Liposomal Platform Research Articles

Developing new assays (Fluorosomes) for characterization of drug interactions of ABC multidrug transporters

ABCB1/Pgp and ABCG2/BCRP multidrug transporters are important in developing multidrug resistance and significantly modify ADME‐Tox parameters of several therapeutic compounds. Regulatory agencies (EMA, FDA) state in their guidelines that drug candidates be tested for in vitro inhibition of Pgp and ABCG2. Only the application of different assays can reveal complex drug interactions of these polyspecific transporters. We investigate these interactions either in a native cell membrane environment or by using purified proteins reconstituted in a liposomal platform. ABC transporters extrude drugs at the expense of ATP hydrolysis, thus drug interactions can be followed in proteoliposomes (PL) by measuring ATPase activity or transport of labeled compounds. Providing appropriate lipid environment for ABCG2 was a key step in establishing new assays. PLs of ABCG2, in contrast to those containing ABCB1, have to contain cholesterol. In addition, phosphatidylethanolamine‐containing E.coli lipids support the ABCG2 activity better than phosphatidylcholine‐based lipids. In our international collaboration we are working on new assays similar to that developed earlier for the Pgp (the Fluorosome‐trans‐pgp assay). This method involves PLs that contain a fluorescent drug sensing probe in the interior of the PL, and measures in real time changes in fluorescence which reflect the Pgp‐ or ABCG2‐mediated transport. With Fluorosome‐trans‐abcg2/pgp assay, and direct liposomal assay observing the inhibition of uptake of fluorescently labeled substrate drugs we examine transporter‐drug interactions. The results are analyzed in correlation with the ATPase activity measurements.

Reversal of Chemoresistance in Ovarian Cancer by Co‐Delivery of MDR Inhibitors and Paclitaxel Using a Targeted Liposomal Platform

The over-expression of P-gp drug efflux pumps has led to the emergence of multidrug resistance in a variety of tumors. With this in mind, folate-targeted liposomes co-loaded with paclitaxel (PCT) and P-gp inhibitor tariquidar (XR) were developed. Aim To allow for the effective reversal of chemo-resistance in ovarian cancer cells using targeted liposomes co-loaded with tariquidar and paclitaxel. Methods: Folic acid was conjugated to the distal end of the PEG chain and post-inserted onto liposomes composed of ePC, cholesterol and DOTAP. Evaluation of drug resistance of ovarian cancer lines SKOV3, HeyA8 and Tynku was carried out by quantifying MDR1 and MRP1 receptor expression followed by a rhodamine123 exclusion assay. The cytotoxicity of the formulations was evaluated using the Promega viability assay. The cellular effects of the formulations were evaluated using the iCyte imaging cytometer, M4 phase holographic imager (PHI) and fluorescence microscopy. Results As compared to MRP1 higher expression of MDR1 was seen in the ovarian cancer cells. In vitro cytotoxicity experiments demonstrated that the XR+PCT liposomes was able to effectively overcome resistance to PCT even at low PCT doses of 50 nM. Using live cell imaging, paclitaxel-induced arrest of cell cycle progression and onset of apoptosis following was visible from 1.5 µM to 50 nM only with the co-loaded liposomes. The cell cycle arrest was further visualized using time-lapse PHI. The paclitaxel-mediated microtubule dysfunction was then visualized by the presence of polymerized microtubules around the cell nucleus by fluorescence microscopy. Conclusions The co-loaded liposomes were able to effectively reverse chemoresistance in a variety of drug-resistant ovarian cancers.

Abstract T P99: Development of Liposomes Loaded With Computed Tomogratphy Contrast Agent for Thrombus Imaging

Introduction: Liposomes are the most established nanocarriers that can be loaded with contrast agents as well as tailored for targeting to the desired tissue. Targeted CT liposomes represent a novel approach for rapid thrombus imaging. They allow specific and selective accumulation in thrombus providing significant contrast enhancement (expressed as HU) over the surrounding tissue. Hypothesis: We hypothesize that preparation technology will produce homogenous liposomes with sufficient level of loaded CT contrast agent. Methods: Liposomes were composed of distearoyl phosphatidylcholine, cholesterol, and distearoyl phosphatidylethanolamine - polyethylene glycol 2000 (molar ratio, 55/45/5). CT liposomes were prepared by lipid film hydration with iohexol (Omnipaque 350, GE Healthcare) followed by freeze-thaw extrusion (100 nm). Subsequently, they were purified by dialysis (Slide-A-Lyzer, cut-off 10 kDa) to remove un-loaded ioxehol. After purification, liposome-associated iohexol was determined by UV/VIS spectrophotometry and CT. Size distribution of final CT liposomes was assessed by dynamic light scattering. Results: Preparation technology produced homogenous liposome population having appropriate size distribution (90-110 nm and PD index, 0.05-0.10). Iohexol that remains associated in final CT liposomes represented only 5% of its initial level entering the preparation process. Almost 95% of iohexol was released and its final content was found to be 5 mg iodine/ml. Conclusions: Liposomal CT formulation was prepared with satisfactory size distribution and homogenity. Although the significant portion of iohexol was released, these CT liposomes were still detectable in vitro by CT. This basic liposomal platform will be optimized to achieve higher iohexol loading and will be further developed for thrombus imaging such as targeted CT liposomes.

Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells

The goal of the current research is to evaluate the potential of photodynamic therapy (PDT) in the treatment of triple negative breast cancer (TNBC) with the development of a theranostic thermosensitive liposome platform to deliver indocyanine green (ICG) as the near-infrared (NIR) photosensitizer excited by an 808 nm diode laser. In the PDT protocol, an optimized thermosensitive liposome formulation is investigated to formulate ICG as the photosensitizer, which is exited by laser light at the wavelength of 808 nm delivered by a fiber-coupled laser system. ICG in both free solution and thermosensitive liposomal formulation were evaluated as the NIR photosensitizer and compared in the PDT treatment on a panel of triple negative breast cancer cell lines along with the nontumorigenic mammary epithelial cell line MCF-10A. In addition to cytotoxicity, and clonogenic survival assessment, the role of DNA double strand break damage was evaluated. Both MTT and clonogenic assays revealed that PDT using ICG inhibited the growth of several TNBC cell lines as well as the non-tumorigenic human breast epithelial cell line MCF-10A; and the liposomal formulation of ICG did not compromise the in vitro treatment potency, though free ICG performed slightly more effective in certain cell lines, but was not statistically significant. Cell viability was dose dependent in regards to ICG concentration and irradiation energy. Interestingly, PDT using the described protocol was more potent to inhibit the growth of MDA-MB-468 and HCC-1806 cells, coinciding with the observation that these cells are more sensitive toward DNA damaging agents. In comparison, cell lines HCC-70, BT-549, and MCF-10A were found to have less of an inhibitory effect. Furthermore, substantial DNA double strand breaks (DSBs) were observed 30 min after the PDT treatment via a γ-H2AX staining assay. PDT induced DNA damage has the potential to lead to mutagenicity, which may have various responses depending on the repair capabilities of the cells. Our results suggest that PDT using indocyanine green loaded liposomes were effective in inhibiting tumor cell growth to varying extents with higher responses observed for MDA-MB-468 and HCC-1806 cells.

Intravesical liposome and antisense treatment for detrusor overactivity and interstitial cystitis/painful bladder syndrome.

Purpose. The following review focuses on the recent advancements in intravesical drug delivery, which brings added benefit to the therapy of detrusor overactivity and interstitial cystitis/painful bladder syndrome (IC/PBS). Results. Intravesical route is a preferred route of administration for restricting the action of extremely potent drugs like DMSO for patients of interstitial cystitis/painful bladder syndrome (IC/PBS) and botulinum toxin for detrusor overactivity. Patients who are either refractory to oral treatment or need to mitigate the adverse effects encountered with conventional routes of administration also chose this route. Its usefulness in some cases can be limited by vehicle (carrier) toxicity or short duration of action. Efforts have been underway to overcome these limitations by developing liposome platform for intravesical delivery of biotechnological products including antisense oligonucleotides. Conclusions. Adoption of forward-thinking approaches can achieve advancements in drug delivery systems targeted to future improvement in pharmacotherapy of bladder diseases. Latest developments in the field of nanotechnology can bring this mode of therapy from second line of treatment for refractory cases to the forefront of disease management.

Liposomal Encapsulation Enhances In Vivo Near Infrared Imaging of Exposed Phosphatidylserine in a Mouse Glioma Model

We have previously demonstrated that exposed phosphatidylserine (PS) on tumor vascular endothelial cells is highly tumor specific, and development of the PS targeted near infrared (NIR) optical probe enables successful in vivo optical imaging of U87 gliomas in a mouse model. Liposomes have been widely used as a nanovector for delivery of chemotherapeutics and imaging contrast agents due to their high payload and longer circulation time. In the current study, we have fabricated PS-targeted liposomal nanoprobes encapsulating a NIR dye, IRDye® 800CW, aiming to enhance PS-targeted tumor imaging. Hydrophilic 800CW dye was packed into the core of polyethylene glycol (PEG)-coated liposomes functionalized with F(ab’)2 fragments of PGN635, a fully human monoclonal antibody that binds PS. As expected, in vivo dynamic NIR imaging revealed significantly improved tumor/normal contrast (TNR = 20 ± 3; p < 0.01) of subcutaneous U87 gliomas in mice after injection of the liposomal nanoprobes. Markedly enhanced TNR was observed after the tumors were irradiated to increase PS exposure (TNR = 48 ± 6; p < 0.05). Intriguingly, the liposomal nanoprobes, PGN-L-800CW showed distinct biodistribution and pharmacokinetics compared to the 800CW-PGN probes used in our previous study. Our data further suggest the usefulness of PS-targeted imaging probes for sensitive tumor detection and the potential of utilizing liposomal platform for glioma theranostics.

The physiology of cardiovascular disease and innovative liposomal platforms for therapy

Heart disease remains the major cause of death in males and females, emphasizing the need for novel strategies to improve patient treatment and survival. A therapeutic approach, still in its infancy, is the development of site-specific drug-delivery systems. Nanoparticle-based delivery systems, such as liposomes, have evolved into robust platforms for site-specific delivery of therapeutics. In this review, the clinical impact of cardiovascular disease and the pathophysiology of different subsets of the disease are described. Potential pathological targets for therapy are introduced, and promising advances in nanotherapeutic cardiovascular applications involving liposomal platforms are presented.

Assessment of Therapeutic Efficacy of Liposomal Nanoparticles Mediated Gene Delivery by Molecular Imaging for Cancer Therapy

The inadequate treatment efficacy, suboptimal cancer detection and disease monitoring in anticancer therapies have led to the quest for clinically relevant, innovative multifaceted solutions such as development of targeted and traceable approaches. Molecular imaging technologies with the versatility of liposomal nanoparticles platform offer tangible options to better guide treatment delivery and monitor outcome. In this study, we introduced noninvasive, quantitative and functional imaging techniques with reporter gene methods to probe breast cancer processes with liposomal nanoparticles by bioluminescence imaging (BLI). A breast cancer model was applied for therapy by injecting 5.0 x 10(5) 4T1 cells carrying a reporter system encoding a double fusion reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into BALB/c mice. Liposomal nanoparticles loaded with a triple fusion gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk) and renilla luciferase (Rluc) and red fluorescent protein (RFP) were applied by in situ injection for monitoring and evaluating gene therapy. The BALB/c mice were subsequently treated with ganciclovir (GCV) and the growth status of tumor was monitored by bioluminescence imaging of Fluc and the treatment delivery of liposomal nanoparticle was efficiently tracked by Rluc imaging. In fact, TF plasmids were shown to be useful for monitoring and evaluating targeting efficacy and gene therapy by non-invasive molecular imaging. In conclusion, the combination of noninvasive imaging techniques and liposomal nanoparticle can provide a practical and clinically useful way for gene delivery and monitoring the level of gene expression over time and treatment response in patients undergoing gene therapy.

Mimicking Adhesive Functionalities of Blood Platelets using Ligand-Decorated Liposomes

Platelet transfusion is used for treating a variety of bleeding complications. Natural platelet-based transfusion products have very short storage life (3-7 days) and high risks of biological contamination and side effects. Consequently, there is significant clinical interest in synthetic platelet-mimetic constructs that can promote hemostasis, while allowing convenient large-scale production, easy portability, long storage life, and minimal biological risks. To this end, research efforts are being directed toward particles that can amplify aggregation of activated platelets or can mimic platelet's ability to undergo adhesion to various vascular matrix proteins. Here, we report on a synthetic construct design that combines the mimicry of platelet's shear-dependent adhesion to vWF and shear-independent adhesion to collagen under flow, on a single particle. For this, we have used 150-nm-diameter liposomes as model particles and have decorated their surface simultaneously with vWF-binding and collagen-binding recombinant protein fragments or synthetic peptide motifs. We demonstrate in vitro that these surface-modified liposomes are able to adhere onto vWF surfaces in a shear-dependent fashion and onto collagen surfaces in a shear-independent fashion under flow. Moreover, when the vWF-binding and the collagen-binding were integrated on a single liposomal platform, the resultant heteromultivalent liposomes showed significantly enhanced adhesion to a vWF/collagen mixed surface compared to liposomes bearing vWF-binding or collagen-binding ligands only, as long as the ligand motifs did not spatially interfere with each other. Altogether, our results establish the feasibility of efficiently mimicking platelet's dual adhesion mechanisms on synthetic particles.

Development and validation of an HPLC/UV assay for separation and quantification of peptide antigens from a liposomal vaccine delivery platform

The development and validation of an HPLC method for the quantification of eight peptide antigens from the therapeutic cancer vaccine DPX-0907 is described. The antigens were formulated in DepoVax™, a patented liposomal vaccine delivery platform used in a phase 1 study for breast, ovarian, and prostate cancers. A gradient reversed-phase method with UV detection was optimized for separating and quantifying the peptide mixture. Several extraction methods investigated to extract the peptides from the lipids led to poor recovery of one or more of the peptides. A simple, reproducible, and high-recovery extraction procedure for the simultaneous quantification of hydrophilic and hydrophobic peptides was discovered using a liquid-liquid extraction with water-saturated n-butanol and sodium bicarbonate (0.1 M). The method was found to be specific, linear, accurate, precise, and reliable within the range of 50-150% of the nominal concentration for DPX-0907. The validated method was successfully applied to the assay of peptide content in pre-clinical and clinical batches of DPX-0907.

Induction of antigen-specific B cell tolerance through the development of a versatile platform to target siglecs (45.4)

Abstract CD22 and SiglecG are proposed to be negative regulators of B cell receptor signaling through their ability to recognize sialic acid-containing glycans. Recent studies have shown that multivalent presentation of high affinity CD22 and SiglecG ligands in cis with a T-independent antigen can tolerize B cells (Duong et al., J. Exp. Med., 2010). Here we describe studies aimed at showing the generality of this phenomenon. To realize this aim, a novel liposomal nanoparticle platform was developed for presentation of both high affinity Siglec ligands and antigen. Chemical conjugation of the Siglec ligand and antigen to this platform is facile and, importantly, this platform is highly compatible with in vivo studies. Using this new platform, robust B cell tolerance is achieved toward both T-independent and T-dependent antigens in mice. Mechanistic studies provide clear evidence that: i) CD22 and SiglecG are the targets; ii) apoptosis of B cells is the mode of tolerance; iii) tolerization is highly selective to B cells containing a BCR that is specific for the targeted antigen. These studies reinforce the hypothesis that CD22 and SiglecG are critical components of a B cell that allows it to distinguish self from non-self. This method has clear potential for treatment of autoimmune diseases and allergies by antigen specific tolerization of B cells.

Paramagnetic and fluorescent liposomes for target-specific imaging and therapy of tumor angiogenesis

Angiogenesis is essential for tumor growth and metastatic potential and for that reason considered an important target for tumor treatment. Noninvasive imaging technologies, capable of visualizing tumor angiogenesis and evaluating the efficacy of angiostatic therapies, are therefore becoming increasingly important. Among the various imaging modalities, magnetic resonance imaging (MRI) is characterized by a superb spatial resolution and anatomical soft-tissue contrast. Revolutionary advances in contrast agent chemistry have delivered versatile angiogenesis-specific molecular MRI contrast agents. In this paper, we review recent advances in the preclinical application of paramagnetic and fluorescent liposomes for noninvasive visualization of the molecular processes involved in tumor angiogenesis. This liposomal contrast agent platform can be prepared with a high payload of contrast generating material, thereby facilitating its detection, and is equipped with one or more types of targeting ligands for binding to specific molecules expressed at the angiogenic site. Multimodal liposomes endowed with contrast material for complementary imaging technologies, e.g., MRI and optical, can be exploited to gain important preclinical insights into the mechanisms of binding and accumulation at angiogenic vascular endothelium and to corroborate the in vivo findings. Interestingly, liposomes can be designed to contain angiostatic therapeutics, allowing for image-supervised drug delivery and subsequent monitoring of therapeutic efficacy.

“Clickable”, polymerized liposomes as a versatile and stable platform for rapid optimization of their peripheral compositions

A versatile and stable liposomal platform is developed for rapid optimization of its peripheral composition. The platform is based on polydiacetylene lipids terminated with alkynyl groups. Conditions for copper-catalyzed azide-alkyne cycloaddition (a "click" reaction) are optimized for rapid attachment of azides with controlled composition onto the liposomes.

A Tumor-Targeted Nanodelivery System to Improve Early MRI Detection of Cancer

The development of improvements in magnetic resonance imaging (MRI) that would enhance sensitivity, leading to earlier detection of cancer and visualization of metastatic disease, is an area of intense exploration. We have devised a tumor-targeting, liposomal nanodelivery platform for use in gene medicine. This systemically administered nanocomplex has been shown to specifically and efficiently deliver both genes and oligonucleotides to primary and metastatic tumor cells, resulting in significant tumor growth inhibition and even tumor regression. Here we examine the effect on MRI of incorporating conventional MRI contrast agent Magnevist into our anti-transferrin receptor single-chain antibody (TfRscFv) liposomal complex. Both in vitro and in an in vivo orthotopic mouse model of pancreatic cancer, we show increased resolution and image intensity with the complexed Magnevist. Using advanced microscopy techniques (scanning electron microscopy and scanning probe microscopy), we also established that the Magnevist is in fact encapsulated by the liposome in the complex and that the complex still retains its nanodimensional size. These results demonstrate that this TfRscFv-liposome-Magnevist nanocomplex has the potential to become a useful tool in early cancer detection.

Pros and Cons of the Liposome Platform in Cancer Drug Targeting

Coating of liposomes with polyethylene-glycol (PEG) by incorporation in the liposome bilayer of PEG-derivatized lipids results in inhibition of liposome uptake by the reticulo-endothelial system and significant prolongation of liposome residence time in the blood stream. Parallel developments in drug loading technology have improved the efficiency and stability of drug entrapment in liposomes, particularly with regard to cationic amphiphiles such as anthracyclines. An example of this new generation of liposomes is a formulation of pegylated liposomal doxorubicin known as Doxil® or Caelyx®, whose clinical pharmacokinetic profile is characterized by slow plasma clearance and small volume of distribution. A hallmark of these long-circulating liposomal drug carriers is their enhanced accumulation in tumors. The mechanism underlying this passive targeting effect is the phenomenon known as enhanced permeability and retention (EPR) which has been described in a broad variety of experimental tumor types. Further to the passive targeting effect, the liposome drug delivery platform offers the possibility of grafting tumor-specific ligands on the liposome membrane for active targeting to tumor cells, and potentially intracellular drug delivery. The pros and cons of the liposome platform in cancer targeting are discussed vis-à-vis nontargeted drugs, using as an example a liposome drug delivery system targeted to the folate receptor.

A Pegylated Liposomal Platform: Pharmacokinetics, Pharmacodynamics, and Toxicity in Mice Using Doxorubicin as a Model Drug

Aims were to observe pharmacokinetics, pharmacodynamics, and toxicity for constructing a Sino-pegylated liposomal platform. Human hepatocarcinoma cells (Bel7402) and murine hepatocarcinoma cells (H22) were used for the cytotoxicity assay and the in vivo solid xenograft tumor model in mice, respectively. Pharmacokinetic results in mice showed that the pegylated liposomal doxorubicin markedly prolonged the blood circulation of doxorubicin. Elimination half-time (T1/2,γ) of pegylated, regular liposomal doxorubicin and free doxorubicin were 46.09 ± 14.44, 26.04 ± 3.34, and 23.72 ± 5.13 h, respectively. The area under the concentration-time curves (AUC0–∞) (h × μg/g) of the pegylated and regular liposomal doxorubicin were 6.8- and 2.6-fold higher than that of free doxorubicin, respectively. Cytotoxicity and antitumor activity in vivo indicated that activity of the pegylated liposomal doxorubicin was higher than that of the regular or the free one, respectively. After two weeks of tail intravenous injection of the pegylated liposomal doxorubicin at a single dose of 10 mg/kg, no significant damage was observed in gastric, intestinal mucosa, and heart muscle, but pronounced damages were found in the control group after dosing free doxorubicin. The results demonstrate that the pegylated liposomes improve the efficacy of toxics and reduce the toxicity, therefore providing favorable evidence for building a pegylated liposomal platform.