABSTRACT
The aim of this study was to develop biocompatible, water-soluble (153) Sm-labeled chitosan nanoparticles (NPs) containing folate and polyethyleneimine functionalities i.e. chitosan-graft-PEI-folate (CHI-DTPA-g-PEI-FA), suitable for targeted therapy. The physicochemical properties of the obtained NPs were characterized by dynamic light-scattering analysis for their mean size, size distribution, and zeta potential; scanning electron microscopy for surface morphology; and (1) H-NMR, FT-IR analyses for molecular dispersity of folate in the NPs. NPs were spherical with mean diameter below 250 nm, polydispersity of below 0.15, and positive zeta potential values. The NP complex ((153) Sm-CHI-DTPA-g-PEI-FA) was stable at 25 °C (6-8 h, >90% radiochemical purity, instant thin layer chromatography (ITLC)). Binding studies using fluorescent NPs for internalization also demonstrated significant uptake in MCF-7 cells. MCF-7 cell internalization was significantly greater for 4T1. In blocking studies, both MCF-7 and 4T1 cell lines demonstrated specific folate receptor (FR) binding (decreasing 45%). In vivo biodistribution studies indicated major excretion of NPs metabolites and/or free (153) Sm through the kidneys. The preliminary imaging studies in 4T1 tumor-bearing mice showed minor uptake up to 96 h. The present folic acid that functionalized chitosan NP is a candidate material for folate receptor therapy.
Subject(s)
Breast Neoplasms/drug therapy , Chitosan/analogs & derivatives , Folic Acid/chemistry , Nanoparticles/administration & dosage , Polyethyleneimine/analogs & derivatives , Radiopharmaceuticals/chemistry , Samarium/chemistry , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Survival/drug effects , Chitosan/chemistry , Drug Carriers , Female , Humans , MCF-7 Cells , Mice , Mice, Inbred BALB C , Molecular Targeted Therapy , Nanoparticles/chemistry , Organometallic Compounds , Organophosphorus Compounds , Polyethyleneimine/chemistry , Radiopharmaceuticals/pharmacokinetics , Samarium/pharmacokinetics , Spectroscopy, Fourier Transform Infrared , Tissue DistributionABSTRACT
We fabricated a targeted delivery system for doxorubicin (Dox) using ß-1,3-glucan (Glu) as a carrier and decorated by trastuzumab antibody having the status of targeting agent against Her2+ breast tumors. Glu-Dox conjugates were also functionalized with polyethylenimine (PEI) intended for increasing specific cellular uptake of prepared nanoparticles. The self-assembled nanoparticles were prepared through conjugation of Dox- [Glu-Dox-] using succinic anhydride (Sa) in place of a linker. Nanoparticles had spherical morphology with positive zeta potential. In-vitro cell viability assay on two breast cancer cell lines demonstrated acceptable toxicity against tested cell lines. Confocal microscopic images demonstrated the remarkable cytoplasmic uptake of the nanoparticles in Her2-overexpressing 4T1 cells. A controlled release of Dox from Glu-Dox nanoparticles was investigated. In-vivo studies were performed on female Balb/C mice. The volume of the induced tumors was calculated following intravenous administration of nanoparticles. The tumor volume diminished efficiently and more rapidly after administration of nanoparticles containing Dox. Based on survival results, the formulation of Dox targeted nanoparticles appeared very promising for the treatment of tumors. It could be concluded that Glu-Dox targeted nanoparticles have potential advantages for delivering anticancer drugs to the target tissue.
