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Roger Shen,1 Jane J Kim,2 Mingyi Yao,2,3 Tamer A Elbayoumi2,3 1Department of Family Medicine, Northeastern Health Systems-Tahlequah City Hospital, Tahlequah, OK, USA; 2Department of Pharmaceutical Sciences, College of Pharmacy-Glendale, Midwestern University, 3Nanomedicine Center of Excellence in Translational Nanomedicine, Midwestern University, Glendale, AZ, USA Abstract: Berberine (Brb) is an active alkaloid occurring in various common plant species, with well-recognized potential for cancer therapy. Brb not only augments the efficacy of antineoplastic chemotherapy and radiotherapy but also exhibits direct antimitotic and proapoptotic actions, along with distinct antiangiogenic and antimetastatic activities in a variety of tumors. Despite its low systemic toxicity, several pharmaceutical challenges limit the application of Brb in cancer therapy (ie, extremely low solubility and permeability, very poor pharmacokinetics (PKs), and oral bioavailability). Among lipid-based nanocarriers investigated recently for Brb, stealth amphiphilic micelles of polymeric phospholipid conjugates were studied here as a promising strategy to improve Brb delivery to tumors. Specifically, physicochemically stable micelles made of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-PE) mixed with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (PEG-succinate ester of vitamin E), in a 3:1 M ratio, increased Brb solubilization by 300%. Our PEG-PE/TPGS-mixed micelles firmly retained the incorporated Brb, displaying extended-release profile in simulated media, with up to 30-fold projected improvement in simulated PKs of Brb. Owing to the markedly better uptake of Brb-containing mixed micelles in vitro, our Brb-mixed micelles nanoformulation significantly amplified apoptosis and overall cytotoxic effectiveness against monolayer and spheroid cultures of human prostate carcinomas (16- to 18-fold lower half-maximal inhibitory concentration values in PC3 and LNPaC, respectively), compared to free Brb. Mixed PEG-PE/TPGS micelles represent a promising delivery platform for the sparingly soluble anticancer agent, Brb, encouraging further pharmaceutical development of this drug for cancer therapy. Keywords: mixed micelles, polymer–phospholipid conjugates, vitamin E TPGS, berberine hydrochloride, apoptosis, prostatic adenocarcinoma

Amy Gavin,1 Jimmy TH Pham,2 Dawei Wang,2 Bill Brownlow,3 Tamer A Elbayoumi3 1College of Dental Medicine, 2Arizona College of Osteopathic Medicine, 3Department of Pharmaceutical Sciences, College of Pharmacy-Glendale, Midwestern University, Glendale, AZ, USA Abstract: Oral cavity and oropharyngeal cancers are considered the eighth most common cancer worldwide, with relatively poor prognosis (62% of patients surviving 5 years, after diagnosis). The aim of this study was to develop a proof-of-concept mucoadhesive lozenge/buccal tablet, as a potential platform for direct sustained delivery of therapeutic antimitotic nanomedicines. Our system would serve as an adjuvant therapy for oral cancer patients undergoing full-scale diagnostic and operative treatment plans. We utilized lipid-based nanocarriers, namely nanoemulsions (NEs), containing mixed-polyethoxylated emulsifiers and a tocopheryl moiety–enriched oil phase. Prototype NEs, loaded with the proapoptotic lipophilic drug genistein (Gen), were further processed into buccal tablet formulations. The chitosan polyelectrolyte solution overcoat rendered NE droplets cationic, by acting as a mucoadhesive interfacial NE layer. With approximate size of 110 nm, the positively charged chitosan-layered NE (+25 mV) vs negatively charged chitosan-free/primary aqueous NE (-28 mV) exhibited a controlled-release profile and effective mucoadhesion for liquid oral spray prototypes. When punch-pressed, porous NE-based buccal tablets were physically evaluated for hardness, friability, and swelling in addition to ex vivo tissue mucoadhesion force and retention time measurements. Chitosan-containing NE tablets were found equivalent to primary NE and placebo tablets in compression tests, yet significantly superior in all ex vivo adhesion and in vitro release assays (P≤0.05). Following biocompatibility screening of prototype chitosan-layered NEs, substantial anticancer activity of selected cationic Gen-loaded NE formulations, against two oropahryngeal carcinomas, was observed. The data strongly indicate the potential of such nanomucoadhesive systems as maintenance therapy for oral cancer patients awaiting surgical removal, or postresection of identified cancerous lesions. Keywords: isoflavone, genistein, chitosan, squamous cell carcinomas

Autoimmune diseases, including multiple sclerosis (MS), are proven to increase the likelihood of developing cardiovascular disease (CVD) due to a robust systemic immune response and inflammation. MS can lead to cardiovascular abnormalities that are related to autonomic nervous system dysfunction by causing inflammatory lesions surrounding tracts of the autonomic nervous system in the brain and spinal cord. CVD in MS patients can affect an already damaged brain, thus worsening the disease course by causing brain atrophy and white matter disease. Currently, the true prevalence of cardiovascular dysfunction and associated death rates in patients with MS are mostly unknown and inconsistent. Treating vascular risk factors is recommended to improve the management of this disease. This review provides an updated summary of CVD prevalence in patients with MS, emphasizing the need for more preclinical studies using animal models to understand the pathogenesis of MS better. However, no distinct studies exist that explore the temporal effects and etiopathogenesis of immune/inflammatory cells on cardiac damage and dysfunction associated with MS, particularly in the cardiac myocardium. To this end, a thorough investigation into the clinical presentation and underlying mechanisms of CVD must be conducted in patients with MS and preclinical animal models. Additionally, clinicians should monitor for cardiovascular complications while prescribing medications to MS patients, as some MS drugs cause severe CVD., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Anthracycline antibiotics, namely, doxorubicin (DOX) and daunorubicin, are among the most widely used anticancer therapies, yet are notoriously associated with severe myocardial damage due to oxidative stress and mitochondrial damage. Studies have indicated the strong pharmacological properties of Berberine (Brb) alkaloid, predominantly mediated via mitochondrial functions and nuclear networks. Despite the recent emphasis on Brb in clinical cardioprotective studies, pharmaceutical limitations hamper its clinical use. A nanoformulation for Brb was developed (mMic), incorporating a cationic lipid, oleylamine (OA), into the TPGS-mixed corona of PEGylated-phosphatidylethanolamine (PEG-PE) micelles. Cationic TPGS/PEG-PE mMic with superior Brb loading and stability markedly enhanced both intracellular and mitochondria-tropic Brb activities in cardiovascular muscle cells. Sub-lethal doses of Brb via cationic OA/TPGS mMic, as a DOX co-treatment, resulted in significant mitochondrial apoptosis suppression. In combination with an intense DOX challenge (up to ~50 µM), mitochondria-protective Brb-OA/TPGS mMic showed a significant 24 h recovery of cell viability ( p ≤ 0.05-0.01). Mechanistically, the significant relative reduction in apoptotic caspase-9 and elevation of antiapoptotic Bcl-2 seem to mediate the cardioprotective role of Brb-OA/TPGS mMic against DOX. Our report aims to demonstrate the great potential of cationic OA/TPGS-mMic to selectively enhance the protective mitohormetic effect of Brb to mitigate DOX cardiotoxicity.

The emerging landscape of nanomedicine includes a wide variety of active pharmaceutical ingredients and drug formulations. Their design provides nanomedicines with unique features leading to improved pharmacokinetics and pharmacodynamics. They are manufactured using conventional or biotechnological manufacturing processes. Their physical characteristics are vastly different from traditional small-molecule drugs. Pharmacists are important members of the multi-disciplinary team of scientists involved in their development and clinical application. Consequently, their training should lead to an understanding of the complexities associated with the production and evaluation of nanomedicines. Therefore, student pharmacists, post-doctoral researchers, and trainees should be given more exposure to this rapidly evolving class of therapeutics. This commentary will provide an overview of nanomedicine education within the selection of pharmacy programs globally, discuss the current regulatory challenges, and describe different approaches to incorporate nanomedicine science in pharmacy programs around the world.

Homeostasis of vascular tone is intricately and delicately maintained systemically and locally, by autonomic nerves and hormones in the blood and by intimal vasoactive substances, respectively. The balance can be acutely or chronically interrupted secondary to many alterations, especially under pathological conditions. Excessive matricellular glycoprotein thrombospondin 1 (TSP1) levels in circulation have been found to play an important role in ischemia-reperfusion injuries of different organs, by acutely suppressing vasorelaxation and chronically remodeling vascular bed. Our laboratory has been interested in identifying new drug moieties, which can selectively and effectively counteract TSP1-induced vascular dysfunction, in order to address associated clinical complications. Preliminary studies using computational docking and molecular models revealed potential drug candidates for further evaluation via vascular functional bioassay to prove the antagonism using an ex vivo vascular model. Herein, we described an efficient screening method for the identification of active drug candidates, by adapting a multiwire myograph system to perform a protocol with different treatments, in the presence of pathological levels of TSP1. We discussed the promising pharmacological evaluation results and suggested suitable modification for versatile applications. We also described the necessity of pre-determination of optimal resting tension to obtain the maximal response, if the experimental test model is different from those with determined optimal resting tension.

The field of nanomedicine is a rapidly growing scientific domain. Nanomedicine encompasses a diverse number of active pharmaceutical ingredients. Submissions of Investigational New Drugs and New Drug Applications have risen dramatically over the last decade. There are over 50 nanomedicines approved for use by the US Food and Drug Administration (FDA). Because of the fundamental role pharmacists will play in therapeutic and administrative decisions regarding nanomedicines, it is imperative for future pharmacists to gain exposure early in their training to this rapidly evolving class of drugs. This commentary describes nanomedicines, discusses current regulatory challenges, and provides recommendations for judicious incorporation of nanomedicine topics into the Doctor of Pharmacy curriculum based on emerging pharmaceutical and clinical science applications., (© 2021 American Association of Colleges of Pharmacy.)

Elevated thrombospondin 1 (TSP1) is a prevalent factor, via cognate receptor CD47, in the pathogenesis of cardiovascular conditions, including ischemia-reperfusion injury (IRI) and pulmonary arterial hypertension (PAH). Moreover, TSP1/CD47 interaction has been found to be associated with platelet hyperaggregability and impaired nitric oxide response, exacerbating progression in IRI and PAH. Pathological TSP1 in circulation arises as a target of our novel therapeutic approach. Our "proof-of-concept" pharmacological strategy relies on recombinant human CD47 peptide (rh-CD47p) as a decoy receptor protein (DRP) to specifically bind TSP1 and neutralize TSP1-impaired vasorelaxation, strongly implicated in IRI and PAH. The binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western blotting and further quantified with modified ELISA, which also revealed a linear molar dose-dependent interaction. Ex vivo, pretreatment protocol with rh-CD47p (rh-CD47p added prior to TSP1 incubation) demonstrated a prophylactic effect against TSP1-impairment of endothelium-dependent vasodilation. Post-treatment set-up (TSP1 incubation prior to rh-CD47p addition), mimicking pre-existing excessive TSP1 in PAH, reversed TSP1-inhibited vasodilation back to control level. Dose titration identified an effective molar dose range (approx. ≥1:3 of tTSP1:rh-CD47p) for prevention of/recovery from TSP1-induced vascular dysfunction. Our results indicate the great potential for proposed novel decoy rh-CD47p-therapy to abrogate TSP1-associated cardiovascular complications, such as PAH.

Creatine kinase (CK) enzyme overexpression has been suggested to play a role in the process of tumorigenesis and metastasis. Cyclocreatine (CCR) is a substrate analog of creatine kinase (CK), where its phosphorylated form is a poor phosphate donor in comparison with native bioenergetic molecule, creatine phosphate (Cr-P). The compound CCR has been shown to markedly inhibit the growth of a broad spectrum of cancers, both in vitro and in vivo. Intracellularly, CCR is phosphorylated by CK to yield a synthetic phosphagen [(N-phosphorylcyclocreatine (CCR ~P)], with thermodynamic and kinetic properties distinct from those of creatine phosphate (Cr-P). Distinct inhibition of tumor growth and metastasis has been attributed to CCR accumulation as CCR ~P in tumor cells, especially in those expressing a high level of CK protein, with minimal adverse effects. Unfortunately, the clinical use of CCR against malignancies is quite limited due to its amphoteric nature, which accounts for most of its extremely low membrane permeability, as well as limited oral bioavailability (BA) and poor systemic pharmacokinetics (PK).Our current work describes the encapsulation of CCR , utilizing freeze and thaw vesicles (FTV )-composed mostly of saturated PC, DOPE, and Chol-into stealth™ liposomes , postcoated with 4.5 M% PEG-PE. Following physicochemical characterization, in vitro release and cellular uptake kinetics confirmed efficient delivery of liposomal CCR (CCR-Lip), leading to intracellular accumulation of its CC-P metabolic product. Successful delivery of CCR to cancer cell effectively depleted low energetic cancer cells of ATP significantly mediating myc-induced metabolic changes. CCR-Lip showed significant antimetastatic and anticancer effectiveness against both MCF-7 and PC-3 human carcinoma models (p < 0.05-0.01), with 4- to 6-fold lower IC50 values vs. closest drug control. Such shift in bioenergetics was coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak , thus inducing a cell-intrinsic mechanism to counteract uncontrolled neoplastic proliferation, in target cancer cells. Our novel liposomal delivery system of the CCR substrate analog demonstrated strong inhibition of malignant cell bioenergetics, leading to significant antineoplastic and proapoptotic actions, against different cancers.

Introduction : A critical mechanism of how hypoxia/ischemia causes irreversible myocardial injury is through the exhaustion of adenosine triphosphate (ATP). Cyclocreatine (CCr) and its water-soluble salt Cyclocreatine-Phosphate (CCrP) are potent bioenergetic agents that preserve high levels of ATP during ischemia. Areas covered : CCr and CCrP treatment prior to the onset of ischemia, preserved high levels of ATP in ischemic myocardium, reduced myocardial cell injury, exerted anti-inflammatory and anti-apoptotic activities, and restored contractile function during reperfusion in animal models of acute myocardial infarction (AMI), global cardiac arrest, cardiopulmonary bypass, and heart transplantation. Medline and Embase (1970 - Feb 2019), the WIPO databank (up to Feb 2019); no language restriction. Expert opinion : This review provides the basis for a number of clinical applications of CCrP and CCr to minimize ischemic injury and necrosis. One strategy is to administer CCrP to AMI patients in the pre-hospital phase, as well as during, or after Percutaneous Coronary Intervention (PCI) procedure to potentially achieve protection of the myocardium, reduce infarcted-size, and, thus, limit the progression to heart failure. Another clinical applications are in predictable myocardial ischemia where pretreatment with CCrP would likely improve outcome and quality of life of patients who will undergo cardiopulmonary bypass for coronary revascularization and end-stage heart failure patients scheduled for heart transplantation.

Lipid-based vesicles, namely cationic liposomal nanocarriers have been recognized early on as one of the most attractive delivery systems for RNA, protein, and oligonucleotides. Despite several advantages of conventional liposomal carriers for therapeutic macromolecules, their flexible and unsupported bilayered membranes can pose some limitations for efficient intracellular delivery of their sensitive cargos. Hence, polymerized liposomes, a concept conceived about 20 years ago, might offer structural solution to current in vivo efficiency concerns affecting traditional cationic phospholipid vectors, especially when adapted to enable superior loading and stability, typically required for effective intracellular delivery of proteins and polynucleotides.Our recent approach attempted to remodel polymerized liposomal vesicles-specifically their semi-rigid membrane structure-to create block-polymerized bilayered vesicles (generally composed of DOTAP: DOPE: Diyne PC in 0.1:1:1 molar ratio). Adopting a modified freeze-dry-rehydration technique allowed modular reassembly of such partially polymerized lipidic vesicles (PPL). Different prototype cationic partially polymerized liposomal preparations (PPLs) were successfully developed (mean particle size range 150-300 nm), demonstrating enhanced physicochemical stability and loading capacity, thus promoting improved intracellular delivery of model RNAi and protein cargos.

Berberine (Brb) is an active isoquinoline alkaloid occurring in various common plant species, with well-known potential for cancer therapy. Earlier reports has shown that Brb not only augments the efficacy of antineoplastic chemotherapy and radiotherapy, but it also exhibits direct anti-mitotic, and pro-apoptotic activities, plus significant anti-angiogenic and anti-metastatic activities in a variety of solid tumors. Notwithstanding its low systemic toxicity, a few pharmaceutical limitations severely hamper the application of Brb in cancer therapy (namely, very slight aqueous solubility and exceedingly low membrane permeability; combined with poor systemic pharmacokinetic, PK, profile).Lipid-based nanocarriers, amphiphilic mixed micelles (Mic) composed of polymeric phospholipid conjugates and PEG-succinate ester of tocopherol were investigated as promising strategy, to improve Brb delivery into tumors. Following physicochemical characterization of micellar Brb, in vitro release studies in simulated physiological media were performed, combined with PK-simulation and in vitro assays of cytotoxicity and direct apoptosis induction in different human prostate cancer cell lines (PC3 and LNPaC).Optimized stealth PEG-PE/TPGS-mixed micelles achieved efficient solubilization of Brb to potentially improve its systemic PK profiles (>30-fold). Our mixed micellar platform resulted in significant enhancement of the pro-apoptotic action and overall anticancer efficacy of Brb, against various in vitro (monolayer and spheroid) models of prostate cancers.

Relatively little is known about the inflammatory mediators and mechanisms that drive the progression of influenza flu infection to cytokine storm, lung dysfunction, organ failure, and ultimately death. Vaccines and antiviral medications cannot control the excessive host inflammatory response associated with severe influenza flu infection. Studies by Elgebaly et al demonstrated the rapid release of a potent inflammatory mediator, recently named Nourin, by local mammalian tissues in response to injury and infection. Nourin is a formyl peptide that acts through the formyl peptide receptor (FPR) on phagocytic leukocytes. As an initial signal in the innate immunity, Nourin stimulates leukocyte chemotaxis, induces acute and chronic inflammation, and stimulates the release of a number of the cytokine storm mediators from monocytes, neutrophils and endothelial cells. Furthermore, Nourin detected in plasma samples from patients with severe influenza infection was much higher compared to moderate influenza. The Nourin antagonist, Cyclosporin H, is a potent anti-inflammatory compound, which acts as a specific competitive antagonist of formyl peptides on the formyl peptide receptor (FPR) on phagocytic: leukocytes. Cyclosporin H completely blocked neutrophil chemotaxis induced by: (a) the standard formyl peptide, f-MLF, (b) the Staphylococcus aureus bacteria-derived formyl peptide Phenol-soluble modulins, such as PSM3a, plus(c) the host-derived Nourin released by: (1) cultured epithelial cells infected with the PR8 HINI influenza virus for 6.to 24 hours, (2),Nourin detected in the serum of mouse model of HINI Swine flu influenza infection for 6 hours , along with (3) Nourin detected in plasma samples collected from severe and moderate influenza pa- tients. Furthermore, in-vivo treatment by Cyclosporin H in the mouse model of HINI Swine flu influenza infection for 5 days markedly reduced lung inflammation and endothelial cell damage. Thus, two clinical applications for Nourin and its antagonist Cyclosporin H are proposed: Diagnostic Application: The blood Nourin test can be used as a key inflammatory biomarker for "early" detection and monitoring of influenza flu patients proceeding to hyperactive inflammation and, thus, permitting early crucial anti-inflammatory therapy. Therapeutic Application: Cyclosporin H will specifically block Nourin as an important initial stimulant of cytokine mediators, and thus can control the development and progression of cytokine storm plus organ inflammation, which usually initiates 3 to 8 days post influenza. Since Cyclosporin H does not target the virus,, it will not develop drug resistance,and will reduce the host uncontrpolled inflammatory response, induced by both new strains of flu viruses and existing viruses with mutations.

The potent anti-proliferative and pro-apoptotic actions of tocotrienols (T3) against cancer, but not normal tissues, have been hampered by their limited systemic bioavailabilty. Recent expansive development of diverse nanoemulsion (NE) vehicles emphasized their vast potential to improve the effective dosing of different clinical and experimental drugs of lipophilic nature, such as T3. The emphasis of the present work is to develop a pharmaceutically scalable, low-energy nano-emulsification approach for optimized incorporation of T3-rich palm oil (Tocomin®), possessing anticancer activity as a potential cutaneous delivery platform for adjunctive therapy of skin carcinomas, either alone or in combination with other chemotherapeutic agents. Different Tocomin®-NEs, obtained with different homogenization strategies, were screened based on physicochemical uniformity (droplet size, charge and polydispersity) and subjected to stress physical stability testing, along with chemical content analysis (≥90% Tocomin® - incorporation efficiency). Adopted hybrid nano-emulsification of Tocomin®, correlated with highest preservation of DPPH-radical scavenging capacity of active T3 in prototype formulation, Tocomin®-NE, which effectively permeated diffusion cell membranes 4-folds higher than propyleneglycol (PG)-admixed Tocomin® control. Against two different cell models of human cutaneous carcinoma, Tocomin®-hybrid NE demonstrated significantly stronger cytotoxic profiles (p ≤ 0.01), visible in both concentration- and time- dependent manners, with at least 5-folds lower IC50 values, compared to those estimated for the closest Tocomin®-control. The proposed hybrid nano-emulsified formulation of Tocomin® provides simple and stable delivery platform, for effective topical application against keratinocyte tumors.

There is a great need for effective protection against cutaneous pathologies arising from chronic exposure to harmful solar UVB radiations. A promising pharmaceutical strategy to improve the efficacy of chemotherapeutic/preventative natural compounds (e.g., soy isoflavone Genistein, Gen) is to enhance their dermal delivery using nanoemulsion (NE) formulations. This report investigates the development of nanoemulsified tocotrienol(T3)-rich fraction of red palm oil (Tocomin®), to yield an optimal NE delivery system for dermal photoprotection (z-average size <150 nm, ζ-potential ≈ -30 mV, polydispersity index < 0.25). Physicochemical characterization and photostability studies indicate NE formulations utilizing surfactant mixture (Smix) of Solutol® HS-15 (SHS15) blended with vitamin E TPGS (TPGS) as cosurfactant was significantly superior to formulations that utilized Lutrol® F68 (LF68) as the cosurfactant. A ratio of 60:40 of SHS15-TPGS-NE was further identified as lead Tocomin® NE topical platform using in vitro pharmaceutical skin reactivity studies that assess cutaneous irritancy and cytotoxicity. Prototype Tocomin® NE loaded with the antiphotocarcinogenic molecule Gen (Gen-Tocomin® NE) showed slow-release profile in both liquid and cream forms. Gen-Tocomin® NE also showed excellent biocompatibility, and provided substantial UVB protection to cultured subcutaneous L929 fibroblasts, indicating the great potential of our Tocomin® NE warranting further prototype development as topical pharmaceutical platform for skin photoprotection applications., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Genistein (Gen), a major soy isoflavone, produces extensive pro-apoptotic anticancer effects, mediated predominantly via induction of mitochondrial damage. Based on several biophysical model criteria, our rational assumptions for the native mitochondrial selectivity of Gen allowed its design as a cationic lipid-based nanocarrier (NC) system. Proof-of-concept nano-formulations, lipidic micelles (Mic), and nanoemulsions (NEs) incorporated Gen, which serves as therapeutic and targeting moieties, specific for mitochondria. Our in vitro experimental data demonstrated superior physicochemical properties and significant cytotoxicity of Gen-NCs (five- to tenfolds lower EC50) compared to all drug controls, in hepatic and colon carcinomas. The established mitochondria-specific accumulation of the various Gen-NCs positively correlated with marked mitochondrial depolarization effects. Within first 24 h, Gen-NC treatments ultimately lead to distinct activation of intrinsic apoptotic pathway markers, such as cytosolic cytochrome c and specific caspase-9 vs. nonspecific caspases-3, 7, and 8. Such mechanistic evidence of the mitochondriotropic activity of our Gen-NC platforms favors their prospective as intracellularly targeted delivery nano-vehicles, to enhance anticancer efficacy of different co-formulated chemotherapeutic agents.

Clinical use of genistein against cancer is limited by its extremely low aqueous solubility, poor bioavailability and pharmacokinetics. Based on structural analogy with steroidal compounds, liposomal vehicle compositions were designed and optimized for maximum incorporation of genistein's flavonoid structure. Model conventional and stealth liposomes of genistein (GenLip)--incorporating unsaturated phospholipids and cholesterol--have demonstrated enhanced drug solubilization (over 350-folds > aqueous drug solution), shelf-life stability, and extended release profile. Owing to effective cellular delivery, preservation of genistein's antioxidant activity was confirmed through marked neutralization of peroxides via GenLip, in both quantitative and microscopic fluorescent-probe oxidation assays. Furthermore, significant broad-spectrum anticancer efficacy of GenLip, in murine and human cancer cell lines (p < 0.05-0.001), was achieved in a concentration and time-dependent manner--approx. 5-7 lower IC50 values versus all non-incorporated drug controls. Indicative of key pro-apoptotic activity, GenLip produced DNA laddering, with 1/3 of free drug solution content, and resulted in the highest induction level of P53-independent apoptotic pathway markers, compared to all treatments, in our assays (namely, mitochondrial polarization, and caspase-3/7 enzymes). Our proof-of-principle pharmaceutical design of genistein-loaded liposomes shows optimal loading capacity and physico-chemical properties, which improved cellular delivery and specific pro-apototic effectiveness of incorporated drug, against various cancers.

Genistein (Gen) soy isoflavone produces extensive pro-apoptotic anticancer effects, mediated predominantly via induction of mitochondrial damages. Rationalization of the native mitochondrial selectivity of Gen, utilizing biophysical model assumptions, led to our design of cationic lipid-based nanocarriers (NC) of Gen. Prototype nanoformulations, lipidic micelles (Mic) and nanoemulsions (NEs) incorporated Gen to serve as both therapeutic and targeting moieties, specific for mitochondria. Both Gen-NCs, showing superior physicochemical properties, produced significant cytotoxicity (5-10-fold lower EC50), compared to all drug controls, in hepatic and colon carcinomas. Owing to the mitochondria-specific accumulation of Gen-NCs, their mitochondrial depolarization effect was most evident, leading to marked activation of intrinsic apoptotic pathway markers--cytosolic cytochrme c and specific caspase-9--thus, confirming the direct mitochondrial action of Gen-NCs. This mechanistic evidence of the mitochondria specificity of our Gen-NE and Gen-Mic strongly indicates their potential as targeted delivery nanosystems to augment anticancer efficacy of many lipophilic chemotherapeutics.

The quinonoid anthracycline, doxorubicin (Adriamycin), is a widely used potent antineoplastic agent, showing the broadest spectrum of antineoplastic activity against various types of solid carcinomas, hematological malignancies, and soft tissue sarcomas. Unfortunately, the clinical use of doxorubicin is associated with cumulative dose-limiting cardiac toxicity, manifested as cardiomyopathy and congestive heart failure, in which mitochondrial damage is primarily implicated. Free radical formation at and inside mitochondria, in particular the rise of reactive oxygen species (ROS), has long been hypothesized as the common mechanism by which doxorubicin causes this severe cardiotoxicity. Concomitant with newly gained insights into the central role of mitochondria in programmed cell death (apoptosis), irreversible destabilization of mitochondrial membrane permeability transition (mMPT), and disruption of mitochondrial Ca(2+) homeostasis have been strongly implicated in triggering myocardial apoptosis, due to accumulated doxorubicin dosing. Hence, our current protocols show the development of mitochondria-targeted nanoemulsions (NEs), based on previous work using nano-vesicle surface modification with mitochondriotropic triphenylphosphonium (TPP) ligands, which have successfully been demonstrated to target drug and DNA-loaded liposomes to mitochondria in living mammalian cells. Our mitochondria-specific TPP-coated therapeutic NEs are prepared using tocopherol oxygen scavengers and are highly loaded with mitochondria-stabilizing therapeutics, namely, cyclosporine A (CsA). Our targeted nano-formulation, proposed as injectable adjuvant therapy, is capable of reaching target affected mitochondria in sufficient therapeutic concentration, in order to revert or at least limit oxidative and non-oxidative doxorubicin-induced mitochondrial damage, manifested in affected cardiac muscle tissues, Based on several encouraging studies using in vitro model rat cardiac muscle, H9C2 cardiomyocytes, and vascular media tunica media, A10, cell cultures, our proof-of principal mitochondriotropic nano-therapy demonstrates strong potential to improve not only the cardiac safety profile, through concurrent rescue administration of targeted nano-encapsulated FDA-approved cyclosporine A (CSA), but also dosing range of the currently available potent adriamycin/doxorubicin-based chemotherapy regimens.

In recent years, diverse nanoemulsion vehicles (NEs) have been developed with vast potential for improving therapeutic index of clinically approved and experimental drugs. Using oils rich in omega-3 and omega-6 polyunsaturated fatty acids (PUFA), several promising nanoemulsion formulations have been developed recently for oral and systemic administration. The aim of our present work is to successfully develop and characterize optimized nanoemulsion platform, using the PUFA-rich argan oil that contain several important anti-inflammatory and antimitotic natural components. Using various emulsifying mixtures of polyethoxylated solutol HS-15 and polyethyleneglucol Vitamin E succinyl ester (TPGS), to form different NEs showing extended shelf-life stability. The physicochemical properties of prototype argan NEs were analyzed and utilizing a 32 full factorial design, followed by biocompatibility screen, using normal vascular myocytes and areolar fibroblasts. While 90-180 day stability of NEs correlated with TPGS:solutol surfactant blend ratios, adverse effects on integrity of test cultures were only noted at high TPGS content in the emulsifier system, exceeding 80%. Finally, the anti-proliferative efficacy of selected stable and acceptably biocompatible nanoscale TPGS-emulsified argan oil formulations was investigated using murine breast and colon carcinoma cells. The IC50 values of the combination of argan oil and TPGS (40-80% wt of emulsifiers) were 5-9 folds lower compared to TPGS-free and argan-oil free control NEs. Argan oil NE, stabilized with Vitamin E TPGS and solutol HS mixtures, demonstrated significant pro-apoptotic effect on both test cancer cell lines, indicating built-in anticancer properties for such NE platform, potentially enhancing overall antineoplastic effects of incorporated candidate chemotherapeutic agents.

An ever increasing number of diverse nanomaterials for biopharmaceutical applications is emerging, ranging from liposomes, niosomes, micelles, nanoemulsions, dendrimers, nanocells, cell ghosts, lipoproteins to solid nanoparticles and quantum dots. Several of these nanomaterials have been brought from the bench into the clinic over the last three decades giving raise to "Nanomedicine", a term implying the application of nanotechnology for therapy and diagnosis. Though pharmaceutical nanoparticular carriers have been successfully established as devices for enhancing the therapeutic index of clinically approved and experimental drugs, their utilization for imaging and monitoring biological functions has just began to materialize. The development of the effective carrier mechanism does not only mean the execution of delivery, but also the positive confirmation of the correct and specific delivery the cargo. Consequently, the ability to track and image the fate of any nanomedicine starting from the systemic down to the sub-cellular level becomes essential. In unison, the importance of monitoring any desired or undesired cellular and subcellular morphological and functional alterations subsequent to or indicative of therapeutic delivery is gaining increasingly attention. Focusing on selected pharmaceutical nanomaterials, this review tries to illustrate the interconnectivity between therapeutic applications of nanocarriers and their use for imaging and monitoring cellular and subcellular functions.

Micelles from the mixture of poly(ethylene glycol)-phosphatidyl ethanolamine conjugate (PEG-PE) and d-alpha-tocopheryl polyetheyene glycol 1000 succinate (TPGS) were prepared loaded with the poorly soluble anticancer drug camptothecin (CPT). The solubilization of CPT by the mixed micelles was more efficient than with earlier described micelles made of PEG-PE alone. CPT-loaded mixed micelles were stable upon storage and dilution and firmly retained the incorporated drug. The cytotoxicity of the CPT-loaded mixed micelles against various cancer cells in vitro was remarkably higher than that of the free drug. PEG-PE/TPGS mixed micelles may serve as pharmaceutical nanocarriers with improved solubilization capacity for poorly soluble drugs.