Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Summary

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Abstract

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 µm size droplets. Highly positive ζ-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.

In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Introduction

Drug-delivery vectors capable of ensuring high antitumor efficacy and reducing side effects are primary goals while remaining a severe chemical-pharmaceutical challenge^1,2. To date, their progress is limited at first by the contrast of an insufficient in situ drug release and a critical level of nonspecific toxicity^3,4,5. In recent years, several drug delivery systems have been implemented to improve the administration of anticancer agents, including liposomes, polymeric micelles, polymersomes

Protocol

NOTE: All the reagents and instruments are listed in the Table of Materials.

1. Fabricating and characterizing microdroplets

1. Preparing Dox-loaded DDAB-MDs
   1. Dissolve the DDAB powder in ethanol to obtain a final concentration of 10 mM and a final volume of 1 mL. Prepare 1 mL of Dox stock solution dissolving 2 mg Dox powder in ethanol.
      CAUTION: Dox is known to have acute oral toxicity, category 4 and carcinogenicity, category 1B. Us.......

Discussion

To improve the efficacy of anthracyclines as antitumor drugs, this work presents the formation of DDAB shelled PFC droplets encapsulating the chemotherapeutic drug doxorubicin (Dox) and the effect of such formulation interacting with the high aggressive triple-negative breast cancer cells, MDA-MB-231.

Building up of DOX@DDAB-MDs
Dox loaded MDs have been formulated by the insonation method with an extremely fast, well reproducible, user-friendly, and efficient protocol. T.......

Materials

Name	Company	Catalog Number	Comments
µ-Petri dish	Ibidi	81156	35mm high, IbiTreat
1,1,1,2,3,4,4,5,5,5-Decafluoropentane	Sigma-Aldrich	138495-42-8	b.p. 55°C
12-well culture plate	Corning
15 ml centrifuge tube	Falcon	89039-664
3D-Petri dishes 12:256	Microtissues (Sigma-Aldrich)	Z764000-6EA	Small
3D-Petri dishes 12:81	Microtissues (Sigma-Aldrich)	Z764019-6EA	Large
5%CO2 culture incubator, 37°C	Thermo Scienific		HERAcell 150i
50 ml centrifuge tube	Falcon	352070
Biological safety cabinet, II level
Calcein	Sigma-Aldrich
Calcein-AM	Sigma-Aldrich	148504-34-1	4mM stock solution in DMSO
cam sCMOS Andor Zyla 4.2	Andor Instruments
Centrifuge Hettich Universal 320R	Hettich Lab. Technology
DAPI	SIgma-Aldrich
Dimethyldioctadecylammonium bromide powder	Sigma-Aldrich	3700-67-2
DMEM (Dulbecco's Modified Eagle Medium)	Corning	15-013-CV
Doxorubicin hydrochloride	Sigma-Aldrich	25316-40-9
DPBS (Dulbecco's Modified PBS)	Corning	21-030-CV	pH 7,4
Ethanol 70%	Sigma-Aldrich
EZ-C1 digital ecliplse	Nikon Instruments		Silver version 3.91
Fetal Bovine Serum (FBS)	Corning	35-079-CV
Goniometer BI-200SM	Brookhaven Instruments Corporations
Laser Ar+	Spectra Physics
Laser He-Ne	Melles-Griot
L-Glutammine	Corning	25-005-CI
Mcroscope Nikon Eclipse Ti	Nikon Instruments
MDA-MB 231 cell line	ATCC
Microsoft Excel	Microsoft
Microplates reader Spark	Tecan group
NanoZetaSizer ZS	Malvern Instruments LTD
Neubauer improved chamber		718605
NIS Elements software	Nikon Instruments		AR 4.30
Pen/Strepto	Corning	30-002-CI
Photocorrelator BI-9000 AT	Brookhaven Instruments Corporations	62927-1
Photometer HC120	Brookhaven Instruments Corporations	N° 1275
Pipettors and tips, various size	Gilson
Propidium Iodide	SIgma-Aldrich
Serological pipets, various size	Corning
Solid-state laser	Suwtech Laser	N° 22368
T25 Flasks	Sarstedt	83.3910.002
T75 Flasks	Sarstedt	83.3911.002
Trypsin/EDTA 0.05%	EuroClone	ECB3052D
Vibra-Cell VCX-400	Sonics & Materials, inc
Water bath			37°C