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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Chemistry

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

Published: March 14th, 2021

DOI:

10.3791/62255

1Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, 2Department of Molecular Medicine, Sapienza University of Rome
* These authors contributed equally

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.

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.

Drug-delivery vectors capable of ensuring high antitumor efficacy and reducing side effects are primary goals while remaining a severe chemical-pharmaceutical challenge1,2. To date, their progress is limited at first by the contrast of an insufficient in situ drug release and a critical level of nonspecific toxicity3,4,5. In recent years, several drug delivery systems have been implemented to improve the administration of anticancer agents, including liposomes, polymeric micelles, polymersomes.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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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.......

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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

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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.......

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This work has received funding from the European Union Horizon 2020 research and innovation program under grant agreement AMPHORA (766456).

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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

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