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Karim S Shalaby,1 Mahmoud E Soliman,1 Luca Casettari,2 Giulia Bonacucina,3 Marco Cespi,3 Giovanni F Palmieri,3 Omaima A Sammour,1 Abdelhameed A El Shamy1,† 1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; 2Department of Biomolecular Sciences, School of Pharmacy, University of Urbino, Urbino, Italy; 3School of Pharmacy, University of Camerino, Camerino, Italy †Abdel Hameed El-Shamy passed away on August 25, 2013 Abstract: In this study, di- and triblock copolymers based on polyethylene glycol and polylactide were synthesized by ring-opening polymerization and characterized by proton nuclear magnetic resonance and gel permeation chromatography. Nanoparticles containing noscapine were prepared from these biodegradable and biocompatible copolymers using the nanoprecipitation method. The prepared nanoparticles were characterized for size and drug entrapment efficiency, and their morphology and size were checked by transmission electron microscopy imaging. Artificial neural networks were constructed and tested for their ability to predict particle size and entrapment efficiency of noscapine within the formed nanoparticles using different factors utilized in the preparation step, namely polymer molecular weight, ratio of polymer to drug, and number of blocks that make up the polymer. Using these networks, it was found that the polymer molecular weight has the greatest effect on particle size. On the other hand, polymer to drug ratio was found to be the most influential factor on drug entrapment efficiency. This study demonstrated the ability of artificial neural networks to predict not only the particle size of the formed nanoparticles but also the drug entrapment efficiency. This may have a great impact on the design of polyethylene glycol and polylactide-based copolymers, and can be used to customize the required target formulations. Keywords: noscapine, polyethylene glycol (PEG), polylactide (PLA), biodegradable nanoparticles, artificial neural networks (ANNs)

The article focuses on the use of different lipid nanovesicle preparations containing Campylobacter jejuni cytolethal distending toxins (CDT) lysate, which inhibit proliferation in tumor intestinal epithelial cells and have potential applications in anti-tumor strategies.

Indocyanine green (ICG) J-aggregates (IJA) are a unique form of aggregation that exhibits superior properties to monomeric ICG. Despite their higher photoacoustic (PA) signals for imaging and heating stability during photothermal therapy (PTT), they exhibit low stability under a biological milieu. Our group previously proposed a simple procedure for in-situ preparation of IJA into liposomes, accelerating their formation and optical properties. To comprehend their potential applications, we systematically investigated the effect of the lipid bilayer composition on ICG J-aggregation and stability. Moreover, their in vitro compatibility and photothermal toxicity in monolayers and cancer spheroids, besides their in vivo biodistribution and clearance were evaluated. Our findings revealed the importance of high cholesterol and PEG-lipid content and low charged lipids (∼ 5 mol %) in liposomes to promote a high IJA/ICG ratio and, thus, high heating stability. More importantly, IJA-liposomes revealed high biocompatibility in monolayer and cancer spheroids with efficient photothermal toxicity. Finally, IJA-liposomes were cleared from the body without toxicity. Interestingly, IJA-liposomes mainly showed lower affinity to the liver than monomeric ICG, resulting in higher renal clearance. Overall, our biodegradable IJA-liposomes could be an excellent alternative to gold-based agents suitable for PA imaging and cancer PTT., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wafa Al-Jamal reports financial support was provided by The Phospholipid Research Center. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug delivery systems efficiently and safely administer therapeutic agents to specific body sites. Liposomes, spherical vesicles made of phospholipid bilayers, have become a powerful tool in this field, especially with the rise of microfluidic manufacturing during the COVID-19 pandemic. Despite its efficiency, microfluidic liposomal production poses challenges, often requiring laborious, optimization on a case-by-case basis. This is due to a lack of comprehensive understanding and robust methodologies, compounded by limited data on microfluidic production with varying lipids. Artificial intelligence offers promise in predicting lipid behaviour during microfluidic production, with the still unexploited potential of streamlining development. Herein we employ machine learning to predict critical quality attributes and process parameters for microfluidic-based liposome production. Validated models predict liposome formation, size, and production parameters, significantly advancing our understanding of lipid behaviour. Extensive model analysis enhanced interpretability and investigated underlying mechanisms, supporting the transition to microfluidic production. Unlocking the potential of machine learning in drug development can accelerate pharmaceutical innovation, making drug delivery systems more adaptable and accessible., Competing Interests: Declaration of competing interest No private study sponsors had any involvement in the study design, data collection, or interpretation of data presented in this manuscript. P.L. declares the following competing interests: has consulted and received research grants from Lipoid GmbH, Sanofi-Aventis Deutschland and DSM Nutritional Products Ltd.; received research grants from PPM Services S.A., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In recent years, nutraceuticals have emerged as a promising strategy for maintaining health and represent a high-growth market in Italy and across Europe. However, the lack of strict regulations regarding formulation requirements and proof of efficacy raises serious concerns about their poor bioavailability and, consequently, their uncertain health benefits. An emblematic example is t-resveratrol (RES), a cardioprotective stilbene polyphenol that undergoes extensive metabolism in the intestine and liver, resulting in a bioavailability of <1 %. This manuscript describes a novel technological matrix developed with the primary goal of improving RES oral bioavailability. This technology can be classified as a lipid-based autoemulsifying drug delivery system (LIBADDS), in which RES is thoroughly solubilized in a hot liquid phase composed of lipids and surfactants, and the mixture is further adsorbed onto a powder composed of polysaccharides and sodium caseinate (N a C), along with inert excipients, and then compressed. In this study, NaC was used for the first time to trigger pancreatin-mediated hydrolysis of an enteric-coated tablet, allowing micellar delivery of RES to the small intestine. The RES-containing tablets were characterized via differential scanning calorimetry (DSC) and X-ray diffraction (PXRD). The digested formulation, with simulated gastric and enteric fluids, was dimensionally assessed via dynamic light scattering (DLS). Finally, calculations of the bioaccessible fraction, dissolution tests, and in vitro permeability experiments using Caco-2 cell monolayers were carried out to preliminarily define the overall efficiency and applicability of this new technology in improving RES intestinal permeability., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Controlled manufacturing and long-term stability are key challenges in the development and translation of nanomedicines. This is exemplified by the mRNA-nanoparticle vaccines against COVID-19, which require (ultra-)cold temperatures for storage and shipment. Various cryogenic protocols have been explored to prolong nanomedicine shelf-life. However, freezing typically induces high mechanical stress on nanoparticles, resulting in aggregation or destabilization, thereby limiting their performance and application. Hence, evaluating the impact of freezing and storing on nanoparticle properties already early-on during preclinical development is crucial. In the present study, we used prototypic π electron-stabilized polymeric micelles based on mPEG-b-p(HPMAm-Bz) block copolymers to macro- and microscopically study the effect of different cryoprotective excipients on nanoformulation properties like size and size distribution, as well as on freezing-induced aggregation phenomena via in-situ freezing microscopy. We show that sucrose, unlike trehalose, efficiently cryoprotected paclitaxel-loaded micelles, and we exemplify the impact of formulation composition for efficient cryoprotection. We finally establish microfluidic mixing to formulate paclitaxel-loaded micelles with sucrose as a cryoprotective excipient in a single production step and demonstrate their stability for 6 months at -20 °C. The pharmaceutical properties and preclinical performance (in terms of tolerability and tumor growth inhibition in a patient-derived triple-negative breast cancer xenograft mouse model) of paclitaxel-loaded micelles were successfully cryopreserved. Together, our efforts promote future pharmaceutical development and translation of π electron-stabilized polymeric micelles, and they illustrate the importance of considering manufacturing and storage stability issues early-on during nanomedicine development., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Vesicants are chemical warfare agents (CWAs) capable of causing severe skin damage and systemic toxicity. Melatonin, known for its anti-inflammatory and antioxidant properties, can mitigate the effects of these agents. Self-nano-emulsifying drug delivery systems (SNEDDS) containing a high melatonin concentration (5 %, 50 mg/g) were optimized using a quality-by-design approach from biocompatible, non-irritant excipients with a particle size of about 100 nm. The melatonin-loaded SNEDDS showed a 43-fold greater permeability than a conventional melatonin cream. Chemical stability at ambient temperature (25 °C) was maintained for one year. The preparation of optimised melatonin-loaded SNEDDS using a simple mixing method was compared to microfluidic micromixers. Mixing was successfully achieved using a 3D-printed (fused deposition modeling or stereolithography) T-shaped toroidal microfluidic chip (with a channel geometry optimized by computational fluid dynamics), resulting in a scalable, continuous process for the first time with a substantial reduction in preparation time compared to other conventional mixing approaches. No statistically significant differences were observed in the key quality attributes, such as particle size and melatonin loading, between mixing method till kinetic equilibrium solubility is reached and mixing using the 3D-printed micromixers. This scalable, continuous, cost-effective approach improves the overall efficiency of SNEDDS production, reduces the cost of quality control for multiple batches, and demonstrates the potential of continuous microfluidic manufacture with readily customizable 3D-printed micromixers at points of care, such as military bases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Dolores Remedios Serrano Lopez reports a relationship with Complutense University of Madrid that includes: employment]., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The valorization of discarded wool from dairy sheep breeding is a challenging issue. The most proposed strategies lie in the processing of keratin extracted from wool without reducing the molecular weight of the protein chains (the high molecular weight-HMW keratin). Here, the HMW keratin has been spun for the first time by solution blow spinning. A screening study of the process carried out with a 2-level full factorial design revealed that keratin filaments can be obtained by using the polyethylene oxide at 900 kDa, a 2 bar air pressure, and a 30 cm needle-collector distance. An annealing at 80 °C for 15 min, at pH 3.5 with citric acid contributes to increasing the viscosity of the keratin solutions thereby allowing the production of defect-free and water-stable filaments having diameters from 1 to 6 μm. A negligible toxic effect was observed after 24 and 48 h on HT29 epithelial cells and normal blood cells displayed behavior similar to the control demonstrating that the patches are hemocompatible. Therefore, the developed SBS process of keratin aqueous solutions could represent a valuable platform for developing patches that need to be blood-contacting and deposited in-situ., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs) are promising natural nanocarriers for the delivery of therapeutic agents. As with any other kind of cell, red blood cells (RBCs) produce a limited number of EVs under physiological and pathological conditions. Thus, RBC-derived extracellular vesicles (RBCEVs) have been recently suggested as next-generation delivery systems for therapeutic purposes. In this paper, we show that thanks to their unique biological and physicochemical features, RBCs can be efficiently pre-loaded with several kinds of molecules and further used to generate RBCEVs. A physical vesiculation method, based on "soft extrusion", was developed, producing an extremely high yield of cargo-loaded RBCEV mimetics. The RBCEVs population has been deeply characterized according to the new guidelines MISEV2023, showing great homogeneity in terms of size, biological features, membrane architecture and cargo. In vitro preliminary results demonstrated that RBCEVs are abundantly internalized by cells and exert peculiar biological effects. Indeed, efficient loading and delivery of miR-210 by RBCEVs to HUVEC has been proven, as well as the inhibition of a known mRNA target. Of note, the bench-scale process can be scaled-up and translated into clinics. In conclusion, this investigation could open the way to a new biomimetic platform for RNA-based therapies and/or other therapeutic cargoes useful in several diseases., (© 2024. The Author(s).)

Atherosclerosis still represents a major driver of cardiovascular diseases worldwide. Together with accumulation of lipids in the plaque, inflammation is recognized as one of the key players in the formation and development of atherosclerotic plaque. Systemic anti-inflammatory treatments are successful in reducing the disease burden, but are correlated with severe side effects, underlining the need for targeted formulations. In this work, curcumin is chosen as the anti-inflammatory payload model and further loaded in lignin-based nanoparticles (NPs). The NPs are then coated with a tannic acid (TA)- Fe (III) complex and further cloaked with fragments derived from platelet cell membrane, yielding NPs with homogenous size. The two coatings increase the interaction between the NPs and cells, both endothelial and macrophages, in steady state or inflamed status. Furthermore, NPs are cytocompatible toward endothelial, smooth muscle and immune cells, while not inducing immune activation. The anti-inflammatory efficacy is demonstrated in endothelial cells by real-time quantitative polymerase chain reaction and ELISA assay where curcumin-loaded NPs decrease the expression of Nf-κb, TGF-β1, IL-6, and IL-1β in lipopolysaccharide-inflamed cells. Overall, due to the increase in the cell-NP interactions and the anti-inflammatory efficacy, these NPs represent potential candidates for the targeted anti-inflammatory treatment of atherosclerosis., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Optimizing current therapies is among next steps in metastatic melanoma (MM) treatment landscape. The innovation of this study is the design of production process by microfluidics of cell membrane (CM)-modified nanoparticles (NPs), as an emerging biomimetic platform that allows for reduced immune clearance, long blood circulation time and improved specific tumor targeting. To achieve melanoma selectivity, direct membrane fusion between synthetic liposomes and CMs extracted from MM cell line was performed by microfluidic sonication approach, then the hybrid liposomes were loaded with cobimetinib (Cob) or lenvatinib (Lenva) targeting agents and challenged against MM cell lines and liver cancer cell line to evaluate homotypic targeting and antitumor efficacy. Characterization studies demonstrated the effective fusion of CM with liposome and the high encapsulation efficiency of both drugs, showing the proficiency of microfluidic-based production. By studying the targeting of melanoma cells by hybrid liposomes versus liposomes, we found that both NPs entered cells through endocytosis, whereas the former showed higher selectivity for MM cells from which CM was extracted, with 8-fold higher cellular uptake than liposomes. Hybrid liposome formulation of Cob and Lenva reduced melanoma cells viability to a greater extent than liposomes and free drug and, notably, showed negligible toxicity as demonstrated by bona fide haemolysis test. The CM-modified NPs presented here have the potential to broaden the choice of therapeutic options in MM treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Lyotropic Liquid Crystalline (LLC) nanoparticles represent an emerging class of smart, biocompatible, and biodegradable systems for the delivery of drugs. Among these, structures with complex 3D architectures such as cubosomes are of particular interest. These are non- lamellar assemblies having hydrophobic and hydrophilic portions able to carry drugs of different nature. They can further be modulated including suitable additives to control the release of the active payload, and to promote an active targeting. Starting from monoolein (GMO) cubic phase, different concentrations of mannose-based esters were added, and the eventual structural modifications were monitored to ascertain the effects of the presence of glycolipids. Moreover, the structural properties of these nanosystems loaded with Dexamethasone (DEX), a very well-known anti-inflammatory steroid, were also studied. Experiments were carried out by synchrotron Small Angle X-ray Scattering (SAXS), Raman Microspectroscopy (RMS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) measurements. The drug delivery potential (i.e. entrapment efficiency and release properties) of the obtained nanoparticles was evaluated. Finally, in vitro cytocompatibility and anti-inflammatory activity studies of the prepared formulations were carried out. Inclusion of mannose-based surfactants up to 10 mol% influenced the structural parameters of Im3m cubic phase and swollen cubic phases were obtained with the different glycolipids with lattice parameters significantly higher than GMO. A complete cytocompatibility and an increased DEX activity were observed, thus suggesting the possibility to use GMO/glycolipids nanoparticles to formulate innovative drug delivery systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Saquinavir mesylate (SQV) is a protease inhibitor commonly employed for the treatment of human immunodeficiency virus-1 infection. It is generally administered orally as tablets in combination with other antiviral drugs. Another promising route of administration can be represented by the vaginal one through topically applied formulations. This delivery can reduce the first-pass effect in the case of systemic drug adsorption or prevent HIV infection. We propose the formulation of a Carbopol® 974 (C974) hydrogel containing biodegradable mPEG-PL(L)GA nanoparticles (NPs) for the vaginal delivery of SQV, intended both as a prevention and a therapeutic strategy. mPEG-PL(L)GA NPs were incorporated into the C974 polymeric matrix, leading to a reduction of the hydrogel consistency dependent on NPs and C974 concentrations. Despite the moderate drug loading into NPs, the presence of the NPs had an impact on the in vitro release of the drug from the hydrogel at pH 5.5 using immersion cells. A higher amount of the drug was released, probably due to the effect of NPs in promoting the incorporation of the drug into the hydrogel at a high SQV dose. These findings can be useful for the development of topically applied hydrogels for SQV delivery, possibly having improved in vivo therapeutic outcomes., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Fungal infections of the skin, nails, and hair are a common health concern affecting a significant proportion of the population worldwide. The current treatment options include topical and systematic agents which have low permeability and prolonged treatment period, respectively. Consequently, there is a growing need for a permeable, effective, and safe treatment. Keratin nanoparticles are a promising nanoformulation that can improve antifungal agent penetration, providing sustainable targeted drug delivery. In this study, keratin nanoparticles were prepared using a custom-made 3D-printed microfluidic chip and the manufacturing process was optimized using the design of experiments (DoE) approach. The total flow rate (TFR), flow rate ratio (FRR), and keratin concentration were found to be the most influential factors of the size and polydispersity index (PDI) of the nanoparticles. The crosslinking procedure by means of tannic acid as safe and biocompatible compound was also optimized. Keratin nanoparticles loaded with a different amount of tioconazole showed a size lower than 200 nm, a PDI lower than 0.2 and an encapsulation efficiency of 91 ± 1.9 %. Due to their sustained drug release, the formulations showed acceptable in vitro biocompatibility. Furthermore, a significant inhibitory effect compared to the free drug against Microsporum canis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Diabetic foot ulcers (DFUs) are a crucial complication of diabetes, as in a diabetic wound, each step of the physiological healing process is affected. This entails a more easily infectable wound, and delayed tissue regeneration due to the inflammation that occurs, leading to a drastic decrease in the overall patient's quality of life. As a strategy to manage DFUs, skin alternatives and wound dressings are currently receiving a lot of attention as they keep the wound environment "under control", while providing bioactive compounds that help to manage infection and inflammation and promote tissue repair. This has been made possible thanks to the advent of emerging technologies such as 3D Bioprinting to produce skin resembling constructs or microfluidics (MFs) that allows the manufacture of nanoparticles (NPs) that act as drug carriers, in a prompt and less expensive way. In the present proof-of-concept study, the possibility of combining two novel and appealing techniques in the manufacturing of wound dressings has been demonstrated for first time. The novelty of this work consists in the combination of liposomes (LPs) encapsulating the active pharmaceutical ingredient (API) into a hydrogel that is further printed into a three-dimensional scaffold for wound dressing; to the knowledge of the authors this has never been done before. A grid-shaped scaffold has been produced through the coaxial 3D bioprinting technique which has allowed to combine, in one single filament, two different bioinks. The inner core of the filament is a nanocomposite hydrogel consisting of hydroxyethyl cellulose (HEC) and PEGylated LPs encapsulated with thyme oil (TO) manufactured via MFs for the first time. The outer shell of the filament, instead, is represented by a hybrid hydrogel composed of sodium alginate/cellulose nanocrystals (SA/CNC) and enriched with free TO. This provides a combination of two different release ratios of the API, a bulk release for the first 24 h thanks to the free TO in the shell of the filament and a sustained release for up to 10 days provided from the API inside the LPs. Confocal Microscopy verified the actual presence of the LPs inside the scaffold after printing and evaluation using the zone of inhibition test proved the antibacterial activity of the manufactured scaffolds against both Gram-positive and Gram-negative bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

LEDs development has attracted attention over conventional mercury lamps for the tiny size, high efficiency, long lifetime, low operating temperature. The antimicrobial effectiveness of traditional UV-lamps radiation (wavelength of 254 nm) compared to UV-C LEDs (LED1 wavelength range 275-286 nm and LED2 range 260-270 nm) was carried out, for possible applications to automated sterile drug compounding. The UV lamp and the tested UV-LED devices remarkably reduced microbial load, following a time-dose response, but the best performance was evidenced by LED1, which guaranteed the complete inactivation of high concentrations of bacteria, yeasts, and spores at doses between 200 and 2000 J/m 2 ., Competing Interests: Declaration of Competing Interest None to declare, (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Breast-conserving surgery (BCS) is the primary strategy for treating early-stage breast cancer; however, the incidence of local recurrence and breast tissue loss negatively impacts patients and survivors. Furthermore, radiotherapy and/or systemic therapies are frequently advised to avoid recidivism and increase the patient's chance of survival, resulting in longer duration of treatments, and unpleasant systemic side effects. Given the poor prognosis and the heterogeneity between individuals and tumors, a patient-centered approach is fundamental. Herein we developed a multipurpose 4D printed implant made of a blend of carboxymethyl cellulose sodium salt (CMC) and cellulose nanocrystals (CNC), loaded with doxorubicin (DOX). To predict printability performance, full rheological characterization was carried out. The smart device was programmed to change size, under swelling, to better fit in the tissue cavity, resulting in a great potential for personalization, thus improving the aesthetic outcomes. The influence of the formulation and printing parameters on the morpho transformation was investigated through the swelling test, confirming the possibility to program the 4D shape. The manufactured implants were characterized by a variety of methods, including in vitro release studies. Lastly, the anticancer activity was conducted in vitro, on MDA-MB-231 cells. Implants promoted an anticancer effect of -58% viability after 72 h incubation, even when tested 4 weeks after the printing process. Overall, the morpho transformation and the in vitro studies have shown that the implant could represent a potential strategy for breast cancer following resection, to fill the void in the breast resulting from the surgery and provide an anticancer effect to avoid recurrence., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

To simulate the deposition of drugs in the oro-pharynx region, several in vitro models are available such as the United States Pharmacopeia-Induction Port (USP-IP) throat and the Virginia Commonwealth University (VCU) models. However, currently, there is no such in vitro model that incorporates a biological barrier to elucidate drug transport across the pharyngeal cells. Cellular models such as in vitro air-liquid interface (ALI) models of human respiratory epithelial cell lines are extensively used to study drug transport. To date, no studies have yet been performed to optimise the ALI culture conditions of the human pharyngeal cell line Detroit 562 and determine whether it could be used for drug transport. Therefore, this study aimed to develop a novel 3D-printed throat model integrated with an ALI cellular model of Detroit 562 cells and optimise the culture conditions to investigate whether the combined model could be used to study drug transport, using Lidocaine as a model drug. Differentiating characteristics specific to airway epithelia were assessed using 3 seeding densities (30,000, 60,000, and 80,000 cells/well (c/w), respectively) over 21 days. The results showed that Detroit 562 cells completely differentiates on day 18 of ALI for both 60,000 and 80,000 c/w with significant mucus production, showing response to bacterial and viral stimuli and development of functional tight junctions and Lidocaine transport with no significant differences observed between the ALI models with the 2 cell seeding densities. Results showed the suitability of the Low density (60,000 c/w or 1.8 × 10 5 cells/cm 2 ) ALI model to study drug transport. Importantly, the developed novel 3D-printed throat model integrated with our optimised in vitro Detroit 562 ALI model showed transport of Lidocaine throat spray. Overall, the study highlights the potential of the novel 3D-printed bio-throat integrated model as a promising in vitro system to investigate the transport of inhalable drug therapies targeted at the oro-pharyngeal region., (© 2023. Controlled Release Society.)

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core-crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLac n ) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the α v β 3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the α v β 3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of α v β 3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application., (© 2022. The Author(s).)

Respiratory tract infections (RTIs) are reported to be the leading cause of death worldwide. Delivery of liposomal antibiotic nano-systems via the inhalation route has drawn significant interest in RTIs treatment as it can directly target the site of infection and reduces the risk of systemic exposure and side effects. Moreover, this formulation system can improve pharmacokinetics and biodistribution and enhance the activity against intracellular pathogens. Microfluidics is an innovative manufacturing technology that can produce nanomedicines in a homogenous and scalable way. The objective of this study was to evaluate the antibiofilm efficacy of two liposomal ciprofloxacin formulations with different vesicle sizes manufactured by using a 3D-printed microfluidic chip. Each formulation was characterised in terms of size, polydispersity index, charge and encapsulation. Moreover, the aerosolisation characteristics of the liposomal formulations were investigated and compared with free ciprofloxacin solution using laser diffraction and cascade impaction methods. The in vitro drug release was tested using the dialysis bag method. Furthermore, the drug transport and drug release studies were conducted using the alveolar epithelial H441 cell line integrated next-generation impactor in vitro model. Finally, the biofilm eradication efficacy was evaluated using a dual-chamber microfluidic in vitro model. Results showed that both liposomal-loaded ciprofloxacin formulations and free ciprofloxacin solution had comparable aerosolisation characteristics and biofilm-killing efficacy. The liposomal ciprofloxacin formulation of smaller vesicle size showed significantly slower drug release in the dialysis bag technique compared to the free ciprofloxacin solution. Interestingly, liposomal ciprofloxacin formulations successfully controlled the release of the drug in the epithelial cell model and showed different drug transport profiles on H441 cell lines compared to the free ciprofloxacin solution, supporting the potential for inhaled liposomal ciprofloxacin to provide a promising treatment for respiratory infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Disclosure Statement No potential conflict of interest was reported by the authors., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In recent years, 3D printing has attracted great interest in the pharmaceutical field as a promising tool for the on-demand manufacturing of patient-centered pharmaceutical forms. Among the existing 3D printing techniques, direct powder extrusion (DPE) resulted as the most practical approach thanks to the possibility to directly process excipients and drugs in a single step. The main goal of this work was to determine whether different grades of ethylene vinyl acetate (EVA) copolymer might be employed as new feedstock materials for the DPE technique to manufacture transdermal patches. By selecting two model drugs with different thermal behavior, (i.e., ibuprofen and diclofenac sodium) we also wanted to pay attention to the versatility of EVA excipient in preparing patches for customized transdermal therapies. EVA was combined with 30 % (w/w) of each model drugs. The physicochemical composition of the printed devices was investigated through Fourier-transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analyses. FT-IR spectra confirmed that the starting materials were effectively incorporated into the final formulation, and thermal analyses demonstrated that the extrusion process altered the crystalline morphology of the raw polymers inducing the formation of crystals at lower thicknesses. Lastly, the drug release and permeation profile of the printed systems was evaluated for 48 h and showed to be dependent on the VA content of the EVA grade (74.5 % of ibuprofen released from EVA 4030AC matrix and 12.6 % of diclofenac sodium released from EVA1821A matrix). Hence, this study demonstrated that EVA and direct powder extrusion technique could be promising tools for manufacturing transdermal patches. By selecting the EVA grade with the appropriate VA content, drugs with dissimilar melting points could be printed preserving their thermal stability. Moreover, the desired drug release and permeation profile of the drug can be achieved, representing an important advantage in terms of personalized medicine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Gamma oryzanol (ORZ) is a nutraceutical that is poorly water soluble with poor intestinal absorption. In the current work, ORZ was nanoformulated into uncoated and chitosan coated micelles based on methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone) (mPEG-PCL) and poly(ε-caprolactone)-b-methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone) (PCL-PEG-PCL) copolymers for augmenting ORZ oral delivery. The physicochemical properties, morphological study, in-vitro release and safety of the nanoplaforms were determined. Importantly, the nephroprotective competence of the nanoplaforms was analyzed against acute kidney injury (AKI) rat model and the sirtuin-1 associated machineries were assessed. The results revealed that the micelles exerted particle size (PS) from 97.9 to 117.8 nm that was markedly increased after chitosan coating. The reversal of zeta potential from negative to highly positive further confirmed efficient coating. In vitro release profiles demonstrated prolonged release pattern. The nanoforms conferred higher cell viability values than free ORZ on Vero cell line. The designed micelles displayed augmented nephroprotection compared to free ORZ with the supremacy of CS coated micelles over uncoated ones in restoring kidney parameters to normal levels. The attenuated AKI was fulfilled via the modulation of sirtuin-1 signaling pathways translated by restoring the histological features, increasing renal antioxidant states, renal autophagy and decreasing renal inflammation and renal apoptosis. These outcomes confirmed that surface modification with chitosan had a considerable leverage on micelles safety, release behavior and in vivo performance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

After two decades of research in the field of nanomedicine, nanoscale delivery systems for biologicals are becoming clinically relevant tools. Microfluidic-based fabrication processes are replacing conventional techniques based on precipitation, emulsion, and homogenization. Here, the focus is on solid lipid nanoparticles (SLNs) for the encapsulation and delivery of lysozyme (LZ) as a model biologic. A thorough analysis was conducted to compare conventional versus microfluidic-based production techniques, using a 3D-printed device. The efficiency of the microfluidic technique in producing LZ-loaded SLNs (LZ SLNs) was demonstrated: LZ SLNs were found to have a lower size (158.05 ± 4.86 nm vs 180.21 ± 7.46 nm) and higher encapsulation efficacy (70.15 ± 1.65 % vs 53.58 ± 1.13 %) as compared to particles obtained with conventional methods. Cryo-EM studies highlighted a peculiar turtle-like structure on the surface of LZ SLNs. In vitro studies demonstrated that LZ SLNs were suitable to achieve a sustained release over time (7 days). Enzymatic activity of LZ entrapped into SLNs was challenged on Micrococcus lysodeikticus cultures, confirming the stability and potency of the biologic. This systematic analysis demonstrates that microfluidic production of SLNs can be efficiently used for encapsulation and delivery of complex biological molecules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Developing targeted drug delivery systems is an urgent need to decrease the side effects and increase the drug's efficiency. Most cancer cells show an increased sugar consumption compared to healthy cells due to the deregulation of sugar transporters. Consequently, liposomes, as a biocompatible nanocarrier, could be surface decorated by sugars to enhance drug targeting into cancer cells. Our work outlines a new strategy to easily manufacture sucrose decorated liposomes using sucrose stearate, a biocompatible and biodegradable non-ionic surfactant, with a scalable microfluidic approach. Sucrose decorated liposomes were loaded with berberine hydrochloride, a well-known phytochemical compound to investigate its effects on triple-negative breast cancer cells (MDA-MB-231). Using the microfluidic manufacturing system, we prepared berberine-loaded liposomes using a mixture of phosphatidylcholine and cholesterol with and without sucrose stearate with a size up to 140 nm and narrow polydispersity. Stability was confirmed for 90 days, and the in vitro release profile was evaluated. The formulations showed acceptable in vitro biocompatibility and significantly higher anti-proliferative effect on MDA-MB-231 cell line. These results have been confirmed by an increased uptake evaluated by flow cytometry and confocal microscopy. Taken together, our findings represent an innovative, easy, and scalable approach to obtain sugar decorated liposomal formulations without any surface-chemistry reactions. They can be potentially used as an anticancer targeted drug delivery system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Breast cancer is the second most common cancer worldwide, characterized by a high incidence and mortality rate. Despite the advances achieved in cancer management, improvements in the quality of life of breast cancer survivors are urgent. Moreover, considering the heterogeneity that characterizes tumors and patients, focusing on individuality is fundamental. In this context, 3D printing (3DP) and 4D printing (4DP) techniques allow for a patient-centered approach. At present, 3DP applications against breast cancer are focused on three main aspects: treatment, tissue regeneration, and recovery of the physical appearance. Scaffolds, drug-loaded implants, and prosthetics have been successfully manufactured; however, some challenges must be overcome to shift to clinical practice. The introduction of the fourth dimension has led to an increase in the degree of complexity and customization possibilities. However, 4DP is still in the early stages; thus, research is needed to prove its feasibility in healthcare applications. This review article provides an overview of current approaches for breast cancer management, including standard treatments and breast reconstruction strategies. The benefits and limitations of 3DP and 4DP technologies are discussed, as well as their application in the fight against breast cancer. Future perspectives and challenges are outlined to encourage and promote AM technologies in real-world practice.

During the past decades, 3D printing has revolutionised different areas of research. Despite the considerable progress achieved in 3D printing of pharmaceuticals, the limited choice of suitable materials remains a challenge to overcome. The growing search for sustainable excipients has led to an increasing interest in biopolymers. Poly(3-hydroxybutyrate) (PHB) is a biocompatible and biodegradable biopolymer obtained from bacteria that could be efficiently employed in the pharmaceutical field. Here we aimed to demonstrate its potential application as a thermoplastic material for personalised medicine through 3D printing. More specifically, we processed PHB by using direct powder extrusion, a one-step additive manufacturing technique. To assess and denote the feasibility and versatility of the process, a 3D square model was manufactured in different dimensions (sidexheight: 12x2 mm; 18x2 mm; 24x2 mm) and loaded with increasing percentages of a model drug (up to 30% w/w). The manufacturing process was influenced by the drug content, and indeed, an increase in the amount of the drug determined a reduction in the printing temperature, without affecting the other parameters (such as the layer height). The composition of the model squares was investigated using Fourier-transform infrared spectroscopy, the resulting spectra confirmed that the starting materials were successfully incorporated into the final formulations. The thermal behaviour of the printed systems was characterized by differential scanning calorimetry, and thermal gravimetric analysis. Moreover, the sustained drug release profile of the formulations was performed over 21 days and showed to be dependent on the dimensions of the printed object and on the amount of loaded drug. Indeed, the formulation with 30% w/w in the dimension 24x2 mm released the highest amount of drug. Hence, the results suggested that PHB and direct powder extrusion technique could be promising tools for the manufacturing of prolonged release and personalised drug delivery forms., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Insomnia is a chronic disorder with a mean prevalence ranged from 6% to 15% worldwide. The usual pharmacologic treatment for insomnia has been benzodiazepines and barbiturates. More recently, z-drugs were introduced in the therapeutic arsenal to maximize benefits and minimize treatment damage. Zolpidem tartrate, whose primary indication is for sleep initiation problems, is conventionally used at a recommended dose of 5 mg for women as well as elderly patients (<65 years-old) and 10 mg for non-elderly men. However, it was demonstrated that the dose of zolpidem should be adjusted according to the gender, age, condition of the patient and the presence of polypharmacy to decrease the occurrence of adverse events. Faced with the therapeutic limitations inherent to marketed products, magistral preparations offer medical and legal alternatives to mass treatment. The use of a semi-automatic technique, with standardized protocol, such as 3D printing should be advantageously implemented as an alternative to standard compounding procedures. In this work, the pressure-assisted microsyringes method was selected as it allows the tridimensional printing, and so the customization of the dose, by easily extruding a viscous semi-liquid material, called "slurry", through a syringe at room temperature. It has been demonstrated that this methodology allows obtaining printlets that responded to the zolpidem-containing tablets monograph of the US pharmacopoeia Edition 42. The compounding preparations proposed in this work therefore have the same criteria of requirements as a commercial form., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The antimicrobial activity of several essential oils (EOs) and their related microemulsions (MEs) was investigated. EOs were obtained from Cannabis sativa L. cv CS (C. sativa), Carum carvi L. (C. carvi), Crithmum maritimum L. (C. maritimum), Cuminum cyminum L. (C. cyminum), x Cupressocyparis leylandii A.B. Jacks & Dallim. (C. leylandii), Cupressus arizonica Greene (C. arizonica), Ferula assa-foetida L. (F. assa-foetida)., Ferula gummosa Boiss. (F. gummosa), Juniperus communis L. (J. communis), Juniperus x pfitzeriana (Spath) P.A. Schmidt (J. pfitzeriana), Pimpinella anisum L (P. anisum). Preliminary screening revealed that Cuminum cyminum, Crithmum maritimum, and Pimpinella anisum (10% v/v) were effective against all tested microorganisms (Escherichia coli ATCC 35218, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC 29213, Pseudomonas fluorescens DSM 4358, and Candida albicans ATCC 10231), with growth inhibition diameter from 10 to 25 mm. These EOs were used to formulate the MEs with an average size < 50 nm and a good stability over 30 days. EOs’ antimicrobial activity was further enhanced in the MEs, with a generalized lowering of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. C. cyminum-ME reached, in most cases, MIC two times lower (0.312%) than the corresponding EO (0.625%) and even eight times lower against S. aureus (0.156 vs. 1.25%). A more remarkable microbicide effect was noted for C. cyminum-ME, with MBC values eight times lower (from 0.312 to 0.625%) than the corresponding EO (from 2.5 to 5%). Overall, MEs resulted in an efficient system for EOs encapsulation, enhancing solubility and lowering concentration to exert antimicrobial efficacy.

In recent years, researchers are exploring innovative green materials fabricated from renewable natural substances to meet formulation needs. Among them, biopolymers like chitosans and biosurfactants such as sugar fatty acid esters are of potential interest due to their biocompatibility, biodegradability, functionality, and cost-effectiveness. Both classes of biocompounds possess the ability to be efficiently employed in wound dressing to help physiological wound healing, which is a bioprocess involving uncontrolled oxidative damage and inflammation, with an associated high risk of infection. In this work, we synthesized two different sugar esters (i.e., lactose linoleate and lactose linolenate) that, in combination with chitosan and sucrose laurate, were evaluated in vitro for their cytocompatibility, anti-inflammatory, antioxidant, and antibacterial activities and in vivo as wound care agents. Emphasis on Wnt/β-catenin associated machineries was also set. The newly designed lactose esters, sucrose ester, and chitosan possessed sole biological attributes, entailing considerable blending for convenient formulation of wound care products. In particular, the mixture composed of sucrose laurate (200 µM), lactose linoleate (100 µM), and chitosan (1%) assured its superiority in terms of efficient wound healing prospects in vivo together with the restoring of the Wnt/β-catenin signaling pathway, compared with the marketed wound healing product (Healosol®), and single components as well. This innovative combination of biomaterials applied as wound dressing could effectively break new ground in skin wound care., (Copyright © 2022 Elsevier B.V. All rights reserved.)