One of the challenges of modern biotechnology is to find new routes to mitigate the resistance to conventional antibiotics. Antimicrobial peptides (AMPs) are an alternative type of biomolecules, naturally present in a wide variety of organisms, with the capacity to overcome the current microorganism resistance threat. Here, we reviewed our recent efforts to develop a new library of non-rationally produced AMPs that relies on bacterial genome inherent diversity and compared it with rationally designed libraries. Our approach is based on a four-stage workflow process that incorporates the interplay of recent developments in four major emerging technologies: artificial intelligence, molecular dynamics, surface-display in microorganisms, and microfluidics. Implementing this framework is challenging because to obtain reliable results, the in silico algorithms to search for candidate AMPs need to overcome issues of the state-of-the-art approaches that limit the possibilities for multi-space data distribution analyses in extremely large databases. We expect to tackle this challenge by using a recently developed classification algorithm based on deep learning models that rely on convolutional layers and gated recurrent units. This will be complemented by carefully tailored molecular dynamics simulations to elucidate specific interactions with lipid bilayers. Candidate AMPs will be recombinantly-expressed on the surface of microorganisms for further screening via different droplet-based microfluidic-based strategies to identify AMPs with the desired lytic abilities. We believe that the proposed approach opens opportunities for searching and screening bioactive peptides for other applications.

Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.

We describe the manufacture of low-cost microfluidic systems to produce nanoscale liposomes with highly uniform size distributions (i.e., low polydispersity indexes (PDI)) and acceptable colloidal stability. This was achieved by exploiting a Y-junction device followed by a serpentine micromixer geometry to facilitate the diffusion between the mixing phases (i.e., continuous and dispersed) via advective processes. Two different geometries were studied. In the first one, the microchannels were engraved with a laser cutting machine on a polymethyl methacrylate (PMMA) sheet and covered with another PMMA sheet to form a two-layer device. In the second one, microchannels were not engraved but through-hole cut on a PMMA sheet and encased by a top and a bottom PMMA sheet to form a three-layer device. The devices were tested out by putting in contact lipids dissolved in alcohol as the dispersed phase and water as the continuous phase to self-assemble the liposomes. By fixing the total flow rate (TFR) and varying the flow rate ratio (FRR), we obtained most liposomes with average hydrodynamic diameters ranging from 188 ± 61 to 1312 ± 373 nm and 0.30 ± 0.09 PDI values. Such liposomes were obtained by changing the FRR from 5:1 to 2:1. Our results approached those obtained by conventional bulk synthesis methods such as a thin hydration bilayer and freeze-thaw, which produced liposomes with diameters ranging from 200 ± 38 to 250 ± 38 nm and 0.30 ± 0.05 PDI values. The produced liposomes might find several potential applications in the biomedical field, particularly in encapsulation and drug delivery.

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.