Liposomal Delivery of Cyclocreatine Impairs Cancer Cell Bioenergetics Mediating Apoptosis.

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.

Tocotrienol Nanoemulsion Platform of Curcumin Elicit Elevated Apoptosis and Augmentation of Anticancer Efficacy against Breast and Ovarian Carcinomas.

Vitamin E (VE) tocotrienols (T3), recognized for their cancer-specific anti-proliferative and pro-apoptotic activities, have been previously fabricated into bio-active nanoemulsion (NE) formulations. Here, our viscosity-adapted δ-T3 NE platform was developed to additionally incorporate curcumin (CUR), which is known for its potent suppression of signaling pathways involved in malignant cell growth, survival and metastasis. Thanks to efficient 70:30 wt % surfactant mix of Lutrol F-127:VE-TPGS, in conjunction with optimal CUR loading, a prototype CUR in δ-T3 NE was successfully prepared. Model CUR/δ-T3 NE demonstrated excellent nano-scale aspects (mean particle size = 261 nm, PDI = 0.27, and ζ-potential = -35 mV), pharmaceutical stability, and controlled release properties. Suitability for systemic administration was also verified via standardized in vitro biocompatibility and hemocompatibility assays. In two human cancer cells (MCF-7 and OVCAR-8), our CUR/δ-T3 NE prominently suppressed constitutive NF-κB activation, and significantly induced apoptosis. Finally, the combined CUR/δ-T3 NE produced superior cytotoxicity profiles, in concentration- and time-dependent manners (p ≤ 0.05), at least three to four folds lower IC50 than in closest CUR control. The strong synergism, estimated in both cultured carcinomas, revealed the augmented therapeutic efficacy of our CUR/δ-T3 NE combined platform, supporting its strong potential towards pharmaceutical development for cancer therapy.

Development and evaluation of vitamin E d-α-tocopheryl polyethylene glycol 1000 succinate-mixed polymeric phospholipid micelles of berberine as an anticancer nanopharmaceutical.

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.

Enhanced effectiveness of tocotrienol-based nano-emulsified system for topical delivery against skin carcinomas.

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.

Development and In Vitro Evaluation of Vitamin E-Enriched Nanoemulsion Vehicles Loaded with Genistein for Chemoprevention Against UVB-Induced Skin Damage.

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.

Mitochondria-specific nano-emulsified therapy for myocardial protection against doxorubicin-induced cardiotoxicity.

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.

Development and evaluation of tocopherol-rich argan oil-based nanoemulsions as vehicles possessing anticancer activity.

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.