Droplet Microfluidics for Tumor Drug-Related Studies and Programmable Artificial Cells

Abstract

Anticancer drug development is a crucial step toward cancer treatment, that requires realistic predictions of malignant tissue development and sophisticated drug delivery. Tumors often acquire drug resistance and drug efficacy, hence cannot be accurately predicted in 2D tumor cell cultures. On the other hand, 3D cultures, including multicellular tumor spheroids (MCTSs), mimic the in vivo cellular arrangement and provide robust platforms for drug testing when grown in hydrogels with characteristics similar to the living body. Microparticles and liposomes are considered smart drug delivery vehicles, are able to target cancerous tissue, and can release entrapped drugs on demand. Microfluidics serve as a high-throughput tool for reproducible, flexible, and automated production of droplet-based microscale constructs, tailored to the desired final application. In this review, it is described how natural hydrogels in combination with droplet microfluidics can generate MCTSs, and the use of microfluidics to produce tumor targeting microparticles and liposomes. One of the highlights of the review documents the use of the bottom-up construction methodologies of synthetic biology for the formation of artificial cellular assemblies, which may additionally incorporate both target cancer cells and prospective drug candidates, as an integrated "droplet incubator" drug assay platform.

Keywords: artificial cells; droplet microfluidics; drug screening; tumor spheroids.

Figure 1

A schematic diagram describing how in vitro drug screening and delivery systems can be utilised as constituents toward novel microfluidic generated drug screening platforms, termed as “droplet incubators”. Multicellular tumor spheroids (MCTSs) in natural hydrogels as in vitro 3D models.

Figure 2

a) Fluorescence microscopy images of MCTS co‐cultures of fibroblasts (green) and cancer cells (red). Tumor invasion occurs only by MCTSs surrounded by the collagen‐alginate 3D hydrogel. Reproduced with permission.^{[ 33 ]} Copyright 2018, Elsevier. b) Microfluidic assisted formation of liver‐in‐a‐droplet. Microfluidic production of liver‐in‐a‐droplet, where hepatocytes and fibroblasts were encapsulated in the core and shell of the capsule, respectively. Reproduced with permission.^{[ 62 ]} Copyright 2016, Royal Society of Chemistry. c) A microfluidics device used for production of collagen core and alginate shell capsules used for 3D tumor vascularization experiments. 3D vascularized tumors expressed increased drug resistance in the presence of a commonly used chemotherapeutic drug, although this resistance reduced when treated with drug carrying nanoparticles. Reproduced with permission.^{[ 63 ]} Copyright 2017, American Chemical Society. d) The rationale of combining microfluidics, hydrogels and cancer cell encapsulation in order to produce high throughput personalized drug treatments. Reproduced with permission.^{[ 46 ]} Copyright 2019, John Wiley and Sons.^{[ 64 ]} Copyright 2020, American Chemical Society.

Figure 3

a) Magnetically responsive drug‐laden chitosan capsules fabricated using microfluidics. The chitosan droplets are loaded with superparamagnetic iron oxide nanoparticles (SPIO NPs) and chemotherapeutic drug, vinblastine (VBL), which is released by a pulsatile magnetic field. Reproduced with permission.^{[ 88 ]} Copyright 2019, MDPI. b) Droplet microfluidics fabricated near‐infrared (NIR) responsive 1‐tetradecanol microparticles, carrying doxorubicin (DOX)/ IR780 (photothermal agent). DOX release is achieved under NIR light pulses. Reproduced with permission.^{[ 91 ]} Copyright 2019, Elsevier. c) Graphic diagrams and microscopy images of possible polymer microparticles fabricated using microfluidics. Reproduced with permission.^{[ 106 ]} Copyright 2019, John Wiley and Sons. d) Lipid‐polymer Janus microparticles fabricated using microfluidics and solvent evaporation. Different structures achieved by altering the concentrations of the phases. a) Janus, b) Janus‐patchy, c) Triple, d) Quadruple, e,f) Core‐shell and sustained paclitaxel release from Janus microparticles. Reproduced with permission.^{[ 113 ]} Copyright 2019, Elsevier.

Figure 4

a) Representation of intravenous delivery of encapsulated drug in nanocarriers. (Top) Passive targeting of liposomal nanocarriers through the EPR effect, (Bottom) Ligand carrying liposomes for active tumor targeting. Reproduced with permission.^{[ 9 ]} Copyright 2019, John Wiley and Sons. b) An example of drug carrying tumor targeting liposome through a hyaluronic acid conjugate recognized by CD44 receptor. The liposome is endocytosed and acidic pH within a transformed cell causes the release of the docetaxel anticancer drug. Reproduced with permission.^{[ 175 ]} Copyright 2018, Elsevier. c) Presentation of multiple approaches to design stimuli responsive liposomes, while incorporating targeting ligands to achieve selective drug release. Reproduced with permission.^{[ 180 ]} Copyright 2017, John Wiley and Sons.

Figure 5

a) MHF device, with alcohol/lipids and water as inlets. Reciprocal diffusion at the interface of the two phases causes lipid hydration and vesicle formation, due to the self‐assembling nature of lipids. Reproduced with permission.^{[ 185 ]} Copyright 2021, Royal Society of Chemistry. b) Double emulsion of water in octanol/lipid and automated octanol extraction leads to formation of liposomes. Reproduced with permission.^{[ 193 ]} Copyright 2016, Nature Communications. c) Microfluidic device that combines a Y‐junction and SHM for liposome generation. Reproduced with permission.^{[ 194 ]}Copyright 2019, Elsevier. d) Microfluidic device for liposome formation followed by post treatment using buffer for organic solvent removal. This microfluidic integrated post treating step avoids fusion of liposomes and does not affect encapsulation efficacy. Reproduced with permission.^{[ 202 ]} Copyright 2020, American Chemical Society.

Figure 6

a) Guest‐host protocell construct for activation of synergistic or antagonistic behaviors. Glucose oxidase (GOx) containing proteinosome (guest protocell), trapped in a fatty acid micelle coacervate (host protocell). Depending on the glucose concentration of the surroundings, pathway 1 (synergistic) or 2 (antagonistic) is initiated. Low glucose concentration causes the coacervate to become fluorescent and high glucose concentration decreases pH and induces the formation of fatty acid vesicles. Reproduced with permission.^{[ 211 ]} Copyright 2018, Springer Nature. b) Schematic illustration for the formation of DIBs, multicore artificial cell constructs produced by droplet microfluidics and one or multiple DIBs and aqueous solutions encapsulated in a hydrogel capsule. Reproduced with permission.^{[ 213 ]} Copyright 2016, John Wiley and Sons. c) Highly compartmentalized capsules produced using novel bat‐wing junction, a–o) 2–15 water droplets encapsulated within solid semi‐permeable capsules, p–r) Different water droplets trapped in solid TMPTA solid capsules, s,t) encapsulated DIB networks in TMPTA/water/squalene. Reproduced with permission.^{[ 240 ]} Copyright 2017, Royal Society of Chemistry. d) Enzymatic reaction pathway in a natural eukaryotic cell and cell inspired hollow hydrogels encapsulating inverse opal particles with immobilized enzymes fabricated using microfluidics. Reproduced with permission.^{[ 218 ]} Copyright 2018, AAAS. e) Encapsulation of DIBs in alginate shells using non‐planar droplet microfluidic device. The outermost shell contains components such as magnetic particles, which offer mobility to the artificial cell construct in the presence of a magnet. Reproduced with permission.^{[ 127 ]}. Copyright 2019, John Wiley and Sons. f) Vesicle‐cell engineered hybrid, where hydrolysis of lactose to glucose within the hybrid causes a chemical reaction that produces fluorescence. Reproduced with permission.^{[ 230 ]} Copyright 2018, Springer Nature.

Figure 7

Schematic illustration of the stages required to perform anticancer drug screening using droplet incubators. a) A representation of a droplet incubator. The outer hydrogel shell of the droplet incubator hosts cancer cells and over incubation develop tumor spheres. The core is divided into three sections suggesting drug encapsulation and release systems. DIBs section proposes a cascade of reactions (A→E), that involves lipid bilayers and protein pores aiming the release of certain drug(s), to the shell. Another pie section describes possible smart nanocarriers (tumor targeting liposomes and microparticles) for the release of chemotherapeutic drugs under the influence of internal or external effects. Drug screening is possible by incorporating programmability within the context of artificial cells in the presence of tumorspheres. b) Hydrophilic Drug A and Drug B dilutions are performed within a microfluidic device. [Chip (shown) fabricated by 3D printing (unpublished data by the authors) for Worldcare Technologies Inc, using the design from,^{[ 245} , ^{246 ]})] c) The candidate drug(s) flow into a device with droplet forming junctions which can generate triple emulsions (i.e., droplet incubator) at the outlet. d) Droplet incubators collected into a 24‐well plate for further analysis. Each well may host a single droplet incubator to generate a concentration gradient and drug ratio array, depending on the dilutions from the microfluidic chip in b).