Co-delivery of 5-Fluorouracil and Curcumin Nanohybrid Formulations for Improved Chemotherapy Against Oral Squamous Cell Carcinoma.

PURPOSE: The chemotherapeutics agent, 5-fluorouracil (5-FU), and curcumin (Cur), a natural antioxidant, has a wide pharmacological window to treat oral carcinoma; however, both drugs have limited bioavailability. This research study designs to develop a nanoemulsions (NEs) formulation by combining 5-FU and Cur to improve anticancer activity against oral cavity squamous cell carcinoma (OSCC) cells from the diversified origin for in vitro analysis, SCC090 (human tongue) and SCC152 (human hypo-pharynx). METHODOLOGY: NEs formulated through homogenization, applying high-energy ultrasonication technique. The prepared 5-FUNE/Cur-NE/5-FU-Cur-NE were characterized and optimized by different in vitro assays to evaluate release system and treatment of OSCC cells to monitor cellular acceptability, such as in vitro anticancer activity by MTT assay, cell uptake studies and protein expression associated apoptotic study. RESULTS: 5-FUNE/Cur-NE/5-FU-Cur-NE successfully formulated and show mean-value of the particle size (150-200 nm), surface charge (- 25.70 to - 37.91 mV), and PDI (0.194). In vitro release of 5-FUNE/Cur-NE/5-FU-Cur-NEs was monitored over a course of 04 days, where acidic pH shows higher release as compared to alkaline pH, along with acceptable stability data. Cytotoxicity study has shown higher-dose-dependent anticancer effect with a reduced IC50 value of NEs as compared to BLNE. Cellular uptake study of 5-FUNE/Cur-NE/5-FU-Cur-NEs upgraded many folds, comparatively BLNE and show potential cell arrest. Additionally, the cell protein (Blc2, Bax, P53, and P21) expression was revised and raised cell apoptosis. CONCLUSION: The combinational loaded, 5-FU and Cur in nanoformulation system have proven their potency to deliver improved anticancer activity, against oral cancer.

Synchronized Ratiometric Codelivery of Metformin and Topotecan through Engineered Nanocarrier Facilitates In Vivo Synergistic Precision Levels at Tumor Site.

The combination of metabolic modulators with chemotherapy holds vast promise for effective inhibition of tumor progression and invasion. Herein, a ratiometric codelivery platform is developed for metformin (MET), a known metabolic modulator and topotecan (TPT), a chemotherapeutic drug, by engineering lipid bilayer-camouflaged mesoporous silica nanoparticles (LB-MSNs). In an attempt to deliver and maintain high tumor site concentrations of MET and TPT, a novel ion pairing-assisted loading procedure is developed using pamoic acid (PA) as an in situ trapping agent. PA, a hydrophobic counterion, increases the hydrophobicity of MET and TPT and facilitates MSNs with exceptionally high payload capacity (>40 and 32 wt%, respectively) and controlled release profile. Further, the synergy between MET and TPT determined by a modeling approach helps to afford synchronized delivery of both the drugs. Coloaded MET and TPT LB-MSNs present synergistic cytotoxicity against MDA-MB-231/4T1 cells and effectively promote apoptosis via mitochondrial membrane depolarization and cell cycle arrest. Extended pharmacokinetic profiles in preclinical models with fourfold to sevenfold longer circulation half-life and 7.5-100 times higher tumor site concentrations correspond to a significant increase in pharmacodynamic efficacy. Taken together, the developed codelivery approach effectively addresses the challenges in the chemotherapeutic efficacy of MET and TPT collectively.

In Depth Analysis of Pressure-Sensitive Adhesive Patch-Assisted Delivery of Memantine and Donepezil Using Physiologically Based Pharmacokinetic Modeling and in Vitro/in Vivo Correlations.

The objective of this work was to evaluate the feasibility of transdermal delivery of two widely prescribed dementia drugs for the Alzheimer's disease. In this regard, the drug in adhesive patches of memantine (ME) co-loaded with donepezil (DO) was prepared using an ethylene vinyl acetate polymer and characterized for drug content, the crystallinity of drugs in the polymer matrix, and in vitro permeation. To understand the different physical and chemical processes underlying the percutaneous absorption, it is required to employ a comprehensive model that accounts for the anatomy and physiology of the skin. A transdermal physiologically based pharmacokinetic (TPBPK) model was developed and was integrated in a compartmental pharmacokinetic model to predict the plasma drug concentrations in rats. The model predictions showed a good fit with the experimental data, as evaluated by the prediction error calculated for both drugs. It was evident from the simulations that the drug diffusivity and partition coefficient in the polymer matrix are the critical parameters that affect the drug release from the vehicle and subsequently influence the in vivo pharmacokinetic profile. Moreover, a correlation function was built between the in vitro permeation data and in vivo absorption for both ME and DO. A good point-to-point in vitro/in vivo correlation (IVIVC, Level A correlation) was achieved by predicting the plasma concentrations with convolution for the entire study duration. The results of our study suggested that the implementation of mechanistic modeling along with IVIVC can be a valuable tool to evaluate the relative effects of formulation variables on the bioavailability from transdermal delivery systems.

Multifunctional Glycoconjugate Assisted Nanocrystalline Drug Delivery for Tumor Targeting and Permeabilization of Lysosomal-Mitochondrial Membrane.

Nanotechnology has emerged as the most successful strategy for targeting drug payloads to tumors with the potential to overcome the problems of low concentration at the target site, nonspecific distribution, and untoward toxicities. Here, we synthesized a novel polymeric conjugate comprising chondroitin sulfate A and polyethylene glycol using carbodiimide chemistry. We further employed this glycoconjugate possessing the propensity to provide stability, stealth effects, and tumor targeting via CD44 receptors, all in one, to develop a nanocrystalline system of docetaxel (DTX@CSA-NCs) with size < 200 nm, negative zeta potential, and 98% drug content. Taking advantage of the enhanced permeability and retention effect coupled with receptor mediated endocytosis, the DTX@CSA-NCs cross the peripheral tumor barrier and penetrate deeper into the cells of tumor mass. In MDA-MB-231 cells, this enhanced cellular uptake was observed to exhibit a higher degree of cytotoxicity and arrest in the G2 phase in a time dependent fashion. Acting via a mitochondrial-lysosomotropic pathway, DTX@CSA-NCs disrupted the membrane potential and integrity and outperformed the clinically used formulation. Upon intravenous administration, the DTX@CSA-NCs showed better pharmacokinetic profile and excellent 4T1 induced tumor inhibition with significantly less off target toxicity. Thus, this glycoconjugate stabilized nanocrystalline formulation has the potential to take nano-oncology a step forward.

Anisamide-Anchored Lyotropic Nano-Liquid Crystalline Particles with AIE Effect: A Smart Optical Beacon for Tumor Imaging and Therapy.

The prospective design of nanocarriers for personalized oncotherapy should be an ensemble of targeting, imaging, and noninvasive therapeutic capabilities. Herein, we report the development of the inverse hexagonal nano-liquid crystalline (NLC) particles that are able to host formononetin (FMN), a phytoestrogen with known anticancer activity, and tetraphenylethene (TPE), an iconic optical beacon with aggregation-induced emission (AIE) signature, simultaneously. Ordered three-dimensional mesoporous internal structure and high-lipid-volume fraction of NLC nanoparticles (NLC NPs) frame the outer compartment for the better settlement of payloads. Embellishment of these nanoparticles by anisamide (AA), a novel sigma receptor targeting ligand using carbodiimide coupling chemistry ensured NLC's as an outstanding vehicle for possible utility in surveillance of tumor location as well as the FMN delivery through active AIE imaging. The size and structural integrity of nanoparticles were evaluated by quasi-elastic light scattering, cryo field emission scanning electron microscopy small-angle X-ray scattering. The existence of AIE effect in the nanoparticles was evidenced through the photophysical studies that advocate the application of NLC NPs in fluorescence-based bioimaging. Moreover, confocal microscopy illustrated the single living cell imaging ability endowed by the NLC NPs. In vitro and in vivo studies supported the enhanced efficacy of targeted nanoparticles (AA-NLC-TF) in comparison to nontargeted nanoparticles (NLC-TF) and free drug. Apparently, this critically designed multimodal NLC NPs may establish a promising platform for targeted and image-guided chemotherapy for breast cancer.

Long Acting Ionically Paired Embonate Based Nanocrystals of Donepezil for the Treatment of Alzheimer's Disease: a Proof of Concept Study.

PURPOSE: The aim of the present study was to prepare a patient friendly long acting donepezil (D) nanocrystals (NCs) formulation, with a high payload for i.m administration. As the native D hydrochloride salt has high aqueous solubility it is necessary to increase its hydrophobicity prior to the NCs formation. METHODS: D was ionically paired with embonic acid (E) in aqueous media and was successfully characterized using techniques like DSC, PXRD, FT-IR, NMR etc. Later, we converted the bulk ion pair into NCs using high pressure homogenization technique to study further in-vitro and in-vivo. RESULTS: The bulk ion pair has a drug content of 66% w/w and an 11,000 reduced solubility in comparison to native D hydrochloride. Also, its crystalline nature was confirmed by DSC and PXRD. The possible interaction sites responsible for the ion pair formation were identified though NMR. The prepared NCs has mean particle size 677.5 ± 72.5 nm and PDI 0.152 ± 0.061. In-vitro release showed a slow dissolution of NCs. Further, excellent bio compatibility of NCs were demonstrated in 3T3 cells. Following i.m administration of single dose of NCs, the D plasma level was found to be detectable up to 18 days. In vivo pharmacodynamic studies revealed that the single dose NCs i.m injection improved spatial memory learning and retention in ICV STZ model. CONCLUSION: Our results suggest that the developed formulation has a potential to replace the current daily dosing regimen to a less frequent dosing schedule. Graphical Abstract Improved pharmacokinetic and pharmacodynamic profile after administration of single dose donpezil embonate nanocrystals in Rats.

P-gp modulatory acetyl-11-keto-ß-boswellic acid based nanoemulsified carrier system for augmented oral chemotherapy of docetaxel.

In spite of being a very potent and promising drug against many types of cancer, docetaxel suffers the disadvantage of low solubility and poor bioavailability rendering it unsuitable for oral administration. Also, the available marketed formulation for intravenous administration has its inherent drawbacks owing to the presence of polysorbate 80. Here, we exploited the anticancer and P-gp inhibitory potential of naturally occurring frankincense oil to fabricate a stable docetaxel loaded nanoemulsified carrier system for oral delivery. The nanoemulsion possessing desirable particle size (122±12nm), polydispersity (0.086±0.007) and zeta potential (-29.8±2.1mV) was stable against all type of physical stresses and simulated physiological conditions tested. The formulation showed higher uptake in Caco-2 cells and inhibited P-gp transporter significantly (P<0.05). In MDA-MB-231 cells, it showed less IC50, arrested cells in G2-M phase and exhibited higher degree of apoptosis than marketed formulation Taxotere®. The 182.58±4.16% increment in relative oral bioavailability led to higher in vivo anti-proliferative activity manifesting 19% more inhibition than Taxotere®. Conclusively, it is revealed that the developed nanoemulsion will be a propitious approach towards alternative docetaxel therapy.

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.

Vitamin B6 Tethered Endosomal pH Responsive Lipid Nanoparticles for Triggered Intracellular Release of Doxorubicin.

This study reports the development of Vitamin B6 (VitB6) modified pH sensitive charge reversal nanoparticles for efficient intracellular delivery of Doxorubicin (DOX). Herein, VitB6 was conjugated to stearic acid, and the nanoparticles of the lipid were formulated by solvent injection method (DOX-B6-SA-NP). Because of the pKa (5.6) of VitB6, DOX-B6-SA-NP showed positive charge and enhanced release of DOX at pH 5. Confocal microscopy illustrated that DOX-B6-SA-NP treatment kept higher DOX accumulation inside the cells than conventional pH insensitive lipid nanoparticles (DOX-SA-NP). The cationic charge of nanoparticles subsequently facilitated the endosomal escape and promoted the nuclear accumulation of DOX. Furthermore, in vitro cytotoxicity, apoptosis, cell cycle arrest, and mitochondrial membrane depolarization studies supported the enhanced efficacy of DOX-B6-SA-NP in comparison to free DOX and DOX-SA-NP. Intravenous pharmacokinetics and biodistribution investigations indicated that pH sensitive nanoparticles can significantly prolong the blood circulation time of DOX in biological system and increase the drug accumulation to tumor site. Consequent to this, DOX-B6-SA-NP also exhibited much enhanced therapeutic efficacy and lower toxicity in tumor-bearing rats compared to free DOX. The reduction in toxicity was confirmed by histological and survival analysis. In conclusion, these results suggest that the VitB6 modified charge reversal nanoparticles can be a novel platform for the successful delivery of anticancer drugs.

Dual functioning microspheres embedded crosslinked gelatin cryogels for therapeutic intervention in osteomyelitis and associated bone loss.

In the present research,we simultaneously addressed the condition of osteomyelitis and osteoporosis by developing a gelatin based chemically cross linked cryogel system embedded with CaCO3 microspheres and ciprofloxacin hydrochloride was incorporated in both the microspheres and the 3D matrix of cryogel. The fabricated cryogel was characterized for the swelling ratio, swelling kinetics, porosity, pore volume, compression strength and in vitro rate of degradation which were found to be dependent on the concentration of gelatin, duration of freezing and number of freeze-thaw cycles. The sustained release of drug was obtained up to 21days after the initial burst, and the concentration was maintained above the MIC for the entire duration of the study. The in vitro antibacterial study in Staphylococcus aureus and Escherichia coli exhibited 33mm, 30mm, 28mm, 27mm and 43mm, 37mm, 37mm, and 36mm zone of inhibition respectively at day 1, 3, 5 and 7. The cell viability, number of cells in the growth phase and alkaline phosphatase levels were found to be significantly higher in rat osteoblasts cultured in cryogel as compared to 2D surface. All these results demonstrate the propitious potential of this microsphere incorporated, ciprofloxacin-loaded, industrially scalable cryogel system for therapeutic intervention in osteoporosis and associated osteomyelitis.

Investigation of salt formation between memantine and pamoic acid: Its exploitation in nanocrystalline form as long acting injection.

In the present work, we prepared memantine-pamoic acid (MEM-PAM) salt by counter ion exchange in the aqueous phase to reduce the water solubility of MEM hydrochloride (native form) to make it suitable for long acting injection. The ratio of MEM to PAM in salt formation was optimized to maximize the loading efficiency and complexation efficiency. The 2:1 molar ratio of MEM to PAM salt form displayed nearly 95% complexation efficiency and 50% drug loading. The solubility was decreased by a ∼1250 folds. Thermo Gravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), and Powder X-ray Diffraction Analysis (PXRD) studies revealed the formation of new solid phase. Additionally, Nuclear Magnetic Resonance (NMR) spectroscopy confirmed the anhydrous nature of the salt form. Through Fourier transformation infrared spectroscopy (FT-IR) we identified the molecular interactions. Further, the microcrystals of the salt were transformed into nanocrystals (NCs) using high pressure homogenization. The particle size distribution and atomic force microscopy confirmed the monodispersed and spherical shape of the NCs. The in vitro dissolution studies were performed under sink condition in phosphate buffer saline pH 6.8. The results of MTT assay in murine fibroblast 3T3 cell line show that the NCs were less cytotoxic and more tolerable than plain MEM HCl. The in vivo performance of NCs administered as i.m. injection at three different doses in female Sprague-Dawley rats showed that the plasma levels lasted till the 24th day of the study. The pharmacokinetic parameters AUC0-∞ and Cmax increased linearly with increasing dose. Therefore, the results suggest that injectable NCs could represent a therapeutic alternative for the treatment of AD.

Preparation and characterization of isoniazid and lamivudine co-loaded polymeric microspheres.

Context The rate of co-infection of HIV/Tuberculosis is increasing alarmingly. This calls for a drug delivery approach targeting both diseases. Objective The study aims to investigate co-loading of isoniazid, an antitubercular drug and lamivudine an antiretroviral drug, into polymeric microspheres for simultaneous treatment of both diseases. Materials and methods Microspheres were prepared by o/o emulsion solvent evaporation method by employing ethylcellulose and eudragit RS 100 as polymers. The prepared formulation was suitably characterized for FTIR, DSC, percent yield, loose surface crystals, entrapment efficiency, and in vitro studies. The surface morphology of microspheres was observed using digital microscope and scanning electron microscope. Cell viability study was done on Caco-2 cells. Results and discussion FTIR and DSC studies demonstrated compatibility and stability of excipients. Microscopy studies revealed that particles were spherical in shape and distributed over a range of 120-270 µm. Percent yield, LSC and %EE have shown promising results. In vitro release showed biphasic release pattern with sustained release up to 12 h. Mechanism of drug release followed Higuchi Kinetics and non-fickian release behaviour. The formulation containing drug/polymer ratio 1:2 and EU/EC of 1:1 showed optimum response in context to achievement of controlled release. The cell viability studies showed that the prepared system had no toxic effect on intestinal epithelial Caco-2 cells. Conclusion Polymeric microspheres were prepared and suitably characterized for simultaneous delivery of two drugs. This matrix system could be used for better therapeutic outcome in this deadly co-infection.

Investigating the role of Pluronic-g-Cationic polyelectrolyte as functional stabilizer for nanocrystals: Impact on Paclitaxel oral bioavailability and tumor growth.

Paclitaxel (PTX) is a potent anticancer drug which suffers limitations of extremely low oral bioavailability due to low solubility, rapid metabolism and efflux by P-gp transporters. The main objective of this study was to overcome the limitation of PTX by designing delivery systems that can enhance the absorption using multiple pathways. A novel Pluronic-grafted chitosan (Pl-g-CH) copolymer was developed and employed as a functional stabilizer for nanocrystals (NCs) and hypothesized that it would improve PTX absorption by several mechanisms and pathways. Pl-g-CH was synthesized and characterized using (1)H NMR and then used as a stabilizer during nanocrystal development. To establish our proof of concept the optimized formulation having a particle size 192.7 ± 9.2 nm and zeta potential (+) 38.8 ± 3.12 mV was studied extensively on in vitro Caco-2 model. It was observed that nanocrystals rendered higher PTX accumulation inside the cell than Taxol™. P-gp inhibitory potential of Pl-g-CH was proved by flow cytometry and fluorescence microscopy where the much enhanced fluorescence intensity of Rhodamine 123 (Rho-123, P-gp substrate) was observed in the presence of Pl-g-CH. In addition, a significant decrease in Trans Epithelial Electrical Resistance (TEER) of Caco-2 cell monolayers was observed with nanocrystals as well as with Taxol™ (in the presence of free Pl-g-CH compared to only Taxol™). This supports the role of the stabilizer in reversible opening of tight junctions between cells which can allow paracellular transport of drug. The in vivo results were in complete corroboration with in vitro results. Nanocrystals resulted in much enhanced absorption with 12.6-fold improvement in relative bioavailability to that of Taxol™. Concomitantly efficacy data in B16 F10 murine melanoma model also showed a significant reduction in tumor growth with nanocrystals compared to Taxol™ and control. Based on the results it can be suggested that nanocrystals with functional stabilizers can be a promising approach for the oral delivery of anticancer drugs which are P-gp substrates STATEMENT OF SIGNIFICANCE: Nanocrystals are currently one of the most explored novel drug delivery systems especially for oral delivery of drugs because of ease in synthesis and high drug loading. But their use is still limited for oral delivery of anticancer drugs which are P-gp substrates. This particular study aims at widening the scope of nanocrystals by using a functional stabilizer which participates in enhancing the oral absorption of anticancer drugs and controlling the tumor growth.

An insight into functionalized calcium based inorganic nanomaterials in biomedicine: Trends and transitions.

Over the recent years the use of biocompatible and biodegradable nanoparticles in biomedicine has become a significant priority. Calcium based ceramic nanoparticles like calcium phosphate (CaP) and calcium carbonate (CaCO3) are therefore considered as attractive carriers as they are naturally present in human body with nanosize range. Their application in tissue engineering and localized controlled delivery of bioactives for bones and teeth is well established now, but recently their use has increased significantly as carrier of bioactives through other routes also. These delivery systems have become most potential alternatives to other commonly used delivery system because of their cost effectiveness, biodegradability, chemical stability, controlled and stimuli responsive behaviour. This review comprehensively covers their characteristic features, method of preparation and applications but the thrust is to focus their recent development, functionalization and use in systemic delivery. On the same platform mineralization of other nanoparticulate delivery system which has widened their application drug delivery will be discussed. The emphasis has been given on their pH dependent properties which make them excellent carriers for tumour targeting and intracellular delivery. Finally this review also attempts to discuss their drawback which limits their clinical utility.

Therapeutic interventions in sepsis: current and anticipated pharmacological agents.

Sepsis is a clinical syndrome characterized by a multisystem response to a pathogenic assault due to underlying infection that involves a combination of interconnected biochemical, cellular and organ-organ interactive networks. After the withdrawal of recombinant human-activated protein C (rAPC), researchers and physicians have continued to search for new therapeutic approaches and targets against sepsis, effective in both hypo- and hyperinflammatory states. Currently, statins are being evaluated as a viable option in clinical trials. Many agents that have shown favourable results in experimental sepsis are not clinically effective or have not been clinically evaluated. Apart from developing new therapeutic molecules, there is great scope for for developing a variety of drug delivery strategies, such as nanoparticulate carriers and phospholipid-based systems. These nanoparticulate carriers neutralize intracorporeal LPS as well as deliver therapeutic agents to targeted tissues and subcellular locations. Here, we review and critically discuss the present status and new experimental and clinical approaches for therapeutic intervention in sepsis.