Nanocarrier - Mediated Salinomycin Delivery Induces Apoptosis and Alters EMT Phenomenon in Prostate Adenocarcinoma.

Salinomycin (Sal) has been recently discovered as a novel chemotherapeutic agent against various cancers including prostate cancer which is one of the most commonly diagnosed cancers affecting male populations worldwide. Herein we designed salinomycin nanocarrier (Sal-NPs) to extend its systemic circulation and to increase its anticancer potential. Prepared nanoform showed high encapsulation and sustained release profile for salinomycin. The present study elucidated the cytotoxicity and mechanism of apoptotic cell death of Sal-NPs against prostate cancer both in vitro and in vivo. At all measured concentrations, Sal-NPs showed more significant cytotoxicity to DU145 and PC3 cells than Sal alone. This effect was mediated by apoptosis, as confirmed by ROS generation, loss of MMP and cell cycle arrest at the G1 phase in both cells. Sal-NPs efficiently inhibited migration of PC3 and DU145 cells via effectively downregulating the epithelial mesenchymal transition. Also, the results confirmed that Sal-NPs can effectively inhibit the induction of Prostate adenocarcinoma in male Wistar rats. Sal-NPs treatment exhibited a decrease in tumour sizes, a reduction in prostate weight, and an increase in body weight, which suggests that Sal-NPs is more effective than salinomycin alone. Our results suggest that the molecular mechanism underlying the Sal-NPs anticancer effect may lead to the development of a potential therapeutic strategy for treating prostate adenocarcinoma.

The impact of different levels of functional oil supplementation in combination with salinomycin on growth performance and intestinal microbiota of broilers undergoing Eimeria challenge: An analysis of dynamics.

The effect of salinomycin sodium alone and in combination with functional oils on performance and microbiota of broiler infected Eimeria were evaluated. 512 broilers were randomly assigned to 4 treatments (8 replicates, 16 birds/pen): a Control group (any additives); Ionophore group: salinomycin supplementation at 66 ppm (SS66); Ionophore +0.075% Functional oil (FO) group (SS66 + FO supplementation at 750 ppm); and Ionophore +0.10% FO group (SS66 + FO supplementation at 1000 ppm). At 14 days of age, birds were gavaged with 1 mL of a saline solution containing sporulated oocysts of E. tenella, E. acervulina and E. maxima. Performance indices were measured weekly. At 28 days, intestinal content was collected for microbiota analysis. Broilers of Control group presented the worst performance indices. Broilers of Ionophore + FO (0.075% and 0.10%) groups exhibited a higher BW at 28 days of age. The supplementation of Ionophore +0.075% FO resulted in a higher relative proportion of Firmicutes and a lower proportion of Actinobacteria in the ileum-jejunum. Lactobacillaceae was the dominant family in the jejunal, and ileal microbiotas of broilers fed diets supplemented with Ionophore, Ionophore +0.075% FO and Ionophore +0.10% FO. The supplementation of ionophore yielded higher numbers of Lactobacillaceae, Enterobactereaceae and Cloritridiaceae in the cecal. Ionophore associated with FO controlled the Lactobacillaceae, Enterobactereaceae and Cloritridiaceae families present in the cecum. Therefore, the combination of salinomycin with functional oil showed synergistic effect on performance and modulation of intestinal microbiota of broilers challenged with Eimeria.

Evaluation of bexagliflozin in cats with poorly regulated diabetes mellitus.

The aim of this study was to investigate the effect of bexagliflozin on glycemic control in poorly regulated diabetic cats and to evaluate for adverse events associated with this medication. Sodium-glucose cotransporter 2 inhibitors are a newer class of drugs used in the management of humans with type 2 diabetes mellitus. The objective of this study was to evaluate the effect of the orally administered drug, bexagliflozin in a group of poorly regulated diabetic cats over a 4-week study period. Five client-owned cats with poorly controlled diabetes mellitus receiving insulin therapy were enrolled. Bexagliflozin was administered once daily. Serum fructosamine, serum biochemistry profile, and 10-hour blood glucose curves were assessed at baseline (Day 0), Day 14, and Day 28. All cats had a significant reduction in insulin dose requirement (P = 0.015) and insulin was discontinued in 2 cats. There was a significant decrease in blood glucose concentration obtained from blood glucose concentration curves during the study period (P = 0.022). Serum fructosamine decreased in 4 of the 5 cats with a median decrease of 152 µmol/L (range: 103 to 241 µmol/L), which was not statistically significant (P = 0.117). No cats had any documented episodes of hypoglycemia. Adverse effects were mild. The addition of bexagliflozin significantly improved diabetic management in this group of cats.

Le but de cette étude était d'étudier l'effet de la bexagliflozine sur la maitrise de la glycémie chez les chats diabétiques mal régulés et d'évaluer les événements indésirables associés à ce médicament. Les inhibiteurs du cotransporteur sodium-glucose 2 sont une nouvelle classe de médicaments utilisés dans la prise en charge des personnes atteintes de diabète de type 2. L'objectif de cette étude était d'évaluer l'effet du médicament administré par voie orale, la bexagliflozine, dans un groupe de chats diabétiques mal régulés sur une période d'étude de 4 semaines. Cinq chats appartenant à des clients atteints de diabète sucré mal maitrisé et recevant une insulinothérapie ont été inclus. La bexagliflozine a été administrée une fois par jour. La fructosamine sérique, le profil biochimique sérique et les courbes de glycémie sur 10 heures ont été évalués au départ (jour 0), au jour 14 et au jour 28. Tous les chats ont présenté une réduction significative de la dose d'insuline requise (P = 0,015) et l'insuline a été interrompue chez deux chats. Il y avait une diminution significative de la concentration de glucose dans le sang obtenue à partir des courbes de concentration de glucose dans le sang au cours de la période d'étude (P = 0,022). La fructosamine sérique a diminué chez 4 des 5 chats avec une diminution médiane de 152 µmol/L (plage : 103 à 241 µmol/L), ce qui n'était pas statistiquement significatif (P = 0,117). Aucun chat n'a eu d'épisodes documentés d'hypoglycémie. Les effets indésirables étaient légers. L'ajout de bexagliflozine a considérablement amélioré la gestion du diabète dans ce groupe de chats.(Traduit par Docteur Serge Messier).

Specnuezhenide reduces carbon tetrachloride-induced liver injury in mice through inhibition of oxidative stress and hepatocyte apoptosis.

OBJECTIVES: This study aimed to investigate the hepatoprotective effects of specnuezhenide against carbon tetrachloride (CCl4)-induced liver injury in mice. METHODS: Male C57BL/6 mice were intraperitoneally injected with 10 ml/kg body weight of CCl4 (0.5% diluted in arachis oil) for acute liver injury after oral administration of specnuezhenide for 7 days. Twenty-four hours after the final CCl4 injection, mice were euthanized and plasma and liver samples were collected. KEY FINDINGS: The results showed that specnuezhenide markedly and dose-dependently reduced serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) activity and relative liver weight, as well as ameliorated histopathological damage caused by CCl4 and decreased malondialdehyde (MDA) levels, and increased the activity of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Moreover, specnuezhenide promoted the expression and nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the mRNA and protein expression of Nrf2 signalling-related genes heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Finally, TUNEL staining and immunohistochemistry indicated that specnuezhenide prevented CCl4-induced hepatocytic apoptosis by up-regulating B-cell lymphoma 2 (Bcl-2) expression and downregulating Bcl-2-associated X (Bax) expression. CONCLUSIONS: Specnuezhenide reduced CCl4-induced liver injury in mice by inhibiting oxidative stress via activation of Nrf2 signalling and decreasing hepatocyte apoptosis.

Discovery of Chromane-6-Sulfonamide Derivative as a Potent, Selective, and Orally Available Novel Retinoic Acid Receptor-Related Orphan Receptor γt Inverse Agonist.

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays a dominant role in inflammation, autoimmunity, and host defense. RORγt is a key transcription factor mediating T helper 17 (Th17) cell differentiation and IL-17 production, which is able to activate CD8+ T cells and elicit antitumor efficacy. A series of sulfonamide derivatives as novel RORγt inverse agonists were designed and synthesized. Using GSK2981278 (phase II) as a starting point, we engineered structural modifications that significantly improved the activity and pharmacokinetic profile. In animal studies, oral administration of compound d3 showed a robust and dose-dependent inhibition of the IL-17A cytokine expression in a mouse imiquimod-induced skin inflammation model. Docking analysis of the binding mode revealed that the compound d3 occupied the active pocket suitably. Thus, compound d3 was selected as a clinical compound for the treatment of Th17-driven autoimmune diseases.

Accuracy in recognising happy facial expressions is associated with antidepressant response to a NOP receptor antagonist but not placebo treatment.

BACKGROUND: Clinical trials with putative antidepressants can be difficult to execute as it can take up to 8 weeks before differences emerge between drug and placebo, and long expensive trials often fail. Implementation of early response biomarkers could aid this process significantly with potential to identify new treatments. AIMS: In a secondary analysis, we examined the association of early effects on emotional processing with later clinical outcome following treatment with the novel NOP antagonist LY2940094 versus placebo. We hypothesised that early induction of positive bias would be associated with reduced severity of depression after 8 weeks of treatment. METHODS: This was a multicentre, randomised, double-blind, parallel-group, fixed-dose, placebo-controlled, 8 week study to assess sensitivity of the facial emotional recognition task (FERT) to early changes in emotional bias induced by LY2940094. Patients who met diagnostic criteria for major depression were randomised to receive LY2940094 (N = 70) or placebo (N = 66). At week 1 and 6, the FERT was completed by 33 patients in the LY2940094 group and 34 in the placebo group. RESULTS: Patients identified happy faces with higher accuracy (Wald χ2(1,33) = 14.25, p < 0.001) after 1 week treatment with LY290094 compared to placebo (Wald χ2(1,32) = 0.83, p = 0.36) and this correlated with eventual treatment response measured by the Hamilton Depression Rating Scale 7 weeks later. CONCLUSION: These data suggest that emotional processing biomarkers may be sensitive to early effects of antidepressant treatment indicative of later clinical response. Further studies in this area may be useful in developing new treatments and clinical trial designs for predicting antidepressant response.

Local administration of ReveromycinA ointment suppressed alveolar bone loss in mice.

ReveromycinA (RMA) was developed and is a unique agent for inhibiting osteoclast activity. In a previous study, we experimentally induced periodontal disease in a high-turnover osteoporosis osteoprotegerin-knockout mice (OPG KO) model and found that intraperitoneal administration of RMA inhibited alveolar bone resorption. We prepared a novel RMA-containing ointment for topical non-invasive administration in the oral cavity, in preparation for possible future clinical application. And we investigated whether this ointment can inhibit alveolar bone resorption in an experimental mouse model of periodontal disease. We examined wild-type (WT) and OPG KO mice ligated with wire around contact points on the left first and second molars to cause food impaction and induce experimental periodontal disease. RMA was administered three times a day. Using micro-computed tomography, we measured the volume of alveolar bone loss and also performed histological analysis. Our findings showed that localized administration of RMA containing ointment resulted in suppressed alveolar bone resorption, reduced osteoclast count, and lower immunostaining scores of inflammation sites compared with controls in both OPG KO and WT mice. Localized application of the specific osteoclast suppressor RMA in ointment form in the oral cavity could be a novel treatment for periodontitis that inhibits alveolar bone resorption locally.

EVOO Polyphenols Relieve Synergistically Autophagy Dysregulation in a Cellular Model of Alzheimer's Disease.

(1) Background: Autophagy, the major cytoplasmic process of substrate turnover, declines with age, contributing to proteostasis decline, accumulation of harmful protein aggregates, damaged mitochondria and to ROS production. Accordingly, abnormalities in the autophagic flux may contribute to many different pathophysiological conditions associated with ageing, including neurodegeneration. Recent data have shown that extra-virgin olive oil (EVOO) polyphenols stimulate cell defenses against plaque-induced neurodegeneration, mainly, through autophagy induction. (2) Methods: We carried out a set of in vitro experiments on SH-SY5Y human neuroblastoma cells exposed to toxic Aß1-42 oligomers to investigate the molecular mechanisms involved in autophagy activation by two olive oil polyphenols, oleuropein aglycone (OleA), arising from the hydrolysis of oleuropein (Ole), the main polyphenol found in olive leaves and drupes and its main metabolite, hydroxytyrosol (HT). (3) Results: Our data show that the mixture of the two polyphenols activates synergistically the autophagic flux preventing cell damage by Aß1-42 oligomers., in terms of ROS production, and impairment of mitochondria. (4) Conclusion: Our results support the idea that EVOO polyphenols act synergistically in autophagy modulation against neurodegeneration. These data confirm and provide the rationale to consider these molecules, alone or in combination, as promising candidates to contrast ageing-associated neurodegeneration.

GE11 Modified PLGA/TPGS Nanoparticles Targeting Delivery of Salinomycin to Breast Cancer Cells.

Salinomycin (Sal) is a potent inhibitor with effective anti-breast cancer properties in clinical therapy. The occurrence of various side effect of Sal greatly limits its application. The epidermal growth factor receptor (EGFR) family is a family of receptors highly expressed in most breast cancer cells. GE11 is a dodecapeptide which shows excellent EGFR affinity. A series of nanoparticles derivatives with GE11 peptide conjugated PLGA/TPGS were synthesized. Nanoprecipitation method was used to prepare the Sal loaded nanoparticles at the optimized concentration. The characterization, targeting efficacy, and antitumor activity were detected both in vitro and in vivo. Encapsulation of Sal in GE11 modified PLGA/TPGS nanoparticles shows an improved therapy efficacy and lower systemic side effect. This represents the delivery system a promising strategy to enhance the therapeutic effect against EGFR highly expressed breast cancer.

Towards Novel Treatments for Schizophrenia: Molecular and Behavioural Signatures of the Psychotropic Agent SEP-363856.

Schizophrenia is a complex psychopathology whose treatment is still challenging. Given the limitations of existing antipsychotics, there is urgent need for novel drugs with fewer side effects. SEP-363856 (SEP-856) is a novel psychotropic agent currently under phase III clinical investigation for schizophrenia treatment. In this study, we investigated the ability of an acute oral SEP-856 administration to modulate the functional activity of specific brain regions at basal levels and under glutamatergic or dopaminergic-perturbed conditions in adult rats. We found that immediate-early genes (IEGs) expression was strongly upregulated in the prefrontal cortex and, to a less extent, in the ventral hippocampus, suggesting an activation of these regions. Furthermore, SEP-856 was effective in preventing the hyperactivity induced by an acute injection of phencyclidine (PCP), but not of d-amphetamine (AMPH). The compound effectively normalized the PCP-induced increase in IEGs expression in the PFC at all doses tested, whereas only the highest dose determined the major modulations on AMPH-induced changes. Lastly, SEP-856 acute administration corrected the cognitive deficits produced by subchronic PCP administration. Taken together, our data provide further insights on SEP-856, suggesting that modulation of the PFC may represent an important mechanism for the functional and behavioural activity of this novel compound.

Combined delivery of salinomycin and docetaxel by dual-targeting gelatinase nanoparticles effectively inhibits cervical cancer cells and cancer stem cells.

Intra-tumor heterogeneity is widely accepted as one of the key factors, which hinders cancer patients from achieving full recovery. Especially, cancer stem cells (CSCs) may exhibit self-renewal capacity, which makes it harder for complete elimination of tumor. Therefore, simultaneously inhibiting CSCs and non-CSCs in tumors becomes a promising strategy to obtain sustainable anticancer efficacy. Salinomycin (Sal) was reported to be critical to inhibit CSCs. However, the poor bioavailability and catastrophic side effects brought about limitations to clinical practice. To solve this problem, we previously constructed gelatinase-stimuli nanoparticles composed of nontoxic, biocompatible polyethylene glycol-polycaprolactone (PEG-PCL) copolymer with a gelatinase-cleavable peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG) inserted between the two blocks of the copolymer. By applying our "smart" gelatinase-responsive nanoparticles for Sal delivery, we have demonstrated specific accumulation in tumor, anti-CSCs ability and reduced toxicity of Sal-NPs in our previous study. In the present study, we synthesized Sal-Docetaxel-loaded gelatinase-stimuli nanoparticles (Sal-Doc NP) and confirmed single emulsion as the optimal method of producing Sal-Doc NPs (Sal-Doc SE-NP) in comparison with nanoprecipitation. Sal-Doc SE-NPs inhibited both CSCs and non-CSCs in mice transplanted with cervical cancer, and might be associated with enhanced restriction of epithelial-mesenchymal transition (EMT) pathway. Besides, the tumorigenic capacity and growing speed were obviously suppressed in Sal-Doc-SE-NPs-treated group in rechallenge experiment. Our results suggest that Sal-Doc-loaded gelatinase-stimuli nanoparticles could be a promising strategy to enhance antitumor efficacy and reduce side effects by simultaneously suppressing CSCs and non-CSCs.

Ovarian activity and antioxidant indices during estrous cycle of Barki ewes under effect of thyme, celery and salinomycin as feed additives.

This research aimed to examine the effects of thyme, celery and salinomycin on ovarian sex hormones, reproductive traits and antioxidant status during the estrous cycle. Seventy-five mature Barki ewes aged 2-3 years with an average weight of 40 ± 1.5 kg were assigned randomly into five groups (15 head/group). Group 1 was kept as the control; groups 2 and 3 received 20 g/head/day thyme (T) and celery (C) as dried herbs, respectively. Group 4 (T×C) received 10 g thyme + 10 g celery/head/day, and group 5 was treated with salinomycin 1 g/head/day. Blood samples were collected during follicular and luteal phases of the estrous cycle. Thyme and celery and the mixture of T×C increased (P < 0.01) estradiol-17ß (E2) during the follicular phase of the estrous cycle, while only the celery group showed a marked (P < 0.001) increase in progesterone (P4) during the luteal phase compared with the control. Salinomycin supplementation decreased (P < 0.05) E2 concentrations during the follicular and luteal phases of the estrous cycle. Supplementation with thyme and celery enhanced (P < 0.001) antioxidant capacity in the luteal phase compared with the follicular stage. The salinomycin group showed increased (P < 0.01) levels of reduced glutathione (GSH) and decreased malondialdehyde (MDA) levels compared with the control group throughout luteal phase. For the interaction between estrous phases and treatments, thyme, celery, and T×C supplementation revealed an increase (P < 0.05) in superoxide dismutase (SOD), GSH, and glutathione disulfide (GSSG) levels compared with the control group during the follicular and luteal phases. Thyme and celery supplementation improved the number of services per conception and fertilization from 1st and 2nd inseminations, respectively. In conclusion, the applied treatment had significant effects on reproductive performance and antioxidant status in ewes throughout the estrous cycle.

Salinomycin inhibits proliferative vitreoretinopathy formation in a mouse model.

Proliferative vitreoretinopathy (PVR) is a progressive disease that develops in a subset of patients who undergo surgery for retinal detachment repair, and results in significant vision loss. PVR is characterized by the migration of retinal pigment epithelial (RPE) cells into the vitreous cavity, where they undergo epithelial-to-mesenchymal transition and form contractile membranes within the vitreous and along the retina, resulting in recurrent retinal detachments. Currently, surgical intervention is the only treatment for PVR and there are no pharmacological agents that effectively inhibit or prevent PVR formation. Here, we show that a single intravitreal injection of the polyether ionophore salinomycin (SNC) effectively inhibits the formation of PVR in a mouse model with no evidence of retinal toxicity. After 4 weeks, fundus photography and optical coherence tomography (OCT) demonstrated development of mean PVR grade of 3.5 (SD: 1.3) in mouse eyes injected with RPE cells/DMSO (vehicle), compared to mean PVR grade of 1.6 (SD: 1.3) in eyes injected with RPE cells/SNC (p = 0.001). Additionally, immunohistochemistry analysis showed RPE cells/SNC treatment reduced both fibrotic (αSMA, FN1, Vim) and inflammatory (GFAP, CD3, CD20) markers compared to control RPE cells/DMSO treatment. Finally, qPCR analysis confirmed that Tgfß, Tnfα, Mcp1 (inflammatory/cytokine markers), and Fn1, Col1a1 and Acta2 (fibrotic markers) were significantly attenuated in the RPE cells/SNC group compared to RPE/DMSO control. These results suggest that SNC is a potential pharmacologic agent for the prevention of PVR in humans and warrants further investigation.

The Effects of a Combination of Quillaja saponaria and Yucca schidigera on Eimeria spp. in Broiler Chickens.

A series of studies was carried out to determine the anticoccidial effects of a product derived from plant material sourced from Quillaja saponaria and Yucca schidigera. These plants are known to contain high concentrations of triterpenoid and steroidal saponins, substances that are known to display an array of biological effects. Battery tests involving individual Eimeria acervulina, Eimeria maxima, and Eimeria tenella infections and graded levels of a quillaja/yucca combination (QY) (0, 200, 250, and 300 ppm) were conducted. Body weight gain, coccidial lesion scores, and total oocysts per gram of feces (OPG) were used to evaluate anticoccidial effects. In addition, three floor pen trials evaluated the effects of 250 ppm QY in the control coccidial infections. The first pen trial measured the effects of 250 ppm QY, both alone and in combination with 66 ppm salinomycin (Sal), in a 2 3 2 factorial treatment arrangement. Two additional 42-day pen studies assessed the effects 250 ppm QY in birds vaccinated for coccidiosis. Data from the three battery trials indicated that at doses of 250 ppm QY or more, weight gain was improved, E. acervulina and E. tenella lesion scores were reduced, and OPG was lowered. In general, OPG was reduced by about 50% across all species by 250 and 300 ppm QY. Results of the pen study indicated that 250 ppm QY and Sal, when fed individually, reduced OPG and lesion scores and improved final performance. However, when QY and Sal were administered concurrently, further significant reductions in OPG occurred. The final performance of broilers vaccinated for coccidiosis was also improved at 250 ppm QY, as was OPG at both 21 and 28 days. Thus, at QY doses of 250 ppm or more, anticoccidial activity was evident but lacked the potency exhibited by many standard anticoccidials. When combined with either Sal or a live coccidiosis vaccine, QY improved the anticoccidial effects and performance of these anticoccidial methods.

Salinomycin-Loaded Small-Molecule Nanoprodrugs Enhance Anticancer Activity in Hepatocellular Carcinoma.

BACKGROUND: There is currently no effective treatment for advanced hepatocellular carcinoma (HCC), and chemotherapy has little effect on long-term survival of HCC patients, largely due to the cancer stem cell (CSC) chemoresistance of HCC. METHODS: We constructed a small-molecule nanometer-sized prodrug (nanoprodrug) loaded with salinomycin (SAL) for the treatment of HCC. SAL was encapsulated by the prodrug LA-SN38 (linoleic acid modified 7-ethyl-10-hydroxycamptothecin) to construct a self-assembled nanoprodrug further PEGylated with DSPE-PEG2000. We characterized this codelivered nanoprodrug and its antitumor activity both in vitro in human HCC cell lines and in vivo in mice. RESULTS: Delivery of the SAL- and LA-SN38-based nanoprodrugs effectively promoted apoptosis of HCC cells, exerted inhibition of HCC tumor-sphere formation as well as HCC cell motility and invasion, and reduced the proportion of CD133+ HCC-CSC cells. In nude mice, the nanoprodrug suppressed growth of tumor xenografts derived from human cell lines and patient. CONCLUSION: Our results show that SAL-based nanoprodrugs are a promising platform for treating patients with HCC and a novel strategy for combination therapy of cancers.

Pulmonary delivery of nanostructured lipid carriers for effective repurposing of salinomycin as an antiviral agent.

Coronavirus disease outbreak caused a severe public health burden all over the world. Salinomycin (SAL) is a broad-spectrum antibiotic that had drawn attention in selective targeting of cancer and viral infections. Recent drug screen identified SAL as a potent antiviral agent against SARS-CoV-2. In this hypothesis, we discuss the potential of pulmonary delivery of SAL using nanostructured lipid carriers (NLCs) against SARS-CoV-2.

Salinomycin-loaded PLA nanoparticles: drug quantification by GPC and wave voltammetry and biological studies on osteosarcoma cancer stem cells.

A new straightforward gel permeation chromatography (GPC) method was developed to calculate the drug encapsulation efficiency and loading content of Poly(lactic acid) nanoparticles (PLA NPs) loaded with Salinomycin (Sal), exploiting the capability of this technique to separate a macromolecular/molecular mixture on the basis of the molecular weight of each component. The proposed GPC method allowed Sal detection until 1% of Sal content in PLA NPs, avoiding sample pre-treatments. The method was validated by wave voltammetry (SW) technique, using a slightly modified literature procedure, useful to detect Sal in the concentration range 0.4 ≤ C/µmol/L ≤ 12 (linear concentration range). PLA-based NPs were prepared by nanoprecipitation with either native and functionalized PLA. Specifically, folate-decorated PLA NPs (PLA-FA NPs) were obtained by CuAAC click functionalization of alkyne-grafted PLA with azide-folate. Sal-loaded NPs were characterized physicochemically and morphologically. They exhibited adequate physicochemical properties, good drug encapsulation efficiency (98 ± 0.5% and 99 ± 0.5%), and loading content (8.8 ± 0.1% and 8.9 ± 0.1% for PLA/Sal and PLA-FA/Sal NPs, respectively). The size of empty PLA NPs resulted smaller (90 ± 3.2 nm and 680 ± 15.3 nm, for PLA NPs and PLA-FA NPs respectively) than the correspondent drug-loaded NPs (110 ± 3.8 nm and 875 ± 20.5 nm, respectively). Their biological activity was assessed on osteosarcoma bulk cells MG63, healthy osteoblast cell line (hFOB1.19), and enriched osteosarcoma cancer stem cells (CSCs), showing cell-depending effect. Entrapped Sal maintained its cytotoxic effect on CSCs and MG63 cells, with a potency comparable to the free drug and no evident benefit was detected for folate-decorated PLA NPs respect to native PLA NPs. Graphical abstract.

Enhanced and Prolonged Antitumor Effect of Salinomycin-Loaded Gelatinase-Responsive Nanoparticles via Targeted Drug Delivery and Inhibition of Cervical Cancer Stem Cells.

BACKGROUND: Cervical cancer stem cells (CCSCs) represent a subpopulation of tumor cells that possess self-renewal capacity and numerous intrinsic mechanisms of resistance to conventional chemotherapy and radiotherapy. These cells play a crucial role in relapse and metastasis of cervical cancer. Therefore, eradication of CCSCs is the primary objective in cervical cancer therapy. Salinomycin (Sal) is an agent used for the elimination of cancer stem cells (CSCs); however, the occurrence of several side effects hinders its application. Nanoscale drug-delivery systems offer great promise for the diagnosis and treatment of tumors. These systems can be used to reduce the side effects of Sal and improve clinical benefit. METHODS: Sal-loaded polyethylene glycol-peptide-polycaprolactone nanoparticles (Sal NPs) were fabricated under mild and non-toxic conditions. The real-time biodistribution of Sal NPs was investigated through non-invasive near-infrared fluorescent imaging. The efficacy of tumor growth inhibition by Sal NPs was evaluated using tumor xenografts in nude mice. Flow cytometry, immunohistochemistry, and Western blotting were used to detect the apoptosis of CSCs after treatment with Sal NPs. Immunohistochemistry and Western blotting were used to examine epithelial-mesenchymal transition (epithelial interstitial transformation) signal-related molecules. RESULTS: Sal NPs exhibited antitumor efficacy against cervical cancers by inducing apoptosis of CCSCs and inhibiting the epithelial-mesenchymal transition pathway. Besides, tumor pieces resected from Sal NP-treated mice showed decreased reseeding ability and growth speed, further demonstrating the significant inhibitory ability of Sal NPs against CSCs. Moreover, owing to targeted delivery based on the gelatinase-responsive strategy, Sal NPs was more effective and tolerable than free Sal. CONCLUSION: To the best of our knowledge, this is the first study to show that CCSC-targeted Sal NPs provide a potential approach to selectively target and efficiently eradicate CCSCs. This renders them a promising strategy to improve the therapeutic effect against cervical cancer.

Stabilization of indocyanine green dye in polymeric micelles for NIR-II fluorescence imaging and cancer treatment.

One of the most commonly used near infrared (NIR) dyes is indocyanine green (ICG), which has been extensively used for NIR bioimaging, photothermal and photodynamic therapy. However, upon excitation this dye can react with molecular oxygen to form singlet oxygen (SO), which can then cleave ICG to form non-fluorescent debris. In order to reduce the reaction between ICG and oxygen, we used energy transfer (ET) between the former and the NIR dye IR-1061. The two dyes were encapsulated in micelles composed of biocompatible poly(ethylene glycol)-block-poly(ε-caprolactone) (PCL-PEG). Micelles were characterized for their size using dynamic light scattering (DLS) and were found to measure about 35 nm in diameter. Fluorescence emission measurements were conducted to show that the stability of ICG against photodecomposition is increased. Moreover, this increased stability allows the encapsulated dye to generate more heat and for a longer time, compared to its free form. Studies with a SO indicator showed that as more IR-1061 is added to the micelles, less SO is produced. These results show how by changing the amount of added IR-1061 it is possible to tune the heat and SO generated by the system. Cell viability studies demonstrated that while particles were nontoxic under physiological conditions, upon 808 nm irradiation they become potent at eradicating MCF7 cancer cells. Moreover, it was demonstrated that both the increase of temperature and the creation of decomposition debris play a role in the cytotoxic efficacy of the micelles. Dye-loaded micelles that were injected to live mice showed bright fluorescence in the over 1000 nm NIR (OTN-NIR) region, allowing for visualization of blood vessels and internal organs. Most importantly, the encapsulated dyes remained stable for over 30 minutes, gradually accumulating in the liver and spleen. The presence of IR-1061 in addition to the heat-generating dye ICG allowed for simultaneous temperature modification and monitoring. We were able to assess the change in temperature by measuring the change in the fluorescence intensity of IR-1061 in the OTN-NIR region, a range with deep penetration of living tissues. These features illustrate the potential use of ICG/IR-1061 in PCL-PEG micelles as promising candidates for cancer treatment and diagnosis.

The stem cell inhibitor salinomycin decreases colony formation potential and tumor-initiating population in docetaxel-sensitive and docetaxel-resistant prostate cancer cells.

BACKGROUND: Prostate cancer (PCa) is one of the most frequently diagnosed tumors in men. In general, therapies for localized PCa are curative. However, treatment of advanced PCa is considered palliative since development of therapy resistance occurs rapidly. It has been shown that tumor-initiating cells are likely involved in therapy resistance. They are not eliminated by conventional therapies and thereby lead to tumor progression and relapse. The aim of this study was to evaluate the effects of the known stem cell inhibitor salinomycin on this critical subpopulation of cells. METHODS: Expression of the cell surface markers CD24 and CD44 was assessed by immunofluorescence and fluorescence-activated cell sorting. Colony formation efficiency and classification of colony types with varying tumor-initiating potential (holoclones, meroclones, and paraclones) were analyzed in an automated way by the newly developed CATCH-colonies software in the absence or presence of salinomycin. RESULTS: Automated high-resolution colony formation analysis consistently identified the various colony types in a broad range of PCa cell lines. Serial clonogenic assays confirmed that holoclones show the highest colony formation potential and maintain their tumor-initiating capacity over multiple rounds. Furthermore, holoclones showed high expression of CD44, while CD24 was not expressed in these clones, thus representing the well-described tumor-initiating CD24- /CD44high population. Salinomycin decreased the CD24- /CD44high population in both docetaxel-sensitive PC3 and docetaxel-resistant (DR) PC3-DR. Moreover, treatment of PC3, DU145, PC3-DR, and DU145-DR with salinomycin led to a significant reduction in the colony formation potential by targeting the colonies with high tumor-initiating potential. CONCLUSIONS: Taken together, we demonstrated that salinomycin specifically targets the tumor-initiating cell population in docetaxel-sensitive and docetaxel-resistant PCa cells and may represent a potential therapeutic approach for the treatment of advanced PCa.