Lipid Cubic Systems for Sustained and Controlled Delivery of Antihistamine Drugs.

Antihistamines are capable of blocking mediator responses in allergic reactions including allergic rhinitis and dermatological reactions. By incorporating various H1 receptor antagonists into a lipid cubic phase network, these active ingredients can be delivered locally over an extended period of time owing to the mucoadhesive nature of the system. Local delivery can avoid inducing unwanted side effects, often observed after systematic delivery. Lipid-based antihistamine delivery systems are shown here to exhibit prolonged release capabilities. In vitro drug dissolution studies investigated the extent and release rate of two model first-generation and two model second-generation H1 antagonist antihistamine drugs from two monoacyglycerol-derived lipid models. To optimize the formulation approach, the systems were characterized macroscopically and microscopically by small-angle X-ray scattering and polarized light to ascertain the mesophase accessed upon an incorporation of antihistamines of varying solubilities and size. The impact of encapsulating the antihistamine molecules on the degree of mucoadhesivity of the lipid cubic systems was investigated using multiparametric surface plasmon resonance. With the ultimate goal of developing therapies for the treatment of allergic reactions, the ability of the formulations to inhibit mediator release utilizing RBL-2H3 mast cells with the propensity to release histamine upon induction was explored, demonstrating no interference from the lipid excipient on the effectiveness of the antihistamine molecules.

Lipid-bilayer-coated nanogels allow for sustained release and enhanced internalization.

Nanoparticle drug delivery system improves the therapeutic efficacy of a drug; however, achieving sustained release from nanoparticles is challenging, owing to the increase of surface area and pronounced burst release. In this study, by incorporating an organogel of 12-hydroxystearic acid (12-HSA) into lipid-bilayers, a gel-liposomal formulation was developed to sustain drug release over time. The lipid-bilayer-coated nanogels (LBCNs) with a particle size of approximately 200 nm and with a core-shell structure had an entrapment efficiency of up to 80% for paclitaxel. LBCNs could continually release both hydrophobic and water-soluble drugs over time. Interestingly, the incorporation of organogel enhanced the cellular uptake of liposomes significantly and, accordingly, enabled improved cytotoxicity of chemotherapy agent against the cancer cells. In conclusion, by formulating the organogel into the lipid bilayers, gel-liposomes were developed, allowing for sustained drug release, improved internalization and the resultant enhancement of cytotoxicity of chemotherapy agent to cancer cells.

Lipid bilayer-coated mesoporous silica nanoparticles carrying bovine hemoglobin towards an erythrocyte mimic.

Hemoglobin (Hb)-loaded mesoporous silica nanoparticles (MSNs) coated with a lipid bilayer (LB-MSNs) were investigated as an erythrocyte mimic. MSNs with a large average pore size (10 nm) act as a rigid core and provide a protective environment for Hb encapsulated inside the pores. The colloidal stability of Hb-loaded MSNs was enhanced upon the application of a lipid bilayer, through fusion of PEGylated liposomes onto the exterior surface of Hb-loaded MSNs. The morphology and mesostructure of the MSNs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and surface area analysis. The Hb loading capacity (mg/g) in MSNs was studied by thermogravimetric analysis (TGA). UV-Vis absorption spectroscopy revealed that Hb inside MSNs had an identical, but slightly broadened peak in the Soret region compared to free Hb. Furthermore the encapsulated Hb exhibits similar peroxidase-like activity in catalyzing the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with hydrogen peroxide. The introduction of a supported lipid bilayer (LB) demonstrated the potential to prevent premature Hb release (the burst release decreased from 25.50 ±â€¯0.33% to 6.73 ±â€¯0.83%) and increased the colloidal stability of the Hb-loaded MSNs (hydrodynamic diameter remained ∼250 nm for at least one week). The in vivo systemic circulation and biodistribution of LB-MSNs were studied in optically transparent zebrafish embryos, revealing that LB-MSNs have the potential to act as an erythrocyte mimic in transfusion therapy.

The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes.

[Preliminary study on pH-sensitive lipid bilayer-coated mesoporous silica nanoparticles as a novel drug carrier for antitumor drug].

This study plans to prepare lipid bilayer-coated mesoporous silica nanoparticles (LMSNs) which are pH sensitive with core-shell structure to improve the tumor cell lethality of antitumor drug. The lipid coated mesoporous silica nanoparticles loaded with irinotecan (CPT-11) (CPT-11-LMSNs) were prepared by hot water-film hydration method, and the characterized its morphology, particle size and release in vitro. Meanwhile, the intracellular uptake and cell toxicity of CPT-11-LMSNs and intracellular accumulation of CPT-11 were evaluated on human breast carcinoma cell line (MCF-7). The results indicated that the mean diameter of the spherical LMSNs was (120.27 +/- 5.91) nm. The slow release in simulated normal physiological conditions and a rapid release under simulated intracellular condition demonstrated the pH sensitivity of CPT-11-MSNs in vitro. Moreover, the CPT-11-LMSN could improve the intracellular CPT-11 cumulant 2.1 times and reduce half maximal inhibitory concentration (IC50) values of CPT-11 1.4 times compared with CPT-11-MSNs, demonstrating a stronger cell lethality.

Chemically reducible lipid bilayer coated mesoporous silica nanoparticles demonstrating controlled release and HeLa and normal mouse liver cell biocompatibility and cellular internalization.

A controlled release system composed of mesoporous silica nanoparticles with covalently bound dipalmitoyl moieties supporting phosphorylated lipids has been successfully synthesized and characterized. This MSN system demonstrates controlled release of fluorescein molecules under disulfide reducing conditions. Flow cytometry analyses confirm increased biocompatibility of the resulting lipid bilayer MSNs (LB-MSNs) from nonfunctional MSNs. Fluorescently labeled LB-MSNs are examined via confocal fluorescent microscopy ex vivo and were found to enter both normal and cancer cell lines. The LB-MSNs presented here have potential to be used as rapid and diverse functionalized, stable liposome analogues for drug delivery.

Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction.

For subunit vaccines, adjuvants play a key role in shaping immunological memory. Nanoparticle (NP) delivery systems for antigens and/or molecular danger signals are promising adjuvants capable of promoting both cellular and humoral immune responses, but in most cases the mechanisms of action of these materials are poorly understood. Here, we studied the immune response elicited by NPs composed of multilamellar "stapled" lipid vesicles carrying a recombinant Plasmodium vivax circumsporozoite antigen, VMP001, both entrapped in the aqueous core and anchored to the lipid bilayer surfaces. Immunization with these particles and monophosphoryl lipid A (MPLA), a US Food and Drug Administration-approved immunostimulatory agonist for Toll-like receptor-4, promoted high-titer, high-avidity antibody responses against VMP001, lasting more than 1 y in mice at 10-fold lower doses than conventional adjuvants. Compared to soluble VMP001 mixed with MPLA, VMP001-NPs promoted broader humoral responses, targeting multiple epitopes of the protein and a more balanced Th1/Th2 cytokine profile from antigen-specific T cells. To begin to understand the underlying mechanisms, we examined components of the B-cell response and found that NPs promoted robust germinal center (GC) formation at low doses of antigen where no GC induction occurred with soluble protein immunization, and that GCs nucleated near depots of NPs accumulating in the draining lymph nodes over time. In parallel, NP vaccination enhanced the expansion of antigen-specific follicular helper T cells (T(fh)), compared to vaccinations with soluble VMP001 or alum. Thus, NP vaccines may be a promising strategy to enhance the durability, breadth, and potency of humoral immunity by enhancing key elements of the B-cell response.

Intelligent design of multifunctional lipid-coated nanoparticle platforms for cancer therapy.

Nanotechnology is rapidly evolving and dramatically changing the paradigms of drug delivery. The small sizes, unique chemical properties, large surface areas, structural diversity and multifunctionality of nanoparticles prove to be greatly advantageous for combating notoriously therapeutically evasive diseases such as cancer. Multifunctional nanoparticles have been designed to enhance tumor uptake through either passive or active targeting, while also avoiding reticuloendothelial system uptake through the incorporation of PEG onto the surface. First-generation nanoparticle systems, such as liposomes, are good carriers for drugs and nucleic acid therapeutics, although they have some limitations. These lipid bilayers are now being utilized as excellent carriers for drug-loaded, solid core particles such as iron oxide, mesoporus silica and calcium phosphate. In this article, their design, as well as their multifunctional role in cancer therapy are discussed.

Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor.

Calcium phosphate (CaP) nanoparticles (NP) with an asymmetric lipid bilayer coating have been designed for targeted delivery of siRNA to the tumor. An anionic lipid, dioleoylphosphatydic acid (DOPA), was employed as the inner leaflet lipid to coat the nano-size CaP cores, which entrap the siRNA, such that the coated cores were soluble in organic solvent. A suitable neutral or cationic lipid was used as the outer leaflet lipid to form an asymmetric lipid bilayer structure verified by the measurement of NP zeta potential. The resulting NP was named LCP-II with a size of about 25 to 30nm in diameter and contained a hollow core as revealed by TEM imaging. PEGylation of NP was done by including a PEG-phospholipid conjugate, with or without a targeting ligand anisamide, in the outer leaflet lipid mixture. The sub-cellular distribution studied in the sigma receptor positive human H460 lung cancer cells indicated that LCP-II could release more cargo to the cytoplasm than our previous lipid/protamine/DNA (LPD) formulation, leading to a significant (~40 fold in vitro and ~4 fold in vivo) improvement in siRNA delivery. Bio-distribution study showed that LCP-II required more PEGylation for MPS evasion than the previous LPD, probably due to increased surface curvature in LCP-II.

Artificial envelopment of nonenveloped viruses: enhancing adenovirus tumor targeting in vivo.

Recombinant adenovirus (Ad) is a powerful tool in gene therapy. However, the ability to deliver Ad by systemic administration is limited due to rapid clearance from blood circulation, transfection of nontarget tissues, toxicity, and immunogenicity. To address these limitations, we developed an artificially enveloped Ad vector prepared by self-assembly of lipid bilayers around the Ad capsid. The physicochemical and structural features of the enveloped Ad vector can be altered according to the type of lipid used without the need for genetic modification or conjugation chemistry. Here we engineered 4 different types of artificially enveloped Ad using cationic, neutral, fusogenic, and PEGylated lipids to form the envelopes and obtained extended blood circulation times following i.v. administration and reduced vector immunogenicity. Moreover, the PEGylated lipid-enveloped Ad was capable of specifically delivering genes via the systemic circulation to solid tumors subcutaneously implanted in the absence of high levels of gene transfer to the liver and spleen. This provides the basis for the development of a novel vector platform for systemic delivery of Ad to disseminated targets. Furthermore, the artificial envelopment of Ad presented herein is an illustration of a general strategy to modulate the biological function of nonenveloped viruses and may have implications broader than gene therapy.

Nanodisk-associated amphotericin B clears Leishmania major cutaneous infection in susceptible BALB/c mice.

Nanometer-scale, apolipoprotein-stabilized phospholipid bilayer disk complexes (nanodisks [ND]) harboring the toxic and poorly soluble antileishmanial agent amphotericin B (AMB) were examined for efficacy in treatment of Leishmania major-infected BALB/c mice (Mus musculus). L. major-infected mice were intraperitoneally (i.p.) treated with AMB-ND in 0-, 1-, and 5-mg/kg doses at 24 h, 48 h, and 4, 7, 14, and 21 days postinfection in two experiments. L. major-infected mice were i.p. treated with phosphate-buffered saline, 5 mg/kg AMB-ND, or 5 mg/kg lipid-associated amphotericin B (liposomal amphotericin B, AmBisome) at 24 h, 48 h, and 10, 20, 30, and 40 days postinfection in one experiment. Parasite numbers, footpad lesion size progression, and development of cytokine responses were assayed at days 7, 15, 30, 50, 140, and 250 or at days 14, 30, 50, 95, and 140 postinfection. Mice administered AMB-ND in 1- or 5-mg/kg doses were significantly protected from L. major, displaying decreases in lesion size and parasite burden, particularly at the 5-mg/kg dosage level. In contrast to the i.p. treated AmBisome group, BALB/c mice treated with i.p. AMB-ND completely cleared an L. major infection by 140 to 250 days postinfection, with no lesions remaining and no parasites isolated from infected animals. Restimulated mixed lymphocyte culture cytokine responses (interleukin-4 [IL-4], IL-12, IL-10, NO, and gamma interferon) were unchanged by AMB-ND administration compared to controls. The marked clearance of Leishmania parasites from a susceptible strain of mice without an appreciable change in the cytokine response suggests that AMB-ND represent a potentially useful formulation for treatment of intrahistiocytic organisms.

A new approach for treatment of deep skin infections by an ethosomal antibiotic preparation: an in vivo study.

OBJECTIVES: Dermal and subdermal bacterial infections, caused mainly by Staphylococcus aureus, are currently treated by systemic antibiotics. The aim of the present study was to investigate a new approach to treat deep skin and soft tissue bacterial infections by dermal application of erythromycin in an ethosomal carrier. METHODS: A model for deep dermal S. aureus infection in mice was developed. The efficiency of ethosomal erythromycin applied to the skin-infected site was compared with intraperitoneal erythromycin administration and with local application of hydroethanolic erythromycin solution. The parameters evaluated were the development of dermal wound, histological sections and bacterial count of the infected tissue. RESULTS: The in vivo experiments demonstrated a very efficient healing of S. aureus-induced deep dermal infections when the mice were treated with ethosomal erythromycin. Bacterial counts and histological evaluation of the skin treated with ethosomal antibiotic revealed no bacterial growth and normal skin structure. On the contrary, no subdermal healing was observed in infected animals treated with topical hydroethanolic erythromycin solution. In this group, animals developed deep dermal abscesses and the dermal structures were destroyed where S. aureus colonies were present. Bacterial counts of the infected tissues were 1.06 x 10(7) and 0.27 x 10(7) cfu/g of tissue, respectively, on days 7 and 10. CONCLUSIONS: Therapy with ethosomal erythromycin applied to the skin of S. aureus-infected mice was as effective as systemically administered erythromycin, suggesting a new possibility to treat deep dermal infections by local application of antibiotic in ethosomal carrier.

A novel gene delivery system for mammalian cells.

Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Non-viral efficacy of delivery remains quite poor. We are investigating the feasibility of a novel lipid-based delivery system, cochleates, to deliver transgenes to mammalian cells. Rhodamine-labelled empty cochleates were incubated with two cell-lines (4T1 adenocarcinoma and H36.12 macrophage hybridoma) and primary macrophages in vitro and in vivo. Cochleates containing green fluorescent protein (GFP) expression plasmid were incubated with 4T1 adenocarcinoma cells. Cellular uptake of labelled cochleates or transgene GFP expression were visualised with fluorescence microscopy. 4T1 and H36.12 lines showed 39% and 23.1% uptake of rhodamine-cochleates, respectively. Human monocyte-derived macrophages and mouse peritoneal macrophages had 48+/-5.38% and 51.46+/-15.6% uptake of rhodamine-cochleates in vitro. In vivo 25.69+/-0.127% of peritoneal macrophages were rhodamine-positive after intra-peritoneal injection of rhodamine-cochleates. 19.49+/-10.12% of 4T1 cells expressed GFP. Cochleates may therefore be an effective, non-toxic and non-immunogenic method to introduce transgenes in vitro and in vivo.

Elongated supramolecular assemblies in drug delivery.

This review presents different lipid-based elongated microstructures: tubules, cochleate cylinders and ribbons. Their composition, process of preparation and the mechanism behind their formation is discussed as well as their use as a drug delivery system.

Lipid-coated microgels for the triggered release of doxorubicin.

We have systematically engineered a polymeric, multi-component drug delivery system composed of a lipid-coated hydrogel microparticle (microgel). The design of this delivery system was motivated by the recent elucidation of the mechanism of regulated secretion from the secretory granule and the compositional and structural features that underlie its ability to store and release endogenous drug-like compounds. The present work describes the assembly and response of a prototype construct which displays several important features of the secretory granule, including its high drug loading capacity, and triggered microgel swelling, resulting in the burst release of drug. To achieve this, ionic microgels were synthesized, and loaded with doxorubicin via ion exchange. These microgels were then coated with a lipid bilayer, and the release of doxorubicin was triggered from the gels using either lipid-solubilizing surfactants or electroporation. The use of a microanalytical technique is featured utilizing micropipette manipulation that allows the study of the behavior of individual microparticles. The lipid-coated microgels were electroporated in saline solution; they swelled and disrupted their bilayer coating over a period of several seconds and exchanged doxorubicin with the external plasma saline over a period of several minutes. It is envisioned that this system will ultimately find utility in drug delivery systems that are designed to release chemotherapeutic agents and peptides by the application of a triggering signal.

Influence of cholesterol on liposome fluidity by EPR. Relationship with percutaneous absorption.

The influence of liposome composition on bilayer fluidity and its effect on the percutaneous absorption into the skin were investigated. Liposomes formed with saturated or unsaturated phospholipids (H-PC or PC) with varying amounts of cholesterol were prepared and their penetration behaviour into the stratum corneum was followed up by means of the stripping method. The order and dynamics of the hydrophobic domain of the vesicles were studied using electron paramagnetic resonance (EPR) methodology. Phospholipid composition and the amount of cholesterol exert a considerable influence on the penetration behaviour of the probe encapsulated in the liposomes. This behaviour is closely related to the fluidity characteristics of these liposomes studied by EPR. Therefore, a penetration mechanism of the vesicles into the skin, based on the incorporation of lipids into the skin lipids and on fluidity behaviour, is suggested.

Liposome-incorporated dexamethasone palmitate inhibits in-vitro lymphocyte response to mitogen.

The use of liposomes for the pulmonary delivery of corticosteroid is an area that is under active investigation. We have recently developed a novel liposomal corticosteroid preparation based on the incorporation of dexamethasone palmitate (DMP) within the bilayer of small unilamellar vesicles (SUVs) made of egg yolk phosphatidylcholine (EPC) and cholesterol; molar ratio EPCC:cholesterol: DMP, 4:3:0.3. In the present study, the biological activity of DMP-SUVs was evaluated using the lymphocyte transformation test with peripheral blood mononuclear cells (PBMCs) and a gamma-interferon production assay. Results showed that DMP-SUVs (but not empty SUVs) inhibited [3H]thymidine uptake and gamma-interferon production by concanavalin A-stimulated PBMCs by 94 and 96%, respectively, at a concentration corresponding to 10(-6) M dexamethasone. The inhibition by DMP-SUVs was found to require a 24-h pre-incubation with unstimulated PBMCs, suggesting that interaction of SUVs with lymphocytes may be altered by mitogen stimulation. We conclude that our DMP liposomal preparation is biologically active and may be considered a promising alternative to conventional local glucocorticoid therapy.

Functional drug targeting to erythrocytes in vivo using antibody bearing liposomes as drug vehicles.

Covalent attachment of anti-erythrocyte F(ab')2 to the liposome surface has recently been shown to considerably enhance the liposome binding to erythrocytes in vivo. These antibody bearing liposomes have now been found quite effective as vehicles for delivering the antimalarial drug, chloroquine, to erythrocytes in Plasmodium berghei-infected mice. This demonstrates the usefulness of antibody targeted liposomes as carriers for site-specific drug delivery.

Biologically active, recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome-encapsulated DNA.

DNA entrapped in liposomes containing lactosylceramide in the bilayers is found to be associated with clathrin-coated vesicles isolated from the rat livers after intravenous injection of these liposomes. The presence of the exogenous DNA in the coated vesicles was detected by Southern blotting. The amount of DNA present in the coated vesicles does not appear to vary up to 4 h after injection of the liposomes into the animals. The recognition of the lactosyl group present in the liposome by the galactose receptor present on the surface of the different liver cells may lead to their internalization in a way analogous to receptor-mediated endocytosis of various macromolecules. DNA present in the lumen of the coated vesicles is found to be biologically active as evidenced by its replication in bacterial cells and mouse fibroblasts.

Liposomes as drug carriers in cancer therapy.

Liposomes, microscopic particles composed of lipid bilayer membranes, can carry water-soluble drugs in their aqueous compartments and lipid soluble drugs in their bilayers. Over the last 10 years, they have been studied more intensively than any other type of drug carrier. The first impulse, as with other pharmaceuticals, has been to inject them iv. However, since they do not leave the vascular system readily except in the sinusoids of liver and spleen, many of the projected applications to nonhematogenous cancers are unlikely to succeed. Nonetheless, the last few years have brought a great increase in sophistication with respect to the physical chemistry, biochemistry, and pharmacology of liposomes. Routes other than the vascular are being explored, and iv injection is being exploited for access to targets within the vascular space. The most likely clinical applications to be successful soon are in parasitology and immunology; practical applications in oncology are distinctly possible, but still speculative.