Postpolymerization Modification of Poly(2-vinyl-4,4-dimethyl azlactone) as a Versatile Strategy for Drug Conjugation and Stimuli-Responsive Release.

Postpolymerization modification of highly defined "scaffold" polymers is a promising approach for overcoming the existing limitations of controlled radical polymerization such as batch-to-batch inconsistencies, accessibility to different monomers, and compatibility with harsh synthesis conditions. Using multiple physicochemical characterization techniques, we demonstrate that poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) scaffolds can be efficiently modified with a coumarin derivative, doxorubicin, and camptothecin small molecule drugs. Subsequently, we show that coumarin-modified PVDMA has a high cellular biocompatibility and that coumarin derivatives are liberated from the polymer in the intracellular environment for cytosolic accumulation. In addition, we report the pharmacokinetics, biodistribution, and antitumor efficacy of a PVDMA-based polymer for the first time, demonstrating unique accumulation patterns based on the administration route (i.e., intravenous vs oral), efficient tumor uptake, and tumor growth inhibition in 4T1 orthotopic triple negative breast cancer (TNBC) xenografts. This work establishes the utility of PVDMA as a versatile chemical platform for producing polymer-drug conjugates with a tunable, stimuli-responsive delivery.

Physicochemical comparison of chitin characteristics in three major stored-product beetle pests: Implications for biofumigant toxicity.

The present study investigates the chitin properties of stored-product insect pests and their association with the fumigant toxicity of garlic essential oil. Chitin isolates of Callosobruchus maculatus, Sitophilus oryzae, and Tribolium castaneum adults were characterized using FT-IR, XRD, EA, SEM-EDS, and NMR techniques. Fumigant toxicity assay was performed under airtight condition in glass vial. The S. oryzae contains highest chitin content (19 %), followed by T. castaneum (10 %) and C. maculatus (8 %). The degree of crystallinity was lower in C. maculatus (67.13 %) than in S. oryzae (77.05 %) and T. castaneum (76.56 %). Morphologically, C. maculatus chitin displayed a flat lamellar surface with pores, while S. oryzae and T. castaneum exhibited densely arranged microfibrils based surfaces. Fumigant toxicity assays revealed varied susceptibility levels, C. maculatus exhibited higher susceptibility (0.27 µL/L air of LC50) compared to S. oryzae and T. castaneum (14.35 and 3.74 µL/L air of LC50, respectively) to garlic essential oil. The higher chitin content, greater crystallinity, and densely arranged structures in S. oryzae might contribute to its tolerance towards fumigant. Additionally, physico-chemical properties and penetration potentiality of the bioactive constituents might be linked to the toxicity in insects. Understanding these relations can enrich knowledge of chitin's role in fumigant toxicity mechanism.

Correction Notice: Microscopy and Plate Reader-based Methods for Monitoring the Interaction of Platelets and Tumor Cells in vitro.

Platelets and their activation status play an essential role in cancer metastasis. Therefore, the anti-metastatic potential of antiplatelet drugs has been investigated for many years. However, the initial screening of these antiplatelet drugs to determine which agents can inhibit the interactions of platelets and tumor cells is very limited due to reliance upon expensive, time-consuming, and low-throughput animal experiments for screening. In vitro models of the platelet-tumor cell interaction can be a useful tool to rapidly screen multiple antiplatelet drugs and compare their ability to disrupt platelet-tumor cell interactions, while also identifying optimal concentrations to move forward for in vivo validation. Hence, we adopted methods used in platelet activation research to isolate and label platelets before mixing them with tumor cells (MDA-MB-231-RFP cells) in vitro in a static co-culture model. Platelets were isolated from other blood components by centrifugation, followed by fluorescent labeling using the dye CMFDA (CellTrackerTM Green). Labeling platelets allows microscopic observation of the introduced platelets with tumor cells grown in cell culture dishes. These methods have facilitated the study of platelet-tumor cell interactions in tissue culture. Here, we provide details of the methods we have used for platelet isolation from humans and mice and their staining for further interaction with tumor cells by microscopy and plate reader-based quantification. Moreover, we show the utility of this assay by demonstrating decreased platelet-tumor cell interactions in the presence of the T-Prostanoid receptor (TPr) inhibitor ifetroban. The methods described here will aid in the rapid discovery of antiplatelet agents, which have potential as anti-metastatic agents as well. Key features • Analysis of platelet-tumor cell binding dynamics. • In vitro methods developed for measuring platelet-tumor cell binding to enable rapid testing of antiplatelet and other compounds. • Complementary analysis of platelet-tumor cell binding by imaging and fluorimetry-based readings. • Representative results screening the effect of the antiplatelet drug, ifetroban, on platelet-tumor cell binding using the protocol. • Validation results were presented with both a TPr agonist and ifetroban (antagonist).

Lutein loaded double-layered polymer nanocarrier modulate H2O2 and CoCl2 induced oxidative and hypoxia damage and angiogenic markers in ARPE-19 cells.

Lutein plays a crucial role in the protection of retina by diminishing oxidative stress in diabetic retinopathy (DR). However, its poor aqueous solubility, chemical instability and low bioavailability edge its application. Also, beneficial effects of lutein supplementation and lower lutein levels in the serum and retina of DR patients created an interest in nanopreparation. Hence, lutein-loaded chitosan­sodium alginate nanocarrier comprising oleic acid core (LNCs) was developed and examined its protective effect on hyperglycemia-mediated changes in oxidative stress and angiogenesis in ARPE-19 cells. Results showed that the LNCs have smaller size and a smooth spherical morphology and did not affect the ARPE-19 cell viability (up to 20 µM) and showed higher cellular uptake in both normal and H2O2-induced stress conditions. LNCs pre-treatment suppressed the H2O2-induced oxidative stress and CoCl2-induced hypoxia-mediated elevation of intracellular reactive oxygen species, protein carbonyl and malondialdehyde levels by restoring antioxidant enzymes in ARPE-19 cells. Further, LNCs protected H2O2-mediated down-regulation of Nrf2 and its downstream antioxidant enzymes. LNCs also restored the H2O2-altered angiogenic (Vascular endothelial growth factor (VEGF), X-box binding protein 1 (XBP-1) and Hypoxia-inducible factor 1-alpha (HIF-1α)), endoplasmic reticulum stress (activating transcription factor-4 (ATF4)) and tight junction (Zona occludens 1 (ZO-1)) markers. To conclude, we could successfully develop biodegradable LNCs to improve the cellular uptake of lutein to treat DR by curtailing oxidative stress in retina.

Chitosan-sodium alginate-fatty acid nanocarrier system: Lutein bioavailability, absorption pharmacokinetics in diabetic rat and protection of retinal cells against H2O2 induced oxidative stress in vitro.

Aiming to enhance therapeutic efficiency of lutein, lutein loaded chitosan-sodium alginate (CS-SA) based nanocarrier system (LNCs) were prepared and evaluated for lutein bioavailability and pharmacokinetics in diabetic rats in comparison to micellar lutein (control). Further, cytotoxicity, cellular uptake and protective activity against H2O2 induced oxidative stress in ARPE-19 cells were studied. Results revealed that LNCs displayed maximal lutein AUC in plasma, liver and eye respectively in normal (3.1, 2.7 and 5.2 folds) and diabetic (7.3, 3.4 and 2.8 folds) rats. Lutein from LNCs exhibited a higher half-life time, mean residence time and slow clearance from the plasma, indicating prolonged circulation compared to control. In ARPE-19 cells, pre-treatment with LNCs (10 µM) have significantly attenuated H2O2 induced cell death, intracellular ROS and mitochondrial membrane potential compared to control. In conclusion, LNCs improve the lutein bioavailability in conditions like diabetes, diabetic retinopathy and cataract to curtail oxidative stress in retinal cells.

Biodegradable chitosan-sodium alginate-oleic acid nanocarrier promotes bioavailability and target delivery of lutein in rat model with no toxicity.

Efficient delivery of macular carotenoid lutein to target retinal tissue is possible with enhanced intestinal uptake remains a major challenge owing to the polarity, sensitivity to light, heat and solubility. In this study, to overcome such constraints, biodegradable polymers chitosan-sodium alginate-oleic acid based nano-carrier loaded with lutein (LNCs) was prepared and safety efficacy was examined in vivo. Acute-toxicity of LNCs (0.1, 1, 10 and 100 mg/kg body weight) revealed that the LD50 of LNCs was higher than 100 mg/kg body weight. In subacute-toxicity of LNCs (1 and 10 mg/kg body weight) revealed no mortality with no morphological and clinical changes in rats. Histology, haematology and biochemical analysis of urine and plasma confirmed no toxicity of LNCs compared to control. Post-prandial plasma and tissue (retina) levels of lutein from LNCs were higher. Results demonstrate increased bioavailability of lutein from LNCs with no toxicity suggests applications in food and pharma.

Chitosan-oleic acid-sodium alginate a hybrid nanocarrier as an efficient delivery system for enhancement of lutein stability and bioavailability.

Lutein is a hydrophobic antioxidant carotenoid with proven retinal and macular protection against oxidative stress. However, low aqueous solubility and bioavailability limit its clinical application. Hence, focus of the study was to improve the solubility and bioavailability of lutein by using a chitosan-oleic acid-sodium alginate-based nano-carrier system (LNCs). LNCs were prepared by ionic gelation and optimized by Plackett-Burman factorial algorithm. The size and zeta potential of LNCs were characterized by electron microscopy and dynamic light scattering. LNCs were within a size range of 40-160 nm with a desired zeta potential of +45 ± 5 mV and PDI of 0.174 ± 0.02. Further, LNCs displayed 1000-fold higher aqueous solubility with controlled and sustained release kinetics in vitro. Compared to micellar lutein, higher intracellular transport (40%) of lutein from LNCs in Caco-2 cells. Oral gavage of single dose of LNCs in rats resulted in higher (128.3%) bioavailability of lutein compared to micellar lutein. Further, a dose-dependent increase in plasma (135.20, 165.30 nmol/mL) and eyes (1.51 & 3.98 µg/g) was observed upon oral gavage of LNCs (10 and 100 mg/kg BW). Results demonstrate higher solubility and bioavailability of lutein from LNCs and hence could be an efficient therapeutic tool to conquer macular degeneration and retinopathy.