RESUMO
Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.
Assuntos
Endocitose/genética , Técnicas de Transferência de Genes , Lipídeos/genética , RNA Interferente Pequeno/genética , Animais , Ouro/administração & dosagem , Ouro/química , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Lipídeos/administração & dosagem , Lipídeos/química , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Camundongos , Microscopia Eletrônica , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/químicaRESUMO
Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8(+) T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Inativação Gênica , Receptor de Morte Celular Programada 1/imunologia , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/imunologia , Eletroporação , Humanos , Imunoterapia , Leucócitos Mononucleares/imunologia , Lipídeos/imunologia , Ativação Linfocitária/imunologia , Nanopartículas , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Practical methods for enhancing protein production in vivo remain a challenge. RNA activation (RNAa) is emerging as one potential solution by using double-stranded RNA (dsRNA) to increase endogenous gene expression. This approach, although related to RNA interference (RNAi), facilitates a response opposite to gene silencing. Duplex dsP21-322 and its chemically modified variants are examples of RNAa-based drugs that inhibit cancer cell growth by inducing expression of tumor suppressor p21(WAF1/CIP1) (p21). In this study, we investigate the therapeutic potential of dsP21-322 in an orthotopic model of bladder cancer by formulating a 2'-fluoro-modified derivative (dsP21-322-2'F) into lipid nanoparticles (LNP) for intravesical delivery. LNP composition is based upon clinically relevant formulations used in RNAi-based therapies consisting of PEG-stabilized unilamellar liposomes built with lipid DLin-KC2-DMA. We confirm p21 induction, cell-cycle arrest, and apoptosis in vitro following treatment with LNP-formulated dsP21-322-2'F (LNP-dsP21-322-2'F) or one of its nonformulated variants. Both 2'-fluoro modification and LNP formulation also improve duplex stability in urine. Intravesical delivery of LNP-dsP21-322-2'F into mouse bladder results in urothelium uptake and extends survival of mice with established orthotopic human bladder cancer. LNP-dsP21-322-2'F treatment also facilitates p21 activation in vivo leading to regression/disappearance of tumors in 40% of the treated mice. Our results provide preclinical proof-of-concept for a novel method to treat bladder cancer by intravesical administration of LNP-formulated RNA duplexes.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Nanopartículas/administração & dosagem , RNA de Cadeia Dupla/genética , Neoplasias da Bexiga Urinária/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Administração Intravesical , Apoptose/genética , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Antígeno Ki-67/metabolismo , Nanopartículas/química , RNA de Cadeia Dupla/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Tumoral/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.
Assuntos
RNA/administração & dosagem , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Administração Intravesical , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Medições Luminescentes/métodos , Camundongos , Camundongos Nus , Nanopartículas , RNA/genética , Ultrassonografia , Neoplasias da Bexiga Urinária/diagnóstico por imagem , Neoplasias da Bexiga Urinária/genéticaRESUMO
An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.
Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Polaridade Celular , Células Cultivadas , Endocitose , Técnicas de Silenciamento de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Isoenzimas/biossíntese , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoproteínas LDL/metabolismo , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Camundongos , Corpos Multivesiculares/metabolismo , Especificidade de Órgãos , Biossíntese de Proteínas , Interferência de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Tempo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/biossíntese , Proteínas rab5 de Ligação ao GTP/deficiência , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
There is a clear need for methods to provide a safe controlled release of therapeutic proteins, either to achieve and maintain high local protein concentrations, or for sustained systemic delivery. We have developed a protein delivery system that combines in situ cross-linkable polysaccharide hydrogels with gelatin. This formulation is injectable, easy to apply, and obviates the need for organic solvents or potentially toxic cross-linking agents in the formulation process. The cross-linked polysaccharides themselves (comprising hyaluronic acid, dextran and/or carboxymethylcellulose) provided prolonged release of fluorescently labeled albumin (FITC-albumin). The duration of release was markedly extended by the incorporation of gelatin into the formulation: FITC-albumin and interleukin-2 (IL-2) were released over the course of more than 3 weeks. The IL-2 maintained >70% activity throughout that time. Gelatin also accelerated the gelation time of the hydrogels, and reduced their swelling in phosphate-buffered saline. The composite hydrogel (dextran-carboxymethylcellulose-gelatin) showed minimal cytotoxicity in vitro, and benign tissue reaction after subcutaneous injection in rats.
Assuntos
Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/síntese química , Hidrogéis/química , Proteínas/administração & dosagem , Proteínas/química , Animais , Reagentes de Ligações Cruzadas/química , Difusão , Composição de Medicamentos/métodos , Injeções Subcutâneas , Masculino , Teste de Materiais , CamundongosRESUMO
Circulating γδ T cells are cytotoxic lymphocytes that are unique to primates. Recent -studies have shown that amino-bisphosphonates (nBP) activate γδ T cells to kill tumor cells in an indirect mechanism, which requires antigen presenting cells (APC). We hypothesized that selective targeting of nBP to monocytes would result in a more potent γδ T cells activation in circulation, and in tissue associated macrophages (TAM) following monocytes-laden drug extravasation and liposomes accumulation at the tumor site. In addition, inhibition of TAM by alendronate liposomes (ALN-L) is expected. ALN was targeted exclusively to monocytes, but not to lymphocytes, by encapsulating it in negatively-charged liposomes. The proportion of human γd-T cells in the CD3(+) population following treatment with ALN-L or the free drug was increased, from 5.6 ± 0.4% to 50.9 ;± 12.2% and 49.5 ± 12.9%, respectively. ALN solution and liposomes treatments resulted in an increased, and in a dose dependent manner, TNFα secretion from h-PBMC. Preliminary results showed that ALN-L inhibited tumor growth in a nude mouse breast tumor model. It is suggested that enhanced activation of γδ T cells could be obtained due to interaction with circulating monocytes as well as by TAM endocytosing liposomal nBP leading to a potentiated anti-tumor effect of nBP. It should be noted that this could be validated only in primates/humans since γδ T cells are unique in these species.
Assuntos
Alendronato/farmacologia , Antineoplásicos/farmacologia , Lipossomos/farmacologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Alendronato/química , Alendronato/farmacocinética , Análise de Variância , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipossomos/química , Lipossomos/farmacocinética , Ativação Linfocitária/efeitos dos fármacos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos Nus , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.
Assuntos
Inativação Gênica , Inflamação/terapia , Macrófagos/efeitos dos fármacos , Nanopartículas , RNA Interferente Pequeno/uso terapêutico , Receptores CCR2/antagonistas & inibidores , Animais , Aterosclerose/terapia , Glicemia , Diabetes Mellitus/cirurgia , Diabetes Mellitus/terapia , Modelos Animais de Doenças , Sobrevivência de Enxerto/genética , Humanos , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Infarto do Miocárdio/prevenção & controle , Infarto do Miocárdio/terapia , Nanopartículas/química , Receptores CCR2/genéticaRESUMO
Partial inactivation and transient depletion of monocytes/macrophages by liposomal bisphosphonates (LIP-BPs) is widely experimented in various inflammatory disorders including restenosis. Previous studies on activation of cytokines by LIP-BPs are limited to certain cell lines. Moreover, the correlation between in vitro and in vivo studies and complement (C) activation has not been reported. We report here a comprehensive study on the bioactivity of LIP-BPs on various cells' internalization and proliferation, mechanism of cell death, cytokines (in vitro and in vivo) and C activation (in the rat, rabbit and pig). The role of the following parameters has been determined i) drug type (clodronate/alendronate); ii) vesicles size (60-800nm); iii) charge (neutral/negative/ positive); and iv) cell culture type (various cell lines and primary cultures). It was found that monocyte/macrophage inhibition and cytokine activation depend on the cell type, with a limited correlation to the bioactivity obtained in the rat and rabbit models of restenosis. Negatively charged liposomes (85+/-20nm) effectively depleted rabbit's monocytes (67% depletion), with a minor activation of cytokines and no C activation. It is concluded that cell culture studies are insufficient for assessing cytokine activation, and that by controlling LIP-BP properties (size, charge and drug type) optimal bioactivity could be achieved.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Reestenose Coronária/tratamento farmacológico , Citocinas/imunologia , Difosfonatos/administração & dosagem , Difosfonatos/uso terapêutico , Lipossomos/química , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Reestenose Coronária/imunologia , Difosfonatos/imunologia , Difosfonatos/farmacologia , Humanos , Lipossomos/imunologia , Masculino , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Coelhos , RatosRESUMO
Here we develop an injectable composite system based for repeated ultrasound-triggered on-demand drug delivery. An in situ-cross-linking hydrogel maintains model drug (dye)-containing liposomes in close proximity to gas-filled microbubbles that serve to enhance release events induced by ultrasound application. Dye release is tunable by varying the proportions of the liposomal and microbubble components, as well as the duration and intensity of the ultrasound pulses in vitro. Dye is minimal at baseline. The composite shows minimal cytotoxicity in vitro, and benign tissue reaction after subcutaneous injection in rats. Ultrasound application also triggers drug release for two weeks after injection in vivo.
Assuntos
Hidrogéis/administração & dosagem , Hidrogéis/química , Lipossomos/química , Lipossomos/efeitos da radiação , Sonicação , Animais , Hidrogéis/efeitos da radiação , Masculino , Camundongos , Microbolhas , RatosRESUMO
Injectable local anesthetics that would last for many days could have a marked impact on periprocedural care and pain management. Formulations have often been limited in duration of action, or by systemic toxicity, local tissue toxicity from local anesthetics, and inflammation. To address those issues, we developed liposomal formulations of saxitoxin (STX), a compound with ultrapotent local anesthetic properties but little or no cytotoxicity. In vitro, the release of bupivacaine and STX from liposomes depended on the lipid composition and on whether dexamethasone was incorporated. In cell culture, bupivacaine, but not STX, was myotoxic (to C2C12 cells) and neurotoxic (to PC12 cells) in a concentration- and time-dependent manner. Liposomal formulations containing combinations of the above compounds produced sciatic nerve blockade lasting up to 7.5 days (with STX + dexamethasone liposomes) in male Sprague-Dawley rats. Systemic toxicity only occurred where high loadings of dexamethasone increased the release of liposomal STX. Mild myotoxicity was only seen in formulations containing bupivacaine. There was no nerve injury on Epon-embedded sections, and these liposomes did not up-regulate the expression of 4 genes associated with nerve injury in the dorsal root ganglia. These results suggest that controlled release of STX and similar compounds can provide very prolonged nerve blocks with minimal systemic and local toxicity.