Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the incorporation and time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for > 3 mol% PTXL (low), days for ≈ 3 mol% PTXL (moderate), and ≥ 20 days for < 3 mol% PTXL (long-term). Cell viability experiments on human cancer cell lines using CLPTXL nanoparticles (NPs) in the distinct CLPTXL solubility regimes reveal an unexpected dependence of efficacy on PTXL content in NPs. Remarkably, formulations with lower PTXL content and thus higher stability show higher efficacy than those formulated at the membrane solubility limit of ≈ 3 mol% PTXL (which has been the focus of most previous physicochemical studies and clinical trials of PTXL-loaded CLs). Furthermore, an additional high-efficacy regime is seen on occasion for liposome compositions with PTXL ≥ 9 mol% applied to cells at short time scales (hours) after formation. At longer time scales (days), CLPTXL NPs with ≥ 3 mol% PTXL lose efficacy while formulations with 1–2 mol% PTXL maintain high efficacy. Our findings underscore the importance of understanding the relationship of the kinetic phase behavior and physicochemical properties of CLPTXL NPs to efficacy.

Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new Synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for > 3 mol% PTXL (low), days for ≈ 3 mol% PTXL (moderate), and ≥ 20 days for < 3 mol% PTXL (long-term). Cell viability experiments on human cancer cell lines using CL PTXL nanoparticles (NPs) in the distinct CL PTXL solubility regimes reveal an unexpected nonmonotonic dependence of efficacy on PTXL content in NPs delivered at short time scales (hours) after liposome hydration, where we see two distinct high-efficacy regimes at low (˂ 2 mol%) and high (9 mol%) drug loading. These newly identified high-efficacy regimes flank the membrane solubility . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/154948 doi: bioRxiv preprint first posted online Jun. 23, 2017; 2 limit (≈ 3 mol%, where efficacy declines), which has been the focus of most previous physicochemical studies (and clinical trials) of PTXL-loaded CLs. At longer times scales (days), CL PTXL NPs with ≥ 3 mol% PTXL lose efficacy while formulations with 1-2 mol% PTXL maintain high efficacy. Our findings underscore the importance of understanding the relationship of the kinetic phase behavior and physicochemical properties of CL PTXL NPs to efficacy.

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Problem statement: Curcumin the active component of turmeric is known for its wide biological actions. Extensive studies on curcumin highlighted its anti-inflammatory, anti-oxidant, antimicrobial, anti-carcinogenic and anti-coagulant activity. The anti-inflammatory activities of curcumin have been demonstrated both in vitro and in vivo. Though curcumin and its anti-inflammatory properties are well documented, the exact mechanism of action and effective in vivo dosage required for potential anti-inflammatory activity of curcumin are yet to be determined. The current work reflects in identfying the the role curcumin in the inflmmatory cascade and arriving at an optimal effective dose (ED 50 ). Approach: The objective of the current study is to understand and establish the role of curcumin in the treatment of inflammatory condition, through in-silico, in-vitro and in-vivo studies. The specificity and binding affinity of curcumin to major inflammatory mediators such as, cytokines/chemokines, signaling proteins and transcription factors were evaluated using molecular docking. Subsequently, in-vitro experiments were conducted to establish the role of curcumin in reducing the release of histamine and β-hexosaminidase form U937 human monocytes cell lines. Further, the Effective Dose (ED 50 ) of curcumin was established for its potent in vivo anti-inflammatory activity. Results: Our study confirmed a strong affinity of curcumin to various inflammatory mediators (ERK, PKC, P38 MAP Kinase, NFkB and Lipoxygenase). Curcumin when studied for its affinity towards chemokines/cytokines and TNF-α was found to be ineffective. Proportionate reduction in histamine and β-hexosaminidase release in U937 cells in vitro and inhibition of paw edema in carrageenan induced inflammation in rats affirmed the dose dependent anti-inflammatory activity of curcumin. This in vivo study elucidated the ED 50 value to be 570.6 mg kg −1 body weight for curcumin, which apparently shall be the potent dose to screen its anti-inflammatory activity. Conclusion: Overall results suggest that, curcumin mediates its anti-inflammatory activity by its direct effect on multitarget inflammatory mediators while others were mediated by the downstream effects of curcumin. Curcumin can be a potent molecule in treatment of various diseases associated with inflammation, with its multi-target potency and high safety profile.

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