A complementary metaproteomic approach to interrogate microbiome cultivated from clinical colon biopsies.

The human gut microbiome plays a vital role in preserving individual health and is intricately involved in essential functions. Imbalances or dysbiosis within the microbiome can significantly impact human health and are associated with many diseases. Several metaproteomics platforms are currently available to study microbial proteins within complex microbial communities. In this study, we attempted to develop an integrated pipeline to provide deeper insights into both the taxonomic and functional aspects of the cultivated human gut microbiomes derived from clinical colon biopsies. We combined a rapid peptide search by MSFragger against the Unified Human Gastrointestinal Protein database and the taxonomic and functional analyses with Unipept Desktop and MetaLab-MAG. Across seven samples, we identified and matched nearly 36,000 unique peptides to approximately 300 species and 11 phyla. Unipept Desktop provided gene ontology, InterPro entries, and enzyme commission number annotations, facilitating the identification of relevant metabolic pathways. MetaLab-MAG contributed functional annotations through Clusters of Orthologous Genes and Non-supervised Orthologous Groups categories. These results unveiled functional similarities and differences among the samples. This integrated pipeline holds the potential to provide deeper insights into the taxonomy and functions of the human gut microbiome for interrogating the intricate connections between microbiome balance and diseases.

Optimizing MS-Based Multi-Omics: Comparative Analysis of Protein, Metabolite, and Lipid Extraction Techniques.

Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

Unveiling Neuroprotection and Regeneration Mechanisms in Optic Nerve Injury: Insight from Neural Progenitor Cell Therapy with Focus on Vps35 and Syntaxin12.

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increases in ischemia and expression of genes linked to neuronal regeneration. Post NPC injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, the proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved the mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/ß-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/ß-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Bottom-Up Proteomics: Advancements in Sample Preparation.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics is a powerful technique for profiling proteomes of cells, tissues, and body fluids. Typical bottom-up proteomic workflows consist of the following three major steps: sample preparation, LC-MS/MS analysis, and data analysis. LC-MS/MS and data analysis techniques have been intensively developed, whereas sample preparation, a laborious process, remains a difficult task and the main challenge in different applications. Sample preparation is a crucial stage that affects the overall efficiency of a proteomic study; however, it is prone to errors and has low reproducibility and throughput. In-solution digestion and filter-aided sample preparation are the typical and widely used methods. In the past decade, novel methods to improve and facilitate the entire sample preparation process or integrate sample preparation and fractionation have been reported to reduce time, increase throughput, and improve reproducibility. In this review, we have outlined the current methods used for sample preparation in proteomics, including on-membrane digestion, bead-based digestion, immobilized enzymatic digestion, and suspension trapping. Additionally, we have summarized and discussed current devices and methods for integrating different steps of sample preparation and peptide fractionation.

Recent Advances in Intranasal Liposomes for Drug, Gene, and Vaccine Delivery.

Liposomes are safe, biocompatible, and biodegradable spherical nanosized vesicles produced from cholesterol and phospholipids. Recently, liposomes have been widely administered intranasally for systemic and brain delivery. From the nasal cavity, liposome-encapsulated drugs and genes enter the systemic circulation primarily via absorption in the respiratory region, whereas they can be directly transported to the brain via the olfactory pathway. Liposomes can protect drugs and genes from enzymatic degradation, increase drug absorption across the nasal epithelium, and prolong the residence time in the nasal cavity. Intranasal liposomes are also a potential approach for vaccine delivery. Liposomes can be used as a platform to load antigens and as vaccine adjuvants to induce a robust immune response. With the recent interest in intranasal liposome formulations, this review discusses various aspects of liposomes that make them suitable for intranasal administration. We have summarized the latest advancements and applications of liposomes and evaluated their performance in the systemic and brain delivery of drugs and genes administered intranasally. We have also reviewed recent advances in intranasal liposome vaccine development and proposed perspectives on the future of intranasal liposomes.

The Role of Extracellular Vesicles in Optic Nerve Injury: Neuroprotection and Mitochondrial Homeostasis.

Stem cell therapies hold great promise as alternative treatments for incurable optic nerve disorders. Although mesenchymal stem cells exhibit various tissue regeneration and recovery capabilities that may serve as valuable therapies, the clinical applications remain limited. Thus, we investigated the utility of extracellular vesicles (EVs) from human placenta-derived mesenchymal stem cells (hPSCs) in this context. Hypoxically preconditioned hPSCs (HPPSCs) were prepared via short-term incubation under 2.2% O2 and 5.5% CO2. The EVs were then isolated. R28 cells (retinal precursor cells) were exposed to CoCl2 and treated with EVs for 24 h. Cell proliferation and regeneration were measured using a BrdU assay and immunoblotting; ATP quantification revealed the extent of the mitochondrial function. The proteome was determined via liquid chromatography-tandem mass spectroscopy. Differentially expressed proteins (DEPs) were detected and their interactions identified. HPPSC_EVs functions were explored using animal models of optic nerve compression. HPPSC_EVs restored cell proliferation and mitochondrial quality control in R28 cells damaged by CoCl2. We identified DEPs (p < 0.05) that aided recovery. The mitochondrial DEPs included LONP1; PARK7; VDAC1, 2, and 3; HSPD1; and HSPA9. EVs regulated the levels of mitophagic proteins in R28 cells injured by hypoxia; the protein levels did not increase in LONP1 knockdown cells. LONP1 is a key mediator of the mitophagy that restores mitochondrial function after hypoxia-induced optic nerve injury.

A review of suspension trapping digestion method in bottom-up proteomics.

The standard bottom-up proteomic workflow is comprised of sample preparation, data acquisition, and data analysis. While the latter two parts have made considerable advances in the last decade, sample preparation has remained an important challenge within the workflow due to the multi-step nature of complex biological samples, and still requires much development. Several sample preparation methods have been developed and used in the last two decades, including in-gel, in-solution, on-bead, filter-aided sample preparation, and suspension trapping, to improve reproducibility, efficiency, scalability, and reduce the handling time of this process. One of the most recent methods developed and applied in proteomics studies in recent years is suspension trapping, which combines rapid detergent removal, reactor-type protein digestion, and peptide clean-up in a tip or spin column. Suspension trapping is a simple, rapid, and reproducible digestion method that can effectively handle proteins in low microgram or sub-microgram amounts. This review discusses the benefits of the suspension trapping digestion method in relation to its development and application in bottom-up proteomics studies. We also discuss recent applications of suspension trapping digestion to different sample types and the features of the suspension trapping digestion method compared with other sample preparation methods.

Pharmaceutical Formulations with P-Glycoprotein Inhibitory Effect as Promising Approaches for Enhancing Oral Drug Absorption and Bioavailability.

P-glycoprotein (P-gp) is crucial in the active transport of various substrates with diverse structures out of cells, resulting in poor intestinal permeation and limited bioavailability following oral administration. P-gp inhibitors, including small molecule drugs, natural constituents, and pharmaceutically inert excipients, have been exploited to overcome P-gp efflux and enhance the oral absorption and bioavailability of many P-gp substrates. The co-administration of small molecule P-gp inhibitors with P-gp substrates can result in drug-drug interactions and increased side effects due to the pharmacological activity of these molecules. On the other hand, pharmaceutically inert excipients, including polymers, surfactants, and lipid-based excipients, are safe, pharmaceutically acceptable, and are not absorbed from the gut. Notably, they can be incorporated in pharmaceutical formulations to enhance drug solubility, absorption, and bioavailability due to the formulation itself and the P-gp inhibitory effects of the excipients. Different formulations with inherent P-gp inhibitory activity have been developed. These include micelles, emulsions, liposomes, solid lipid nanoparticles, polymeric nanoparticles, microspheres, dendrimers, and solid dispersions. They can bypass P-gp by different mechanisms related to their properties. In this review, we briefly introduce P-gp and P-gp inhibitors, and we extensively summarize the current development of oral drug delivery systems that can bypass and inhibit P-gp to improve the oral absorption and bioavailability of P-gp substrates. Since many drugs are limited by P-gp-mediated efflux, this review is helpful for designing suitable formulations of P-gp substrates to enhance their oral absorption and bioavailability.

Discovery of Post-Translational Modifications in Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.

Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents.

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5-50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.

Preparation of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Drug Delivery and the Effects of Preparation Parameters of Solvent Injection Method.

Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have emerged as potential drug delivery systems for various applications that are produced from physiological, biodegradable, and biocompatible lipids. The methods used to produce SLNs and NLCs have been well investigated and reviewed, but solvent injection method provides an alternative means of preparing these drug carriers. The advantages of solvent injection method include a fast production process, easiness of handling, and applicability in many laboratories without requirement of complicated instruments. The effects of formulations and process parameters of this method on the characteristics of the produced SLNs and NLCs have been investigated in several studies. This review describes the methods currently used to prepare SLNs and NLCs with focus on solvent injection method. We summarize recent development in SLNs and NLCs production using this technique. In addition, the effects of solvent injection process parameters on SLNs and NLCs characteristics are discussed.

UBA2 activates Wnt/ß-catenin signaling pathway during protection of R28 retinal precursor cells from hypoxia by extracellular vesicles derived from placental mesenchymal stem cells.

BACKGROUND: Stem cell transplantation has been proposed as an alternative treatment for intractable optic nerve disorders characterized by irrecoverable loss of cells. Mesenchymal stem cells, with varying tissue regeneration and recovery capabilities, are being considered for potential cell therapies. To overcome the limitations of cell therapy, we isolated exosomes from human placenta-derived mesenchymal stem cells (hPMSCs) and investigated their therapeutic effects in R28 cells (retinal precursor cells) exposed to CoCl2. METHOD: After 9 h of exposure to CoCl2, the hypoxic damaged R28 cells were divided into the non-treatment group (CoCl2 + R28 cells) and treatment group (CoCl2 + R28 cells treated with exosome). Immunoblot analysis was performed for Pcna, Hif-1α, Vegf, Vimentin, Thy-1, Gap43, Ermn, Neuroflament, Wnt3a, ß-catenin, phospo-GSK3ß, Lef-1, UBA2, Skp1, ßTrcp, and ubiquitin. The proteomes of each group were analyzed by liquid chromatography/tandem mass (LC-MS/MS) spectrometry. Differentially expressed proteins (DEPs) were detected by label-free quantification, and the interactions of the proteins were examined through signal transduction pathway and gene ontology analysis. RESULT: We observed that exosome could significantly recover proliferation damaged by CoCl2 treatment. In addition, the treatment group presented the decreased expression of Hif-1α protein (P < 0.05) and increased expression of proliferation marker, Pcna, and nerve regeneration-related factors such as Vimentin, Thy-1, and Neuroflament (P < 0.05) compared with the non-treatment group. In total, 200 DEPs were identified in the non-treatment group and treatment group (fold change ≥ 2, p < 0.05). Catenin and ubiquitin systems (UBA2, UBE2E3, UBE2I) were found in both the DEP lists of downregulated proteins from the non-treatment group and upregulated proteins from the treatment group. The mRNA expressions of ubiquitin systems were significantly decreased under hypoxic conditions. Moreover, UBA2 and Wnt/ß-catenin protein were associated with the rescue of the hypoxic damaged R28 cells. Using a siRNA system, we could find it out that hPMSC exosomes could not repair altered expressions of target proteins by CoCl2 in lacking UBA2 R28 cells. CONCLUSION: This study reported that hypoxic damaged expression of regeneration markers in R28 cells was significantly recovered by hPMSC exosomes. We could also demonstrate that UBA2 played a key role in activating the Wnt/ß-catenin signaling pathway during protection of hypoxic damaged R28 cells, induced by hPMSC exosomes.

Proteomic Profiling of Emiliania huxleyi Using a Three-Dimensional Separation Method Combined with Tandem Mass Spectrometry.

Emiliania huxleyi is one of the most abundant marine planktons, and it has a crucial feature in the carbon cycle. However, proteomic analyses of Emiliania huxleyi have not been done extensively. In this study, a three-dimensional liquid chromatography (3D-LC) system consisting of strong cation exchange, high- and low-pH reversed-phase liquid chromatography was established for in-depth proteomic profiling of Emiliania huxleyi. From tryptic proteome digest, 70 fractions were generated and analyzed using liquid chromatography-tandem mass spectrometry. In total, more than 84,000 unique peptides and 10,000 proteins groups were identified with a false discovery rate of ≤0.01. The physicochemical properties of the identified peptides were evaluated. Using ClueGO, approximately 700 gene ontology terms and 15 pathways were defined from the identified protein groups with p-value ≤0.05, covering a wide range of biological processes, cellular components, and molecular functions. Many biological processes associated with CO2 fixation, photosynthesis, biosynthesis, and metabolic process were identified. Various molecular functions relating to protein binding and enzyme activities were also found. The 3D-LC strategy is a powerful approach for comparative proteomic studies on Emiliania huxleyi to reveal changes in its protein level and related mechanism.

Determination of KD025 (SLx-2119), a Selective ROCK2 Inhibitor, in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry and its Pharmacokinetic Application.

KD025 (SLx-2119), the first specific Rho-associated protein kinase 2 (ROCK2) inhibitor, is a potential new drug candidate currently undergoing several phase 2 clinical trials for psoriasis, idiopathic pulmonary fibrosis, chronic graft-versus-host disease, and systemic sclerosis. In this study, a bio-analytical method was developed and fully validated for the quantification of KD025 in rat plasma and for application in pharmacokinetic studies. KD025 and GSK429286A (the internal standard) in rat plasma samples were analyzed by high-performance liquid chromatography-tandem mass spectrometry with m/z transition values of 453.10 → 366.10 and 433.00 → 178.00, respectively. The method was fully validated according to the United State Food and Drug Administration guidelines in terms of selectivity, linearity, accuracy, precision, sensitivity, matrix effects, extraction recovery, and stability. The method enabled the quantification of KD025 levels in rat plasma following oral administration of 5 mg/kg KD025 and intravenous administration of 2 mg/kg KD025 to rats, respectively. Our findings suggest that the developed method is practical and reliable for pharmacokinetic studies of KD025 in preclinical animals.

Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics.

Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.

Preparation of an oil suspension containing ondansetron hydrochloride as a sustained release parenteral formulation.

Ondansetron hydrochloride (ODS) is a selective 5-hydroxytryptamine type 3 antagonist for nausea and emesis prevention in neoplastic patients. To reduce dosing frequency and side effects and improve patient compliance, a sustained release parenteral formulation of ODS was developed. Microparticles of methylcellulose (MC) and ODS were prepared using the spray-drying method and suspended in oils to form oil suspensions. The formulations were evaluated for residual moisture, drug content, size distribution, DSC, XRD, FTIR, SEM, drug release, and pharmacokinetic studies. The effects of polymers and oils on the drug release were evaluated. MC showed the most prominent sustained release effect among various polymers examined with the optimum MC/ODS ratio of 2:1 (w/w). The particle size of the produced microparticles was in the mean diameter of approximately 3 µm. Physicochemical characterization suggested that ODS existed in an amorphous matrix within the microparticles and interacted with MC via hydrogen bonds. Corn oil was selected as the appropriate oil for suspension due to the sustained release of ODS and the appropriate viscosity. The optimized sustained release formulation of ODS was the corn oil suspension of spray-dried microparticles containing MC and ODS (2:1, w/w). It showed an in vitro drug sustained release up to 120 h, while the oil suspension of ODS without any polymer released the drug within 2 h. Following subcutaneous administration in rats, the optimized formulation could prolong the drug release until 72 h with the enhanced bioavailability in comparison with the ODS solution. The oil suspension of spray-dried microparticles might be an efficient approach for prolongation of the drug effect in the management of nausea and emesis. Graphical abstract.

Data on optimization and drug release kinetics of nanostructured lipid carriers containing ondansetron hydrochloride prepared by cold high-pressure homogenization method.

Nanostructured lipid carriers (NLCs), the second generation of lipid nanoparticles could enhance the drug loading capacity and minimize the drug expulsion during storage [1,2]. They are prepared from mixtures of solid and liquid lipids [3,4]. The article described the data for the preparation, optimization, and drug release studies of NLCs loaded with ondansetron hydrochloride (OSH), a water-soluble drug. The OSH-loaded NLCs were prepared using a modified cold high-pressure homogenization method. The NLCs were optimized for various parameters of formulation and preparation process on the basis of particle size (PS), polydispersity index (PI), entrapment efficiency (EE), and drug loading (DL). The dataset presented here supports "Nanostructured lipid carriers containing ondansetron hydrochloride by cold high-pressure homogenization method: Preparation, characterization, and pharmacokinetic evaluation" [5].

Preparation of Ondansetron Hydrochloride-Loaded Nanostructured Lipid Carriers Using Solvent Injection Method for Enhancement of Pharmacokinetic Properties.

PURPOSE: This study aimed to incorporate ondansetron hydrochloride (ODS), a water-soluble drug into nanostructured lipid carriers (NLCs) to improve the pharmacokinetic properties of the drug. METHODS: NLCs were produced by solvent injection method. Various parameters of formulation and process were assessed to enhance the drug incorporation into NLCs. Physicochemical analyses, in vitro drug release, and pharmacokinetic studies were performed. RESULTS: Entrapment efficiency (EE) of ODS was considerably improved (>90%) by increasing pH of the aqueous phase. The use of an appropriate level of liquid lipid resulted in small, monodispersed NLCs with the enhanced EE and drug loading (DL). The optimized NLCs formulation exhibited particle size of 185.2 ± 1.9 nm, polydispersity index of 0.214 ± 0.006, EE of 93.2 ± 0.5%, and DL of 10.43 ± 0.05% as well as an in vitro sustained-release profile of ODS. Differential scanning calorimetry and X-ray powder diffraction suggested the amorphous state of ODS in the NLCs. The pharmacokinetic study in rats exhibited the sustained-release characteristic of the optimized ODS-loaded NLCs following subcutaneous administration with an extended Tmax and mean residence time as well as the enhanced systemic exposure compared to the ODS solution. CONCLUSIONS: The ODS-loaded NLCs appear potential for prolongation of drug action and reduction in dosing frequency.