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Cancer is the second leading cause of death globally after heart diseases. Currently used highly cytotoxic anti-cancer drugs not only kill cancer cells but also often kill non-cancerous healthy body cells, causing adverse side effects. Efforts are now being directed towards developing tumor-selective chemotherapy. Tumor/tumor endothelial cell selective peptide ligands are being covalently grafted onto the exo-surfaces of drug carriers such as liposomes, polymers, etc. A number of prior studies used conjugation of tumor/tumor endothelial cell-selective RGDK- or CGKRK-peptide ligands on the outer surfaces of liposomes, metal-based nanoparticles, single walled carbon nanotubes (SWNTs), etc. However, studies aimed at examining the relative cell membrane fusogenicities and the relative degrees of cellular uptake for the RGDK- and CGKRK-ligand-grafted nanometric drug carriers have not yet been undertaken. Herein, using the widely used liposomes of DOPC, DOPE, DOPS and cholesterol (45 : 25 : 20 : 15, w/w ratio) as the model biomembranes and the fluorescence resonance energy transfer (FRET) assay for measuring membrane fusogenicities, we show that the liposomes of the RGDK-lipopeptide are more biomembrane fusogenic than the liposomes of the CGKRK-lipopeptide. Notably, such FRET assay-derived relative biomembrane fusogenicities of the liposomes of RGDK- and CGKRK-lipopeptides were found to be consistent with their relative degrees of cellular uptake in cultured cancer cells. The present findings open the door for undertaking in-depth in vivo studies aimed at evaluating the relative therapeutic potential of different nanocarriers of drugs/genes/siRNA having tumor-targeting RGDK- and CGKRK-peptides on their exo-surfaces.
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Liposomas , Liposomas/química , Humanos , Lipopéptidos/química , Lipopéptidos/farmacología , Oligopéptidos/química , Membrana Celular/metabolismo , Membrana Celular/química , Transferencia Resonante de Energía de Fluorescencia , Portadores de Fármacos/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Colesterol/química , Colesterol/metabolismo , Fosfatidilcolinas/química , Antineoplásicos/química , Antineoplásicos/farmacologíaRESUMEN
To ensure effective immune response in genetic immunizations, DNA/mRNA vaccines need to be delivered to body's antigen presenting cells (APCs) which is a challenging task. This is primarily due to presence of high concentrations of various degradative enzymes inside them. To this end, mannose receptor (over expressed in APCs) selective cationic liposomes have been used in the past for delivering antigen-encoded plasmid DNA to APCs. APCs also express integrin receptors on their cell surfaces. However, studies aimed at delivering DNA vaccines into APCs via integrin receptors have not yet been undertaken. Herein, we report on the use of cationic liposomes of a priorly disclosed α5ß1 integrin receptor selective RGDK-lipopeptide for macrophage transfection. In this study, we have used pCMV-GFP (as model DNA vaccine) and RAW 264.7 cells (mouse macrophages cells) as model APC. We show that the liposomes of RGDK-lipopeptide containing a previously reported endosome-disrupting histidinylated lipid and DOPE (as co-lipid) in 0.5:0.5:1.0 mole ratio are the most competent in transfecting macrophage cells (44%). Findings in the fluorescence resonance energy transfer based membrane fusogencity assay revealed that the enhanced macrophage transfection efficiency of the liposomes containing RGDK-lipopeptide, endosome-disrupting histidinylated and DOPE may originate from its higher membrane fusogenicity than that for liposomes containing only RGDK-lipopeptide and DOPE. The presently described biologically safe liposomal formulations of RGDK-lipopeptide are expected to find biomedical applications in future for combating cancer and infectious diseases through genetic immunizations.
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Despite remarkable progress during the past decade, eradication of established tumors by targeted cancer therapy and cancer immunotherapy remains an uphill task. Herein, we report on a combination approach for eradicating established mouse melanoma. Our approach employs the use of tumor selective chemotherapy in combination with in vivo dendritic cell (DC) targeted DNA vaccination. Liposomes of a newly synthesized lipopeptide containing a previously reported tumor-targeting CGKRK-ligand covalently grafted in its polar head-group region were used for tumor selective delivery of cancer therapeutics. Liposomally co-loaded STAT3siRNA and WP1066 (a commercially available inhibitor of the JAK2/STAT3 pathway) were used as cancer therapeutics. In vivo targeting of a melanoma antigen (MART-1) encoded DNA vaccine (p-CMV-MART1) to dendritic cells was accomplished by complexing it with a previously reported mannose-receptor selective in vivo DC-targeting liposome. Liposomes of the CGKRK-lipopeptide containing encapsulated FITC-labeled siRNA, upon intravenous administration in B16F10 melanoma bearing mice, showed remarkably higher accumulation in tumors 24 h post i.v. treatment, compared to their degree of accumulation in other body tissues including the lungs, liver, kidneys, spleen and heart. Importantly, the findings in tumor growth inhibition studies revealed that only in vivo DC-targeted genetic immunization or only tumor-selective chemotherapy using the presently described systems failed to eradicate the established mouse melanoma. The presently described combination approach is expected to find future applications in combating various malignancies (with well-defined surface antigens).
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Melanoma , Vacunas de ADN , Animales , Ratones , Liposomas/metabolismo , Células Dendríticas , Antígenos de Neoplasias , Melanoma/metabolismo , Lipopéptidos , Ratones Endogámicos C57BLRESUMEN
[This corrects the article DOI: 10.1021/acsomega.7b00935.].
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Combating brain tumors (glioblastoma multiforme or GBM) is a formidable challenge because of the existence of blood-brain barrier (BBB), a tight cellular junction that separates the central nervous system (CNS) and systemic circulation. Such a selectively permeable barrier prevents the entry of therapeutic molecules from blood circulation to brain parenchyma. Towards enhancing the efficacy of brain tumor-selective drug delivery without perturbing the BBB integrity, nanometric drug carriers are increasingly becoming an efficient therapeutic modality in preclinical studies. Psychostimulant drugs such as amphetamine and methylated amphetamine (METH) are known to penetrate the BBB. Still, little effort has been made to exploit them in nano-drug delivery, largely due to their toxicities. Herein, for the first time, we design, synthesize, and formulate three different ß-amphetaminylated cationic lipid nanoparticles. We show that the ß-amphetaminylated cationic lipid nanoparticles are nontoxic and can cross the BBB presumably through active transcytosis. The BBB penetrating ability also depends on the hydrophilic-hydrophobic balance of the lipids, with hexadecyl lipid (16-BACL) nanoparticle showing maximum accumulation in the brain. The lipid nanoparticle of 16-BACL can simultaneously encapsulate paclitaxel and PDL1-siRNA. The dual drug-loaded lipid nanoparticles showed apoptosis driven cellular cytotoxicity against GL261 cells and improved the overall survivability of orthotopic glioblastoma bearing mice compared to their non-targeting counterpart. The present work describes a new class of BBB-crossing lipid nanoparticles and delineates their therapeutic promise against glioblastoma.
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Anfetamina/química , Antineoplásicos/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Lípidos/química , Nanopartículas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Barrera Hematoencefálica/patología , Neoplasias Encefálicas/patología , Cationes/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Glioblastoma/patología , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Paclitaxel/química , Paclitaxel/farmacología , Tamaño de la Partícula , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
Glioblastoma multiforme (GBM) is one of the most aggressive tumors with a median survival of only 15 months. Effective therapeutics need to overcome the formidable challenge of crossing the blood-brain barrier (BBB). Receptors and transporters overexpressed on BCECs are being used for designing liposomes, polymers, polymeric micelles, peptides, and dendrimer-based drug carriers for combating brain tumors. Herein, using the orthotopic mouse glioblastoma model, we show that codelivering a small-molecule inhibitor of the JAK/STAT pathway (WP1066) and STAT3siRNA with nanometric (100-150 nm) α5ß1 integrin receptor-selective liposomes of RGDK-lipopeptide holds therapeutic promise in combating glioblastoma. Rh-PE (red)-labeled liposomes of RGDK-lipopeptide were found to be internalized in GL261 cells via integrin α5ß1 receptors. Intravenously administered near-infrared (NIR)-dye-labeled α5ß1 integrin receptor-selective liposomes of RGDK-lipopeptide were found to be accumulated preferentially in the mouse brain tumor tissue. Importantly, we show that iv injection of WP1066 (a commercially sold small-molecule inhibitor of the JAK/STAT pathway) and STAT3siRNA cosolubilized within the liposomes of RGDK-lipopeptide leads to significant inhibition (>350% compared to the untreated mice group) of orthotopically growing mouse glioblastoma. The present strategy may find future use in combating GBM.
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Glioblastoma/metabolismo , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Barrera Hematoencefálica/metabolismo , Células CHO , Línea Celular Tumoral , Cricetulus , Glioblastoma/genética , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Liposomas/química , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/genéticaRESUMEN
Since mannose receptors (MRs) are expressed on the surfaces of dendritic cells (DCs), the most professional antigen presenting cells in our body, DNA vaccine carriers containing either covalently grafted mannosyl- or mannose-mimicking shikimoyl-ligands are being increasingly used in ex vivo DC-transfection based DNA vaccination. To this end, we have recently demonstrated that ex vivo immunization of mice with liposomes of shikimoylated cationic amphiphiles containing a 6-amino hexanoic acid spacer group in the head-group region in complexation with melanoma antigen (MART1) encoded DNA vaccine (pCMV-MART1) induces long lasting anti-melanoma immune responses (C. Voshavar, et al., J. Med. Chem., 2017, 60, 1605-1610). This finding prompted us to examine, in the present investigation, the efficacies of gold nanoparticles conjugated to the mannose-mimicking shikimoyl ligand (SL) via a 6-amino hexane thiol spacer (AuNPs-SL) for use in ex vivo DC-transfection based genetic immunization. Herein, we report on the design, synthesis, physico-chemical characterization and bioactivities of AuNPs-SL. Dynamic light scattering and transmission electron microscopy studies revealed the hydrodynamic diameters of theAuNPs-SL nanoconjugates to be within the range of 23-44 nm and their surface potentials within the range of 9-28 mV. MTT-assay showed the non-cytotoxic nature of AuNPs-SL and the findings in the electrophoretic gel retardation assays revealed strong DNA binding properties of the AuNPs-SL. Importantly, subcutaneous immunization of C57BL/6J mice with DCs ex vivo transfected with an electrostatic complex of AuNPs-SL & melanoma antigen (MART1) encoded DNA vaccine (p-CMV-MART1) induced a long lasting (100 days) anti-tumor immune response in immunized mice upon subsequent challenge with a lethal dose of melanoma. Notably, mice immunized with either autologous mbmDCs ex vivo pre-transfected with nanoplexes of shikimoylated AuNPs-SL & an irrelevant pCMV-SPORT-ß-gal plasmid (without having encoded melanoma antigen) or untransfected DCs showed no lasting protection against subsequent tumor challenge. The presently described shikimoyl-decorated gold nanoparticles (AuNPs-SL) are expected to find future use in ex vivo DC-transfection based genetic immunization against cancer and other infectious diseases.
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Vacunas contra el Cáncer/inmunología , Oro/química , Nanopartículas del Metal/química , Nanoestructuras/química , Safrol/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Inmunidad Celular , Interferón gamma/metabolismo , Ligandos , Antígeno MART-1/genética , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/mortalidad , Ratones , Ratones Endogámicos C57BL , Nanoestructuras/uso terapéutico , Nanoestructuras/toxicidad , Plásmidos/genética , Plásmidos/metabolismo , Tasa de Supervivencia , Trasplante HomólogoRESUMEN
The clinical success of dendritic cell (DC)-based genetic immunization remains critically dependent on the availability of effective and safe nano-carriers for targeting antigen-encoded DNA vaccines to DCs, the most potent antigen-presenting cells in the human body in vivo. Recent studies revealed the efficacies of mannose receptor-mediated in vivo DC-targeted genetic immunization by liposomal DNA vaccine carriers containing both mannose-mimicking shikimoyl and transfection enhancing guanidinyl functionalities. However, to date, the efficacies of this approach have not been examined for metal-based nanoparticle DNA vaccine carriers. Herein, we report for the first time, the design, synthesis, physico-chemical characterization and bioactivities of gold nanoparticles covalently functionalized with a thiol ligand containing both shikimoyl and guanidinyl functionalities (Au-SGSH). We show that Au-SGSH nanoparticles can deliver DNA vaccines to mouse DCs under in vivo conditions. Subcutaneous administration of near infrared (NIR) dye-labeled Au-SGSH showed significant accumulation of the NIR dye in the DCs of the nearby lymph nodes compared to that for the non-targeting NIR-labeled Au-GSH nanoconjugate containing only a covalently tethered guanidinyl group, not the shikimoyl-functionality. Under prophylactic settings, in vivo immunization (s.c.) with the Au-SGSH-pCMV-MART1 nanoplex induced a long-lasting (180 days) immune response against murine melanoma. Notably, mannose receptor-mediated in vivo DC-targeted immunization (s.c.) with the Au-SGSH-MART1 nanoplex significantly inhibited established melanoma growth and increased the overall survivability of melanoma-bearing mice under therapeutic settings. The Au-SGSH nanoparticles reported herein have potential use for in vivo DC-targeted genetic immunization against cancer and infectious diseases.
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Células Dendríticas/metabolismo , Oro/química , Nanopartículas del Metal/química , Nanoconjugados/química , Vacunas de ADN/inmunología , Animales , Células de la Médula Ósea/citología , Línea Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Femenino , Colorantes Fluorescentes/química , Inmunidad Activa , Antígeno MART-1/química , Antígeno MART-1/inmunología , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/prevención & control , Nanopartículas del Metal/toxicidad , Ratones , Ratones Endogámicos C57BL , Plásmidos/química , Plásmidos/metabolismo , Compuestos de Sulfhidrilo/química , Vacunas de ADN/químicaRESUMEN
Limited tumor tissue penetration is one of the key impeding factors retarding successful in vivo exploitations of anti-angiogenic cancer therapy. Herein we report on the design of a cell penetrating peptide-decorated lipid nano-assembly which, upon systemic administration, induces significant mouse tumor growth inhibition via enhanced tumor infiltration of encapsulated anti-angiogenic siRNA.
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Inhibidores de la Angiogénesis/administración & dosificación , Antineoplásicos/administración & dosificación , Péptidos de Penetración Celular/administración & dosificación , Lípidos/administración & dosificación , Melanoma/tratamiento farmacológico , Nanopartículas/administración & dosificación , Neovascularización Patológica/tratamiento farmacológico , Tratamiento con ARN de Interferencia , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Inyecciones Intraperitoneales , Lípidos/química , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patologíaRESUMEN
Numerous prior studies on fighting cancer have been based on using inhibitors of JAK-STAT pathway (signal transducer and activator of transcription 3 (STAT3) inhibitor in particular), a signaling pathway responsible for progression of many types of cancer cells. However, recent studies have shown that STAT3 activation leads to upregulation of program death receptor-ligand 1 (PD-L1, an immune checkpoint protein that plays a major role behind evasion of immune systems by growing tumors) expression levels in tumor cells, leading to enhanced immune suppression. This is why global efforts are being witnessed in combating cancer through use of immune checkpoint inhibitors. Herein, we report on the design, synthesis, physicochemical characterizations, and bioactivity evaluation of novel tumor- and tumor-vasculature-targeting noncytotoxic Au-CGKRK nanoconjugates (17-80 nm) for combating tumor. Using a syngeneic mouse tumor model, we show that intraperitoneal (i.p.) administration of the Au-CGKRK nanoparticles (NPs) complexed with both PD-L1siRNA (the immune checkpoint inhibitor) and STAT3siRNA (the JAK-STAT pathway inhibitor) results in significant (>70%) enhancement in overall survivability (OS) in melanoma-bearing mice (n = 5) when compared to the OS in the untreated mice group. The expression levels of CD8 and CD4 proteins in the tumor lysates of differently treated mice groups (by Western blotting) are consistent with the observed OS enhancement being a T-cell-driven process. Biodistribution study using near-infrared dye-loaded Au-CGKRK nanoconjugates revealed selective accumulation of the dye in mouse tumor. Notably, the overall survival benefits were significantly less (â¼35%) when melanoma-bearing mice were treated (i.p.) with Au-CGKRK NPs complexed with only PD-L1siRNA or with STAT3siRNA alone. The presently described Au-CGKRK nanoconjugates are expected to find future use in therapeutic RNA-interference-based cancer immunotherapy.
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Therapeutic agents harboring both wound healing and antibacterial activities have much demand in biomedical applications. Development of such candidates with clinically approved materials adds more advantages toward these applications. Recently, silver metal complex nanomaterials have been playing a major role in medical uses especially for antibacterial activity and wound healing. In this report, we designed and synthesized silver nitroprusside complex nanoparticles (abbreviated as AgNNPs) using sodium nitroprusside and silver nitrate (both are FDA approved precursors). The nanoparticles (AgNNPs) were thoroughly characterized by various physicochemical techniques such as XRD, FTIR, TGA, DLS, EDAX, Raman, ICP-OES, HRTEM, and FESEM. The cell viability assay in normal cells (EA.hy 926 cells, NIH 3T3) using MTT reagents and CEA assay (CEA: Chick embryo angiogenesis assay) in fertilized eggs demonstrate the biocompatibility of AgNNPs. These nanoparticles show effective antibacterial activity against both Gram positive and Gram negative bacteria through membrane and DNA damage. Additionally, AgNNPs accelerate the wound healing in C57BL6 mice by altering the macrophages from M1 to M2. Considering the results together, the current study may offer the development of new silver nanocomplex nanomaterials that shows synergistic effect on antibacterial activity and wound healing (2-in-1-system). To the best of our knowledge, this is the first report for the synthesis, characterization, and biomedical applications of silver nitroprusside nanoparticles.
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Despite significant progress in neurosurgery and radiation therapy during the past decade, overall survivability (OS) of glioblastoma patients continues to be less than 2 years. The scope of systemic chemotherapy is greatly limited by poor drug transport across the blood brain barrier (BBB) and, thereby, suboptimal drug accumulation in glioma tissue. To this end, use of large amino acid transporter-1 (LAT1) overexpressed both on brain capillary endothelial cells (BCECs) and glioma cells has begun. Prior reports on the use of LAT1 mediated delivery of model drugs showed their brain accumulations. However, in depth in vivo glioblastoma regression studies aimed at examining the therapeutic potential of LAT1 mediated delivery of potent chemotherapeutics to brain tumor tissues have not yet been undertaken. Herein, we report on the development of a nanometric (100-135 nm) promising LAT1 selective liposomal drug carrier prepared from a novel l-3,4-dihydroxyphenylalanine (l-DOPA) functionalized amphiphile (Amphi-DOPA). In vitro studies using Rh-PE labeled liposomes of Amphi-DOPA both in untreated glioma (GL261) cells and in GL261cells preincubated with LAT1 antibody revealed LAT1 mediated cellular uptake. Intravenously administered NIR-dye labeled liposomes of Amphi-DOPA in glioblastoma-bearing mice showed preferential accumulation of the dye in brain tissue. Notably iv administration of WP1066-loaded liposomes of Amphi-DOPA enhanced the overall survivability of C57BL/6J mice bearing orthotopically established mouse glioblastoma by â¼60% compared to that for the untreated mouse group. Furthermore, we show that the OS of established glioblastoma-bearing mice can be significantly enhanced (by >300% compared to that for the untreated mouse group) when the presently described LAT1 mediated targeted chemotherapy with WP1066-loaded liposomes of Amphi-DOPA is combined with in vivo DC-targeted DNA vaccination using a survivin (a glioblastoma antigen) encoded DNA vaccine. The present findings open a new door for LAT1 mediated systemic chemotherapy of glioblastoma.
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Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Glioblastoma/metabolismo , Glioblastoma/terapia , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Levodopa/química , Liposomas/química , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Femenino , Citometría de Flujo , Transportador de Aminoácidos Neutros Grandes 1/genética , Ratones , Ratones Endogámicos C57BL , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Herein we report the design and development of α5 ß1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against αv ß3 , αv ß5 , and α5 ß1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α5 ß1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexinâ V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors.
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Curcumina/administración & dosificación , Curcumina/farmacología , Portadores de Fármacos/química , Integrina alfa5beta1/química , Nanotubos de Carbono/química , Oligopéptidos/química , Animales , Anticuerpos Monoclonales/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Curcumina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/inmunología , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Células 3T3 NIH , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Tamaño de la Partícula , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl headgroup are promising DNA vaccine carriers for dendritic cell (DC) transfection. The present structure-activity study reports on the influence of spacer length (between mannose-mimicking headgroups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the anti-melanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57BL/6J mice under prophylactic settings.
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Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/inmunología , Portadores de Fármacos/química , Manosa/análogos & derivados , Melanoma/prevención & control , Transfección/métodos , Vacunas de ADN/administración & dosificación , Animales , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Células Dendríticas/metabolismo , Femenino , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Melanoma/inmunología , Ratones Endogámicos C57BL , Tensoactivos/química , Vacunación/métodos , Vacunas de ADN/inmunología , Vacunas de ADN/uso terapéuticoRESUMEN
Despite significant recent progress in the area of translational genomics of neuroblastoma, the overall survival rates for children with high-risk NB continue to be not more than 5 years due to tumor relapse and/or drug-resistant tumors. Herein we report on the development of a neuroblastoma targeting nanometric (130-150 nm) circulation stable liposomal system prepared from a novel nipecotic acid-derived cationic amphiphile (NACA). The size ranges of liposomes (130-150 nm) were confirmed by both dynamic light scattering and transmission electron microscopy. The findings in the gel electrophoresis assay revealed that siRNAs encapsulated within the liposomes of NACA (with 90% entrapment efficiency) are protected from attack by RNase. Cellular uptake experiments using FAM-siRNA loaded liposomes of NACA showed the liposomal entry in human neuroblastoma cells (IMR-32) to be mediated via the GABAA receptor. CDC20siRNA-loaded liposomes of NACA caused significantly higher CDC20 gene silencing efficiency in IMR-32 cells compared to CDC20 gene knockdown efficiency mediated by CDC20siRNA-loaded control non-targeting liposomes (NTL). The findings in the annexin-V binding based flow cytometric apoptosis assay and MTT-based cellular cytotoxicity assay support the notion that pronounced (80%) neuroblastoma cell death upon treatment with CDC20siRNA & PTX co-loaded liposomes of NACA presumably originates from enhanced apoptosis of cells. Importantly, intravenously administered CDC20siRNA & PTX co-loaded liposomes of NACA significantly inhibited growth of xenografted human neuroblastoma in athymic nude mice. The presently disclosed strategy of co-delivering potent anticancer siRNA and small molecule chemotherapeutics using liposomes of NACA opens a new door for combating the dreaded disease of neuroblastoma.
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Proteínas Cdc20/genética , Liposomas , Neuroblastoma/tratamiento farmacológico , Ácidos Nipecóticos/química , Paclitaxel/administración & dosificación , ARN Interferente Pequeño/genética , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Accomplishing significantly enhanced overall survivability (OS) remains a formidable challenge in combating glioblastoma. The presence of the blood-brain barrier acts as the major biological barrier in delivering drugs to the brain. Herein, liposomal formulations of two novel nicotinylated amphiphiles are reported for targeting potent anticancer drugs to orthotopic mouse glioblastoma. It is shown that intravenous administration of the potent signal transducer and activator of transcription 3 (STAT3) inhibitor (WP-1066)-loaded liposomes of nicotinylated amphiphiles in combination with in vivo dendritic cell (DC)-targeted subcutaneous genetic immunization (using tyrosinase-related protein-2 encoded DNA vaccine) markedly enhances the OS of orthotopic glioblastoma-bearing mice (by >500% compared to the OS for the control group). Notably, the overall survival benefits in orthotopic-brain-tumor-bearing mice treated with only targeted chemotherapy or with only in vivo DC-targeted genetic immunization are found to be significantly less. The presently described simple approach avoids the need of isolation of any autologous immune cells. In summary, the preclinical findings described herein open the door for combating glioblastoma in humans through harnessing synergistic effects of targeted chemotherapy and in vivo DC-targeted genetic immunization.
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Cationic lipid-guided nucleic acid delivery holds great promise in gene therapy and genome-editing applications for treating genetic diseases. However, the major challenge lies in achieving therapeutically relevant efficiencies. Prior findings, including our own, demonstrated that asymmetry in the hydrophobic core of cationic lipids imparted superior transfection efficiencies. To this end, we have developed a lipid nanocarrier system with an asymmetric hydrophobic core (PS-Lips) derived from a mixture of fatty acids of food-grade palmstearin and compared its efficiency with symmetric palmitic acid-based nanocarrier system (P-Lip). PS-Lips exhibited superior transfection efficiencies with both plasmid DNA (pDNA) and mRNA in multiple cultured cells than the control P-Lip. More importantly, PS-Lips exhibited 2-fold superior transfections with linear nucleic acid, green fluorescent protein (GFP) mRNA in hematopoietic cells, when compared with the commercial control lipofectamine RNAiMAX. PS-Lips was also found to be effective in delivering genome-editing tools (CRISPR/Cas9, sgRNA encoded pDNA with a reporter GFP construct) than P-Lip in HEK-293 cells. In the present study, we report that cationic liposomes derivatized from natural food-grade fat palmstearin with a natural hydrophobic core asymmetry are efficient in delivering both linear and circular nucleic acids. In particular, PS-Lips is efficient in delivering mRNA to hematopoietic cells. These findings can be further exploited in the genome-editing approach for treating ß-globinopathies.
RESUMEN
Numerous prior studies have been reported on the use of pH-sensitive drug carriers such as micelles, liposomes, peptides, polymers, nanoparticles, etc. that are sensitive to the acidic (pH = â¼6.5) microenvironments of tumor tissues. Such systems have been primarily used in the past as effective drug/gene/microRNA carriers for releasing their anti-cancer payloads selectively to tumor cells/tissues. Herein, we report on the development of new liposomal drug carriers prepared from glutamic acid backbone-based cationic amphiphiles containing both endosomal pH-sensitive histidine as well as cellular uptake & solubility enhancing guanidine moieties in their polar head-group regions. The most efficient one among the four presently described endosomal pH-sensitive liposomal drug carriers not only effectively delivers potent anti-cancer drugs (curcumin & paclitaxel) to mouse tumor, but also significantly contributes to inhibiting mouse tumor growth. The findings in the in vitro mechanistic studies are consistent with apoptosis of tumor cells being mediated through increased cell cycle arrest in the G2/M phase. Findings in the FRET assay and in vitro drug release studies conducted with the liposomes of the most efficient pH-sensitive lipid demonstrated its pH dependent fusogenic and controlled curcumin release properties. Importantly, the presently described liposomal formulation of curcumin & paclitaxel enhanced overall survivability of tumor bearing mice. To the best of our knowledge, the presently described system (curcumin, paclitaxel and liposomal carrier itself) is the first of its kind pH-sensitive liposomal formulation of potent chemotherapeutics in which the liposomal drug itself exhibits significant mouse tumor growth inhibition properties.
Asunto(s)
Antineoplásicos/farmacología , Curcumina/química , Portadores de Fármacos/química , Endosomas/química , Liposomas/química , Liposomas/farmacología , Paclitaxel/química , Animales , Antineoplásicos/química , Línea Celular Tumoral , Curcumina/farmacología , Sistemas de Liberación de Medicamentos , Endosomas/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Ratones , Micelas , Paclitaxel/farmacología , Polímeros/químicaRESUMEN
A major limiting factor retarding the clinical success of dendritic cell (DC)-based genetic immunizations (DNA vaccination) is the scarcity of biologically safe and effective carrier systems for targeting the antigen-encoded DNA vaccines to DCs under in vivo settings. Herein, we report on a potent, mannose receptor selective in vivo DC-targeting liposomes of a novel cationic amphiphile with mannose-mimicking shikimoyl head-group. Flow cytometric experiments with cells isolated from draining lymph nodes of mice s.c. immunized with lipoplexes of pGFP plasmid (model DNA vaccine) using anti-CD11c antibody-labeled magnetic beads revealed in vivo DC-targeting properties of the presently described liposomal DNA vaccine carrier. Importantly, s.c. immunizations of mice with electrostatic complex of the in vivo DC-targeting liposome and melanoma antigen-encoded DNA vaccine (p-CMV-MART1) induced long-lasting antimelanoma immune response (100 days post melanoma tumor challenge) with remarkable memory response (more than 6 months after the second tumor challenge). The presently described direct in vivo DC-targeting liposomal DNA vaccine carrier is expected to find future exploitations toward designing effective vaccines for various infectious diseases and cancers.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/inmunología , Neoplasias/tratamiento farmacológico , Vacunas de ADN/administración & dosificación , Animales , Biomimética , Antígeno CD11c/genética , Vacunas contra el Cáncer/inmunología , Femenino , Inmunoterapia Activa , Liposomas , Ratones , Neoplasias/inmunología , Vacunas de ADN/inmunologíaRESUMEN
Prior studies reported significant anticancer activities of ceramides. However, anticancer activities of homoserine based ceramides have not been tested. With a view to compare the anticancer activity of ceramides and homoceramides, in the present study, we have synthesized four serine based and four homoserine based C8-ceramide analogues. Since many cancer cells have shown resistance to ceramides, curcumin is now being used in combination with ceramides because of its ability to reverse multidrug resistance. Aimed at targeting curcumin-ceramide combination to tumor endothelial cells, herein we have used a tumor vasculature targeting liposomes of a newly synthesized pegylated RGDGWK-lipopeptide. Importantly, the liposomal formulations of the homoserine based C8-ceramide analogue containing oleyl chain showed more promising antineoplastic activities under both in vitro and systemic settings than the liposomal formulations of commercially available C8-ceramide. Findings in the mouse tumor growth inhibition study revealed synergistic therapeutic benefit from simultaneous delivery of curcumin and a homoserine based ceramide containing oleyl chain to tumor vasculature. Results in RT-PCR and Western blot experiments suggest that inhibition of solid tumor growth is mediated via inhibition of PI3K-Akt signaling pathway. The present structure-activity study is the first report to demonstrate therapeutic promise of curcumin-homoserine based ceramide combination in antiangiogenic cancer therapy.