Transporter targeted-carnitine modified pectin-chitosan nanoparticles for inositol hexaphosphate delivery to the colon: An in silico and in vitro approach.

Orally targeted delivery systems have attracted ample interest in colorectal cancer management. In this investigation, we developed Inositol hexaphosphate (IHP) loaded Tripolyphosphate (Tr) crosslinked Pectin (Pe) Chitosan (Ch) nanoparticles (IHP@Tr*Pe-Ch-NPs) and modified them with l-Carnitine (CE) (CE-IHP@Tr*Pe-Ch-NPs) to improve uptake in colon cells. The formulated CE-IHP@Tr*Pe-Ch-NPs displayed a monodisperse distribution with 219.3 ± 5.5 nm diameter and 30.17 mV surface charge. Cell-line studies revealed that CE-IHP@Tr*Pe-Ch-NPs exhibited excellent biocompatibility in J774.2 and decreased cell viability in DLD-1, HT-29, and MCF7 cell lines. More cell internalization was seen in HT-29 and MCF7 due to overexpression of the OCTN2 and ATB0,+ transporter (CE transporters) compared to DLD-1. The cell cycle profile, reactive oxygen species, apoptosis, and mitochondrial membrane potential assays were performed to explore the chemo-preventive mechanism of CE-IHP@Tr*Pe-Ch-NPs. Moreover, the in-silico docking studies revealed enhanced interactive behavior of CE-IHP@Tr*Pe-Ch-NPs, thereby proving their targeting ability. All the findings suggested that CE-IHP@Tr*Pe-Ch-NPs could be a promising drug delivery approach for colon cancer targeting.

Regulatory safety pharmacology and toxicity assessments of a standardized stem extract of Cassia occidentalis Linn. in rodents.

Cassia occidentalis Linn (CO) is an annual/perennial plant having traditional uses in the treatments of ringworm, gastrointestinal ailments and piles, bone fracture, and wound healing. Previously, we confirmed the medicinal use of the stem extract (ethanolic) of CO (henceforth CSE) in fracture healing at 250 mg/kg dose in rats and described an osteogenic mode of action of four phytochemicals present in CSE. Here we studied CSE's preclinical safety and toxicity. CSE prepared as per regulations of Current Good Manufacturing Practice for human pharmaceuticals/phytopharmaceuticals and all studies were performed in rodents in a GLP-accredited facility. In acute dose toxicity as per New Drug and Clinical Trial Rules, 2019 (prior name schedule Y), in rats and mice and ten-day dose range-finding study in rats, CSE showed no mortality and no gross abnormality at 2500 mg/kg dose. Safety Pharmacology showed no adverse effect on central nervous system, cardiovascular system, and respiratory system at 2500 mg/kg dose. CSE was not mutagenic in the Ames test and did not cause clastogenicity assessed by in vivo bone marrow genotoxicity assay. By a sub chronic (90 days) repeated dose (as per OECD, 408 guideline) study in rats, the no-observed-adverse-effect-level was found to be 2500 mg/kg assessed by clinico-biochemistry and all organs histopathology. We conclude that CSE is safe up to 10X the dose required for its osteogenic effect.

Inhibition of cartilage degeneration and subchondral bone deterioration by Spinacia oleracea in human mimic of ACLT-induced osteoarthritis.

Osteoarthritis (OA) is an aging disorder characterized by degenerated cartilage and sub-chondral bone alteration in affected knee joints. Globally, millions of people suffer from this disease. However, there is a lack of safe and promising therapeutics, making the exploration and development of leads from natural sources urgent. Accordingly, food as medicine may be the most suitable approach for the treatment of this degenerative disease. Herein, we elucidated the protective role of Spinacia oleracea extract (SOE) in an anterior cruciate ligament transection (ACLT) model of osteoarthritis as a mimic of the human condition. ACL transection was done in the tibio-femoral joints of rats. SOE was orally administered at the dosage of 125 and 250 mg kg-1 day-1 for four weeks. It was shown that the animals with SOE treatment had better joint morphology than the ACLT animals, as evident by the shiny appearance of their cartilage. Hematoxylin and safranin-o staining showed that the number of chondrocytes was significantly reduced in the OA model, which was prevented with SOE treatment. The reduction in the cartilage thickness was well observed by toluidine blue staining. The reduced stain by safranin-o and toluidine blue, indicated proteoglycan loss in the ACLT-induced osteoarthritis model. The proteoglycan content and cartilage thickness were restored in the SOE group upon treatment at an SOE dosage of 125 and 250 mg kg-1 day-1. The micro-CT parameters of subchondral bone (SCB) and cartilage degradation markers in the serum corroborated our findings of the protective effects of SOE. In summary, our study suggests that SOE has therapeutic potential, which if taken regularly as a food supplement, can have beneficial effects.

Theranostic lyotropic liquid crystalline nanostructures for selective breast cancer imaging and therapy.

Herein, we report the development of theranostic lyotropic liquid crystalline nanostructures (LCN's) loaded with unique MnO nanoparticles (MNPs) for selective cancer imaging and therapy. MNPs serves as a fluorescent agent as well as a source of manganese (Mn2+) and enables localized oxidative stress under the hallmarks of cancer (acidosis, high H2O2 level). In pursuit of synergistic amplification of Mn2+ antitumor activity, betulinic acid (BA) is loaded in LCN's. In this investigation, nano-architecture of LCN's phase interface is established via SAXS, Cryo-TEM and Cryo-FESEM. Intriguing in vitro studies showed that the LCN's triggered hydroxyl radical production and exhibited greater selective cytotoxicity in cancer cells, ensuring the safety of normal cells. Significant tumor ablation is realized by the 96.5 % of tumor growth inhibition index of LCN's as compared to control group. Key insights into on-site drug release, local anti-cancer response, and tumor location are gained through precise guidance of fluorescent MNPs. In addition, comprehensive assessment of the safety, pharmacokinetics and tumor distribution behavior of LCN's is performed in vivo or ex vivo. This work emphasizes the promise of modulating tumor microenvironment with smart endogenous stimuli sensitive nano systems to achieve advanced comprehensive cancer nano-theranostics without any external stimulus. STATEMENT OF SIGNIFICANCE: Effective diagnosis and treatment approaches with maximum anti-cancer activity and minimal side-effects are critical to ameliorate cancer therapy. Compared to radiation, photodynamic and photothermal therapy, the specific and selective activation of tumor microenvironmental endogenous stimuli for the logical generation of cytotoxic OH· free radicals serves as an efficient therapeutic strategy for chemodynamic-cancer treatment. In this investigation, MnO nanoparticles fulfill two needs (fluorescence-based optical imaging and a source of Mn2+ based chemodynamic therapy) in one unit. This approach also ensures the safety of normal cells, as the toxic OH· free radical activity is substantially suppressed under the mild alkaline/H2O2 conditions in normal cell microenvironment.

Prevention of articular cartilage degeneration in a rat model of monosodium iodoacetate induced osteoarthritis by oral treatment with Withaferin A.

Withaferin A (WFA), a highly oxygenated withanolide is used for anti-osteoporotic, fracture healing, obesity control as medicine and dietary supplement in Ayurveda and Unani medicine but its potential remains to be investigate for the osteoarthritis studies. In the present study, chondro-protective effects of WFA, under in vitro and in vivo conditions were evaluated. In-vitro pharmacological activity of WFA was tested on rat articular chondrocytes through MTT, DPPH, different staining, FACS and translation studies. In-vivo studies of WFA were evaluated through monosodium iodoacetate (MIA) induced osteoarthritis studies. DPPH assay, alcian blue and toluidine blue staining indicated the chondrogenic potential of WFA. Similarly, WFA enhance chondrogenesis through up-regulation of SOX9 protein. In addition, WFA reduced the ROS generation, mitochondrial depolarization and apoptosis induced by inflammatory cytokines IL-1ß and TNF-α. Furthermore, WFA treatment in MIA treated rats alleviated cartilage erosion and improvement in sub-chondral bone micro-architecture by decrease in Tissue volume (∼32%), and trabecular bone pattern factor (∼28%). Taken together, our study provides convincing evidence for the candidature of WFA (10 mg kg-1 day-1) as a potential agent for the treatment of cartilage degenerative diseases like osteoarthritis.

Heartwood extract from Dalbergia sissoo promotes fracture healing and its application in ovariectomy-induced osteoporotic rats.

OBJECTIVES: This study was undertaken to investigate the effects of a heartwood ethanolic extract (HEE) made from the Dalbergia sissoo on facture healing and in the prevention of pathological bone loss resulting from estrogen deficiency in ovariectomized (Ovx) rats. METHODS: Heartwood ethanolic extract (250, 500 and 1000 mg/kg per day) was administered orally immediately next day after drill-hole injury and continued for 2 weeks. Ovx rats received HEE at same doses for 12 weeks and compared with 17-ß estradiol (E2; 100 µg/kg for 5 days/week subcutaneously) group. Confocal imaging for fracture healing, micro-architecture of long bones, biomechanical strength, formation of mineralized nodule by bone marrow osteoprogenitor cells, bone turnover markers and gene expression were studied. One-way ANOVA was used to test significance. KEY FINDINGS: Heartwood ethanolic extract treatment promoted fracture healing, formation of new bone at the drill-hole site and stimulated osteogenic genes at callus region. HEE administration to the Ovx rats exhibited better micro-architectural parameters at various anatomical positions, better bone biomechanical strength and more osteoprogenitor cells in the bone marrow compared with Ovx + vehicle group. HEE exhibited no uterine estrogenicity. CONCLUSIONS: Oral administration of HEE was found to promote fracture healing and exhibited osteoprotective effect by possibly stimulation of osteoblast function.

Dried and free flowing granules of Spinacia oleracea accelerate bone regeneration and alleviate postmenopausal osteoporosis.

OBJECTIVE: The aim of this study was to demonstrate the efficacy of extract derived from Spinacia oleracea extract (SOE) in reversing bone loss induced by ovariectomy and bone healing properties in a drill-hole fracture model in rats. METHODS: SOE was administered orally for 12 weeks in adult ovariectomized Sprague Dawley rats after inducing osteopenic condition. Bone micro-architecture, expressions of osteogenic and resorptive gene markers, biomechanical strength, new bone formation, and bone turnover markers were studied. Uterine histomorphometry was used to assess estrogenicity. Bone regeneration potential of SOE was assessed in a drill-hole fracture model. Fracture healing was assessed by calcein intensity and micro-CT analysis of callus at fracture region. RESULTS: SOE prevented ovariectomy-induced bone loss as evident from 122% increase in bone volume/tissue volume (BV/TV) and 29% decline in Tb.Sp in femoral trabecular micro-architecture. This was corroborated by the more than twofold stimulation in the expression of osteogenic genes runt-related transcription factor 2, osterix, osteocalcin, bone morphogenetic protein 2, collagen-1. Furthermore in the fracture healing model, we observed a 25% increase in BV/TV and enhancement in calcein intensity at the fractured site. The extract when converted into dried deliverable Spinaceae oleracea granule (SOG) form accelerated bone regeneration at fracture site, which was more efficient as evident by a 39% increase in BV/TV. Transforming SOE into dried granules facilitated prolonged systemic availability, thus providing enhanced activity for a period of 14 days. CONCLUSIONS: SOE treatment effectively prevents ovariectomy-induced bone loss and stimulated fracture healing in adult rats. The dried granular form of the extract of Spinaceae oleracea was effective in fracture healing at the same dose.

Characterization and evaluation of amphotericin B loaded MDP conjugated poly(propylene imine) dendrimers.

This paper describes a novel strategy for targeted delivery of amphotericin B (AmB) to macrophages with muramyl dipeptide (MDP) conjugated multimeric poly(propyleneimine) (PPI) dendrimers. Synergistic antiparasitic activity due to immunostimulation by multimeric presentation of MDP on dendrimers was anticipated. MDP conjugated 5.0G PPI (MdPPI) dendrimers were synthesized and characterized. Therapeutic activity and toxicity of dendrimeric formulation of AmB (MdPPIA) were compared with marketed formulations of AmB. Highly significant (P<0.01) reduction in toxicity was observed in hemolytic toxicity and cytotoxicity studies in erythrocytes and J774A.1 macrophage cells, respectively. Formulation MdPPIA showed appreciable macrophage targeting potential and higher or equivalent antiparasitic activity against parasite infected macrophage cell lines and in vivo infection in Balb/c mice. These results suggest the developed MDP conjugated dendrimeric formulation of AmB as a promising immunostimulant targeted drug delivery system and a safer alternative to marketed formulations. From the clinical editor: Parasitic infections remain a significant issue in the clinical setting. The authors in this article studied the use of ligand anchored dendrimeric formulation of Amphotericin B to target infected macrophages and showed reduced toxicity, high anti-leishmanial activity. This may add another treatment option to available formulations in the future.

Moxifloxacin-loaded nanoemulsions having tocopheryl succinate as the integral component improves pharmacokinetics and enhances survival in E. coli-induced complicated intra-abdominal infection.

In the present work, a novel nanoemulsion laden with moxifloxacin has been developed for effective management of complicated intra-abdominal infections. Moxifloxacin nanoemulsion fabricated using high pressure homogenization was evaluated for various pharmaceutical parameters, pharmacokinetics (PK) and pharmacodynamics (PD) in rats with E. coli-induced peritonitis and sepsis. The developed nanoemulsion MONe6 (size 168 ± 28 nm and zeta potential (ZP) 24.78 ± 0.45 mV, respectively) was effective for intracellular delivery and sustaining the release of MOX. MONe6 demonstrated improved plasma (AUC(MONe6/MOX) = 2.38-fold) and tissue pharmacokinetics of MOX (AUC(MONe6/MOX) = 2.63 and 1.47 times in lung and liver, respectively). Calculated PK/PD index correlated well with a reduction in bacterial burden in plasma as well as tissues. Enhanced survival on treatment with MONe6 (65.44%) and as compared to the control group (8.22%) was a result of reduction in lipid peroxidation, neutrophil migration, and cytokine levels (TNF-α and IL6) as compared to untreated groups in the rat model of E. coli-induced sepsis. Parenteral nanoemulsions of MOX hold a promising advantage in the therapy of E. coli-induced complicated intra-abdominal infections and is helpful in the prevention of further complications like septic shock and death.

Optimization of novel tocopheryl acetate nanoemulsions for parenteral delivery of curcumin for therapeutic intervention of sepsis.

OBJECTIVE: The objective of this study is to develop a nanostructured parenteral delivery system, laden with curcumin (CUR), for the therapeutic intervention of sepsis and associated pathologies. METHODS: Nanoemulsions were fabricated using sonication and speed homogenization. Size and zeta potential were evaluated by dynamic light scattering and transmission electron microscopy analysis. Pharmacodynamic and pharmacokinetic studies were performed on a rat model of lipopolysaccharide (LPS)-induced sepsis. RESULTS: The drug content of optimized nanoemulsion (F5) formulation (particle size 246 ± 08 nm, polydispersity index (PDI) of 0.120, zeta potential of -41.1 ± 1.2 mV) was found to be 1.25 mg/ml. In vitro release studies demonstrated that F5 was able to sustain the release of CUR for up to 24 h. Minimal hemolysis and cellular toxicity demonstrated its suitability for intravenous administration. Significant reduction of inflammatory mediator levels was mediated through enhanced uptake by in RAW 264.7 and THP-1 in absence/presence of LPS. Nanoemulsion resulted in an improvement of plasma concentration (AUCF5/AUC CUR = 8.80) and tissue distribution of CUR in rats leading to a reduction in LPS-induced lung and liver injury due to less neutrophil migration, reduced TNF-α levels and oxidative stress (demonstrated by levels of lipid peroxides as well as carbonylated proteins) as confirmed by histopathological studies. CONCLUSION: The findings suggest that the therapeutic performance (i.e., reduction in oxidative damage in tissues) of CUR can be enhanced by employing tocol acetate nanoemulsions (via improving pharmacokinetics and tissue distribution) as a platform for drug delivery in sepsis-induced organ injury.

Cationic nanoemulsions bearing ciprofloxacin surf-plexes enhances its therapeutic efficacy in conditions of E. coli induced peritonitis and sepsis.

PURPOSE: Chitosan (CH) coated ciprofloxacin-sodium deoxycholate surfplex (CFn-SDC) loaded nanoemulsion (LE-CH-CFn-SDC) developed in order to improve tissue penetration of the CFn as well as to mop up the endotoxin (Lipopolysaccharides or LPS) released from bacteria during antibiotic treatment. METHODS: Size and zeta potential was evaluated for nanoemulsions prepared by high-speed homogenization and sonication. Drug analysis in samples was done by HPLC equipped with fluorescence detector. All formulations were evaluated for any change in LPS induced NO and TNF-α release and ROS generation in J774 macrophages. The formulations were also evaluated for in-vitro killing efficiency on E-Coli. The efficacy of formulations in terms of survival and pharmacokinetics and inhibition of induction of cytokines was carried out in E-coli induced peritonitis model in rats. LE-CH-CFn-SDC interacted with LPS both by electrostatic and hydrophobic interactions. RESULTS: LE-CH-CFn-SDC resulted in reduction of endotoxin release and MIC values for E. coli. LE-CH-CFn-SDC also reduced NO and TNF-α as well as ROS generation by reducing the uptake of LPS in J774 macrophages. LE-CH-CFn-SDC improved CFn pharmacokinetics and tissue distribution, by reducing the bacterial burden, LPS and cytokines (TNF-α and IL-6) thereby improving survival in a rat model of E. coli induced peritonitis. CONCLUSION: In conclusion, this work highlights the effectiveness of the chitosan-coated nanoemulsion as intracorporeal approach for therapeutic intervention of E. coli induced peritonitis as well as in sepsis.

Investigations into an alternate approach to target mannose receptors on macrophages using 4-sulfated N-acetyl galactosamine more efficiently in comparison with mannose-decorated liposomes: an application in drug delivery.

In this study the potential of 2 different ligands, i.e., palmitoyl mannose (Man-Lip) and 4-SO(4)GalNAc (Sulf-Lip) to target resident macrophages was investigated after surface decoration of Amphotericin B (AmB) loaded liposomes. In the case of Sulf-Lip, the 4-SO(4)GalNAc was adsorbed through electrostatic interaction on cationic liposomes, which was confirmed by change in zeta potential from +48.2 ± 3.7 mV for Lip to +12.2 ± 1.3 mV for Sulf-Lip. The mean particle size of Sulf-Lip and Man-Lip was found to be 139.4 ± 7.4 nm and 147.4 ± 8.6 nm, respectively. Flow cytometric data reveal enhanced uptake of Sulf-Lip in both J774 and RAW cell lines in comparison with the uptake of Man-Lip. Intracellular localization studies indicate that the fluorescence intensity of Sulf-Lip was much higher in comparison with that of Man-Lip and Lip formulations. Sulf-Lip and Man-Lip showed significantly higher localization of AmB at all time points in comparison with Lip (P < 0.05) after intravenous (IV) administration. The studies provide evidence that 4-SO(4)GalNAc possesses a promising feature for targeting resident macrophages and its application in the conditions of leishmaniasis is in the offing. FROM THE CLINICAL EDITOR: This in vivo study compares two different ligands to deliver Amphotericin B l(AmB) loaded liposomes to resident macrophages. Targeted approaches showed significantly higher localization of AmB at all time points in comparison to non-targeted liposomes, and future applications in leishmaniasis are already under preparation.