RESUMEN
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.
Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Humanos , Placa Aterosclerótica/metabolismo , Ácidos Docosahexaenoicos/uso terapéutico , Ácidos Docosahexaenoicos/farmacología , Liposomas/uso terapéutico , Aterosclerosis/metabolismo , Antiinflamatorios/uso terapéutico , Apolipoproteínas E/genéticaRESUMEN
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.
Asunto(s)
Liposomas , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Furina/metabolismo , Vía de Señalización Wnt , Sistemas de Liberación de Medicamentos , Células Madre Neoplásicas/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Barrera Hematoencefálica/diagnóstico por imagen , Sistemas de Liberación de Medicamentos/métodos , Ultrasonografía/métodos , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Encefalopatías/tratamiento farmacológico , Colorantes Fluorescentes/administración & dosificación , Liposomas/administración & dosificación , Ratones , Ratones Desnudos , Microburbujas , Nanomedicina/métodos , Imagen Óptica/métodosRESUMEN
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. 10â¯nm poly(N-(2-hydroxypropyl)methacrylamide) polymers, 100â¯nm PEGylated liposomes and 2000â¯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.
Asunto(s)
Cirrosis Hepática/metabolismo , Macrófagos/metabolismo , Animales , Sistemas de Liberación de Medicamentos , Citometría de Flujo , Inmunohistoquímica , Liposomas/química , Linfocitos/metabolismo , Masculino , Ratones , Microburbujas , Microscopía Fluorescente , Nanomedicina , Polímeros/química , Microtomografía por Rayos XRESUMEN
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 24â¯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 (4â¯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.
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Antineoplásicos Hormonales/administración & dosificación , Dexametasona/administración & dosificación , Glucocorticoides/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Animales , Peso Corporal/efectos de los fármacos , Huesos/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Liposomas , Ratones Noqueados , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Distribución Tisular , Carga Tumoral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Células Madre Adultas/efectos de los fármacos , Colesterol/química , Medios de Cultivo Condicionados/farmacología , Organoides/efectos de los fármacos , Fosfolípidos/química , Técnicas de Cultivo de Tejidos , Proteína Wnt3A/farmacología , Células Madre Adultas/metabolismo , Células Madre Adultas/patología , Biopsia , Carcinoma Hepatocelular/patología , Enfermedad Hepática en Estado Terminal/patología , Hepatitis C/patología , Degeneración Hepatolenticular/patología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Yeyuno/patología , Liposomas/administración & dosificación , Liposomas/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patología , Organoides/metabolismo , Organoides/patologíaRESUMEN
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.
Asunto(s)
Liposomas/química , Microburbujas/uso terapéutico , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Femenino , Colorantes Fluorescentes/química , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Nanopartículas/química , Imagen Óptica/métodos , Permeabilidad , Polímeros/química , Propiedades de Superficie , Tomografía Computarizada por Rayos X , Ondas UltrasónicasRESUMEN
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.
Asunto(s)
Enfermedades del Pie/veterinaria , Enfermedades de los Caballos/inducido químicamente , Liposomas/química , Oligosacáridos/toxicidad , Polietilenglicoles/química , Exametazima de Tecnecio Tc 99m/farmacocinética , Animales , Enfermedades del Pie/diagnóstico por imagen , Enfermedades del Pie/metabolismo , Pezuñas y Garras/patología , Enfermedades de los Caballos/diagnóstico por imagen , Caballos , Inflamación/inducido químicamente , Inflamación/diagnóstico por imagen , Inflamación/veterinaria , Masculino , Radiofármacos/farmacocinéticaRESUMEN
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.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Liposomas/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Linfocitos T Citotóxicos/efectos de los fármacos , Anticuerpos Antineoplásicos/biosíntesis , Anticuerpos Antineoplásicos/genética , Presentación de Antígeno/efectos de los fármacos , Antígenos de Neoplasias/química , Linfocitos T CD8-positivos/efectos de los fármacos , Citocinas/biosíntesis , Sistemas de Liberación de Medicamentos , Humanos , Macrófagos/efectos de los fármacos , Melanoma Experimental/genética , Receptor Toll-Like 4/efectos de los fármacos , Antígeno gp100 del Melanoma/efectos de los fármacosRESUMEN
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.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Piel/efectos de los fármacos , Biopsia con Aguja , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/farmacología , Células Cultivadas , Reactividad Cruzada , Citometría de Flujo , Humanos , Inmunohistoquímica , Inmunoterapia/métodos , Liposomas/farmacología , Piel/citología , Piel/patología , Receptores Toll-Like/inmunologíaRESUMEN
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.
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Antineoplásicos Hormonales/administración & dosificación , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Dexametasona/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Neoplasias Óseas/patología , Línea Celular Tumoral , Humanos , Liposomas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Próstata/patología , Ratas , Ratas Sprague-DawleyRESUMEN
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.
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Antígenos CD/inmunología , Antígenos/inmunología , Moléculas de Adhesión Celular/inmunología , Reactividad Cruzada , Glicoconjugados/inmunología , Lectinas Tipo C/inmunología , Liposomas/inmunología , Lectinas de Unión a Manosa/inmunología , Polisacáridos/inmunología , Receptores de Superficie Celular/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Antígenos/química , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Secuencia de Carbohidratos , Células Dendríticas/inmunología , Sistemas de Liberación de Medicamentos , Glicoconjugados/química , Glicoesfingolípidos/química , Glicoesfingolípidos/inmunología , Humanos , Células de Langerhans/inmunología , Liposomas/química , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Polisacáridos/químicaRESUMEN
Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings. We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.
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Exosomas , Liposomas/análisis , Nanopartículas/análisis , Materiales Biomiméticos , Línea Celular Tumoral , Citometría de Flujo , HumanosRESUMEN
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.
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Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Citocinas/inmunología , Dexametasona/administración & dosificación , Dexametasona/farmacología , Macrófagos/efectos de los fármacos , Movimiento Celular , Células Cultivadas , Humanos , Liposomas , Macrófagos/citología , Macrófagos/inmunologíaRESUMEN
PURPOSE: Magnetic resonance imaging (MRI) with targeted contrast agents provides a promising means for diagnosis and treatment monitoring after cerebrovascular injury. Our goal was to demonstrate the feasibility of this approach to detect the neuroinflammatory biomarker intercellular adhesion molecule-1 (ICAM-1) after stroke and to establish a most efficient imaging procedure. PROCEDURES: We compared two types of ICAM-1-functionalized contrast agent: T 1-shortening gadolinium chelate-containing liposomes and T2(*)-shortening micron-sized iron oxide particles (MPIO). Binding efficacy and MRI contrast effects were tested in cell cultures and a mouse stroke model. RESULTS: Both ICAM-1-targeted agents bound effectively to activated cerebrovascular cells in vitro, generating significant MRI contrast-enhancing effects. Direct in vivo MRI-based detection after stroke was only achieved with ICAM-1-targeted MPIO, although both contrast agents showed similar target-specific vascular accumulation. CONCLUSIONS: Our study demonstrates the potential of in vivo MRI of post-stroke ICAM-1 upregulation and signifies target-specific MPIO as most suitable contrast agent for molecular MRI of cerebrovascular inflammation.
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Medios de Contraste , Molécula 1 de Adhesión Intercelular/genética , Imagen por Resonancia Magnética , Material Particulado , Accidente Cerebrovascular/diagnóstico , Regulación hacia Arriba/genética , Animales , Encéfalo/irrigación sanguínea , Encéfalo/patología , Línea Celular , Células Endoteliales/metabolismo , Compuestos Férricos , Gadolinio , Inflamación/patología , Molécula 1 de Adhesión Intercelular/metabolismo , Liposomas , Ratones , Ratones Endogámicos C57BL , Tamaño de la Partícula , Cambios Post Mortem , ARN Mensajero/genética , ARN Mensajero/metabolismo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patologíaRESUMEN
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.
Asunto(s)
Medios de Contraste/análisis , Compuestos Férricos/análisis , Imagen por Resonancia Magnética , Nanopartículas/análisis , Animales , Materiales Biocompatibles/análisis , Materiales Biocompatibles/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Contraste/toxicidad , Compuestos Férricos/toxicidad , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Ratones , Microscopía Fluorescente , Nanopartículas/toxicidad , Nanopartículas/ultraestructura , Polietilenglicoles/análisis , Polietilenglicoles/toxicidadRESUMEN
Cancer immunotherapy requires potent tumor-specific CD8(+) and CD4(+) T-cell responses, initiated by dendritic cells (DCs). Tumor antigens can be specifically targeted to DCs in vivo by exploiting their expression of C-type lectin receptors (CLR), which bind carbohydrate structures on antigens, resulting in internalization and antigen presentation to T-cells. We explored the potential of glycan-modified liposomes to target antigens to DCs to boost murine and human T-cell responses. Since DC-SIGN is a CLR expressed on DCs, liposomes were modified with DC-SIGN-binding glycans Lewis (Le)(B) or Le(X). Glycan modification of liposomes resulted in increased binding and internalization by BMDCs expressing human DC-SIGN. In the presence of LPS, this led to 100-fold more efficient presentation of the encapsulated antigens to CD4(+) and CD8(+) T-cells compared to unmodified liposomes or soluble antigen. Similarly, incubation of human moDC with melanoma antigen MART-1-encapsulated liposomes coated with Le(X) in the presence of LPS led to enhanced antigen-presentation to MART-1-specific CD8(+) T-cell clones. Moreover, this formulation drove primary CD8(+) T-cells to differentiate into high numbers of tetramer-specific, IFN-γ-producing effector T-cells. Together, our data demonstrate the potency of a glycoliposome-based vaccine targeting DC-SIGN for CD4(+) and CD8(+) effector T-cell activation. This approach may offer improved options for treatment of cancer patients and opens the way to in situ DC-targeted vaccination.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular/inmunología , Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Polisacáridos/inmunología , Receptores de Superficie Celular/inmunología , Animales , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/genética , Células Dendríticas/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Lectinas Tipo C/genética , Liposomas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Polisacáridos/administración & dosificación , Receptores de Superficie Celular/genéticaRESUMEN
Over the past few decades, many different types of nanomedicines have been evaluated, both in vitro and in vivo. In general, nanomedicines are designed to improve the in vivo properties of low-molecular-weight (chemo-) therapeutic drugs, i.e. their biodistribution and the target site accumulation, and to thereby improve the balance between their efficacy and toxicity. A significant number of studies have also addressed the in vitro properties of nanomedicines, showing e.g. their ability to overcome cellular multidrug resistance (MDR). Particularly promising results in this regard have been reported for 'pharmacologically active' carrier materials, such as Pluronics, which are able to directly inhibit drug efflux pumps and other cellular detoxification mechanisms. In the present report, we have set out to evaluate the ability of classical (and pharmacologically inactive) carrier materials to overcome MDR. To this end, four different drug-sensitive and drug-resistant cancer cell lines were treated with increasing concentrations of free doxorubicin, of polymer-bound doxorubicin, of micellar doxorubicin and of liposomal doxorubicin, and resistance indices (IC(50) in resistant cells/IC(50) in sensitive cells) were determined. In addition, the cellular uptake of the four formulations was evaluated using fluorescence microscopy. It was found that the carrier materials did manage to overcome MDR to some extent, but that the overall benefit was quite small; only for polymer-bound doxorubicin in A431 cells, a significant (4-fold) reduction in the resistance index was observed. These findings indicate that the ability of classical nanomedicines to overcome cellular MDR should not be overestimated.
Asunto(s)
Acrilamidas/administración & dosificación , Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , 1,2-Dipalmitoilfosfatidilcolina/administración & dosificación , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/química , Humanos , Liposomas , Ratones , Micelas , Nanomedicina , Fosfatidiletanolaminas/administración & dosificación , Fosfatidiletanolaminas/química , Polietilenglicoles/administración & dosificación , Polietilenglicoles/químicaRESUMEN
Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.