Pharmaceutics

Background: Gadolinium-based contrast agents (GBCAs) are routinely used in magnetic resonance imaging (MRI). Although macrocyclic GBCAs were initially considered biologically inert, it is now known that a fraction of patients retains gadolinium (Gd) for prolonged periods in tissues such as blood, bone, and brain. Because the first cellular interactions of GBCAs occur in the bloodstream, this study aimed to elucidate the uptake mechanism but also the intracellular persistence and release dynamics of gadoterate meglumine, one of the most widely used macrocyclic agents, in white blood cells (WBCs). Methodology and principal findings: WBCs and K562 cells were incubated with gadoterate meglumine under different conditions to investigate its cellular entry mechanisms. Uptake of the contrast agent was quantified by measuring intracellular Gd using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Time and concentration-dependent incubation of K562 cells revealed saturable uptake kinetics consistent with a Michaelis-Menten model which is independent of the phase of the cell cycle. Gadoterate meglumine uptake in both WBCs and K562 cells was shown to be an active process, as uptake was strongly reduced or abolished at low temperature (16C and 4C) and in the presence of metabolic inhibitors (sodium azide and 2-deoxyglucose). Co-incubation with multiple endocytosis inhibitors (Dyngo 4a, Dynole 2-24 and chlorpromazine) did not significantly decrease intracellular Gd levels in K562 cells and caused only a slight reduction in WBCs, indicating that endocytosis is not the main entry pathway for gadoterate meglumine in these cells. Furthermore, we assessed the retention time of the Gd inside the cells, showing that only after 24 hours post incubation 80% percent of the intracellular Gd was released through an active process. Finally, we demonstrate that one of the mechanisms of Gd release from WBCs involves extracellular vesicles, which may substantially increase its potential for downstream accumulation in different tissues, including immunoprivileged tissues like brain. Significance: The observed time-dependent accumulation, temperature and energy dependence of gadoterate meglumine uptake demonstrate that active cellular mechanisms are primarily responsible for GBCA internalization. Furthermore, our results indicate that macropinocytosis, phagocytosis, and clathrin-mediated endocytosis are not the primary routes of gadoterate meglumine entry. Hereby, we also describe that Gd externalization is an active process involving extracellular vesicles which may influence the Gd distribution in different tissues and its consequent long-term retention. Further studies are required to explore strategies to block this process in order to mitigate potential long-term gadolinium retention.

This work describes a new approach for rapid and reproducible formulation of drug loaded biodegradable nanoparticles based on polyester copolymers, including poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) and poly(caprolactone)-poly(ethylene glycol) (PCL-PEG). The new approach, termed Solvent-free Nanoparticle Assembly Protocol (SNAP), carries several advantages over conventional polyester formulation strategies, including very rapid formulation (minutes) and the ability to use nanoparticles immediately without lengthy solvent evaporation or washing steps. Altering polyester molecular weight and concentration, alongside the introduction of specific functional groups yielded precise control of nanoparticle properties, including size, shape, surface charge, drug release and loading. We examined loading of multiple therapeutic compounds, including diclofenac, loperamide, bortezomib, CT179, panobinostat, docetaxel, methotrexate, and camptothecin. The SNAP protocol facilitated the rapid production of stable, drug-loaded nanoparticles with a narrow size distribution and generally good drug loading. Using Fluorescence Resonance Energy Transfer (FRET) and size exclusion chromatography (SEC) with a focus on the model agent Rhodamine B, we were able to carefully examine stability of the nanoparticle and assess the distribution of small molecules within the polymer as well as nanoparticle stability. In vivo evaluation of fluorescently labeled nanoparticles using real-time, intravital microscopy showed that, after direct administration to cerebrospinal fluid (CSF) via the intrathecal cisterna magna (IT-CM) route, the dynamic accumulation of nanoparticles within the perivascular space (PVS) depends on the size of the vessel that is imaged. Nanoparticles accumulated steadily within the PVS of large vessels, while accumulating more slowly and exhibiting clearance from medium-sized and smaller vessels over the course of several hours. In sum, these studies present a new platform for facile production of polyester nanoparticles, demonstrate their ability to encapsulate a variety of hydrophobic small molecules, and expand our knowledge on the development of nanocarriers for intrathecal administration. Taken together, these data open new opportunities for development safer and more effective nanoparticle-based therapies.

Vodobatinib (VBN) is a weakly basic (pKa {approx} 2.3), anticancer treatment with poor enteric solubility and low oral bioavailability. This study demonstrates how an emerging polymeric amorphization technique, slurry conversion, can yield amorphous drug-polymer salts with enhanced dissolution rates. The technique had not previously been applied to a weakly basic drug, so design rules for this class of active were unknown. Two acidic polymers, poly(styrene sulfonic acid) (PSSA) and poly(acrylic acid) (PAA), were individually evaluated for salt formation with VBN. Formulation involved blending the drug and polymer in a 1:2 (v/v) ratio of a protic liquid to solvent and a 1:9 (w/w) ratio of solid to solvent. Design rules for effective combinations of solvents and protic liquids were developed and optimized to thread the needle between dissolution of all species and acid-base interactions, both of which were required to form amorphous salts. Drug loadings of 10%, 20%, and 40% by mass were tested. X-ray photoelectron spectroscopy was employed to evaluate protonation of the quinoline nitrogen atoms on VBN, a key indicator of successful salt formation. Powder X-ray diffraction was used to confirm that the resulting slurry contained amorphous VBN, and 1H NMR spectroscopy indicated residual solvent remained after drying, which remains an area for improvement. In dissolution kinetics tests in FeSSIF, the lead drug-polymer salt formulation achieved a concentration of dissolved VBN up to 140 {micro}g/mL, an improvement of >35-fold compared to <4 {micro}g/mL (LLD) for crystalline VBN. These results demonstrate that slurry conversion is a viable polymeric amorphization technique even for weakly basic drugs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/734800v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1812ceforg.highwire.dtl.DTLVardef@1ad06dcorg.highwire.dtl.DTLVardef@9d8bb7org.highwire.dtl.DTLVardef@13fcbe8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extrahepatic delivery of lipid nanoparticles (LNPs) to non-phagocytic cells is a major challenge, with the leading strategy involving surface functionalization with target-specific monoclonal antibody (mAb) ligands. We investigate the stability of mAb-conjugated LNPs using two anchoring systems: the commonly used DSPE-PEG2kDa-maleimide and a block copolymer, PCL5kDa-b-PEG2kDa -maleimide, with the hypothesis that conjugation to a 150,000 Da antibody could overwhelm the relatively small ~600 Da aliphatic anchor on the PEG-lipid in vivo. Shedding of the mAB would compromise targeting. Conjugation integrity following IV injection was assessed by tagging LNPs and mAbs with metal ion tracers that could be quantified by ICP-MS. Results show that DSPE-PEG-mAb rapidly (within 1h) dissociates from LNPs in blood, leading to accelerated LNP clearance. In contrast, mAbs conjugated using PCL-b-PEG remained stably associated with the LNP over the 24h circulation and clearance of the construct. Results are connected to a thermodynamic model that reproduces experimental findings for PEG-anchor(-mAb) shedding in vitro and in vivo. This study identifies anchoring strength as a critical, unconsidered parameter for in vivo performance when conjugating mAbs to LNPs for extrahepatic delivery.

Background: Natural essential oils exhibit antimicrobial and wound-healing properties, but their therapeutic application is limited by poor water solubility, volatility, and instability. This study developed and characterized a nanoemulsion of Ocimum gratissimum essential oil (OGNe) and evaluated its physicochemical properties, dermal safety, antibacterial activity, and wound-healing potential. Methods: Essential oil was obtained by hydrodistillation and formulated into nanoemulsions by high-speed stirring emulsification. Physicochemical properties, including pH, droplet size, polydispersity index, and storage stability, were determined. Acute dermal toxicity was assessed in Wistar rats following OECD Test Guideline 402. Antibacterial activity was evaluated using broth microdilution, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays. Wound-healing efficacy was investigated using an excision wound model over 21 days using distilled water and trolamine serving as controls. Results: OGNe exhibited a stable milky appearance, near-neutral pH, and droplet sizes ranging from 26 to 224 nm. No signs of dermal toxicity or behavioral abnormalities were observed after topical administration. The nanoemulsion showed selective antibacterial activity, with the highest susceptibility against Acinetobacter baumannii (MIC = 1.125 L/mL), whereas Escherichia coli remained resistant. Time-kill assays demonstrated concentration-dependent bacteriostatic activity. In vivo, OGNe significantly accelerated wound contraction from day 3 onward (p < 0.0001), achieving healing rates comparable to or exceeding those of trolamine during the inflammatory and proliferative phases. Conclusion: Ocimum gratissimum nanoemulsions represent stable, biocompatible topical formulations that combine selective antibacterial activity with enhanced wound healing, supporting their potential as phytopharmaceutical nanoformulations for the management of acute skin wounds.

IntroductionIn this study, we developed a modular in vitro platform that integrates advanced polymer-drug conjugation chemistry with stepwise cytotoxicity screening in both 2D (monolayer) and 3D (spheroids) glioblastoma (GBM) models. Buparlisib was selected as the model therapeutic due to its well-characterised mechanism of action, high blood-brain barrier permeability, and relevance to PI3K-targeted therapy. MethodsTwo mechanistically distinct conjugation strategies were explored using N-(2-hydroxypropyl)methacrylamide-based copolymers. The first strategy was based on a redox-sensitive disulphide linkage designed for intracellular glutathione-triggered release, whereas the second used an azide-bearing derivative compatible with strain-promoted azide-alkyne cycloaddition. Drug release was assessed by high-performance liquid chromatography. Biological activity was systematically evaluated in U87MG, U118MG, and T98G cells under 2D conditions using a resazurin-based metabolic activity assay. Subsequently, the more promising disulphide-based formulations were assessed in 3D spheroids by metabolic activity measurements and live-cell monitoring of spheroid growth dynamics. ResultsFree Buparlisib showed the strongest inhibitory effect, while its modification and polymer conjugation reduced the apparent activity. Nevertheless, the disulphide-based derivative and polymer conjugate retained concentration-dependent activity, whereas the azide-based polymer conjugate showed minimal effects. Moreover, treatment responses differed between cell lines and between 2D and 3D models. DiscussionOverall, linker chemistry, cell-line-specific behaviour, and model dimensionality strongly influenced the biological performance of the polymeric Buparlisib formulations. The redox-sensitive polymer conjugate therefore represents the more promising strategy for further development.

PurposeExtracellular vesicles (EVs) are increasingly explored as natural vehicles for drug delivery and gene therapy approaches. However, reproducible yield and scalability of EV production still pose major challenges in the clinical translation of EV-based therapies. In this study, we sought to quantify and characterize EVs released by suspension-cultured HEK293 cells (Expi293F cells) grown in shaker flasks or small-scale bioreactors, to investigate how the culturing environment affects EV production yield. MethodsExpi293F cells were cultivated (N=3) in either shaker flasks or a bioreactor system, and total cell density, viability, and size were monitored. Supernatants were drawn daily post-cell seeding and were analyzed for EV quantity, size, morphology, and CD63 expression. ResultsNo significant differences were observed in terms of total cell density, viability, and cell size between both cultivation settings. However, cultivation of Expi293F cells in the bioreactor environment significantly increased EV yield by 3-fold compared to shaker flask cultivation (p < 0.01). Other parameters such as average nanoparticle size, EV morphology, and CD63 expression remained comparable between both cultivation methods. ConclusionThese results demonstrate that Expi293F-derived EV yield can be increased by culturing cells in a scalable bioreactor system. These findings pave the way towards the production of therapeutic-based EVs in a scalable and reproducible manner suitable for future (pre-)clinical applications.

BackgroundReliable tracking of extracellular vesicles (EVs), key biological nanocarriers in nanomedicine, remains a major technical challenge due to the limitations of conventional lipophilic dyes, including aggregation, micelle formation, and nonspecific background signals that compromise biodistribution analyses. MethodsHere, we present a fluorogenic labeling strategy based on Aco-600, a water-soluble probe exhibiting a "light-on" activation in hydrophobic environments. Medium/large EVs (m/lEVs) derived from murine BV2 microglial cells were labeled and intranasally administered to adult C57BL/6 mice. EV biodistribution and brain uptake were quantitatively assessed by ex vivo fluorescence imaging on brain cryosections at multiple time points (5-1440 min), focusing on the cortex and hippocampus. ResultsAco-600 labeling enabled high signal-to-noise detection with minimal background and no evidence of dye aggregation artifacts. Quantitative analysis revealed a consistent spatiotemporal distribution profile across brain regions, with peak signal intensity at 60 minutes post-administration, followed by progressive clearance. This approach provided reproducible and sensitive tracking of EV biodistribution following a clinically relevant intranasal delivery route. ConclusionsOur findings establish fluorogenic labeling as a robust and artifact-minimizing strategy for in vivo EV tracking. This method enhances the accuracy of biodistribution studies and supports the development of EV-based nanomedicine platforms, particularly for central nervous system delivery applications.

Leptomeningeal disease (LD), involving the metastasis of cells to the leptomeningeal membranes in the central nervous system (CNS), can be a deadly complication of several different types of cancer originating in the periphery or CNS, including breast cancer (BC) and pediatric medulloblastoma (MB). Targeted therapy represents a promising new approach to improve overall survival for LD patients. To this date, angiopep-2 (Ang2) and transactivating transcriptional activator (TAT), two well-known peptides for their brain delivery capability, have been reported to transport therapeutic cargos into the CNS for treatment of disease. Current administration strategies, however, still rely on oral delivery or intravenous injection (IV), where the substances need to travel through complex biological barriers to reach the subarachnoid space (SAS), which is the primary location of LD. Our research group has focused on the intrathecal (IT) route of administration as an alternative approach that can potentially enable high exposure of drug to CSF exposed tissues. However, there is a major field gap in understanding how targeting peptides can access (or not access) LD as a function of their route of administration. Therefore, our work was focused on comparing the targeting capability of Ang2 vs TAT by IT vs IV routes of administration. We first generated two xenograft models of LD by directly infusing breast cancer cells (MDA-MB231) or medulloblastoma cells (HDMB03) into the SAS via intracisternal magna injection (ICM) to form BC-LD and MB-LD models, respectively. These tumor models were characterized for overall survival, tumor growth patterns, and presence of hydrocephalus. Second, we further administered fluorescently labeled Ang2 or TAT peptides either IV or ICM into tumor bearing mice. Neuraxial fluorescence images were examined to evaluate the targeting ability of these two peptides based on colocalization between peptide signal and tumor tissues ex vivo. We discovered that the median survival of both models was negatively related to the number of the cells infused. While HDMB03 cells tended to metastasize preferentially to the brain region, MDA-MB231 cells tended to metastasize preferentially to the spinal cord. Both models present hydrocephalus as one of the common clinical symptoms in LD patients. Compared to the healthy control, MB-LD yielded a 7.3-fold increase and BC-LD a 26.5-fold increase in ventricular volume. Furthermore, targeting achieved by TAT was significantly higher than targeting achieved by Ang2 in thoracic spine for the MB-LD model. For BC-LD model, TAT signal was found to be significantly higher than Ang2 signal in the olfactory bulbs, brain stem, thoracic spine, and lumbar spine regions. While both peptides showed a strong signal at 2 hours post ICM injection, signal was not detectable 24 hours after administration, reflecting washout or degradation. Significantly, these data provide evidence that ICM will be a preferable route of administration over IV for the purpose of maximally targeting LD.

Intrathecal (IT) drug delivery, i.e., the infusion of substances directly into cerebrospinal fluid (CSF) by lumbar, ventricular, or cisternal access points, is one method that can be used to bypass the blood brain barrier (BBB), however, IT-administered substances also suffer from rapid turnover and poor tissue penetration. Although nanoparticles and colloids can circulate within the subarachnoid space to sustain the levels of encapsulated drug in CSF, their access to deep tissue regions remains incomplete. Here, we present a new method for enhancing CNS delivery of IT-administered nanoparticles. CSF Flow Enhancement (CFE) refers to the manipulation of CSF production, distribution, and clearance for therapeutic purposes. We tested the overarching hypothesis that infusion of hypertonic fluid adjacent to the choroid plexus would enhance fluid production and movement to improve the CNS delivery of IT-administered nanoparticles. Model polystyrene nanoparticles (100nm) were solubilized in aCSF of increasing tonicity (1-9X tonicity) and infused into the cisterna magna, after which tissues were removed to examine delivery to CNS tissues and peripheral organs. Our results demonstrate that an infusion of up to 4X hypertonic aCSF in 10uL is well tolerated and yields significant improvements in CNS localization of co-administered nanoparticles, more than doubling the delivery of nanoparticles to the ventral surfaces of the brain and sometimes dramatic (up to 10-fold) increases in delivery to specific tissue regions and surfaces of the CNS. Significantly, we provide early evidence that modulation of tonicity can define the parenchymal fate of IT administered colloids: while nanoparticles were not detected in the brain parenchyma of mice that received a standard infusion, parenchymal delivery was observed for the 2X condition, and extensive perivascular infiltration of nanoparticles was observed for the 4X condition. Lastly, we show that the delivery improvements achieved by CFE are generalizable across multiple sizes of polystyrene nanoparticle (20, 40, or 100nm). Collectively, this work describes a tonicity-based approach for achieving CFE by the intrathecal route, which we posit is a useful and potentially generalizable approach for improving CNS drug delivery.

Retinal and choroidal vascular diseases are major causes of vision loss that require frequent intravitreal anti-VEGF therapy. Anti-angiogenic peptide AXT107 demonstrated efficacy in preclinical studies and was advanced to the clinical stage. To provide for sustained delivery of the peptide and avoid complications with intravitreal injection, we evaluated suprachoroidal delivery of AXT107 microparticles (MP-AXT107). The original, soluble AXT107 formulation was ineffective at inhibiting laser-induced choroidal neovascularization (CNV) in our rat model and was consequently reformulated as microparticles. MP-AXT107 demonstrated high peptide incorporation efficiency, reproducible morphology, and physical and chemical stability for at least 9 months under refrigerated storage. In the rat CNV model, suprachoroidal MP-AXT107 significantly reduced neovascular area by approximately 60% relative to vehicle controls. Safety and durability were evaluated in a 9-month GLP toxicology study in Gottingen minipigs following a single suprachoroidal injection of vehicle or MP-AXT107 (0.125-1.25 mg/eye). Transient increases in IOP and mild ocular inflammatory findings were observed immediately following administration but resolved rapidly without lasting effects. No treatment-related adverse ocular findings were observed during the remainder of the study, and the highest tested dose (1.25 mg/eye) was established as the no-observed-adverse-effect level. Bioanalysis at study completion demonstrated persistent AXT107 localization primarily within choroid/RPE and scleral tissues, with no signs of systemic exposure. Collectively, these findings demonstrate that suprachoroidal delivery of MP-AXT107 enables sustained anti-angiogenic activity with favorable ocular safety and prolonged tissue retention, supporting further clinical development as a durable therapy for retinal and choroidal vascular diseases.

Animal cell-cultured ingredients are entering the EU and UK pet food markets under frameworks that do not require pre-market, ingredient-level safety assessments, creating an ethical need for transparent safety disclosure. We present the first public safety dossier for this sector, describing the proprietary mouse embryonic stem cell line PE25 and its derived, non-viable cellular and conditioned media ingredient produced in food and feed-grade media. PE25 characterization confirmed Mus musculus identity, sterility, absence of mycoplasma and replication-competent retroviruses, and stable growth. Doxorubicin-induced p53 stress testing, CD44/BMI1 profiling, and soft agar assays showed no cancer-like traits and a non-tumorigenic profile; the final ingredient contains no viable cells. Independent OECD TG 471 and 487 assays confirmed non-genotoxicity. Heavy metals, biogenic amines, solvents, and chemical residues were below regulatory limits. Given process variability, we recommend case-by-case safety evaluation and propose this dossier as a model for responsible commercialization.

Efficient delivery of molecular therapies to the central nervous system (CNS) remains a major barrier to treating neurogenetic disorders such as Niemann Pick type C (NPC) disease. Focused ultrasound-mediated blood-brain barrier opening (FUS-BBBO) has emerged as a non-invasive strategy to enhance delivery of systemically administered therapeutics. In this study, we evaluated whether FUS-BBBO could enable delivery of lipid nanoparticle (LNP)-packaged modified mRNA (modRNA) to the cerebellum in an NPC mouse model. A pilot study in wild-type mice demonstrated successful FUS-mediated BBB opening, delivery of LNP-packaged GFP mRNA, and subsequent protein expression in the cerebellum. We then performed a controlled study in NPC mice comparing delivery of LNP-GFP and LNP-NPC modRNA using intravenous administration with and without FUS-BBBO. BBB opening was confirmed by contrast-enhanced MRI in FUS-treated animals. Quantitative PCR revealed the presence of GFP mRNA in the cerebellum following FUS-BBBO, whereas NPC mRNA was minimal or undetectable across groups. However, no GFP or NPC1 protein expression was detected in the cerebellum by western blot in any experimental group. Consistent with this, no therapeutic effect on Purkinje cell survival was observed. These results demonstrate that while FUS-BBBO reliably induces BBB opening and can facilitate limited delivery of LNP-packaged mRNA to the brain, this did not translate into detectable protein expression or therapeutic benefit in the NPC model under the conditions tested. This discrepancy between successful delivery in wild-type mice and lack of efficacy in diseased animals points to potential important biological and/or formulation-dependent barriers that must be addressed to enable effective CNS delivery of LNP-based mRNA therapies.

The Next Generation Impactor (NGI) is one of the regulatory gold standards for characterizing aerodynamic particle size distributions (APSDs) of orally inhaled drug products (OIDPs); however, its reliance on complex, resource-intensive in vitro testing under tightly controlled environmental conditions limits experimental flexibility and introduces variability. In alignment with the growing regulatory emphasis on New Approach Methodologies (NAMs) for drug development, this study presents a rigorously validated computational fluid particle dynamics (CFPD) based virtual NGI (vNGI) as an in silico method complementary to conventional testing. The vNGI replicates a significant portion of the NGI geometry and airflow physics, enabling high-resolution spatiotemporal analysis of aerosol transport and deposition mechanisms that are otherwise inaccessible experimentally. A comprehensive verification and validation framework was implemented, including mesh and particle independence studies, turbulence model assessment, and comparison of stagewise deposition efficiencies with available in vitro data at 30 L/min. The model's capabilities were further extended to low and high flow rates, and two bio-relevant mouth-throat models and polydisperse particle laden aerosol were added. The model demonstrates strong predictive capability for a few stages and provides mechanistic insight into discrepancies in other stages, depending on the type of analysis. Importantly, this work establishes the vNGI as a fit-for-purpose according to NAM by (i) defining a clear context of use for APSD prediction and inhaler performance evaluation, (ii) capturing physically and biologically relevant air-particle interactions, and (iii) demonstrating technical robustness and reproducibility through systematic validation. The platform can potentially further enable simulation of environmental and physiological conditions, such as humidity effects, that are difficult to control experimentally, thereby improving human relevance and reducing reliance on costly and time-consuming in vitro testing. This study positions the vNGI as a scalable, regulatory aligned NAM capable of supporting early stage drug device combination product development, device optimization, and an alternative bioequivalence assessment, contributing to ongoing efforts to enhance predictive performance, reduce experimental burden, and transition toward human centric, inhalation product evaluation.

Favipiravir (FPV) is an RdRp inhibitor developed and licensed in Japan for influenza but which has shown promising in-vitro activity against a range of RNA viruses. A physiologically-based pharmacokinetic model was developed for oral FPV and its metabolite M1 in order to optimise the dose regimen against plasma concentration targets for a number of viral pathogens. The model was validated using clinical data and was able to capture the variability in plasma concentrations for a population of individuals. FPV doses predicted to cause in-vivo exposures exceeding in-vitro IC90 targets against influenza, Ebola, Lassa fever, CCHF, SFTS, Andes virus and SARS-CoV-2, lie within the window of observed safe dosing, with SARS-CoV-2 requiring predicted doses of 2400 mg twice daily due to lower in-vitro potency. Simulations showed that a loading dose on day one of treatment should allow plasma targets to be exceeded on day one. Simulations of chronic kidney disease (CKD) showed no change to FPV plasma concentration in individuals with CKD3 and CKD5 compared to healthy individuals. Clinical data suggested active renal efflux of M1 which led to a predicted 2.2 and 11.5 fold increase in the maximum plasma concentrations of M1 in individuals with CKD3 and CKD5 respectively in comparison with healthy individuals.

Background: Chronic and non-healing wounds remain a major clinical challenge with limited therapeutic options. Angiogenesis and inflammation are central to tissue repair, and mesenchymal stem cells (MSC) contribute to these processes through their trophic and immunomodulatory secretome. Cannabidiol (CBD) exhibits antioxidant and immunomodulatory properties. However, whether CBD-rich Cannabis sativa extract stimulate MSC toward a pro-angiogenic secretome remains unclear. Purpose: This study aims to determine whether purified CBD or a phytochemically CBD-rich full spectrum extract stimulate umbilical cord-derived human MSC (UC-hMSC) to secrete pro-angiogenic factors and enhance endothelial responses relevant to wound healing. Methods: UC-hMSC were preconditioned with either purified CBD or a CBD-rich full-spectrum extract. Transcriptional changes were assessed by qPCR. The functional impact of the resulting secretome was evaluated in vitro using HUVEC-based proliferation and tube formation assays, and in vivo through the chick chorioallantoic membrane assay. To explore underlying mechanisms, we examined HIF-1 stabilization and VEGFA release in UC-hMSC, and VEGFR-2/ERK signaling in HUVEC. Results: Purified CBD and full-spectrum CBD extract preconditioned UC-hMSC secretomes, increased HUVEC proliferation, tube formation, and enhanced vascular branching in the CAM assay. Mechanistic analyses indicated activation of the HIF-1/VEGF axis in UC-hMSC, and ERK1/2 activation in HUVEC that was sensitive to VEGFR-2 blockade. Conclusion: Purified CBD and CBD-rich full-spectrum extract prime UC-hMSC toward a pro-angiogenic secretome that promotes endothelial activation and neovascularization. These findings suggest that cannabinoid-based preconditioning of UC-hMSC involves the HIF-1/VEGF axis and VEGFR-2/ERK signaling pathways in endothelial cells, supporting further investigation of this approach in wound healing and regenerative therapies.

Lipid Nanoparticles (LNPs) are widely used as delivery systems for nucleic acid therapeutics, where transfection efficiency is determined by both the identities of constituent lipid components and their composition ratios. While prior studies have focused on learning molecular representations for individual components, modeling how multiple components and their ratios jointly influence LNP performance remains underexplored. In this work, we propose STRATA, a framework that models molecule interaction between LNP components, which is known to contribute to LNP transfection efficiency. Our approach is built on two complementary views: (1) a ratio-centric view that captures interaction patterns induced by composition ratios through a transformer with a Ratio-induced Positional Embedding, and (2) a molecule-centric view that incorporates interaction-induced effects into structure-based molecule embeddings. By jointly training and aligning these views, our model integrates molecular structure and composition ratio within a unified framework that captures interaction-driven effects. Experiments demonstrate that our method improves prediction accuracy and generalization to unseen molecules and ratios, highlighting the effectiveness of our approach. Implementation code will be available after acceptance.

Introduction: Conventional substance-level disproportionality analysis may miss safety patterns specific to a dose form, route, or intended site. More granular analyses are hindered by incomplete, inconsistent reporting of product information. The Pharmaceutical Product Identifier (PhPID), representing products by substance, strength, and dose form, may support more granular analyses. Objective: To explore the use of PhPID-like dose form information for site-specific disproportionality analysis in dexamethasone. Methods: We evaluated VigiBase reports (January 1, 2001 - December 31, 2024) for completeness of dose form and route data. We standardized dexamethasone entries to PhPID Level 3 standards, representing substance and administrable dose form. Through disproportionality analysis (Information Component, IC) we compared substance-level and site-specific results. Results: Among 56.4 million suspected/interacting drugs, dose form was reported in 47.7%, route in 69.4%. Among 109,248 dexamethasone entries, 703 dose form and 80 route variations were mapped to 53 and 44 standard codes respectively; about half could be mapped unambiguously. Site-specific analyses revealed biologically plausible patterns not apparent in substance-level analyses. Ocular use showed higher ICs for glaucoma and cataract, while systemic use showed higher IC for psychiatric and endocrine events (e.g., depression, agitation, Cushing's syndrome). IC time-trends suggested that some signals (e.g., cataract with Ocular use) could emerge earlier in site-specific analyses. Conclusion: More granular product information, aligned with PhPID, may improve signal detection and characterization of site-specific safety issues. These findings support granular identifiers in pharmacovigilance while highlighting the need for better capture and standardization of dose form and route of administration data.

Modulation of immune cells as therapeutic tools has gained significant clinical relevance in the treatment of cancer. Among them, macrophages represent a promising immunotherapeutic platform not only because they can internalize tumor material, but also because they profoundly shape the tumor microenvironment through cytokine production, antigen presentation, metabolic regulation, and modulation of other immune and stromal populations. Lipid Nanoparticles (LNPs) have enabled RNA therapies to the bedside and are thus considered the gold standard for gene delivery. However, optimizing LNPs for RNA delivery to macrophages remains an active area of investigation. Here, we propose the surface modification of unPEGylated LNPs using the Layer-by-Layer (LbL) approach for enhanced RNA delivery to macrophages. Specifically, we show that fucoidan, a sulfated polysaccharide, when at the outermost layer in the LbL process provides two physicochemical advantages to unPEGylated LNPs: (1) stability in PBS and (2) resistance to lyophilization in the presence of cryoprotectant. Additionally, fucoidan improves macrophage targeting and RNA transfection efficiency compared to previously synthesized hyaluronan-decorated LbL LNPs. Fucoidan LbL LNPs (Fuc-LNPs) preferentially accumulated in CD11b+ macrophages when co-cultured with U87 glioblastoma cells, which was not observed for control PEGylated LNPs. Furthermore, Fuc-LNPs induced a higher transfection of mRNA in primary human macrophages when compared to PEGylated control LNPs. Using the model mRNA encoding CAR@CD19, Fuc-LNPs generated CAR macrophages which mediated CD19 cell ablation in vitro. Altogether, these findings highlight the potential of the LbL strategy to modulate the targeting properties of LNPs, improving RNA delivery to human macrophages and encouraging further studies using LbL LNPs for the generation of CAR-Macrophages in the context of solid tumors.

Innate immune agonists are promising therapeutic agents to induce immune responses against cancer. However, these agents have been limited by toxicity associated with systemic accumulation and activity in off-target cells. In this work, a targeted nanoparticle (NP) platform to encapsulate and protect the Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly(I:C)) and promote its specific delivery to antigen presenting cells (APCs), macrophages and dendritic cells, for activation of this cell population was designed. To determine NP physiochemical properties that promote APC delivery, we developed a library of NP surface chemistries formed by electrostatic adsorption of polyanion coatings onto liposomes using layer-by-layer (LbL) assembly and screened the particles on APCs and off-target cells. Dextran sulfate was identified as a promising coating to enhance specific APC delivery. We applied these design parameters to develop a poly(I:C)-loaded NP for an APC-targeted immunotherapy. In a model of metastatic ovarian cancer, the LbL NP prolonged poly(I:C) retention in the peritoneal space--with 2-fold remaining 24-48hr after administration compared to free poly(I:C)--ultimately reducing systemic accumulation and associated toxicities. Compared to free drug, the NP reduced the increase in serum levels of TNF, IL-6, and CXCL10 by 9-, 4-, and 31-fold respectively. NP-treated mice experienced lower weight loss and recovered more quickly at a higher poly(I:C) dose, indicating a widening of the therapeutic window. The NP formulation enhanced accumulation of poly(I:C) in the tumor 2-fold and activation of the target APC population compared to free drug, and ultimately slowed tumor growth and extended survival in combination with doxorubicin chemotherapy. Overall, this work demonstrates a modular NP delivery strategy to improve the delivery, safety, and therapeutic window of a TLR3 agonist.