Experimental and Numerical Integrated Strategy for the Optimization of Microfluidic Parameters for Eudragit L100 Nanoparticles and Microparticles.

Oral immunization offers a minimally invasive administration, inducing local and systemic immune responses and facilitating mass immunization without needle-related risks. However, the gastrointestinal environment poses challenges, compromising vaccine effectiveness through enzymatic degradation and poor absorption by Peyer's patches. Advances in nanoparticle and microparticle (NP/MP) technology protect vaccines from degradation and enhance targeted release. The aim of this study was to develop pH-controlled polymeric carriers for the oral delivery of protein vaccines in order to target the antigen-presenting cells and M cells in the region of Peyer's patches. Here, myoglobin was chosen as a model protein vaccine. This study focuses on Eudragit L100, a pH-responsive polymer stable in acidic conditions and dissolving at higher pH, to develop carriers for controlled myoglobin release in the intestinal tract. A microfluidic-based manufacturing process for Eudragit L100 NPs and MPs is optimized using a comprehensive experimental and computational approach to obtain NPs and MPs through the same setup. Integrating in silico and experimental methods highlights the potential of numerical simulations to streamline final product development. This approach improves the efficiency and cost-effectiveness of NP/MP production, demonstrating how the combination of design of experiment and numerical simulations can optimize production parameters and refine manufacturing processes for advanced drug delivery systems.

Smart Electrospun Nanofibers from Short Peptidomimetics Based on Pyrrolo-pyrazole Scaffold.

We prepared a small library of short peptidomimetics based on 3-pyrrolo-pyrazole carboxylate, a non-coded γ-amino acid, and glycine or alanine. The robust and eco-friendly synthetic approach adopted allows to obtain the dipeptides in two steps from commercial starting materials. This gives the possibility to shape these materials by electrospinning into micro- and nanofibers, in amounts required to be useful for coating surfaces of biomedical relevance. To promote high quality of electrospun fibers, different substitution patterns were evaluated, all for pure peptide fibers, free of any polymer or additive. The best candidate, which affords a homogeneous fibrous matrix, was prepared in larger amounts, and its biocompatibility was verified. This successful work is the first step to develop a new biomaterial able to produce pristine peptide-based nanofibers to be used as helpful component or stand-alone scaffolds for tissue engineering or for the surface modification of medical devices.

Influence of Electrospun Fibre Secondary Morphology on Antibiotic Release Kinetic and Its Impact on Antimicrobic Efficacy.

Vascular graft infections are a severe complication in vascular surgery, with a high morbidity and mortality. Prevention and treatment involve the use of antibiotic- or antiseptic-impregnated artificial vascular grafts, but currently, there are no commercially available infection-proof small-diameter vascular grafts (SDVGs). In this work we investigated the antimicrobic activity of two SDVGs prototypes loaded with tobramycin and produced via the electrospinning of drug-doped PLGA (polylactide-co-glycolide) solutions. Differences in rheological and conductivity properties of the polymer solutions resulted in non-identical fibre morphology that deeply influenced the hydration profile and consequently the in vitro cumulative drug release, which was investigated by using a spectrofluorimetric technique. Using DDSolver Excel add-in, modelling of the drug release kinetic was performed to evaluate the release mechanism involved: Prototype 1 showed a sustained and diffusive driven drug release, which allowed for the complete elution of tobramycin within 2 weeks, whereas Prototype 2 resulted in a more extended drug release controlled by both diffusion and matrix relaxation. Time-kill assays performed on S. aureus and E. coli highlighted the influence of burst drug release on the decay rate of bacterial populations, with Prototype 1 being more efficient on both microorganisms. Nevertheless, both prototypes showed good antimicrobic activity over the 5 days of in vitro testing.

Tobramycin Nanoantibiotics and Their Advantages: A Minireview.

Nowadays, antimicrobial resistance (AMR) represents a challenge for antibiotic therapy, mostly involving Gram-negative bacteria. Among the strategies activated to overcome AMR, the repurposing of already available antimicrobial molecules by encapsulating them in drug delivery systems, such as nanoparticles (NPs) and also engineered NPs, seems to be promising. Tobramycin is a powerful and effective aminoglycoside, approved for complicated infections and reinfections and indicated mainly against Gram-negative bacteria, such as Pseudomonas aeruginosa, Escherichia coli, Proteus, Klebsiella, Enterobacter, Serratia, Providencia, and Citrobacter species. However, the drug presents several side effects, mostly due to dose frequency, and for this reason, it is a good candidate for nanomedicine formulation. This review paper is focused on what has been conducted in the last 20 years for the development of Tobramycin nanosized delivery systems (nanoantibiotics), with critical discussion and comparison. Tobramycin was selected as the antimicrobial drug because it is a wide-spectrum antibiotic that is effective against both Gram-positive and Gram-negative aerobic bacteria, and it is characterized by a fast bactericidal effect, even against multidrug-resistant microorganisms (MDR).

Liposome Formulation and In Vitro Testing in Non-Physiological Conditions Addressed to Ex Vivo Kidney Perfusion.

This work focuses on formulating liposomes to be used in isolated kidney dynamic machine perfusion in hypothermic conditions as drug delivery systems to improve preservation of transplantable organs. The need mainly arises from use of kidneys from marginal donors for transplantation that are more exposed to ischemic/reperfusion injury compared to those from standard donors. Two liposome preparation techniques, thin film hydration and microfluidic techniques, are explored for formulating liposomes loaded with two model proteins, myoglobin and bovine serum albumin. The protein-loaded liposomes are characterized for their size by DLS and morphology by TEM. Protein releases from the liposomes are tested in PERF-GEN perfusion fluid, 4 °C, and compared to the in vitro protein release in PBS, 37 °C. Fluorescent liposome uptake is analyzed by fluorescent microscope in vitro on epithelial tubular renal cell cultures and ex vivo on isolated pig kidney in hypothermic perfusion conditions. The results show that microfluidics are a superior technique for obtaining reproducible spherical liposomes with suitable size below 200 nm. Protein encapsulation efficiency is affected by its molecular weight and isoelectric point. Lowering incubation temperature slows down the proteins release; the perfusion fluid significantly affects the release of proteins sensitive to ionic media (such as BSA). Liposomes are taken up by epithelial tubular renal cells in two hours' incubation time.

Tobramycin Supplemented Small-Diameter Vascular Grafts for Local Antibiotic Delivery: A Preliminary Formulation Study.

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/wPLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 µm and its internal diameter was 2.61 ± 0.05 mm. The graft's antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.

Tissue Engineered Esophageal Patch by Mesenchymal Stromal Cells: Optimization of Electrospun Patch Engineering.

Aim of work was to locate a simple, reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited, namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size, pore orientation, porosity, and pore distribution were produced using two different techniques, such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity, seeding efficiency, and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method, as these conditions successfully improved the cellularization of polymeric patches. Furthermore, the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results, it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover, the protocol turned out to be simple, repeatable, and reproducible.

Improvement of the Firocoxib Dissolution Performance Using Electrospun Fibers Obtained from Different Polymer/Surfactant Associations.

An electrospinning process was optimized to produce fibers of micrometric size with different combinations of polymeric and surfactant materials to promote the dissolution rate of an insoluble drug: firocoxib. Scanning Electron Microscopy (SEM) showed that only some combinations of the proposed carrier systems allowed the production of suitable fibers and further fine optimization of the technique is also needed to load the drug. Differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD) suggest that the drug is in an amorphous state in the final product. Drug amorphization, the fine dispersion of the active in the carriers, and the large surface area exposed to water interaction obtained through the electrospinning process can explain the remarkable improvement in the dissolution performance of firocoxib from the final product developed.

Staggered Herringbone Microfluid Device for the Manufacturing of Chitosan/TPP Nanoparticles: Systematic Optimization and Preliminary Biological Evaluation.

Chitosan nanoparticles (CS NPs) showed promising results in drug, vaccine and gene delivery for the treatment of various diseases. The considerable attention towards CS was owning to its outstanding biological properties, however, the main challenge in the application of CS NPs was faced during their size-controlled synthesis. Herein, ionic gelation reaction between CS and sodium tripolyphosphate (TPP), a widely used and safe CS cross-linker for biomedical application, was exploited by a microfluidic approach based on a staggered herringbone micromixer (SHM) for the synthesis of TPP cross-linked CS NPs (CS/TPP NPs). Screening design of experiments was applied to systematically evaluate the main process and formulative factors affecting CS/TPP NPs physical properties (mean size and size distribution). Effectiveness of the SHM-assisted manufacturing process was confirmed by the preliminary evaluation of the biological performance of the optimized CS/TPP NPs that were internalized in the cytosol of human mesenchymal stem cells through clathrin-mediated mechanism. Curcumin, selected as a challenging model drug, was successfully loaded into CS/TPP NPs (EE% > 70%) and slowly released up to 48 h via the diffusion mechanism. Finally, the comparison with the conventional bulk mixing method corroborated the efficacy of the microfluidics-assisted method due to the precise control of mixing at microscales.

Hyaluronic Acid-Decorated Chitosan Nanoparticles for CD44-Targeted Delivery of Everolimus.

Bronchiolitis obliterans syndrome (BOS), caused by lung allograft-derived mesenchymal cells' abnormal proliferation and extracellular matrix deposition, is the main cause of lung allograft rejection. In this study, a mild one-step ionotropic gelation method was set up to nanoencapsulate the everolimus, a key molecule in allograft organ rejection prevention, into hyaluronic acid-decorated chitosan-based nanoparticles. Rationale was the selective delivery of everolimus into lung allograft-derived mesenchymal cells; these cells are characterized by the CD44-overexpressing feature, and hyaluronic acid has proven to be a natural selective CD44-targeting moiety. The optimal process conditions were established by a design of experiment approach (full factorial design) aiming at the control of the nanoparticle size (≤200 nm), minimizing the size polydispersity (PDI 0.171 ± 0.04), and at the negative ζ potential maximization (-30.9 mV). The everolimus was successfully loaded into hyaluronic acid-decorated chitosan-based nanoparticles (95.94 ± 13.68 µg/100 mg nanoparticles) and in vitro released in 24 h. The hyaluronic acid decoration on the nanoparticles provided targetability to CD44-overexpressing mesenchymal cells isolated from bronchoalveolar lavage of BOS-affected patients. The mesenchymal cells' growth tests along with the nanoparticles uptake studies, at 37 °C and 4 °C, respectively, demonstrated a clear improvement of everolimus inhibitory activity when it is encapsulated in hyaluronic acid-decorated chitosan-based nanoparticles, ascribable to their active uptake mechanism.

Intra-Articular Formulation of GE11-PLGA Conjugate-Based NPs for Dexamethasone Selective Targeting-In Vitro Evaluation.

Selectively targeted nanoscale drug delivery systems have recently emerged as promising intravenously therapeutic option for most chronic joint diseases. Here, a newly synthetized dodecapeptide (GE11)-polylactide-co-glycolide (PLGA)-based conjugate was used to prepare smart nanoparticles (NPs) intended for intra-articular administration and for selectively targeting Epidermal Growth Factor Receptor (EGFR). GE11-PLGA conjugate-based NPs are specifically uptaken by EGFR-overexpressed fibroblast; such as synoviocytes; which are the primarily cellular component involved in the development of destructive joint inflammation. The selective uptake could help to tune drug effectiveness in joints and to decrease local and systemic side effects. Dexamethasone (DXM) is a glucorticoid drug commonly used in joint disease treatment for both systemic and local administration route. In the present research; DXM was efficiently loaded into GE11-PLGA conjugate-based NPs through an eco-friendly nanoprecipitation method set up for this purpose. DXM loaded GE11-PLGA conjugate-based NPs revealed satisfactory ex vivo cytocompatibility; with proper size (≤150 nm) and good dimensional stability in synovial fluid. Intra-articular formulation was developed embedding DXM loaded GE11-PLGA conjugate-based NPs into thermosetting chitosan-based hydrogel; forming a biocompatible composite hydrogel able to quickly turn from liquid state into gel state at physiological temperature; within 15 min. Moreover; the use of thermosetting chitosan-based hydrogel extends the local release of active agent; DXM.

CNA-loaded PLGA nanoparticles improve humoral response againstS. aureus-mediated infections in a mouse model: subcutaneous vs. nasal administration strategy.

The aim of this work was the assessment of the "in vivo" immune response of a poly(lactide-co-glycolide)-based nanoparticulate adjuvant for a sub-unit vaccine, namely, a purified recombinant collagen-binding bacterial adhesion fragment (CNA19), against Staphylococcus aureus-mediated infections. "In vivo" immunogenicity studies were performed on mice: immunisation protocols encompassed subcutaneous and intranasal administration of CNA19 formulated as nanoparticles (NPs) and furthermore, CNA19-loaded NPs formulated in a set-up thermosetting chitosan-ß-glycerolphosphate (chitosan-ß-GP) solution for intranasal route in order to extend antigen exposure to nasal mucosa. CNA19 loaded NPs (mean size of about 195 nm, 9.04 ± 0.37µg/mg as CNA19 loading capacity) confirmed as suitable vaccine for subcutaneous administration with a more pronounced adjuvant effect (about 3-fold higher) with respect to aluminium, recognised as "reference" adjuvant. CNA19 loaded NPs formulated in an optimised thermogelling chitosan-ß-GP solution showed promising results for eliciting an effective humoral response and a good chance as intranasal boosting dose.

Antibiotic-Loaded Nano-Sized Delivery Systems: An Insight into Gentamicin and Vancomycin.

The fight against infectious disease has remained an ever-evolving challenge in the landscape of healthcare. The ability of pathogens to develop resistance against conventional drug treatments has decreased the effectiveness of therapeutic interventions, and antibiotic resistance is recognized as one of the main challenges of our time. The goal of this systematic review paper is to provide insight into the research papers published on innovative nanosized drug delivery systems (DDSs) based on gentamycin and vancomycin and to discuss the opportunity of their repurposing through nano DDS formulations. These two antibiotics are selected because (i) gentamicin is the first-line drug used to treat suspected or confirmed infections caused by Gram-negative bacterial infections and (ii) vancomycin is used to treat serious Gram-positive bacterial infections. Moreover, both antibiotics have severe adverse effects, and one of the purposes of their formulation as nanosized DDSs is to overcome them. The review paper includes an introduction focusing on the challenges of infectious diseases and traditional therapeutic treatments, a brief description of the chemical and pharmacological properties of gentamicin and vancomycin, case studies from the literature on innovative nanosized DDSs as carriers of the two antibiotic drugs, and a discussion of the results found in the literature.

Computational-Aided Approach for the Optimization of Microfluidic-Based Nanoparticles Manufacturing Process.

In the last few years, the microfluidic production of nanoparticles (NPs) is becoming a promising alternative to conventional industrial approaches (e.g., nanoprecipitation, salting out, and emulsification-diffusion) thanks to the production efficiency, low variability, and high controllability of the production parameters. Nevertheless, the development of new formulations and the switching of the production process toward microfluidic platforms requires expensive and time-consuming number of experiments for the tuning of the formulation to obtain NPs with specific morphological and functional characteristics. In this work, we developed a computational fluid dynamic pipeline, validated through an ad hoc experimental strategy, to reproduce the mixing between the solvent and anti-solvent (i.e., acetonitrile and TRIS-HCl, respectively). Moreover, beyond the classical variables able to describe the mixing performances of the microfluidic chip, novel variables were described in order to assess the region of the NPs formation and the changing of the amplitude of the precipitation region according to different hydraulic conditions. The numerical approach proved to be able to capture a progressive reduction of the nanoprecipitation region due to an increment of the flow rate ratio; in parallel, through the experimental production, a progressive increment of the NPs size heterogeneity was observed with the same fluid dynamic conditions. Hence, the preliminary comparison between numerical and experimental evidence proved the effectiveness of the computational strategy to optimize the NPs manufacturing process.

Stereochemical Behavior of Pyrrolo-Pyrazole Peptidomimetics Promoting Phase-Selective Supramolecular Organogels.

Supramolecular gels were developed by taking advantage of an assembly of small dipeptides containing pyrrolo-pyrazole scaffolds. The dipeptides were prepared through a robust and ecofriendly synthetic approach from the commercially available starting materials of diazoalkanes and maleimides. By playing with the functionalization of the scaffold, the choice of the natural amino acid, and the stereochemistry, we were able to obtain phase-selective gels. In particular, one peptidomimetic showed gelation ability and thermoreversibility in aromatic solvents at very low concentrations. Rheology tests showed a typical viscoelastic solid profile, indicating the formation of strong gels that were stable under high mechanical deformation. NMR studies were performed, allowing us to determine the conformational and stereochemical features at the base of the supramolecular interactions.

Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids.

Lipid-based nanocarriers have emerged as helpful tools to deliver sensible biomolecules such as proteins and oligonucleotides. To have a fast and robust microfluidic-based nanoparticle synthesis method, the setup of versatile equipment should allow for the rapid transfer to scale cost-effectively while ensuring tunable, precise and reproducible nanoparticle attributes. The present work aims to assess the effect of different micromixer geometries on the manufacturing of lipid nanocarriers taking into account the influence on the mixing efficiency by changing the fluid-fluid interface and indeed the mass transfer. Since the geometry of the adopted micromixer varies from those already published, a Design of Experiment (DoE) was necessary to identify the operating (total flow, flow rate ratio) and formulation (lipid concentration, lipid molar ratios) parameters affecting the nanocarrier quality. The suitable application of the platform was investigated by producing neutral, stealth and cationic liposomes, using DaunoXome®, Myocet®, Onivyde® and Onpattro® as the benchmark. The effect of condensing lipid (DOTAP, 3-10-20 mol%), coating lipids (DSPE-PEG550 and DSPE-PEG2000), as well as structural lipids (DSPC, eggPC) was pointed out. A very satisfactory encapsulation efficiency, always higher than 70%, was successfully obtained for model biomolecules (myoglobin, short and long nucleic acids).

Shaping the future from the small scale: dry powder inhalation of CRISPR-Cas9 lipid nanoparticles for the treatment of lung diseases.

INTRODUCTION: Most lung diseases are serious conditions resulting from genetic and environmental causes associated with high mortality and severe symptoms. Currently, treatments available have a palliative effect and many targets are still considered undruggable. Gene therapy stands as an attractive approach to offering innovative therapeutic solutions. CRISPRCas9 has established a remarkable potential for genome editing with high selectivity to targeted mutations. To ensure high efficacy with minimum systemic exposure, the delivery and administration route are key components that must be investigated. AREAS COVERED: This review is focused on the delivery of CRISPRCas9 to the lungs, taking advantage of lipid nanoparticles (LNPs), the most clinically advanced nucleic acid carriers. We also aim to highlight the benefits of pulmonary administration as a local delivery route and the use of spray drying to prepare stable nucleic-acid-based dry powder formulations that can overcome multiple lung barriers. EXPERT OPINION: Exploring the pulmonary administration to deliver CRISPRCas9 loaded in LNPs as a dry powder increases the chances to achieve high efficacy and reduced adverse effects. CRISPRCas9 loaded in LNP-embedded microparticles has not yet been reported in the literature but has the potential to reach and accumulate in target cells in the lung, thus, enhancing overall efficacy and safety.

Electrospun Fibers Loaded with Pirfenidone: An Innovative Approach for Scar Modulation in Complex Wounds.

Hypertrophic scars (HTSs) are pathological structures resulting from chronic inflammation during the wound healing process, particularly in complex injuries like burns. The aim of this work is to propose Biofiber PF (biodegradable fiber loaded with Pirfenidone 1.5 w/w), an electrospun advanced dressing, as a solution for HTSs treatment in complex wounds. Biofiber has a 3-day antifibrotic action to modulate the fibrotic process and enhance physiological healing. Its electrospun structure consists of regular well-interconnected Poly-L-lactide-co-poly-ε-caprolactone (PLA-PCL) fibers (size 2.83 ± 0.46 µm) loaded with Pirfenidone (PF, 1.5% w/w), an antifibrotic agent. The textured matrix promotes the exudate balance through mild hydrophobic wettability behavior (109.3 ± 2.3°), and an appropriate equilibrium between the absorbency % (610.2 ± 171.54%) and the moisture vapor transmission rate (0.027 ± 0.036 g/min). Through its finer mechanical properties, Biofiber PF is conformable to the wound area, promoting movement and tissue oxygenation. These features also enhance the excellent elongation (>500%) and tenacity, both in dry and wet conditions. The ancillary antifibrotic action of PF on hypertrophic scar fibroblast (HSF) for 3 days downregulates the cell proliferation over time and modulates the gene expression of transforming growth factor ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) at 48-72 h. After 6 days of treatment, a decrement of α-SMA protein levels was detected, proving the potential of biofiber as a valid therapeutic treatment for HTSs in an established wound healing process.

Electrospun Naringin-Loaded Fibers for Preventing Scar Formation during Wound Healing.

Hypertrophic scars (HTSs) are aberrant structures that develop where skin is injured complexly and represent the result of a chronic inflammation as a healing response. To date, there is no satisfactory prevention option for HTSs, which is due to the complexity of multiple mechanisms behind the formation of these structures. The present work aimed to propose Biofiber (Biodegradable fiber), an advanced textured electrospun dressing, as a suitable solution for HTS formation in complex wounds. Biofiber has been designed as a 3-day long-term treatment to protect the healing environment and enhance wound care practices. Its textured matrix consists of homogeneous and well-interconnected Poly-L-lactide-co-poly-ε-caprolactone (PLA-PCL) electrospun fibers (size 3.825 ± 1.12 µm) loaded with Naringin (NG, 2.0% w/w), a natural antifibrotic agent. The structural units contribute to achieve an optimal fluid handling capacity demonstrated through a moderate hydrophobic wettability behavior (109.3 ± 2.3°), and a suitable balance between absorbency (389.8 ± 58.16%) and moisture vapor transmission rate (MVTR, 2645 ± 60.43 g/m2 day). The flexibility and conformability of Biofiber to the body surfaces is due to its innovative circular texture, that also allow it to obtain finer mechanical properties after 72 h in contact with Simulated Wound Fluid (SWF), with an elongation of 352.6 ± 36.10%, and a great tenacity (0.25 ± 0.03 Mpa). The ancillary action of NG results in a prolonged anti-fibrotic effect on Normal Human Dermal Fibroblasts (NHDF), through the controlled release of NG for 3 days. The prophylactic action was highlighted at day 3 with the down regulation of the major factors involved in the fibrotic process: Transforming Growth Factor ß1 (TGF-ß1), Collagen Type 1 alpha 1 chain (COL1A1), and α-smooth muscle actin (α-SMA). No significant anti-fibrotic effect has been demonstrated on Hypertrophic Human Fibroblasts derived from scars (HSF), proving the potential of Biofiber to minimize HTSs in the process of early wound healing as a prophylactic therapy.

Microfluidic mixing as platform technology for production of chitosan nanoparticles loaded with different macromolecules.

In recent decades, biotechnological drugs have emerged as relevant therapeutic tools. However, therapeutic molecules can exert their activity only if properly formulated and delivered into the body. In this regard, nano-sized drug delivery systems have been shown to provide protection, stability, and controlled release of payloads, increasing their therapeutic efficacy. In this work, a microfluidic mixing technique for the preparation of chitosan-based nanoparticles was established with the capability of easily exchanging macromolecular biological cargos such as the model protein ß-Galactosidase, mRNA, and siRNA. The nanoparticles obtained showed hydrodynamic diameters ranging from 75 nm to 105 nm, low polydispersity of 0.15 to 0.22 and positive zeta potentials of 6 mV to 17 mV. All payloads were efficiently encapsulated (>80 %) and the well-known cytocompatibility of chitosan-based nanoparticles was confirmed. Cell culture studies demonstrated increased cellular internalization of loaded nano-formulations compared to free molecules as well as successful gene silencing with nano-formulated siRNA, suggesting the ability of these nanoparticles to escape the endosome.