Docetaxel-trastuzumab stealth immunoliposome: development and in vitro proof of concept studies in breast cancer

Abstract

BackgroundTrastuzumab plus docetaxel is a mainstay to treat HER2-positive breast cancers. However, developing nanoparticles could help to improve the efficacy/toxicity balance of this doublet by improving drug trafficking and delivery to tumors. This project aimed to develop an immunoliposome in breast cancer, combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab, and comparing its performances on human breast cancer cell lines with standard combination of docetaxel plus trastuzumab.MethodsSeveral strategies to engraft trastuzumab to pegylated liposomes were tested. Immunoliposomes made of natural (antibody nanoconjugate-1 [ANC-1]) and synthetic lipids (ANC-2) were synthesized using standard thin film method and compared in size, morphology, docetaxel encapsulation, trastuzumab engraftment rates and stability. Antiproliferative activity was tested on human breast cancer models ranging from almost negative (MDA-MB-231), positive (MDA-MB-453) to overexpressing (SKBR3) HER2. Finally, cell uptake of ANC-1 was studied by electronic microscopy.ResultsANC-1 showed a greater docetaxel encapsulation rate (73%±6% vs 53%±4%) and longer stability (up to 1 week) as compared with ANC-2. Both ANC presented particle size ≤150 nm and showed similar or higher in vitro antiproliferative activities than standard treatment, ANC-1 performing better than ANC-2. The IC50s for docetaxel combined to free trastuzumab were 8.7±4, 2±0.7 and 6±2 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. The IC50s for ANC-1 were 2.5±1, 1.8±0.6 and 3.4±0.8 nM and for ANC-2 were 1.8±0.3 nM, 2.8±0.8 nM and 6.8±1.8 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. Cellular uptake appeared to depend on HER2 expression, the higher the expression, the higher the uptake.ConclusionIn vitro results suggest that higher antiproliferative efficacy and efficient drug delivery can be achieved in breast cancer models using nanoparticles.

From 3D spheroids to tumor bearing mice: efficacy and distribution studies of trastuzumab-docetaxel immunoliposome in breast cancer

“…This new entity showed promising features such as optimal size (ie, ,150 nm), high docetaxel encapsulation rate (ie, .90%), and trastuzumab engraftment rate (ie, .30%). 6 In vitro efficacy studies were previously performed on 2D monolayer assays, on a limited but representative panel of human breast cancer cells (ie, MDA- 6 We then hypothesized that 2D monolayer models were not suitable to actually evaluate the antiproliferative efficacy of nanoparticles and that better mimicking tumor organization and microenvironment were necessary. To this end, we developed 3D spheroid models.…”

“…As previously described, liposomes were synthesized using the thin film method. 6 PC, Chol, PG, docetaxel, and Mal-PEG were mixed in a 50:19:15:1.7:1 molar ratio. Briefly, lipids were dissolved in methanol.…”

“…Stability studies were previously performed in PBS at 4°C, protected from light. 6 Docetaxel leakage from immunoliposomes and liposomes was also evaluated in biological environment using RPMI culture media (Thermo Fisher Scientific) at 37°C, after 5 minutes, 30 minutes, 1 hour, 24 hours, and 1 week incubation with gentle stirring.…”

“…After stabilization, docetaxel leakage was similar to the PBS matrix condition previously published. 6 In vitro cytotoxicity studies On MDA-MB-453 low-density spheroids, early incubation led to concentration-dependent cytotoxicity (Figure 2A); the higher the concentration, the higher the antiproliferative effect. Free drugs, docetaxel liposomes + free trastuzumab, and immunoliposomes showed marked efficacy as cell growth was reduced by 95%, 94%, and 94% when compared to the control group, respectively, but no difference was observed between treatments (P=0.599, one-way ANOVA with multiple comparison testing).…”

“…When first tested in vitro using monolayer models, this immunoliposome proved to perform better than free docetaxel + free trastuzumab. 6 However, and although results were promising, the issue of tumor uptake and tissue diffusion that cannot be properly described with standard two-dimensional (2D) monolayer models makes in vitro efficacy data hardly transposable to in vivo experiments. Indeed, only three-dimensional (3D) models better mimicking cell-cell junctions, cell-matrix interactions, cell polarity, and oxygen gradient can closely predict biological responses, provided that the right culturing conditions are met.…”