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Background: In glioblastoma (GBM) therapy research, tumour treating fields by the company Novocure™, have shown promise for increasing patient overall survival. When used with the chemotherapeutic agent temozolomide, they extend median survival by five months. However, there is a space to design alternative systems that will be amenable for wider use in current research. Therefore, we sought to establish a custom-built alternating electric field device to investigate the effect of electrode design on the responsiveness of cancer cells to this therapy., Methods: A 96-well microtiter plate modified with an electrode array was fabricated to investigate its application as an in vitro alternating electric field device. This was initially performed with patient-derived GCE 31 and GIN 31 cell lines found in the core and invasive margin of the GBM tumour, respectively. We sought to establish the effect of the application of low-intensity (3 V/ cm) electric fields with an application duration of 4-48 h, using intermediate frequency (300 kHz) alternating currents (AC). To demonstrate that electric fields were entering the cell, GCE 31 and GIN 31 cells were treated with the inorganic, non-conductive zinc oxide (ZnO) nanoparticles (NP), previously demonstrated to enhance the efficacy of TTFs. After a 4-h exposure to NP, cells were then exposed to alternating electric fields or currents and their metabolic activity was assessed. To better understand how the position and morphology of cells can affect cell therapy responsiveness to alternating electric fields or currents, GBM results were compared to those from the semi-adherent brain tumour cell line, D425., Results: Contrary to previous findings, there was no significant difference between the GIN 31 and GCE 31 cells exposed to alternating electric fields or currents treated with or without NP compared to cells untreated and unstimulated. D425 cells exposed to alternating electric fields exhibited a pronounced metabolic increase (1.8-fold), while those exposed to alternating electric currents with or without ZnO had a reduced metabolism relative to the untreated control., Conclusions: The initial hypothesis for the lack of effect of electrical stimulation on the adherent cells was that, due to only a single pair of electrodes being used, the proportion of cells that were in the correct orientation for electric field effects was limited. However, the dramatic shift in cell behaviour of the semi-adherent cells shows that cell morphology plays an important role in the responsiveness of cancer cells to AC electric fields. This study highlights the lack of understanding of the complex mechanisms by which electric fields exert effects on cancer cells. We propose that, for the therapy to be enhanced for patients, research should first focus on the underlying mechanisms of action, specifically on how individual cancer cell types respond to this therapy., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Resistance mechanisms in brain tumors, such as medulloblastoma and glioblastoma, frequently involve the entrapment of chemotherapeutic agents within endosomes and the extracellular expulsion of drugs. These barriers to effective treatment are exacerbated in nanotechnology-based drug delivery systems, where therapeutic nanoparticles often remain confined within endosomes, thus diminishing their therapeutic efficacy. Addressing this challenge necessitates the development of novel strategies to enhance the efficiency of cancer therapies. This study tests the hypothesis that external electrical stimuli can modulate intracellular trafficking of chemotherapeutic drugs in common malignant brain tumors in children (medulloblastoma) and adults (glioblastoma) by using gold nanoparticles (GNPs). In our experiments, alternating current (AC) stimulation ranging from 1 kHz to 5 MHz and at a strength of 1 V/cm significantly reduced cell viability in drug-resistant medulloblastoma and enhanced delivery of GNPs in glioblastoma. Low-frequency AC resulted in a 50% increase in apoptosis compared to controls and an 8-fold increase in cell death in cisplatin-resistant medulloblastoma cells, accompanied by a substantial reduction in EC 50 from 2.5 to 0.3 μM. Similarly, vincristine-resistant cells demonstrated a 4-fold enhancement in drug sensitivity. Furthermore, high-frequency AC facilitated a significant increase from 20 to 75% in the endosomal escape of GNPs in glioblastoma cells. These findings underscore the potential of AC to selectively disrupt cancer cell resistance mechanisms and bolster the efficacy of nanoparticle-based therapies. The results indicate the effectiveness of AC stimulation in circumventing the limitations inherent in current nanotechnology-based drug delivery systems but also illustrates its transformative potential for treating aggressive, drug-resistant brain tumors., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB., (© 2023. The Author(s).)

Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Oral specially coated formulations have the potential to improve treatment outcomes of a range of diseases in distal intestinal tract whilst limiting systemic drug absorption and adverse effects. Their development is challenging, partly because of limited knowledge of the physiological and pathological distal gastrointestinal factors, including colonic chyme fluid distribution and motor function. Recently, non-invasive techniques such as magnetic resonance imaging (MRI) have started to provide novel important insights. In this feasibility study, we formulated a coated capsule consisting of a hydroxypropyl methylcellulose (HPMC) shell, coated with a synthetic polymer based on polymethacrylate-based copolymer (Eudragit ® ) that can withstand the upper gastrointestinal tract conditions. The capsule was filled with olive oil as MRI-visible marker fluid. This allowed us to test the ability of MRI to track such a coated capsule in the gastrointestinal tract and to assess whether it is possible to image its loss of integrity by exploiting the ability of MRI to image fat and water separately and in combination. Ten healthy participants were administered capsules with varying amounts of coating and underwent MRI imaging of the gastrointestinal tract at 45 min intervals. The results indicate that it is feasible to track the capsules present in the gastrointestinal tract at different locations, as they were detected in all 10 participants. By the 360 min endpoint of the study, in nine participants the capsules were imaged in the small bowel, in eight participants in the terminal ileum, and in four in the colon. Loss of capsule integrity was observed in eight participants, occurring predominantly in distal intestinal regions. The data indicate that the described approach could be applied to assess performance of oral formulations in undisturbed distal gastrointestinal regions, without the need for ionizing radiation or contrast agents.

Dietary lipids and some pharmaceutical lipid excipients can facilitate the targeted delivery of drugs to the intestinal lymphatics. Here, the feasibility of magnetic resonance imaging (MRI) for imaging lipid uptake into the intestinal lymphatics was assessed, shedding light on which lymph nodes can be targeted using this approach. Three healthy male volunteers were scanned at 3.0 T at baseline, 120, 180, 240, and 300 min post high-fat meal. A sagittal multi-slice image was acquired using a diffusion-weighted whole-body imaging sequence with background suppression (DWIBS) (pre inversion TI = 260 ms). Changes in area, major, and minor axis length were compared at each time point. Apparent diffusion coefficient (ADC) was calculated (b = 0 and 600 s/mm 2 ) across eight slices. An average of 22 nodes could be visualised across all time points. ADC increased at 120 and 180 min compared to the baseline in all three participants by an average of 9.2% and 6.8%, respectively. In two participants, mean node area and major axis lengths increased at 120 and 180 min relative to the baseline. In conclusion, the method described shows potential for repeated lymph node measurements and the tracking of lipid uptake into the lymphatics. Further studies should focus on methodology optimisation in a larger cohort.

Helicobacter pylori ( H. pylori ) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA 47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20-49%), LabA shares an L-shaped fold with SabA and BabA. The 'head' region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain ('crown' region), the 'J99-type' (insertion ~31 ​amino acids), and the H. pylori '26695 type' (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding., Competing Interests: 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., (© 2020 The Author(s).)

Mucus is the first biological component inhaled drugs encounter on their journey towards their pharmacological target in the upper airways. Yet, how mucus may influence drug disposition and efficacy in the lungs has been essentially overlooked. In this study, a simple in vitro system was developed to investigate the factors promoting drug interactions with airway mucus in physiologically relevant conditions. Thin layers of porcine tracheal mucus were prepared in Transwell ® inserts and initially, the diffusion of various fluorescent dyes across those layers was monitored over time. A deposition system featuring a MicroSprayer ® aerosolizer was optimized to reproducibly deliver liquid aerosols to multiple air-facing layers and then exploited to compare the impact of airway mucus on the transport of inhaled bronchodilators. Both the dyes and drugs tested were distinctly hindered by mucus with high logP compounds being the most affected. The diffusion rate of the bronchodilators across the layers was in the order: ipratropium glycopyronnium > formoterol > salbutamol > indacaterol, suggesting hydrophobicity plays an important role in their binding to mucus but is not the unique parameter involved. Testing of larger series of compounds would nevertheless be necessary to better understand the interactions of inhaled drugs with airway mucus., Competing Interests: The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Trans-plasma membrane electron transfer (tMPET) is a process by which reducing equivalents, either electrons or reductants like ascorbic acid, are exported to the extracellular environment by the cell. TPMET is involved in a number of physiological process and has been hypothesised to play a role in the redox regulation of cancer metabolism. Here, we use a new electrochemical assay to elucidate the 'preference' of cancer cells for different trans tPMET systems. This aids in proving a biochemical framework for the understanding of tPMET role, and for the development of novel tPMET-targeting therapeutics. We have delineated the mechanism of tPMET in 3 lung cancer cell models to show that the external electron transfer is orchestrated by ascorbate mediated shuttling via tPMET. In addition, the cells employ a different, non-shuttling-based mechanism based on direct electron transfer via Dcytb. Results from our investigations indicate that tPMETs are used differently, depending on the cell type. The data generated indicates that tPMETs may play a fundamental role in facilitation of energy reprogramming in malignant cells, whereby tPMETs are utilised to supply the necessary energy requirement when mitochondrial stress occurs. Our findings instruct a deeper understanding of tPMET systems, and show how different cancer cells may preferentially use distinguishable tPMET systems for cellular electron transfer processes., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

All eukaryotic organisms require iron to function. Malfunctions within iron homeostasis have a range of physiological consequences, and can lead to the development of pathological conditions that can result in an excess of non-transferrin bound iron (NTBI). Despite extensive understanding of iron homeostasis, the links between the "macroscopic" transport of iron across biological barriers (cellular membranes) and the chemistry of redox changes that drive these processes still needs elucidating. This review draws conclusions from the current literature, and describes some of the underlying biophysical and biochemical processes that occur in iron homeostasis. By first taking a broad view of iron uptake within the gut and subsequent delivery to tissues, in addition to describing the transferrin and non-transferrin mediated components of these processes, we provide a base of knowledge from which we further explore NTBI uptake. We provide concise up-to-date information of the transplasma electron transport systems (tPMETSs) involved within NTBI uptake, and highlight how these systems are not only involved within NTBI uptake for detoxification but also may play a role within the reduction of metabolic stress through regeneration of intracellular NAD(P)H/NAD(P) + levels. Furthermore, we illuminate the thermodynamics that governs iron transport, namely the redox potential cascade and electrochemical behavior of key components of the electron transport systems that facilitate the movement of electrons across the plasma membrane to the extracellular compartment. We also take account of kinetic changes that occur to transport iron into the cell, namely membrane dipole change and their consequent effects within membrane structure that act to facilitate transport of ions.

Despite research efforts full potential of siRNA-based therapeutics has not yet been fully realized due to a need for suitable, effective delivery formulations. Here, we examine a potential of a new class of water-soluble chitosans as siRNA platform for pulmonary delivery. The system is based on piperazine-substituted chitosans, a material designed to integrate established, safe application of chitosan for mucosal administration with novel properties: the piperazine-substituted chitosans are freely water-soluble at physiological pH, possess low cytotoxicity (no significant reduction in cell viability up to 0.1 mg/ml), and provide efficient incorporation of siRNA into sub-300 nm colloidal complexes (at relatively low polymer/siRNA ratio of 5:1). In vitro, the complexes achieved silencing of a model gene at a level of 40-80%, when tested in a panel of lung epithelial cells. Considering the formulation 'developability', there were no significant changes in the complexes' size and integrity on aerosolisation by microsprayer (PenCentury™) device. Following intratracheal aerolisation, the complexes deposited throughout the lung, although relatively inhomogeneously, as judged from IVIS imaging of the isolated mouse lung (visualizing DY647-siRNA). In vivo data illustrate absence of adverse effects on repeated administration of complexes and significant tumor reduction in atopical lung cancer model in mice. Altogether, the data illustrates potential of substituted chitosan derivatives to be utilized as a safe system for inhalation delivery of siRNA.

Permeability enhancers are receiving increased attention arising from their ability to increase transepithelial permeability and thus, bioavailability of orally or pulmonary administered biopharmaceutics. Here we present the synthesis and the in vitro assaying of a series of lactose-based non-ionic surfactants, highlighting the relationship between their structure and biological effect. Using tensiometric measurements the critical micelle concentrations (CMCs) of the surfactants were determined and demonstrate that increasing hydrophobic chain length reduces surfactant CMC. In vitro testing on Caco-2 intestinal and Calu-3 airway epithelia revealed that cytotoxicity, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, is presented for most of the surfactants at concentrations greater than their CMCs. Further biological study demonstrates that application of cytotoxic concentrations of the surfactants is associated with depolarizing mitochondrial membrane potential, increasing nuclear membrane permeability and activation of effector caspases. It is, therefore, proposed that when applied at cytotoxic levels, the surfactants are inducing apoptosis in both cell lines tested. Importantly, through the culture of epithelial monolayers on Transwell ® supports, the surfactants demonstrate the ability to reversibly modulate transepithelial electrical resistance (TEER), and thus open tight junctions, at non-toxic concentrations, emphasizing their potential application as safe permeability enhancers in vivo.

Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement - a stage between initial cellular internalization and final gene silencing of siRNA delivery systems.

Helicobacter pylori is a leading cause of peptic ulceration and gastric cancer worldwide. To achieve colonization of the stomach, this Gram-negative bacterium adheres to Lewis(b) (Le(b)) antigens in the gastric mucosa using its outer membrane protein BabA. Structural information for BabA has been elusive, and thus, its molecular mechanism for recognizing Le(b) antigens remains unknown. We present the crystal structure of the extracellular domain of BabA, from H. pylori strain J99, in the absence and presence of Le(b) at 2.0- and 2.1-Å resolutions, respectively. BabA is a predominantly α-helical molecule with a markedly kinked tertiary structure containing a single, shallow Le(b) binding site at its tip within a β-strand motif. No conformational change occurs in BabA upon binding of Le(b), which is characterized by low affinity under acidic [K D (dissociation constant) of ~227 μM] and neutral (K D of ~252 μM) conditions. Binding is mediated by a network of hydrogen bonds between Le(b) Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions. The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Le(b), respectively. Knowledge of the molecular basis of Le(b) recognition by BabA provides a platform for the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa.

Using microemulsions (µEs) as preservation media for cells was pursued in the 1990s; however, the difficulty in formulating biocompatible µEs and keeping unacclimatized cells alive for more than three days hindered developments in this area. This work explores the use of fully dilutable self-microemulsifying delivery systems (SMEDS) formulated with lecithin (Le) and polyglycerol-10-caprylate (PG10C) at a ratio of 2/5. This surfactant blend was mixed with ethyl oleate (EOL) at a ratio of 60 surfactant/40 EOL to produce a D60 dilution line. This D60 SMEDS was diluted with 0.9% w/v NaCl solution to produce lecithin-linker µEs (LLMs). The properties of the resulting LLMs were predicted using the hydrophilic–lipophilic-difference (HLD) and net-average curvature (NAC) model, indicating that LLMs with aqueous content from 5% to 60% are bicontinuous, confirmed via viscosity and conductivity. The largest yeast activity and viability obtained with LLMs were achieved with 30% aqueous content, resulting from the balance between having enough water for the effective transport of metabolites, enough SMEDS to contribute nutrients and lipids, and a low enough water to limit the partition of PG10C that, when present in the aqueous phase, inhibited yeast activity. For SMEDS, its low water activity ensured that the yeast remained dormant, keeping them alive for at least 10 weeks. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the usefulness of human embryonic kidney HEK293 cells as a model of normal cells in biotin-mediated therapy. The expression and role of sodium multivitamin transporter (SMVT) in the uptake and accumulation of free biotin, as well as cationic and neutral biotinylated PAMAM dendrimers of the fourth generation synthesized in our laboratory, were assessed in HEK293 cells in comparison to other immortalized (HaCaT) and cancer cells (HepG2, U-118 MG). The obtained data showed that a higher level of SMVT in HEK293 cells was not associated with a stronger uptake of biotin and biotinylated PAMAM dendrimers. Biotinylation increased the selective uptake of neutral dendrimers in an inversely proportional manner to the concentration used; however, the accumulation in HEK293 cells was lower than that in cells of other cell lines. The time-dependent biotin and biotinylated dendrimers uptake profiles differed significantly. Therefore, it should be assumed that the efficiency of biotinylated nanoparticles' uptake depends on multiple cellular transport mechanisms. Toxicity tests showed significantly higher sensitivity to PAMAM conjugates for HEK293 cells than for HepG2 and HaCaT cells. Molecular modeling studies and the profile of biotin uptake suggest that not only SMVT but also monocarboxylate transporter 1 (MCT-1) may play an important role in the selective transport of biotin and biotinylated nanoparticles into cells. Due to the complexity of the problem, further studies are necessary. In summary, HEK293 cells can be considered a valuable model of normal cells in the study of biotin- targeted therapy using nanoparticles based on PAMAM dendrimers. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is one of the most common diseases of the central nervous system in the middle-aged and elderly population. It is a neurodegenerative disorder, and its main clinical symptoms include the loss of established memories, a decline in learning capacity, and the buildup of β-amyloid peptides. The disease is often accompanied by neurodegenerative changes and the formation of neurofibrillary tangles. However, the number of drugs available for the clinical treatment of AD remains limited. Currently, existing medications are not effective in completely curing the disease or stopping its progression. Due to their excellent biocompatibility and biodegradability, polymers have been widely used as drug delivery carriers in various fields including cancer therapy and wound healing. The use of polymers enables targeted drug delivery and prolonged release profiles. In recent years, researchers have made significant progress in utilizing polymers such as polyethylene glycol, poly (lactic-co-glycolic acid) (PLGA), and chitosan (CS) to deliver drugs and blood-brain barrier receptor ligands for the treatment of AD. Moreover, many polymers with inherent therapeutic properties have been developed, including the already marketed GV-971 as well as experimental polymers such as PLGA and CS oligosaccharide. This review summarizes the applications of polymers in AD treatment over the past few years and highlights their current limitations to help researchers better understand current advancements in polymer development and identify future research directions. [ABSTRACT FROM AUTHOR]

Preclinical studies have demonstrated that nanoparticles (NPs) hold significant potential for advancing cancer therapy by enhancing therapeutic efficacy while reducing side effects. Their effectiveness in solid tumors is, however, often constrained by insufficient accumulation and penetration. Understanding how the physicochemical properties of NPs – such as size, shape, and surface charge – influence their interaction with cells within the tumor is critical for optimizing NP design. In this study, we addressed the challenge of inconsistent NP behavior by systematically evaluating NP uptake in both 2D and 3D tumor models, and NP penetration in spheroids. Our results showed that larger NPs exhibited higher internalization rates in 2D models but limited penetration in 3D spheroids. Furthermore, negatively charged NPs consistently achieved superior accumulation and deeper penetration than neutral and positively charged NPs. Spherical NPs outperformed rod-shaped NPs in tumor accumulation and penetration. These findings underscore the importance of carefully tailoring NP properties to the complex tumor microenvironment for improved therapeutic outcomes in real tumors. [ABSTRACT FROM AUTHOR]

Cell microencapsulation is one of the most studied strategies to overcome the challenges associated with the implementation of mesenchymal stem/stromal cells (MSCs) in vivo. This approach isolates/shields donor MSCs from the host immune system using a semipermeable membrane that allows for the diffusion of gases, nutrients, and therapeutics, but not host immune cells. As a result, microencapsulated MSCs survive and engraft better after infusion, and they can be delivered specifically to the targeted site. Additionally, microencapsulation enables the co-culture of MSCs with different types of cells in a three-dimensional (3D) environment, allowing for better cellular interaction. Alginate, collagen, and cellulose are the most popular materials, and air jet extrusion, microfluidics, and emulsion are the most used techniques for MSC cell encapsulation in the literature. These materials and techniques differ in the size range of the resultant microcapsules and their compatibility with the applied materials. This review discusses various materials and techniques used for the microencapsulation of MSCs. We also shed light on the recent findings in this field, the advantages and drawbacks of using encapsulated MSCs, and the in vivo translation of the microencapsulated MSCs in cell therapy. [ABSTRACT FROM AUTHOR]

Background/Objectives: Parkinson's disease (PD) is a progressive neuro-degenerative disorder characterized by α-synuclein aggregation, which promotes neuronal death and accelerates neurodegeneration. Small interfering RNA (siRNA) can reduce α-synuclein levels, but its therapeutic potential is limited by poor stability and delivery challenges. Similarly, Selegiline (Sel), a monoamine oxidase-B (MAO-B) inhibitor, has low bioavailability, restricting its effectiveness. This study aims to develop an intranasal (IN) albumin-coated liposomal system (C-LipSel-siSNCA2) for the co-delivery of Sel and α-synuclein-targeting siRNA (siSNCA2) to enhance brain targeting and therapeutic efficacy. Methods: Liposomes were prepared using the ethanol injection method and optimized via D-optimal design for size, charge, and encapsulation efficiency (EE%). The optimized formulation was coated with human serum albumin (HSA) and characterized for stability, cellular uptake, and gene silencing. In vivo pharmacokinetics and pharmacodynamics were assessed in a rotenone-induced PD rat model to evaluate the motor function, biochemical markers, and brain-targeting efficiency. Results: Optimized liposomes had a particle size of 113.5 ± 6.8 nm, zeta potential of 6.2 ± 0.8 mV, and high EE% (Sel: 92.35%; siRNA: 78.66%). Albumin coating increased size to 136.5 ± 10.3 nm and shifted zeta potential to −13.5 ± 1.4 mV, enhancing stability and targeting. IN administration achieved a 3-fold increase in brain area under the concentration-time curve (AUC) versus intravenous delivery. In PD rats, C-LipSel-siSNCA2 improved motor and non-motor functions, restored dopamine levels, enhanced catalase activity, and reduced MAO-B levels, mitigating dopamine degradation and α-synuclein aggregation. Conclusions: This non-invasive, dual-action nanoplatform offers a targeted therapy for PD, combining siRNA gene silencing and MAO-B inhibition, with the potential for clinical translation in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

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Background/Objectives: Mirtazapine (MRZ) is a psychotropic drug prescribed to manage serious sorts of depression. By virtue of its extensive initial-pass metabolic process with poor water solubility, the ultimate bioavailability when taken orally is a mere 50%, necessitating repeated administration. The current inquiry intended to fabricate nose-to-brain chitosan-grafted cationic leciplexes of MRZ (CS-MRZ-LPX) to improve its pharmacokinetic weaknesses and boost the pharmacodynamics aspects. Methods: Primarily, MRZ-loaded leciplexes (MRZ-LPXs) were fabricated and tailored employing a central composite design (CCD). Vesicle diameter size (VS), entrapment efficiency (EE %), cumulative MRZ release percentage (CMRZR %), and total quantity penetrating after twenty-four hours (Q24) were the four parameters assessed. Then, the determined optimum formulation was coated with chitosan (CS-MRZ-LPX) and utilized in pharmacodynamics investigations and in vivo biologic distribution studies in Wistar male rats. Results: The customized MRZ-LPX formulation had a diameter size of 186.2 ± 3.5 nm and drug EE of 45.86 ± 0.76%. Also, the tailored MRZ-LPX formulation had a cumulative amount of MRZ released of 76.66 ± 3.06% and the total Q24 permeated was 383.23 ± 13.08 µg/cm2. Intranasal delivery of the tailored CS-MRZ-LPX revealed notably superior pharmacokinetic attributes inside the brain and circulation compared to the orally administered MRZ suspension and the intranasal free drug suspension (p < 0.05); the relative bioavailability was 370.9% and 385.6% for plasma and brain, respectively. Pharmacodynamics' and immunohistopathological evaluations proved that optimum intranasal CS-MRZ-LPX boosted antidepressant activity compared to the oral and free nasal drug administration. Conclusions: CS-MRZ-LPX tailored formulation can potentially be regarded as a prospective nano platform to boost bioavailability and enhance pharmacodynamics efficacy. Ultimately, intranasal CS-MRZ-LPX can be considered a promising avenue for MRZ targeted brain delivery as an antidepressant. [ABSTRACT FROM AUTHOR]

Glioblastoma (GB) is one of the most aggressive and treatment-resistant cancers due to its complex tumor microenvironment (TME). We previously showed that GB progression is dependent on the aberrant induction of chaperone-mediated autophagy (CMA) in pericytes (PCs), which promotes TME immunosuppression through the PC secretome. The secretion of extracellular matrix (ECM) proteins with anti-tumor (Lumican) and pro-tumoral (Osteopontin, OPN) properties was shown to be dependent on the regulation of GB-induced CMA in PCs. As biomarkers are rarely studied in TME, in this work, we aimed to validate Lumican and OPN as prognostic markers in the perivascular areas of the peritumoral niche of a cohort of GB patients. Previously, we had validated their expression in GB xenografted mice presenting GB infiltration (OPN) or GB elimination (Lumican) dependent on competent or deficient CMA PCs, respectively. Then, patient sample classification by GB infiltration into the peritumoral brain parenchyma was related to GB-induced CMA in microvasculature PCs, analyzing the expression of the lysosomal receptor, LAMP-2A. Our results revealed a correlation between GB-induced CMA activity in peritumoral PCs and GB patients' outcomes, identifying three degrees of severity. The perivascular expression of both immune activation markers, Iba1 and CD68, was related to CMA-dependent PC immune function and determined as useful for efficient GB prognosis. Lumican expression was identified in perivascular areas of patients with less severe outcome and partially co-localizing with PCs presenting low CMA activity, while OPN was primarily found in perivascular areas of patients with poor outcome and partially co-localizing with PCs presenting high CMA activity. Importantly, we found sex differences in the incidence of middle-aged patients, being significantly higher in men but with worse prognosis in women. Our results confirmed that Lumican and OPN in perivascular areas of the GB peritumoral niche are effective predictive biomarkers for evaluating prognosis and monitoring possible therapeutic immune responses dependent on PCs in tumor progression. [ABSTRACT FROM AUTHOR]

Large skeletal muscle injuries such as volumetric muscle loss (VML) disrupt native tissue structures, including biophysical and biochemical signaling cues that promote the regeneration of functional skeletal muscle. Various biofabrication strategies have been developed to create engineered skeletal muscle constructs that mimic native matrix and cellular microenvironments to enhance muscle regeneration; however, there remains a need to create scalable engineered tissues that provide mechanical stability as well as structural and spatiotemporal signaling cues to promote cell-mediated regeneration of contractile skeletal muscle. We describe a novel strategy for bioprinting multifunctional myoblast-loaded fibrin microthreads (myothreads) that recapitulate the cellular microniches to drive myogenesis and aligned myotube formation. We characterized myoblast alignment, myotube formation, and tensile properties of myothreads as a function of cell-loading density and culture time. We showed that increasing myoblast loading densities enhances myotube formation. Additionally, alignment analyses indicate that the bioprinting process confers myoblast alignment in the constructs. Finally, tensile characterizations suggest that myothreads possess the structural stability to serve as a potential platform for developing scalable muscle scaffolds. We anticipate that our myothread biofabrication approach will enable us to strategically investigate biophysical and biochemical signaling cues and cellular mechanisms that enhance functional skeletal muscle regeneration for the treatment of VML. [ABSTRACT FROM AUTHOR]

Interleukin-4-inducing principle from schistosome eggs (IPSE/alpha-1) is a protein produced exclusively by the eggs of the trematode Schistosoma mansoni. IPSE/alpha-1 is a secretory glycoprotein which activates human basophils via an IgE-dependent but non-antigen-specific mechanism. Sequence analyses revealed a potential nuclear localization signal (NLS) at the C terminus of IPSE/alpha-1. Here we show that this sequence (125-PKRRRTY-131) is both necessary and sufficient for nuclear localization of IPSE or IPSE-enhanced green fluorescent protein (EGFP) fusions. While transiently expressed EGFP-IPSE/alpha-1 was exclusively nuclear in the Huh7 and U-2 OS cell lines, a mutant lacking amino acids 125 to 134 showed both nuclear and cytoplasmic staining. Moreover, insertion of the IPSE/alpha-1 NLS into a tetra-EGFP construct rendered the protein nuclear. Alanine scanning mutagenesis revealed a requirement for the KRRR residues. Fluorescence microscopy depicted, and Western blotting further confirmed, that recombinant IPSE/alpha-1 protein added exogenously is rapidly internalized by CHO cells and accumulates in nuclei in an NLS-dependent manner. A mutant protein in which the NLS motif was disrupted by triple mutation (RRR to AAA) was able to penetrate CHO cells but did not translocate to the nucleus. Furthermore, the uptake of native glycosylated IPSE/alpha-1 was confirmed in human primary monocyte-derived dendritic cells and was found to be a calcium- and temperature-dependent process. Live-cell imaging showed that IPSE/alpha-1 is not targeted to lysosomes. In contrast, peripheral blood basophils do not take up IPSE/alpha-1 and do not require the presence of an intact NLS for activation. Taken together, our results suggest that IPSE/alpha-1 may have additional nuclear functions in host cells.

This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.

Advances in biotechnology have now created a capacity to produce therapeutically active proteins on a commercial scale, opening the potential for their application in an array of disease conditions. The process of translation of the variety of different therapeutic proteins into the medicines used in clinics is now occurring. To assist in this translation, new formulations to deliver proteins could play an important role. These new formulations need to more adequately address the pharmacological and therapeutic requirement for each particular protein/peptide and, in that way, either improve present therapies or extend with new entries the current list of protein based medicines used in clinic.

Nanomedicine, the most promising approach for regulated and targeted drug delivery, is frequently applied in cancer treatment. Essentially, accumulating evidence indicates that nanomedicine has positive results in the treatment of breast cancer (BC), with many BC patients benefiting from nanomedicine-related treatments. Currently, nanodrug delivery systems based on stimulus responses are gaining popularity because of their additional ability to manage drug release depending on the interior environment of the cancer. This review includes a synopsis of several types of internal (pH, redox, enzyme, reactive oxygen species, and hypoxia) stimuli-responsive nanoparticle drug delivery systems as well as perspectives for forthcoming times. Stimulus-responsive nanoparticles can remain stable under physiological conditions while being rapidly activated to release drugs in response to specific stimuli, prolonging blood circulation and increasing cancer cellular uptake, resulting in excellent therapeutic performance and improved biosafety. In this paper, we discuss tumor microenvironment responsive Nanoformulation for breast cancer treatment. [ABSTRACT FROM AUTHOR]

The objective of this study was to compare the properties of core–shell nanoparticles with a PLGA core and shells composed of different types of polymers, focusing on their structural integrity. The core PLGA nanoparticles were prepared either through a high-pressure homogenization–solvent evaporation technique or nanoprecipitation, using poloxamer 188 (P188), a copolymer of divinyl ether with maleic anhydride (DIVEMA), and human serum albumin (HSA) as the shell-forming polymers. The shells were formed through adsorption, interfacial embedding, or conjugation. For dual fluorescent labeling, the core- and shell-forming polymers were conjugated with Cyanine5, Cyanine3, and rhodamine B. The nanoparticles had negative zeta potentials and sizes ranging from 100 to 250 nm (measured using DLS) depending on the shell structure and preparation technique. The core–shell structure was confirmed using TEM and fluorescence spectroscopy, with the appearance of FRET phenomena due to the donor–acceptor properties of the labels. All of the shells enhanced the cellular uptake of the nanoparticles in Gl261 murine glioma cells. The integrity of the core–shell structures upon their incubation with the cells was evidenced by intracellular colocalization of the fluorescent labels according to the Manders' colocalization coefficients. This comprehensive approach may be useful for the selection of the optimal preparation method even at the early stages of the core–shell nanoparticle development. [ABSTRACT FROM AUTHOR]

Microbial fuel cells (MFCs) are a candidate for green energy sources due to microbes' ability to generate charge in their metabolic processes. The main problem in MFCs is slow charge transfer between microorganisms and electrodes. Several methods to improve charge transfer have been used until now: modification of microorganisms by conductive polymers, use of lipophilic mediators, and conductive nanomaterials. We created an MFC with a graphite anode, covering it with 9,10-phenatrenequinone and polypyrrole-modified Saccharomyces cerevisiae with and without 10 nm sphere gold nanoparticles. The MFC was evaluated using cyclic voltammetry and power density measurements. The peak current from cyclic voltammetry measurements increased from 3.76 mA/cm2 to 5.01 mA/cm2 with bare and polypyrrole-modified yeast, respectively. The MFC with polypyrrole- and nanoparticle-modified yeast reached a maximum power density of 150 mW/m2 in PBS with 20 mM Fe(III) and 20 mM glucose, using a load of 10 kΩ. The same MFC with the same load in wastewater reached 179.2 mW/m2. These results suggest that this MFC configuration can be used to improve charge transfer. [ABSTRACT FROM AUTHOR]

Both commercially pure titanium and titanium alloys are established biomaterials for implantation in bone and are widely used today in dentistry. Titanium particulates have been shown in some patient clusters to induce cellular immune mediators responsible for type I and IV hypersensitivity reactions, causing amplified corrosion, osteolysis, and increased odds of implant failure. Systemically, titanium particles were found to affect varying organ tissues and cause potentially harmful effects. In vivo and vitro studies have shown that titanium dental implant corrosion can be induced by factors relating to bio-tribocorrosion. In this literature review, the consequences of titanium implant corrosion and particulate dissemination are discussed and later juxtaposed against a promising novel implant material, zirconia. Zirconia offers characteristics similar to titanium along with additional advantages such as being non-corrosive and having a lower propensity for inducing immune responses. From the mounting evidence discussed in this article, metal allergy testing would be advantageous for choosing an appropriate implant material to minimize potential adverse effects on cellular functions of local and diffuse tissues. Objective: This literature review aims to elucidate and describe mechanisms in which titanium implants may become corroded and induce cellular aberrations both locally and systemically in vivo. Implications of this study provide supportive evidence regarding the selection of appropriate biomaterials for implant patients susceptible to mounting a hypersensitivity reaction to titanium. [ABSTRACT FROM AUTHOR]

Asenapine maleate (ASPM) is a second-generation atypical antipsychotic that is approved for treating acute schizophrenia and bipolar disorder in adults by the US FDA. The major downside of ASPM therapy is rapid, extensive first-pass hepatic metabolism following its oral administration with a very low oral bioavailability of < 2%. In this work, we developed ASPM nanoformulations conjugated with ligands such as arginine-glycine-aspartic acid (RGD) and peptide dendrimers (PDs) with the intention of improving the oral bioavailability of the drug by targeting it to the intestinal lymphatic system (ILS). Peptide dendrimers (PDs), both lipidated and nonlipidated, were synthesized by Fmoc solid phase peptide synthesis (SPPS). Reverse phase high performance chromatography (RP-HPLC) was used to purify the synthesized PDs, and the PDs were characterized by differential scanning calorimetry (DSC) electrospray ionization mass spectroscopy (ESI+-MS), Nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy. The thin film hydration method was used to prepare liposomes, and the process variables affecting the liposome parameters were optimized using the Box‒Behnken design (BBD).Liposomes were PEGylated using DSPE-PEG-COOH2000 and further conjugated with ligands (RGD, PD-1 and PD-2) using EDC-NHS chemistry. The formulation was characterized using different spectroscopic techniques. In vitro, cell line studies, such as cytotoxicity, cell uptake, uptake mechanism, and receptor saturation studies, were performed on both Caco2 and Raji-B cells. The pharmacokinetic parameters of the developed liposomal formulation were evaluated using pharmacokinetic studies on Sprague- Dawley (SD) rats. The psychostimulant-induced hyperactivity model was used to evaluate the pharmacodynamic performance of the developed formulations by measuring the reversal of hyperlocomotor activity induced by levodopa-carbidopa. [ABSTRACT FROM AUTHOR]

Erythropoietin-producing hepatocellular A2 (EphA2) is a member of the Eph tyrosine kinase receptor family that has been linked to various biological processes. In tumors, EphA2 overexpression is associated with noncanonical pathway activation, tumor progression, and a poor prognosis, which has emphasized its importance as a marker of malignancy. Studies on numerous cancer models have highlighted EphA2's dual and often contradictory action, which can be attributed to EphA2′s interactions involving multiple pathways and different ligands, as well as the heterogeneity of the tumor microenvironment. In this review, we summarize the main mechanisms underlying EphA2 dysregulation in cancer, highlighting its molecular complexity. Then, we analyze therapies that have been developed over time to counteract its action. We discuss the limitations of the described approaches, emphasizing the fact that the goal of new options is high specificity without losing therapeutic efficacy. For this reason, immunotherapy or the emerging field of targeted protein degradation with proteolysis-targeting chimeras (PROTACs) may represent a promising solution that can be developed based on a deeper understanding of the molecular mechanisms sustaining EphA2 oncogenic activity. [ABSTRACT FROM AUTHOR]

Chitosan (CS) derivatives have been extensively investigated to enhance the physicochemical and biological properties of CS, such as its solubility, biocompatibility, and bioactivity, which are required in various areas of pharmacy and medicine. The present work emphasizes the ongoing research and development in this field, suggesting that the further exploration of CS derivatives could lead to innovative solutions that benefit society. The physicochemical properties, biological activities, methods of preparation, advantages, limitations, intended application areas, and realized practical implementations of particular CS derivatives are summarized and discussed herein. Despite the numerous promising attributes of CS derivatives as reported in this paper, however, challenges like target selectivity, standardization (purity, chitosan structural variability), and cost-effectiveness still need addressing for widespread implementation, especially in drug delivery. Therefore, basic research studies still prevail in CS drug delivery systems. However, for specific applications such as wound healing and tissue engineering, implementations of CS derivatives in practice are found to be more frequent. To obtain a more complex view of the topic, information from the scientific papers reviewed is supplemented with information from actual patents and clinical studies. Both basic research advances and the most successful and important medical implementations of CS derivatives are discussed concerning further challenges and future perspectives. [ABSTRACT FROM AUTHOR]

To overcome chondrosarcoma's (CHS) high chemo- and radioresistance, we used polyethylene glycol-encapsulated iron oxide nanoparticles (IONPs) for the controlled delivery of the chemotherapeutic doxorubicin (IONPDOX) to amplify the cytotoxicity of proton radiation therapy. Human 2D CHS SW1353 cells were treated with protons (linear energy transfer (LET): 1.6 and 12.6 keV/µm) with and without IONPDOX. Cell survival was assayed using a clonogenic test, and genotoxicity was tested through the formation of micronuclei (MN) and γH2AX foci, respectively. Morphology together with spectral fingerprints of nuclei were measured using enhanced dark-field microscopy (EDFM) assembled with a hyperspectral imaging (HI) module and an axial scanning fluorescence module, as well as scanning electron microscopy (SEM) coupled with energy-dispersive X-Ray spectroscopy (EDX). Cell survival was also determined in 3D SW3153 spheroids following treatment with low-LET protons with/without the IONPDOX compound. IONPDOX increased radiosensitivity following proton irradiation at both LETs in correlation with DNA damage expressed as MN or γH2AX. The IONPDOX–low-LET proton combination caused a more lethal effect compared to IONPDOX–high-LET protons. CHS cell biological alterations were reflected by the modifications in the hyperspectral images and spectral profiles, emphasizing new possible spectroscopic markers of cancer therapy effects. Our findings show that the proposed treatment combination has the potential to improve the management of CHS. [ABSTRACT FROM AUTHOR]

Fucose, fucose-containing oligosaccharides, and fucose-containing polysaccharides have been widely applied in the fields of food and medicine, including applications in Helicobacter pylori eradication and renal function protection. Fucose-containing carbohydrates (FCCs) derived from marine organisms such as seaweed, invertebrates, microalgae, fungi, and bacteria have garnered growing attention due to their diverse bioactivities and potential therapeutic applications. Marine-derived FCCs characterized by high fucose residue content and extensive sulfate substitution, including fucoidan, fucosylated chondroitin sulfate, and fucose-rich microbial exopolysaccharides, have demonstrated significant potential in promoting gastrointestinal health. This review describes the unique structural features of FCCs and summarizes their health benefits, including regulation of gut microbiota, modulation of microbial metabolism, anti-adhesion activities against H. pylori and gut pathogens, protection against inflammatory injuries, and anti-tumor activities. Additionally, this review discusses the structural characteristics that influence the functional properties and the limitations related to the activity research and preparation processes of FCCs, providing a balanced perspective on the application potential and challenges of FCCs with specific structures for the regulation of gastrointestinal health and diseases. [ABSTRACT FROM AUTHOR]

Imipramine hydrochloride (IMP), a tricyclic antidepressant used for major depression, enuresis, and neuropathic pain, is limited by gastrointestinal complications, low oral bioavailability (44%), and complex dosing requirements. This study aimed to explore a novel non-invasive nasal delivery system using chitosan nanoparticles (Cs NPs) embedded in an in situ gel to address the limitations of oral IMP administration. Cs NPs loaded with IMP were synthesized via ionic gelation and assessed for precision in drug concentration using a validated HPLC method. The particles were integrated into a thermoresponsive polymer, Pluronic F127, to form an in situ gel suitable for nasal administration. The formulation was characterized for gelation temperature, duration, viscosity, mucoadhesive strength, and overall gel robustness. Drug release kinetics and the controlled release mechanism were studied using ex vivo permeation tests with Franz diffusion cells and nasal sheep mucosa. The optimized nanoparticle formulation (F4-50) exhibited a consistent PS of 141.7 ± 2.2 nm, a zeta potential (ZP) of 16.79 ± 2.1 mV, and a high encapsulation efficiency of 67.71 ± 1.9%. The selected in situ gel formulation, F4-50-P1, demonstrated a gelation temperature of 33.6 ± 0.94 °C and a rapid gelation time of 48.1 ± 0.7 s. Transform-attenuated total reflectance infrared spectroscopy (ATR-IR) confirmed the compatibility and effective encapsulation of IMP within the formulation. The release profile of F4-50 included an initial burst release followed by a sustained release phase, with F4-50-P1 showing improved control over the burst release. The flux rates were 0.50 ± 0.01 mg/cm2/h for F4-50 and 0.33 ± 0.06 mg/cm2/h for F4-50-P1, indicating effective permeation. The developed chitosan nanoparticle-based in situ gel formulation provides a promising approach for the controlled release of IMP, enhancing therapeutic efficacy and patient compliance while mitigating the disadvantages associated with oral delivery. [ABSTRACT FROM AUTHOR]