Liposomal delivery of dexamethasone attenuates prostate cancer bone metastatic tumor growth in vivo.

BACKGROUND: The inflammatory tumor microenvironment, and more specifically the tumor-associated macrophages, plays an essential role in the development and progression of prostate cancer towards metastatic bone disease. Tumors are often characterized by a leaky vasculature, which - combined with the prolonged circulation kinetics of liposomes - leads to efficient tumor localization of these drug carriers, via the so-called enhanced permeability and retention (EPR) -effect. In this study, we evaluated the utility of targeted, liposomal drug delivery of the glucocorticoid dexamethasone in a model of prostate cancer bone metastases. METHODS: Tumor-bearing Balb-c nu/nu mice were treated intravenously with 0.2-1.0-5.0 mg/kg/week free- and liposomal DEX for 3-4 weeks and tumor growth was monitored by bioluminescent imaging. RESULTS: Intravenously administered liposomes localize efficiently to bone metastases in vivo and treatment of established bone metastases with (liposomal) dexamethasone resulted in a significant inhibition of tumor growth up to 26 days after initiation of treatment. Furthermore, 1.0 mg/kg liposomal dexamethasone significantly outperformed 1.0 mg/kg free dexamethasone, and was found to be well-tolerated at clinically-relevant dosages that display potent anti-tumor efficacy. CONCLUSIONS: Liposomal delivery of the glucocorticoid dexamethasone inhibits the growth of malignant bone lesions. We believe that liposomal encapsulation of dexamethasone offers a promising new treatment option for advanced, metastatic prostate cancer which supports further clinical evaluation.

Liposomal encapsulation of dexamethasone modulates cytotoxicity, inflammatory cytokine response, and migratory properties of primary human macrophages.

The encapsulation of drugs into liposomes aims to enhance their efficacy and reduce their toxicity. Corticosteroid-loaded liposomes are currently being evaluated in patients suffering from rheumatoid arthritis, atherosclerosis, colitis, and cancer. Here, using several different fluorophore-labeled formulations, we comprehensively studied the impact of liposome encapsulation of the prototypic corticosteroid dexamethasone on various primary human cells in vitro. Liposomal dexamethasone targeted several primary cell types in a dose and time-dependent manner, but specifically reduced cytotoxicity against human fibroblasts and macrophages in comparison to the solute drug. Furthermore, macrophage maturation and polarization markers were altered. Interestingly, liposomal dexamethasone induced proinflammatory cytokine secretion (specifically TNF, IL1ß, IL6) in unstimulated cells, but reduced this response under inflammatory conditions. Monocyte and macrophage migration was significantly inhibited by dexamethasone-loaded liposomes. The findings indicate that the encapsulation of dexamethasone into liposomes modulates their cellular mechanism of action, and provides important indications for follow-up in vivo investigations. FROM THE CLINICAL EDITOR: This study investigates mechanism of action of liposomal dexamethason in the treatment of inflammatory conditions. It is concluded that liposomal dexamethasone actually induces proinflammatory cytokine secretion in unstimulated cells, but reduces the same response under inflammatory conditions. Monocyte and macrophage migration was also inhibited. The findings indicate that liposomal dexamethasone may have different mechanisms of action than its native counterpart.

RSPO3 Furin domain-conjugated liposomes for selective drug delivery to LGR5-high cells.

The transmembrane receptor LGR5 potentiates Wnt/ß-catenin signaling by binding both secreted R-spondin (RSPOs) and the Wnt tumor suppressors RNF43/ZNRF3, directing clearance of RNF43/ZNRF3 from the cell surface. Besides being widely used as a stem cell marker in various tissues, LGR5 is overexpressed in many types of malignancies, including colorectal cancer. Its expression characterizes a subpopulation of cancer cells that play a crucial role in tumor initiation, progression and cancer relapse, known as cancer stem cells (CSCs). For this reason, ongoing efforts are aimed at eradicating LGR5-positive CSCs. Here, we engineered liposomes decorated with different RSPO proteins to specifically detect and target LGR5-positive cells. Using fluorescence-loaded liposomes, we show that conjugation of full-length RSPO1 to the liposomal surface mediates aspecific, LGR5-independent cellular uptake, largely mediated by heparan sulfate proteoglycan binding. By contrast, liposomes decorated only with the Furin (FuFu) domains of RSPO3 are taken up by cells in a highly specific, LGR5-dependent manner. Moreover, encapsulating doxorubicin in FuFuRSPO3 liposomes allowed us to selectively inhibit the growth of LGR5-high cells. Thus, FuFuRSPO3-coated liposomes allow for the selective detection and ablation of LGR5-high cells, providing a potential drug delivery system for LGR5-targeted anti-cancer strategies.

Injectable liposomal docosahexaenoic acid alleviates atherosclerosis progression and enhances plaque stability.

Atherosclerosis is a chronic inflammatory vascular disease that is characterized by the accumulation of lipids and immune cells in plaques built up inside artery walls. Docosahexaenoic acid (DHA, 22:6n-3), an omega-3 polyunsaturated fatty acid (PUFA), which exerts anti-inflammatory and antioxidant properties, has long been purported to be of therapeutic benefit to atherosclerosis patients. However, large clinical trials have yielded inconsistent data, likely due to variations in the formulation, dosage, and bioavailability of DHA following oral intake. To fully exploit its potential therapeutic effects, we have developed an injectable liposomal DHA formulation intended for intravenous administration as a plaque-targeted nanomedicine. The liposomal formulation protects DHA against chemical degradation and increases its local concentration within atherosclerotic lesions. Mechanistically, DHA liposomes are readily phagocytosed by activated macrophages, exert potent anti-inflammatory and antioxidant effects, and inhibit foam cell formation. Upon intravenous administration, DHA liposomes accumulate preferentially in atherosclerotic lesional macrophages and promote polarization of macrophages towards an anti-inflammatory M2 phenotype, resulting in attenuation of atherosclerosis progression in both ApoE-/- and Ldlr-/- experimental models. Plaque composition analysis demonstrates that liposomal DHA inhibits macrophage infiltration, reduces lipid deposition, and increases collagen content, thus improving the stability of atherosclerotic plaques against rupture. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) further reveals that DHA liposomes can partly restore the complex lipid profile of the plaques to that of early-stage plaques. In conclusion, DHA liposomes offer a promising approach for applying DHA to stabilize atherosclerotic plaques and attenuate atherosclerosis progression, thereby preventing atherosclerosis-related cardiovascular events.

Nanoclusters of iron oxide: effect of core composition on structure, biocompatibility, and cell labeling efficacy.

Inorganic nanocrystals have a variety of applications in medicine. They may serve as contrast agents, therapeutics, and for in vitro diagnostics. Frequently, the synthesis route yields hydrophobically capped nanocrystals, which necessitates their subsequent coating to render a water-soluble and biocompatible probe. Biocompatibility is crucial for cellular imaging applications, which require large quantities of diagnostically active nanoparticles to be loaded into cells. We have previously reported the design and synthesis of a fluorescent and magnetic resonance imaging-detectable core-shell nanoparticle that encapsulates hydrophobically coated iron oxide nanocrystals. The core of soybean oil and iron oxide is covered by a shell mixture of phospholipids, some of which contained polyethylene glycol. Despite the biocompatibility of these components, we hypothesize that we can improve this formulation with respect to in vitro toxicity. To this aim, we measured the effect of six different core compositions on nanoparticle structure, cell labeling efficacy, and cell viability, as well as cell tracking potential. We methodically investigated the causes of toxicity and conclude that, even when combining biocompatible materials, the resulting formulation is not guaranteed to be biocompatible.

Betulinic acid delivered in liposomes reduces growth of human lung and colon cancers in mice without causing systemic toxicity.

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid with potent anticancer capacity that targets the mitochondrial pathway of apoptosis. BetA has a broad efficacy in vitro against prevalent cancer types, including lung, colorectal, prostate, cervix and breast cancer, melanomas, neuroblastomas, and leukemias. The cytotoxic effects of the compound against healthy cells are minimal, rendering BetA a promising potential anticancer drug. However, because of the weak hydrosolubility of BetA, it has been difficult to study its efficacy in vivo and a pharmaceutical formulation is not yet available. We report the development of a liposome formulation of BetA and show its successful application in mice. Large liposomes, assembled without cholesterol to reduce their rigidity, efficiently incorporated BetA. Nude mice xenografted with human colon and lung cancer tumors were treated intravenously with the BetA-containing liposomes. Tumor growth was reduced to more than 50% compared with the control treatment, leading to an enhanced survival of the mice. Oral administration of the liposomal formulation of BetA also slowed tumor growth. Any signs of systemic toxicity caused by BetA treatment were absent. Thus, liposomes are an efficient formulation vehicle for BetA, enabling its preclinical development as a nontoxic compound for the treatment of cancers.

Photodynamic Therapy Targeting Macrophages Using IRDye700DX-Liposomes Decreases Experimental Arthritis Development.

Macrophages play a crucial role in the initiation and progression of rheumatoid arthritis (RA). Liposomes can be used to deliver therapeutics to macrophages by exploiting their phagocytic ability. However, since macrophages serve as the immune system's first responders, it is inadvisable to systemically deplete these cells. By loading the liposomes with the photosensitizer IRDye700DX, we have developed and tested a novel way to perform photodynamic therapy (PDT) on macrophages in inflamed joints. PEGylated liposomes were created using the film method and post-inserted with micelles containing IRDye700DX. For radiolabeling, a chelator was also incorporated. RAW 264.7 cells were incubated with liposomes with or without IRDye700DX and exposed to 689 nm light. Viability was determined using CellTiterGlo. Subsequently, biodistribution and PDT studies were performed on mice with collagen-induced arthritis (CIA). PDT using IRDye700DX-loaded liposomes efficiently induced cell death in vitro, whilst no cell death was observed using the control liposomes. Biodistribution of the two compounds in CIA mice was comparable with excellent correlation of the uptake with macroscopic and microscopic arthritis scores. Treatment with 700DX-loaded liposomes significantly delayed arthritis development. Here we have shown the proof-of-principle of performing PDT in arthritic joints using IRDye700DX-loaded liposomes, allowing locoregional treatment of arthritis.

Multimodal clinical imaging to longitudinally assess a nanomedical anti-inflammatory treatment in experimental atherosclerosis.

Atherosclerosis is an inflammatory disease causing great morbidity and mortality in the Western world. To increase the anti-inflammatory action and decrease adverse effects of glucocorticoids (PLP), a nanomedicinal liposomal formulation of this drug (L-PLP) was developed and intravenously applied at a dose of 15 mg/kg PLP to a rabbit model of atherosclerosis. Since atherosclerosis is a systemic disease, emerging imaging modalities for assessing atherosclerotic plaque are being developed. (18)F-Fluoro-deoxy-glucose positron emission tomography and dynamic contrast enhanced magnetic resonance imaging, methods commonly used in oncology, were applied to longitudinally assess therapeutic efficacy. Significant anti-inflammatory effects were observed as early as 2 days that lasted up to at least 7 days after administration of a single dose of L-PLP. No significant changes were found for the free PLP treated animals. These findings were corroborated by immunohistochemical analysis of macrophage density in the vessel wall. In conclusion, this study evaluates a powerful two-pronged strategy for efficient treatment of atherosclerosis that includes nanomedical therapy of atherosclerotic plaques and the application of noninvasive and clinically approved imaging techniques to monitor delivery and therapeutic responses. Importantly, we demonstrate unprecedented rapid anti-inflammatory effects in atherosclerotic lesions after the nanomedical therapy.

Multimodal and multiscale optical imaging of nanomedicine delivery across the blood-brain barrier upon sonopermeation.

Rationale: The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. Sonopermeation, which relies on the combination of ultrasound and microbubbles, has emerged as a powerful tool to permeate the BBB, enabling the extravasation of drugs and drug delivery systems (DDS) to and into the central nervous system (CNS). When aiming to improve the treatment of high medical need brain disorders, it is important to systematically study nanomedicine translocation across the sonopermeated BBB. To this end, we here employed multimodal and multiscale optical imaging to investigate the impact of DDS size on brain accumulation, extravasation and penetration upon sonopermeation. Methods: Two prototypic DDS, i.e. 10 nm-sized pHPMA polymers and 100 nm-sized PEGylated liposomes, were labeled with fluorophores and intravenously injected in healthy CD-1 nude mice. Upon sonopermeation, computed tomography-fluorescence molecular tomography, fluorescence reflectance imaging, fluorescence microscopy, confocal microscopy and stimulated emission depletion nanoscopy were used to study the effect of DDS size on their translocation across the BBB. Results: Sonopermeation treatment enabled safe and efficient opening of the BBB, which was confirmed by staining extravasated endogenous IgG. No micro-hemorrhages, edema and necrosis were detected in H&E stainings. Multimodal and multiscale optical imaging showed that sonopermeation promoted the accumulation of nanocarriers in mouse brains, and that 10 nm-sized polymeric DDS accumulated more strongly and penetrated deeper into the brain than 100 nm-sized liposomes. Conclusions: BBB opening via sonopermeation enables safe and efficient delivery of nanomedicine formulations to and into the brain. When looking at accumulation and penetration (and when neglecting issues such as drug loading capacity and therapeutic efficacy) smaller-sized DDS are found to be more suitable for drug delivery across the BBB than larger-sized DDS. These findings are valuable for better understanding and further developing nanomedicine-based strategies for the treatment of CNS disorders.

Liver fibrosis affects the targeting properties of drug delivery systems to macrophage subsets in vivo.

Myeloid immune cells promote inflammation and fibrosis in chronic liver diseases. Drug delivery systems, such as polymers, liposomes and microbubbles, efficiently target myeloid cells in healthy liver, but their targeting properties in hepatic fibrosis remain elusive. We therefore studied the biodistribution of three intravenously injected carrier material, i.e. 10 nm poly(N-(2-hydroxypropyl)methacrylamide) polymers, 100 nm PEGylated liposomes and 2000 nm poly(butyl cyanoacrylate) microbubbles, in two fibrosis models in immunocompetent mice. While whole-body imaging confirmed preferential hepatic uptake even after induction of liver fibrosis, flow cytometry and immunofluorescence analysis revealed markedly decreased carrier uptake by liver macrophage subsets in fibrosis, particularly for microbubbles and polymers. Importantly, carrier uptake co-localized with immune infiltrates in fibrotic livers, corroborating the intrinsic ability of the carriers to target myeloid cells in areas of inflammation. Of the tested carrier systems liposomes had the highest uptake efficiency among hepatic myeloid cells, but the lowest specificity for cellular subsets. Hepatic fibrosis affected carrier uptake in liver and partially in spleen, but not in other tissues (blood, bone marrow, lung, kidney). In conclusion, while drug carrier systems target distinct myeloid cell populations in diseased and healthy livers, hepatic fibrosis profoundly affects their targeting efficiency, supporting the need to adapt nanomedicine-based approaches in chronic liver disease.

Liposomal dexamethasone inhibits tumor growth in an advanced human-mouse hybrid model of multiple myeloma.

Glucocorticoids are the cornerstone in the clinic for treatment of hematological malignancies, including multiple myeloma. Nevertheless, poor pharmacokinetic properties of glucocorticoids require high and frequent dosing with the off-target adverse effects defining the maximum dose. Recently, nanomedicine formulations of glucocorticoids have been developed that improve the pharmacokinetic profile, limit adverse effects and improve solid tumor accumulation. Multiple myeloma is a hematological malignancy characterized by uncontrolled growth of plasma cells. These tumors initiate increased angiogenesis and microvessel density in the bone marrow, which might be exploited using nanomedicines, such as liposomes. Nano-sized particles can accumulate as a result of the increased vascular leakiness at the bone marrow tumor lesions. Pre-clinical screening of novel anti-myeloma therapeutics in vivo requires a suitable animal model that represents key features of the disease. In this study, we show that fluorescently labeled long circulating liposomes were found in plasma up to 24 h after injection in an advanced human-mouse hybrid model of multiple myeloma. Besides the organs involved in clearance, liposomes were also found to accumulate in tumor bearing human-bone scaffolds. The therapeutic efficacy of liposomal dexamethasone phosphate was evaluated in this model showing strong tumor growth inhibition while free drug being ineffective at an equivalent dose (4 mg/kg) regimen. The liposomal formulation slightly reduced total body weight of myeloma-bearing mice during the course of treatment, which appeared reversible when treatment was stopped. Liposomal dexamethasone could be further developed as monotherapy or could fit in with existing therapy regimens to improve therapeutic outcomes for multiple myeloma.

Human regulatory T cells control xenogeneic graft-versus-host disease induced by autologous T cells in RAG2-/-gammac-/- immunodeficient mice.

PURPOSE: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4+CD25+ regulatory T cells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell-dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2-/-gammac-/- mice by i.v. administration of human peripheral blood mononuclear cells (PBMC). EXPERIMENTAL DESIGN: Human PBMC, depleted of or supplemented with autologous CD25+ Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of human T cells, and secretion of human cytokines were monitored at weekly intervals. RESULTS: Depletion of CD25+ cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25+ cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4+ and CD8+ T cells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-gamma, suggesting the de novo development of TR1 cells. CONCLUSIONS: These results show, for the first time, the potent in vivo capacity of naturally occurring human Tregs to control GvHD-inducing autologous T cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring human Tregs.

Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells.

Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein.

Sonoporation enhances liposome accumulation and penetration in tumors with low EPR.

The Enhanced Permeability and Retention (EPR) effect is a highly variable phenomenon. To enhance EPR-mediated passive drug targeting to tumors, several different pharmacological and physical strategies have been evaluated over the years, including e.g. TNFα-treatment, vascular normalization, hyperthermia and radiotherapy. Here, we systematically investigated the impact of sonoporation, i.e. the combination of ultrasound (US) and microbubbles (MB), on the tumor accumulation and penetration of liposomes. Two different MB formulations were employed, and their ability to enhance liposome accumulation and penetration was evaluated in two different tumor models, which are both characterized by relatively low levels of EPR (i.e. highly cellular A431 epidermoid xenografts and highly stromal BxPC-3 pancreatic carcinoma xenografts). The liposomes were labeled with two different fluorophores, enabling in vivo computed tomography/fluorescence molecular tomography (CT-FMT) and ex vivo two-photon laser scanning microscopy (TPLSM). In both models, in spite of relatively high inter- and intra-individual variability, a trend towards improved liposome accumulation and penetration was observed. In treated tumors, liposome concentrations were up to twice as high as in untreated tumors, and sonoporation enhanced the ability of liposomes to extravasate out of the blood vessels into the tumor interstitium. These findings indicate that sonoporation may be a useful strategy for improving drug targeting to tumors with low EPR.

Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.

Enhanced engraftment of human cells in RAG2/gammac double-knockout mice after treatment with CL2MDP liposomes.

OBJECTIVE: The ability of human cells to repopulate the bone marrow of nonobese diabetic immunodeficient mice (NOD/SCID) is commonly used as a standard assay to quantify the primitive human hematopoietic stem cell population. We studied the applicability of the immunodeficient RAG2(-/-)gammac(-/-) double-knockout mouse for this purpose. METHODS: RAG2(-/-)gammac(-/-) mice and NOD/SCID mice were injected intravenously (i.v.) with umbilical cord blood-derived CD34(+) cells and engraftment was quantified by determining the human CD45+ cell chimerism in bone marrow at several time points. RAG2(-/-)gammac(-/-) were pretreated with total-body irradiation and depleted of macrophages in liver, spleen, and bone marrow by i.v. injection of clodronate diphosphonate containing liposomes. RESULTS: We demonstrated that the frequency of chimerism and the level of engraftment in macrophage-depleted RAG2(-/-)gammac(-/-) largely resemble that in NOD/SCID mice. Also similar is the multilineage differentiation pattern in the two mouse strains at 7 weeks after transplantation, with a prominent outgrowth in RAG2(-/-)gammac(-/-) of CD19+ cells (88% +/- 10%). Cells of other lineages were clearly less frequent: 9% +/- 2% myeloid cells and 0.1% +/- 0.1% erythroid cells. As for immature progenitors, 6% +/- 1% of the human cells express the CD34 antigen and 0.4% +/- 0.1% have the CD34+,CD33,38,71(-) phenotype. The presence of human committed progenitors (i.e., CFU-GM/BFU-E) was evident. The persistence of human cells at 4 months after transplantation shows that the RAG2(-/-)gammac(-/-) support long-term maintenance of human hematopoiesis. CONCLUSION: Our findings indicate that macrophage-depleted RAG2(-/-)gammac(-/-) are a suitable model for studying human hematopoiesis including multipotential stem cells, and long-term repopulation.

Distribution of technetium-99m PEG-liposomes during oligofructose-induced laminitis development in horses.

Liposomes are phospholipid nanoparticles used for targeted drug delivery. This study aimed to determine whether intravenous liposomes accumulate in lamellar tissue during laminitis development in horses so as to assess their potential for targeted lamellar drug delivery. Polyethylene-glycol (PEG) coated liposomes were prepared according to the film hydration method and labelled using (99m)Tc-hexamethyl-propylene-amine-oxime. Six horses received 10 g/kg oligofructose via nasogastric tube to induce laminitis, and four control horses received water via nasogastric tube. All horses received 300 µmol (99m)Tc-PEG-liposomes (5.5 GBq) plus 5.5 µmol/kg PEG-liposomes by slow intravenous infusion. Scintigraphic imaging was performed at 0, 6 and 12 h post-infusion. Technetium-99m liposome uptake was measured in regions of interest over the hoof, fetlock and metacarpus. At the study end-point horses were euthanased, tissue samples collected and tissue liposome levels were calculated as the percentage of the injected dose of (99m)Tc-liposomes per kilogram of tissue. Data were analysed non-parametrically. All horses receiving oligofructose developed clinical and histological signs of laminitis. Technetium-99m liposome uptake in the hoof increased with time in laminitis horses (P = 0.04), but decreased with time in control horses (P = 0.01). Technetium-99m liposome levels in lamellar tissue from laminitis horses were 3.2-fold higher than controls (P = 0.02) and were also higher in laminitis vs. control skin, muscle, jejunum, colon, and kidney (P < 0.05). Liposomes accumulated in lamellar tissue during oligofructose-induced laminitis development and demonstrated potential for targeted lamellar drug delivery in acute laminitis. This study provides further evidence that lamellar inflammation occurs during laminitis development. Liposome accumulation also occurred in the skin, muscle, jejunum, colon and kidneys, suggesting systemic inflammation in this model.

In situ Delivery of Tumor Antigen- and Adjuvant-Loaded Liposomes Boosts Antigen-Specific T-Cell Responses by Human Dermal Dendritic Cells.

Dendritic cells (DCs) have an important role in tumor control via the induction of tumor-specific T-cell responses and are therefore an ideal target for immunotherapy. The human skin is an attractive site for tumor vaccination as it contains various DC subsets. The simultaneous delivery of tumor antigen with an adjuvant is beneficial for cross-presentation and the induction of tumor-specific T-cell responses. We therefore developed liposomes that contain the melanoma-associated antigen glycoprotein 100280-288 peptide and Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) as adjuvant. These liposomes are efficiently taken up by monocyte-derived DCs, and antigen presentation to CD8(+) T cells was significantly higher with MPLA-modified liposomes as compared with non-modified liposomes or the co-administration of soluble MPLA. We used a human skin explant model to evaluate the efficiency of intradermal delivery of liposomes. Liposomes were efficiently taken up by CD1a(+) and especially CD14(+) dermal DCs. Induction of CD8(+) T-cell responses by emigrated dermal DCs was significantly higher when MPLA was incorporated into the liposomes as compared with non-modified liposomes or co-administration of soluble MPLA. Thus, the modification of antigen-carrying liposomes with TLR ligand MPLA significantly enhances tumor-specific T-cell responses by dermal DCs and is an attractive vaccination strategy in human skin.

MPLA incorporation into DC-targeting glycoliposomes favours anti-tumour T cell responses.

Dendritic cells (DC) are attractive targets for cancer immunotherapy as they initiate strong and long-lived tumour-specific T cell responses. DC can be effectively targeted in vivo with tumour antigens by using nanocarriers such as liposomes. Cross-presentation of tumour antigens is enhanced with strong adjuvants such as TLR ligands. However, often these adjuvants have off-target effects, and would benefit from a DC-specific targeting strategy, similar to the tumour antigen. The goal of this study was to develop a strategy for specifically targeting DC with tumour antigen and adjuvant by using glycoliposomes. We have generated liposomes containing the glycan Lewis(Le)(X) which is highly specific for the C-type lectin receptor DC-SIGN expressed by DC. Le(X)-modified liposomes were taken up by human monocyte-derived DC in a DC-SIGN-specific manner. As adjuvants we incorporated the TLR ligands Pam3CySK4, Poly I:C, MPLA and R848 into liposomes and compared their adjuvant capacity on DC. Incorporation of the TLR4 ligand MPLA into glycoliposomes induced DC maturation and production of pro-inflammatory cytokines, in a DC-SIGN-specific manner, and DC activation was comparable to administration of soluble MPLA. Incorporation of MPLA into glycoliposomes significantly enhanced antigen cross-presentation of the melanoma tumour antigen gp100280-288 peptide to CD8(+) T cells compared to non-glycosylated MPLA liposomes. Importantly, antigen cross-presentation of the gp100280-288 peptide was significantly higher using MPLA glycoliposomes compared to the co-administration of soluble MPLA with glycoliposomes. Taken together, our data demonstrates that specific targeting of a gp100 tumour antigen and the adjuvant MPLA to DC-SIGN-expressing DC enhances the uptake of peptide-containing liposomes, the activation of DC, and induces tumour antigen-specific CD8(+) T cell responses. These data demonstrate that adjuvant-containing glycoliposome-based vaccines targeting DC-SIGN(+) DC represent a powerful new approach for CD8(+) T cell activation.

Cross-presentation through langerin and DC-SIGN targeting requires different formulations of glycan-modified antigens.

Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based antigen targeting to both the human DC-specific C-type lectin receptor (CLR) DC-SIGN and the LC-specific CLR langerin. Since DC-SIGN and langerin are able to recognize the difucosylated oligosaccharide Lewis Y (Le(Y)), we prepared neoglycoconjugates bearing this glycan epitope to allow targeting of both lectins. Le(Y)-modified liposomes, with an approximate diameter of 200nm, were significantly endocytosed by DC-SIGN(+) DCs and mediated efficient antigen presentation to CD4(+) and CD8(+) T cells. Surprisingly, although langerin bound to Le(Y)-modified liposomes, LCs exposed to Le(Y)-modified liposomes could not endocytose liposomes nor mediate antigen presentation to T cells. However, LCs mediated an enhanced cross-presentation when antigen was delivered through langerin using Le(Y)-modified synthetic long peptides. In contrast, Le(Y)-modified synthetic long peptides were recognized by DC-SIGN, but did not trigger antigen internalization nor antigen cross-presentation. These data demonstrate that langerin and DC-SIGN have different size requirements for antigen uptake. Although using glycans remains an interesting option in the design of anti-cancer vaccines targeting multiple CLRs, aspects such as molecule size and conformation need to be taken in consideration.