INVESTIGATION OF LIGAND SIZE ON TARGETED PLGA NANOPARTICLES FOR HER2 BREAST CANCER: TRASTUZUMAB AND ITS ScFv ANTI-HER2 ANTIBODY
Date
2018-06-21
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-3866-3972
Type
Thesis
Degree Level
Masters
Abstract
This research focuses on assessing the effect of various formulation parameters on targeting human epidermal growth factor receptor-2 (HER2) specifically in breast cancer. Poly (D, L-lactide-co-glycolide) (PLGA) polymer, which is approved by FDA, was used to form nanoparticles (NPs) encapsulating docetaxel (DOC) as chemotherapy. HER2 antibody moieties, either whole IgG (TrAb) or single chain fragment variable (ScFv), were decorated on the PLGA NPs surface and evaluated in terms of their ability to target HER2 breast cancer cells. We observed the effects of these NPs against different cell lines (MCF-7 and SK-BR-3). Thus, ligand modified structurally concealed PLGA NPs could be a promising delivery tool for targeting HER2 breast tumor in vitro that improves the release of chemotherapy while reducing the systemic side effects.
A solvent evaporation procedure was adjusted to form NP formulations using both ester and carboxylic acid terminated PLGA. Incorporation of ligands (TrAb or ScFv) was conducted through chemical covalent conjugation processes by using different cross-linkers bis(sulfo-succinimidyl) (BS3) or N-hydroxysuccinimide esters (NHS) and 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The size, zeta potential, polydispersity index, which were determined for the physicochemical characterizations. The physicochemical characterizations of formulations were executed to assess the effects of different ligands in vitro for drug targeting. Also, Fourier Transform Infrared (FTIR) was used to conform the covalent bond insertion between the linkers and ligands (TrAb or ScFv). DOC loading was quantified by a fully validated mass spectrometry method and antibody (Ab) quantification was performed by using the bicinchoninic acid (BCA) assay. Moreover, in vitro drug release profile was assessed. In vitro cellular targeting was examined by measuring the level of HER2 expression through Fluorescence-activated cell sorting (FACS) in two cell lines as well as western blot analysis. Cytotoxicity assay was performed in SK-BR-3 cell line for all the formulations.
Modified PLGA NPs showed a mean diameter particle size below 400 nm with approximately neutral zeta potential; for example, ScFv-DOC-Ester PLGA NPs formulation size was 312 8.769 nm, and their zeta potential was 0.024 0.075 mV. The average size of the acidic PLGA TrAb-DOC NPs was 382.5 21.5 nm, and their zeta potential was 0.045 0.037 mV. DOC encapsulation efficiency reached up to 65% to 85% depending on the type of the NPs formulations, and the amount of anti-HER2 attachment efficiency exceeded 40%. The cellular targeting of nanoparticles was studied using two cell lines MCF-7 (low HER2 expression) and SK-BR-3 (high HER2 expression), and different levels of HER2 expression were evaluated. The significant reduction in the level of HER2 expression was observed for all modified NPs formulations in HER2 overexpressed SK-BR-3 cells.
Overall, in vitro targeting further demonstrated that modified NPs accumulated DOC in tumor cell line more efficiently than conventional medication. The TrAb conjugated to DOC modified NPs formulations were able to increase the HER2 targeting for DOC compared to ScFv modified NPs. In SK-BR-3 cells, the cytotoxicity of modified NPs was more potent than the conventional DOC due to the targeting, and slow release of DOC from the modified NPs. This system has the potential of improving the targeting and the release of chemotherapeutic drugs into the tumor cells while reducing the side effects caused by affecting healthy tissues.
Our data demonstrated that the high affinity for anti-HER2 modified PLGA NPs formulations to efficiently and explicitly target DOC to the HER2 overexpressing cancer cells can be exploited as a potential strategy for chemotherapeutic drug delivery system for HER2 overexpressing breast cancers. Thus, ligand modified structurally concealed PLGA NPs could be a promising delivery tool for targeting HER2 breast tumor in vivo that improves the release of chemotherapy while reducing the systemic side effects.
Description
Keywords
Targeting Drug Delivery, Nanoparticles, HER2 Receptor, Breast Cancer, PLGA Polymer, Docetaxel, HER2 Antibody
Citation
Degree
Master of Science (M.Sc.)
Department
Pharmacy and Nutrition
Program
Pharmacy