RESUMEN
Despite tremendous global efforts since the beginning of the COVID-19 pandemic, still only a limited number of prophylactic and therapeutic options are available. Although vaccination is the most effective measure in preventing morbidity and mortality, there is a need for safe and effective post-infection treatment medication. In this study, we explored a pipeline of 21 potential candidates, examined in the Calu-3 cell line for their antiviral efficacy, for drug repurposing. Ralimetinib and nafamostat, clinically used drugs, have emerged as attractive candidates. Due to the inherent limitations of the selected drugs, we formulated targeted liposomes suitable for both systemic and intranasal administration. Non-targeted and targeted nafamostat liposomes (LipNaf) decorated with an Apolipoprotein B peptide (ApoB-P) as a specific lung-targeting ligand were successfully developed. The developed liposomal formulations of nafamostat were found to possess favorable physicochemical properties including nano size (119-147 nm), long-term stability of the normally rapidly degrading compound in aqueous solution, negligible leakage from the liposomes upon storage, and a neutral surface charge with low polydispersity index (PDI). Both nafamostat and ralimetinib liposomes showed good cellular uptake and lack of cytotoxicity, and non-targeted LipNaf demonstrated enhanced accumulation in the lungs following intranasal (IN) administration in non-infected mice. LipNaf retained its anti-SARS-CoV 2 activity in Calu 3 cells with only a modest decrease, exhibiting complete inhibition at concentrations >100 nM. IN, but not intraperitoneal (IP) treatment with targeted LipNaf resulted in a trend to reduced viral load in the lungs of K18-hACE2 mice compared to targeted empty Lip. Nevertheless, upon removal of outlier data, a statistically significant 1.9-fold reduction in viral load was achieved. This observation further highlights the importance of a targeted delivery into the respiratory tract. In summary, we were able to demonstrate a proof-of-concept of drug repurposing by liposomal formulations with anti-SARS-CoV-2 activity. The biodistribution and bioactivity studies with LipNaf suggest an IN or inhalation route of administration for optimal therapeutic efficacy.
Asunto(s)
COVID-19 , Humanos , Ratones , Animales , Liposomas , Reposicionamiento de Medicamentos , Pandemias , Distribución Tisular , Pulmón , SARS-CoV-2RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer-related death, and it is highly resistant to therapy owing to its unique extracellular matrix. VAV1 protein, overexpressed in several cancer diseases including pancreatic cancer (PC), increases tumor proliferation and enhances metastases formation, which are associated with decreased survival. We hypothesized that an additive anti-tumor effect could be obtained by co-encapsulating in PLGA nanoparticles (NPs), the negatively charged siRNA against VAV1 (siVAV1) with the positively charged anti-tumor LL37 peptide, as a counter-ion. Several types of NPs were formulated and were characterized for their physicochemical properties, cellular internalization, and bioactivity in vitro. NPs' biodistribution, toxicity, and bioactivity were examined in a mice PDAC model. An optimal siVAV1 formulation (siVAV1-LL37 NPs) was characterized with desirable physicochemical properties in terms of nano-size, low polydispersity index (PDI), neutral surface charge, high siVAV1 encapsulation efficiency, spherical shape, and long-term shelf-life stability. Cell assays demonstrated rapid engulfment by PC cells, a specific and significant dose-dependent proliferation inhibition, as well as knockdown of VAV1 mRNA levels and migration inhibition in VAV1+ cells. Treatment with siVAV1-LL37 NPs in the mice PDAC model revealed marked accumulation of NPs in the liver and in the tumor, resulting in an increased survival rate following suppression of tumor growth and metastases, mediated via the knockdown of both VAV1 mRNA and protein levels. This proof-of-concept study validates our hypothesis of an additive effect in the treatment of PC facilitated by co-encapsulating siVAV1 in NPs with LL37 serving a dual role as a counter ion as well as an anti-tumor agent.
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Nanopartículas , Neoplasias Pancreáticas , Animales , Ratones , Catelicidinas , Línea Celular Tumoral , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Distribución Tisular , Neoplasias PancreáticasRESUMEN
Over-activation of the endocannabinoid/CB1R system is a hallmark feature of obesity and its related comorbidities, most notably type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD). Although the use of drugs that widely block the CB1R was found to be highly effective in treating all metabolic abnormalities associated with obesity, they are no longer considered a valid therapeutic option due to their adverse neuropsychiatric side effects. Here, we describe a novel nanotechnology-based drug delivery system for repurposing the abandoned first-in-class global CB1R antagonist, rimonabant, by encapsulating it in polymeric nanoparticles (NPs) for effective hepatic targeting of CB1Rs, enabling effective treatment of NAFLD and T2D. Rimonabant-encapsulated NPs (Rimo-NPs) were mainly distributed in the liver, spleen, and kidney, and only negligible marginal levels of rimonabant were found in the brain of mice treated by iv/ip administration. In contrast to freely administered rimonabant treatment, no CNS-mediated behavioral activities were detected in animals treated with Rimo-NPs. Chronic treatment of diet-induced obese mice with Rimo-NPs resulted in reduced hepatic steatosis and liver injury as well as enhanced insulin sensitivity, which were associated with enhanced cellular uptake of the formulation into hepatocytes. Collectively, we successfully developed a method of encapsulating the centrally acting CB1R blocker in NPs with desired physicochemical properties. This novel drug delivery system allows hepatic targeting of rimonabant to restore the metabolic advantages of blocking CB1R in peripheral tissues, especially in the liver, without the negative CB1R-mediated neuropsychiatric side effects.
Asunto(s)
Cannabinoides , Diabetes Mellitus Tipo 2 , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Rimonabant/uso terapéutico , Antagonistas de Receptores de Cannabinoides/uso terapéutico , Antagonistas de Receptores de Cannabinoides/farmacología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Cannabinoides/uso terapéuticoRESUMEN
Hepatitis B virus has infected a third of the world's population, and 296 million people are living with chronic infection. Chronic infection leads to progressive liver disease, including hepatocellular carcinoma and liver failure, and there remains no reliable curative therapy. These gaps in our understanding are due, in large part, to a paucity of animal models of HBV infection. Here, we show that rhesus macaques regularly clear acute HBV infection, similar to adult humans, but can develop long-term infection if immunosuppressed. Similar to patients, we longitudinally detected HBV DNA, HBV surface antigen, and HBV e antigen in the serum of experimentally infected animals. In addition, we discovered hallmarks of HBV infection in the liver, including RNA transcription, HBV core and HBV surface antigen translation, and covalently closed circular DNA biogenesis. This pre-clinical animal model will serve to accelerate emerging HBV curative therapies into the clinic.
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Hepatitis B Crónica , Hepatitis B , Neoplasias Hepáticas , Animales , Antígenos de Superficie , Virus de la Hepatitis B/genética , Humanos , Macaca mulattaRESUMEN
Herpes simplex virus-1 (HSV-1) is highly contagious, and there is a need for a therapeutic means to eradicate it. We have identified an siRNA (siHSV) that knocks down gene expression of the infected cell protein 0 (ICP0), which is important in the regulation of HSV infection. The selected siHSV was encapsulated in liposomes to overcome its poor stability, increase cell permeability, and prolonging siRNA circulation time. Several siRNAs against ICP0 have been designed and identified. We examined the role of various parameters, including formulation technique, lipids composition, and ratio. An optimal liposomal siHSV formulation (LipDOPE-siHSV) was characterized with desirable physiochemical properties, in terms of nano-size, low polydispersity index (PDI), neutral surface charge, high siHSV loading, spherical shape, high stability in physiologic conditions in vitro, and long-term shelf-life stability (>1 year, 4 °C). The liposomes exhibited profound internalization by human keratinocytes, no cytotoxicity in cell cultures, no detrimental effect on mice liver enzymes, and a gradual endo-lysosomal escape. Mice biodistribution studies in intact mice revealed accumulation, mainly in visceral organs but also in the trigeminal ganglion. The therapeutic potential of siHSV liposomes was demonstrated by significant antiviral activity both in the plaque reduction assay and in the 3D epidermis model, and the mechanism of action was validated by the reduction of ICP0 expression levels.
RESUMEN
Quantum dots offer superior optical features and hold a great potential as an imaging tool in comparison to 'conventional' fluorescent dyes. However, in vivo application in inflammatory-associated disorders is limited due to potential toxicity following systemic administration. Vascular inflammation contributes to cardiovascular diseases such as restenosis (re-narrowing of the artery following angioplasty), and poor prognosis is associated with the increased number of monocytes-derived macrophages (MDMs) in the arterial wall. Local administration of a suitable delivery system targeting MDMs could provide effective fluorescent imaging while minimizing systemic exposure and toxicity. We report here on the physicochemical characteristics and the structural stability of MDMs-targeted liposomal QDs (LipQDs), cellular uptake and cytotoxicity, the systemic biodistribution of LipQDs following local intra-luminal administration of LipQDs in carotid-injured rats vs. systemic administration, and imaging of QDs in the arterial tissue. The local treatment with LipQDs was found to be a suitable approach for targeting QDs to MDMs in the injured artery. In contrast to free QDs, the LipQDs formulation exhibited unique properties including structural and fluorescent stability, increased accumulation and retention for up to 24 h, and targeting properties enabling imaging of MDMs. MDMs imaging by targeted nanoparticles (NPs) could potentially serve for the detection of MDMs density in the injured artery for diagnostic purposes.
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Puntos Cuánticos , Animales , Arterias , Liposomas , Macrófagos , Ratas , Distribución TisularRESUMEN
The majority of developed and approved anticancer nanomedicines have been designed to exploit the dogma of the enhanced permeability and retention (EPR) effect, which is based on the leakiness of the tumor's blood vessels accompanied by impeded lymphatic drainage. However, the EPR effect has been under scrutiny recently because of its variable manifestation across tumor types and animal species and its poor translation to human cancer therapy. To facilitate the EPR effect, systemically injected NPs should overcome the obstacle of rapid recognition and elimination by the mononuclear phagocyte system (MPS). We hypothesized that circulating monocytes, major cells of the MPS that infiltrate the tumor, may serve as an alternative method for achieving increased tumor accumulation of NPs, independent of the EPR effect. We describe here the accumulation of liposomal quantum dots (LipQDs) designed for active delivery via monocytes, in comparison to LipQDs designed for passive delivery (via the EPR effect), following IV administration in a mammary carcinoma model. Hydrophilic QDs were synthesized and entrapped in functionalized liposomes, conferring passive ("stealth" NPs; PEGylated, neutral charge) and active (monocyte-mediated delivery; positively charged) properties by differing in their lipid composition, membrane PEGylation, and charge (positively, negatively, and neutrally charged). The various physicochemical parameters affecting the entrapment yield and optical stability were examined in vitro and in vivo. Biodistribution in the blood, various organs, and in the tumor was determined by the fluorescence intensity and Cd analyses. Following the treatment of animals (intact and mammary-carcinoma-bearing mice) with disparate formulations of LipQDs (differing by their lipid composition, neutrally and positively charged surfaces, and hydrophilic membrane), we demonstrate comparable tumor uptake of QDs delivered by the passive and the active routes (mainly by Ly-6Chi monocytes). Our findings suggest that entrapping QDs in nanosized liposomal formulations, prepared by a new facile method, imparts superior structural and optical stability and a suitable biodistribution profile leading to increased tumor uptake of fluorescently stable QDs.
Asunto(s)
Liposomas/farmacología , Neoplasias Mamarias Animales/tratamiento farmacológico , Sistema Mononuclear Fagocítico/química , Puntos Cuánticos/química , Animales , Vasos Sanguíneos/efectos de los fármacos , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Lípidos/química , Lípidos/farmacología , Liposomas/química , Neoplasias Mamarias Animales/patología , Ratones , Nanomedicina , Células Neoplásicas Circulantes , Permeabilidad/efectos de los fármacosRESUMEN
Non-viral, polymeric-based, siRNA nanoparticles (NPs) have been proposed as promising gene delivery systems. Encapsulating siRNA in targeted NPs could confer improved biological stability, extended half-life, enhanced permeability, effective tumor accumulation, and therapy. In this work, a peptide derived from apolipoprotein B100 (ApoB-P), the protein moiety of low-density lipoprotein, was used to target siRNA-loaded PEGylated NPs to the extracellular matrix/proteoglycans (ECM/PGs) of a mammary carcinoma tumor. siRNA against osteopontin (siOPN), a protein involved in breast cancer development and progression, was encapsulated into PEGylated poly(d,l-lactic-co-glycolic acid) (PLGA) NPs using the double emulsion solvent diffusion technique. The NPs obtained possessed desired physicochemical properties including ~200 nm size, a neutral surface charge, and high siOPN loading of ~5 µg/mg. ApoB-P-targeted NPs exhibited both enhanced binding to isolated ECM and internalization by MDA-MB-231 human mammary carcinoma cells, in comparison to non-targeted NPs. Increased accumulation of the targeted NPs was achieved in the primary mammary tumor of mice xenografted with MDA-MB-231 mammary carcinoma cells as well as in the lungs, one of the main sites affected by metastases. siOPN NPs treatment resulted in significant inhibition of tumor growth (similar bioactivity of both formulations), accompanied with significant reduction of OPN mRNA levels (~40% knockdown of mRNA levels). We demonstrated that targeted NPs possessed enhanced tumor accumulation with increased therapeutic potential in mice models of mammary carcinoma.
RESUMEN
Major advances have been achieved in understanding the mechanisms and risk factors leading to cardiovascular disorders and consequently developing new therapies. A strong inflammatory response occurs with a substantial recruitment of innate immunity cells in atherosclerosis, myocardial infarction, and restenosis. Monocytes and macrophages are key players in the healing process that ensues following injury. In the inflamed arterial wall, monocytes, and monocyte-derived macrophages have specific functions in the initiation and resolution of inflammation, principally through phagocytosis, and the release of inflammatory cytokines and reactive oxygen species. In this review, we will focus on delivery systems, mainly nanoparticles, for modulating circulating monocytes/monocyte-derived macrophages. We review the different strategies of depletion or modulation of circulating monocytes and monocyte subtypes, using polymeric nanoparticles and liposomes for the therapy of myocardial infarction and restenosis. We will further discuss the strategies of exploiting circulating monocytes for biological targeting of nanocarrier-based drug delivery systems for therapeutic and diagnostic applications.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Monocitos/inmunología , Animales , Enfermedades Cardiovasculares/inmunología , HumanosRESUMEN
The innate immunity system plays a critical role in vascular repair and restenosis development. Liposomes encapsulating bisphosphonates (LipBPs), but not free BPs, suppress neointima formation following vascular injury mediated in part by monocytes. The objective of this study was to elucidate the role of monocyte subpopulations on vascular healing following LipBP treatment. The potency- and dose-dependent treatment effect of clodronate (CLOD) and alendronate (ALN) liposomes on restenosis inhibition, total monocyte depletion, and monocytes subpopulation was studied. Rats subjected to carotid injury were treated by a single IV injection of LipBPs at the time of injury. Low- and high-dose LipALN treatment (3 and 10 mg/kg, respectively) resulted in a dose-dependent effect on restenosis development after 30 days. Both doses of LipALN resulted in a dose-dependent inhibition of restenosis, but only high dose of LipALN depleted monocytes (-60.1 ± 4.4%, 48 h post injury). Although LipCLOD treatment (at an equivalent potency to 3 mg/kg alendronate) significantly reduced monocyte levels (72.1 ± 6%), no restenosis inhibition was observed. The major finding of this study is the correlation found between monocyte subclasses and restenosis inhibition. Non-classical monocyte (NCM) levels were found higher in LipALN-treated rats, but lower in LipCLOD-treated rats, 24 h after injury and treatment. We suggest that the inhibition of circulating monocyte subpopulations is the predominant mechanism by which LipBPs prevent restenosis. The effect of LipBP treatment on the monocyte subpopulation correlates with the dose and potency of LipBPs.
Asunto(s)
Alendronato/administración & dosificación , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Ácido Clodrónico/administración & dosificación , Reestenosis Coronaria/prevención & control , Monocitos/inmunología , Lesiones del Sistema Vascular/tratamiento farmacológico , Animales , Traumatismos de las Arterias Carótidas/inmunología , Liposomas , Masculino , Ratas , Lesiones del Sistema Vascular/inmunologíaRESUMEN
siRNA-loaded nanoparticles (NPs) administered systemically can overcome the poor stability and rapid elimination of free double-stranded RNA in circulation, resulting in increased tumor accumulation and efficacy. siRNA against osteopontin (siOPN), a protein involved in breast cancer development, was encapsulated in poly(D,L-lactic-co-glycolic acid) NPs by a double emulsion solvent diffusion (DESD) technique. We also compared the effect of polyethylenimine (PEI) molecular weight (800 Da and 25 kDa), used as the counter-ion for siRNA complexation, on the physicochemical properties of the NPs, cytotoxicity, and cellular uptake. NPs prepared by the DESD technique were obtained at the desired size (â¼170 nm) using both types of PEIs, and were characterized with a neutral surface charge, high encapsulation yield (up to â¼60%), siOPN concentration of 5.6-8.4 µg/mg, stability in physiologic conditions in vitro and in vivo, and long-term shelf-life stability (> 3 years). The NPs prepared using both PEIs exhibited no cytotoxicity in primary smooth muscle culture, and no detrimental effect on mice liver enzymes following their IV administration. Following cellular uptake and biodistribution studies, the therapeutic potential of the NPs was demonstrated by a significant decrease of tumor progression and size in an ectopic xenograft model of mammary carcinoma in mice.
Asunto(s)
Emulsiones/química , Ácido Láctico/química , Neoplasias Mamarias Experimentales/terapia , Nanopartículas/química , Ácido Poliglicólico/química , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/toxicidad , Solventes/química , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Difusión , Modelos Animales de Enfermedad , Endocitosis/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Humanos , Ratones Endogámicos BALB C , Peso Molecular , Nanopartículas/ultraestructura , Osteopontina/metabolismo , Tamaño de la Partícula , Polietileneimina/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Suero , Electricidad Estática , Distribución Tisular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Even though some progress in diagnosis and treatment has been made over the years, there is still no definitive treatment available for Glioblastoma multiforme (GBM). Convection-enhanced delivery (CED), a continuous infusion-mediated pressure gradient via intracranial catheters, studied in clinical trials, enables in situ drug concentrations several orders of magnitude greater than those achieved by systemic administration. We hypothesized that the currently limited efficacy of CED could be enhanced by a liposomal formulation, thus achieving enhanced drug localization to the tumor site with minimal toxicity. We hereby describe a novel approach for treating GBM by CED of liposomes containing the known chemotherapeutic agent, temozolomide (TMZ). A new technique for encapsulating TMZ in hydrophilic (PEGylated) liposomes, characterized by nano-size (121nm), low polydispersity index (<0.13) and with near-neutral charge (-Ê,0.2mV), has been developed. Co-infusion of PEGylated Gd-DTPA liposomes and TMZ-liposomes by CED in GBM bearing rats, resulted in enhanced tumor detection with longer residence time than free Gd-DTPA. Treatment of GBM-bearing rats with either TMZ solution or TMZ-liposomes resulted in greater tumor inhibition and significantly higher survival. However, the longer survival and smaller tumor volumes exhibited by TMZ liposomal treatment in comparison to TMZ in solution were insignificant (p<0.053); and only significantly lower edema volumes were observed. Thus, there are no clear-cut advantages to use a liposomal delivery system of TMZ via CED over a drug solution.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Sistemas de Liberación de Medicamentos , Glioblastoma/tratamiento farmacológico , Animales , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/farmacología , Convección , Dacarbazina/administración & dosificación , Dacarbazina/farmacocinética , Dacarbazina/farmacología , Gadolinio DTPA/administración & dosificación , Liposomas , Masculino , Nanopartículas , Tamaño de la Partícula , Polietilenglicoles/química , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Tasa de Supervivencia , Temozolomida , Carga TumoralRESUMEN
Quantum dots (QDs), semiconductor nanocrystals, are fluorescent nanoparticles of growing interest as an imaging tool of a diseased tissue. However, a major concern is their biocompatibility, cytotoxicity, and fluorescence instability in biological milieu, impeding their use in biomedical applications, in general, and for inflammation imaging, in particular. In addition, for an efficient fluorescent signal at the desired tissue, and avoiding systemic biodistribution and possible toxicity, targeting is desired. We hypothesized that phagocytic cells of the innate immunity system (mainly circulating monocytes) can be exploited as transporters of specially designed liposomes containing QDs to the inflamed tissue. We developed a liposomal delivery system of QDs (LipQDs) characterized with high encapsulation yield, enhanced optical properties including far-red emission wavelength and fluorescent stability, high quantum yield, and protracted fluorescent decay lifetime. Treatment with LipQDs, rather than free QDs, exhibited high accumulation and retention following intravenous administration in carotid-injured rats (an inflammatory model). QD-monocyte colocalization was detected in the inflamed arterial segment only following treatment with LipQDs. No cytotoxicity was observed following LipQD treatment in cell cultures, and changes in liver enzymes and gross histopathological changes were not detected in mice and rats, respectively. Our results suggest that the LipQD formulation could be a promising strategy for imaging inflammation.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inflamación/diagnóstico por imagen , Monocitos/química , Monocitos/metabolismo , Imagen Óptica , Puntos Cuánticos/química , Animales , Compuestos de Cadmio/química , Células Cultivadas , Liposomas/química , Liposomas/farmacocinética , Masculino , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Ratas , Ratas Endogámicas , Compuestos de Selenio/química , Sulfuros/química , Distribución Tisular , Compuestos de Zinc/químicaRESUMEN
Cationic antimicrobial peptides (AMPs) are part of the innate immunity, and act against a wide variety of pathogenic microorganisms by perturbation of the microorganism's plasma membrane. Although attractive for clinical applications, these agents suffer from limited stability and activity in vivo, as well as non-specific interaction with host biological membranes, leading to cytotoxic adverse effects. We hypothesized that encapsulation of AMPs within liposomes could result in reduced cytotoxicity, and with enhanced stability as well as bioactivity against herpes simplex virus 1 (HSV-1). We formulated nano-sized liposomal formulations of LL-37 and indolicidin, and their physicochemical properties, cellular uptake, in vitro cytotoxicity and antiviral efficacy have been determined. Lower cytotoxicity of LL-37 liposomes was found in comparison to indolicidin liposomes attributed to the superior physicochemical properties, and to the different degree of interaction with the liposomal membrane. The disc-like shaped LL-37 liposomes (106.8±10.1nm, shelf-life stability of >1year) were taken up more rapidly and to a significantly higher extent than the free peptide by human keratinocyte cell line (HaCaT), remained intact within the cells, followed by release of the active peptide within the cytoplasm and migration of the vesicles' lipids to the plasma membrane. LL-37 liposomes were found significantly less toxic than both the free agent and liposomal indolicidin. In the new 3D epidermis model (immortalized primary keratinocytes) liposomal LL-37 treatment (>20µM), but not free LL-37, efficiently protected the epidermis, inhibiting HSV-1 infection. This positive antiviral effect was obtained with no cytotoxicity even at very high concentrations (400µM). Thus, the antiviral activity of encapsulated LL-37 was significantly improved, expanding its therapeutic window. Liposomal LL-37 appears to be a promising delivery system for HSV therapy.
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Péptidos Catiónicos Antimicrobianos/administración & dosificación , Antivirales/administración & dosificación , Herpesvirus Humano 1/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Células Cultivadas , Epidermis/virología , Prepucio/citología , Humanos , Queratinocitos/virología , Lípidos/química , Liposomas , Masculino , CatelicidinasRESUMEN
Nonhuman primates are critical animal models for the study of human disorders and disease and offer a platform to assess the role of immune cells in pathogenesis via depletion of specific cellular subsets. However, this model is currently hindered by the lack of reagents that safely and specifically ablate myeloid cells of the monocyte/macrophage Lin. Given the central importance of macrophages in homeostasis and host immunity, development of a macrophage-depletion technique in nonhuman primates would open new avenues of research. Here, using LA at i.v. doses as low as 0.1 mg/kg, we show a >50% transient depletion of circulating monocytes and tissue-resident macrophages in RMs by an 11-color flow cytometric analysis. Diminution of monocytes was followed rapidly by emigration of monocytes from the bone marrow, leading to a rebound of monocytes to baseline levels. Importantly, LA was well-tolerated, as no adverse effects or changes in gross organ function were observed during depletion. These results advance the ex vivo study of myeloid cells by flow cytometry and pave the way for in vivo studies of monocyte/macrophage biology in nonhuman primate models of human disease.
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Alendronato/farmacología , Separación Celular/métodos , Modelos Animales de Enfermedad , Citometría de Flujo/métodos , Macaca mulatta/inmunología , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Alendronato/administración & dosificación , Alendronato/toxicidad , Animales , Médula Ósea/efectos de los fármacos , Recuento de Células , Movimiento Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Humanos , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Liposomas , Células Mieloides/citología , Células Mieloides/efectos de los fármacosRESUMEN
Bone sialoprotein (BSP) and osteopontin (OPN) are important factors in the metastasis of breast cancer, which were examined as targets for antineoplastic therapy by siRNA. In addition, the effect of gene silencing on their transcription factor Runx2 and their interaction partners integrin ß(3) and matrix metalloproteinase 2 was studied. The effect of siRNAs directed against these genes was assessed by monitoring expression levels followed by functional assays in cell culture as well as skeletal metastases caused by human MDA-MB-231(luc) breast cancer cells in nude rats. Upon silencing of the targets, cell migration was profoundly impaired (p < 0.001 for BSP-siRNA), but the impact on proliferation was low. Systemic administration by osmotic mini-pumps of BSP-siRNA but not OPN-siRNA decreased osteolytic lesions (p = 0.067). Extraosseous tumour growth was not affected. As an alternative approach, non-viral, polymeric based formulations of siRNAs in nanoparticles (NP) were developed. Locoregional administration of the two siRNAs targeting OPN and BSP encapsulated in these biodegradable NP reduced skeletal lesions even more efficiently (p = 0.03). Compared to systemic administration, this treatment caused not only a more pronounced anti-osteolytic effect at a 25-fold lower total siRNA dose, but also had a slight reducing effect on tumour incidence (p = 0.095). In conclusion, the siRNA treatment had a small effect on cellular proliferation but a significant efficacy against migration of and osteolysis induced by MDA-MB-231 cells. Our data underline that siRNA mediated knockdown is a powerful tool for identifying targets for pharmacological intervention. In addition, encapsulation of siRNA into biodegradable NP is a strategy, which promises well for using siRNA.
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Neoplasias Óseas/prevención & control , Neoplasias de la Mama/prevención & control , Movimiento Celular , Sialoproteína de Unión a Integrina/metabolismo , Osteólisis/prevención & control , Osteopontina/metabolismo , Animales , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Humanos , Sialoproteína de Unión a Integrina/antagonistas & inhibidores , Sialoproteína de Unión a Integrina/genética , Osteólisis/metabolismo , Osteólisis/patología , Osteopontina/antagonistas & inhibidores , Osteopontina/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Desnudas , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Tumorales CultivadasRESUMEN
The current treatment for coronary restenosis following balloon angioplasty involves the use of a mechanical or drug eluting stent (DES). The advent of DES systems has effectively allayed much of the challenge of restenosis that has plagued the success of percutaneous coronary interventions (PCI). However, there are certain limitations to DES use, among which is late stent thrombosis. Innate immunity and inflammation are of major importance in the overreaction of the wound healing response to PCI-induced vascular injury, which leads to restenosis. Liposomes containing alendronate have been shown to deplete circulating monocytes and reduce experimental restenosis. This review presents a unique systemic approach for treating restenosis with alendronate liposomal nano-carriers and reports on its formulation development, formulation variables affecting monocyte/macrophage targeting, pharmacokinetics (PK) and biodistribution, in vitro and in vivo anti-inflammatory effect, and the recent results of the phase II clinical trial.
Asunto(s)
Alendronato/administración & dosificación , Conservadores de la Densidad Ósea/administración & dosificación , Reestenosis Coronaria/tratamiento farmacológico , Alendronato/farmacocinética , Animales , Conservadores de la Densidad Ósea/farmacocinética , Reestenosis Coronaria/inmunología , Humanos , Liposomas , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunologíaRESUMEN
Circulating γδ T cells are cytotoxic lymphocytes that are unique to primates. Recent -studies have shown that amino-bisphosphonates (nBP) activate γδ T cells to kill tumor cells in an indirect mechanism, which requires antigen presenting cells (APC). We hypothesized that selective targeting of nBP to monocytes would result in a more potent γδ T cells activation in circulation, and in tissue associated macrophages (TAM) following monocytes-laden drug extravasation and liposomes accumulation at the tumor site. In addition, inhibition of TAM by alendronate liposomes (ALN-L) is expected. ALN was targeted exclusively to monocytes, but not to lymphocytes, by encapsulating it in negatively-charged liposomes. The proportion of human γd-T cells in the CD3(+) population following treatment with ALN-L or the free drug was increased, from 5.6 ± 0.4% to 50.9 ;± 12.2% and 49.5 ± 12.9%, respectively. ALN solution and liposomes treatments resulted in an increased, and in a dose dependent manner, TNFα secretion from h-PBMC. Preliminary results showed that ALN-L inhibited tumor growth in a nude mouse breast tumor model. It is suggested that enhanced activation of γδ T cells could be obtained due to interaction with circulating monocytes as well as by TAM endocytosing liposomal nBP leading to a potentiated anti-tumor effect of nBP. It should be noted that this could be validated only in primates/humans since γδ T cells are unique in these species.
Asunto(s)
Alendronato/farmacología , Antineoplásicos/farmacología , Liposomas/farmacología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Alendronato/química , Alendronato/farmacocinética , Análisis de Varianza , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Liposomas/química , Liposomas/farmacocinética , Activación de Linfocitos/efectos de los fármacos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratones , Ratones Desnudos , Monocitos/efectos de los fármacos , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Following recent successes with percutaneous coronary intervention (PCI) for treating coronary artery disease (CAD), many challenges remain. In particular, mechanical injury from the procedure results in extensive endothelial denudation, exposing the underlying collagen IV-rich basal lamina, which promotes both intravascular thrombosis and smooth muscle proliferation. Previously, we reported the engineering of collagen IV-targeting nanoparticles (NPs) and demonstrated their preferential localization to sites of arterial injury. Here, we develop a systemically administered, targeted NP system to deliver an antiproliferative agent to injured vasculature. Approximately 60-nm lipid-polymeric NPs were surface functionalized with collagen IV-targeting peptides and loaded with paclitaxel. In safety studies, the targeted NPs showed no signs of toxicity and a ≥3.5-fold improved maximum tolerated dose versus paclitaxel. In efficacy studies using a rat carotid injury model, paclitaxel (0.3 mg/kg or 1 mg/kg) was i.v. administered postprocedure on days 0 and 5. The targeted NP group resulted in lower neointima-to-media (N/M) scores at 2 wk versus control groups of saline, paclitaxel, or nontargeted NPs. Compared with sham-injury groups, an â¼50% reduction in arterial stenosis was observed with targeted NP treatment. The combination of improved tolerability, sustained release, and vascular targeting could potentially provide a safe and efficacious option in the management of CAD.