RESUMO
MicroRNAs (miRNAs) regulate gene expression by post-transcriptional inhibition of target genes. Proangiogenic small extracellular vesicles (sEVs; popularly identified with the name "exosomes") with a composite cargo of miRNAs are secreted by cultured stem cells and present in human biological fluids. Lipid nanoparticles (LNPs) represent an advanced platform for clinically approved delivery of RNA therapeutics. In this study, we aimed to (1) identify the miRNAs responsible for sEV-induced angiogenesis; (2) develop the prototype of bioinspired "artificial exosomes" (AEs) combining LNPs with a proangiogenic miRNA, and (3) validate the angiogenic potential of the bioinspired AEs. We previously reported that human sEVs from bone marrow (BM)-CD34+ cells and pericardial fluid (PF) are proangiogenic. Here, we have shown that sEVs secreted from saphenous vein pericytes and BM mesenchymal stem cells also promote angiogenesis. Analysis of miRNA datasets available in-house or datamined from GEO identified the let-7 family as common miRNA signature of the proangiogenic sEVs. LNPs with either hsa-let-7b-5p or cyanine 5 (Cy5)-conjugated Caenorhabditis elegans miR-39 (Cy5-cel-miR-39; control miRNA) were prepared using microfluidic micromixing. let-7b-5p-AEs did not cause toxicity and transferred functionally active let-7b-5p to recipient endothelial cells (ECs). let-7b-AEs also improved EC survival under hypoxia and angiogenesis in vitro and in vivo. Bioinspired proangiogenic AEs could be further developed into innovative nanomedicine products targeting ischemic diseases.
Assuntos
Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Lipossomos/química , MicroRNAs/metabolismo , Nanopartículas/química , Neovascularização Fisiológica , Líquido Pericárdico/fisiologia , Animais , Exossomos/genética , Vesículas Extracelulares/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Camundongos , MicroRNAs/genéticaRESUMO
Ovarian cancer is an aggressive tumor owing to its ability to metastasize from stage II onward. Herein, lipid nanoparticles (LNPs) that encapsulate combination of small interfering RNAs (siRNAs), polo-like kinase-1 (PLK1), and eukaryotic translation-initiation factor 3c (eIF3c), to target different cellular pathways essential for ovarian cancer progression are generated. The LNPs are further modified with hyaluronan (tNPs) to target cluster of differentiation 44 (CD44) expressing cells. Interestingly, hyaluronan-coated LNPs (tNPs) prolong functional activity and reduce growth kinetics of spheroids in in vitro assay as compared to uncoated LNPs (uNPs) due to ≈1500-fold higher expression of CD44. Treatment of 2D and 3D cultured ovarian cancer cells with LNPs encapsulating both siRNAs result in 85% cell death and robust target gene silencing. In advanced orthotopic ovarian cancer model, intraperitoneal administration of LNPs demonstrates CD44 specific tumor targeting of tNPs compared to uNPs and robust gene silencing in tissues involved in ovarian cancer pathophysiology. At very low siRNA dose, enhanced overall survival of 60% for tNPs treated mice is observed compared to 10% and 20% for single siRNA-, eIF3c-tNP, and PLK1-tNP treatment groups, respectively. Overall, LNPs represent promising platform in the treatment of advanced ovarian cancer by improving median- and overall-survival.
Assuntos
Nanopartículas , Neoplasias Ovarianas , Animais , Feminino , Inativação Gênica , Humanos , Lipídeos , Camundongos , RNA Interferente PequenoRESUMO
Despite progress in systemic small interfering RNA (siRNA) delivery to the liver and to solid tumors, systemic siRNA delivery to leukocytes remains challenging. The ability to silence gene expression in leukocytes has great potential for identifying drug targets and for RNAi-based therapy for leukocyte diseases. However, both normal and malignant leukocytes are among the most difficult targets for siRNA delivery as they are resistant to conventional transfection reagents and are dispersed in the body. We used mantle cell lymphoma (MCL) as a prototypic blood cancer for validating a novel siRNA delivery strategy. MCL is an aggressive B-cell lymphoma that overexpresses cyclin D1 with relatively poor prognosis. Down-regulation of cyclin D1 using RNA interference (RNAi) is a potential therapeutic approach to this malignancy. Here, we designed lipid-based nanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies that are specifically taken up by human MCL cells in the bone marrow of xenografted mice. When loaded with siRNAs against cyclin D1, CD38-targeted LNPs induced gene silencing in MCL cells and prolonged survival of tumor-bearing mice with no observed adverse effects. These results highlight the therapeutic potential of cyclin D1 therapy in MCL and present a novel RNAi delivery system that opens new therapeutic opportunities for treating MCL and other B-cell malignancies.
Assuntos
Linfócitos B/imunologia , Linfoma de Células B/terapia , Linfoma de Célula do Manto/terapia , Nanomedicina/métodos , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , ADP-Ribosil Ciclase 1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Ciclina D1/genética , Regulação para Baixo , Inativação Gênica , Humanos , Lipídeos , Linfoma de Células B/imunologia , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/imunologia , Camundongos , Nanopartículas , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The immunocompability of polyelectrolyte capsules synthesized by layer-by-layer deposition has been investigated. Capsules of different architecture and composed of either non-degradable or biodegradable polymers, with either positively or negatively charged outer surface, and with micrometer size, have been used, and the capsule uptake by different cell lines has been studied and quantified. Immunocompatibility studies were performed with peripheral blood mononuclear cells (PBMCs). Data demonstrate that incubation with capsules, at concentrations relevant for practical applications, did not result in a reduced viability of cells, as it did not show an increased apoptosis. Presence of capsules also did not result in an increased expression of TNF-α, as detected with antibody staining, as well as at mRNA level. It also did not result in increased expression of IL-6, as detected at mRNA level. These results indicate that the polyelectrolyte capsules used in this study are immunocompatible.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Polieletrólitos/efeitos adversos , Células A549 , Apoptose/efeitos dos fármacos , Cápsulas , Linhagem Celular , Células Cultivadas , Humanos , Leucócitos Mononucleares/metabolismo , Polieletrólitos/farmacocinética , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines have emerged as an effective vaccination strategy. Although currently applied toward viral pathogens, data concerning the platform's effectiveness against bacterial pathogens are limited. Here, we developed an effective mRNA-LNP vaccine against a lethal bacterial pathogen by optimizing mRNA payload guanine and cytosine content and antigen design. We designed a nucleoside-modified mRNA-LNP vaccine based on the bacterial F1 capsule antigen, a major protective component of Yersinia pestis, the etiological agent of plague. Plague is a rapidly deteriorating contagious disease that has killed millions of people during the history of humankind. Now, the disease is treated effectively with antibiotics; however, in the case of a multiple-antibiotic-resistant strain outbreak, alternative countermeasures are required. Our mRNA-LNP vaccine elicited humoral and cellular immunological responses in C57BL/6 mice and conferred rapid, full protection against lethal Y. pestis infection after a single dose. These data open avenues for urgently needed effective antibacterial vaccines.
Assuntos
Vacina contra a Peste , Peste , Yersinia pestis , Camundongos , Animais , Peste/prevenção & controle , Vacina contra a Peste/genética , Proteínas de Bactérias/genética , Camundongos Endogâmicos C57BL , Yersinia pestis/genética , Antígenos de Bactérias/genéticaRESUMO
The potential of microtubule-associated protein targets for cancer therapeutics remains largely unexplored due to the lack of target-specific agents. Here, we explored the therapeutic potential of targeting cytoskeleton-associated protein 5 (CKAP5), an important microtubule-associated protein, with CKAP5-targeting siRNAs encapsulated in lipid nanoparticles (LNPs). Our screening of 20 solid cancer cell lines demonstrated selective vulnerability of genetically unstable cancer cell lines in response to CKAP5 silencing. We identified a highly responsive chemo-resistant ovarian cancer cell line, in which CKAP5 silencing led to significant loss in EB1 dynamics during mitosis. Last, we demonstrated the therapeutic potential in an in vivo ovarian cancer model, showing 80% survival rate of siCKAP5 LNPs-treated animals. Together, our results highlight the importance of CKAP5 as a therapeutic target for genetically unstable ovarian cancer and warrants further investigation into its mechanistic aspects.
Assuntos
Nanopartículas , Neoplasias Ovarianas , Humanos , Animais , Feminino , Inativação Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética , Microtúbulos/metabolismo , Neoplasias Ovarianas/genéticaRESUMO
Ionizable lipid-based nanoparticles (LNPs) are the most advanced non-viral drug delivery systems for RNA therapeutics and vaccines. However, cell type-specific, extrahepatic mRNA delivery is still a major hurdle, hampering the development of novel therapeutic modalities. Herein, a novel ionizable lipid library is synthesized by modifying hydrophobic tail chains and linkers. Combined with other helper lipids and utilizing a microfluidic mixing approach, stable LNPs are formed. Using Luciferase-mRNA, mCherry mRNA, and Cre mRNA together with a TdTomato animal model, superior lipids forming LNPs for potent cell-type specific mRNA delivery are identified. In vitro assays concluded that combining branched ester tail chains with hydroxylamine linker negatively affects mRNA delivery efficiency. In vivo studies identify Lipid 23 as a liver-trophic, superior mRNA delivery lipid and Lipid 16 as a potent cell type-specific ionizable lipid for the CD11bhi macrophage population without an additional targeting moiety. Finally, in vivo mRNA delivery efficiency and toxicity of these LNPs are compared with SM-102-based LNP (Moderna's LNP formulation) and are shown to be cell-specific compared to SM-102-based LNPs. Overall, this study suggests that a structural combination of tail and linker can drive a novel functionality of LNPs in vivo.
Assuntos
Nanopartículas , Animais , RNA Mensageiro/genética , Nanopartículas/química , Lipídeos/químicaRESUMO
Multiple myeloma (MM) is a cancer of differentiated plasma cells that occurs in the bone marrow (BM). Despite the recent advancements in drug development, most patients with MM eventually relapse and the disease remains incurable. RNA therapy delivered via lipid nanoparticles (LNPs) has the potential to be a promising cancer treatment, however, its clinical implementation is limited due to inefficient delivery to non-hepatic tissues. Here, targeted (t)LNPs designed for delivery of RNA payload to MM cells are presented. The tLNPs consist of a novel ionizable lipid and are coated with an anti-CD38 antibody (αCD38-tLNPs). To explore their therapeutic potential, it is demonstrated that LNPs encapsulating small interference RNA (siRNA) against cytoskeleton-associated protein 5 (CKAP5) lead to a ≈90% decrease in cell viability of MM cells in vitro. Next, a new xenograft MM mouse model is employed, which clinically resembles the human disease and demonstrates efficient homing of MM cells to the BM. Specific delivery of αCD38-tLNPs to BM-residing and disseminated MM cells and the improvement in therapeutic outcome of MM-bearing mice treated with αCD38-tLNPs-siRNA-CKAP5 are shown. These results underscore the potential of RNA therapeutics for treatment of MM and the importance of developing effective targeted delivery systems and reliable preclinical models.
Assuntos
Mieloma Múltiplo , Humanos , Animais , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Medula Óssea , Recidiva Local de Neoplasia , RNA Interferente Pequeno/uso terapêuticoRESUMO
Understanding the interactions of nanomaterials with the immune system is essential for the engineering of new macromolecular systems for in vivo applications. Systematic study of immune activation is challenging due to the complex structure of most macromolecular probes. We present here the use of engineered gold nanoparticles to determine the sole effect of hydrophobicity on the immune response of splenocytes. The gene expression profile of a range of cytokines (immunological reporters) was analyzed against the calculated log P of the nanoparticle headgroups, with an essentially linear increase in immune activity with the increase in hydrophobicity observed in vitro. Consistent behavior was observed with in vivo mouse models, demonstrating the importance of hydrophobicity in immune system activation.
Assuntos
Citocinas/imunologia , Ouro/química , Nanopartículas Metálicas/química , Baço/imunologia , Animais , Citocinas/genética , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Baço/citologiaRESUMO
Chemo-immunotherapy is a combination of "standard-of-care" chemotherapy with immunotherapy and it is considered the most advanced therapeutic modality for various types of cancers. However, many cancer patients still poorly respond to current regimen of chemo-immunotherapy and suggest nanotherapeutics as a boosting agent. Recently, heme oxygenase-1 (HO1) is shown to act as an immunotherapeutic molecule in tumor myeloid cells, in addition to general chemoresistance function in cancer cells suggesting that HO1-targeted therapeutics can become a novel, optimal strategy for boosting chemo-immunotherapy in the clinic. Currently the available HO1-inhibitors demonstrate serious adverse effects in clinical use. Herein, tumor myeloid cell- and cancer cell-dual targeted HO1-inhibiting lipid nanotherapeutic boost (T-iLNTB) is developed using RNAi-loaded lipid nanoparticles. T-iLNTB-mediated HO1-inhibition sensitizes cancer cells to "standard-of-care" chemotherapeutics by increasing immunogenic cell death, and directly reprograms tumor myeloid cells with distinguished phenotype. Furthermore, tumor myeloid cell reprogramming by T-iLNTB induces CD8+ cytotoxic T cell recruitment, which drives "Cold-to-Hot" transition and correlates with improved responsiveness to immune checkpoint inhibitor in combination therapy. Finally, ex vivo study proves that HO1-inhibition directly affects tumor macrophage differentiation. This study demonstrates the potential of T-iLNTB as a novel therapeutic modality for boosting chemo-immunotherapy.
Assuntos
Nanopartículas , Neoplasias , Humanos , Imunoterapia , Lipídeos , Lipossomos , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.
Assuntos
Portadores de Fármacos/toxicidade , Fibroblastos/efeitos dos fármacos , Lipídeos/toxicidade , Nanopartículas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia , HumanosRESUMO
The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4ß7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.
Assuntos
Colite/terapia , Inativação Gênica , Nanopartículas/química , RNA Interferente Pequeno/farmacologia , Animais , Colite/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfa4/química , Integrina alfa4/genética , Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/genética , Lipídeos/química , Lipídeos/farmacologia , Fígado/efeitos dos fármacos , Camundongos , Nanopartículas/uso terapêutico , RNA Interferente Pequeno/genéticaRESUMO
Currently there are no specific therapies addressing the distinctive biology of human papillomavirus (HPV)-induced cancer approved for clinical use. Short interfering RNA (siRNA) has much potential for therapeutic manipulation of HPV E6/E7 oncoproteins. Lipid-based nanoparticles (LNPs) can be utilized for systemic transportation and delivery of siRNA at target site. We recently developed a recombinant protein linker that enables uniform conjugation of targeting antibodies to the LNPs. Herein, we demonstrate the therapeutic efficacy of anti-E6/E7 siRNA delivered via targeted LNPs (tLNPs) in a xenograft HPV-positive tumor model. We show that anti-epidermal growth factor receptor (EGFR) antibodies, anchored to the LNPs as targeting moieties, facilitate cargo delivery but also mediate anti-tumor activity. Treatment with siE6 via tLNPs resulted in 50% greater reduction of tumor volume compared to treatment with siControl encapsulated in isoLNPs (coated with isotype control antibodies). We demonstrate superior suppression of HPV oncogenes and higher induction of apoptosis by the tLNPs both in vitro and in vivo. Altogether, the coupling of inhibitory siE6 with anti-EGFR antibodies, that further elicited anti-tumor effects, successfully restricted tumor progression. This system that combines potent siRNA and therapeutically functional tLNPs can be modulated against various cancer models.
Assuntos
Neoplasias de Cabeça e Pescoço , Nanopartículas , Proteínas Oncogênicas Virais , Neoplasias do Colo do Útero , Linhagem Celular Tumoral , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Lipídeos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , RNA Interferente Pequeno , Proteínas RepressorasRESUMO
The failure of cancer therapies in clinical settings is often attributed to the lack of a relevant tumor model and pathological heterogeneity across tumor types in the clinic. The objective of this study was to develop a robust in vivo tumor model that better represents clinical tumors for the evaluation of anti-cancer therapies. We successfully developed a simple mouse tumor model based on 3D cell culture by injecting a single spheroid and compared it to a tumor model routinely used by injecting cell suspension from 2D monolayer cell culture. We further characterized both tumors with cellular markers for the presence of myofibroblasts, pericytes, endothelial cells and extracellular matrix to understand the role of the tumor microenvironment. We further investigated the effect of chemotherapy (doxorubicin), nanomedicine (Doxil®), biological therapy (Avastin®) and their combination. Our results showed that the substantial blood vasculature in the 3D spheroid model enhances the delivery of Doxil® by 2.5-fold as compared to the 2D model. Taken together, our data suggest that the 3D tumors created by simple subcutaneous spheroid injection represents a robust and more vascular murine tumor model which is a clinically relevant platform to test anti-cancer therapy in solid tumors.
Assuntos
Bevacizumab/farmacologia , Doxorrubicina/análogos & derivados , Neoplasias Experimentais , Neovascularização Patológica , Neoplasias Ovarianas , Esferoides Celulares , Animais , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Feminino , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Polietilenoglicóis/farmacologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-alpha blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-alpha in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-alpha suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the anti-inflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-alpha suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.
Assuntos
Ceramidas/imunologia , Interleucina-10/biossíntese , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Anti-Inflamatórios/imunologia , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-10/genética , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Camundongos , RNA Mensageiro/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Interferon regulatory factor 8 (IRF8) protein plays a critical role in the differentiation, polarization, and activation of mononuclear phagocytic cells. In light of previous studies, we explored the therapeutic potential of IRF8 inhibition as immunomodulatory therapy for inflammatory bowel disease (IBD). To this end, we utilized siRNA-loaded lipid-based nanoparticles (siLNPs) and demonstrated a â¼90% reduction of IRF8 mRNA levels in vitro (PV < 0.0001), alongside a notable reduction in IRF8 protein. Moreover, silencing IRF8 ex vivo in splenocytes lead to a profound downregulation of IRF8 protein, followed by an immunomodulatory effect, as represented by a decrease in the secretion of TNFα, IL6 and IL12/IL23 (IL12p40) proinflammatory cytokines (PV = 0.0045, 0.0330, <0.0001, respectively). In order to silence IRF8 in vivo, selectively in inflammatory leukocytes, we used siLNPs that were coated with anti-Ly6C antibodies via our recently published ASSET targeting approach. Through this strategy, we have demonstrated a selective binding of the targeted-LNPs (T-LNPs) to Ly6C + inflammatory leukocytes. Finally, an immunomodulatory effect was demonstrated in vivo in an IBD mouse model with a profound decrease of TNFα, IL6, IL12/IL23, and IL1ß pro-inflammatory cytokines (n = 5, PV < 0.0001, <0.0001, <0.0001, 0.02, respectively) and an improvement of colon-morphology as assessed by colon-length measurements and colonoscopy (PV < 0.0001). Overall, using antibody-targeted siLNPs, we showed a notable reduction of IRF8 mRNA and protein and demonstrated a targeted immunomodulation therapeutic effect ex vivo and in vivo, in the DSS colitis model. We claim that a selective silencing of IRF8 in inflammatory leukocytes (such as Ly6C+) may serve as a therapeutic approach for treating inflammatory disorders.
Assuntos
Anti-Inflamatórios/metabolismo , Doenças Inflamatórias Intestinais/terapia , Fatores Reguladores de Interferon/genética , Leucócitos/metabolismo , Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Colesterol/química , Modelos Animais de Doenças , Feminino , Terapia Genética , Humanos , Imunomodulação , Fatores Reguladores de Interferon/metabolismo , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Células RAW 264.7 , Propriedades de Superfície , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Therapeutic alteration of gene expression in vivo can be achieved by delivering nucleic acids (e.g., mRNA, siRNA) using nanoparticles. Recent progress in modified messenger RNA (mmRNA) synthesis facilitated the development of lipid nanoparticles (LNPs) loaded with mmRNA as a promising tool for in vivo protein expression. Although progress have been made with mmRNA-LNPs based protein expression in hepatocytes, cell specificity is still a major challenge. Moreover, selective protein expression is essential for an improved therapeutic effect, due to the heterogeneous nature of diseases. Here, we present a precision protein expression strategy in Ly6c+ inflammatory leukocytes in inflammatory bowel disease (IBD) induced mice. We demonstrate a therapeutic effect in an IBD model by targeted expression of the interleukin 10 in Ly6c+ inflammatory leukocytes. A selective mmRNA expression strategy has tremendous therapeutic potential in IBD and can ultimately become a novel therapeutic modality in many other diseases.
Assuntos
Sistemas de Liberação de Medicamentos , Interleucina-10/uso terapêutico , Leucócitos/metabolismo , Animais , Antígenos Ly/metabolismo , Colite/tratamento farmacológico , Colite/patologia , Modelos Animais de Doenças , Feminino , Lipídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Nanopartículas/ultraestrutura , Células RAW 264.7 , RNA Mensageiro/metabolismoRESUMO
Previous studies have identified relevant genes and signalling pathways that are hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as short interfering RNAs1-3 (siRNAs), opens up opportunities for personalized medicine. Yet, although major progress has been made in developing siRNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting4-8, their clinical translation has not occurred. This is in part because of the massive development and production requirements and the high batch-to-batch variability of current technologies, which rely on chemical conjugation. Here we present a self-assembled modular platform that enables the construction of a theoretically unlimited repertoire of siRNA targeted carriers. The self-assembly of the platform is based on a membrane-anchored lipoprotein that is incorporated into siRNA-loaded lipid nanoparticles that interact with the antibody crystallizable fragment (Fc) domain. We show that a simple switch of eight different mAbs redirects the specific uptake of siRNAs by diverse leukocyte subsets in vivo. The therapeutic potential of the platform is demonstrated in an inflammatory bowel disease model by targeting colon macrophages to reduce inflammatory symptoms, and in a Mantle Cell Lymphoma xenograft model by targeting cancer cells to induce cell death and improve survival. This modular delivery platform represents a milestone in the development of precision medicine.
Assuntos
Colite/terapia , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi/métodos , Animais , Anticorpos Monoclonais/química , Colite/genética , Feminino , Lipoproteínas/química , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/uso terapêuticoRESUMO
Glioblastoma multiforme (GBM) is one of the most infiltrating, aggressive, and poorly treated brain tumors. Progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, yet the vast majority of these leading drug candidates for the treatment of GBM are ineffective, mainly due to restricted passages across the blood-brain barrier. Nanoparticles have been emerged as a promising platform to treat different types of tumors due to their ability to transport drugs to target sites while minimizing adverse effects. Herein, we devised a localized strategy to deliver RNA interference (RNAi) directly to the GBM site using hyaluronan (HA)-grafted lipid-based nanoparticles (LNPs). These LNPs having an ionized lipid were previously shown to be highly effective in delivering small interfering RNAs (siRNAs) into various cell types. LNP's surface was functionalized with hyaluronan (HA), a naturally occurring glycosaminoglycan that specifically binds the CD44 receptor expressed on GBM cells. We found that HA-LNPs can successfully bind to GBM cell lines and primary neurosphers of GBM patients. HA-LNPs loaded with Polo-Like Kinase 1 (PLK1) siRNAs (siPLK1) dramatically reduced the expression of PLK1 mRNA and cumulated in cell death even under shear flow that simulate the flow of the cerebrospinal fluid compared with control groups. Next, a human GBM U87MG orthotopic xenograft model was established by intracranial injection of U87MG cells into nude mice. Convection of Cy3-siRNA entrapped in HA-LNPs was performed, and specific Cy3 uptake was observed in U87MG cells. Moreover, convection of siPLK1 entrapped in HA-LNPs reduced mRNA levels by more than 80% and significantly prolonged survival of treated mice in the orthotopic model. Taken together, our results suggest that RNAi therapeutics could effectively be delivered in a localized manner with HA-coated LNPs and ultimately may become a therapeutic modality for GBM.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Glioblastoma/genética , Glioblastoma/terapia , Ácido Hialurônico/química , Lipídeos/química , Nanopartículas/química , Terapêutica com RNAi/métodos , Animais , Transporte Biológico , Proteínas de Ciclo Celular/deficiência , Morte Celular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Gradação de Tumores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Proto-Oncogênicas/deficiência , Quinase 1 Polo-LikeRESUMO
Modulating T cell function by down-regulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity, and viral infections. Hematopoietic cells, in general, and primary T lymphocytes, in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Herein, we describe a novel strategy to specifically deliver siRNAs to murine CD4(+) T cells using targeted lipid nanoparticles (tLNPs). To increase the efficacy of siRNA delivery, these tLNPs have been formulated with several lipids designed to improve the stability and efficacy of siRNA delivery. The tLNPs were surface-functionalized with anti-CD4 monoclonal antibody to permit delivery of the siRNAs specifically to CD4(+) T lymphocytes. Ex vivo, tLNPs demonstrated specificity by targeting only primary CD4(+) T lymphocytes and no other cell types. Systemic intravenous administration of these particles led to efficient binding and uptake into CD4(+) T lymphocytes in several anatomical sites including the spleen, inguinal lymph nodes, blood, and the bone marrow. Silencing by tLNPs occurs in a subset of circulating and resting CD4(+) T lymphocytes. Interestingly, we show that tLNP internalization and not endosome escape is a fundamental event that takes place as early as 1 h after systemic administration and determines tLNPs' efficacy. Taken together, these results suggest that tLNPs may open new avenues for the manipulation of T cell functionality and may help to establish RNAi as a therapeutic modality in leukocyte-associated diseases.