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^6,7,8,9,10. These systems exhibit potential in increasing circulation time and selectivity of drugs, while reducing distribution and accumulation in healthy organs and tissues. Anyway, the encapsulated formulations of antineoplastic chemotherapy drugs, such as anthracyclines, led to a significantly reduced drug internalization efficiency. Recently, stimuli-responsive micron and submicron carriers such as microbubbles¹¹, microdroplets, hybrid gold nanoparticles¹², nano-hydrogels¹³, PLGA scaffolds, and mesoporous platforms¹⁴, have been gaining pharmacological interest for their high versatility in targeting and exerting tumor inhibitory effects using doxorubicin (Dox) and docetaxel. Pioneering experiments to turn these carriers into efficient anticancer soldiers for multimodal tasking (i.e., chemotherapeutic, photothermal, and gene synergistic approaches) and molecular imaging¹⁵ have paved the way for personalized theranostic nanomedicine.

In this scenario, phase-change perfluorocarbon microdroplets (MDs) have been evaluated through the key opportunity they offer to conjugate high drug cargo loading, chemical versatility of the MDs shell addressing biological barriers, colloidal stability and synthesis efficiency^11,12. As an additional asset, the echogenicity of the MDs promoted by acoustic or optical vaporization of the perfluorocarbon (PFC) core allows to gain in situ imaging and promising therapeutic efficacy. Moreover, MDs core vaporization obtained by the energy release of ionizing particle beams can be exploited for beam tracking and radiation dosimetry.

The present study is aimed to develop decafluoropentane (DFP) microdroplets stabilized by a multiple usable shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant. DDAB shelled-MDs meet both physico-chemical and biological expectations. DFP based microdroplets have been demonstrated to be valuable phase-change contrast agents to achieve biocompatible and stable perfluorocarbon MDs¹⁶. DDAB crystalline gel saturates long-chains at physiological temperature, deeply penetrating the hydrophobic core, stabilizing the droplet and the drug cargo therein. Moreover, the high positive ζ-potential at the water interface enhances the colloidal stability of the MDs. Biological attractiveness of DDAB shell surface lies in the ability to cause the death of bacteria and fungi, at concentrations that barely affect mammalian cells, and to bind plasma membranes, negatively charged antigenic proteins, nucleotides, DNA, or nanoparticles. The above-mentioned features can be exploited to generate a remarkable immunoadjuvant, gene therapy and antitumor action within mammalian cells¹⁷.

Dox-loaded DDAB-MDs (Dox@DDAB-MDs) described herein promote the drug release against highly aggressive, invasive, and poorly differentiated triple-negative breast cancer cells. A simple and rapid protocol is described below based on high power probe insonation to obtain stable and high-density DDAB-MDs with a narrow size distribution with a high loading efficiency of Dox in a one-step formulation. Such characteristics are competitive even for other preparation methods like microfluidic devices and high shear homogenizers¹⁶.

The other major limiting issue in designing efficient drug delivery vectors is that the activity of a drug is a function of various parameters (e.g., absorption, distribution, concentrations) obtainable in an actual biological target, which cannot be considered by monolayer cell models¹⁸. For this reason, the history of the development of novel antitumor formulations is studded with in vitro studies that unfortunately have resulted to be ineffective already at the level of preclinical models in animals¹⁹.

Particularly, the need to move from cell cultures to a more complex and reliable system than in vivo and ex vivo studies is linked to the inherent limitations of pharmacological studies on 2D cultures. In this context, the in vitro 3D systems are included, such as spheroids, organoids, organ-on-chip, which simulate the morphology, activity, and physiological response of more complex structures than the 2D monolayers²⁰. In a preclinical view, 3D cell models mimicking the cellular microenvironment offer the possibility to better understand complex biology in a physiologically more pertinent frame in which traditional monolayer cultures are not effective^21,22.

After proving that DDAB-MDs can interact with the cell membrane of human breast cancer cells, favoring drug internalization and cell death at very low (nanomolar) Dox concentration, the efficacy of such methodology against 3D spheroids of mammalian tumor cells, MDA-MB-231, has been tested.

Protocol

NOTE: All the reagents and instruments are listed in the Table of Materials. 1. Fabricating and characterizing microdroplets Preparing Dox-loaded DDAB-MDs 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…

Representative Results

Dox@DDAB-MDs were developed according to protocol (Section 1) as schematically described in Figure 1. The obtained MDs are made of a monolayer of DDAB encapsulating the DFP core (Figure 1A). The cationic charge of DDAB and the sonication procedure avoid the formation of DDAB multilamellar layers stacked at the DFP and water interface23. The CLSM micrograph (Figure 1B</stron…

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

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