RESUMO
A key initiating step in atherosclerosis is the accumulation and retention of apolipoprotein B complexing lipoproteins within the artery walls. In this work, we address this exact initiating mechanism of atherosclerosis, which results from the oxidation of low-density lipoproteins (oxLDL) using therapeutic nanogels. We present the development of biocompatible polyethylene glycol (PEG) cross-linked nanogels formed from a single simultaneous cross-linking and co-polymerization step in water without the requirement for an organic solvent, high temperature, or shear stress. The nanogel synthesis also incorporates in situ noncovalent electrostatically driven template polymerization around an innate anti-inflammatory and anti-oxidizing paraoxonase-1 (PON-1) enzyme payload-the release of which is triggered because of matrix metalloproteinase responsive elements instilled in the PEG cross-linker monomer. The results obtained demonstrate the potential of triggered release of the PON-1 enzyme and its efficacy against the production of ox-LDL, and therefore a reduction in macrophage foam cell and reactive oxygen species formation.
Assuntos
Lipoproteínas LDL , Polietilenoglicóis , Nanogéis , Polimerização , ÁguaRESUMO
Bone is a favorable microenvironment for tumor growth and a frequent destination for metastatic cancer cells. Targeting cancers within the bone marrow remains a crucial oncologic challenge due to issues of drug availability and microenvironment-induced resistance. Herein, we engineered bone-homing polymeric nanoparticles (NPs) for spatiotemporally controlled delivery of therapeutics to bone, which diminish off-target effects and increase local drug concentrations. The NPs consist of poly(D,L-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and bisphosphonate (or alendronate, a targeting ligand). The engineered NPs were formulated by blending varying ratios of the synthesized polymers: PLGA-b-PEG and alendronate-conjugated polymer PLGA-b-PEG-Ald, which ensured long circulation and targeting capabilities, respectively. The bone-binding ability of Ald-PEG-PLGA NPs was investigated by hydroxyapatite binding assays and ex vivo imaging of adherence to bone fragments. In vivo biodistribution of fluorescently labeled NPs showed higher retention, accumulation, and bone homing of targeted Ald-PEG-PLGA NPs, compared with nontargeted PEG-PLGA NPs. A library of bortezomib-loaded NPs (bone-targeted Ald-Bort-NPs and nontargeted Bort-NPs) were developed and screened for optimal physiochemical properties, drug loading, and release profiles. Ald-Bort-NPs were tested for efficacy in mouse models of multiple myeloma (MM). Results demonstrated significantly enhanced survival and decreased tumor burden in mice pretreated with Ald-Bort-NPs versus Ald-Empty-NPs (no drug) or the free drug. We also observed that bortezomib, as a pretreatment regimen, modified the bone microenvironment and enhanced bone strength and volume. Our findings suggest that NP-based anticancer therapies with bone-targeting specificity comprise a clinically relevant method of drug delivery that can inhibit tumor progression in MM.
Assuntos
Antineoplásicos , Neoplasias Ósseas/tratamento farmacológico , Ácidos Borônicos , Sistemas de Liberação de Medicamentos , Ácido Láctico , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas , Polietilenoglicóis , Ácido Poliglicólico , Pirazinas , Microambiente Tumoral/efeitos dos fármacos , Alendronato/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Ácido Láctico/síntese química , Ácido Láctico/química , Ácido Láctico/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Nanopartículas/química , Nanopartículas/ultraestrutura , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Ácido Poliglicólico/síntese química , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Pirazinas/química , Pirazinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Protein therapeutics have gained attention recently for treatment of a myriad of human diseases due to their high potency and unique mechanisms of action. We present the development of a novel polymeric thermosponge nanoparticle for efficient delivery of labile proteins using a solvent-free polymer thermo-expansion mechanism with clinical potential, capable of effectively delivering a range of therapeutic proteins in a sustained manner with no loss of bioactivity, with improved biological half-lives and efficacy in vivo.
Assuntos
Anti-Inflamatórios/administração & dosagem , Preparações de Ação Retardada/química , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Interleucina-10/administração & dosagem , Nanopartículas/química , Polímeros/química , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Linhagem Celular , Sistemas de Liberação de Medicamentos , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Insulina/farmacocinética , Insulina/farmacologia , Interleucina-10/farmacocinética , Interleucina-10/farmacologia , Camundongos , Nanopartículas/ultraestrutura , TemperaturaRESUMO
One limitation of current biodegradable polymeric nanoparticles is their inability to effectively encapsulate and sustainably release proteins while maintaining protein bioactivity. Here we report the engineering of PLGA-polycation nanoparticles with a core-shell structure that act as a robust vector for the encapsulation and delivery of proteins and peptides. The optimized nanoparticles can load high amounts of proteins (>20 % of nanoparticles by weight) in aqueous solution without organic solvents through electrostatic interactions by simple mixing, thereby forming nanospheres in seconds with diameters <200â nm. The relationship between nanosphere size, surface charge, PLGA-polycation composition, and protein loading is also investigated. The stable nanosphere complexes contain multiple PLGA-polycation nanoparticles, surrounded by large amounts of protein. This study highlights a novel strategy for the delivery of proteins and other relevant molecules.
Assuntos
Nanopartículas , Polímeros/química , Proteínas/química , Microscopia Eletrônica de TransmissãoRESUMO
Hyperthermia and chemotherapy represent potential modalities for cancer treatments. However, hyperthermia can be invasive, while chemotherapy drugs often have severe side effects. Recent clinical investigations have underscored the potential synergistic efficacy of combining hyperthermia with chemotherapy, leading to enhanced cancer cell killing. In this context, magnetic iron oxide nanogels have emerged as promising candidates as they can integrate superparamagnetic iron oxide nanoparticles (IONPs), providing the requisite magnetism for magnetic hyperthermia, with the nanogel scaffold facilitating smart drug delivery. This review provides an overview of the synthetic methodologies employed in fabricating magnetic nanogels. Key properties and designs of these nanogels are discussed and challenges for their translation to the clinic and the market are summarised.
Assuntos
Sistemas de Liberação de Medicamentos , Hipertermia Induzida , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Nanogéis/química , Nanopartículas Magnéticas de Óxido de Ferro/química , Animais , Polietilenoimina/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapêutico , Compostos Férricos/química , Polietilenoglicóis/químicaRESUMO
Polymeric materials have been used in a range of pharmaceutical and biotechnology products for more than 40 years. These materials have evolved from their earlier use as biodegradable products such as resorbable sutures, orthopaedic implants, macroscale and microscale drug delivery systems such as microparticles and wafers used as controlled drug release depots, to multifunctional nanoparticles (NPs) capable of targeting, and controlled release of therapeutic and diagnostic agents. These newer generations of targeted and controlled release polymeric NPs are now engineered to navigate the complex in vivo environment, and incorporate functionalities for achieving target specificity, control of drug concentration and exposure kinetics at the tissue, cell, and subcellular levels. Indeed this optimization of drug pharmacology as aided by careful design of multifunctional NPs can lead to improved drug safety and efficacy, and may be complimentary to drug enhancements that are traditionally achieved by medicinal chemistry. In this regard, polymeric NPs have the potential to result in a highly differentiated new class of therapeutics, distinct from the original active drugs used in their composition, and distinct from first generation NPs that largely facilitated drug formulation. A greater flexibility in the design of drug molecules themselves may also be facilitated following their incorporation into NPs, as drug properties (solubility, metabolism, plasma binding, biodistribution, target tissue accumulation) will no longer be constrained to the same extent by drug chemical composition, but also become in-part the function of the physicochemical properties of the NP. The combination of optimally designed drugs with optimally engineered polymeric NPs opens up the possibility of improved clinical outcomes that may not be achievable with the administration of drugs in their conventional form. In this critical review, we aim to provide insights into the design and development of targeted polymeric NPs and to highlight the challenges associated with the engineering of this novel class of therapeutics, including considerations of NP design optimization, development and biophysicochemical properties. Additionally, we highlight some recent examples from the literature, which demonstrate current trends and novel concepts in both the design and utility of targeted polymeric NPs (444 references).
Assuntos
Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Polímeros/química , HumanosRESUMO
We have synthesized a bimodal lipidic molecule bearing both fluorophore and contrast agent signatures on the same structure in order to create a robust bimodal liposome for both magnetic resonance imaging (MRI) and fluorescence microscopy utility. The dual-modality concept considered in the synthesis of this new paramagnetic and fluorescent lipid is valuable in that anatomical information (MRI) as well as very sensitive localization (ex vivo fluorescence microscopy) of signal, and therefore liposome biodistribution, is obtainable. Bimodal cationic and neutral PEGylated liposomes were formulated using this novel lipid probe and used to label cells in vitro and image human ovarian xenografts in vivo. Tumour signal enhancement was increased by over 6-fold post-administration of the neutral PEGylated liposomes, and was maintained at this level up to the 24 h end-point. Our results showed this lipid to be more effective and sensitive than the single signature paramagnetic lipid Gd.DOTA.DSA at cellular labelling and tumour MRI.
Assuntos
Meios de Contraste/síntese química , Corantes Fluorescentes/síntese química , Lipídeos/química , Lipossomos , Imageamento por Ressonância Magnética/métodos , Neoplasias Ovarianas/diagnóstico , Adenocarcinoma/diagnóstico , Linhagem Celular Tumoral , Meios de Contraste/química , Feminino , Corantes Fluorescentes/química , Humanos , Lipídeos/síntese química , Lipossomos/análise , Microscopia de Fluorescência/métodos , Estrutura MolecularRESUMO
In this review we discuss the development of paramagnetic liposomes incorporating MRI contrast agents and show how these are utilized in cellular imaging in vitro. Bi-functional, bi-modal imaging paramagnetic liposome systems are also described. Next we discuss the upgrading of paramagnetic liposomes into bi-modal imaging neutral nanoparticles for in vivo imaging applications. We discuss the development of such systems and show how paramagnetic liposomes and imaging nanoparticles could be developed as platforms for future multi-functional, multi-modal imaging theranostic nanodevices tailor-made for the combined imaging of early stage disease pathology and functional drug delivery.
Assuntos
Lipossomos/química , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Meios de Contraste/química , Humanos , Lipossomos/metabolismo , Imageamento por Ressonância Magnética , Microscopia de Fluorescência , RadiografiaRESUMO
Folate-targeted bimodal paramagnetic and fluorescent liposomes were developed and showed enhanced accumulation in a folate receptor expressing tumor model. These bimodal liposomes were composed of both a paramagnetic and a fluorescent lipid, and utilized a PEG-lipid amphiphile for prolonged in vivo circulation. The particles were formulated to ensure a size distribution of approximately 100 nm with a low polydispersity index. IGROV-1 cells were used to induce tumors in nude Balb/c mice, and the folate-targeted liposomes were injected intravenously. Rapid accumulation of the folate-targeted liposomes within the tumor tissue compared to nontargeted liposomes was observed. Furthermore, folate-labeled liposomes showed a 4-fold increase in tumor T(1) signal intensity at just 2 h postinjection with similar results being obtained for the nontargeted liposomes only 24 h postinjection. In addition, the folate-targeted liposomes were injected at half the nontargeted liposome dose, further demonstrating their effectiveness. Histological analysis of sectioned tumor slices revealed distinct fluorescence patterns between the targeted and nontargeted systems, with a more localized and hyperintense fluorescence signal observed from tumor sections post-folate-targeted liposome injections. These results demonstrate the effectiveness of folate targeting for dynamic real-time solid tumor MRI and provide insight into kinetics of targeted and nontargeted nanoparticles to solid tumors.
Assuntos
Proteínas de Transporte/metabolismo , Corantes Fluorescentes/metabolismo , Lipossomos/metabolismo , Imageamento por Ressonância Magnética/métodos , Magnetismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Receptores de Folato com Âncoras de GPI , Gadolínio/química , Gadolínio/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Microscopia de Fluorescência , Neoplasias/genética , Neoplasias/patologiaRESUMO
Growth factors have great therapeutic potential for various disease therapy and tissue engineering applications. However, their clinical efficacy is hampered by low bioavailability, rapid degradation in vivo and non-specific biodistribution. Nanoparticle based delivery systems are being evaluated to overcome these limitations. Herein, we have developed a thermosensitive heparin nanosponge (Hep-NS) by a one step photopolymerization reaction between diacrylated pluronic and thiolated heparin molecules. The amount of heparin in Hep-NS was precisely controlled by varying the heparin amount in the reaction feed. Hep-NS with varying amounts of heparin showed similar size and shape properties, though surface charge decreased with an increase in the amount of heparin conjugation. The anticoagulant activity of the Hep-NS decreased by 65% compared to free heparin, however the Hep-NS retained their growth factor binding ability. Four different growth factors, bFGF, VEGF, BMP-2, and HGF were successfully encapsulated into Hep-NS. In vitro studies showed sustained release of all the growth factors for almost 60 days and the rate of release was directly dependent on the amount of heparin in Hep-NS. The released growth factors retained their bioactivity as assessed by a cell proliferation assay. This heparin nanosponge is therefore a promising nanocarrier for the loading and controlled release of growth factors.
Assuntos
Heparina/química , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Nanoestruturas/química , Animais , Proteína Morfogenética Óssea 2/metabolismo , Reagentes de Ligações Cruzadas/química , Preparações de Ação Retardada/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Nanopartículas , Nanoestruturas/uso terapêutico , Poloxâmero/química , Distribuição TecidualRESUMO
The pharmacological manipulation of liver X receptors (LXRs) has been an attractive therapeutic strategy for atherosclerosis treatment as they control reverse cholesterol transport and inflammatory response. This study presents the development and efficacy of nanoparticles (NPs) incorporating the synthetic LXR agonist GW3965 (GW) in targeting atherosclerotic lesions. Collagen IV (Col IV) targeting ligands are employed to functionalize the NPs to improve targeting to the atherosclerotic plaque, and formulation parameters such as the length of the polyethylene glycol (PEG) coating molecules are systematically optimized. In vitro studies indicate that the GW-encapsulated NPs upregulate the LXR target genes and downregulate proinflammatory mediator in macrophages. The Col IV-targeted NPs encapsulating GW (Col IV-GW-NPs) successfully reaches atherosclerotic lesions when administered for 5 weeks to mice with preexisting lesions, substantially reducing macrophage content (≈30%) compared to the PBS group, which is with greater efficacy versus nontargeting NPs encapsulating GW (GW-NPs) (≈18%). In addition, mice administered the Col IV-GW-NPs do not demonstrate increased hepatic lipid biosynthesis or hyperlipidemia during the treatment period, unlike mice injected with the free GW. These findings suggest a new form of LXR-based therapeutics capable of enhanced delivery of the LXR agonist to atherosclerotic lesions without altering hepatic lipid metabolism.
Assuntos
Benzoatos/farmacologia , Benzilaminas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores X do Fígado/agonistas , Nanomedicina , Receptores de LDL/genética , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/patologia , Benzoatos/química , Benzoatos/uso terapêutico , Benzilaminas/química , Benzilaminas/uso terapêutico , Células Cultivadas , Colesterol/sangue , Colágeno Tipo IV/química , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Portadores de Fármacos/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas/química , Nanopartículas/metabolismo , Polietilenoglicóis/química , Receptores de LDL/deficiência , Triglicerídeos/sangueRESUMO
Therapeutic nanoparticles (TNPs) have shown heterogeneous responses in human clinical trials, raising questions of whether imaging should be used to identify patients with a higher likelihood of NP accumulation and thus therapeutic response. Despite extensive debate about the enhanced permeability and retention (EPR) effect in tumors, it is increasingly clear that EPR is extremely variable; yet, little experimental data exist to predict the clinical utility of EPR and its influence on TNP efficacy. We hypothesized that a 30-nm magnetic NP (MNP) in clinical use could predict colocalization of TNPs by magnetic resonance imaging (MRI). To this end, we performed single-cell resolution imaging of fluorescently labeled MNPs and TNPs and studied their intratumoral distribution in mice. MNPs circulated in the tumor microvasculature and demonstrated sustained uptake into cells of the tumor microenvironment within minutes. MNPs could predictably demonstrate areas of colocalization for a model TNP, poly(d,l-lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG), within the tumor microenvironment with >85% accuracy and circulating within the microvasculature with >95% accuracy, despite their markedly different sizes and compositions. Computational analysis of NP transport enabled predictive modeling of TNP distribution based on imaging data and identified key parameters governing intratumoral NP accumulation and macrophage uptake. Finally, MRI accurately predicted initial treatment response and drug accumulation in a preclinical efficacy study using a paclitaxel-encapsulated NP in tumor-bearing mice. These approaches yield valuable insight into the in vivo kinetics of NP distribution and suggest that clinically relevant imaging modalities and agents can be used to select patients with high EPR for treatment with TNPs.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Óxido Ferroso-Férrico/metabolismo , Fibrossarcoma/tratamento farmacológico , Imageamento por Ressonância Magnética/métodos , Magnetismo/métodos , Nanomedicina/métodos , Nanopartículas , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/administração & dosagem , Polietilenoglicóis/metabolismo , Poliglactina 910/metabolismo , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Linhagem Celular Tumoral , Química Farmacêutica , Dano ao DNA , Progressão da Doença , Feminino , Óxido Ferroso-Férrico/química , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Humanos , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/química , Paclitaxel/metabolismo , Tamanho da Partícula , Polietilenoglicóis/química , Poliglactina 910/química , Valor Preditivo dos Testes , Fatores de Tempo , Distribuição Tecidual , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Liver X receptors (LXRs) attenuate inflammation by modulating the expression of key inflammatory genes, making LXRs and their ligands particularly attractive candidates for therapeutic intervention in cardiovascular, metabolic, and/or inflammatory diseases. Herein, enhanced proresolving activity of polymeric nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (LXR-NPs) is demonstrated, developed from a combinatorial library of more than 70 formulations with variations in critical physicochemical parameters. In vitro studies on peritoneal macrophages confirm that LXR-NPs are significantly more effective than the free agonist at downregulating pro-inflammatory mediators (MCP-1 and TNFα), as well as inducing the expression of LXR target genes (ABCA1 and SREBP1c). Through a zymosan-induced acute peritonitis in vivo model, LXR-NPs are found to be more efficient than free GW3965 at limiting the recruitment of polymononuclear neutrophils (50% vs 17%), suppressing the gene expression and secretion of pro-inflammatory factors MCP-1 and TNFα in peritoneal macrophages, and decreasing the resolution interval up to 4 h. Furthermore, LXR-NPs suppress the secretion of MCP-1 and TNFα by monocytes and macrophages more efficiently than the commercial drug dexamethasone. Overall, these findings demonstrate that LXR-NPs are capable of promoting resolution of inflammation and highlight the prospect of LXR-based nanotherapeutics for inflammatory diseases.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Nanopartículas/uso terapêutico , Polímeros/uso terapêutico , Animais , Anti-Inflamatórios/química , Benzoatos/química , Benzoatos/uso terapêutico , Benzilaminas/química , Benzilaminas/uso terapêutico , Modelos Animais de Doenças , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Receptores Nucleares Órfãos/metabolismo , Peritonite/tratamento farmacológico , Polímeros/químicaRESUMO
RNA interference (RNAi) is being widely explored as a means of tumour therapy due to the specific and potent silencing of targeted genes. However, in vivo delivery of RNAi effectors, such as small interfering RNA (siRNA) and detection of delivery is fraught with problems. Here, we describe novel theranostic PEGylated siRNA nanoparticles termed liposome-entrapped siRNA (LEsiRNA) nanoparticles. Our LEsiRNA nanoparticles are MR sensitive, contain labels for fluorescence microscopy/histology and promote functional siRNA delivery to tumours in mice leading to a significant reduction in both Survivin expression and tumour growth. LEsiRNA nanoparticles, administered by intravenous injection, were shown to accumulate in xenograft tumours by MR contrast image enhancements 24h post-administration. Fluorescence microscopy was used to corroborate the MR results and simultaneously demonstrate co-localisation of nanoparticles and siRNA within the tumours. The LEsiRNA nanoparticle-mediated delivery of the anti-cancer Survivin siRNA causes significant reduction in tumour growth when compared to controls. Our results suggest that LEsiRNA nanoparticles can be valuable as an in vivo delivery agent for siRNA therapy to tumours.
Assuntos
Proteínas Inibidoras de Apoptose/genética , Nanopartículas/química , Neoplasias/terapia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Proteínas Repressoras/genética , Animais , Western Blotting , Química Farmacêutica , Meios de Contraste/administração & dosagem , Estabilidade de Medicamentos , Feminino , Corantes Fluorescentes/administração & dosagem , Lipossomos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologia , SurvivinaRESUMO
We report the syntheses of novel cationic lipids comprised of cholesteryl-moieties linked to guanidinium functional groups, and also cationic lipids comprising a dialkylglycylamide moiety conjugated with a polyamine or a guanidinium functional group. In plasmid DNA (pDNA) transfection studies, these cationic lipids were formulated into cationic liposomes with the neutral co-lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) or with a recently reported neutral lipophosphoramidate derivative of histamine (MM27). We observe that cationic liposomes prepared from the cationic lipid N',N'-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide (DODAG) and DOPE frequently mediate the highest levels of transfection in vitro in all three different cell lines studied (OVCAR-3, IGROV-1 and HeLa) both in the presence or absence of serum. In addition, in vitro cellular toxicity was found to be minimal. Alternatively, we observe that DODAG alone forms lipoplex nanoparticles with small interfering RNA (siRNA) that are able to mediate the functional delivery of two previously validated anti-hepatitis B virus (HBV)--siRNAs to murine liver in vivo with minimal observable liver toxicity and immune stimulation. Specific knock-down of HBV infection parameters (virion and hepatic mRNA levels) is observed that is at least equivalent to the impact of extensive treatment with lamivudine (a licensed antiviral drug).
Assuntos
DNA/administração & dosagem , Dipeptídeos/química , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Transfecção , Animais , Cátions/química , Linhagem Celular , Sobrevivência Celular , Vírus da Hepatite B/genética , Humanos , Lipossomos/química , Camundongos , Camundongos Transgênicos , Nanopartículas/química , RNA Interferente Pequeno/genéticaRESUMO
A novel bimodal fluorescent and paramagnetic liposome is described for cellular labeling. In this study, we show the synthesis of a novel gadolinium lipid, Gd.DOTA.DSA, designed for liposomal cell labeling and tumor imaging. Liposome formulations consisting of this lipid were optimized in order to allow for maximum cellular entry, and the optimized formulation was used to label HeLa cells in vitro. The efficiency of this novel bimodal Gd-liposome formulation for cell labeling was demonstrated using both fluorescence microscopy and magnetic resonance imaging (MRI). The uptake of Gd-liposomes into cells induced a marked reduction in their MRI T 1 relaxation times. Fluorescence microscopy provided concomitant proof of uptake and revealed liposome internalization into the cell cytosol. The optimized formulation was also found to exhibit minimal cytotoxicity and was shown to have capacity for plasmid DNA (pDNA) transfection. A further second novel neutral bimodal Gd-liposome is described for the labeling of xenograft tumors in vivo utilizing the enhanced permeation and retention effect (EPR). Balb/c nude mice were inoculated with IGROV-1 cells, and the resulting tumor was imaged by MRI using these in vivo Gd-liposomes formulated with low charge and a poly(ethylene glycol) (PEG) calyx for long systemic circulation. These Gd-liposomes which were less than 100 nm in size were shown to accumulate in tumor tissue by MRI, and this was also verified by fluorescence microscopy of histology samples. Our in vivo tumor imaging results demonstrate the effectiveness of MRI to observe passive targeting of long-term circulating liposomes to tumors in real time, and allow for MRI directed therapy, wherein the delivery of therapeutic genes and drugs to tumor sites can be monitored while therapeutic effects on tumor mass and/or size may be simultaneously observed, quantitated, and correlated.
Assuntos
Lipossomos/metabolismo , Imageamento por Ressonância Magnética , Neoplasias/diagnóstico , Morte Celular/efeitos dos fármacos , Células HeLa , Compostos Heterocíclicos/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Ligantes , Lipídeos/química , Lipossomos/química , Lipossomos/toxicidade , Microscopia de Fluorescência , Neoplasias/patologia , Compostos Organometálicos/metabolismo , Espectrofotometria Atômica , TransfecçãoRESUMO
Cellular entry of imaging probes, such as contrast agents for magnetic resonance imaging (MRI), is a key requirement for many molecular imaging studies, particularly imaging intracellular events and cell tracking. Here, we describe the successful development and in vitro analysis of MAGfect, a novel liposome formulation containing a lipidic gadolinium contrast agent for MRI, Gd-DOTA-Chol , designed to enter and label cells. Liposome formulation and cell incubation time were optimised for maximum cellular uptake of the imaging probe in a variety of cell lines. MRI analysis of cells incubated with MAGfect showed them to be highly MRI active. This formulation was examined further for cytotoxicity, cell viability and mechanism of cell labelling. One of the key advantages of using MAGfect as a labelling vehicle arises from its potential for additional functions, such as concomitant drug or gene delivery and fluorescent labelling. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA (pDNA) into cells and expression levels were comparable to the commercial transfection agent Trojene.