An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

Blood-Nanoparticle Interactions Create a Brain Delivery Superhighway for Doxorubicin.

Purpose: This study investigated the brain targeting mechanism of doxorubicin-loaded polybutyl cyanoacrylate (PBCA) nanoparticles, particularly their interactions with the blood-brain barrier (BBB). The BBB protects the brain from drugs in the bloodstream and represents a crucial obstacle in the treatment of brain cancer. Methods: An advanced computer model analyzed the brain delivery of two distinct formulations, Doxil® and surfactant-coated PBCA nanoparticles. Computational learning was combined with in vitro release and cell interaction studies to comprehend the underlying brain delivery pathways. Results: Our analysis yielded a surprising discovery regarding the brain delivery mechanism of PBCA nanoparticles. While Doxil® exhibited the expected behavior, accumulating in the brain through extravasation in tumor tissue, PBCA nanoparticles employed a unique and previously uncharacterized mechanism. They underwent cell hitchhiking, resulting in a remarkable more than 1000-fold increase in brain permeation rate compared to Doxil® (2.59 × 10-4 vs 0.32 h-1). Conclusion: The nonspecific binding to blood cells facilitated and intensified interactions of surfactant-coated PBCA nanoparticles with the vascular endothelium, leading to enhanced transcytosis. Consequently, the significant increase in circulation time in the bloodstream, coupled with improved receptor interactions, contributes to this remarkable uptake of doxorubicin into the brain.

Understanding the In Vitro-In Vivo Nexus: Advanced correlation models predict clinical performance of liposomal doxorubicin.

In the century of precision medicine and predictive modeling, addressing quality-related issues in the medical supply chain is critical, with 62 % of the disruptions being attributable to quality challenges. This study centers on the development and safety of liposomal doxorubicin, where animal studies alone often do not adequately explain the complex interplay between critical quality attributes and in vivo performances. Anchored in our aim to elucidate this in vitro-in vivo nexus, we compared TLD-1, a novel liposomal doxorubicin delivery system, against the established formulations Doxil® and Lipodox®. Robust in vitro-in vivo correlations (IVIVCs) with excellent coefficients of determination (R2 > 0.98) were obtained in the presence of serum under dynamic high-shear conditions. They provided the foundation for an advanced characterization and benchmarking strategy. Despite the smaller vesicle size and reduced core crystallinity of TLD-1, its release behavior closely resembled that of Doxil®. Nevertheless, subtle differences between the dosage forms observed in the in vitro setting were reflected in the bioavailabilities observed in vivo. Data from a Phase-I clinical trial facilitated the development of patient-specific IVIVCs using the physiologically-based nanocarrier biopharmaceutics model, enabling a more accurate estimation of doxorubicin exposure. This advancement could impact clinical practice by allowing for more precise dose estimation and aiding in the assessment of the interchangeability of generic liposomal doxorubicin.

Bromocriptine protects perilesional spinal cord neurons from lipotoxicity after spinal cord injury.

Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity, damaging the neurons. However, how lipids are metabolized by spinal cord neurons after spinal cord injury remains unclear. Herein, we investigated lipid metabolism by spinal cord neurons after spinal cord injury and identified lipid-lowering compounds to treat spinal cord injury. We found that lipid droplets accumulated in perilesional spinal cord neurons after spinal cord injury in mice. Lipid droplet accumulation could be induced by myelin debris in HT22 cells. Myelin debris degradation by phospholipase led to massive free fatty acid production, which increased lipid droplet synthesis, ß-oxidation, and oxidative phosphorylation. Excessive oxidative phosphorylation increased reactive oxygen species generation, which led to increased lipid peroxidation and HT22 cell apoptosis. Bromocriptine was identified as a lipid-lowering compound that inhibited phosphorylation of cytosolic phospholipase A2 by reducing the phosphorylation of extracellular signal-regulated kinases 1/2 in the mitogen-activated protein kinase pathway, thereby inhibiting myelin debris degradation by cytosolic phospholipase A2 and alleviating lipid droplet accumulation in myelin debris-treated HT22 cells. Motor function, lipid droplet accumulation in spinal cord neurons and neuronal survival were all improved in bromocriptine-treated mice after spinal cord injury. The results suggest that bromocriptine can protect neurons from lipotoxic damage after spinal cord injury via the extracellular signal-regulated kinases 1/2-cytosolic phospholipase A2 pathway.

Modeling the Drug delivery Lifecycle of PLG Nanoparticles Using Intravital Microscopy.

Polylactide-co-glycolide (PLG) nanoparticles hold immense promise for cancer therapy due to their enhanced efficacy and biodegradable matrix structure. Understanding their interactions with blood cells and subsequent biodistribution kinetics is crucial for optimizing their therapeutic potential. In this study, three doxorubicin-loaded PLG nanoparticle systems are synthesized and characterized, analyzing their size, zeta potential, morphology, and in vitro release behavior. Employing intravital microscopy in 4T1-tumor-bearing mice, real-time blood and tumor distribution kinetics are investigated. A mechanistic pharmacokinetic model is used to analyze biodistribution kinetics. Additionally, flow cytometry is utilized to identify cells involved in nanoparticle hitchhiking. Following intravenous injection, PLG nanoparticles exhibit an initial burst release (<1 min) and rapidly adsorb to blood cells (<5 min), hindering extravasation. Agglomeration leads to the clearance of one carrier species within 3 min. In stable dispersions, drug release rather than extravasation remains the dominant pathway for drug elimination from circulation. This comprehensive investigation provides valuable insights into the interplay between competing kinetics that influence the lifecycle of PLG nanoparticles post-injection. The findings advance the understanding of nanoparticle behavior and lay the foundation for improved cancer therapy strategies using nanoparticle-based drug delivery systems.

Adrenomedullin has a pivotal role in trophoblast differentiation: A promising nanotechnology-based therapeutic target for early-onset preeclampsia.

Early-onset preeclampsia (EOPE) is a severe pregnancy complication associated with defective trophoblast differentiation and functions at implantation, but manifestation of its phenotypes is in late pregnancy. There is no reliable method for early prediction and treatment of EOPE. Adrenomedullin (ADM) is an abundant placental peptide in early pregnancy. Integrated single-cell sequencing and spatial transcriptomics confirm a high ADM expression in the human villous cytotrophoblast and syncytiotrophoblast. The levels of ADM in chorionic villi and serum were lower in first-trimester pregnant women who later developed EOPE than those with normotensive pregnancy. ADM stimulates differentiation of trophoblast stem cells and trophoblast organoids in vitro. In pregnant mice, placenta-specific ADM suppression led to EOPE-like phenotypes. The EOPE-like phenotypes in a mouse PE model were reduced by a placenta-specific nanoparticle-based forced expression of ADM. Our study reveals the roles of trophoblastic ADM in placental development, EOPE pathogenesis, and its potential clinical uses.

SpatialDM for rapid identification of spatially co-expressed ligand-receptor and revealing cell-cell communication patterns.

Cell-cell communication is a key aspect of dissecting the complex cellular microenvironment. Existing single-cell and spatial transcriptomics-based methods primarily focus on identifying cell-type pairs for a specific interaction, while less attention has been paid to the prioritisation of interaction features or the identification of interaction spots in the spatial context. Here, we introduce SpatialDM, a statistical model and toolbox leveraging a bivariant Moran's statistic to detect spatially co-expressed ligand and receptor pairs, their local interacting spots (single-spot resolution), and communication patterns. By deriving an analytical null distribution, this method is scalable to millions of spots and shows accurate and robust performance in various simulations. On multiple datasets including melanoma, Ventricular-Subventricular Zone, and intestine, SpatialDM reveals promising communication patterns and identifies differential interactions between conditions, hence enabling the discovery of context-specific cell cooperation and signalling.

Fecal Microbiota Transplantation Revealed a Pain-related Gut Microbiota Community in Ovariectomized Mice.

Higher sensitivity to pain is a common clinical symptom in postmenopausal females. The gut microbiota (GM) has recently been identified as participating in various pathophysiological processes and may change during menopause and contribute to multiple postmenopausal symptoms. Here, we investigated the possible correlation between GM alteration and allodynia in ovariectomized (OVX) mice. Results showed that OVX mice exhibited allodynia from 7 weeks after surgery compared with sham-operated (SHAM) mice by comparing pain-related behaviors. Fecal microbiota transplantation (FMT) from OVX mice induced allodynia in normal mice while FMT from SHAM mice alleviated allodynia in OVX mice. Microbiome 16S rRNA sequencing and linear discriminant analysis revealed alteration of the GM after OVX. Furthermore, Spearman's correlation analysis showed associations between pain-related behaviors and genera, and further verification identified the possible pain-related genera complex. Our findings provide new insights into the underlying mechanisms of postmenopausal allodynia, and suggest pain-related microbiota community as a promising therapeutic target. PERSPECTIVE: This article provided the evidence of gut microbiota playing essential roles in postmenopausal allodynia. This work intended to offer a guidance for further mechanism investigation into gut-brain axis and probiotics screening for postmenopausal chronic pain.

Simulate SubQ: The Methods and the Media.

For decades, there has been a growing interest in injectable subcutaneous formulations to improve the absorption of drugs into the systemic circulation and to prolong their release over a longer period. However, fluctuations in the blood plasma levels together with bioavailability issues often limit their clinical success. This warrants a closer look at the performance of long-acting depots, for example, and their dependence on the complex interplay between the dosage form and the physiological microenvironment. For this, biopredictive performance testing is used for a thorough understanding of the biophysical processes affecting the absorption of compounds from the injection site in vivo and their simulation in vitro. In the present work, we discuss in vitro methodologies including methods and media developed for the subcutaneous route of administration on the background of the most relevant absorption mechanisms. Also, we highlight some important knowledge gaps and shortcomings of the existing methodologies to provide the reader with a better understanding of the scientific evidence underlying these models.

Phytotoxicity and the molecular response in yttrium oxide nanoparticle-treated Arabidopsis thaliana seedlings.

Due to the widespread application of rare earth oxide nanoparticles in various fields, their release into the environment is inevitable, and their potential toxicity and ecological impact have become a concern. Yttrium oxide nanoparticles are important rare earth oxide nanoparticles; however, their impact on plants and the molecular mechanism underlying their influence on plant growth and development are unclear. In this study, we found that yttrium oxide nanoparticles at concentrations exceeding 2 mM significantly inhibited the growth of Arabidopsis seedlings. Using Arabidopsis marker lines for auxin signaling, we found that the application of yttrium oxide nanoparticles resulted in disordered auxin signaling in root cells. Auxin signaling in the cells of the quiescent center and columella stem cells decreased, while auxin signaling in the cells of the stele was enhanced. In addition, trypan blue staining showed that yttrium oxide nanoparticles induced root cell death. Transcriptome analysis showed that the nanoparticles specifically inhibited the expression of lignin synthesis-related genes, activated the MAPK signaling pathway, and enhanced the ethylene and abscisic acid signaling pathways in plants. This study demonstrates the phytotoxicity of yttrium oxide nanoparticles at the molecular level in Arabidopsis, and it provides a new perspective on how plants respond to rare earth oxide stress.

Modeling digestion, absorption, and ketogenesis after administration of tricaprilin formulations to humans.

At present, tricaprilin is used as a ketogenic source for the management of mild to moderate Alzheimer's disease. After administration of the medium-chain triglyceride, tricaprilin is hydrolyzed to octanoic acid and further metabolized to ketones, acting as an alternative energy substrate for the brain. In this investigation, we developed a physiologically-based biopharmaceutics model simulating in vivo processes following the peroral administration of tricaprilin. The model includes multiple data sources to establish a partially verified framework for the simulation of plasma profiles. The input parameters were identified based on existing literature data and in vitro digestion studies. Model validation was conducted using the data from a phase I clinical trial. A partial parameter sensitivity analysis elucidated various influences on the plasma ketone levels that are mainly responsible for the therapeutic effects of tricaprilin. Based on our findings, we concluded that dispersibility and lipolysis of tricaprilin together with the gastric emptying patterns are limiting ketogenesis, while other steps such as the conversion of octanoic acid to ketone bodies play a minor role only.

Human early syncytiotrophoblasts are highly susceptible to SARS-CoV-2 infection.

Direct in vivo investigation of human placenta trophoblast's susceptibility to SARS-CoV-2 is challenging. Here we report that human trophoblast stem cells (hTSCs) and their derivatives are susceptible to SARS-CoV-2 infection, which reveals heterogeneity in hTSC cultures. Early syncytiotrophoblasts (eSTBs) generated from hTSCs have enriched transcriptomic features of peri-implantation trophoblasts, express high levels of angiotensin-converting enzyme 2 (ACE2), and are productively infected by SARS-CoV-2 and its Delta and Omicron variants to produce virions. Antiviral drugs suppress SARS-CoV-2 replication in eSTBs and antagonize the virus-induced blockage of STB maturation. Although less susceptible to SARS-CoV-2 infection, trophoblast organoids originating from hTSCs show detectable viral replication reminiscent of the uncommon placental infection. These findings implicate possible risk of COVID-19 infection in peri-implantation embryos, which may go unnoticed. Stem cell-derived human trophoblasts such as eSTBs can potentially provide unlimited amounts of normal and genome-edited cells and facilitate coronavirus research and antiviral discovery.

Can HER2 1+ Breast Cancer Be Considered as HER2-Low Tumor? A Comparison of Clinicopathological Features, Quantitative HER2 mRNA Levels, and Prognosis among HER2-Negative Breast Cancer.

Background: Human epidermal growth factor receptor 2 (HER2)-low tumor is a new entity defined as HER2 immunohistochemistry (IHC) 1+ or 2+/fluorescence in situ hybridization (FISH)-negative. We aimed to evaluate whether HER2 mRNA levels tested by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) could better define HER2-low tumors. Patients and methods: Consecutive breast cancer patients with hormonal receptor-positive, HER2-negative diseases, and HER2 mRNA results were included. Clinicopathologic features, HER2 mRNA expression level, and prognosis were compared among HER2 0, 1+ and 2+/FISH− groups. Concordance of the HER2 category between qRT-PCR and IHC/FISH was analyzed for each group. Results: 2296 patients were included: 368 (16.0%) HER2 0, 911 (39.7%) 1+, and 1017 (44.3%) 2+/FISH− tumors. HER2 1+ cases shared similarities with HER2 0 tumors in terms of clinicopathologic features (all p > 0.05), whereas IHC 2+/FISH− cases were less often non-IDC (p = 0.045), node-negative (p = 0.044), and Ki-67 < 14% (p <0.001). The mRNA expression was similar between HER2 0 and 1+ cases (p = 0.063), and both were lower than 2+/FISH− cases (p < 0.001). A poor concordance rate was found between IHC/FISH and qRT-PCR for HER2 0 and HER2-low cases (Cohen's kappa 0.126, p < 0.001). No survival difference was observed among these groups, whether stratified by HER2 IHC/FISH status or mRNA level (all p > 0.05). Conclusions: HER2 1+ cases had similar clinicopathological features to HER2 0 breast cancers, and both were different from HER2 2+/FISH− cases. HER2 mRNA levels were comparable between HER2 0 and 1+ tumors, and both were significantly lower than IHC 2+/FISH− tumors. Neither IHC nor qRT-PCR may be optimal to quantify HER2-low expression, especially for HER2 1+ patients.

Accuracy of ultrasonographic changes during neoadjuvant chemotherapy to predict axillary lymph node response in clinical node-positive breast cancer patients.

Purpose: To evaluate whether changes in ultrasound features during neoadjuvant chemotherapy (NAC) could predict axillary node response in clinically node-positive breast cancer patients. Methods: Patients with biopsy-proven node-positive disease receiving NAC between February 2009 and March 2021 were included. Ultrasound (US) images were obtained using a 5-12-MHz linear array transducer before NAC, after two cycles, and at the completion of NAC. Long and short diameter, cortical thickness, vascularity, and hilum status of the metastatic node were retrospectively reviewed according to breast imaging-reporting and data system (BI-RADS). The included population was randomly divided into a training set and a validation set at a 2:1 ratio using a simple random sampling method. Factors associated with node response were identified through univariate and multivariate analyses. A nomogram combining clinical and changes in ultrasonographic (US) features was developed and validated. The receiver operating characteristic (ROC) and calibration plots were applied to evaluate nomogram performance and discrimination. Results: A total of 296 breast cancer patients were included, 108 (36.5%) of whom achieved axillary pathologic complete response (pCR) and 188 (63.5%) had residual nodal disease. Multivariate regression indicated that independent predictors of node pCR contain ultrasound features in addition to clinical features, clinical features including neoadjuvant HER2-targeted therapy and clinical response, ultrasound features after NAC including cortical thickness, hilum status, and reduction in short diameter ≥50%. The nomogram combining clinical features and US features showed better diagnostic performance compared to clinical-only model in the training cohort (AUC: 0.799 vs. 0.699, P=0.001) and the validation cohort (AUC: 0.764 vs. 0.638, P=0.027). Conclusions: Ultrasound changes during NAC could improve the accuracy to predict node response after NAC in clinically node-positive breast cancer patients.

An in vitro gel-based system for characterizing and predicting the long-term performance of PLGA in situ forming implants.

In situ forming implants are exposed to an extracellular matrix resembling a gel rather than aqueous solution upon subcutaneous administration. The aim of study was to develop a gel-based release testing system for characterizing the long-term in vitro behavior of in situ forming implants. The gel-based system consisted of an agarose gel mimicking the subcutaneous injection site and a receiver layer comprising phosphate buffer. Poly(D,L-lactide-co-glycolide) in situ forming implants containing leuprolide acetate as the model peptide and N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO) or triacetin as co-solvent were investigated. The gel-based release testing system discriminated between the formulations. Accelerated release data obtained at elevated temperatures were able to predict real-time release applying the Arrhenius equation. Monitoring of the microenvironmental pH of the implants was performed by UV-Vis imaging in the gel-based system at 50 °C. A pH drop (from pH 7.4 to 6.7 for the NMP and DMSO implants, to pH 5.5 for the triacetin implants) within the first day was observed, followed by an increase to pH âˆ¼7.4. The gel-based system coupled with UV imaging offered opportunity for detailed evaluation and prediction of the in vitro performance of long-acting injectables, facilitating future development of in situ depot forming delivery systems.

Contralateral S1 nerve root transfer for motor function recovery in the lower extremity among patients with central nervous system injury: study protocol for a randomized controlled trial.

BACKGROUND: Central nervous system injury (CNSI) comprises a series of common diseases that severely affect patients' motor function and quality of life and is associated with high disability and mortality rates. Previous studies have shown that contralateral lumbosacral nerve root transfer significantly improved the function of the paralyzed limb in rat models of CNSI. These studies showed that severing the sacral 1 nerve root (S1) did not damage the function of the ipsilateral lower extremity. Thus, we speculate that contralateral S1 nerve root transfer can improve the recovery of a paralyzed limb. Because no associated rigorously designed randomized controlled trial has evaluated the effectiveness of contralateral S1 nerve transfer thus far, we designed this clinical trial to compare the effects of this new treatment approach with those of traditional treatments in paralyzed patients after chronic CNSI. METHODS: This is a single-center, prospective, randomized controlled trial. Forty patients, who meet the inclusion criteria and have hemiplegia caused by chronic CNSI, will be randomly divided into the surgical or non-surgical group. The treatment effect in the 2 groups will be assessed before and 3, 6, 9, 12, 18, and 24 months after intervention by using numerous scales and resting-state functional magnetic resonance imaging. The primary outcome will be the Fugl-Meyer score for the lower limbs 24 months after treatment. The secondary outcomes include the modified Ashworth spasm scale, the modified Barthel scale, 10-m walking speed measurement results, three-dimensional gait analysis, muscle strength testing, electromyography, and resting-state functional magnetic resonance imaging findings. Safety outcomes and adverse events will be observed simultaneously. DISCUSSION: We expect that the surgery will improve the sensorimotor functions of the paralyzed limb, and the results of this trial will provide high-quality clinical evidence for a new efficient treatment strategy for disability after CNSI. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR1800014414, registration date: 12 January 2018.

Long Non-coding RNA MSTRG.24008.1 Regulates the Regeneration of the Sciatic Nerve via the miR-331-3p-NLRP3/MAL Axis.

Peripheral nerve injury (PNI) is a common clinical problem, which can cause severe disability and dramatically affect a patient's quality of life. Neural regeneration after PNI is a complex biological process that involves a variety of signaling pathways and genes. Emerging studies demonstrated that long non-coding RNAs (lncRNAs) were abnormally expressed after PNI and played pivotal roles in peripheral nerve regeneration. Based on the rat sciatic nerve injury model, we found that the expression levels of several lncRNAs were increased significantly in the sciatic nerve after injury. Software prediction prompted us to focus on one up-regulated lncRNA, MSTRG.24008.1. Dual-luciferase reporter assay, RNA pull-down assay and RNA interference approach verified that MSTRG.24008.1 regulated neuroregeneration via the miR-331-3p/nucleotide-binding oligomerization domain-like pyrin domain containing 3 (NLRP3)/myelin and lymphocyte protein (MAL) axis in vitro. Subsequently, we performed gastrocnemius muscle gravity and sciatic functional index experiments to evaluate the recovery of injured sciatic nerves after MSTRG.24008.1 siRNA interference in vivo. In conclusion, knockdown of MSTRG.24008.1 promotes the regeneration of the sciatic nerve via the miR-331-3p/NLRP3/MAL axis, which may provide a new strategy to evaluate and repair injured peripheral nerves clinically.

Towards functional characterization of excipients for oral solid dosage forms using UV-vis imaging. Liberation, release and dissolution.

The purpose of this study was to investigate whole-dosage form UV-vis imaging as a potential tool for functional characterization of excipients used in solid oral dosage forms. To this end, tablets (average mass 260.0 mg, 224.5 mg and 222.1 mg) containing theophylline anhydrate (20 % w/w), 1% (w/w) magnesium stearate, and 79 % (w/w) of either microcrystalline cellulose (MCC, Avicel PH 101) or hydroxypropyl methylcellulose (HPMC, Methocel K15 M or K100 M) were prepared as model systems. Drug liberation from tablets was studied in 0.01 M HCl at 37 °C using a Sirius SDi2 equipped with a USP IV type flow cell comprising a UV-vis imaging detector operating at 255 nm and 520 nm. The effluent from the flow cell was passed through a downstream spectrophotometer, and UV-vis spectra in the wavelength range 200-800 nm were recorded every 2 min. The erosion and swelling behavior of the MCC tablets and HPMC K15 M and K100 M tablets were visualized in real time. The swelling of HPMC K15 M and K100 M containing tablets was assessed quantitatively as changes in tablet diameter measured at 520 nm, and was clearly distinguished from the swelling of the MCC tablets. Namely, an increment of 2.5 mm in diameter was determined for the HPMC tablets while the MCC tablets increased by 0.5-1 mm in diameter. Gel layers of variable thickness were observed only for the HPMC K15 M and K100 M tablets. In addition, a relatively high initial liberation rate of theophylline was found for the MCC tablets as compared to the HPMC tablets. UV-vis imaging revealed features of liberation not revealed by simply measuring drug concentration in the dissolution media or by visual assessment. It may be sufficiently sensitive to be further developed for functional characterization of excipients and provide insights into drug-excipient interactions likely to be useful in formulation development.

Initial Leuprolide Acetate Release from Poly(d,l-lactide-co-glycolide) in Situ Forming Implants as Studied by Ultraviolet-Visible Imaging.

The initial drug release from in situ forming implants is affected by factors such as the physicochemical properties of the active pharmaceutical ingredient, the type of the excipients utilized, and the surrounding environment. The feasibility of UV-vis imaging for characterization of the initial behavior of poly(d,l-lactide-co-glycolide) (PLGA)/1-methyl-2-pyrrolidinone (NMP) in situ forming implants was investigated. The in vitro release of leuprolide acetate (LA) and implant formation in real time were monitored using dual-wavelength imaging at 280 and 525 nm, respectively, in matrices based on agarose gel and hyaluronic acid (HA) solution emulating the subcutaneous matrix. Three hours upon injection of the pre-formulation, approximately 15% of the total amount of LA administered was found in the agarose gel, while 5% was released from the implant into the HA solution. Concurrently, more extensive swelling of the implants in the HA solution as compared to implants in the agarose gel was observed. Transport of both LA and the solvent NMP was investigated using UV-vis imaging in a small-scale cell where the geometry of the formulation was controlled, showing a linear correlation between drug release and solvent escape. Light microscopy showed that the microstructures of the resulting implants in agarose gel and HA solution were different, which may be attributed to the different solvent exchange rates. UV imaging was also used to examine the interaction of LA with the release medium by characterizing the diffusion of LA in agarose gel, HA solution, and phosphate buffered saline. The reduced LA diffusivity in HA solution as compared to agarose gel and the LA distribution coefficient in the agarose gel-HA system indicated the presence of interactions between LA and HA. Our findings show that the external environment affects the solvent exchange kinetics for in situ forming implants in vitro, resulting in different types of initial release behavior. UV-vis imaging in combination with biorelevant matrices may offer an interesting approach in the development of in situ forming implant delivery systems.