Carbon nanomaterials in oncology: an expanding horizon.

Carbon nanomaterials have been attracting attention in oncology for the development of safe and effective cancer nanomedicines in increasing improved patient compliance for generally recognized as safe (GRAS) prominence. Toxicity, safety and efficacy of carbon nanomaterials are the major concerns in cancer theranostics. Various parameters such as particle size and shape or surface morphology, surface charge, composition, oxidation and nonoxidative-stress-related mechanisms are prone to toxicity of the carbon nanomaterials. Currently, few cancer-related products have been available on the market, although some are underway in preclinical and clinical phases. Thus, our main aim is to provide comprehensive details on the carbon nanomaterials in oncology from the past two decades for patient compliance and safety.

Topical delivery of enoxaparin using nanostructured lipid carrier.

Topical application of enoxaparin (ENX; low molecular weight heparin) prevents the occurrence of thrombosis at traumatic anastomosis site. Particulate carrier system like nanostructured lipid carriers (NLCs) could notably improve skin penetration of ENX. ENX-loaded NLCs were prepared by the solvent diffusion technique. The effect of formulation and process variables on the physicochemical properties of prepared NLCs was studied and characterized. In vitro skin permeation studies revealed better passage of enoxaparin by NLCs than of plain drug. The in vivo skin retention was monitored by fluorescence microscopy. The prepared NLCs when stored for 120 days were found to be more stable at 4 ± 2 °C than room temperature. The overall results of the study demonstrated the importance of carrier composition on the physicochemical properties, morphology, skin irritation and consequently the effectiveness of particulate system as a vehicle for topical delivery of enoxaparin.

Surface structured liposomes for site specific delivery of an antiviral agent-indinavir.

The present investigation was aimed at targeting indinavir, a protease inhibitor to cells of mononuclear phagocyte system (MPS) via mannosylated liposomes. ß-d-1-thiomannopyranoside residues were covalently coupled with dimyristoyl phosphatidylethanolamine (DMPE) to generate mannosylated-DMPE (Man-DMPE) conjugate which was further incorporated with disteroyl phosphatidyl choline and cholesterol to prepare mannosylated liposomes. The optimized mannosylated liposomes were nanometric in size (142 ± 2.8 nm) with optimum entrapment efficiency (88.7 ± 2.3%). Less than 20% cumulative drug release was observed from the prepared formulations in 24 h in phosphate buffer saline (pH 7.4). Cellular uptake studies performed on J774.A1 macrophage cell line via flow cytometric analysis depicted enhanced uptake of mannosylated liposomes as compared to plain liposomes. Annexin-V-fluorescein isothiocyanate/propidium iodide apoptosis assay delineated marginal cytotoxicity in macrophages from the developed formulation. Plasma and tissue distribution studies performed to assess the drug reach to macrophage rich regions depicted a significant level (P < 0.05) of indinavir in macrophage rich tissues like liver, spleen, and lungs from mannosylated liposomes as compared to plain liposomes and free drug. The conducted studies suggest the potential of indinavir loaded mannosylated liposomes for anti-human immunodeficiency virus therapy.

PEGylated nanocarriers for systemic delivery.

In this chapter, we outline the protocols for PEGylation of some drug carriers, such as dendrimer, polymeric nanoparticles, liposomes, for systemic delivery. PEGylation simply refers to the modification of particle surface by covalently grafting, entrapping, or adsorbing PEG chains of vivid length. However, limitation of simple adsorption being easy displacement of the coating layer in vivo, covalent mode for PEGylation of nanoparticles is mostly preferred, and outlined herein. Derivatization and activation of polyethylene glycol is an important step during PEGylation and its chemistry chiefly relies on availability as well as type of functional groups on carrier periphery. A summarized set of protocols for PEGylation of widely explored nanocarriers for systemic delivery is presented.

Enhanced transdermal delivery of an anti-HIV agent via ethanolic liposomes.

Indinavir, as a protease inhibitor with a short biological half life, variable pH-dependent oral absorption, and extensive first-pass metabolism, presents a challenge with respect to its oral administration. The current work aims to formulate and characterize indinavir-bearing ethanolic liposomes (ethosomes), and to investigate their enhanced transdermal delivery potential. The prepared ethanolic liposomes were characterized to be spherical, unilamellar structures having low polydispersity, nanometric size range, and improved entrapment efficiency over other delivery formulations. Permeation studies of indinavir across human cadaver skin resulted in enhanced transdermal flux from ethanolic liposomes that was significantly (P < 0.05) greater than that with ethanolic drug solution, conventional liposomes, or plain drug solution. Additionally, the ethanolic liposomes showed the shortest lag time for indinavir, thus presenting a suitable approach for transdermal delivery of this protease inhibitor. From the clinical editor: This study characterizes indinavir bearing ethanolic liposomes (ethosomes), and investigate their enhanced transdermal delivery potential, demonstrating a potentially a suitable approach for transdermal delivery of this protease inhibitor for HIV treatment, which typically has been associated with limited bioavailability via the oral route.

Evaluation of solid lipid nanoparticles as carriers for delivery of hepatitis B surface antigen for vaccination using subcutaneous route.

PURPOSE: Solid lipid nanoparticles (SLN) have emerged as carriers for therapeutic peptides, proteins, antigens and bioactive molecules. We have explored the potential of SLN as carrier for Hepatitis B surface antigen (HBsAg) by surface modifications to enhance their loading efficiency and the cellular uptake, using subcutaneous route. METHODS: Four different formulations of SLN were prepared by solvent injection method and characterized for various physical properties: particle size, surface morphology, shape, zeta potential, polydispersity, X-ray diffraction analysis, release profile and entrapment efficiency. HBsAg loaded SLN were studied for their functional characteristics, in vitro cellular uptake and internalization studies by human dendritic cells, macrophages and fibroblasts, T cell proliferation and TH1/TH2 response. Humoral immune response elicited by subcutaneously administered HBsAg containing SLN formulations were studied in vivo in mice. RESULTS: Compared to soluble HBsAg; SLN, particularly the mannosylated formulation, showed better cellular uptake, lesser cytotoxicity and induction of greater TH1 type of immune response. They also showed better immunological potential by producing sustained antibody titer. CONCLUSION: Mannosylated SLN appears to be promising as carrier for vaccine delivery against hepatitis B as ascertained by in vitro and in vivo studies, however further investigations on humans are required to establish their potential as vaccines against hepatitis B infection.

Comparative evaluation of hepatitis B surface antigen-loaded elastic liposomes and ethosomes for human dendritic cell uptake and immune response.

The aim of the present study was to evaluate two vesicular carrier systems, ethosomes and elastic liposomes loaded with hepatitis B surface antigen, for in vitro qualitative and quantitative uptake by human dendritic cells (DCs) and ability to stimulate T lymphocytes. Quantitative uptake of antigen-loaded carriers was documented by flow cytometry, and internalization of the systems by the DCs was studied using spectral bioimaging. Ability of antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes and levels of TH1/TH2 cytokines were also examined using flow cytometry. Both vesicular carrier systems as antigen delivery modules and DCs as antigen-presenting cells were able to generate a protective immune response. However, ethosomes were found to have higher internalizing ability and immunogenicity in comparison with elastic liposomes. These properties of ethosomes coupled with their skin-navigating potential, make it an attractive vehicle for development of a transcutaneous vaccine against hepatitis B in preference to elastic liposomes. FROM THE CLINICAL EDITOR: Two carrier systems for more potent vaccine administration - ethosomes and elastic liposomes loaded with hepatitis B surface antigen - are compared. Ethosomes demonstrated higher internalizing ability and immunogenicity. Due to their known skin-navigating potential, ethosomes may represent an attractive vehicle for development of a transcutaneous vaccine against hepatitis B.

In vitro evaluation of surface functionalized gelatin nanoparticles for macrophage targeting in the therapy of visceral leishmaniasis.

The present study evaluates the potential of surface functionalized gelatin nanoparticles (f-GNPs) for efficient macrophage-specific delivery of amphotericin B (AmB) in the treatment of visceral leishmaniasis (VL). Further, the effect of spacer for macrophage targeting was also evaluated. Gelatin was functionalized either through conjugation to mannose via direct coupling (mGelatin) or via PEG spacer (m-Gelatin), and the synthesis was confirmed by FTIR. AmB-loaded f-GNPs, that is, mGNPs and m-GNPs prepared from mGelatin and m-Gelatin conjugates, respectively, were characterized. In vitro concanavalin A (Con-A) agglutination assay confirmed the availability of mannose on the surface of these f-GNPs. Kinetics of cellular uptake of AmB-loaded f-GNPs by J774A.1 macrophage cells assessed through flow cytometry demonstrated a steady increase and maximum cell-associated fluorescence was observed at 4h for m-GNPs and 6 h for m-GNPs. Measurement of cytotoxicity using Annexin-V-FITC/PI apoptosis assay delineated marginal changes (7-9%) in treated macrophages following 48 h incubation, establishing the safety of f-GNPs. m-GNPs showed a 5.4-fold reduction in IC(50) in comparison with plain AmB suggesting significant enhancement of antileishmanial activity. Our results indicate that f-GNPs could be a promising carrier for specific delivery of AmB to macrophages for effective treatment of VL. Furthermore, spacer contributed significantly in reducing the cytotoxicity as well as increasing the uptake and activity of f-GNPs.

Preparation, characterization and evaluation of targeting potential of amphotericin B-loaded engineered PLGA nanoparticles.

PURPOSE: The objective of present work was to develop a mannose-anchored, engineered nanoparticulate system for efficient delivery of amphotericin B to macrophages. Furthermore, the effect of spacer on macrophage targeting was also evaluated. METHODS: PLGA was conjugated to mannose via direct coupling (M-PLGA) and via PEG spacer (M-PEG-PLGA), and engineered PLGA nanoparticles (M-PNPs and M-PEG-PNPs) were prepared from respective conjugates. These prepared engineered PNPs were characterized for size, polydispersity index (PDI), surface charge, and drug entrapment efficiency (% DEE). Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were employed to study the shape and surface morphology of engineered PNPs. Macrophage targeting was evaluated via cellular uptake, ex vivo antileishmanial activity and in vivo biodisposition pattern of engineered PNPs in macrophage-rich organs. RESULTS: The developed engineered PNPs were found to be of nanometric size (<200 nm) and to have low PDI (<0.162) and good entrapment efficiency (%DEE >53.0%). AFM and TEM revealed that both M-PNPs and M-PEG-PNPs had smooth surface and spherical topography. Engineered PNPs with spacer showed enhanced uptake, potential antileishmanial activity and higher disposition in macrophage-rich organs, suggesting improved macrophage targeting. CONCLUSIONS: The results suggest that engineering of nanoparticles could lead to development of efficient carrier for macrophage targeting.

Carbohydrate-conjugated multiwalled carbon nanotubes: development and characterization.

This work presents a novel cascade of chemical functionalization of multiwalled carbon nanotubes (MWCNTs) through chemical modification by a carbohydrate, D-galactose. Galactose-conjugated or galactosylated MWCNTs were synthesized involving the sequential steps of carboxylation, acylation, amine modification, and finally, galactose conjugation. The modification of MWCNTs with galactose was investigated by elemental analysis, x-ray diffraction analysis, Fourier transform-infrared spectroscopy, Raman spectroscopy, and zeta potential measurements, at every sequential step of functionalization. Size and surface characteristics of chemically modified MWCNTs were monitored by transmission electron microscopy and scanning electron microscopy. That galactosylation improved the dispersibility of MWCNTs in aqueous solvents was confirmed by investigation of their dispersion characteristics at different pH values. Thus, the galactosylated MWCNTs as developed could be used for delivery of different bioactive(s) as well as active ligand (galactose)-based targeting to hepatic tissue. FROM THE CLINICAL EDITOR: This work presents a novel cascade of functionalization of multiwalled carbon nanotubes (MWCNTs) through chemical modification by a carbohydrate. Galactosylation improves the dispersibility of MWCNTs in aqueous solvents. The galactosylated MWCNTs could be used for delivery of different bioactive(s) as well as active ligand-based targeting to hepatic tissue.

Lymphatic targeting of zidovudine using surface-engineered liposomes.

The present investigation was aimed at lymphatic targeting of zidovudine (ZDV)-loaded surface-engineered liposomes (SE liposomes). Surface of liposomes was engineered by incorporation of charges (positive or negative) and site-specific ligand (mannose) in order to enhance localization to lymphatics, specifically to lymph node and spleen. Positively and negatively charged nanosized SE liposomes (120 +/- 10 nm) were prepared using stearylamine (SA) and dicetyl phosphate (DCP), respectively, while ligand-coated SE liposomes were prepared using mannose-terminated SA (mannose conjugate). The SE liposomes were characterized for shape and surface morphology, size, entrapment efficiency, and in vitro drug release. All the SE liposomes formulations showed biphasic ZDV release, whereas mannose-coated liposomes (MAN-Lip) significantly reduced (p < 0.05) drug release compared with conventional liposome (Lip). The organ distribution pattern of the SE liposomes exhibited significant reduction in free ZDV concentration in serum, whereas significantly increased quantity was detected in the spleen and lymph nodes (p < 0.05). Fluorescent microscopy suggested enhanced uptake and localization of the SE liposomes in the lymph nodes and spleen, which were in the order: mannose coated > negatively charged > positively charged > Lip. Thus, the SE liposomes appeared to be promising novel vesicular system for enhanced targeting of ZDV to lymphatics, in AIDS chemotherapy.

Elastic liposomes mediated transdermal delivery of an anti-jet lag agent: preparation, characterization and in vitro human skin transport study.

In order to get across the intact skin, drug-laden carriers have to pass through narrow, confining pores of 50 nm or less diameter, under the influence of a suitable transdermal gradient. Novel ultradeformable carriers, the elastic liposomes achieve this target via its deforming and self-optimizing property. The main goal of this work was to prepare and characterize, elastic liposomes bearing melatonin, an anti-jet lag agent for its efficient transdermal delivery. Elastic liposomes bearing melatonin were prepared by modified extrusion method and characterized for shape, lamellarity, size distribution, percent drug loading, turbidity profile by Transmission electron microscopy (TEM), Dynamic light scattering (DLS), Mini-column centrifugation and Nephelometric techniques. The effect of different formulation variables like type of surfactant and concentration of surfactant on the deformability of vesicles, turbidity changes, transdermal flux across human cadaver skin, amount of drug deposited into the skin were investigated. Confocal laser scanning (CLS) micrographs revealed that probe (Rhodamine Red) loaded elastic liposomes were able to penetrate much deeper than the probe loaded conventional rigid liposomes. Out of the three surfactants utilized namely, Span 80, Sodium cholate and Sodium dodecylsulphate, formulation bearing Span 80 at an optimum lipid: surfactant ratio of 85:15% w/w proved to be the best in all parameters studied. The optimum skin permeation profile including greater transdermal flux and lower lag time of melatonin from optimized elastic liposomes via human cadaver skin was observed. Our results of the present study demonstrated the feasibility of elastic liposomal system for transdermal delivery of this anti- jet lag agent, which provides better transdermal flux, higher entrapment efficiency, greater skin drug deposition and possesses the ability of a self-penetration enhancer as compared to conventional liposomes.

Challenges in the use of carbon nanotubes for biomedical applications.

Carbon nanotubes (CNTs) are considered for use in numerous technological applications, including as biocompatible modules for the delivery of bioactives. However, there are unique properties of CNTs that limit their use as vehicles for various purposes. This review highlights the various challenges to a pharmaceutical scientist while exploring CNTs as bioactive delivery vehicles. The lack of solubility, nonbiodegradability, circulation half-life of 3-3.5 hours, biocompatibility, and immunogenicity limitations of CNTs are discussed in this review. These limitations indicate the need for modifications in order to explore the feasibility of CNTs as delivery vehicles.

Development, characterization, and toxicity evaluation of amphotericin B-loaded gelatin nanoparticles.

Our aim in the present investigation was to develop a nanoparticulate carrier of amphotericin B (AmB) for controlled delivery as well as reduced toxicity. Nanoparticles of different gelatins (GNPs) (type A or B) were prepared by two-step desolvation method and optimized for temperature, pH, amount of cross-linker, and theoretical drug loading. AmB-loaded GNPs were characterized for size, polydispersity index (PI), shape, morphology, surface charge, drug release, and hemolysis. The developed GNPs (GNP(A300)) were found to be of nanometric size (213 +/- 10 nm), having low PI (0.092 +/- 0.015) and good entrapment efficiency (49.0 +/- 2.9%). All GNPs showed biphasic release characterized by an initial burst followed by controlled release. The in vivo hematological toxicity results suggest nonsignificant reduction (P > .05) in hemoglobin concentration and hematocrit. Nephrotoxicity results showed that there was a nonsignificant (P > .05) increase in blood urea nitrogen and serum creatinine levels. The results confirm that developed GNPs could optimize AmB delivery in terms of cost and safety, and type A gelatin with bloom number 300 was found suitable for such preparation.

Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes.

We have evaluated the efficiency of novel modified liposomes (ethosomes) for transcutaneous immunization (TCI) against Hepatitis B. Antigen-loaded ethosomes were prepared and characterized for shape, lamellarity, fluidity, size distribution, and entrapment efficiency. Spectral bio-imaging and flow cytometric studies showed efficient uptake of Hepatitis B surface antigen (HBsAg)-loaded ethosomes by murine dendritic cells (DCs) in vitro, reaching a peak by 180 min. Transcutaneous delivery potential of the antigen-loaded system using human cadaver skin demonstrated a much higher skin permeation of the antigen in comparison to conventional liposomes and soluble antigen preparation. Topically applied HBsAg-loaded ethosomes in experimental mice showed a robust systemic and mucosal humoral immune response compared to intramuscularly administered alum-adsorbed HBsAg suspension, topically applied plain HBsAg solution and hydroethanolic (25%) HBsAg solution. The ability of the antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes was demonstrated by BrdU assay and a predominantly TH1 type of immune response was observed by multiplex cytometric bead array analysis. HBsAg-loaded ethosomes are able to generate a protective immune response and their ability to traverse and target the immunological milieu of the skin may find a potential application in the development of a transcutaneous vaccine against Hepatitis B virus (HBV).

Vesicles as tools for the modulation of skin permeability.

Human skin is a remarkably efficient barrier designed to keep our insides in and the outside out. The modulation of this efficient barrier's properties, including its permeability to chemicals, drugs and biologically active agents is the prime target for various dermal, transdermal, drug, antigen and gene delivery approaches. Therefore, several methods have been attempted to enhance the permeation rate of biologically active agents, temporarily and locally. One of the approaches is the application of drug-laden vesicular formulations. This review presents various mechanisms involved in increasing drug transport across the skin via different vesicular approaches, such as liposomes, elastic vesicles and ethosomes, along with compiling the research work conducted in this field.

Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes.

The aim of the current investigation is to evaluate the transdermal potential of novel vesicular carrier, ethosomes, bearing methotrexate (MTX), an anti-psoriatic, anti-neoplastic, highly hydrosoluble agent having limited transdermal permeation. MTX loaded ethosomes were prepared, optimized and characterized for vesicular shape and surface morphology, vesicular size, entrapment efficiency, stability, in vitro human skin permeation and vesicle-skin interaction. The formulation (EE(9)) having 3% phospholipid content and 45% ethanol showing the greatest entrapment (68.71+/-1.4%) and optimal nanometric size range (143+/-16 nm) was selected for further transdermal permeation studies. Stability profile of prepared system assessed for 120 days revealed very low aggregation and growth in vesicular size (8.8+/-1.2%). MTX loaded ethosomal carriers also provided an enhanced transdermal flux of 57.2+/-4.34 microg/cm(2)/h and decreased lag time of 0.9 h across human cadaver skin. Skin permeation profile of the developed formulation further assessed by confocal laser scanning microscopy (CLSM) revealed an enhanced permeation of Rhodamine Red (RR) loaded formulations to the deeper layers of the skin (170 microm). Also, the formulation retained its penetration power after storage. Vesicle skin interaction study also highlighted the penetration enhancing effect of ethosomes with some visual penetration pathways and corneocytes swelling, a measure of retentive nature of formulation. Our results suggests that ethosomes are an efficient carrier for dermal and transdermal delivery of MTX.

Functional polymeric nanoparticles: an efficient and promising tool for active delivery of bioactives.

Nanotechnology is a multidisciplinary field and has achieved breakthroughs in bioengineering, molecular biology, diagnostics, and therapeutics. A recent advance in nanotechnology is the development of a functional nanosystem by incorporation, adsorption, or covalent coupling of polymers, carbohydrates, endogenous substances/ligands, peptides, proteins, nucleic acids, and polysaccharides to the surface of nanoparticles. Functionalization confers a wide array of interesting properties such as stealth characteristics, a bioadhesive property, and that it prevents aggregation of nanoparticles, imparts biostability and solubility, reduces toxicity, and provides site-specific delivery. This makes the nanosystem an intelligent tool for diagnostics, prognostics, and controlled and sustained delivery of protein, peptide, pDNA, and other therapeutic agents to specific targets (tissue, cell, and intracellular). Various types of functional nanosystems, such as carbon nanotubes, quantum dots, polymeric micelles, dendrimers, metallic nanoparticles, and liposomes, are being extensively explored. However, high tissue accumulation of nonbiodegradable nanoparticles has caused toxicity problems and rendered them as not-so-popular therapeutic and diagnostic systems. The toxicity and safety of nonbiodegradable nanoparticles are subject to future research. Polymeric nanoparticles have offered attractive alternative modules due to biocompatibility, nonimmunogenicity, nontoxicity, biodegradability, simple preparation methods, high physical stability, possibility of sustained drug release, and higher probability for surface functionalization. Depending on properties that have been modified, polymeric nanoparticles can be grouped in to four classes, namely, stealth, polysaccharide decorated biomimetic, bioadhesive, and ligand-anchored functional polymeric nanoparticles (f-PNPs). This review explores the ligand-anchored f-PNP as a carrier for active delivery of bioactives, envisaged to date. This review also details the ligands available for conjugation, their method of coupling to nanoparticles, and applications of f-PNPs in anticancer drug delivery, oral delivery, gene delivery, vaccine delivery, and intracellular delivery; site-specific delivery to liver, macrophages, lymphatics, and brain; and miscellaneous applications. This review also addresses formidable challenges encountered, and proposes some future strategies for development of a promising site-specific active delivery system.