RESUMEN
Chondrosarcoma (ChS), a malignant cartilage-producing tumor, is the second most frequently diagnosed osseous sarcoma after osteosarcoma. It represents a very heterogeneous group of malignant chemo- and radiation-resistant neoplasms, accounting for approximately 20% of all bone sarcomas. The majority of ChS patients have a good prognosis after a complete surgical resection, as these tumors grow slowly and rarely metastasize. Conversely, patients with inoperable disease, due to the tumor location, size, or metastases, represent a great clinical challenge. Despite several genetic and epigenetic alterations that have been described in distinct ChS subtypes, very few therapeutic options are currently available for ChS patients. Therefore, new prognostic factors for tumor progression as well as new treatment options have to be explored, especially for patients with unresectable or metastatic disease. Recent studies have shown that a correlation between immune infiltrate composition, tumor aggressiveness, and survival does exist in ChS patients. In addition, the intra-tumor microvessel density has been proven to be associated with aggressive clinical behavior and a high metastatic potential in ChS. This review will provide an insight into the ChS microenvironment, since immunotherapy and antiangiogenic agents are emerging as interesting therapeutic options for ChS patients.
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Condrosarcoma , Microambiente Tumoral , Humanos , Condrosarcoma/patología , Condrosarcoma/genética , Condrosarcoma/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/terapia , Neoplasias Óseas/metabolismo , Neoplasias Óseas/genética , Inmunoterapia , Inhibidores de la Angiogénesis/uso terapéutico , Inhibidores de la Angiogénesis/farmacologíaRESUMEN
Nanoparticle's success as drug delivery systems for cancer treatment has been achieved through passive targeting mechanisms. However, tumor heterogeneity and rapid drug clearance limit the treatment efficacy. Improved outcomes and selective drug release can be achieved by grafting ligands at the surface of nanocarriers that bind molecules overexpressed in the tumor microenvironment (TME). In this work, we developed a docetaxel-loaded nanoemulsions (NEs) binding an anti-netrin-1 monoclonal antibody (NP137) to selectively target the netrin-1 protein overexpressed in many different tumors. The goal is to refine a combined approach utilizing NP137 and docetaxel as an improved tumor-targeting chemotherapeutic agent for addressing triple-negative breast cancer (TNBC). Several factors have been considered for the optimization of the active targeted drug delivery system via the click-chemistry conjugation, as the impact of PEGylated surfactant that stabilize the NEs shell on conjugation efficiency, cytocompatibility with EMT6 cell line and colloidal stability over time of NEs. Results showed that a 660 Da PEG chain length contributed to NEs colloidal stability and had no impact on cell viability or on the antibody binding ability for its ligand after surface conjugation. Moreover, docetaxel was encapsulated into the oily core of NEs, with an encapsulation efficiency of 70 %. To validate our treatment strategy in vivo, the 4T1 murine breast cancer model was used. As a result, the comparison of active-targeted and non-targeted NEs revealed that only active-targeted NE could decrease the tumor growth rate.
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Docetaxel , Nanopartículas , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Animales , Femenino , Nanopartículas/química , Línea Celular Tumoral , Ratones , Docetaxel/farmacología , Docetaxel/uso terapéutico , Docetaxel/administración & dosificación , Humanos , Antineoplásicos/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
Alpha-melanocyte stimulating hormone (α-MSH) and its receptor, melanocortin 1 receptor (MC1R), have been proposed as potential target for anti-cancer strategies in melanoma research, due to their tissue specific expression and involvement in melanocyte homeostasis. However, their role in prevention and treatment of melanoma is still debated and controversial. Although a large body of evidence supports α-MSH in preventing melanoma development, some preclinical findings suggest that the α-MSH downstream signalling may promote immune escape and cancer resistance to therapy. Additionally, in metastatic melanoma both MC1R and α-MSH have been reported to be overexpressed at levels much higher than normal cells. Furthermore, targeted therapy (e.g. BRAF inhibition in BRAFV600E mutant tumours) has been shown to enhance this phenomenon. Collectively, these data suggest that targeting MC1R could serve as an approach in the treatment of metastatic melanoma. In this review, we explore the molecular biology of α-MSH with particular emphasis into its tumor-related properties, whilst elaborating the experimental evidence currently available regarding the interplay between α-MSH/MC1R axis, melanoma and antitumor strategies.
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Melanoma , Receptor de Melanocortina Tipo 1 , alfa-MSH , Humanos , Relevancia Clínica , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Receptor de Melanocortina Tipo 1/genéticaRESUMEN
In this study, a circular conjugate of granulocyte colony-stimulating factor (G-CSF) was prepared by conjugating the two end-chains of poly(ethylene glycol) (PEG) to two different sites of the protein. For the orthogonal conjugation, a heterobifunctional PEG chain was designed and synthesized, bearing the dipeptide ZGln-Gly (ZQG) at one end-chain, for transglutaminase (TGase) enzymatic selective conjugation at Lys41 of G-CSF, and an aldehyde group at the opposite end-chain, for N-terminal selective reductive alkylation of the protein. The cPEG-Nter/K41-G-CSF circular conjugate was characterized by physicochemical methods and compared with native G-CSF and the corresponding linear monoconjugates of G-CSF, PEG-Nter-G-CSF, and PEG-K41-G-CSF. The results demonstrated that the circular conjugate had improved physicochemical and thermal stability, prolonged pharmacokinetic interaction, and retained the biological activity of G-CSF. The PEGylation strategy employed in this study has potential applications in the design of novel protein-based therapeutics.
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Aldehídos , Factor Estimulante de Colonias de Granulocitos , Alquilación , Fenómenos Químicos , DipéptidosAsunto(s)
Fenómenos Bioquímicos , Neoplasias , Humanos , Inmunoterapia , Neoplasias/terapia , Microambiente TumoralRESUMEN
Reactive oxygen species, including RNS, contribute to the control of multiple immune cell functions within the tumor microenvironment (TME). Tumor-infiltrating myeloid cells (TIMs) represent the archetype of tolerogenic cells that actively contribute to dismantle effective immunity against cancer. TIMs inhibit T cell functions and promote tumor progression by several mechanisms including the amplification of the oxidative/nitrosative stress within the TME. In tumors, TIM expansion and differentiation is regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced by cancer and immune cells. Nevertheless, the role of GM-CSF in tumors has not yet been fully elucidated. In this study, we show that GM-CSF activity is significantly affected by RNS-triggered post-translational modifications. The nitration of a single tryptophan residue in the sequence of GM-CSF nourishes the expansion of highly immunosuppressive myeloid subsets in tumor-bearing hosts. Importantly, tumors from colorectal cancer patients express higher levels of nitrated tryptophan compared to non-neoplastic tissues. Collectively, our data identify a novel and selective target that can be exploited to remodel the TME and foster protective immunity against cancer.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Biomarcadores , Diferenciación Celular , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Inmunomodulación , Ratones , Neoplasias/patología , Especies de Nitrógeno Reactivo/metabolismo , Transducción de Señal , Microambiente Tumoral/inmunologíaRESUMEN
Immune checkpoint inhibitors are becoming standard treatments in several cancer types, profoundly changing the prognosis of a fraction of patients. Currently, many efforts are being made to predict responders and to understand how to overcome resistance in non-responders. Given the crucial role of myeloid cells as modulators of T effector cell function in tumors, it is essential to understand their impact on the clinical outcome of immune checkpoint blockade and on the mechanisms of immune evasion. In this review we focus on the existing clinical evidence of the relation between the presence of myeloid cell subsets and the response to anti-PD(L)1 and anti-CTLA-4 treatment. We highlight how circulating and tumor-infiltrating myeloid populations can be used as predictive biomarkers for immune checkpoint inhibitors in different human cancers, both at baseline and on treatment. Moreover, we propose to follow the dynamics of myeloid cells during immunotherapy as pharmacodynamic biomarkers. Finally, we provide an overview of the current strategies tested in the clinic that use myeloid cell targeting together with immune checkpoint blockade with the aim of uncovering the most promising approaches for effective combinations.
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Biomarcadores , Inhibidores de Puntos de Control Inmunológico/farmacología , Proteínas de Punto de Control Inmunitario/metabolismo , Células Mieloides/metabolismo , Animales , Estudios Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Terapia Molecular Dirigida , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Resultado del TratamientoRESUMEN
Tumor-associated macrophages (TAM) are regulators of extracellular matrix (ECM) remodeling and metastatic progression, the main cause of cancer-associated death. We found that disabled homolog 2 mitogen-responsive phosphoprotein (DAB2) is highly expressed in tumor-infiltrating TAMs and that its genetic ablation significantly impairs lung metastasis formation. DAB2-expressing TAMs, mainly localized along the tumor-invasive front, participate in integrin recycling, ECM remodeling, and directional migration in a tridimensional matrix. DAB2+ macrophages escort the invasive dissemination of cancer cells by a mechanosensing pathway requiring the transcription factor YAP. In human lobular breast and gastric carcinomas, DAB2+ TAMs correlated with a poor clinical outcome, identifying DAB2 as potential prognostic biomarker for stratification of patients with cancer. DAB2 is therefore central for the prometastatic activity of TAMs. SIGNIFICANCE: DAB2 expression in macrophages is essential for metastasis formation but not primary tumor growth. Mechanosensing cues, activating the complex YAP-TAZ, regulate DAB2 in macrophages, which in turn controls integrin recycling and ECM remodeling in 3-D tissue matrix. The presence of DAB2+ TAMs in patients with cancer correlates with worse prognosis.This article is highlighted in the In This Issue feature, p. 1611.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Neoplasias/genética , Macrófagos Asociados a Tumores/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
Poly(L-glutamic acid)-co-poly(ethylene glycol) block copolymers (PLE-PEG) are here investigated as polymers for conjugation to therapeutic proteins such as granulocyte colony stimulating factor (G-CSF) and human growth hormone (hGH). PLE-PEG block copolymers are able to stabilize and protect proteins from degradation and to prolong their residence time in the blood stream, features that are made possible thanks to PEG's intrinsic properties and the simultaneous presence of the biodegradable anionic PLE moiety. When PLE-PEG copolymers are selectively tethered to the N-terminus of G-CSF and hGH, they yield homogeneous monoconjugates that preserve the protein's secondary structure. During the current study the pharmacokinetics of PLE10-PEG20k-G-CSF and PLE20-PEG20k-G-CSF derivatives and their ability to induce granulopoiesis were, respectively, assessed in Sprague-Dawley rats and in C57BL6 mice. Our results show that the bioavailability and bioactivity of the derivatives are comparable to or better than those of PEG20k-Nter-G-CSF (commercially known as Pegfilgrastim). The therapeutic effects of PLE10-PEG20k-hGH and PLE20-PEG20k-hGH derivatives tested in hypophysectomized rats demonstrate that the presence of a negatively charged PLE block enhances the biological properties of the conjugates additionally with respect to PEG20k-Nter-hGH.
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Ácido Glutámico , Polietilenglicoles , Animales , Ratones , Ratones Endogámicos C57BL , Polímeros , Ratas , Ratas Sprague-DawleyRESUMEN
This work here presented provides insights over a novel biodegradable polymeric nanosystem made of hyaluronic acid and polyarginine for diaminocyclohexane-platinum (DACHPt) encapsulation. Using mild conditions based on ionic gelation technique, monodispersed blank and DACHPt-loaded nanoparticles (NP) with a size of around 200 nm and negative ζ potential (-35 mV) were obtained. The freeze-drying process was optimized to improve the stability and shelf-life of the developed nanoparticles. After reconstitution, nanoparticles maintained their size showing an association efficiency of around 70% and a high drug loading (8%). In vitro cytotoxicity studies revealed that DACHPt-loaded nanoparticles had a superior anticancer activity compared with oxaliplatin solution. The IC50 was reduced by a factor of two in HT-29 cells (IC50 39 µM vs 74 µM, respectively), and resulted almost 1.3 fold lower in B6KPC3 cells (18 µM vs 23 µM respectively). Whereas toxic effects of both drug and DACHPt-loaded nanoparticles were comparable in the A549 cell line (IC50 11 µM vs 12 µM). DACHPt-loaded nanoparticles were also able to modulate immunogenic cell death (ICD) in vitro. After incubation with B6KPC3 cells, an increase in HMGB1 (high-mobility group box 1) production associated with ATP release occurred. Then, in vivo pharmacokinetic studies were performed after intravenous injection (IV) of DACHPt-loaded nanoparticles and oxaliplatin solution in healthy mice (35.9 µg of platinum equivalent/mouse). An AUC six times higher (24 h * mg/L) than the value obtained following the administration of oxaliplatin solution (3.76 h * mg/L) was found. Cmax was almost five times higher than the control (11.4 mg/L for NP vs 2.48 mg/L). Moreover, the reduction in volume of distribution and clearance clearly indicated a more limited tissue distribution. A simulated repeated IV regimen was performed in silico and showed no accumulation of platinum from the nanoparticles. Overall, the proposed approach discloses a novel nano-oncological treatment based on platinum derivative with improved antitumor activity in vitro and in vivo stability as compared to the free drug.
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Antineoplásicos/administración & dosificación , Portadores de Fármacos , Ácido Hialurónico/química , Nanopartículas , Oxaliplatino/administración & dosificación , Péptidos/química , Células A549 , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Composición de Medicamentos , Estabilidad de Medicamentos , Femenino , Células HT29 , Humanos , Inyecciones Intravenosas , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones Endogámicos C57BL , Modelos Biológicos , Oxaliplatino/química , Oxaliplatino/farmacocinética , Distribución TisularRESUMEN
BACKGROUND: Myeloid derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are two of the major players involved in the inhibition of anti-tumor immune response in cancer patients, leading to poor prognosis. Selective targeting of myeloid cells has therefore become an attractive therapeutic strategy to relieve immunosuppression and, in this frame, we previously demonstrated that lipid nanocapsules (LNCs) loaded with lauroyl-modified gemcitabine efficiently target monocytic MDSCs in melanoma patients. In this study, we investigated the impact of the physico-chemical characteristics of LNCs, namely size and surface potential, towards immunosuppressive cell targeting. We exploited myeloid cells isolated from glioblastoma patients, which play a relevant role in the immunosuppression, to demonstrate that tailored nanosystems can target not only tumor cells but also tumor-promoting cells, thus constituting an efficient system that could be used to inhibit their function. RESULTS: The incorporation of different LNC formulations with a size of 100 nm, carrying overall positive, neutral or negative charge, was evaluated on leukocytes and tumor-infiltrating cells freshly isolated from glioblastoma patients. We observed that the maximum LNC uptake was obtained in monocytes with neutral 100 nm LNCs, while positively charged 100 nm LNCs were more effective on macrophages and tumor cells, maintaining at low level the incorporation by T cells. The mechanism of uptake was elucidated, demonstrating that LNCs are incorporated mainly by caveolae-mediated endocytosis. CONCLUSIONS: We demonstrated that LNCs can be directed towards immunosuppressive cells by simply modulating their size and charge thus providing a novel approach to exploit nanosystems for anticancer treatment in the frame of immunotherapy.
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Antimetabolitos Antineoplásicos/química , Desoxicitidina/análogos & derivados , Glioblastoma/tratamiento farmacológico , Inmunosupresores/química , Lípidos/química , Macrófagos/metabolismo , Células Supresoras de Origen Mieloide/metabolismo , Nanocápsulas/química , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Desoxicitidina/química , Desoxicitidina/farmacología , Composición de Medicamentos , Endocitosis , Humanos , Inmunosupresores/farmacología , Inmunoterapia/métodos , Leucocitos/metabolismo , Tamaño de la Partícula , Transducción de Señal , Propiedades de Superficie , GemcitabinaRESUMEN
PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy. Delineating mechanisms of secondary resistance to this strategy is increasingly important. Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb). Sustained IFNß transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC). Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation. Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature. Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy.
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Anticuerpos Monoclonales/farmacología , Interferón Tipo I/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Células Dendríticas/citología , Células Dendríticas/metabolismo , Resistencia a Antineoplásicos , Humanos , Estimación de Kaplan-Meier , Melanoma/tratamiento farmacológico , Melanoma/mortalidad , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Neoplasias/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Cancer immunotherapy relies on either restoring or activating the function of adaptive immune cells, mainly CD8+ T lymphocytes. Despite impressive clinical success, cancer immunotherapy remains ineffective in many patients due to the establishment of tumor resistance, largely dependent on the nature of tumor microenvironment. There are several cellular and molecular mechanisms at play, and the goal is to identify those that are clinically significant. Among the hematopoietic-derived cells, monocytes are endowed with high plasticity, responsible for their pro- and anti-tumoral function. Indeed, monocytes are involved in several cancer-associated processes such as immune-tolerance, metastatic spread, neoangiogenesis, and chemotherapy resistance; on the other hand, by presenting cancer-associated antigens, they can also promote and sustain anti-tumoral T cell response. Recently, by high throughput technologies, new findings have revealed previously underappreciated, profound transcriptional, epigenetic, and metabolic differences among monocyte subsets, which complement and expand our knowledge on the monocyte ontogeny, recruitment during steady state, and emergency hematopoiesis, as seen in cancer. The subdivision into discrete monocytes subsets, both in mice and humans, appears an oversimplification, whereas continuum subsets development is best for depicting the real condition. In this review, we examine the evidences sustaining the existence of a monocyte heterogeneity along with functional activities, at the primary tumor and at the metastatic niche. In particular, we describe how tumor-derived soluble factors and cell-cell contact reprogram monocyte function. Finally, we point out the role of monocytes in preparing and shaping the metastatic niche and describe relevant targetable molecules altering monocyte activities. We think that exploiting monocyte complexity can help identifying key pathways important for the treatment of cancer and several conditions where these cells are involved.
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Monocitos/fisiología , Animales , Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Plasticidad de la Célula , Humanos , Inmunoterapia , Monocitos/inmunología , Células Supresoras de Origen Mieloide/fisiología , Neoplasias/inmunología , Neoplasias/terapiaRESUMEN
This unit presents methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo in mice, as well as in biological samples from cancer patients. These methods could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover, they could be useful to assess the influence exerted by genetic modifications, chemical inhibitors, and drugs on immune suppressive pathways © 2018 by John Wiley & Sons, Inc.
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Células Supresoras de Origen Mieloide/inmunología , Animales , Citometría de Flujo , Humanos , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Supresoras de Origen Mieloide/efectos de los fármacosRESUMEN
Myeloid-Derived Suppressor Cells (MDSCs), immunosuppressive cells that promote tumor growth, represent an attractive target in cancer immunotherapy. However, the clinical success of this strategy is limited by the lack of efficient drug delivery vehicles targeting this cell compartment. The objective of this work was to develop a delivery carrier, multilayer polymer nanocapsules, with the capacity to co-encapsulate two types of immunomodulatory drugs, a chemokine and an RNAi sequence, aimed at reverting MDSC-mediated immunosuppression. The chemokine CCL2, intended to attract monocyte-macrophage MDSCs, was encapsulated within the L2 inverse micellar aqueous domains of the lipid core of these nanocapsules. On the other hand, two different RNAi sequences that modulate the CCAAT/enhancer-binding protein beta (C/EBPß) pathway, shC/EBPß and miR 142-3p, were successfully associated to their polymer shell. These RNAi sequences were covered by subsequent layers of polyarginine and hyaluronic acid, thereby creating multi-layered assemblies that protected them and facilitated their targeted delivery. The in vitro studies performed in primary MDSCs cultures showed the capacity of miR 142-3p-loaded nanocapsules to reduce the highly immunosuppressive monocyte-macrophage subset. Additionally, the encapsulation of CCL2 within the nanocapsules induced a potent monocyte-macrophage chemoattraction that could be used to direct the therapy to these cell subsets. Finally, in vitro and in vivo studies showed the capacity of shC/EBPß-loaded nanocapsules to downregulate C/EBPß levels in MDSCs and to reduce monocyte differentiation into tumor-associated macrophages in an MCA-203 fibrosarcoma mice model. In conclusion, the multilayer polymer nanocapsules described here are efficient vehicles for the co-delivery of proteins and RNA, and are potential candidates as nanomedicines for the modulation of MDSCs.
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Quimiocina CCL2/administración & dosificación , Factores Inmunológicos/administración & dosificación , Células Supresoras de Origen Mieloide/efectos de los fármacos , Nanocápsulas/química , Péptidos/química , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular Tumoral , Células Cultivadas , Quimiocina CCL2/farmacología , Humanos , Factores Inmunológicos/farmacología , Ratones , Ratones Endogámicos C57BL , Células Supresoras de Origen Mieloide/inmunología , Células RAW 264.7 , Interferencia de ARN , ARN Interferente Pequeño/farmacologíaRESUMEN
In this work, a novel lipophilic 5-fluorouracil (5-FU) derivative was synthesised and encapsulated into lipid nanocapsules (LNC). 5-FU was modified with lauric acid to give a lipophilic mono-lauroyl-derivative (5-FU-C12, MW of about 342 g/mol, yield of reaction 70%). 5-FU-C12 obtained was efficiently encapsulated into LNC (encapsulation efficiency above 90%) without altering the physico-chemical characteristics of LNC. The encapsulation of 5-FU-C12 led to an increased stability of the drug when in contact with plasma being the drug detectable until 3 h following incubation. Cytotoxicity assay carried out using MTS on 2D cell culture showed that 5-FU-C12-loaded LNC had an enhanced cytotoxic effect on glioma (9L) and human colorectal (HTC-116) cancer cell line in comparison with 5-FU or 5-FU-C12. Then, HCT-116 tumour spheroids were cultivated and the reduction of spheroid volume was measured following treatment with drug-loaded LNC and drugs alone. Similar reduction on spheroids volume was observed following the treatment with drug-loaded LNC, 5-FU-C12 and 5-FU alone, while blank LNC displayed a reduction in cell viability only at high concentration. Globally, our data suggest that the encapsulation increased the activity of the 5-FU-C12. However, in-depth evaluations of LNC permeability into spheroids are needed to disclose the potential of these nanosystems for cancer treatment.
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Antineoplásicos/administración & dosificación , Portadores de Fármacos/química , Fluorouracilo/análogos & derivados , Fluorouracilo/administración & dosificación , Lípidos/química , Nanocápsulas/química , Esferoides Celulares/efectos de los fármacos , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Composición de Medicamentos , Sistemas de Liberación de Medicamentos/métodos , Fluorouracilo/farmacología , Células HCT116 , Humanos , Esferoides Celulares/patologíaRESUMEN
Peptides represent a promising therapeutic class with the potential to alleviate many severe diseases. A key limitation of these active molecules relies on the difficulties for their efficient oral administration. The objective of this work has been the rational design of polymer nanocapsules (NCs) intended for the oral delivery of peptide drugs. For this purpose, we selected insulin glulisine as a model peptide. The polymer shell of the NCs was made of a single layer of protamine, a cationic polypeptide selected for its cell penetration properties, or a double protamine/polysialic acid (PSA) layer. Insulin glulisine-loaded protamine and protamine/PSA NCs, prepared by the solvent displacement method, exhibited a size that varied in the range of 200-400â¯nm and a neutral surface charge (from +8â¯mV to -6â¯mV), depending on the formulation. The stability of the encapsulated peptide was assessed using circular dichroism and an in vitro cell activity study. Colloidal stability studies were also performed in simulated intestinal media containing enzymes and the results indicated that protamine NCs were stable and able to protect insulin from the harsh intestinal environment, and that this capacity could be further enhanced with a double PSA-Protamine layer. These NCs were freeze-dried and stored at room temperature without alteration of the physicochemical properties. When the insulin-loaded protamine NCs were administered intra-intestinally to diabetic rats (12â¯h fasting) it resulted in a prolonged glucose reduction (60%) as compared to the control insulin solution. This work raises prospects that protamine NCs may have a potential as oral peptide delivery nanocarriers.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Insulina/análogos & derivados , Nanocápsulas/química , Protaminas/química , Ácidos Siálicos/química , Administración Oral , Animales , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Células Hep G2 , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/administración & dosificación , Insulina/uso terapéutico , Masculino , Nanocápsulas/ultraestructura , Ratas Sprague-DawleyRESUMEN
Myeloid cells play a key role in tumor progression and metastasis by providing nourishment and immune protection, as well as facilitating cancer invasion and seeding to distal sites. Although advances have been made in understanding the biology of these tumor-educated myeloid cells (TEMCs), their intrinsic plasticity challenges our further understanding of their biology. Indeed, in vitro experiments only mimic the in vivo setting, and current gene-knockout technologies do not allow the simultaneous, temporally controlled, and cell-specific silencing of multiple genes or pathways. In this article, we describe the 4PD nanoplatform, which allows the in vivo preferential transfection and in vivo tracking of TEMCs with the desired RNAs. This platform is based on the conjugation of CD124/IL-4Rα-targeting peptide with G5 PAMAM dendrimers as the loading surface and can convey therapeutic or experimental RNAs of interest. When injected i.v. in mice bearing CT26 colon carcinoma or B16 melanoma, the 4PD nanoparticles predominantly accumulate at the tumor site, transfecting intratumoral myeloid cells. The use of 4PD to deliver a combination of STAT3- and C/EBPß-specific short hairpin RNA or miR-142-3p confirmed the importance of these genes and microRNAs in TEMC biology and indicates that silencing of both genes is necessary to increase the efficacy of immune interventions. Thus, the 4PD nanoparticle can rapidly and cost effectively modulate and assess the in vivo function of microRNAs and mRNAs in TEMCs.
Asunto(s)
Dendrímeros/metabolismo , Silenciador del Gen , Células Mieloides/metabolismo , Nanotecnología/métodos , Animales , Línea Celular Tumoral , Neoplasias del Colon , Dendrímeros/administración & dosificación , Subunidad alfa del Receptor de Interleucina-4/inmunología , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Melanoma Experimental , Ratones , MicroARNs , Células Mieloides/inmunología , Nanopartículas/administración & dosificación , Nanopartículas/metabolismo , Nanotecnología/normas , Receptores de Interleucina-4/inmunología , Receptores de Interleucina-4/metabolismoRESUMEN
Bone marrow microenvironment is fundamental for hematopoietic homeostasis. Numerous efforts have been made to reproduce or manipulate its activity to facilitate engraftment after hematopoietic stem cell transplantation but clinical results remain unconvincing. This probably reflects the complexity of the hematopoietic niche. Recent data have demonstrated the fundamental role of stromal and myeloid cells in regulating hematopoietic stem cell self-renewal and mobilization in the bone marrow. In this study we unveil a novel interaction by which bone marrow mesenchymal stromal cells induce the rapid differentiation of CD11b+ myeloid cells from bone marrow progenitors. Such an activity requires the expression of nitric oxide synthase-2. Importantly, the administration of these mesenchymal stromal cell-educated CD11b+ cells accelerates hematopoietic reconstitution in bone marrow transplant recipients. We conclude that the liaison between mesenchymal stromal cells and myeloid cells is fundamental in hematopoietic homeostasis and suggests that it can be harnessed in clinical transplantation.
Asunto(s)
Células de la Médula Ósea/metabolismo , Diferenciación Celular , Hematopoyesis , Células Madre Mesenquimatosas/metabolismo , Células Mieloides/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Células de la Médula Ósea/citología , Antígeno CD11b/metabolismo , Autorrenovación de las Células , Trasplante de Células Madre Hematopoyéticas , Homeostasis , Humanos , Células Madre Mesenquimatosas/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/citología , Óxido Nítrico Sintasa de Tipo II/genéticaRESUMEN
The aim of this work was to rationally design and characterize nanocapsules (NCs) composed of an oily core and a polyarginine (PARG) shell, intended for oral peptide delivery. The cationic polyaminoacid, PARG, and the oily core components were selected based on their penetration enhancing properties. Insulin was adopted as a model peptide to assess the performance of the NCs. After screening numerous formulation variables, including different oils and surfactants, we defined a composition consisting of oleic acid, sodium deoxycholate (SDC) and Span 80. This selected NCs composition, produced by the solvent displacement technique, exhibited the following key features: (i) an average size of 180nm and a low polydispersity (0.1), (ii) a high insulin association efficacy (80-90% AE), (iii) a good colloidal stability upon incubation in simulated intestinal fluids (SIF, FaSSIF-V2, FeSSIF-V2), and (iv) the capacity to control the release of the associated insulin for >4h. Furthermore, using the Caco-2 model cell line, PARG nanocapsules were able to interact with the enterocytes, and reversibly modify the TEER of the monolayer. Both cell adhesion and membrane permeabilization could account for the pronounced transport of the NCs-associated insulin (3.54%). This improved interaction was also visualized by confocal fluorescent microscopy following oral administration of PARG nanocapsulesto mice. Finally, in vivo efficacy studies performed in normoglycemic rats showed a significant decrease in their plasma glucose levels after treatment. In conclusion, here we disclose key formulation elements for making possible the oral administration of peptides.