(483h) Liposomal Encapsulation of Therapeutic Nanobioconjugates Enabled By Microfluidic Devices: In silico Analysis Via Comsol Multiphysics

Over the past two decades, encapsulation of bioactive compounds have gained considerable attention as they represent an avenue for maintaining the activity and stability of such compounds even under harsh conditions [1]. The produced encapsulates have found application in several fields ranging from more active and stable nutraceuticals to the delivery of pharmacological compounds. In this particular case, novel methods to increase bioavailability have been developed for the treatment of various conditions including different types of cancer, neurodegenerative diseases, and even the control of antibiotic-resistant bacteria [2],[3]. Some of the encapsulating materials include polymeric gels and capsules, liposomes, micelles, and mesoporous materials [4],[5]. Liposomes have been particularly attractive for biomedical applications due to their low cytotoxicity and immune response, ease of synthesis and high-delivery rates. Despite these attributes, a major challenge is to assure a homogeneous size distribution, which is critical to maintain enough stability under physiological conditions [6]–[9]. To achieve this, strategies based on microfluidics have been particularly successful mainly due to the possibility of controlling the ratio of the interacting aqueous and non-aqueous phases. Also, the prevalence of laminar flow regimes make the mixing processes in such systems highly-controllable [10],[6],[11]. Moreover, the low volumes required facilitate rapid prototyping in an inexpensive and reproducible manner [12]. Despite the important opportunities for implementing encapsulation processes based on microfluidics, the underlying phenomena and mechanistic description of such approaches remain largely unexplored [13],[14]. In addition to encapsulation techniques, the last ten years have seen the exponential increase in the number on nanoplatforms for the efficient transport and delivery of cargoes at the intracellular level. This includes a large arsenal of materials with different topologies and from different families (e.g., metallic oxides, carbon-based, and polymeric). Over the past three years, we have developed a few cell-penetrating nanobioconjugates by interfacing magnetite nanoparticles with translocating molecules such as the Outer Membrane Protein A (OmpA) of Escherichia coli, and the antimicrobial peptide Buforin II (BUF-II) [15]–[17] . The potent cell-penetrating abilities, high biocompatibility, and the high levels of endosome escape of our nanobioconjugates make them suitable for the delivery of pharmacological cargoes. For this reason, we are currently exploring the delivery of small molecules and gene therapies for the treatment of neurodegenerative diseases. A possible concern upon administration, is that the nanobioconjugates might induce immunogenic responses, and consequently we propose to encapsulate them in liposomal systems.

This work is therefore dedicated to designing microfluidic devices for the liposomal encapsulation of our therapeutic nanobioconjugates. We designed microfluidic devices and tested them in silico with the aid of COMSOL. In our first approach, we put in contact a ferrofluid containing the nanobioconjugates with a suspension of the liposomes in isopropyl alcohol. This interaction was simulated by applying a mixture model for the ferrofluid motion, which was coupled to a convection-diffusion model of diluted species for the mixing with the liposome suspension. Figure 1 shows the results for the flow of the dispersed phase in the microfluidic device. The dispersed phase achieves a complete mixing with the continuous aqueous phase when it arrives at the second loop after about 15 seconds. This is followed by a regime where of a constant volume fraction of around 0.2 under laminar conditions after 30 seconds. Figure 2 illustrates the concentration fraction of the ferrofluid as it mixes with the liposomes’ suspension. As for the dispersed phase in Figure 1, complete mixing is achieved when the fluids reach the second loop of the mixing chamber. As a result, we are certain that the loops of the serpentine chamber contribute to the interaction between both fluids and allow a uniform mixing pattern within a few seconds. To some extent, this can be explained by the effect of Dean vortices inducing chaotic advection [18].

Alternatively, we decided to explore the possibility of increasing the chances for encapsulation by subjecting the device to an acoustic field, which will be generated by an ultrasonic bath in a subsequent experimental testing stage. In this context, ultrasound is used as an external energy source capable of altering the mixing and promoting interactions through acoustic streaming [19]. This new in silico study builds upon the two previously coupled modules by adding an acoustics model with ultrasonic frequencies in the range of 20 kHz-42 kHz. Both studies are applied for the designed Y-type micromixer (inlets angle of 45°) with a serpentine mixing chamber (see Figure 2).

Figures 3a and 3b show the 2D surface sound pressure and acoustic pressure field of ultrasound in the presence of the designed microfluidic device. The same fields in the absence of the microfluidic device are shown in Figures 4a and 4b. Figure 4c shows the changes in both the acoustic field pressure and sound pressure level when the microfluidic device is immersed in the ultrasonic bath. This shows transmission and reflection of the incident ultrasound waves generated by the acoustic field after they impact the solid structure material of the microfluidic device, which was built in COMSOL by using the properties of polymethyl methacrylate (PMMA). This will be the actual situation as we have perfected a low-cost manufacturing technique that is based on laser cutting and engraving on PMMA sheets.

Based on the results obtained thus far, our current ongoing work focuses on coupling the mixture model, transport of diluted species and pressure acoustics. This with the purpose of estimating whether the ultrasonic vibration facilitates encapsulation. Finally, details of the encapsulation process will be attempted by modeling the translocation of the ferrofluid through the lipid bilayer of liposomes after setting up their boundaries as reactive interfaces. For this model, optimal values for relevant parameters such as the velocity field and concentrations can be obtained from previous simulations and subsequently introduced into this multiphysics study.

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