RESUMEN
Nanomedicine aims to develop smart approaches for treating cancer and other diseases to improve patient survival and quality of life. Novel nanoparticles as nanodiamonds (NDs) represent promising candidates to overcome current limitations. In this study, NDs were functionalized with a 200 kDa hyaluronic acid-phospholipid conjugate (HA/DMPE), enhancing the stability of the nanoparticles in water-based solutions and selectivity for cancer cells overexpressing specific HA cluster determinant 44 (CD44) receptors. These nanoparticles were characterized by diffuse reflectance Fourier-transform infrared spectroscopy, Raman spectroscopy, and photoluminescence spectroscopy, confirming the efficacy of the functionalization process. Scanning electron microscopy was employed to evaluate the size distribution of the dry particles, while dynamic light scattering and zeta potential measurements were utilized to evaluate ND behavior in a water-based medium. Furthermore, the ND biocompatibility and uptake mediated by CD44 receptors in three different models of human adenocarcinoma cells were assessed by performing cytofluorimetric assay and confocal microscopy. HA-functionalized nanodiamonds demonstrated the advantage of active targeting in the presence of cancer cells expressing CD44 on the surface, suggesting higher drug delivery to tumors over non-tumor tissues. Even CD44-poorly expressing cancers could be targeted by the NDs, thanks to their good passive diffusion within cancer cells.
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Receptores de Hialuranos , Ácido Hialurónico , Nanodiamantes , Humanos , Nanodiamantes/química , Ácido Hialurónico/química , Receptores de Hialuranos/metabolismo , Línea Celular Tumoral , Fosfolípidos/química , Imagen Óptica , Neoplasias/diagnóstico por imagen , Neoplasias/patología , Neoplasias/metabolismoRESUMEN
Biodegradable nanocarriers possess enormous potential for use as drug delivery systems that can accomplish controlled and targeted drug release, and a wide range of nanosystems have been reported for the treatment and/or diagnosis of various diseases and disorders. Of the various nanocarriers currently available, liposomes and polymer nanoparticles have been extensively studied and some formulations have already reached the market. However, a combination of properties to create a single hybrid system can give these carriers significant advantages, such as improvement in encapsulation efficacy, higher stability, and active targeting towards specific cells or tissues, over lipid or polymer-based platforms. To this aim, this work presents the formulation of poly(lactic-co-glycolic) acid (PLGA) nanoparticles in the presence of a hyaluronic acid (HA)-phospholipid conjugate (HA-DPPE), which was used to anchor HA onto the nanoparticle surface and therefore create an actively targeted hybrid nanosystem. Furthermore, ionic interactions have been proposed for drug encapsulation, leading us to select the free base form of pentamidine (PTM-B) as the model drug. We herein report the preparation of hybrid nanocarriers that were loaded via ion-pairing between the negatively charged PLGA and HA and the positively charged PTM-B, demonstrating an improved loading capacity compared to PLGA-based nanoparticles. The nanocarriers displayed a size of below 150 nm, a negative zeta potential of -35 mV, a core-shell internal arrangement and high encapsulation efficiency (90%). Finally, the ability to be taken up and exert preferential and receptor-mediated cytotoxicity on cancer cells that overexpress the HA specific receptor (CD44) has been evaluated. Competition assays supported the hypothesis that PLGA/HA-DPPE nanoparticles deliver their cargo within cells in a CD44-dependent manner.
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Receptores de Hialuranos , Ácido Hialurónico , Nanopartículas , Pentamidina , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Humanos , Ácido Hialurónico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Receptores de Hialuranos/metabolismo , Nanopartículas/química , Nanopartículas/administración & dosificación , Pentamidina/química , Pentamidina/administración & dosificación , Portadores de Fármacos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos , Lípidos/química , Sistemas de Liberación de MedicamentosRESUMEN
Nanotechnology's potential in revolutionising cancer treatments is evident in targeted drug delivery systems (DDSs) engineered to optimise therapeutic efficacy and minimise toxicity. This study examines a novel nanocarrier constructed with carbon nano-onions (CNOs), engineered and evaluated for its ability to selectively target cancer cells overexpressing the hyaluronic acid receptor; CD44. Our results highlighted that the CNO-based nanocarrier coupled with hyaluronic acid as the targeting agent demonstrated effective uptake by CD44+ PANC-1 and MIA PaCa-2 cells, while avoiding CD44- Capan-1 cells. The CNO-based nanocarrier also exhibited excellent biocompatibility in all tested pancreatic ductal adenocarcinoma (PDAC) cells, as well as healthy cells. Notably, the CNO-based nanocarrier was successfully loaded with chemotherapeutic 4-(N)-acyl- sidechain-containing prodrugs derived from gemcitabine (GEM). These prodrugs alone exhibited remarkable efficacy in killing PDAC cells which are known to be GEM resistant, and their efficacy was amplified when combined with the CNO-based nanocarrier, particularly in targeting GEM-resistant CD44+ PDAC cells. These findings demonstrate the potential of CNOs as promising scaffolds in advancing targeted DDSs, signifying the translational potential of carbon nanoparticles for cancer therapy.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Profármacos , Humanos , Gemcitabina , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Cebollas , Ácido Hialurónico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Línea Celular TumoralRESUMEN
Biodegradable nanocarriers represent promising tools for controlled drug delivery. However, one major drawback related to their use is the long-term stability, which is largely influenced by the presence of water in the formulations, so to solve this problem, freeze-drying with cryoprotectants has been proposed. In the present study, the influence of the freeze-drying procedure on the storage stability of poly(lactide-co-glycolide) (PLGA) nanoparticles and liposomes was evaluated. In particular, conventional cryoprotectants were added to PLGA nanoparticle and liposome formulations in various conditions. Additionally, hyaluronic acid (HA), known for its ability to target the CD44 receptor, was assessed as a cryoprotective excipient: it was added to the nanocarriers as either a free molecule or conjugated to a phospholipid to increase the interaction with the polymer or lipid matrix while exposing HA on the nanocarrier surface. The formulations were resuspended and characterized for size, polydispersity index, zeta potential and morphology. It was demonstrated that only the highest percentages of cryoprotectants allowed the resuspension of stable nanocarriers. Moreover, unlike free HA, HA-phospholipid conjugates were able to maintain the particle mean size after the reconstitution of lyophilized nanoparticles and liposomes. This study paves the way for the use of HA-phospholipids to achieve, at the same time, nanocarrier cryoprotection and active targeting.
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The development of novel nanoconstructs for biomedical applications requires the assessment of their biodistribution, metabolism and clearance in single cells, organs and entire organisms in a living environment. To reduce the number of in vivo experiments performed and to refine the methods used, in accordance with the 3Rs principle, this work proposes an ex vivo experimental system to monitor, using fluorescence microscopy, the distribution of nanoparticles in explanted murine skeletal muscle maintained in a bioreactor that can preserve the structural and functional features of the organ for long periods of time. Fluorescently-labelled liposomes and poly(lactide-co-glycolide) (PLGA)-based nanoparticles were injected into the intact soleus muscle (in the distal region close to the tendon) immediately after explants, and their distribution was analysed at increasing incubation times in cross cryosections from the proximal region of the belly. Both nanocarriers were clearly recognized in the muscle and were found to enter and migrate inside the myofibres, whereas their migration in the connective tissue seemed to be limited. In addition, some fluorescent signals were observed inside the macrophages, demonstrating the physiological clearance of the nanocarriers that did not enter the myofibres. Our ex vivo system therefore provides more information than previous in vitro experiments on cultured muscle cells, highlighting the need for the appropriate functionalization of nanocarriers if myofibre targeting is to be improved.
Asunto(s)
Nanopartículas , Ratones , Animales , Distribución Tisular , Nanopartículas/química , Músculo Esquelético , Células Cultivadas , Colorantes Fluorescentes/químicaRESUMEN
Boron/nitrogen co-doped carbon nano-onions (BN-CNOs) are spherical nanoparticles that consist of multiple inter-nestled fullerene layers, giving them an onion-like internal structure. They have potential as nanocarriers due to their small size, aqueous dispersibility, and biocompatibility. The non-covalent attachment of a biocompatible polymer to BN-CNOs is a simple and effective method of creating a scaffold for a novel nanocarrier system as it allows for increased aqueous dispersibility whilst preventing the immune system from recognising the particle as a foreign object. The non-covalent approach also preserves the electronic and structural properties of the BN-CNOs. In this study, we attached a hyaluronic acid-phospholipid (HA-DMPE) conjugate polymer to the BN-CNO's surface to improve its hydrophilicity and provide targetability toward HA-receptor overexpressing cancer cells. To this end, various ratios of HA-DMPE to BN-CNOs were investigated. The resulting supramolecular systems were characterised via UV-Vis absorption and FTIR spectroscopy, dynamic light scattering, and zeta potential techniques. It was found that the HA-DMPE conjugate polymer was permanently wrapped around the BN-CNO nanoparticle surface. Moreover, the resulting BN-CNO/HA-DMPE supramolecular systems displayed enhanced aqueous solubility compared to unfunctionalised BN-CNOs, with excellent long-term stability observed in aqueous dispersions.
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Pentamidine (PTM), which is a diamine that is widely known for its antimicrobial activity, is a very interesting drug whose mechanism of action is not fully understood. In recent years, PTM has been proposed as a novel potential drug candidate for the treatment of mental illnesses, myotonic dystrophy, diabetes, and tumors. Nevertheless, the systemic administration of PTM causes severe side effects, especially nephrotoxicity. In order to efficiently deliver PTM and reduce its side effects, several nanosystems that take advantage of the chemical characteristics of PTM, such as the presence of two positively charged amidine groups at physiological pH, have been proposed as useful delivery tools. Polymeric, lipidic, inorganic, and other types of nanocarriers have been reported in the literature for PTM delivery, and they are all in different development phases. The available approaches for the design of PTM nanoparticulate delivery systems are reported in this review, with a particular emphasis on formulation strategies and in vitro/in vivo applications. Furthermore, a critical view of the future developments of nanomedicine for PTM applications, based on recent repurposing studies, is provided. Created with BioRender.com.
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Nanopartículas , Pentamidina , Administración Cutánea , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Nanomedicina , Nanopartículas/uso terapéutico , Preparaciones FarmacéuticasRESUMEN
Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Ácido Hialurónico/farmacología , Liposomas/administración & dosificación , Macrófagos/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Polietilenglicoles/química , Animales , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Humanos , Ácido Hialurónico/química , Liposomas/química , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/inmunología , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
Muscular dystrophies are a group of rare genetic pathologies, encompassing a variety of clinical phenotypes and mechanisms of disease. Several compounds have been proposed to treat compromised muscles, but it is known that pharmacokinetics and pharmacodynamics problems could occur. To solve these issues, it has been suggested that nanocarriers could be used to allow controlled and targeted drug release. Therefore, the aim of this study was to prepare actively targeted poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the treatment of muscular pathologies. By taking advantage of the high affinity for carnitine of skeletal muscle cells due to the expression of Na+-coupled carnitine transporter (OCTN), NPs have been actively targeted via association to an amphiphilic derivative of L-carnitine. Furthermore, pentamidine, an old drug repurposed for its positive effects on myotonic dystrophy type I, was incorporated into NPs. We obtained monodispersed targeted NPs, with a mean diameter of about 100 nm and a negative zeta potential. To assess the targeting ability of the NPs, cell uptake studies were performed on C2C12 myoblasts and myotubes using confocal and transmission electron microscopy. The results showed an increased uptake of carnitine-functionalized NPs compared to nontargeted carriers in myotubes, which was probably due to the interaction with OCTN receptors occurring in large amounts in these differentiated muscle cells.
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Carnitina , Nanopartículas , Carnitina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Transporte Biológico , Portadores de Fármacos/metabolismoRESUMEN
This work provides information on the features of low molecular weight hyaluronic acid (HA)-decorated liposomes to target resveratrol (RSV) in the skin. Deformable liposomes were made of soy-phosphatidylcholine with Tween 80 as the fluidizing agent. For HA conjugation, three different phosphoethanolamines were tested: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The different phosphoethanolamine-HA conjugates were inserted into the liposome bilayer by hydration (HA on both faces of the bilayer) or by the postinsertion method (HA only on the external face of the bilayer). The effect of these variables on deformability was experimentally assessed by an in-house method (K value, the lower the value, the higher the deformability) and molecular dynamics (MD) simulations. The results showed that the K values of HA-liposomes obtained by hydration were higher than the K values of HA-liposomes prepared by postinsertion, and both were at least 10-fold higher than the K values of the corresponding plain liposomes. The nature of the lipid anchor played a key role in deformability (DMPE > DOPE > DPPE) with high variability in the case of DOPE formulations. These data were justified by the trends found in silico for the bilayer bending modulus and the HA end-to-end distance. In addition to liposome flexibility, the HA extent seems to be the key factor governing the skin penetration of RSV. When the extent is higher, the amount of the drug retained in the skin is larger. Regarding skin permeation, a parabolic trend was recorded, and the optimal amount to favor skin permeation was an approximately 30 HA/phospholipid (µg/mmol) ratio. This study reports the first piece of evidence that it is possible to control drug delivery in the skin by tuning the amount of HA on the vesicle surface.
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Diseño de Fármacos/métodos , Epidermis/metabolismo , Ácido Hialurónico/química , Resveratrol/administración & dosificación , Administración Cutánea , Dermatitis Atópica/tratamiento farmacológico , Humanos , Liposomas , Simulación de Dinámica Molecular , Tamaño de la Partícula , Permeabilidad , Psoriasis/tratamiento farmacológico , Resveratrol/farmacocinéticaRESUMEN
Chronic lung allograft dysfunction (CLAD) and interstitial lung disease associated with collagen tissue diseases (CTD-ILD) are two end-stage lung disorders in which different chronic triggers induce activation of myo-/fibroblasts (LFs). Everolimus, an mTOR inhibitor, can be adopted as a potential strategy for CLAD and CTD-ILD, however it exerts important side effects. This study aims to exploit nanomedicine to reduce everolimus side effects encapsulating it inside liposomes targeted against LFs, expressing a high rate of CD44. PEGylated liposomes were modified with high molecular weight hyaluronic acid and loaded with everolimus (PEG-LIP(ev)-HA400kDa). Liposomes were tested by in vitro experiments using LFs derived from broncholveolar lavage (BAL) of patients affected by CLAD and CTD-ILD, and on alveolar macrophages (AM) and lymphocytes isolated, respectively, from BAL and peripheral blood. PEG-LIP-HA400kDa demonstrated to be specific for LFs, but not for CD44-negative cells, and after loading everolimus, PEG-LIP(ev)-HA400kDa were able to arrest cell cycle arrest and to decrease phospho-mTOR level. PEG-LIP(ev)-HA400kDa showed anti-inflammatory effect on immune cells. This study opens the possibility to use everolimus in lung fibrotic diseases, demonstrating that our lipids-based vehicles can vehicle everolimus inside cells exerting the same drug molecular effect, not only in LFs, but also in immune cells.
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Everolimus/farmacología , Ácido Hialurónico/farmacología , Liposomas/química , Fibrosis Pulmonar/tratamiento farmacológico , Antiinflamatorios/química , Antiinflamatorios/farmacología , Lavado Broncoalveolar/métodos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células Cultivadas , Sistemas de Liberación de Medicamentos/métodos , Everolimus/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/química , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Enfermedades Pulmonares Intersticiales/tratamiento farmacológico , Enfermedades Pulmonares Intersticiales/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Polietilenglicoles/química , Fibrosis Pulmonar/metabolismoRESUMEN
In this work hyaluronic acid (HA) oligosaccharides with degree of polymerization (DP) 4, 6 and 8, obtained by enzymatic depolymerization of HA, were conjugated to a PEG-phospholipid moiety. The products (HA-DP4, HA-DP6 and HA-DP8) were used to prepare decorated liposomes. The cellular uptake of HA-DP4, HA-DP6 and HA-DP8-decorated fluorescently labelled liposomes was significantly higher (12 to 14-fold) in lung cancer cell lines with high CD44 expression than in those with low CD44 expression, suggesting a receptor-mediated entry of HA-conjugated formulations. Competition assays showed that the uptake followed this rank order: HA-DP8>HA-DP6>HA-DP4 liposomes. Moreover, they are capable of a faster interaction with CD44, followed by phagocytosis, than HA liposomes obtained from HA of higher molecular weight (4800 and 14800 Da). HA-DP4, HA-DP6 and HA-DP8-liposomes did not show cytotoxicity or inflammatory effects. Overall, we propose our new HA-DP oligosaccharides as biocompatible and effective tools for a potential drug delivery to CD44-positive cells.
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Ácido Hialurónico/síntesis química , Liposomas/síntesis química , Oligosacáridos/síntesis química , Polimerizacion , Células A549 , Unión Competitiva , Línea Celular Tumoral , Humanos , Receptores de Hialuranos/química , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Liposomas/química , Liposomas/metabolismo , Neoplasias Pulmonares/metabolismo , Modelos Químicos , Estructura Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Unión ProteicaRESUMEN
Disulfiram (DSF) is an inhibitor of P-glycoprotein (Pgp), the main obstacle limiting the success of doxorubicin (DOX), but it has poor solubility and stability. With the aim to overcome these limitations we prepared liposomes coencapsulating DSF and DOX (LipoDSF-DOX). Liposome stability, drugs release profile, effects on DOX cytotoxicity, Pgp activity and expression in breast cancer cells were evaluated. We observed that LipoDSF-DOX with a 1:3 weight ratio, with DSF in lipid bilayer and DOX in aqueous core, released DSF faster than DOX. LipoDSF-DOX increased DOX intracellular accumulation and cytotoxicity in Pgp-expressing breast cancer cells, with an efficacy superior to the mixture of free DSF and DOX, thanks to a differential kinetics of release of DSF and DOX when carried by liposomes. The mechanism of the increased DOX retention relied on the DSF-induced sulfhydraton of Pgp and followed by its ubiquitination. These events reduced Pgp expression and catalytic activity in LipoDSF-DOX-treated cells. Our results show that LipoDSF-DOX effectively reversed DOX resistance in Pgp-expressing breast cancer cells, exploiting the temporally different kinetics of release of DSF and DOX, optimized to decrease expression and activity of Pgp.