Magnetite nanoparticles (MNPs) have gained significant attention in several applications for drug delivery. However, there are some issues related to cell penetration, especially in the transport of cargoes that show limited membrane passing. A widely studied strategy to overcome this problem is the encapsulation of the MNPs into liposomes to form magnetoliposomes (MLPs), which are capable of fusing with membranes to achieve high delivery rates. This study presents a low-cost microfluidic approach for the synthesis and purification of MLPs and their biocompatibility and functional testing via hemolysis, platelet aggregation, cytocompatibility, internalization, and endosomal escape assays to determine their potential application in gastrointestinal delivery. The results show MLPs with average hydrodynamic diameters ranging from 137 ± 17 nm to 787 ± 45 nm with acceptable polydispersity index (PDI) values (below 0.5). In addition, we achieved encapsulation efficiencies between 20% and 90% by varying the total flow rates (TFRs), flow rate ratios (FRRs), and MNPs concentration. Moreover, remarkable biocompatibility was attained with the obtained MLPs in terms of hemocompatibility (hemolysis below 1%), platelet aggregation (less than 10% with respect to PBS 1×), and cytocompatibility (cell viability higher than 80% in AGS and Vero cells at concentrations below 0.1 mg/mL). Additionally, promising delivery results were obtained, as evidenced by high internalization, low endosomal entrapment (AGS cells: PCC of 0.28 and covered area of 60% at 0.5 h and PCC of 0.34 and covered area of 99% at 4 h), and negligible nuclear damage and DNA condensation. These results confirm that the developed microfluidic devices allow high-throughput production of MLPs for potential encapsulation and efficient delivery of nanostructured cell-penetrating agents. Nevertheless, further in vitro analysis must be carried out to evaluate the prevalent intracellular trafficking routes as well as to gain a detailed understanding of the existing interactions between nanovehicles and cells.

One of the main routes to ensure that biomolecules or bioactive agents remain active as they are incorporated into products with applications in different industries is by their encapsulation. Liposomes are attractive platforms for encapsulation due to their ease of synthesis and manipulation and the potential to fuse with cell membranes when they are intended for drug delivery applications. We propose encapsulating our recently developed cell-penetrating nanobioconjugates based on magnetite interfaced with translocating proteins and peptides with the purpose of potentiating their cell internalization capabilities even further. To prepare the encapsulates (also known as magnetoliposomes (MLPs)), we introduced a low-cost microfluidic device equipped with a serpentine microchannel to favor the interaction between the liposomes and the nanobioconjugates. The encapsulation performance of the device, operated either passively or in the presence of ultrasound, was evaluated both in silico and experimentally. The in silico analysis was implemented through multiphysics simulations with the software COMSOL Multiphysics 5.5^® (COMSOL Inc., Stockholm, Sweden) via both a Eulerian model and a transport of diluted species model. The encapsulation efficiency was determined experimentally, aided by spectrofluorimetry. Encapsulation efficiencies obtained experimentally and in silico approached 80% for the highest flow rate ratios (FRRs). Compared with the passive mixer, the in silico results of the device under acoustic waves led to higher discrepancies with respect to those obtained experimentally. This was attributed to the complexity of the process in such a situation. The obtained MLPs demonstrated successful encapsulation of the nanobioconjugates by both methods with a 36% reduction in size for the ones obtained in the presence of ultrasound. These findings suggest that the proposed serpentine micromixers are well suited to produce MLPs very efficiently and with homogeneous key physichochemical properties.

The delivery of bioactive compounds is often improved by their encapsulation within systems based on different materials, such as polymers and phospholipids. In this regard, one of the most attractive vehicles are liposomes, which can be produced by the self-assembly of phospholipids in aqueous buffered systems. Encapsulation of therapeutic magnetite nanoparticles (MNPs) within liposomes can be accomplished by direct translocation of their lipid bilayer by surface conjugation of potent translocating peptides (and proteins) such as Buforin-II and OmpA. Here, we put forward the notion that to achieve reproducibility and optimize this process, it is possible to develop microfluidic systems that use flow-focusing methods to manipulate the interaction of suspended MNPs (ferrofluids) with the liposomes. With that in mind, we have developed an in silico approach to predict the performance of microfluidic devices specifically designed for the encapsulation process. This was done by running multiphysics simulations in COMSOL to evaluate the macroscopic flow of liposomes and suspended MNPs via a multiphase mixture model. Moreover, we estimated the corresponding interaction using a chemical reaction model based on embedding the Michaelis–Menten equation within the diluted species module’s transport. In this case, the enzymes-substrate interaction was considered similar to that of the MNPs-liposome. As a result, we were able to approach saturation kinetics that resemble that obtained experimentally for the uptake of functionalized MNPs. Future work will be directed towards refining the model by considering more details on the possible stages during the interaction of the involved intermediates.

Cell spheroids represent a scaffold-free route to form cell aggregates through maximizing cell-cell interactions. Spheroids have gained significant attention for the engineering of multilayer tissues as they offer a closer resemblance of physiological conditions observed in vivo, compared with traditional 2D models where cells are grown on flat surfaces. Multilayered tissues are formed by allowing spheroids to interact with themselves within relevant matrices mimicking native conditions of extracellular matrices. However, such conventional fusion methods provide little control over spheroid–spheroid interactions, making their reproducibility very limited. Additionally, such methods are lengthy, which is undesirable from the scalability and economic viewpoints. We propose a methodology to accelerate spheroid fusion by applying magnetic forces externally on spheroids previously magnetized by the internalization of iron oxide nanoparticles. A base mathematical model of spheroids assembly was implemented in COMSOL Multiphysics and involved a laminar two-phase field approach, which was validated by employing a novel segmentation algorithm. The magnetic effect was introduced by an applied volumetric magnetic force, which was generated by the action of two neodymium permanent magnets positioned perpendicular to the computational domain. Our simulations showed that magnetized spheroids fusion can be accelerated by about 55% after applying a magnetic force. In addition, we successfully tested a new modeling approach that allowed taking into account interactions between spheroids and the medium, as evidenced by a standard error of only 13% with respect to the experimental results shown in the literature. Importantly, these simulations also showed that the time required to fuse the spheroids is reduced by about 55%. We are currently validating the model experimentally on extracellular-matrix-derived hydrogels embedded with magnetized spheroids. Future work will be dedicated to calibrating the model with the collected experimental data.