RESUMEN
Exogenous antigen proteolysis by proteasomes and amino peptidases is essential for the production of mature major histocompatibility complex class I (MHC-I) peptides to induce cross-presentation. We report here that when liposomes are modified with octaarginine (R8-Lip), a type of cell-penetrating peptide, the production of the mature MHC-I peptide is enhanced by promoting the C-terminal trimming of the antigen peptide. The efficiency of cross-presentation of ovalbumin (OVA) using the R8-Lip was dramatically higher than that by octalysine modified liposomes (K8-Lip) in mouse bone-marrow derived dendritic cells (BMDCs), although the physical characters of both liposomes were comparable. In this study, we investigated the mechanism responsible for the enhancement in cross-presentation by R8-Lip. Although the efficiencies of cellular uptake, endosomal escape, proteolysis of OVA and DC maturation between the two systems were essentially the same, an analysis of peptide trimming to SIINFEKL (mature MHC-I peptide of OVA) by using R8-Lip and K8-Lip encapsulating peptides of various length clearly indicates that the use of R8-Lip enhances the efficiency of the C-terminal cleavage of antigen-derived peptides. This finding provides a new strategy for achieving efficient cross-presentation by using R8 peptide and arginine-rich peptides. Moreover, this result may contribute to the development of a new paradigm regarding the machinery associated with antigen peptide production.
Asunto(s)
Antígenos/química , Reactividad Cruzada , Liposomas/química , Oligopéptidos/química , Animales , Presentación de Antígeno , Arginina/química , Células de la Médula Ósea/citología , Linfocitos T CD8-positivos/citología , Péptidos de Penetración Celular/química , Cloroquina/química , Células Dendríticas/citología , Endosomas/metabolismo , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/química , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Ovalbúmina/química , Complejo de la Endopetidasa Proteasomal/química , Estructura Terciaria de ProteínaRESUMEN
Recently, much attention has been paid to cell-penetrating peptides (CPPs) as an antigen-delivery tool for presentation through the major histocompatibility complex class I (MHC-I) pathway. However, escape of CPPs from the endosome is inefficient and therefore a bottleneck for antigen delivery. Previously, we showed the importance of topological control of octaarginine (R8) peptides on the liposome surface for regulating cellular uptake as well as intracellular trafficking, especially endosomal escape. In this study, we hypothesized that efficient MHC-I presentation could be achieved by controlled intracellular trafficking of antigen encapsulated in R8-modified liposomes (R8-Lip). The mechanism of uptake of both R8-Lip and cationic liposomes was shown to be by macropinocytosis in dendritic cells. However, confocal laser scanning microscopy (CLSM) revealed that R8-Lip are able to release significantly more antigen to the cytosol than are cationic liposomes. Processing of the antigens delivered by R8-Lip was shown to be proteasome-dependent, which is consistent with selective antigen presentation by R8-Lip via MHC-I. According to antigen-presentation analysis, R8-Lip can induce significantly higher MHC-I presentation at lower doses than either soluble ovalbumin (OVA) or OVA in pH-sensitive or cationic liposomes. Moreover, R8-Lip showed an efficient antitumor effect in vivo. Therefore, R8-Lip is a promising new carrier for MHC-I-specific antigen presentation.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Liposomas/química , Oligopéptidos/química , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Amilorida/farmacología , Animales , Presentación de Antígeno/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Línea Celular Tumoral , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/química , Espacio Intracelular/metabolismo , Liposomas/farmacocinética , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Nocodazol/farmacología , Oligopéptidos/farmacocinética , Carga Tumoral/inmunologíaRESUMEN
We previously reported that transferrin (Tf)-modified liposomes (Tf-L) additionally modified with a cholesterylated pH-sensitive fusogenic peptide (Chol-GALA) can release an encapsulated aqueous phase marker to cytosol via endosomal membrane fusion. However, further obstacles need to be overcome to bring the Tf-L to the level of a viral-like gene delivery system. In this study, we developed a novel packaging method to encapsulate condensed plasmid DNA into PEgylated Tf-L (Tf-PEG-L) to form a core-shell-type nanoparticle. The most difficult challenge was to provide a mechanism of escape for the condensed core from endosome to cytosol in the presence of polyethylene glycol (PEG). We hypothesized that a membrane-introduced Chol-GALA and a PEgylated GALA would interact synergistically to induce membrane fusion between liposome and endosome. By simultaneously incorporating Chol-GALA into the membrane of Tf-PEG-L and GALA at tips of PEG chains, a condensed core was released into cytosol, and transfection activity increased 100-fold. We concluded that topological control was responsible for the synergistic effect of GALA derivatives introduced on Tf-PEG-L.
Asunto(s)
Endosomas , Técnicas de Transferencia de Gen , Fusión de Membrana , Nanopartículas/química , Péptidos/química , Virosomas/química , Colesterol/química , Endosomas/química , Endosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Células K562 , Péptidos/metabolismo , Polietilenglicoles/química , Transferrina/química , Transferrina/metabolismo , Virosomas/metabolismoRESUMEN
Transfection efficiencies using LipofectAMINE varied by more than three orders of magnitude depending on the concentrations of lipid and plasmid DNA (pDNA) used to prepare the lipoplexes. When lipoplexes were formed at lower concentrations a striking positive but non-linear relationship was found between dose and transfection efficiency, while at higher (i.e., normally used) concentrations a linear relationship was maintained. To determine the contribution of intracellular pharmacokinetics (PK) and pharmacodynamics (PD) to the observed nonlinearity, we quantified pDNA in whole cells and nuclei by real-time PCR and compared the results with the transfection efficiencies. There was no significant difference in the efficiency of intracellular PK; however, a remarkable difference was observed in the efficiency of PD. Analysis of individual cells by confocal laser scanning microscopy (CLSM) revealed that the amount of nuclear-delivered pDNA was higher for lipoplexes prepared at the normal concentration (NCL) compared to those of lipoplexes prepared at low concentration (LCL). Moreover, the size of the NCL was larger than that of the LCL. Both the size of the lipoplex particle and the dose appear to contribute to the non-linear efficiency of PD. These results emphasize the need to control not only intracellular PK, but also PD for the rational development of non-viral gene delivery systems.
Asunto(s)
Núcleo Celular/metabolismo , ADN/metabolismo , Expresión Génica , Lípidos/química , Liposomas , Modelos Genéticos , Dinámicas no Lineales , Transfección/métodos , Transporte Activo de Núcleo Celular , Animales , ADN/química , Cinética , Ratones , Células 3T3 NIH , Tamaño de la PartículaRESUMEN
In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.
Asunto(s)
Núcleo Celular/metabolismo , ADN/administración & dosificación , Células Dendríticas/metabolismo , Técnicas de Transferencia de Gen , Señales de Localización Nuclear , Animales , ADN/genética , Células HeLa , Humanos , Liposomas , Luciferasas/genética , Ratones , Células 3T3 NIH , PlásmidosRESUMEN
A multifunctional envelope-type nano device (MEND) for use in the delivery of siRNA expression plasmids is described. The plasmid DNA encoding anti-luciferase short interfering RNA (siRNA) was condensed by poly-L-lysine (PLL) and packaged into the MEND. The silencing effect of the MEND(PLL) showed a 96% inhibition of luciferase activities in a co-transfection study. The silencing effect was maintained at more than 60%, even under the 100-fold diluted conditions. In the luciferase transformed cells, however, the MEND(PLL) showed no significant silencing effect (10%), indicating heterogeneity in transfection by the MEND(PLL). To solve this problem, the DNA condensing agents were optimized by comparing PLL, stearyl octaarginine (STR-R8) and protamine (Prot). No difference in silencing effect (95-97%) was found among these MENDs in a co-transfection study. However, the MEND(Prot) showed a 70% silencing effect in the transformed cells. These results suggest that the MEND(Prot) has less heterogeneity in transfection, while the MEND(PLL) and the MEND(STR-R8) have large heterogeneities. These results demonstrate that MEND(Prot) is a promising gene delivery system for siRNA expression plasmids with less heterogeneity associated with the transfection.
Asunto(s)
Terapia Genética/métodos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Animales , Células COS , Chlorocebus aethiops , Composición de Medicamentos , Electroforesis en Gel de Agar , Excipientes , Genes Reporteros , Luciferasas/genética , Nanoestructuras , Oligopéptidos/química , Plásmidos/genética , Protaminas/química , Estearatos/química , TransfecciónRESUMEN
The development of a multifunctional envelope-type nano device (MEND) for use in a non-viral gene delivery system using a novel lipid film hydration method is described. This packaging method involves three steps: (i) DNA condensation with a polycation, (ii) lipid film hydration for the electrostatic binding of the condensed DNA, and (iii) sonication to package the condensed DNA with lipids. For DNA condensation, the optimum ratio of DNA to poly-L-lysine (PLL) was determined by titrating DNA and PLL. The condensed DNA/PLL complex (DPC) was coated with a lipid bilayer by lipid film hydration followed by sonication, which was confirmed by sucrose density gradient centrifugation. Packaging of DPC with lipids increased the transfection activity 10-fold over that of DPC. MEND, having octaarginine on the envelope as a device for membrane penetration to enhance cellular uptake, showed a 1000-fold higher transfection activity than DPC. The MEND developed in this study represents a promising non-viral gene delivery system.
Asunto(s)
Técnicas de Transferencia de Gen , Lípidos/química , Nanoestructuras/química , Nanotecnología/métodos , Animales , Centrifugación por Gradiente de Densidad , Ésteres del Colesterol/química , Electroforesis en Gel de Agar , Fluoresceína-5-Isotiocianato/química , Transferencia Resonante de Energía de Fluorescencia , Técnica de Fractura por Congelación , Terapia Genética/métodos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Microscopía Electrónica , Células 3T3 NIH , Nanoestructuras/ultraestructura , Oligopéptidos/química , Tamaño de la Partícula , Fosfatidiletanolaminas/química , Plásmidos/química , Plásmidos/genética , Polilisina/química , Rodaminas/química , Sonicación , Electricidad Estática , Estearatos/química , TransfecciónRESUMEN
In a previous study, we reported on the efficient delivery of an antigen to the cytosol and a specific-antigen presentation on MHC class I in dendritic cells by rationally controlling the intracellular trafficking of ovalbumin (OVA), a model antigen, with stearylated octaarginine-modified liposomes (R8-Lip/OVA). However, no significant difference in antitumor effects against E.G7-OVA, OVA expressed lymphoma, was observed between R8-Lip/OVA and an electrostatic complex of R8 and OVA (R8/OVA-Com). In this study, we hypothesized that use of adjuvants clarified the difference in immune responses between R8-Lip/OVA and R8/OVA-Com, and selected polyinosine-polycytidylic acid (polyI:C) as an adjuvant. Cytotoxic T lymphocyte (CTL) activity of the polyI:C and OVA encapsulated R8-Lip (R8-Lip/PIC/OVA) was drastically enhanced compared to R8-Lip/OVA and complete Freund's adjuvant with OVA. Moreover, the incorporation of polyI:C clearly was critical for the difference in antitumor effects and CTL activities between R8-Lip/OVA and R8/OVA-Com. These findings suggest that the carriers that are incorporated polyI:C has a great influence on the induction of cellular immunity in vivo.
Asunto(s)
Linfoma/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Ovalbúmina/administración & dosificación , Poli I-C/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Línea Celular Tumoral , Femenino , Adyuvante de Freund/farmacología , Inmunidad Celular/inmunología , Liposomas , Linfoma/inmunología , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/química , Ovalbúmina/inmunología , Poli I-C/farmacología , Electricidad Estática , Linfocitos T Citotóxicos/inmunologíaRESUMEN
We previously developed octaarginine (R8)-modified lipid envelope-type nanoparticles for siRNA delivery (R8-MEND). Herein, we report on their ex vivo siRNA delivery to primary mouse bone marrow-derived dendritic cells (BMDCs) for potential use as a cancer vaccine. Quantitative imaging analysis of the intracellular trafficking of siRNA revealed that the dissociation process, as well as the rate of endosomal escape limits the siRNA efficiency of the prototype R8-MEND, prepared by the hydration method (R8-MEND(hydo)). Successful endosomal escape was achieved by using a pH-dependent fusogenic peptide (GALA) modified on a lipid mixture that was optimized for endosomal fusion. Furthermore, a modified protocol for the preparation of nanoparticles, mixing the siRNA/STR-R8 complex and small unilamellar vesicles (R8/GALA-MEND(SUV)), results in a more homogenous, smaller particle size, and results in a more efficient intracellular dissociation. Gene knockdown of the suppressor of cytokine signaling 1 (SOCS1), a negative-feedback regulator of the immune response in BMDCs resulted in an enhanced phosphorylation of STAT1, and the production of proinflammatory cytokines. Moreover, SOCS1-silenced BMDCs were more potent in suppressing tumor growth. Collectively, these results show that siRNA loaded in R8/GALA-MEND(SUV) efficiently suppresses endogenous gene expression and consequently enhances dendritic cell-based vaccine potency in vivo.
RESUMEN
We previously developed octaarginine (R8)-modified lipid envelope-type nanoparticles for siRNA delivery (R8-MEND). Herein, we report on their ex vivo siRNA delivery to primary mouse bone marrow-derived dendritic cells (BMDCs) for potential use as a cancer vaccine. Quantitative imaging analysis of the intracellular trafficking of siRNA revealed that the dissociation process, as well as the rate of endosomal escape limits the siRNA efficiency of the prototype R8-MEND, prepared by the hydration method (R8-MEND(hydo)). Successful endosomal escape was achieved by using a pH-dependent fusogenic peptide (GALA) modified on a lipid mixture that was optimized for endosomal fusion. Furthermore, a modified protocol for the preparation of nanoparticles, mixing the siRNA/STR-R8 complex and small unilamellar vesicles (R8/GALA-MEND(SUV)), results in a more homogenous, smaller particle size, and results in a more efficient intracellular dissociation. Gene knockdown of the suppressor of cytokine signaling 1 (SOCS1), a negative-feedback regulator of the immune response in BMDCs resulted in an enhanced phosphorylation of STAT1, and the production of proinflammatory cytokines. Moreover, SOCS1-silenced BMDCs were more potent in suppressing tumor growth. Collectively, these results show that siRNA loaded in R8/GALA-MEND(SUV) efficiently suppresses endogenous gene expression and consequently enhances dendritic cell-based vaccine potency in vivo.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/metabolismo , Nanopartículas/química , Neoplasias/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Endosomas/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Terapia Genética/métodos , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/química , ARN Interferente Pequeño/uso terapéutico , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.
Asunto(s)
Endosomas/metabolismo , Fusión de Membrana , Nanopartículas , Membrana Nuclear/metabolismo , Animales , Secuencia de Bases , Línea Celular , ADN/metabolismo , Cartilla de ADN , Ratones , Plásmidos , Reacción en Cadena de la PolimerasaRESUMEN
A remarkable non-linearity was found between dose and transfection activities of non-viral gene delivery systems, such as a Lipofectamine/DNA complex and an octaarginine-modified multifunctional envelope-type nano device (R8-MEND). We measured the nuclear delivery of pDNA to distinguish the non-linearity in intracellular pharmacokinetics or pharmacodynamics after transfection with R8-MEND at different doses. A remarkable positive non-linearity was found in the pharmacodynamics when the dose was increased. Even dummy pDNA enhanced the efficiency of transcription and/or translation per pDNA in the nucleus, but empty liposomes did not. These results suggest the importance of controlled pharmacodynamics as well as the importance of intracellular pharmacokinetics for the rational design of non-viral gene delivery systems.