Multivesicular microfluidic vesicles for drug delivery and artificial cells: a short review

Multivesicular vesicles (MVVs) are non-concentrically arranged vesicles-in-vesicle systems that can be prepared from bilayer-forming amphiphiles. Although spontaneous formation of MVVs is possible, these hierarchical structures are usually obtained intentionally by guided molecular assembly in a variety of different well-known protocols. This short review discusses the microfluidic method to generate MVVs and current applications as artificial cell-like systems or for drug delivery.

This short review article is based on the article entitled “Multivesicular Vesicles: Preparation and Applications” written by Camila Betterelli Giuliano, Nemanja Cvjetan, Dr. Jessica Ayache, Prof. Dr. Peter Walde and published in the journal “ChemSystemsChem.” This work was supported by the Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (ITN) GA n° 813873, ProtoMet.

Introduction to multivesicular microfluidic vesicles

Liposomes are compartments composed of a membrane made with amphipathic molecules, often phospholipids, forming a bilayer. [1], [2] These compartments can be classified depending on their membrane structure and size (Fig.1). For example, if they have only one bilayer, they are called unilamellar; if they have several, they are called oligolamellar or multilamellar vesicles depending on the number of layers. There is a larger interest in producing unilamellar vesicles, due to their resemblance to the cell membrane and their easier-to-predict membrane transport behaviour. [3] They will be the focus of this short review. Unilamellar vesicles with sizes ranging from 30 to 50 nm in diameter are called “small or sonicated unilamellar vesicles (SUVs)”; from 50 to 500 nm, “large unilamellar vesicles (LUVs)”; and above 500 nm, “giant unilamellar vesicles (GUVs)”.

Figure 1. Cross-sectional representation of different types of liposomes based on membrane structure. Extracted from Giuliano et al, 2020.

Unilamellar vesicles have been widely used in research as models for cell membrane studies and even reached the market as drug delivery systems. [4] Due to their versatility and well-established knowledge base, there has been an increased interest in studying more complex liposome-based systems. Multivesicular microfluidic vesicles are non-concentrically arranged hierarchical compartments composed of smaller liposomes (SUVs or LUVs) inside larger liposomes (LUVs or GUVs), which can be also called multivesicular vesicles (MVVs, Fig. 1). These complex compartments are particularly interesting because, on one hand, they resemble more closely the hierarchical structure of cells [5] and, on the other hand, they allow for modulation of the release profile in drug delivery systems. [6]

Multivesicular microfluidic vesicles production method

There are many ways of obtaining multivesicular vesicles and they are extensively reviewed in Giuliano et al, 2020. Here, we will focus on microfluidic-based production due to the increased control and consequently, reproducibility, provided by the technology. These characteristics are translated and reinforced by the highly monodisperse populations of droplets and the high encapsulation efficiency allowed by microfluidics. [7] The underlying principle of producing liposomes in microfluidics is using water-in-oil-in-water droplets, or double emulsions, as templates. [8]

Double emulsions are usually produced in glass capillaries [9] or in two-junction PDMS chips that are surface treated to be part hydrophobic and part hydrophilic. [8] In the latter, the solution that will be inside the water-in-oil-in-water droplet is pinched in the first junction by the oil phase. The water-in-oil droplet travels in a hydrophobic channel until the second junction, where the oil phase will be pinched by the outer aqueous solution, forming the water-in-oil-in-water droplet (Fig. 2). The lipids are dissolved in the oil phase. Once the double emulsion is formed, there is a need to remove the oil from the membrane, leaving only the lipid bilayer (Fig. 2.b). This removal can be spontaneous or triggered, depending on the characteristics of the molecules involved.

Multivesicular microfluidic vesicles follow a similar production process. The main difference is that the smaller vesicles that will be encapsulated into the larger ones are added to the inner phase.

Figure 2. Multivesicular microfluidic vesicles production. a) Schematic of microfluidic setup for production of double emulsion-templated liposomes. A pressure-driven flow controller drives the inner solution (IA) to the first junction, in which it is enveloped by the oil phase (LO), and then pinched by the outer phase (OA) at the second junction, forming a double emulsion with an ultra-thin oil layer. b) Schematic of multivesicular microfluidic vesicle production with a spontaneous oil layer removal from the templated double emulsions. Extracted from Giuliano et al, 2020.

Multivesicular microfluidic vesicles for drug delivery

Liposomes have been approved and used as drug delivery systems since 1995. [10] They improve the therapeutic index of drugs such as anticancer doxorubicin and daunorubicin but are also known to have limitations in retaining small molecule drugs once in systemic circulation [6]. It is believed that the early release is caused by interactions of the lipid bilayer with enzymes in the blood, causing early degradation of the membrane.

The hierarchical structure of multivesicular vesicles can help solve this problem by providing an extra layer of protection. Also, each vesicle can be tuned to answer to a particular trigger, increasing the level of formulation flexibility. Currently, there is one approved formulation that takes advantage of this property, the DepoFoam. [11] For example, it has been shown that it allows a much higher dose of non-opioid pain relievers without worsening side effects. [12] It is produced by traditional emulsification methods, however, manufacturing challenges resulted in the discontinuation of some of the products that used DepoFoam as a carrier. These challenges could potentially be addressed by a more mature microfluidic technology.

Figure 3. DepoFoam drug delivery system. a) Schematic of DepoFoam hierarchical structure. b) Fluorescent images of the lipid structure (red); the aqueous content (green); and the merged image. c) In vivo comparison of the DepoFoam (Exparel, commercial name) efficacy when compared to different concentrations of the same drug. Extracted from refs [51d] and [94] in Giuliano et al, 2020.

Multivesicular microfluidic vesicles artificial-cell like systems

Multivesicular microfluidic vesicles closely resemble the configuration of living cells, making them suitable models for artificial-cell-like systems. [13] Applications in this field range from confined chemical reaction systems, with the goal of controlling complex chemical reactions in compartments, to mimicking living cells, in which even cell division has been reproduced. [14]

A great example of the use of multivesicular microfluidic vesicles as artificial-cell-like systems is the work of Deng et al, 2016. They reported an artificial cell with a “nucleus” capable of RNA transcription. The artificial cell was a multivesicular vesicle produced with microfluidic glass capillaries.

Figure 4. Multivesicular microfluidic vesicles for artificial-cell-like systems. a) Schematic and representative image of multivesicular microfluidic vesicle production. b) Production of fluorescent RNA in vesicles over time. c) Production of fluorescent RNA in the “nucleus” of the artificial cell. Extracted from reference [53] in Giuliano et al, 2020.

Conclusion and Outlook

Multivesicular microfluidic vesicles are promising tools for the advancement of drug delivery systems and artificial-cell-like systems research. For drug delivery, they provide extra protection for liposomes in the bloodstream while allowing for fine tuning of the system for better release, taking into consideration the properties of the drug and administration route. For artificial-cell-like systems, they closely resemble living cells, allowing for the study of confined reaction systems and more complex cellular behaviors, such as division and RNA transcription.

Microfluidics has advantages over traditional emulsification methods due to improved monodispersity of liposomal populations, which in turn increases the reproducibility of the experiments. It also provides higher control over encapsulation and compartment composition. These characteristics can improve results across the discussed applications and will be particularly important for the pharmaceutical industry once the technology reaches industry scale.

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