Ex-vivo Characterization Research Articles

Intelligent industry-oriented oro-solid formulation of famotidine: Advanced statistical optimization and Ex-Vivo characterization

BackgroundSol-Gel is the conventional approach for in-situ formulation having several limitations, including instability, stringent storage conditions, dose inaccuracy, inconsistent drug release, and patient inconvenience. ObjectivesThe aim of the present investigation was to design and characterize an intelligent oro-solid in-situ targeted dosage form of Famotidine (FTD) incorporating the cross-linked chitosan-vanillin (CrL-CSV) which instantly converted into a gel upon contact with acidic media. Material and MethodsCrL-CSV was designed using microwave-assisted method inculcating FTD and vanillin (VNL). Interaction was observed using FTIR and DSC. QbD was applied to develop intelligent formulation. Taguchi design was accomplished to screen significant critical material attributes (CMAs) and process parameters (CPPs). Box-Behnken design (BBD) was used to correlate the CMAs/CPPs: the amount of CS, VNL, microwave time, and critical quality attributes (CQAs): % FTD release, mucoadhesive strength, and viscosity. CrL-CSV were evaluated using various in-vitro, ex-vivo characterization and stability. Results and DiscussionFTIR and DSC revealed no interaction between the FTD and excipients. The amount of CS, VNL, and microwave time were screened as significant variables due to the high S/N ratio and delta value from the Taguchi design. A significant effect of CMA/CPP on the CQAs was observed in BBD. The optimum CrL-CSV was designed accomplishing 8.74 % chitosan (CS), 0.94 % VNL exposing 80sec in microwave. Immediate gelling (<1min.), desired viscosity (51432.6cps), optimal mucoadhesion strength (0.40 N), better control on drug release (91.5 % in 8 h) and high stability were achieved in the optimum batch of CrL-CSV. ConclusionAn intelligent formulation inculcating the CrL-CSV was developed, which has instant gelling time, high mucoadhesion time, and strength, and controlled release with desired physical properties. Progressive tools like Taguchi and BBD was explored. The formulation can be helpful at industry as it increases patient compliance, processing and stability.

Oral pH triggered colon-specific ketoprofen loaded microspheres for the better management of early morning symptoms associated with rheumatoid arthritis. Part I: Optimization, in-vitro, and ex-vivo characterization

The primary goal of this research was to investigate pH-triggered colon-specific ketoprofen-loaded microspheres (C-SKLM) using Eudragit S-100 as a pH-dependent polymer. The formulation was optimized using the Box-Behnken design. The suggested C-SKLM formulation with the amount of ketoprofen = 189.09 mg, the amount of Eudragit-100 = 600 mg, and the paddle speed = 400 rpm showed a considerable particle size (106 µm), drug encapsulation efficacy (79.67%), and % cumulative drug release at 5th hr (8.62) and 10th hr (92.62) for oral administration. Microphotographs of scanning electron microscopy indicated that the optimized C-SKLM was smooth and spherical. The differential scanning calorimetry and X-ray powder diffraction studies have shown that ketoprofen was dispersed in the polymer in its optimized C-SKLM formulation, and the spectra from the Fourier transform infrared study have shown no significant drug-polymer interaction. Ex-vivo permeation studies on isolated rat gastro-intestinal segments revealed that the maximum amount of ketoprofen permeated after 2 h through the isolated stomach (0.559 ± 0.049 mg), small intestine (1.36 ± 0.226 mg), and colon (0.972 ± 0.068 mg) was observed for ketoprofen pure. In contrast, a significantly lesser amount of the drug was permeated through the stomach (0.1 ± 0.025 mg) and small intestine (0.28 ± 0.1 mg) than the colon (0.697 ± 0.23 mg) from the ketoprofen-loaded microspheres formulation. In-vitro and ex-vivo permeation studies suggest that the developed C-SKLM could be helpful for the effective management of early morning symptoms due to rheumatoid arthritis by delaying the release by targeting the colon.

Superparamagnetic iron oxide nanoparticle enhanced percutaneous microwave ablation: Ex-vivo characterization using magnetic resonance thermometry.

Percutaneous microwave ablation (pMWA) is a minimally invasive procedure that uses a microwave antenna placed at the tip of a needle to induce lethal tissue heating. It can treat cancer and other diseases with lower morbidity than conventional surgery, but one major limitation is the lack of control over the heating region around the ablation needle. Superparamagnetic iron oxide nanoparticles have the potential to enhance and control pMWA heating due to their ability to absorb microwave energy and their ease of local delivery. The purpose of this study is to experimentally quantify the capabilities of FDA-approved superparamagnetic iron oxide Feraheme nanoparticles (FHNPs) to enhance and control pMWA heating. This study aims to determine the effectiveness of locally injected FHNPs in increasing the maximum temperature during pMWA and to investigate the ability of FHNPs to create a controlled ablation zone around the pMWA needle. PMWA was performed using a clinical ablation system at 915MHz in ex-vivo porcine liver tissues. Prior to ablation, 50uL 5mg/mL FHNP injections were made on one side of the pMWA needle via a 23-gauge needle. Local temperatures at the FHNP injection site were directly compared to equidistant control sites without FHNP. First, temperatures were compared using directly inserted thermocouples. Next, temperatures were measured non-invasively using magnetic resonance thermometry (MRT), which enabled comprehensive four-dimensional (volumetric and temporal) assessment of heating effects relative to nanoparticle distribution, which was quantified using dual-echo ultrashort echo time (UTE) subtraction MR imaging. Maximum heating within FHNP-exposed tissues versus control tissues were compared at multiple pMWA energy delivery settings. The ability to generate a controlled asymmetric ablation zone using multiple FHNP injections was also tested. Finally, intra-procedural MRT-derived heat maps were correlated with gold standard gross pathology using Dice similarity analysis. Maximum temperatures at the FHNP injection site were significantly higher than control (without FHNP) sites when measured using direct thermocouples (93.1±6.0°C vs. 57.2±8.1°C, p=0.002) and using non-invasive MRT (115.6±13.4°C vs. 49.0±10.6°C, p=0.02). Temperature difference between FHNP-exposed and control sites correlated with total energy deposition: 66.6±17.6°C, 58.1±8.5°C, and 20.8±9.2°C at high (17.5±2.2kJ), medium (13.6±1.8kJ), and low (8.8±1.1kJ) energies, respectively (all pairwise p<0.05). Each FHNP injection resulted in a nanoparticle distribution within 0.9±0.2cm radially of the injection site and a local lethal heating zone confined to within 1.1±0.4cm radially of the injection epicenter. Multiple injections enabled a controllable, asymmetric ablation zone to be generated around the ablation needle, with maximal ablation radius on the FHNP injection side of 1.6±0.2cm compared to 0.7±0.2cm on the non-FHNP side (p=0.02). MRT intra-procedural predicted ablation zone correlated strongly with post procedure gold-standard gross pathology assessment (Dice similarity 0.9). Locally injected FHNPs significantly enhanced pMWA heating in liver tissues, and were able to control the ablation zone shape around a pMWA needle. MRI and MRT allowed volumetric real-time visualization of both FHNP distribution and FHNP-enhanced pMWA heating that was useful for intra-procedural monitoring. This work strongly supports further development of a FHNP-enhanced pMWA paradigm; as all individual components of this approach are approved for patient use, there is low barrier for clinical translation.

Reduced CCR5 expression among Uganda HIV controllers

BackgroundSeveral mechanisms including reduced CCR5 expression, protective HLA, viral restriction factors, broadly neutralizing antibodies, and more efficient T-cell responses, have been reported to account for HIV control among HIV controllers. However, no one mechanism universally accounts for HIV control among all controllers. In this study we determined whether reduced CCR5 expression accounts for HIV control among Ugandan HIV controllers. We determined CCR5 expression among Ugandan HIV controllers compared with treated HIV non-controllers through ex-vivo characterization of CD4 + T cells isolated from archived PBMCs collected from the two distinct groups.ResultsThe percentage of CCR5 + CD4 + T cells was similar between HIV controllers and treated HIV non-controllers (ECs vs. NCs, P = 0.6010; VCs vs. NCs, P = 0.0702) but T cells from controllers had significantly reduced CCR5 expression on their cell surface (ECs vs. NCs, P = 0.0210; VCs vs. NCs, P = 0.0312). Furthermore, we identified rs1799987 SNP among a subset of HIV controllers, a mutation previously reported to reduce CCR5 expression. In stark contrast, we identified the rs41469351 SNP to be common among HIV non-controllers. This SNP has previously been shown to be associated with increased perinatal HIV transmission, vaginal shedding of HIV-infected cells and increased risk of death.ConclusionCCR5 has a non-redundant role in HIV control among Ugandan HIV controllers. HIV controllers maintain high CD4 + T cells despite being ART naïve partly because their CD4 + T cells have significantly reduced CCR5 densities.

Enhanced Intestinal Permeability of Cefixime by Self-Emulsifying Drug Delivery System: In-Vitro and Ex-Vivo Characterization.

Cefixime (CFX) belongs to a group of third-generation cephalosporin antibiotics with low water solubility and low intestinal permeability, which ultimately leads to significantly low bioavailability. This study aimed to increase solubility, improve drug release, and intestinal permeability of CFX by loading into SEDDS. Suitable excipients were selected based on drug solubility, percent transmittance, and emulsification efficiency. Pseudo-ternary phase diagram was fabricated for the identification of effective self-emulsification region. The best probably optimized formulations were further assessed for encumbered drug contents, emulsification time, cloud point measurement, robustness to dilution, mean droplet size, zeta potential, polydispersity index (PDI), and thermodynamic and chemical stability. Moreover, in vitro drug release studies and ex vivo permeation studies were carried out and apparent drug permeability Papp of different formulations was compared with the marketed brands of CFX. Amongst the four tested SEDDS formulations, F-2 formulation exhibited the highest drug loading of 96.32%, emulsification time of 40.37 ± 3 s, mean droplet size of 19.01 ± 1.12 nm, and demonstrated improved long-term thermodynamic and chemical stability when stored at 4 °C. Release studies revealed a drug release of 97.32 ± 4.82% within 60 min in simulated gastric fluid. Similarly, 97.12 ± 5.02% release of CFX was observed in simulated intestinal fluid within 120 min; however, 85.13 ± 3.23% release of CFX was observed from the marketed product. Ex vivo permeation studies displayed a 2.7-fold increase apparent permeability compared to the marketed product in 5 h. Owing to the significantly improved drug solubility, in vitro release and better antibacterial activity, it can be assumed that CFX-loaded SEDDS might lead to an increased bioavailability and antibacterial activity, possibly leading to improved therapeutic effectiveness.

Abstract 2097: Characterization of mouse and cellular phenotype of ABI1 KO T-cells

Abstract Background: The immune system plays a critical part in cancer development and progression. The increase in our understanding of mechanisms regulating immune-mediated cancer eradication has led to the development of immune-based therapies which have revolutionized treatment of some previously lethal malignancies. T-cell activation requires proper intercellular interactions mediated by cytoskeletal rearrangements and adhesion molecules, along with intracellular signal transduction via phosphorylation by tyrosine kinases (Lck and Fyn). Abelson interactor-1 (ABI1) is an adaptor protein known to regulate actin polymerization, adhesion molecules and tyrosine kinases. Previously, ABI1 has been shown to localize to the immunological synapse in an antigen-dependent manner and has been linked to regulation of IL-2 secretion. Published data from our lab shows that ABI1 has high affinity binding to SH2 domains of Src family kinases, Fyn and Lck, both of which are essential in T-cell activation. Additionally, ABI1 has been linked to hyperactivation of SFKs/STAT3/NF-kB signaling. In this study, we aim to delineate the role of ABI1 in T-cell activation, differentiation and signaling. Methods: We have generated a novel conditional knock-out mouse model to evaluate the effect of a T-cell specific ABI1 KO at the organismal level (ABI1(fl/fl)CD4Cre (ACD4). T-cells isolated from control (Abi1(fl/fl)CD4Cre(-)) and ABI1 KO mice (Abi1(fl/fl)CD4Cre(+)) were used for ex-vivo analysis and further characterization of ABI1 KO cellular phenotype with: Flow Cytometry (CD3+ subpopulations in immune organs), Immunofluorescence (actin polymerization), Western blotting (TCR-mediated T-cell activation) and RNAseq (transcriptional reprogramming). Results: In vitro studies of ABI1 KO primary murine CD3+ T-cells showed increased levels of NF-kB and STAT3. Flow cytometry analysis of CD3+ T cells subpopulations showed a difference in the percentage of CD4+ and CD8+ T cells; percentage of CD8+ T cells was increased, while percentage of CD4+ T cells was decreased in ABI1 KO vs. WT control mice. Furthermore, RNA seq analysis of CD8+ ABI1 KO T cells showed a significant decrease in the mRNA expression of Thpok (Zbtb7b) compared to WT. Conclusion: Our preliminary studies show that ABI1 is a potential modulator of T-cell activation through regulation of Lck/Fyn. This suggests ABI1 may play as a molecular switch negatively regulating T-cell activation by connecting phosphorylation-dependent signal transduction and actin cytoskeletal dynamics. Suggesting ABI1 as a new therapeutic to improve efficacy and longevity of T cell-based cancer immunotherapies. In addition, ABI1 appears to be implicated in the regulation of the percentage of CD4 and CD8 T-cells, a novel discovery which warrants further investigation. Citation Format: Maria Altagracia Ortiz, Andras Perl, Marcin Kortylewski, Leszek Kotula. Characterization of mouse and cellular phenotype of ABI1 KO T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2097.

Differentiation in the expression of toxic effects of polyethylene-microplastics on two freshwater fish species: Size matters

The built up of microplastic (MPs) remains is shaping a new aquatic habitat and imposes the necessity for research of the effects that these relatively new pollutants exert on organisms, environment, and human health. The purpose of the present study was to verify if there is a particle-size dependence of fish response to MPs. Thus, we exposed two freshwater fish species, the zebrafish (Danio rerio) and perch (Perca fluviatilis) for 21 days to polyethylene microplastics (PE-MPs) sized 10–45 μm and 106–125 μm. Thereafter, in the liver and gills tissues, biochemical and molecular parameters and the metabolic profile were examined. Ex-vivo characterization by ATR-FTIR spectroscopy exhibited increased concentration of 10–45 μm PE-MPs in the liver of the two fish species while 106–125 μm PE-MPs mostly concentrated in fish gills. The penetration of PE-MPs to fish and the induced oxidative stress triggered changes in lipid peroxidation, DNA damage and ubiquitination and furthermore stimulated signal transduction pathways leading to autophagy and apoptosis. The smaller PE-MPs were more potent in inducing alterations to all the latter parameters measured than the larger ones. Tissue response in both fish seems to depend on the parameter measured and does not seem to follow a specific pattern. Our results showed that there is no clear sensitivity of one fish species versus the other, against both sizes of PE-MPs they were exposed. In perch the metabolic changes in gills were distinct to the ones observed in liver, following a size dependent pattern, indicating that stress conditions are generated through different mechanisms. All the parameters employed can be suggested further as biomarkers in biomonitoring studies against PE-MPs.

Photodynamic therapy fortified with topical oleyl alcohol-based transethosomal 8-methoxypsoralen for ameliorating vitiligo: Optimization and clinical study

Vitiligo is a common autoimmune skin disorder that is characterized by patchy depigmentation of the skin due to melanocytes and melanin loss. Herein, photodynamic therapy mediated 8-methoxypsoralen (8-MOP), has been used fortified with topical oleyl alcohol-based transethosomes; to overcome the poor solubility and adverse effects associated with 8-MOP oral delivery. A 23 factorial design was used to study the formulation variables. In vitro and ex-vivo characterization besides a clinical study were conducted to assess therapeutic efficacy of the formulation. Results revealed that transethosomes were superior to transfersomes regarding drug protection from degradation. The optimized transethosomal formulation, composed of 50 mg oleyl alcohol, 10 mg Tween 80® and 20% v/v ethanol, exhibited high entrapment efficiency (83.87 ± 4.1%) and drug loading (105.0 ± 0.2%). Moreover, it showed small vesicular size (265.0 ± 2.9 nm) and PDI (0.19). The formulation depicted core and shell structure, high deformability index (12.45 ± 0.7 mL/s) and high ex-vivo skin permeation. The topical application of the developed 8-MOP transethosomal gel enhanced the effect of NB UVB radiation in the treatment of vitiligo patients and exhibited no side effects. Hence, it can be used as a future strategy for delivering 8-MOP without the need of systemic application.

Design, formulation and optimization of topical ethosomes using full factorial design: in-vitro and ex-vivo characterization

The present study aimed to develop lomefloxacin-loaded ethosomal vesicles intended to be applied topically for treating skin infections. Ethosomes were prepared using the cold method. The formulation variables were optimized using 22 factorial design and Design Expert® software for analyzing the data statistically and graphically using response surface plots. Phosphatidylcholine (X1) and ethanol (X2) were chosen as the independent variables, while the dependent variables comprised entrapment efficiency (Y1), vesicles size (Y2) and zeta potential (Y3). The optimized ethosomes were subsequently incorporated into Carbopol® 940 gel and characterized for rheological behaviour, in-vitro release, ex-vivo skin permeation and deposition. The ex-vivo permeation and skin deposition studies showed better results compared to drug solutions. In a nutshell, the ethosomal vesicles were found to be a promising carrier demonstrating enhanced topical delivery of lomefloxacin.

In-vivo and Ex-vivo Characterization of Estrogen Receptor α (ERα)-Mediated Effects on the Pulmonary Vasculature in PH

In-vivo and Ex-vivo Characterization of Estrogen Receptor α (ERα)-Mediated Effects on the Pulmonary Vasculature in PH

Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell-Derived (hESC) Islet-Like Clusters Posttransplantation.

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

Development of rasagiline mesylate loaded solid lipid nanoparticles in a thermosensitive mucoadhesive gel: Formulation design using DoE, in-vitro and ex-vivo characterization

Development of rasagiline mesylate loaded solid lipid nanoparticles in a thermosensitive mucoadhesive gel: Formulation design using DoE, in-vitro and ex-vivo characterization

In-vitro and ex-vivo characterization of novel mannosylated gelatin nanoparticles of linezolid by quality-by-design approach

The present study was aimed to develop and characterize novel linezolid (LNZ) loaded mannosylated gelatin nanoparticles (Mn-GNPs) using the Quality by Design (QbD) approach. The quality target product profile (QTPP) of the nanoparticulate formulation was defined and critical quality attributes (CQAs) were identified. The risk assessment and factor screening was done by Taguchi orthogonal array design to find out the most impacted factors affecting CQAs. A 33 Box-Behnken Design (BBD) was employed to further optimize the identified variables. The optimized LNZ loaded gelatin nanoparticles (GNPs) were then surface-functionalized with mannose (Mn-GNPs) for selective targeting to alveolar macrophages. The particle size of the GNPs and Mn-GNPs were found to be 235.3 ± 11.3 nm with 0.127 ± 0.02 polydispersity index (PDI) and 379 ± 21.7 nm with 0.148 ± 0.03 PDI, while the percent entrapment efficiency (%EE) was 56.8 ± 2.1% and 51.3 ± 3.6%, respectively. Moreover, an in-vitro drug release study was performed and has shown the sustained drug release from both the formulations. This was further confirmed by kinetic studies. The formulations were also screened for ex-vivo haemolytic toxicity study to ensure the in-vivo safety. Thus, these studies revealed the importance and applicability of the QbD approach for the systemic development of nanoparticulate formulations.

Curcumin loaded nanostructured lipid carriers for enhanced skin retained topical delivery: optimization, scale-up, in-vitro characterization and assessment of ex-vivo skin deposition

Nanostructured lipid carriers (NLC) have become a promising drug delivery system for topical delivery of drugs. Delivery of lipophilic drugs with improved stability and entrapment efficiency is one of the foremost benefits of NLC based formulations. The objective of the present study was to improve the permeation of poorly soluble curcumin into topical skin layers for the treatment of chronic inflammatory disorder psoriasis and microbial mediated acne vulgaris. Hot emulsification followed by probe sonication method was employed for the preparation of the curcumin loaded NLC. Further, in-vitro and ex-vivo characterization was performed for designed NLC. The designed NLC showed a mean particle size 96.2 ± 0.9 nm, entrapment efficiency of 70.5 ± 1.65% and zeta potential of -15.2 ± 0.566 mV. Curcumin-NLC showed extended in-vitro release upto 48 hours, whereas free curcumin showed 100% drug release within 4 hours. Ex-vivo skin permeation studies revealed 3.24 fold improved permeation and skin retention in the case of curcumin loaded NLC gel compared to free curcumin gel. The cell viability studies demonstrated the formulation components showed no toxicity towards keratinocyte cells. In keratinocyte cells, improved cell uptake was observed for curcumin-NLC compared to free curcumin dispersion. The results suggested that the NLC based formulation had potential to improve the efficacy of curcumin.

Chronic neural interfacing with cerebral cortex using single-walled carbon nanotube-polymer grids

Objective. The development of electrode arrays able to reliably record brain electrical activity is a critical issue in brain machine interface (BMI) technology. In the present study we undertook a comprehensive physico-chemical, physiological, histological and immunohistochemical characterization of new single-walled carbon nanotubes (SWCNT)-based electrode arrays grafted onto medium-density polyethylene (MD-PE) films. Approach. The long-term electrical stability, flexibility, and biocompatibility of the SWCNT arrays were investigated in vivo in laboratory rats by two-months recording and analysis of subdural electrocorticogram (ECoG). Ex-vivo characterization of a thin flexible and single probe SWCNT/polymer electrode is also provided. Main results. The SWCNT arrays were able to capture high quality and very stable ECoG signals across 8 weeks. The histological and immunohistochemical analyses demonstrated that SWCNT arrays show promising biocompatibility properties and may be used in chronic conditions. The SWCNT-based arrays are flexible and stretchable, providing low electrode-tissue impedance, and, therefore, high compliance with the irregular topography of the cortical surface. Finally, reliable evoked synaptic local field potentials in rat brain slices were recorded using a special SWCNT-polymer-based flexible electrode. Significance. The results demonstrate that the SWCNT arrays grafted in MD-PE are suitable for manufacturing flexible devices for subdural ECoG recording and might represent promising candidates for long-term neural implants for epilepsy monitoring or neuroprosthetic BMI.

UV spectrophotometric method for simultaneous estimation of betamethasone valerate and tazarotene with absorption factor method: Application for in-vitro and ex-vivo characterization of lipidic nanocarriers for topical delivery.

The present study elucidates the development of an accurate, precise and simple simultaneous estimation method for the routine analysis of Betamethasone Valerate (BV) and Tazarotene (TZ). This combination is widely used in the treatment of psoriasis. No method has been reported so far for the simultaneous estimation of BV and TZ in topical dosage forms. The method proposed by this study for the quantification of BV and TZ is the Absorption factor method. The developed method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline. The validated method was found to be linear in a concentration range of 10-38μg/mL and 4-14μg/mL for BV and TZ respectively with a regression coefficient >0.990. The method was validated for accuracy and precision which revealed the recovery of >99.80% with RSD <2.0. The method was found to be precise with RSD <2% for inter and intraday. The developed method was employed for quantification of BV and TZ in lipid based nanocarriers formulation and their in-vitro drug release samples. Further, the developed method was successfully applied for the estimation of BV and TZ in the ex-vivo skin matrix. This showed that the method can sensitively determine the drugs in aqueous and biological samples.

UV Spectrophotometric method for characterization of curcumin loaded nanostructured lipid nanocarriers in simulated conditions: Method development, in-vitro and ex-vivo applications in topical delivery.

Curcumin the extract obtained from the dried rhizome of turmeric, Curcuma longa is a hydrophobic phenol that delivers numerous pharmacological actions like anti-inflammatory, anti-microbial and anti-oxidant, anti-psoriasis, antidiabetic, anticancer. But curcumin has low bioavailability issues that accompany low aqueous solubility, further, when administered orally, >90% of the drug degrades rapidly in the alkaline medium. Administering the drug topically can bypass the problem as well as first-pass metabolism and therefore delivering the drug at the targeted site of action. Encapsulating curcumin in nanostructured lipid nanocarriers (NLC) is an excellent novel strategy. Further, these NLC provides both the controlled release and helps in the enhanced permeation of the drug through the skin's physiological barrier, stratum corneum. For the NLC characterization, a reliable method must be developed that can accurately and precisely determine the drug content in the formulation and also for its in-vitro and ex-vivo characterization. This experiment describes the analytical validation parameters described as per International Conference of Harmonization guidelines to develop a method using the UV-Visible spectroscopy. The method was developed in two solvent systems i.e. methanol and 6.4 pH phosphate buffer with 1.5% polysorbate 80. Methanol solvent was used for the determination of curcumin in the NLC formulation via determining the encapsulation efficiency and 6.4 pH phosphate buffer with 1.5% polysorbate 80 solvent was used for in-vitro and ex-vivo characterization of the developed NLC formulation (cream and gel). These methods were validated in response to linearity, the limit of detection, the limit of quantification, precision, accuracy, repeatability, and specificity.

Development of Tizanidine HCl transdermal patches: In-vitro and Ex-vivo characterization

The purpose of this work was to design and evaluate matrix type transdermal patches of Tizanidine hydrochloride using Hypromellose (HPMC E15) as polymer, dibutyl phthalate as plasticizer and citral as permeation enhancer. The DSC and FTIR results showed the compatibility of the excipients with the drug. These transdermal drug delivery systems were characterized for their thickness, folding endurance, content uniformity, tensile strength and in-vitro release studies of the drug from the polymeric matrix. In-vitro release studies and ex-vivo permeation were carried out with modified Franz diffusion cell using pH 5.8 &amp; pH 7.4 phosphate buffers as receptor medium and it showed controlled release of drug. The results suggest that the formulation of TIZ may be useful in the development of a therapeutic system to deliver TIZ across the skin for a prolonged period, i.e. 24 hr.&#x0D; Keywords: Tizanidine Hydrochloride, Transdermal patch, HPMC E15, in-vitro &amp; ex-vivo.

Intestinal IFN-γ–producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease

IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (TR1) cells but is also produced by CD25+ regulatory T (Treg) cells. We aimed to identify and characterize human intestinal TR1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). CD4+ T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4+ T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed. Intestinal TR1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5+PD-1+ TR1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ+ TR1 cells, but not IL-7 receptor-positive TH cells or CD25+ Treg cells, showed lower IL-10 expression in patients with IBDs. TR1 cells were responsive to IL-23, and IFN-γ+ TR1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25+ Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. We provide the first exvivo characterization of human intestinal TR1 cells. Selective downregulation of IL-10 by IFN-γ+ TR1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.

Surgical stress response and promotion of metastasis in colorectal cancer: a complex and heterogeneous process.

Surgery remains the curative treatment modality for colorectal cancer in all stages, including stage IV with resectable liver metastasis. There is emerging evidence that the stress response caused by surgery as well as other perioperative therapies such as anesthesia and analgesia may promote growth of pre-existing micro-metastasis or potentially initiate tumor dissemination. Therapeutically targeting the perioperative period may therefore reduce the effect that surgical treatments have in promoting metastases, for example by combining β-adrenergic receptor antagonists and cyclooxygenase-2 (COX-2) inhibitors in the perioperative setting. In this paper, we highlight some of the mechanisms that may underlie surgery-related metastatic development in colorectal cancer. These include direct tumor spillage at the time of surgery, suppression of the anti-tumor immune response, direct stimulatory effects on tumor cells, and activation of the coagulation system. We summarize in more detail results that support a role for catecholamines as major drivers of the pro-metastatic effect induced by the surgical stress response, predominantly through activation of β-adrenergic signaling. Additionally, we argue that an improved understanding of surgical stress-induced dissemination, and more specifically whether it impacts on the level and nature of heterogeneity within residual tumor cells, would contribute to the successful clinical targeting of this process. Finally, we provide a proof-of-concept demonstration that ex-vivo analyses of colorectal cancer patient-derived samples using RGB-labeling technology can provide important insights into the heterogeneous sensitivity of tumor cells to stress signals. This suggests that intra-tumor heterogeneity is likely to influence the efficacy of perioperative β-adrenergic receptor and COX-2 inhibition, and that ex-vivo characterization of heterogeneous stress response in tumor samples can synergize with other models to optimize perioperative treatments and further improve outcome in colorectal and other solid cancers.