C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake.

Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4°C and 37°C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox. SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4°C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process.

Pharmacokinetics, Tissue Distribution and Therapeutic Effect of Cationic Thermosensitive Liposomal Doxorubicin Upon Mild Hyperthermia.

PURPOSE: To evaluate pharmacokinetic profile, biodistribution and therapeutic effect of cationic thermosensitive liposomes (CTSL) encapsulating doxorubicin (Dox) upon mild hyperthermia (HT). METHODS: Non-targeted thermosensitive liposomes (TSL) and CTSL were developed, loaded with Dox and characterized. Blood kinetics and biodistribution of Dox-TSL and Dox-CTSL were followed in B16BL6 tumor bearing mice upon normothermia (NT) or initial hyperthermia conditions. Efficacy study in B16BL6 tumor bearing mice was followed with Dox-TSL or Dox-CTSL upon NT or HT. Efficacy study in LLC tumor bearing mice was performed upon two HT conditions. Intravital microscopy was performed on B16BL6 tumors implanted in dorsal-skin fold window-bearing mice. RESULTS: Targeting did not cause faster blood clearance of CTSL compared to TSL. Highest uptake of liposomes was observed in spleen, kidneys and liver. Applying HT prior to CTSL administration increased drug delivery to the tumor and CTSL delivered ~1.7 fold higher Dox concentration compared to TSL. Efficacy in B16BL6 murine melanoma showed that HT had a significant effect on CTSL in tumor suppression and prolonged survival. Efficacy in LLC Lewis lung carcinoma tumor model demonstrates that two HT treatments hold promises for a successful treatment option. CONCLUSION: CTSL have potency to increase drug efficacy in tumors due to their targeted and drug release functions.

Short-chain glycoceramides promote intracellular mitoxantrone delivery from novel nanoliposomes into breast cancer cells.

PURPOSE: To improve therapeutic activity of mitoxantrone (MTO)-based chemotherapy by reducing toxicity through encapsulation in nanoliposomes and enhancing intracellular drug delivery using short-chain sphingolipid (SCS) mediated tumor cell membrane permeabilization. METHODS: Standard (MTOL) and nanoliposomes enriched with the SCS, C8-Glucosylceramide or C8-Galactosylceramide (SCS-MTOL) were loaded by a transmembrane ammonium sulphate gradient and characterized by DLS and cryo-TEM. Intracellular MTO delivery was measured by flow cytometry and imaged by fluorescence microscopy. In vitro cytotoxicity was studied in breast carcinoma cell lines. Additionally, live cell confocal microscopy addressed the drug delivery mechanism by following the intracellular fate of the nanoliposomes, the SCS and MTO. Intratumoral MTO localization in relation to CD31-positive tumor vessels and CD11b positive cells was studied in an orthotopic MCF-7 breast cancer xenograft. RESULTS: Stable SCS-MTOL were developed increasing MTO delivery and cytotoxicity to tumor cells compared to standard MTOL. This effect was much less pronounced in normal cells. The drug delivery mechanism involved a transfer of SCS to the cell membrane, independently of drug transfer and not involving nanoliposome internalization. MTO was detected intratumorally upon MTOL and SCS-MTOL treatment, but not after free MTO, suggesting an important improvement in tumor drug delivery by nanoliposomal formulation. Nanoliposomal MTO delivery and cellular uptake was heterogeneous throughout the tumor and clearly correlated with CD31-positive tumor vessels. Yet, MTO uptake by CD11b positive cells in tumor stroma was minor. CONCLUSIONS: Nanoliposomal encapsulation improves intratumoral MTO delivery over free drug. Liposome bilayer-incorporated SCS preferentially permeabilize tumor cell membranes enhancing intracellular MTO delivery.

Enhanced Specificity and Drug Delivery in Tumors by cRGD-Anchoring Thermosensitive Liposomes.

PURPOSE: To develop RGD-targeted thermosensitive liposomes with increased tumor retention, improving drug release efficiency upon mild hyperthermia (HT) in both tumor and angiogenic endothelial cells. METHODS: Standard termosensitive liposomes (TSL) and TSL containing a cyclic Arg-Gly-Asp (cRGD) pentapeptide with the sequence Arg-Cys-D-Phe-Asp-Gly (RGDf[N-Met]C) were synthetized, loaded with Dox and characterized. Temperature- and time-dependent drug release profiles were assessed by fluorometry. Intracellular Dox delivery was studied by flow cytometry and confocal microscopy. Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC. Intravital microscopy was performed on B16Bl6 tumors implanted in dorsal-skin fold window-bearing mice. Pharmacokinetic and biodistribution of Dox-TSL and Dox-RGD-TSL were followed in B16Bl6 tumor bearing mice upon normothermia or initial hyperthermia conditions. RESULTS: DLS and cryo-TEM revealed particle homogeneity and size of around 85 nm. Doxorubicin loading efficiency was >95%as assessed by spectrofluorometry. Flow cytometry and confocal microscopy showed a specific uptake of RGD-TSL by melanoma and endothelial cells when compared to TSL and an increased doxorubicin delivery. High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature. Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL. Biodistribution studies showed that initial hyperthermia increases Dox uptake in tumors from TSL and RGD-TSL. CONCLUSION: RGD-TSL have potency to increase drug efficacy due to higher uptake by tumor and angiogenic endothelial cells in combination with heat-triggered drug release.

Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration.

The angiogenic potential of solid tumors, or the ability to initiate neovasculature development from pre-existing host vessels, is facilitated by soluble factors secreted by tumor cells and involves breaching of extracellular matrix barriers, endothelial cell (EC) proliferation, migration and reassembly. We evaluated the angiogenic potential of human melanoma cell lines differing in their degree of aggressiveness, based on their ability to regulate directionally persistent EC migration. We observed that conditioned-medium (CM) of the aggressive melanoma cell line BLM induced a high effective migratory response in ECs, while CMs of Mel57 and 1F6 had an inhibitory effect. Further, the melanoma cell lines exhibited a varied expression profile of tissue inhibitor of metalloproteinase-3 (TIMP3), detectable in the CM. TIMP3 expression inversely correlated with aggressiveness of the melanoma cell line, and ability of the respective CMs to induce directed EC migration. Interestingly, TIMP3 expression was found to be silenced in the BLM cell line, concurrent with its role as a tumor suppressor. Treatment with recombinant human TIMP3 and CM of modified, TIMP3 expressing, BLM cells mitigated directional EC migration, while CM of TIMP3 silenced 1F6 cells induced directed EC migration. The functional implication of TIMP3 expression on tumor growth and angiogenic potential in melanoma was evaluated in vivo. We observed that TIMP3 expression reduced tumor growth, angiogenesis and macrophage infiltration of BLM tumors while silencing TIMP3 increased tumor growth and angiogenesis of 1F6 tumors. Taken together, our results demonstrate that TIMP3 expression correlates with inhibition of directionally persistent EC migration and adversely affects the angiogenic potential and growth of melanomas.

Cationic thermosensitive liposomes: a novel dual targeted heat-triggered drug delivery approach for endothelial and tumor cells.

Developing selectively targeted and heat-responsive nanocarriers holds paramount promises in chemotherapy. We show that this can be achieved by designing liposomes combining cationic charged and thermosensitive lipids in the bilayer. We demonstrated, using flow cytometry, live cell imaging, and intravital optical imaging, that cationic thermosensitive liposomes specifically target angiogenic endothelial and tumor cells. Application of mild hyperthermia led to a rapid content release extra- and intracellularly in two crucial cell types in a solid tumor.

Improving intracellular doxorubicin delivery through nanoliposomes equipped with selective tumor cell membrane permeabilizing short-chain sphingolipids.

PURPOSE: To improve nanoliposomal-doxorubicin (DoxNL) delivery in tumor cells using liposome membrane-incorporated short-chain sphingolipids (SCS) with selective membrane-permeabilizing properties. DoxNL bilayers contained synthetic short-chain derivatives of known membrane microdomain-forming sphingolipids; C8-glucosylceramide (C8-GluCer), C8-galactosylceramide (C8-GalCer) or C8-lactosylceramide (C8-LacCer). METHODS: DoxNL enriched with C8-GluCer or C8-GalCer were developed, optimized and characterized with regard to size, stability and drug retention. In vitro cytotoxic activity was studied in a panel of human tumor cell lines and normal cells. Intracellular Dox delivery was measured by flow cytometry and visualized by fluorescence microscopy. For a further understanding of the involved drug delivery mechanism confocal microscopy studies addressed the cellular fate of the nanoliposomes, the SCS and Dox in living cells. RESULTS: C8-LacCer-DoxNL aggregated upon Dox loading. In tumor cell lines SCS-DoxNL with C8-GluCer or C8-GalCer demonstrated strongly increased Dox delivery and cytotoxicity compared to standard DoxNL. Surprisingly, this effect was much less pronounced in normal cells. Nanoliposomes were not internalized, SCS however transfered from the nanoliposomal bilayer to the cell membrane and preceded cellular uptake and subsequent nuclear localization of Dox. CONCLUSION: C8-GluCer or C8-GalCer incorporated in DoxNL selectively improved intracellular drug delivery upon transfer to tumor cell membranes by local enhancement of cell membrane permeability.

The 'nanobig rod' class of gold nanorods: optimized dimensions for improved in vivo therapeutic and imaging efficacy.

Currently, gold nanorods can be synthesized in a wide range of sizes. However, for the intended biological applications gold nanorods with approximate dimensions 50 nm × 15 nm are used. We investigate by computer simulation the effect of particle dimensions on the optical and thermal properties in the context of the specific applications of photoacoustic imaging. In addition we discuss the influence of particle size in overcoming the following biophysical barriers when administrated in vivo: extravasation, avoidance of uptake by organs of the reticuloendothelial system, penetration through the interstitium, binding capability and uptake by the target cells. Although more complex biological influences can be introduced in future analysis, the present work illustrates that larger gold nanorods, designated by us as 'nanobig rods', may perform better at meeting the requirements for successful in vivo applications compared to their smaller counterparts, which are conventionally used.

Multimodality imaging of somatostatin receptor-positive tumors with nuclear and bioluminescence imaging.

Multimodal bioluminescence (BLI) and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging were investigated as means to monitor somatostatin receptor subtype 2 (SST2)-positive neuroendocrine tumors as both a subcutaneously implanted and a liver metastasis animal model in mice and rats. Ultimately, such a model will be of use for studying SST2-targeted peptide receptor radionuclide therapy (PRRT). CA20948 cells were transfected with a green fluorescent protein/luciferase plasmid construct. Cells were inoculated subcutaneously in the shoulder of nude mice: nontransfected cells in the left shoulder and transfected cells in the right shoulder. BLI, SPECT/CT imaging, biodistribution analysis, and ex vivo autoradiography of the tumors were performed. BLI and SPECT/CT imaging were also performed on an intrahepatic tumor model in the rat. Caliper volume measurement of transfected tumors could be correlated with BLI measurements (R2 = .76). SPECT/CT imaging showed high levels of accumulation of 111In-DTPA-octreotide in control and transfected tumors, which was confirmed by biodistribution analysis and autoradiography. Subcapsular inoculation of transfected cells in rat liver resulted in an intrahepatic tumor, which could be visualized by both SPECT/CT and BLI. Transfection of CA20948 tumor cells did not alter the growth properties of the cell line or the expression of SST2. Transfected tumors could be clearly visualized by BLI and SPECT/CT imaging. The transfected SST2-positive tumor cell line could represent a novel preclinical model for tumor monitoring in studies that aim at further optimizing PRRT for neuroendocrine tumors.

Enriching lipid nanovesicles with short-chain glucosylceramide improves doxorubicin delivery and efficacy in solid tumors.

For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.

Development of a liposomal delivery system for temperature-triggered release of a tumor targeting agent, Ln(III)-DOTA-phenylboronate.

Liposomes, capable of temperature-triggered content release at the site of interest, can be of great importance for imaging and therapy of tumors. The delivery of imaging agents or therapeutics can be improved by application of liposomes with a gel-to-liquid phase-transition temperature suitable for mild hyperthermia (41-43°C), and by prolonging their circulation time by incorporation of lipids containing polyethyleneglycol moieties. Still, the rapid wash out of the delivered material from the tumor tissue is a major obstacle for both imaging and therapy. In this study, we developed an optimized temperature sensitive liposomal system to be used with mild hyperthermia: highly stable at physiological temperature and with a sharp transition of the bilayer at 41.5°C, with subsequent rapid release of entrapped compounds such as calcein or tumor cell-targeting contrast agents. Intravital microscopy on calcein/rhodamine containing liposomes was applied to demonstrate the applicability of this system in vivo. The calcein loaded liposomes were injected iv into nude mice with a human BLM melanoma tumor implanted in a dorsal skin-fold window chamber. Arrival of the liposomes at the tumor site and content release after temperature increase were monitored. The results demonstrated not only accumulation of the liposomes at the tumor site, but also a massive release of calcein after increase of the temperature to 41°C. The versatility of the thermosensitive liposomes was further demonstrated by encapsulation of a tumor cell-targeting DOTA-phenylboronate conjugate and its release at elevated temperatures. The DOTA ligand in this system is able to chelate a variety of metals suitable for both diagnostic and therapeutic applications, whereas the phenylboronate function is able to target specifically to tumor cells through a covalent binding with sialic acid moieties over-expressed on their surface upon heat-triggered release from the liposomal carrier.

Hyperthermia and thermosensitive liposomes for improved delivery of chemotherapeutic drugs to solid tumors.

Lipid-based nanocarriers or liposomes have been proven successful in the delivery of chemotherapeutic agents and are currently applied clinically in the treatment of various types of cancer. Liposomes offer the advantage of a high drug payload, decreased drug toxicity and enhanced drug accumulation at tumor sites. Increased accumulation is due to the relatively leaky tumor vasculature that allows liposome extravasation. Between different types of tumors and even within one tumor, vascular permeability and thus liposome extravasation may differ greatly. Furthermore, upon accumulation of liposomes in the tumor area, drug bioavailability is not guaranteed. At present, these are the major issues for clinically used liposomal drugs.Mild hyperthermia (HT), the heating of tumor tissue to temperatures of up to 43 degrees C, has been developed in the past decades as an established and efficacious treatment modality in combination with chemo- and radiotherapy. HT can be used to further improve liposomal chemotherapy in two ways: HT is known to increase vascular permeability in solid tumors and may therefore increase levels of liposome accumulation, and thermosensitive liposomes have been developed that can be triggered to release their contents upon hyperthermia. By applying these two strategies, drug delivery to tumors can be strongly enhanced.

Early in vivo assessment of angiostatic therapy efficacy by molecular MRI.

Noninvasive diagnostic imaging methods to establish the efficacy of angiostatic therapies are becoming increasingly important with the first Food and Drug Administration approvals of such agents. Magnetic resonance molecular imaging is an imaging technique that allows the visualization of pathological processes in vivo with a better spatial resolution as compared with nuclear methods, such as photon emission tomography and single photon emission computed tomography. In this study, we used alpha(v)beta3 targeted bimodal liposomes to quantitate angiogenesis in a tumor mouse model with magnetic resonance imaging (MRI) and to evaluate the therapeutic efficacy of the angiogenesis inhibitors anginex and endostatin. The MRI findings were validated with fluorescence microscopy and showed a very good correlation with the microvessel density. In conclusion, this study provides evidence that molecular MRI can be used to noninvasively measure the efficacy of angiogenesis inhibitors during the course of therapy.

Development and evaluation of an isolated limb infusion model for investigation of drug delivery kinetics to solid tumors by thermosensitive liposomes and hyperthermia.

The combined administration of thermosensitive liposomes (TSLs) and hyperthermia (HT) has been increasingly shown to be a powerful tool for the treatment of solid tumors. At present, it is hypothesized that the circulation of TSLs through the vasculature of a heated tumor results in the rapid release of the entrapped drug, followed by its uptake and distribution within the tumor microenvironment. However, simple questions on the transport kinetics of TSLs through the heated tumor and how much drug is retained upon passage of TSLs through the tumor microcirculation have not been investigated in an experimental setting to-date. The present work describes a novel methodology for investigating these parameters by isolated limb infusion (ILI), developed in a rat model of sarcoma. This approach was used to assess the efficacy of Doxorubicin (Dox) delivery by TSL in a heated (42°C) tumor following a single passage of TSL through the tumor vasculature. Analysis of the effluent post-ILI, whole-tumor histological sections, and tissue homogenates revealed that upon a single passage, Dox delivery by TSL at 42°C did not exceed delivery under conventional (i.e. free Dox) or physiological (i.e. TSL at 37°C, or normothermia; NT) conditions. In fact, mathematical modeling demonstrated that at least thirteen passages are required to obtain the intratumoral Dox levels typically achieved using TSL (i.e. ~5%ID/g). Overall, this work investigates TSL-based determinants for achieving efficacious drug delivery using a model of ILI in tumor-bearing rats and the results bear important implications for TSL disposition in vivo.

Comparing the therapeutic potential of thermosensitive liposomes and hyperthermia in two distinct subtypes of breast cancer.

Local drug delivery of Doxorubicin (Dox) with thermosensitive liposomes (TSL) and hyperthermia (HT) has shown preclinically to achieve high local drug concentrations with good therapeutic efficacy. Currently, this is clinically studied for treatment of chest wall recurrence of breast cancer, however with various outcomes. This study examines the potency of neoadjuvant TSL HT combination therapy in two orthotopic mouse models of human breast cancer, MDA-MB-231 and T-47D, which morphologically correlate to mesenchymal and epithelial phenotypes, respectively. Both cell lines showed improved in vitro chemosensitivity and Dox uptake at HT. Dox-loaded TSL (TSLDox) was stable in vitro in FBS, BALB/c-nu plasma and human plasma, although release of the drug at HT was incomplete for the latter two. Combination treatment with TSLDox and HT in vivo was significantly more effective against MDA-MB-231 tumors, whereas T-47D tumors showed no significant therapeutic response. Ex vivo investigation revealed a higher mean vessel density and poorly differentiated extracellular matrix (ECM) in MDA-MB-231 tumors relative to T-47D tumors. Although in vitro results of the TSLDox and HT treatment were favorable for both cell types, the therapeutic efficacy in vivo was remarkably different. The well-differentiated and slowly-growing T-47D tumors may provide a microenvironment that limits drug delivery to the target cell and therefore renders the therapy ineffective. Mesenchymal and invasive MDA-MB-231 tumors display higher vascularization and less mature ECM, significantly enhancing tumor response to TSLDox and HT treatment. These results yield insight into the efficacy of TSL treatment within different tumor microenvironments, and further advance our understanding of factors that contribute to heterogeneous therapeutic outcomes in clinical trials.

MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.

In oncological research, there is a great need for imaging techniques that specifically identify angiogenic blood vessels in tumors on the basis of differences in the expression level of biomolecular markers. In the angiogenic cascade, different cell surface receptors, including the alphavbeta3-integrin, are strongly expressed on activated endothelial cells. In the present study, we aimed to image angiogenesis by detecting the expression of alphavbeta3 in tumor bearing mice with a combination of magnetic resonance imaging (MRI) and fluorescence microscopy. To that end, we prepared MR-detectable and fluorescent liposomes, which carry approximately 700 alphavbeta3-specific RGD peptides per liposome. RGD competition experiments and RAD-conjugated liposomes were used as controls for specificity. In vivo, both RAD liposomes and RGD liposomes gave rise to signal increase on T1-weighted MR images. It was established by the use of ex vivo fluorescence microscopy that RGD liposomes and RAD liposomes accumulated in the tumor by different mechanisms. RGD liposomes were specifically associated with activated tumor endothelium, while RAD liposomes were located in the extravascular compartment. This study demonstrates that MR molecular imaging of angiogenesis is feasible by using a targeted contrast agent specific for the alphavbeta3-integrin, and that the multimodality imaging approach gave insight into the exact mechanism of accumulation in the tumor.

Radioprotective effect of transferrin targeted citicoline liposomes.

The high level of expression of transferrin receptors (Tf-R) on the surface of endothelial cells of the blood-brain-barrier (BBB) had been widely utilized to deliver drugs to the brain. The primary aim of this study was to use transferrin receptor mediated endocytosis as a pathway for the rational development of holo-transferrin coupled liposomes for drug targeting to the brain. Citicoline is a neuroprotective agent used clinically to treat for instance Parkinson disease, stroke, Alzheimer's disease and brain ischemia. Citicoline does not readily cross the BBB because of its strong polar nature. Hence, citicoline was used as a model drug. (Citicoline liposomes have been prepared using dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC) by dry lipid film hydration-extrusion method). The effect of the use of liposomes composed of DPPC or DSPC on their citicoline encapsulation efficiency and their stability in vitro were studied. Transferrin was coupled to liposomes by a technique which involves the prevention of scavenging diferric iron atoms of transferrin. The coupling efficiency of transferrin to the liposomes was studied. In vitro evaluation of transferrin-coupled liposomes was performed for their radioprotective effect in radiation treated cell cultures. In this study, OVCAR-3 cells were used as a model cell type over-expressing the Tf-R and human umbilical vein endothelial cells (HUVEC) as BBB endothelial cell model. The average diameter of DPPC and DSPC liposomes were 138 +/- 6.3 and 79.0 +/- 3.2 nm, respectively. The citicoline encapsulation capacity of DPPC and DSPC liposomes was 81.8 +/- 12.8 and 54.9 +/- 0.04 microg/micromol of phospholipid, respectively. Liposomes prepared from DSPC showed relatively better stability than DPPC liposomes at 37 degrees C and in the presence of serum. Hence, DSPC liposomes were used for transferrin coupling and an average of 46-55 molecules of transferrin were present per liposome. Free citicoline has shown radioprotective effect at higher doses tested. Interestingly, encapsulation of citicoline in pegylated liposomes significantly improved the radioprotective effect by 4-fold compared to free citicoline in OVCAR-3 but not in HUVEC. Further, citicoline encapsulation in transferrin-coupled liposomes has significantly improved the radioprotective effect by approximately 8-fold in OVCAR-3 and 2-fold in HUVEC cells with respect to the free drug. This is likely due to the entry of citicoline into cells via transferrin receptor mediated endocytosis. In conclusion, our results suggest that low concentrations of citicoline encapsulated in transferrin-coupled liposomes could offer therapeutic benefit in treating stroke compared to free citicoline.

Lanthanide-loaded liposomes for multimodality imaging and therapy.

UNLABELLED: Many advanced molecular imaging agents are currently being investigated preclinically. Especially, liposomes, have proven to be very promising carrier systems for diagnostic agents for use in single-photon emission computed tomography (SPECT) or magnetic resonance imaging (MRI), as well as for therapeutic agents to treat diseases such as cancer. In this study, nanosized liposomes were designed and labeled with the radionuclides, holmium-166 (both a beta- and gamma-emitter and also highly paramagnetic) or technetium-99m, and coloaded with paramagnetic gadolinium allowing multimodality SPECT and MR imaging and radionuclide therapy with one single agent. METHODS: Diethylenetriaminepentaacetic acid bisoctadecylamide (an amphiphilic molecule with a chelating group suitable for labeling with radionuclides) and gadoliniumacetylacetonate (GdAcAc) (a small lipophilic paramagnetic molecule) were incorporated in liposomes. The liposomes were characterized by measuring their mean size and size distribution, gadolinium content, and radiochemical stability after incubation in human serum at 37 degrees C. The MRI properties (in vitro) were determined by use of relaxivity measurements at 1.5 and 3.0 Tesla in order to evaluate their potency as imaging agents. RESULTS: The liposomes were successfully labeled with holmium-166, resulting in a high labeling efficiency (95% +/- 1%) and radiochemical stability (> 98% after 48 hours of incubation), and coloaded with GdAcAc. Labeling of liposomes with technetium-99m was somewhat less efficient (85% +/- 2%), although their radiochemical stability was sufficient (95% +/- 1% after 6 hours of incubation). MRI measurements showed that the incorporation of GdAcAc had a strong effect on the MRI relaxivity. CONCLUSIONS: The synthesized liposomes allow for multimodality imaging and therapy, which makes these new agents highly attractive for future applications.

EGFR targeted thermosensitive liposomes: A novel multifunctional platform for simultaneous tumor targeted and stimulus responsive drug delivery.

The epidermal growth factor receptor (EGFR) is a promising target for anti-cancer therapy. The aim of this study was to design thermosensitive liposomes (TSL), functionalized with anti-EGFR ligands for targeted delivery and localized triggered release of chemotherapy. For targeting, EGFR specific peptide (GE11) and Fab' fragments of cetuximab were used and the effect of ligand density on in vitro tumor targeting was investigated. Ligand conjugation did not significantly change the physicochemical characteristics of liposomes. Fab'-decorated TSL (Fab'-TSL) can specifically and more efficiently bind to the EGFR overexpressed cancer cells as compared to GE11 modified TSL. Calcein labeled Fab'-TSL showed adequate stability at 37°C in serum (<4% calcein released after 1h) and a temperature dependent release at above 40°C. FACS analysis and live cell imaging showed efficient and EGFR mediated cellular association as well as dramatic intracellular cargo release upon hyperthermia. Fab'-conjugation and hyperthermia induced enhanced tumor cell cytotoxicity of doxorubicin loaded TSL. The relative cytotoxicity of Fab'-TSL was also correlated to EGFR density on the tumor cells. These results suggest that Fab'-TSL showed great potential for combinational targeted and triggered release drug delivery.

A Novel Combined Approach of Short-Chain Sphingolipids and Thermosensitive Liposomes for Improved Drug Delivery to Tumor Cells.

Despite the advantages of liposomal drug delivery, the bioavailability of the chemotherapeutic drugs to tumor cells is limited by their slow release from nanocarriers and low drug permeability across cell membranes. Drug encapsulation into stealth thermosensitive liposomes can improve drug delivery to tumors by combining efficient accumulation at tumors and the active release of the payload following remote heat triggering. Short-chain sphingolipids are known to enhance cellular uptake of amphiphilic drugs. We hypothesized that short-chain sphingolipids could be utilized to further improve intracellular drug delivery from a thermoresponsive formulation by enhancing the cell membrane passage of released drug. The following two strategies were investigated: (1) co-delivery of C8-glucosylceramide and doxorubicin within the thermosensitive liposomes and (2) pretreatment with glucosylceramide-enriched drug-free liposomes and subsequent treatment with doxorubicin loaded thermosensitive liposomes. Liposomes were prepared and extensively characterized. Drug uptake, cell cytotoxicity and live cell imaging were performed under normothermic and hyperthermic conditions in melanoma cells. In these studies, hyperthermia improved drug delivery from doxorubicin loaded thermosensitive formulations. However, the results from cell experiments indicated that there was no additional benefit in the co-delivery strategy using doxorubicin loaded glucosylceramide-enriched thermosensitive liposomes. In contrast, cellular studies showed significantly higher doxorubicin internalization in the pretreatment strategy. One-hour exposure of the cells to C8-glucosylceramide before applying hyperthermia caused improved doxorubicin uptake and cytotoxicity as well as an almost instantaneous cellular entry of the doxorubicin released from thermosensitive liposomes. This novel, two-step drug delivery approach can be potentially beneficial for the intracellular delivery of cell impermeable chemotherapeutics.