Bioorthogonal non-canonical amino acid tagging to track transplanted human induced pluripotent stem cell-specific proteome.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo. METHODS: In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts. RESULTS: We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs. CONCLUSION: The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

Fabrication and Optimization of Poly(ε-caprolactone) Microspheres Loaded with S-Nitroso-N-Acetylpenicillamine for Nitric Oxide Delivery.

Heart disease is one of the leading causes of death in the United States and throughout the world. While there are different techniques for reducing or preventing the impact of heart disease, nitric oxide (NO) is administered as nitroglycerin for reversing angina or chest pain. Unfortunately, due to its gaseous and short-lived half-life, NO can be difficult to study or even administer. Therefore, controlled delivery of NO is desirable for therapeutic use. In the current study, the goal was to fabricate NO-releasing microspheres (MSs) using a donor molecule, S-Nitroso-N-Acetyl penicillamine, (SNAP), and encapsulating it in poly(ε-caprolactone) (PCL) using a single-emulsion technique that can provide sustained delivery of NO to cells over time without posing any toxicity risks. Optimization of the fabrication process was performed by varying the duration of homogenization (5, 10, and 20 min) and its effect on entrapment efficiency and size. The optimized SNAP-MS had an entrapment efficiency of ˃50%. Furthermore, we developed a modified method for NO detection by using NO microsensors to detect the NO release from SNAP-MSs in real time, showing sustained release behavior. The fabricated SNAP-MSs were tested for biocompatibility with HUVECs (human umbilical vein endothelial cells), which were found to be biocompatible. Lastly, we tested the effect of controlled NO delivery to human induced pluripotent stem-derived cardiomyocytes (hiPSC-CMs) via SNAP-MSs, which showed a significant improvement in the electrophysiological parameters and alleviated anoxic stress.

A highlight on carbamazepine-induced adverse drug reactions in Saudi Arabia: a retrospective medical records-based study.

The link between human leukocyte antigen (HLA) alleles and carbamazepine-induced cutaneous, respiratory, and gastrointestinal adverse drug reactions (ADR) has created a window of opportunity for preventing certain forms of cutaneous adverse drug reactions (cADRs); however, there is not enough data to make pharmacogenomic recommendations that can be implemented globally. The aim of this study is to assess and document carbamazepine-induced adverse reactions among prescribed Saudi/non-Saudi patients. A retrospective chart review was performed for patients who received carbamazepine (CBZ) in the period between 2016 and 2020, in the Kingdom of Saudi Arabia. Data were gathered and descriptive statistical analyses were performed on the data for the study sample. Comparisons were made using the chi-square test or independent samples' t-test. Statistical significance was considered at p < .05. All statistical analyses were performed using IBM SPSS 21.0 (Armonk, NY; IBM Corp). Results from multivariate logistic regression analyses showed that higher likelihood of carbamazepine-induced adverse reactions was significantly associated with younger age, OR = 0.82, 95% CI (0.74, 0.90); p < 0.001. Patients who were prescribed CBZ for reasons other than epilepsy or seizures were significantly more likely to develop carbamazepine-induced adverse reactions (epilepsy vs. other; OR = 0.63, p = 0.013; seizures vs. other; OR = 0.59, p = 0.018). Gender or medication duration were not related to carbamazepine-induced adverse reactions (p > 0.05). The findings of this study are comparable with those of other studies assessing carbamazepine-associated adverse reactions in children and adults. Recommendations include genetic prescreening, educating patients and parents on the possibility of adverse reactions, and routine laboratory monitoring.

Preclinical Large Animal Porcine Models for Cardiac Regeneration and Its Clinical Translation: Role of hiPSC-Derived Cardiomyocytes.

Myocardial Infarction (MI) occurs due to a blockage in the coronary artery resulting in ischemia and necrosis of cardiomyocytes in the left ventricular heart muscle. The dying cardiac tissue is replaced with fibrous scar tissue, causing a decrease in myocardial contractility and thus affecting the functional capacity of the myocardium. Treatments, such as stent placements, cardiac bypasses, or transplants are beneficial but with many limitations, and may decrease the overall life expectancy due to related complications. In recent years, with the advent of human induced pluripotent stem cells (hiPSCs), newer avenues using cell-based approaches for the treatment of MI have emerged as a potential for cardiac regeneration. While hiPSCs and their derived differentiated cells are promising candidates, their translatability for clinical applications has been hindered due to poor preclinical reproducibility. Various preclinical animal models for MI, ranging from mice to non-human primates, have been adopted in cardiovascular research to mimic MI in humans. Therefore, a comprehensive literature review was essential to elucidate the factors affecting the reproducibility and translatability of large animal models. In this review article, we have discussed different animal models available for studying stem-cell transplantation in cardiovascular applications, mainly focusing on the highly translatable porcine MI model.

Sweat Cortisol and Cortisone Determination in Healthy Adults: UHPLC-MS/MS Assay Validation and Clinical Application.

A simple and effective ultra-high-performance liquid chromatography assay linked to tandem mass spectrometry (UHPLC-MS/MS) for measuring cortisol and cortisone levels in human sweat has been developed and validated. A noninvasive world standard sweat collecting equipment was utilized to collect samples. The samples were analyzed using an Atlantis dC18 (2.1 × 100 mm, 3 µm) column with a 2 mM ammonium acetate and acetonitrile (1 : 1, v : v) mobile phase. In an isocratic condition, the mobile phase was delivered at a flow rate of 0.3 ml/minute. A positive electrospray ionization interface with multiple-reaction monitoring mode was used to provide simultaneous quantification of cortisol, cortisone, and internal standard at transitions of 363.11 to 121.00, 361.18 to 163.11, and 367.19 to 121.24, respectively. The method was validated for cortisol and cortisone determination over a concentration range of 0.5-50 ng/mL The detection limits for cortisol and cortisone in human sweat were 0.3 and 0.2 ng/ml, respectively. The interday coefficients of variation of cortisol and cortisone were ≤8.5% and ≤10.01%, whereas bias was in the range from -7.9% to 2.1% and from -4.3% to 3.0%, respectively. The assay was successfully applied to evaluate the cortisol-to-cortisone ratio in sweat samples collected from healthy adult volunteers.

Modulation of Mitochondrial Bioenergetics by Polydopamine Nanoparticles in Human iPSC-Derived Cardiomyocytes.

Myocardial infarction (MI) leads to the formation of an akinetic scar on the heart muscle causing impairment in cardiac contractility and conductance, leading to cardiac remodeling and heart failure (HF). The current pharmacological approaches for attenuating MI are limited and often come with long-term adverse effects. Therefore, there is an urgent need to develop novel multimodal therapeutics capable of modulating cardiac activity without causing any major adverse effects. In the current study, we have demonstrated the applicability of polydopamine nanoparticles (PDA-NPs) as a bioactive agent that can enhance the contractility and beat propagation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Treatment of hiPSC-CMs with PDA-NPs demonstrated accumulation of the latter into mitochondria and significantly enhanced time-dependent adenosine triphosphate (ATP) production in these cells, indicating improved mitochondrial bioenergetics. Furthermore, the effect of PDA-NPs on hiPSC-CM activity was evaluated by measuring calcium transients. Treatment with PDA-NPs increased the calcium cycling in hiPSC-CMs in a temporal manner. Our results demonstrated a significant reduction in peak amplitude, transient duration, time to peak, and transient decay time in the PDA-NPs-treated hiPSC-CMs as compared to untreated hiPSC-CMs. Additionally, treatment of isolated perfused rat heart ex vivo with PDA-NPs demonstrated cardiotonic effects on the heart and significantly improved the hemodynamic function, suggesting its potential for enhancing whole heart contractility. Lastly, the gene expression analysis data revealed that PDA-NPs significantly upregulated cardiac-specific genes (ACADM, MYL2, MYC, HCN1, MYL7, GJA5, and PDHA1) demonstrating the ability to modulate genetic expression of cardiomyocytes. Taken together, these findings suggest PDA-NPs capability as a versatile nanomaterial with potential uses in next-generation cardiovascular applications.

Identifying stable reference genes in polyethene glycol precipitated urinary extracellular vesicles for RT-qPCR-based gene expression studies in renal graft dysfunction patients.

BACKGROUND: Urinary extracellular vesicles (UEVs) hold RNA in their cargo and are potential sources of biomarkers for gene expression studies. The most used technique for gene-expression studies is quantitative polymerase chain reaction (qPCR). It is critical to use stable reference genes (RGs) as internal controls for normalising gene expression data, which aren't currently available for UEVs. METHODS: UEVs were precipitated from urine of graft dysfunction patients and healthy controls by Polyethylene glycol, Mn6000 (PEG6K). Vesicular characterisation confirmed the presence of UEVs. Gene expression levels of five commonly used RGs, i.e., Beta-2-Microglobulin (B2M), ribosomal-protein-L13a (RPL13A), Peptidylprolyl-Isomerase-A (PPIA), hydroxymethylbilane synthase (HMBS), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were quantified, and their stability was established through the RefFinder. The stability of identified RGs was validated by quantification of Perforin and granzyme B, signature molecules of renal graft dysfunction. RESULTS: Urine precipitated with 12% 6 K PEG yielded round and double-membraned UEVs of size ranging from 30 to 100 nm, as confirmed through transmission electron microscopy. Nanoparticle tracking analysis (59 ± 22 nm) and Dynamic-light-scattering (78 ± 56.5 nm) confirmed their size profile. Semi-quantitative Exocheck antibody array demonstrated the presence of EV protein markers in UEV. Using the comparative ΔCт method and RefFinder analysis, B2M (1.6) and RPL13A (1.8) genes emerged as the most stable reference genes. Validation of target gene expression in renal graft dysfunction patients confirmed the efficiency of B2M and RPL13A through significant upregulation compared to other RGs. CONCLUSIONS: Our study identified and validated B2M and RPL13A as optimal RGs for mRNA quantification studies in the UEVs of patients with renal graft dysfunction.

A One-Stop Protocol to Assess Myocardial Fibrosis in Frozen and Paraffin Sections.

Masson's Trichrome Staining (MTS) is a useful tool for analyzing fibrosis in a plethora of disease pathologies by differential staining of tissue components. It is used to identify collagen fibers in different tissues like heart, lung, skin, and muscles. Especially in cardiac fibrosis, MTS stains the collagen fibers (blue color), which helps in the distinction of scar area versus the healthy area (red color). However, there are several challenges to stain both paraffin-embedded sections and frozen (cryosections) using a single protocol. Therefore, the goal of this study was to develop a simple short protocol to assess cardiac fibrosis in both paraffin-embedded and cryo heart sections. MTS uses three different stains, i.e., Weigert's Iron Hematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue to detect nuclei, cytoplasm, and collagen, respectively. In this study, we developed a simple short protocol that can be adapted by any lab to easily assess cardiac fibrosis in paraffin and frozen heart sections. Furthermore, we have addressed the challenges that are commonly faced during the immunostaining process and troubleshooting techniques. Overall, we have successfully developed a simple one-step protocol to assess myocardial fibrosis in paraffin-embedded and frozen cryosections.

Modeling lay people's ethical views on abortion: A Q-methodology study.

BACKGROUND: It isn't clear how lay people balance the various ethical interests when addressing medical issues. We explored lay people's ethical resolution models in relation to abortion. METHODS: In a tertiary healthcare setting, 196 respondents rank-ordered 42 opinion-statements on abortion following a 9-category symmetrical distribution. Statements' scores were analyzed by averaging-analysis and Q-methodology. RESULTS: Respondents' mean (SD) age was 34.5(10.5) years, 53% were women, 68% Muslims (31% Christians), 28% Saudis (26% Filipinos), and 38% healthcare-related. The most-agreeable statements were "Acceptable if health-benefit to woman large," "Acceptable if congenital disease risk large," and "Woman's right if fetus has congenital disease." The most-disagreeable statements were "State's right even if woman disagrees," "Acceptable even with no congenital disease risk," and "Father's right even if woman disagrees." Q-methodology identified several resolution models that were multi-principled, consequentialism-dominated, and associated with respondents' demographics. The majority of Christian women and men identified with and supported a relatively "fetus rights plus State authority-oriented" model. The majority of Muslim women and men identified with and supported a "conception-oriented" model and "consequentialism plus virtue-oriented" model, respectively. One or more of three motives-related statements received extreme ranks on averaging-analysis and in 33% of the models. CONCLUSIONS: 1) On average, consequentialism, focusing on a woman's health-benefit and congenital disease risk, was the predominant approach. This was followed by the rights approach, favoring a woman's interest but taking context into account. 2) Q-methodology identified various ethical resolution models that were multi-principled and partially associated with respondents' demographics. 3) Motives were important to some respondents, providing empirical evidence against adequacy of principlism.

In Situ Nanotransformable Hydrogel for Chemo-Photothermal Therapy of Localized Tumors and Targeted Therapy of Highly Metastatic Tumors.

Metastasis is one of the predisposing factors for cancer-related mortalities worldwide. Patients with advanced cancers (stage IV) receive palliative care with minimal possibility of achieving complete remission. Antibody-based therapeutic modalities are capable of targeting tumors that are confined to a particular location but are ineffective in targeting distant secondary tumors. In the current study, we have developed a smart nano-transforming hydrogel (NTG) that transforms in situ to polymeric nanoparticles (PA NPs) of 100-150 nm when injected subcutaneously. These nanoparticles targeted the primary and secondary metastatic tumors for up to ∼5 and ∼3 days, respectively. The in situ-formed PA NPs also demonstrated a pH-responsive drug release resulting in about ∼80% release within 100 h at 5.8 pH. When tested in vivo, substantial inhibition of lung metastases was observed compared to chemotherapy, thus demonstrating the efficiency of nanotransforming hydrogels in targeting and inhibiting primary and secondary metastatic tumors.

De novo Drug Delivery Modalities for Treating Damaged Hearts: Current Challenges and Emerging Solutions.

Cardiovascular disease (CVD) is the leading cause of mortality, resulting in approximately one-third of deaths worldwide. Among CVD, acute myocardial infarctions (MI) is the leading cause of death. Current treatment modalities for treating CVD have improved over the years, but the demand for new and innovative therapies has been on the rise. The field of nanomedicine and nanotechnology has opened a new paradigm for treating damaged hearts by providing improved drug delivery methods, specifically targeting injured areas of the myocardium. With the advent of innovative biomaterials, newer therapeutics such as growth factors, stem cells, and exosomes have been successfully delivered to the injured myocardial tissue, promoting improvement in cardiac function. This review focuses on three major drug delivery modalities: nanoparticles, microspheres, and hydrogels, and their potential for treating damaged hearts following an MI.

Self-Assembled Fluorosome-Polydopamine Complex for Efficient Tumor Targeting and Commingled Photodynamic/Photothermal Therapy of Triple-Negative Breast Cancer.

Photodynamic/photothermal therapy (PDT/PTT) that deploys a near-infrared responsive nanosystem is emerging to be a promising modality in cancer treatment. It is highly desirable to have a multifunctional nanosystem that can be used for efficient tumor targeting and inhibiting metastasis/recurrence of cancer. In the current study, self-assembled chlorophyll-rich fluorosomes derived from Spinacia oleracea were developed. These fluorosomes were co-assembled on a polydopamine core, forming camouflaged nanoparticles (SPoD NPs). The SPoD NPs exhibited a commingled PDT/PTT (i.e., interdependent PTT and PDT) that inhibited both normoxic and hypoxic cancer cell growth. These nanoparticles showed stealth properties with enhanced physiological stability and passive tumor targeting. SPoD NPs also exhibited tumor suppression by synergistic PTT and PDT. It also prevented lung metastasis and splenomegaly in tumor-bearing Balb/c mice. Interestingly, treatment with SPoD NPs also caused the suppression of secondary tumors by eliciting an anti-tumor immune response. In conclusion, a co-assembled multifunctional nanosystem derived from S. oleracea showed enhanced stability and tumor-targeting efficacy, resulting in a commingled PDT/PTT effect.

Quality assessment of nine paracetamol 500 mg tablet brands marketed in Saudi Arabia.

OBJECTIVE: To evaluate in-vitro quality of paracetamol 500 mg tablet brands marketed in Saudi Arabia. RESULTS: Two reference (R1 and R2) and seven generic (G1-G7) brands were commercially available. Four brands were single-drug, containing paracetamol only (R1, G1-G3) and five contained additional active ingredients (R2, G4-G7). All brands were immediate-release. Weight variation (n = 20, range as percent difference from mean), active substance content (n = 20, mean (SD) as percent difference from label), breaking force (n = 10, mean (SD)), and friability (n = 20, as percent weight loss) ranged from 97 to 102%, 96.1% (2.9%) to 99.8% (1.1%), 9.9 (0.4) to 21.0 (0.9) kg, and 0.017% to 0.809%, respectively. Disintegration (water medium) time (n = 6, minute: second) ranged from 02:35-03:09 to 12:49-13:10. Dissolution (phosphate buffer, pH 5.8) profile showed a mean release at 30 min of 87% to 97% of label content, with seven brands passing stage-1 (≥ 85% for each of 6 test units) and two passing stage-2 (mean of 12 test units ≥ 85%) criteria. Despite statistically significant differences between R1 and R2 and some of their corresponding generic brands in active substance content, breaking force, and amount dissolved at 30 min, all nine brands met the pre-specified quality standards.

Iontophoresis mediated localized delivery of liposomal gold nanoparticles for photothermal and photodynamic therapy of acne.

Acne is one of the common dermatological skin inflammatory conditions. The current therapeutic modalities for the treatment of acne include the administration of antibiotics and anti-inflammatory agents. The rising instance of antibiotic resistance in acne strains has led to the exploration of alternative therapeutic modalities. In the current study, we have employed a liposomal gold nanoparticle entrapping curcumin (Au Lipos Cur NPs) for dual light-mediated therapy for the treatment of acne. These nanoparticles exerted a positive zeta potential that enabled their localized follicular delivery by iontophoresis. The localized deposition of Au Lipos NPs leads to photothermal transduction causing destruction of sebaceous glands. Furthermore, when the nanoparticles were assessed in vitro by sequential irradiation with NIR and blue light, it resulted in significant inhibition of bacterial growth. Thus the dual light-mediated therapy by Au Lipos Cur NPs can form a potential therapeutic modality for the efficient treatment of recurrent acne.

Eight enteric-coated 50 mg diclofenac sodium tablet formulations marketed in Saudi Arabia: in vitro quality evaluation.

OBJECTIVE: To evaluate in vitro quality of enteric-coated 50 mg diclofenac sodium tablet formulations on Saudi market. RESULTS: A reference and seven generic (G1-7) formulations were commercially available in December 2019/January 2020 and were assessed within 25-75% of manufacture-expiration period. Weight variation (range as% difference from mean, n = 20), active substance content (ASC, mean (SD) as% difference from label, n = 20), hardness (mean (SD), n = 10), and friability (% weight loss, n = 20) were 97-103%, 102.0% (3.4%), 15.4 (1.1) kg, and 0.24%, respectively, for the reference. For G2-7, they were ≤ ±5%, 98.6% (4.0%) to 109.9% (1.8%), 11.9 (0.9) to 18.3 (0.8) kg, and ≤ 0.00 to 0.75%, respectively. G1 ASC, hardness, and friability were 111.3% (1.7%), 20.1 (1.7) kg, and 1.10%, respectively. Disintegration time (n = 6) and dissolution profile (n = 8) were also determined. No formulation disintegrated or released ˃ 0.1% of label ASC in 0.1 N HCl for 2 h. The reference disintegrated in 15:00 min:seconds and released a mean (range) of 100% (99-103%) of label ASC by 45 min in phosphate buffer (pH = 6.8). G1-7 disintegrated in 8:53 to 20:37 min:seconds and released 81% (69-90%) (G1) to 109%. Except for borderline performance of G1, all formulations passed in vitro quality tests according to United States Pharmacopoeia.

NIR-dye based mucoadhesive nanosystem for photothermal therapy in breast cancer cells.

Breast cancer is one of the leading causes of mortality in women, worldwide. The average survival rate of patients suffering from advanced breast cancer is about 27% for five years. Photothermal therapy employing biodegradable nanoparticle are extensively researched for enhanced anticancer therapy in breast cancer treatment. In the current study, we report a chitosan based mucoadherant and biodegradable niosome nanoparticle entrapping near infrared (NIR) dye (IR 806) for the treatment of breast cancer. Niosome entrapping IR 806 (NioIR) showed encapsulation efficacy of about 56 ± 2%. The prepared nanoparticles (NioIR) were further coated with chitosan (NioIR-C) to impart mucoadhesive property to the nanosystem. NioIR-C showed minimal degradation following NIR laser irradiation, thus enhancing its photothermal stability. They also exhibited efficient photothermal transduction, when compared with IR 806 dye. NioIR-C were biocompatible when treated with normal cell lines (NIH 3T3 and L929) and showed cytotoxicity towards breast cancer cell lines (MCF-7 and MDA-MB 231). When triggered with NIR laser, NioIR-C showed photothermal cell death (approximately 93%). The presence of chitosan coating on NioIR led to mucoadherence potential that further enhances the therapeutic effect on breast cancer cells when compared with IR 806 dye and NioIR. Thus NioIR-C can be a promising nanosystem for effective treatment of breast cancer using photothermal therapy.

An improved method for measurement of testosterone in human plasma and saliva by ultra-performance liquid chromatography-tandem mass spectrometry.

The aim of the study was to develop and validate a practical assay of clinically relevant testosterone levels in human plasma and saliva. We performed ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis on Atlantis dC18 steel column using a mobile phase of 2-mM ammonium acetate and acetonitrile (20:80, v: v) that was delivered at 0.3 ml/min. After adding d3-testosterone as an internal standard (IS), we extracted plasma and salivary samples with methyl tert-butyl ether. Mass spectrometry was performed in electrospray positive-ion mode. Targeted ion transitions were examined at m/z 289.18 → 97.04 and 292.24 → 97.04 for testosterone and IS, respectively. We validated the method according to the US Food and Drug Administration guidelines. Elution times for testosterone and IS were both around 1.35 min. Testosterone level was linearly associated (r 2 = 0.9975 and 0.9958) with peak area ratio of testosterone to IS between 0.5-50 ng/ml and 10-400 pg/ml in plasma and saliva, respectively. The coefficient of variation and bias were ≤12.6% and ≤±12.1% in plasma and ≤10.2% and ≤±5.3% in saliva. The extraction recovery of testosterone was ≥92% from plasma and ≥94% from saliva. Testosterone was stable (≥91%) for 24 h at room temperature and for 8 weeks at -20°C in both plasma and salivary samples. We report a simple, validated, UPLC-MS/MS assay that can be used to determine clinically relevant levels of testosterone in human plasma and saliva.

Light-triggered selective ROS-dependent autophagy by bioactive nanoliposomes for efficient cancer theranostics.

Light-responsive nanoliposomes are being reported to induce cancer cell death through heat and reactive oxygen species (ROS). Nanoliposomes (CIR NLPs) encapsulating a near-infrared (NIR) light-sensitive dye, IR780, and a bioactive chlorophyll-rich fraction of Anthocephalus cadamba (CfAc) were synthesized and characterized. These CIR NLPs, when activated by NIR light, displayed localized synergistic cancer cell death under in vitro and in vivo conditions. We demonstrated a NIR light-mediated release of CfAc in cancer cells. The bioactive CfAc was selective in causing ROS generation (leading to autophagic cell death) in cancer cells, while normal healthy cells were unaffected. An increase in the intracellular ROS leading to enhanced lipidation of microtubule-associated protein light chain 3 (LC3-II) was observed only in cancer cells, while normal cells showed no increase in either ROS or LC3-II. In vivo analysis of CIR NLPs in an orthotopic mouse model showed better anti-tumorigenic potential through a combined effect (i.e. via heat and CfAc). We reported for the first time induction of selective and localized, bioactive phyto fraction-mediated autophagic cancer cell death through an NIR light trigger. The synergistic activation of ROS-mediated autophagy by light-triggered nanoliposomes can be a useful strategy for enhancing the anticancer potential of combinational therapies.

The "nano to micro" transition of hydrophobic curcumin crystals leading to in situ adjuvant depots for Au-liposome nanoparticle mediated enhanced photothermal therapy.

Photothermal therapy (PTT) is emerging as a promising treatment for skin cancer. Plasmon-resonant gold-coated liposome nanoparticles (Au Lipos NPs) specifically absorb Near Infra-Red (NIR) light resulting in localized hyperthermia (PTT). In the current study, curcumin (a hydrophobic anticancer agent) was entrapped in Au Lipos NPs as nanocrystals to act as an adjuvant for the PTT of melanoma. NIR light irradiation on Au Lipos Cur NPs triggered the release of curcumin nanocrystals which coalesce to form curcumin microcrystals (CMCs). An in situ"nano to micro" transition in the crystal state of curcumin was observed. This in situ transition leads to the formation of CMCs. These CMCs exhibited sustained release of curcumin for a prolonged duration (>10 days). The localized availability of curcumin aids in enhancing PTT by inhibiting the growth and mobility of cancer cells that escape PTT. In the in vitro modified scratch assay, the Au Lipos Cur NP + Laser group showed >1.5 fold enhanced therapeutic coverage when compared with the Au Lipos NP + Laser group. In vivo PTT studies performed in a B16 tumor model using Au Lipos Cur NPs showed a significant reduction of the tumor volume along with the localized release of curcumin in the tumor environment. It was observed that the localized release of curcumin enables an immediate adjuvant effect resulting in the enhancement of PTT.

A Simple Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Measurement of Cortisol Level in Human Saliva.

A simple ultraperformance liquid chromatography-tandem mass spectrometry assay for measurement of cortisol level in human saliva was developed and validated. Saliva samples containing cortisol were spiked with tolperisone as internal standard (IS) and extracted with a mixture of methyl tert-butyl ether and hexane (8:2, v:v). After solvent evaporation, residue was reconstituted in 100 µl mobile phase. Analysis was performed on Atlantis dC18 column (2.1 × 100 mm, 3 µm particle size) with a mobile phase composed of acetonitrile and 2 mM ammonium acetate (50:50, v:v) and delivered at a flow rate of 0.3 ml/minute. Mass spectrometry acquisition was performed with multiple reaction monitoring in positive-ion mode for cortisol and IS (m/z: 363.1 → 121.0 and 246.0 → 97.9, respectively). Retention times of cortisol and IS were about 1.35 and 2.45 minutes, respectively. The relationship between cortisol level and peak area ratio of cortisol to IS was linear in the range of 0.5-100 ng/ml. Intra- and interday coefficient of variation and bias were ≤ 9.0% and ≤12.0%, respectively. Mean extraction recoveries of cortisol and IS from saliva samples were 92% and 94%, respectively. Using the method, cortisol was found to be ≥ 86% stable in processed (24 hours at room temperature or 48 hours at -20°C) and ≥ 91% stable in unprocessed (24 hours at room temperature or 20 weeks at -20°C) saliva samples. Further, the method was successfully applied to determine daily cortisol profile in saliva samples of a healthy volunteer.