Liposomal Carrier Research Articles

Pre-Targeting and Direct Immunotargeting of Liposomal Drug Carriers to Ovarian Carcinoma

BackgroundEpidermal growth factor receptor (EGFR) is overexpressed in many solid tumor types, such as ovarian carcinoma. Immunoliposome based drug targeting has shown promising results in drug delivery to the tumors. However, the ratio of tumor-to-normal tissue concentrations should be increased to minimize the adverse effects of cytostatic drugs.Methodology/Principal FindingsWe studied the EGFR-targeted doxorubicin immunoliposomes using pre-targeting and local intraperitoneal (i.p.) administration of the liposomes. This approach was used to increase drug delivery to tumors as compared to direct intravenous (i.v.) administration of liposomes. EGFR antibodies were attached on the surface of PEG coated liposomes using biotin-neutravidin binding. Receptor mediated cellular uptake and cytotoxic efficacy of EGFR-targeted liposomes were investigated in human ovarian adenocarcinoma (SKOV-3 and SKOV3.ip1) cells. In vivo distribution of the liposomes in mice was explored using direct and pre-targeting approaches and SPECT/CT imaging. Targeted liposomes showed efficient and specific receptor-mediated binding to ovarian carcinoma cells in vitro, but the difference in cytotoxicity between targeted and non-targeted liposomes remained small. The relatively low cytotoxic efficacy is probably due to insufficient doxorubicin release from the liposomes rather than lack of target binding. Tumor uptake of targeted liposomes in vivo was comparable to that of non-targeted liposomes after both direct and pre-targeting administration. For both EGFR-targeted and non-targeted liposomes, the i.p. administration increased liposome accumulation to the tumors compared to i.v. injections.Conclusions/SignificanceIntraperitoneal administration of liposomes may be a beneficial approach to treat the tumors in the abdominal cavity. The i.p. pre-targeting method warrants further studies as a potential approach in cancer therapy.

Monitoring the subcellular localization of doxorubicin in CHO-K1 using MEKC−LIF: Liposomal carrier for enhanced drug delivery

Doxorubicin (DOX) is an extensively used anthracycline that has proven to be effective against a variety of human malignant tumors, such as ovarian or breast cancer. While DOX was administered into cultured cancer cell targets (such as CHO-K1) in either free drug form or in drug carrier-associated form (i.e., DOX encapsulated in the drug delivery carrier), various action of mechanisms for DOX were initiated, among which, it has been long believed that DOX enters the nucleus, interacts with DNA in numerous ways, and finally halts cell proliferation. Aside from its therapeutic effect, regrettably DOX treatment may be accompanied by the occurrence of cardiac and liver toxicity and drug resistance that are attributed from cellular processes involving the parent drug or its metabolites. Liposomal formulation of DOX, known to be capable of attenuating direct uptake of reticuloendothelial system (RES) and prolonging the circulation time in blood, demonstrated reduced toxic side-effects. We herein report the development of a modified MEKC–LIF (Micellar electrokinetic chromatography–Laser-induced fluorescence) method suitable for analyzing DOX in biological samples. The MEKC migration buffer, consisting of 10mM borate, 100mM sodium dodecyl sulfate (SDS) (pH 9.3), was found to provide an efficient and stable electrophoretic separation and analysis for DOX. Responses were linear in the range of 11.3–725ng/mL; the limit of quantitation (LOQ) was found to be 43.1ng/mL (S/N=10) (equivalent to 74.3nM) and limit of detection (LOD) was calculated as 6.36ng/mL (S/N=3) (equivalent to 11.0nM). This approach was subsequently employed to compare the intracellular accumulation in three subcellular fractions of DOX-treated CHO-K1 cells. These fractions form a pellet at <1400g, 1400–14000g, and >14000g and are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosole components, respectively, resulting from treatment of CHO-K1 cells with 25μM (equivalent to 14.5μg/mL) of two DOX formats (in free drug form or liposomal form synthesized in current study) for different periods of time. Our results indicated that the most abundant DOX was found in the nuclear-enriched fraction of cells treated for 12h and 6h with free and liposomal DOX, respectively, providing direct evidence to confirm the enhanced efficiency of liposomal carriers in delivering DOX into the nucleus. The observations presented herein suggest that subcellular fractionation followed by liquid–liquid extraction and MEKC-LIF could be a powerful diagnostic tool for monitoring intracellular DOX distribution, which is highly relevant to cytotoxicity studies of anthracycline-type anticancer drugs.

Topical Delivery of Thymidine Dinucleotide to Induce p53 Generation in the Skin by Elastic Liposome

Transcription factor p53 has a powerful tumor suppressing function that is associated with many cancers. However, p53 of the molecular weight was higher make the limitation across to skin or cell membrane. Thymidine dinucleotide (pTT), an oligonucleotide, can activate the p53 transcription factor. pTT is a hydrophilic and negative charge oligonucleotide, which delivery in to cell membrane need an appropriate carrier. The aim of this study was to improve the bioavailability of the nucleotide fragment, thymidine dinucleotide (pTT), using elasic liposome carriers to deliver the drug into the skin. The study demonstrate that dioleoylphosphocholine (DOPC) incorporated with sodium cholate at molar ratio 1:1 can archived the particle size about 220 nm. This elastic liposome could penetration through skin from stratum corneum to whole epidermis by confocal laser scanning microscopy (CLSM). Moreover, we observed the the slight increase in generation of p53 by western blot. Keywords—Elastic liposome, Tymidine dinucleotide, p53, Topical delivery

Comparative study on preparative methods of DC-Chol/DOPE liposomes and formulation optimization by determining encapsulation efficiency

Three most commonly used preparative methods, dry-film, reverse phase evaporation and ethanol injection were employed to prepare cationic liposomes composed of DC-Chol and DOPE, respectively. The resulting samples were contrasted through morphology observation, particle size and zeta potential analysis. Sephadex filtration method with high selectivity was developed to determine the encapsulation efficiency of plasmid DNA-loaded cationic vectors, on this basis, cationic liposomes formulation was further optimized by applying Box Behnken design with encapsulation efficiency as evaluation index. The results showed that liposomes prepared by dry-film method were of best quality and stability, moreover, the optimum formulation of cationic liposomes and optimal value of each influencing factors were quantitatively obtained, measured value was highly consistent with predicted results. These findings preliminarily clarified the effect of preparative methods on performance of cationic liposome, as well as formulation factors on encapsulation efficiency, and will provide important methodological reference for further study of liposomes carriers for gene delivery.

Pharmacokinetic behavior and efficiency of acetylcholinesterase inhibition in rat brain after intranasal administration of galanthamine hydrobromide loaded flexible liposomes

Galanthamine hydrobromide (GH) has been approved for symptomatic treatment of Alzheimer's disease (AD) and vascular dementia. Hence, the effects of intranasal administration of GH loaded flexible liposomes have been investigated for the first time on the efficiency of acetylcholinesterase inhibition, as well as the pharmacokinetic behavior of GH in rat brain. The GH loaded flexible liposomes were characterized for shape, entrapment capacity, size distribution and zeta potential by transmission electron microscopy (TEM), ultracentrifugation and dynamic light scattering (DLS), respectively. The inhibition of acetylcholinesterase was investigated using rat brain homogenates as an enzyme resource and microdialysis was used to determine the pharmacokinetic behavior of GH in rats brain. The rat pheochromocytoma PC-12 cell line was used to evaluate the cytotoxicity of GH loaded flexible liposomes. The results revealed that: (i) the efficiency of acetylcholinesterase inhibition of GH was greatly enhanced by intranasal administration compared with oral administration, especially GH loaded in flexible liposomes; (ii) the Cmax and AUC0→10 for intranasal administration of GH loaded flexible liposomes were 3.52 and 3.36 times higher than those of orally administered GH, moreover, the Tmax was greatly shortened from 1.5h for oral administration to 0.75h for intranasal administration of GH loaded flexible liposomes; and (iii) PC-12 cells viability tests showed that the flexible liposome carrier is not toxic to the cultured cells and the cytotoxicity of GH to cells was clearly decreased by loading in flexible liposomes. These results indicate that intranasal administration of GH loaded flexible liposomes could readily transport GH into brain tissues, suggesting some promise for this approach in successful brain–drug targeting in AD treatment.

Nanostructural Hybrid Sensitizers for Photodynamic Therapy

Photodynamic therapy (PDT) is a clinically approved method for treatment of cancer, microbial infections, and some other diseases. PDT has proved effective in the treatment of malignancies of various organs, including lung, bladder, gastrointestinal tract, and skin, and in the therapy of bacterial infection of skin wounds and carious lesions. It employs a combination of light and a drug (photosensitizer, PS) to induce phototoxicity against cancerous cells or bacteria. The efficiency of currently used PSs is limited due to their hydrophobic nature, which causes aggregation of the PS in aqueous media and low tumor selectivity (a low value of the tumor-to-normal tissue ratio). The purpose of this review is to present some aspects of the current state of knowledge on nanostructural carriers for the PS delivery. In this paper we reviewed studies on the development of nanostructural materials for PDT, especially those based on the polymeric and liposomal formulation of PS. We focused mainly on the nanostructural PSs obtained by the covalent attachment of hydrophilic polymer chain to the low-molecular-weight PS, the incorporation of PS into polymeric nanostructures such as micelles, and the solubilization of PS in liposome carriers.

The fractal hologram and elucidation of the structure of liposomal carriers in aqueous and biological media

The present study deals with the physicochemical characterization (size, polydispersity, ζ-potential) of dipalmitoylphosphatidylcholine (DPPC) liposomes and DPPC:cholesterol (chol) (9:1 molar ratio) liposomes, and the determination of their fractal dimension (mass fractal (df) and surface fractal (ds)), in an aqueous (HPLC grade water) and in a biological (fetal bovine serum – FBS) medium. Dynamic, static and electrophoretic light scattering and fluorescence spectroscopy are used as experimental techniques to elucidate the structure and physicochemical parameters of liposomes in an ageing study in two different media, as well as their structural response in changes in concentration and temperature. The extended DLVO theory would be the tool to explain the phenomenology of the colloidal behavior in these systems and of their aggregation process. The fractal dimensionality of DPPC liposomes was decreased while for DPPC:cholesterol (9:1) it remained unaffected in the two dispersion media. The structure of the liposomal systems, the process kinetics, and the fractal dimension are consistent with the diffusion-limited cluster aggregation (DLCA) and reaction-limited cluster aggregation (RLCA) models. On the contrary, hydrodynamic radius (Rh) was found to be stable during the variations of colloidal system conditions, especially due to concentration changes. Finally, we suggest that this study can be a rational road map to design advanced Drug Delivery nano Systems (aDDnSs) with improved pharmacokinetic profile which could be considered as crucial for their effectiveness.

Antitumor efficacy following the intracellular and interstitial release of liposomal doxorubicin

pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.

Evaluation of microemulsion and liposomes as carriers for oral delivery of transforming growth factor alpha in rats

The aim of the present study is to develop microemulsion and liposome carrier systems for oral administration of transforming growth factor alpha (TGF-α) and to investigate the effects of these carrier systems on the gastrointestinal efficiency in rats. Microemulsion (w/o) and liposomes (MLV) were developed and characterised. The carrier systems were administered intragastrically by gastric cannula to male Wistar rats. The highest reduction in the basal acid secretion was observed in the microemulsion containing TGF-α and aprotinin group (TAME).The gastric mucus secretion in microemulsion containing TGF-α (TME) and TAME treatment groups increased significantly compared to the other groups. TGF-α levels in both stomach and duodenum were significantly increased in the TAME group. As a result, it was determined through confocal laser scanning microscope (CLSM) studies that exogenous-applied TGF-α attached to endogenous EGF receptors. The microemulsion formulation was found to be a more suitable carrier system for oral administration of TGF-α.

Ultrasmall superparamagnetic iron oxide (USPIO)-based liposomes as magnetic resonance imaging probes

BackgroundMagnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast.MethodsRegarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the signal enhancement on T1- and T2-weighted sequences of sMLs. To monitor the biodistribution and the clearance of the particles over time in vivo, sMLs were injected in wild type mice.ResultsDLS revealed a mean particle diameter of sMLs in the range between 100 and 200 nm, as confirmed by TEM. An effective iron oxide loading was achieved just for one type of USPIO, with an EE% between 74% and 92%, depending on the initial Fe concentration (being higher for lower amounts of Fe). MRI measurements demonstrated the applicability of these nanostructures as MRI probes.ConclusionOur results show that the development of sMLs is strictly dependent on the physicochemical characteristics of the nanocores. Once established, sMLs can be further modified to enable noninvasive targeted molecular imaging.

Synthesis and characterization of non-viral liposomal carriers for the local application of siRNA molecules and anti-miRNAs in the therapeutic treatment of psoriasis

Background Psoriasis is a common inflammatory skin disease with a multifactorial genetic basis. A dysregulated interplay between keratinocytes and infiltrating immune cells underlies the cutaneous inflammation in psoriasis. Keratinocytes are important producers of antimicrobial peptides such as hBD-2 and LL37 and cytokines such as TNF-alpha, which are essential elements in this process of cell-cell communication [1]. Recently, miRNA203 was identified as an important contributor to this dysfunctional cross talk [2]. We have previously developed a new lipid-based nanosome (SECosome) that enables the effective delivery of siRNA into human skin [3]. The aim of this project is to knockdown mRNA encoding hBD-2, LL37, TNF-alpha and miRNA-203 by tranfection of keratinocytes with SECosomes for the delivery of siRNAs and anti-miRNAs. Ultimately, we want to create a new therapy for psoriasis by intervening at genetic level by means of a topical therapy.

Cationic liposome–nucleic acid complexes: liquid crystal phases with applications in gene therapy

Cationic liposome (CL) carriers of nucleic acids are studied primarily because of their applications in gene delivery and gene silencing with CL-DNA and CL-siRNA (short-interfering RNA) complexes, respectively, and their implications for ongoing clinical gene therapy trials worldwide. A series of synchrotron-based small-angle X-ray scattering studies, dating back to 1997, has revealed that CL-nucleic acid complexes spontaneously assemble into distinct novel liquid crystalline phases of matter. Significantly, transfection efficiency (TE; a measure of expression of an exogenous gene that is transferred into the cell by the lipid carrier) has been found to be dependent on the liquid crystalline structure of complexes, with lamellar complexes showing strong dependence on membrane charge density (σM) and non-lamellar complexes exhibiting TE behaviour independent of σM. The review describes our current understanding of the structures of different liquid crystalline CL-nucleic acid complexes including the recently described gyroid cubic phase of CL-siRNA complexes used in gene silencing. It further makes apparent that the long-term goal of developing optimised liquid crystalline CL-nucleic acid complexes for successful medical applications requires a comprehensive understanding of the nature of the interactions of distinctly structured complexes with cell membranes and events leading to release of active nucleic acids within the cell cytoplasm.

Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size

A major goal in cancer research is to develop carriers that can deliver drugs effectively and without side effects. Liposomal and particulate carriers with diameters of ∼100nm have been widely used to improve the distribution and tumour accumulation of cancer drugs, but so far they have only been effective for treating highly permeable tumours. Here, we compare the accumulation and effectiveness of different sizes of long-circulating, drug-loaded polymeric micelles (with diameters of 30, 50, 70 and 100nm) in both highly and poorly permeable tumours. All the polymer micelles penetrated highly permeable tumours in mice, but only the 30nm micelles could penetrate poorly permeable pancreatic tumours to achieve an antitumour effect. We also showed that the penetration and efficacy of the larger micelles could be enhanced by using a transforming growth factor-β inhibitor to increase the permeability of the tumours.

Liposomes as an alternative delivery system for investigating dietary metal toxicity to Daphnia magna

Dietary metal toxicity studies with invertebrates such as Daphnia magna are often performed using metal-contaminated algae as a food source. A drawback of this approach is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by a reduced essential nutrient content in the metal-contaminated diet, due to prior exposure of the algae to the metal. This hampers the study of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates. The aim of the present study was to develop a technique for producing metal-contaminated liposomes as an alternative delivery system of dietary metals. These liposomes are not vulnerable to metal-induced shifts in nutrient quality. Liposomes were prepared by the hydration of phosphatidylcholine in media containing either 0 (control) or 50 mg Ni/L. The liposomes had average diameters of 19.31 (control) and 10.48 μm (Ni-laden), i.e., a size appropriate for ingestion by D. magna. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt. basis) to make up two diets that differed in Ni content (i.e., 2.0 μg Ni/g dry wt. in the control and 144.2 μg Ni/g dry wt. in the Ni-contaminated diet, respectively). This diet was then fed to D. magna during a 21-day chronic bioassay. The experiment showed that the Ni content and the size distribution of the liposomes were stable for at least 7 days. Also the use of phosphatidylcholine as a liposome component did not affect the reproduction of the daphnids. Exposure to increased level of dietary Ni resulted in 100% mortality after 14 days of exposure and in an increased whole-body Ni concentration in D. magna of 14.9 and 20.4 μg Ni/g dry wt. after 7 and 14 days of exposure, respectively. The Ni-exposed daphnids also exhibited a reduced size (i.e., 30% smaller than the control) after 7 days and a completely halted growth between day 7 and day 14. In terms of reproduction, the size of the first brood (number of juveniles) of the Ni-exposed daphnids was significantly reduced (by 85%) compared to the control. None of the Ni-exposed individuals were able to produce a second brood before dying. The algal ingestion rate – after correction for the indirect effect of a reduced size – was increased (by 68%) by dietary Ni after 6 days of exposure compared to the control, but was severely reduced (by 80% compared to the control) after 13 days. These data suggest that an inhibition of the ingestion process may have contributed to the observed effects of dietary Ni on growth and reproduction beyond 6 days of exposure, although the involvement of other mechanisms cannot be excluded. The mechanism(s) which led to the reduced growth during the first week of exposure remain unclear, although inhibition of the ingestion process can likely be excluded here as an explanation. Overall, this paper demonstrates, using this new method of delivering dietary Ni via liposome carriers and thus excluding potential diet quality shifts, that dietary Ni can indeed induce toxic effects in D. magna. This method may therefore be a promising tool to help further elucidate the mechanisms of dietary metal toxicity to filter-feeding invertebrates.

Development and evaluation of conventional and PEGylated curcumin liposomes, absorption and tissue distribution studies in mice

Curcumin is the main biological active polyphenolic compound present in the rhizomes of turmeric (Curcuma longa Linn.), has a wide biological and pharmacological profile. It has been reported to possess anti-oxidative, anti-inflammatory and anti-carcinogenic properties [1–3]. Many clinical study reports have revealed that curcumin has many beneficial properties in the treatment of various diseases in man such as pancreatic cancer and inflammatory bowel disease [4,5]. Despites these promising effects a poor oral absorption due to its extremely low aqueous solubility and rapid metabolism result in very low oral systemic bioavailability, thus limiting its clinical use. In order to overcome bioavailability drawbacks, this work proposes inclusion of curcumin into liposomal carriers.

Liposomal photosensitizers: potential platforms for anticancer photodynamic therapy

Photodynamic therapy is a well-established and clinically approved treatment for several types of cancer. Antineoplastic photodynamic therapy is based on photosensitizers, i.e., drugs that absorb photons translating light energy into a chemical potential that damages tumor tissues. Despite the encouraging clinical results with the approved photosensitizers available today, the prolonged skin phototoxicity, poor selectivity for diseased tissues, hydrophobic nature, and extended retention in the host organism shown by these drugs have stimulated researchers to develop new formulations for photodynamic therapy. In this context, due to their amphiphilic characteristic (compatibility with both hydrophobic and hydrophilic substances), liposomes have proven to be suitable carriers for photosensitizers, improving the photophysical properties of the photosensitizers. Moreover, as nanostructured drug delivery systems, liposomes improve the efficiency and safety of antineoplastic photodynamic therapy, mainly by the classical phenomenon of extended permeation and retention. Therefore, the association of photosensitizers with liposomes has been extensively studied. In this review, both current knowledge and future perspectives on liposomal carriers for antineoplastic photodynamic therapy are critically discussed.

Liposomal systems as drug delivery vehicles for dermal and transdermal applications

Enhancement strategies are necessary to improve the dermal/transdermal bioavailability of drugs applied to the skin due to its amazing barrier, the stratum corneum. Strategies to overcome this barrier, thus improving drug release to the skin include the use of penetration enhancers, specific delivery systems, supersaturated solutions and physical methods (iontophoresis, electroporation and ultrasound). Delivery of active agents to the skin by liposomal carriers has improved topical therapy in the field of dermatology. The interest in these carriers is based on their potential to enclose various types of biological materials and to deliver them to diverse cell types. Particularly, in recent years liposomes have been shown to be a promising drug-delivery system to the skin. Their use may produce several-fold higher drug concentrations in the epidermis and dermis and lower systemic concentrations when compared to conventional dosage forms. On the other hand, special characteristic vesicles like ethosomes, transfersomes and niosomes may be potential transdermal delivery systems for ionic molecules and polypeptides.

Interaction of curcumin with lipid monolayers and liposomal bilayers

Curcumin shows huge potential as an anticancer and anti-inflammatory agent. However, to achieve a satisfactory bioavailability and stability of this compound, its liposomal form is preferable. Our detailed studies on the curcumin interaction with lipid membranes are aimed to obtain better understanding of the mechanism and eventually to improve the efficiency of curcumin delivery to cells. Egg yolk phosphatidylcholine (EYPC) one-component monolayers and bilayers, as well as mixed systems containing additionally dihexadecyl phosphate (DHP) and cholesterol, were studied. Curcumin binding constant to EYPC liposomes was determined based on two different methods: UV/Vis absorption and fluorescence measurements to be 4.26×10(4)M(-1) and 3.79×10(4)M(-1), respectively. The fluorescence quenching experiment revealed that curcumin locates in the hydrophobic region of EYPC liposomal bilayer. It was shown that curcumin impacts the size and stability of the liposomal carriers significantly. Loaded into the EYPC/DPH/cholesterol liposomal bilayer curcumin stabilizes the system proportionally to its content, while the EYPC/DPH system is destabilized upon drug loading. The three-component lipid composition of the liposome seems to be the most promising system for curcumin delivery. An interaction of free and liposomal curcumin with EYPC and mixed monolayers was also studied using Langmuir balance measurements. Monolayer systems were treated as a simple model of cell membrane. Condensing effect of curcumin on EYPC and EYPC/DHP monolayers and loosening influence on EYPC/DHP/chol ones were observed. It was also demonstrated that curcumin-loaded EYPC liposomes are more stable upon interaction with the model lipid membrane than the unloaded ones.

Conjugation of lipid and CpG-containing oligonucleotide yields an efficient method for liposome incorporation.

For optimal stimulation of T cells, protein-based vaccines must deliver protein antigens to antigen-presenting cells while simultaneously providing immunostimulatory signals. Listeriolysin O (LLO)-containing liposomes have been utilized to efficiently deliver protein antigens to the cytosolic pathway for antigen processing and major histocompatibility complex class I-dependent presentation while codelivering immunostimulatory CpG-oligodeoxyribonuceotides (ODNs). In this report, we describe the synthesis of lipid-CpG-ODN conjugates utilizing maleimide-phosphatidylethanolamine (PE) lipids and 5'-sulfhdryl-containing CpG-ODNs as a method for facile incorporation of CpG-ODNs in liposomal vaccine carriers, an alternative to co-encapsulation inside liposomes and as a means to enhance delivery of CpG-ODNs to their major receptor, Toll-like receptor 9 (TLR9), in the endosome. The characterization and biological evaluation of the vaccine delivery system made of liposomes, which contain the lipid-CpG-ODN conjugates inserted in the liposomal membrane, is described. We demonstrate in vitro in bone marrow derived macrophages that the lipid-CpG-ODN conjugates incorporated onto the liposome bilayers interact with their receptor TLR9 as readily as liposome-encapsulated ODNs and exert their immunostimulatory capabilities. The liposomal vaccine delivery systems were evaluated in mice using ovalbumin (OVA) as a model antigen, and the results indicate equally robust OVA-specific cytotoxic T lymphocyte responses and similar Th1 immune skewing capabilities between liposomes containing lipid-conjugated or encapsulated CpG-ODNs. Overall, this work indicates that conjugating PE lipids and CpG-ODNs results in an efficient method that allows facile incorporation of CpG-ODNs into a liposome-based delivery platform while retaining the immune-stimulating capabilities of CpG-ODNs.

Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic peptide shGALA

An siRNA that specifically silences the expression of mRNA is a potential therapeutic agent for dealing with many diseases including cancer. However, the poor cellular uptake and bioavailability of siRNA remains a major obstacle to clinical development. For efficient delivery to tumor tissue, the pharmacokinetics and intracellular trafficking of siRNA must be rigorously controlled. To address this issue, we developed a liposomal siRNA carrier, a multi-functional nano device (MEND). We describe herein an approach for systemic siRNA delivery to tumors by combining the MEND system with shGALA, a fusogenic peptide. In cultured cell experiments, shGALA-modification enhanced the endosomal escape of siRNA encapsulated in a polyethylene glycol modified MEND (PEG-MEND), resulting in an 82% knockdown of the target gene. In vivo systemic administration clarified that the shGALA-modified MEND (shGALA-MEND) showed 58% gene silencing in tumor tissues at a dose of 4 mg of siRNA/kg body weight. In addition, a significant inhibition of tumor growth was observed only for the shGALA-MEND and no somatic or hepatic toxicity was observed. Given the above data, this peptide-modified delivery system, a shGALA-MEND has great potential for the systemic delivery of therapeutic siRNA aimed at cancer therapy.