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.

Layered nanoemulsions as mucoadhesive buccal systems for controlled delivery of oral cancer therapeutics.

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.

Current trends in liposome research.

Among the several drug delivery systems, liposomes--phospholipid nanosized vesicles with a bilayered membrane structure--have drawn a lot of interest as advanced and versatile pharmaceutical carriers for both low and high molecular weight pharmaceuticals. At present, liposomal formulations span multiple areas, from clinical application of the liposomal drugs to the development of various multifunctional liposomal systems to be used in therapy and diagnostics. This chapter provides a brief overview of various liposomal products currently under development at experimental and preclinical level.

Tumor-targeted nanomedicines: enhanced antitumor efficacy in vivo of doxorubicin-loaded, long-circulating liposomes modified with cancer-specific monoclonal antibody.

PURPOSE: The efficacy of drug delivery systems can be enhanced by making them target specific via the attachment of various ligands. We attempted to enhance tumor accumulation and therapeutic effect of doxorubicin-loaded, long-circulating, polyethylene glycol-coated liposomes (Doxil, ALZA Corp.) by coupling to their surface the anticancer monoclonal antibody (mAb) 2C5 with nucleosome-restricted activity that can recognize the surface of various tumors but not normal cells and specifically targets pharmaceutical carriers to tumor cells in vitro and in vivo. Following earlier in vitro results with various cancer cell lines, the mAb 2C5 liposomes were studied in vivo versus plain and nonspecific-IgG liposomes. EXPERIMENTAL DESIGN: Antibody coupling to Doxil was done via the "postinsertion" technique. Using (111)In-labeled liposomes, the tissue biodistribution and pharmacokinetic profile were studied, as well as their accumulation in tumors in mice, followed by the whole-body gamma-scintigraphic imaging. Therapeutic efficacy of mAb 2C5-targeted Doxil versus nonspecific IgG-modified and original Doxil controls was followed by registering live tumor growth and determining tumor weights upon mice sacrifice. RESULTS: mAb 2C5-targeted liposomes showed enhanced accumulation in tumors, and the in vivo therapeutic activity of the mAb 2C5-Doxil treatment was found to be significantly superior, resulting in final tumor weights of only 25% to 40% compared with all Doxil control treatments, when tested against the s.c. primary murine tumors of 4T1 and C26 and human PC3 tumor in nude mice. CONCLUSIONS: Our results showed the remarkable capability of 2C5-targeted Doxil to specifically deliver its cargo into various tumors, significantly increasing the efficacy of therapy.

Tumor-specific anti-nucleosome antibody improves therapeutic efficacy of doxorubicin-loaded long-circulating liposomes against primary and metastatic tumor in mice.

The efficacy of drug delivery systems can be significantly enhanced by making them target-specific via the attachment of various ligands to their surface. We attempted to enhance tumor accumulation and therapeutic effect of doxorubicin-loaded long-circulating liposomes (Doxil, ALZA Corp.) by coupling to their surface the anticancer monoclonal antibody 2C5 (mAb 2C5) with nuclesome (NS)-restricted activity, that can recognize the surface of various tumor but not normal cells via the surface-bound nucleosomes released from the apoptotically dying neighboring tumor cells and specifically targets pharmaceutical carriers to tumor cells in vitro and in vivo. Antibody coupling to PEGylated doxorubicin-liposomes was performed by the "post-insertion" technique. The pharmacokinetics of plain and immuno-targeted Doxil-mimicking liposomes, as well as their accumulation in primary Lewis lung carcinoma (LLC) tumors in mice was followed by real-time gamma-scintigraphy upon liposomal membrane labeling with (111)In. Therapeutic action of various liposomal formulations was followed by registering primary tumor growth, determining tumor weigh upon mice sacrifice, and by counting the number of metastases in the liver and lungs. 2C5 antibody-targeted liposomes demonstrate significantly enhanced accumulation in LLC tumors. Targeted doxorubicin-loaded PEG-liposomes were significantly more effective in inhibiting tumor growth and metastatic process in the LLC tumor models in mice. Our results clearly show the remarkable capability of 2C5-targeted Doxil to specifically deliver its cargo into various tumor manifestations (solid and metastatic) significantly increasing the efficacy of therapy.

Liposomes for targeted delivery of antithrombotic drugs.

BACKGROUND: Targeted delivery of antithrombotic (thrombolytic) drugs is expected to increase their efficacy and decrease side effects, especially in the case of thrombolytic enzymes. Liposomes, phospholipid nanosized bubbles with a bilayered membrane structure, have drawn a lot of interest as pharmaceutical carriers for drugs and genes. In particular, several attempts have been made to use liposomes as vehicles for antithrombotic agents. OBJECTIVE: This review analyzes the available data on the application of liposomes, including liposomes targeted by specific ligands, for the delivery of antithrombotic/thrombolytic agents in order to increase their efficacy and decrease side effects. METHODS: The papers published on the subject of liposomes loaded with antithrombotic agents, mainly over the last 10 - 15 years, will be discussed. CONCLUSION: Liposomes loaded with various antithrombotic drugs, though they have been the subject of a significant number of experimental papers, can hardly be considered as real candidates for clinical application in the near future.

Tumor-specific antibody-mediated targeted delivery of Doxil reduces the manifestation of auricular erythema side effect in mice.

A mucocutaneous reaction in mice associated with Doxil treatment was identified as auricular erythema (AE). Given that the immuno-targeting of Doxil to tumors was found to influence also its systemic biodistribution pattern, the attempt was made to exploit a specific targeting of Doxil to reduce the manifestation of this adverse reaction. This problem is of general significance, since cutaneous reactions often lead to alterations of Doxil dosing regimen in patients and might subsequently compromise the therapeutic outcome of cancer treatment. Tumor-bearing mice were used to study the biodistribution and skin-tissue accumulation effects of the tumor-targeted Doxil (the clinically used anti-cancer formulation) coupled with the anti-cancer monoclonal 2C5 antibody (mAb 2C5) as well as AE caused by Doxil application. The modification of Doxil with mAb 2C5 resulted in a significant decrease in the normal skin accumulation of doxorubicin compared to original Doxil and substantially reduced AE. The frequency of AE was decreased by three to fourfold with the mAb 2C5-modified doxorubicin-loaded long-circulating liposomes. Thus, targeting of Doxil with the anti-cancer mAb 2C5 not only can increase the tumor-specific accumulation of the drug, but also diminishes the cutaneous side effect of the original Doxil therapy.

Enhanced cytotoxicity of monoclonal anticancer antibody 2C5-modified doxorubicin-loaded PEGylated liposomes against various tumor cell lines.

Doxorubicin-loaded long-circulating liposomes (Doxil, ALZA Corp.) were additionally modified with the nucleosome-specific monoclonal antibody 2C5 (mAb 2C5) recognizing a broad variety of tumor cells via the tumor cell surface-bound nucleosomes. These mAb 2C5-modified PEGylated liposomes demonstrated 3-8-fold increase in the in vitro binding and internalization by multiple cancer cell lines of diverse origins (murine LLC, 4T1, C26 and human BT-20, MCF-7, and PC3), as shown by flow cytometry (FACS) and epi and confocal microscopy. As a result, mAb 2C5-modified Doxil demonstrated significantly higher cytotoxicity towards various cancer cells, including those resistant to doxorubicin, than all control preparations. The specific internalization of the mAb 2C5-Doxil into cytosol, along with the nuclear localization of their drug load, inside the target cancer cells were mainly responsible the superior anticancer activity. The IC50 values of mAb 2C5-Doxil with various murine and human cancer cells were 5-8-fold lower than those of control doxorubicin-loaded liposomes, Doxil or Doxil modified with a nonspecific IgG.

Antinucleosome antibody-modified liposomes and lipid-core micelles for tumor-targeted delivery of therapeutic and diagnostic agents.

Liposomes and lipid-core micelles prepared of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugates have been modified with nucleosome-specific monoclonal antinuclear autoantibody (ANA) 2C5 (mAb 2C5) specifically recognizing a broad variety of cancer cells through the cancer cell surface-bound nucleosomes. mAb 2C5 preserves its specific properties upon the binding with the lipid-based pharmaceutical nanocarriers, and 2C5-modified immunoliposomes and immunomicelles demonstrate an enhanced binding with tumor cells both in vitro and in vivo. We have investigated the delivery of therapeutic and diagnostic agents with such tumor-targeted immunoliposomes and immunomicelles to various tumors in vivo and in vitro. Both lipid-based nanocarriers provided enhanced tumor delivery of imaging agents ((111)In) and antitumor drugs (doxorubicin and photodynamic therapy agents) to tumor cells under different experimental settings. Pharmaceutical lipid-based nanoparticular carriers modified with mAb 2C5 could represent universal systems for tumor-specific delivery of various soluble and insoluble pharmaceuticals.

Enhanced accumulation of long-circulating liposomes modified with the nucleosome-specific monoclonal antibody 2C5 in various tumours in mice: gamma-imaging studies.

PURPOSE: To further improve tumour targeting and delivery of imaging agents by long-circulating liposomes via the coupling of the anti-cancer monoclonal antibody 2C5 with nucleosome-restricted activity, which can recognize the surface of various tumours but not normal cells and can specifically target pharmaceutical carriers to tumour cells in vitro and in vivo. METHODS: The 2C5 antibody was attached to the surface of long-circulating PEG-liposomes (LCL) by the post-insertion technique after antibody modification with a single-terminus activated PEG-lipid derivative to yield nucleosome-specific tumour-targeted liposomes. Tumour cell binding of the targeted liposomes was verified both by fluorescence microscopy and by flow cytometry in several cell lines using fluorescently labelled liposomes. 111In-radiolabelled liposomal formulations (prepared using membrane-anchored chelating groups) were used to examine in vivo biodistribution and tumour accumulation of liposomes by direct gamma scintigraphy. RESULTS: The 2C5 antibody-modified LCL demonstrated a three- to eightfold increase in in vitro specific cell binding to various cancer cell lines of diverse origin. 111In-labelled tumour-targeted liposomes demonstrated prolonged circulation and doubled tumour accumulation compared with that of control formulations. Whole-body gamma scintigraphic imaging of mice implanted with different tumours revealed markedly faster (6 h post injection for 2C5-LCL vs 24 h for non-specific analogues) and superior in vivo tumour visualization with 111In-2C5-LCL than with the 2C5-free formulations in tested tumour models. CONCLUSION: The 2C5 antibody-modified LCL effectively and specifically accumulate in various tumours and can serve as delivery vehicles for imaging agents, allowing for fast and efficient tumour visualization.

Tumor-targeted liposomes: doxorubicin-loaded long-circulating liposomes modified with anti-cancer antibody.

Commercially available doxorubicin-loaded long-circulating liposomes (Doxil, Alza Pharmaceuticals) were modified with the monoclonal nucleosome (NS)-specific 2C5 antibody (mAb 2C5) that recognizes a broad variety of tumors via the tumor cell surface-bound NSs. For incorporation into liposomes, mAb 2C5 was modified with poly(ethylene glycol)-phosphatidyl ethanolamine conjugate (PEG-PE) with the free PEG terminus activated with the p-nitrophenylcarbonyl group (pNP-PEG-PE). Derivatives of mAb 2C5 containing a variable number of PEG-PE residues (10-32) per protein molecule were prepared with a reasonably good preservation of the antibody specific activity even at the highest degree of modification. PEG-PE-modified antibody quantitatively incorporated into the liposomal membrane of doxorubicin-loaded liposomes with a loss of not more than 20% of the encapsulated doxorubicin. 2C5-targeted Doxil liposomes acquired the ability to recognize NSs and specifically bind to various tumor cells. Doxorubicin-loaded long-circulating liposomes modified with the mAb 2C5 kill various tumor cells in vitro with the efficiency higher than non-targeted doxorubicin-loaded liposomes.