Gelatin methacryloyl granular scaffolds for localized mRNA delivery.

mRNA therapy is the intracellular delivery of messenger RNA (mRNA) to produce desired therapeutic proteins. Developing strategies for local mRNA delivery is still required where direct intra-articular injections are inappropriate for targeting a specific tissue. The mRNA delivery efficiency depends on protecting nucleic acids against nuclease-mediated degradation and safe site-specific intracellular delivery.

Monte Carlo simulation-guided design for size-tuned tumor spheroid formation in 3D printed microwells.

Tumor spheroid models have garnered significant attention in recent years as they can efficiently mimic in vivo models, and in addition, they offer a more controlled and reproducible environment for evaluating the efficacy of cancer drugs. In this study, we present the design and fabrication of a micromold template to form multicellular spheroids in a high-throughput and controlled-sized fashion. Briefly, polydimethylsiloxane-based micromolds at varying sizes and geometry were fabricated via soft lithography using 3D-printed molds as negative templates.

Engineering aspects of lipid-based delivery systems: In vivo gene delivery, safety criteria, and translation strategies.

Defects in the genome cause genetic diseases and can be treated with gene therapy. Due to the limitations encountered in gene delivery, lipid-based supramolecular colloidal materials have emerged as promising gene carrier systems. In their non-functionalized form, lipid nanoparticles often demonstrate lower transgene expression efficiency, leading to suboptimal therapeutic outcomes, specifically through reduced percentages of cells expressing the transgene.

Droplet-Based Microfluidics as a Platform to Design Food-Grade Delivery Systems Based on the Entrapped Compound Type.

Microfluidic technology has emerged as a powerful tool for several applications, including chemistry, physics, biology, and engineering. Due to the laminar regime, droplet-based microfluidics enable the development of diverse delivery systems based on food-grade emulsions, such as multiple emulsions, microgels, microcapsules, solid lipid microparticles, and giant liposomes. Additionally, by precisely manipulating fluids on the low-energy-demand micrometer scale, it becomes possible to control the size, shape, and dispersity of generated droplets, which makes microfluidic emulsification an excellent approach for tailoring delivery system properties based on the nature of the entrapped compounds.

Biomimetic Nanovesicles-Sources, Design, Production Methods, and Applications.

Despite all the progress in the field of liposomes and nanoparticles for applications as drug and gene delivery systems, the specific targeting and immune system escape capabilities of these systems are still limited. Biomimetic nanovesicles emerged as a strategy to overcome these and other limitations associated with synthetic carriers, such as short circulation time, cytotoxicity, and difficulty in crossing biological barriers, since many of the desirable abilities of drug delivery systems are innate characteristics of biological vesicles. Thus, the question arises: would biomimetic nanovesicles be responsible for addressing these advances? It is currently known that biomimetic nanovesicles (BNV) can combine the intrinsic advantages of natural materials with the well-known production methods and controllability of synthetic systems.

The diffusion-driven microfluidic process to manufacture lipid-based nanotherapeutics with stealth properties for siRNA delivery.

Our study investigated the manufacturing of lipid-based nanotherapeutics with stealth properties for siRNA delivery by employing a diffusion-driven microfluidic process in one or two-steps strategies to produce siRNA-loaded lipid nanocarriers and lipoplexes, respectively. In the one-step synthesis, siRNA in the aqueous phase is introduced from one inlet, while phospholipids dispersed in anhydrous ethanol are introduced from other inlets, generating the lipid nanocarriers. In the two-steps strategies, the pre-formed liposomes are complexed with siRNA.

Trends in hydrogel-based encapsulation technologies for advanced cell therapies applied to limb ischemia.

Ischemia occurs when blood flow is reduced or restricted, leading to a lack of oxygen and nutrient supply and removal of metabolites in a body part. Critical limb ischemia (CLI) is a severe clinical manifestation of peripheral arterial disease. Atherosclerosis serves as the main cause of CLI, which arises from the deposition of lipids in the artery wall, forming atheroma and causing inflammation.

A modular, reversible sealing, and reusable microfluidic device for drug screening.

Preclinical tests for evaluating potential drug candidates using conventional protocols can be exhaustive and high-cost processes. Microfluidic technologies that can speed up this process and allow fast screening of drugs are promising alternatives. This work presents the design, concept, and operational conditions of a simple, modular, and reversible sealing microdevice useful for drug screening.

Microfluidic encapsulation of nanoparticles in alginate microgels gelled via competitive ligand exchange crosslinking.

Efficient delivery of nanometric vectors complexed with nanoparticles at a target tissue without spreading to other tissues is one of the main challenges in gene therapy. One means to overcome this problem is to confine such vectors within microgels that can be placed in a target tissue to be released slowly and locally. Herein, a conventional optical microscope coupled to a common smartphone was employed to monitor the microfluidic production of monodisperse alginate microgels containing nanoparticles as a model for the encapsulation of vectors.

Microfluidics in Sickle Cell Disease Research: State of the Art and a Perspective Beyond the Flow Problem.

Sickle cell disease (SCD) is the monogenic hemoglobinopathy where mutated sickle hemoglobin molecules polymerize to form long fibers under deoxygenated state and deform red blood cells (RBCs) into predominantly sickle form. Sickled RBCs stick to the vascular bed and obstruct blood flow in extreme conditions, leading to acute painful vaso-occlusion crises (VOCs) - the leading cause of mortality in SCD. Being a blood disorder of deformed RBCs, SCD manifests a wide-range of organ-specific clinical complications of life (in addition to chronic pain) such as stroke, acute chest syndrome (ACS) and pulmonary hypertension in the lung, nephropathy, auto-splenectomy, and splenomegaly, hand-foot syndrome, leg ulcer, stress erythropoiesis, osteonecrosis and osteoporosis.

Hybrid microgels produced via droplet microfluidics for sustainable delivery of hydrophobic and hydrophilic model nanocarriers.

Drug delivery for treatment of chronic diseases relies on the effective delivery of payload materials into the target cells in a long-term release. In this context, the present study investigated hybrid microgels as platforms to carry nanoparticles to drug delivery. Hybrid microgels were produced with silk fibroin (SF) and chondroitin sulfate (CS), and alginate (ALG) by droplet microfluidics.

Bulk and Microfluidic Synthesis of Stealth and Cationic Liposomes for Gene Delivery Applications.

This chapter describes the synthesis of stealth and cationic liposomes and their complexation with plasmid DNA to generate lipoplexes for gene delivery applications. Two techniques are presented: a top-down approach which requires a second step of processing for downsizing the liposomes (i.e.

Perfusion Microfermentor Integrated into a Fiber Optic Quasi-Elastic Light Scattering Sensor for Fast Screening of Microbial Growth Parameters.

This research presents a microfermentor integrated into an optical fiber sensor based on quasi-elastic light scattering (QELS) to monitor and swiftly identify cellular growth kinetic parameters. The system uses a 1310 nm laser light that is guided through single-mode silica optical fibers to the interior of perfusion chambers, which are separated by polycarbonate membranes (470 nm pores) from microchannels, where a culture medium flows in a constant concentration. The system contains four layers, a superior and an inferior layer made of glass, and two intermediate poly(dimethylsiloxane) layers that contain the microchannels and the perfusion chambers, forming a reversible microfluidic device that requires only the sealing of the fibers to the inferior glass cover.

Tracking the Evolution of Transiently Transfected Individual Cells in a Microfluidic Platform.

Transient gene expression (TGE) technology enables the rapid production of large amount of recombinant proteins, without the need of fastidious screening of the producing cells required for stable transfection (ST). However, several barriers must be overcome before reaching the production yields using ST. For optimizing the production yields from suspended cells using TGE, a better understanding of the transfection conditions at the single cell level are required.

High-throughput continuous production of liposomes using hydrodynamic flow-focusing microfluidic devices.

The microfluidic hydrodynamic flow-focusing is a simple technique for nanoscale liposome formation that provides several advantages compared to the traditional manufacturing techniques. This work aimed to perform a systematic study of the liposome formation using planar microfluidic devices with different channel aspect-ratios, as an alternative to enhance the throughput of liposome synthesis. In general, liposomes with a low polydispersity and a precise control of the size were successfully produced from alteration of the flow rate ratio and channel aspect-ratio.

Microencapsulation structures based on protein-coated liposomes obtained through electrospraying for the stabilization and improved bioaccessibility of curcumin.

Novel food-grade hybrid encapsulation structures based on the entrapment of phosphatidylcholine liposomes, within a WPC matrix through electrospraying, were developed and used as delivery vehicles for curcumin. The loading capacity and encapsulation efficiency of the proposed system was studied, and the suitability of the approach to stabilize curcumin and increase its bioaccessibility was assessed. Results showed that the maximum loading capacity of the liposomes was around 1.

Dendritic Cells Stimulated by Cationic Liposomes.

Immunotherapy of cancer aims to harness the immune system to detect and destroy cancer cells. To induce an immune response against cancer, activated dendritic cells (DCs) must present tumor antigens to T lymphocytes of patients. However, cancer patients' DCs are frequently defective, therefore, they are prone to induce rather tolerance than immune responses.

Cationic liposomes produced via ethanol injection method for dendritic cell therapy.

Cationic liposomes can be designed and developed in order to be an efficient gene delivery system for mammalian cells. Dendritic cell (DC) vaccines can be used to treat cancer, as cationic liposomes can deliver tumor antigens to cells while cells remain active. However, most methods used for liposome production are not able to reproduce in large scale the physicochemical and biological properties of liposomes produced in laboratory scale.

Hybrid encapsulation structures based on β-carotene-loaded nanoliposomes within electrospun fibers.

Hybrid encapsulation structures based on β-carotene-loaded nanoliposomes incorporated within the polymeric ultrathin fibers produced through electrospinning were developed to improve the photostability of the antioxidant. These novel materials were intended to incorporate β-carotene into water-based food formulations, overcoming the existing limitations associated with its hydrophobic character. Initially, both empty and antioxidant-loaded nanoliposomes were developed and incorporated into polyvinyl alcohol (PVOH) and polyethylene oxide (PEO) solutions.

Association between cationic liposomes and low molecular weight hyaluronic acid.

This work presents a study of the association between low molecular weight hyaluronic acid (16 kDa HA) and cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The cationic liposome/HA complexes were evaluated to determine their mesoscopic structure, average size, zeta potential, and morphology as a function of the amount of HA in the system. Small angle X-ray scattering results revealed that neighboring cationic liposomes either stick together after a partial coating of low concentration HA or disperse completely in excess of HA, but they never assemble as multilamellar vesicles.

Microfluidic devices for continuous production of pDNA/cationic liposome complexes for gene delivery and vaccine therapy.

To evaluate the process parameters for the production of plasmid DNA/cationic liposome (pDNA/CL) complexes in microfluidic systems, we studied two microfluidic devices: one with simple straight hydrodynamic flow focusing (SMD) and a second one with barriers in the mixing microchannel (patterned walls, PMD). A conventional bulk mixing method was used as a comparison to microfluidic mixing. The CL and the pDNA were combined at a molar positive/negative charge ratio of 6.

Effects of extrusion, lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications.

We developed cationic liposomes containing DNA through a conventional process involving steps of (i) preformation of liposomes, (ii) extrusion, (iii) drying and rehydration and (iv) DNA complexation. Owing to its high prophylactic potentiality against tuberculosis, which had already been demonstrated in preclinical assays, we introduced modifications into the conventional process towards getting a simpler and more economical process for further scale-up. Elimination of the extrusion step, increasing the lipid concentration (from 16 to 64 mM) of the preformed liposomes and using good manufacturing practice bulk lipids (96-98% purity) instead of analytical grade purity lipids (99.

Effectiveness, against tuberculosis, of pseudo-ternary complexes: peptide-DNA-cationic liposome.

We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex.

Surface miscibility of EPC/DOTAP/DOPE in binary and ternary mixed monolayers.

Surface pressure (π)-molecular area (A) curves were used to characterize the packing of pseudo-ternary mixed Langmuir monolayers of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and L-α-dioleoyl phosphatidylethanolamine (DOPE). This pseudo-ternary mixture EPC/DOPE/DOTAP has been successfully employed in liposome formulations designed for DNA non-viral vectors. Pseudo-binary mixtures were also studied as a control.

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes.

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated.