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Residual volumes of infusion solutions vary greatly due to container and dimensional variances. Manufacturers use overfill to compensate, but the exact amounts vary significantly. This variability in overfill - when carrier solutions are used to dilute other parenteral preparations - may lead to variable concentrations and dosing, hence, potential risk for patients. We analyzed the overfill and residual volume of 22 pre-filled infusion containers and evaluated the impact on the (simulated) dosing accuracy of a therapeutic drug product for different handling scenarios. In addition, compendial properties of the diluents (i.e. sub-visible particles, pH, color and opalescence) were assessed. The overfill and residual volume between different containers for the same diluent varied. As container size increased, the relative volume of overfill decreased while the residual volume remained constant. The design and material of the containers (e.g. port systems) defined the residual volume. Different handling scenarios led to differences in dosing accuracy. As a result, no universal approach applicable for all containers can be defined. To ensure the right dose, it is recommended to pre-select the preferred diluent, evaluate fill volumes of carrier solutions, and assess in-use compatibility of the product solution with its diluent in terms of concentration and volume.
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Embalagem de Medicamentos , Humanos , Infusões ParenteraisRESUMO
For the safety and efficacy of frozen cell therapy products, determination of cellular viability is key. However, results of cell viability measurements do not only depend on the cell line or on the inflicted stress, but also on the assay used, making inter-experimental comparisons difficult. The aim of this study was thus to assess commonly used viability assays in clinically relevant human mesenchymal/stromal stem cells and human A549 lung carcinoma cells. Post freeze-thaw stress viability and proliferation were evaluated under different conditions using trypan blue, acridine orange/DAPI stain, alamarBlue, ATP, and neutral red assays. Significant differences in cell viability between metabolic assays were observed, likely due to their distinct intrinsic detection mechanisms. Membrane-integrity based assays generally overestimated cell viabilities in this study. Furthermore, noticeable differences in inter-assay sensitivities were observed. These differences highlight that cell viability methods should be meticulously selected and their associated results carefully interpreted in a relevant context to ensure reliable conclusions. Indeed, although cell membrane integrity based assays are a popular choice to determine cellular quality attributes after freezing and thawing, we demonstrate that metabolic assays may be more suitable in this context.
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Carcinoma , Células-Tronco , Humanos , Congelamento , Sobrevivência Celular , Pulmão , Criopreservação/métodosRESUMO
Polyethlyenimine (PEI) was introduced 1995 as a cationic polymer for nucleic acid delivery. PEI and its derivatives are extensively used in basic research and as reference formulations in the field of polymer-based gene delivery. Despite its widespread use, the number of clinical applications to date is limited. Thus, this review aims to consolidate the past applications of PEI in DNA delivery, elucidate the obstacles that hinder its transition to clinical use, and highlight potential prospects for novel iterations of PEI derivatives. The present review article is divided into three sections. The first section examines the mechanism of action employed by PEI, examining fundamental aspects of cellular delivery including uptake mechanisms, release from endosomes, and transport into the cell nucleus, along with potential strategies for enhancing these delivery phases. Moreover, an in-depth analysis is conducted concerning the mechanism underlying cellular toxicity, accompanied with approaches to overcome this major challenge. The second part is devoted to the in vivo performance of PEI and its application in various therapeutic indications. While systemic administration has proven to be challenging, alternative localized delivery routes hold promise, such as treatment of solid tumors, application as a vaccine, or serving as a therapeutic agent for pulmonary delivery. In the last section, the outcome of completed and ongoing clinical trials is summarized. Finally, an expert opinion is provided on the potential of PEI and its future applications. PEI-based formulations for nucleic acid delivery have a promising potential, it will be an important task for the years to come to introduce innovations that address PEI-associated shortcomings by introducing well-designed PEI formulations in combination with an appropriate route of administration.
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INTRODUCTION: Blood infections from multi-drug-resistant Salmonella pose a major health burden. This is especially true because Salmonella can survive and replicate intracellularly, and the development of new treatment strategies is dependent on expensive and time-consuming in vivo trials. The aim of this study was to develop a Salmonella-infection model that makes it possible to directly observe Salmonella infections of macrophages in vivo and to use this model to test the effect of antimicrobials against intra- and extracellular Salmonella in order to close the gap between in vitro and rodent-infection models. METHODS: We established suitable Salmonella-infection conditions using genetically engineered zebrafish and Salmonella-expressing fluorescent proteins (green fluorescent protein (GFP) and/or mCherry). RESULTS: We detected Salmonella inside and outside zebrafish larvae macrophages. Administration of the cell-impermeable antibiotic tobramycin removed Salmonella residing outside macrophages but did not affect Salmonella in macrophages, whereas ceftriaxone successfully cleared both types of Salmonella. Salmonella inside and outside macrophages experienced substantial DNA damage after administration of fluoroquinolones consistent with the excellent cell penetration of these antibiotics. CONCLUSIONS: The zebrafish-larvae model enables testing of antimicrobials for efficacy against extra- and intracellular Salmonella in a complex in vivo environment. This model thus might serve for antimicrobial lead optimization prior to using rodent models.
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Antibacterianos , Peixe-Zebra , Animais , Larva , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Macrófagos/metabolismo , Salmonella/genéticaRESUMO
Oncotoxic proteins such as the non-structural protein 1 (NS1), a constituent of the rodent parvovirus H1 (H1-PV), offer a novel approach for treatment of tumors that are refractory to other treatments. In the present study, mutated NS1 variants were designed and tested with respect to their oncotoxic potential in human hepatocellular carcinoma cell lines. We introduced single point mutations of previously described important residues of the wild-type NS1 protein and a deletion of 114 base pairs localized within the N-terminal domain of NS1. Cell-viability screening with HepG2 and Hep3B hepatocarcinoma cells transfected with the constructed NS1-mutants led to identification of the single-amino acid NS1-mutant NS1-T585E, which led to a 30% decrease in cell viability as compared to NS1 wildtype. Using proteomics analysis, we could identify new interaction partners and signaling pathways of NS1. We could thus identify new oncotoxic NS1 variants and gain insight into the modes of action of NS1, which is exclusively toxic to human cancer cells. Our in-vitro studies provide mechanistic explanations for the observed oncolytic effects. Expression of NS1 variants had no effect on cell viability in NS1 unresponsive control HepG2 cells or primary mouse hepatocytes. The availability of new NS1 variants in combination with a better understanding of their modes of action offers new possibilities for the design of innovative cancer treatment strategies.
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Parvovirus , Proteínas não Estruturais Virais , Animais , Humanos , Camundongos , Linhagem Celular , Neoplasias Hepáticas/genética , Infecções por Parvoviridae , Parvovirus/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially nonviral gene delivery systems are of increasing interest. However, low transgene expression levels and limited tolerability of these systems remain a challenge. The improvement of nucleic acid delivery using depolymerized chitosan-polyethylenimine DNA complexes (dCS-PEI/DNA) is investigated. The secore complexes are further combined with chitosan-based shells and functionalized with polyethylene glycol (PEG) and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physicochemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-linear PEI/DNA complex with a shell consisting of dCS-PEG-COOH, which results in improved nucleic acid encapsulation, cellular uptake and transfection potency in human hepatoma HuH-7cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induced a high reporter gene signal for three days. It is concluded that ternary coreshell structured nanoparticles comprising functionalized chitosan can be used for in vitro andin vivo gene delivery.
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Quitosana , Nanopartículas , Camundongos , Humanos , Animais , Quitosana/farmacologia , Quitosana/química , Polietilenoimina/farmacologia , Polietilenoimina/química , Transfecção , Técnicas de Transferência de Genes , DNA/genética , Nanopartículas/química , Polietilenoglicóis/farmacologia , Polietilenoglicóis/químicaRESUMO
Introduction: Larch oleoresin has been described regarding several biological activities and medicinal applications, such as wound healing and treatment of ulcers, but little is known about its chemical composition. Material and methods: Eight oleoresins from Larix decidua Mill. obtained from four companies and one adulterated control were therefore investigated to determine their content of essential oils and to verify possible differences in their composition in relation to the harvest and manufacturing processes. Essential oils (EOs) were isolated by distillation and the yield was analysed. Results and discussion: The yield of EO varied among all samples. The yield of the pure larch samples covered a range of 7.8% to 15.5%. A higher yield (19.0%) was observed for adulterated control, which contained oleoresins from different Pinaceae trees. Age of samples had no impact on yield. However, there was a significant statistical variation (p<0.05) in the yields of the mid-summer oleoresins (>10%) compared to early or late summer (<10%), emphasising the importance of the time of collection. Samples were subsequently analysed by GC-MS. EO samples confirmed the presence of various chemical classes, such as monoterpenes, sesquiterpenes, and diterpenes. α-pinene was the compound with the highest concentrations (>50%), followed by ß-pinene (>6%), D-limonene (>2.5%), α-terpineol (>0.9%), ß-myrcene (>0.2%), and 3-carene (>0.05%). Samples were grouped using multivariate data analysis (MVDA) with respect to the chemical variation between the oleoresins' EOs. The resulting four clusters were named low (low yield obtained for the samples), mixed (mixed oleoresin from different Pinaceae species, adulteration control), old (old oleoresin kept in the institute), and normal (other oleoresins) samples, each presenting distinct chemical biomarkers. There were considerable differences between site and time of collection. Essential oil yield did not always meet requirements as defined by the German Homeopathic Pharmacopoeia. In addition, adulterated or aged samples could be identified as compared to pure and fresh larch oleoresins. Conclusion: We conclude that larch oleoresin used for pharmaceutical applications has to be carefully analysed and standardised to guarantee reproducible product quality.
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Malignant ulcerating wounds or neoplastic lesions are a considerable burden for patients suffering from advanced cancer. These wounds have no effective treatment and are very difficult to manage. The present review summarizes evidence in support of a hypothesis put forward in anthroposophic medicine, which suggests a beneficial role of resin from the species Larix decidua Mill. [Pinaceae] for treating such wounds. A systematic search strategy was performed using the databases PubMed, EMBASE and SciFinder. The included publications described the chemical composition of this species, as well as in vitro, in vivo, and ex vivo experiments using plant extracts and isolated compounds. The results show that among the phytochemical classes, terpenoids were the major components of this species, especially in the resin. The summarized biological experiments revealed antimicrobial, antioxidant and anti-inflammatory effects, with promising potential for the extracts and isolated compounds. However, the molecular mechanisms and toxicological effects are as of yet not conclusively evaluated. From the data of our study, we can conclude that L. decidua might indeed have a promising potential for the treatment of malignant wounds, but definitive information that can prove its effectiveness is still lacking. We therefore suggest that future efforts should be dedicated to the evaluation of L. decidua resin's therapeutic use considering its antiseptic action and proposed wound healing properties.
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In vivo micromanipulation using ultrasound is an exciting technology with promises for cancer research, brain research, vasculature biology, diseases, and treatment development. In the present work, we demonstrate in vivo manipulation of gas-filled microparticles using zebrafish embryos as a vertebrate model system. Micromanipulation methods often are conducted in vitro, and they do not fully reflect the complex environment associated in vivo. Four piezoelectric actuators were positioned orthogonally to each other around an off-centered fluidic channel that allowed for two-dimensional manipulation of intravenously injected microbubbles. Selective manipulation of microbubbles inside a blood vessel with micrometer precision was achieved without interfering with circulating blood cells. Last, we studied the viability of zebrafish embryos subjected to the acoustic field. This successful high-precision, in vivo acoustic manipulation of intravenously injected microbubbles offers potentially promising therapeutic options.
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Micropartículas Derivadas de Células , Peixe-Zebra , Acústica , Animais , Encéfalo , Micromanipulação/métodosRESUMO
The essential homeostatic process of dead cell clearance (efferocytosis) is used by viruses in an act of apoptotic mimicry. Among others, virions leverage phosphatidylserine (PS) as an essential "eat me" signal in viral envelopes to increase their infectivity. In a virus-inspired biomimetic approach, we demonstrate that PS can be incorporated into non-viral lipid nanoparticle (LNP) pDNA/mRNA constructs to enhance cellular transfection. The inclusion of the bioactive PS leads to an increased ability of LNPs to deliver nucleic acids in vitro to cultured HuH-7 hepatocellular carcinoma cells resulting in a 6-fold enhanced expression of a transgene. Optimal PS concentrations are in the range of 2.5 to 5% of total lipids. PS-decorated mRNA-LNPs show a 5.2-fold enhancement of in vivo transfection efficiency as compared to mRNA-LNPs devoid of PS. Effects were less pronounced for PS-decorated pDNA-LNPs (3.2-fold increase). Incorporation of small, defined amounts of PS into gene delivery vectors opens new avenues for efficient gene therapy and can be easily extended to other therapeutic systems.
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Nanopartículas , Fosfatidilserinas , Técnicas de Transferência de Genes , Lipossomos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.
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Linfócitos T CD8-Positivos/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Magnésio/metabolismo , Animais , Infecções Bacterianas/imunologia , Restrição Calórica , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Células HEK293 , Humanos , Memória Imunológica , Sinapses Imunológicas/metabolismo , Imunoterapia , Ativação Linfocitária/imunologia , Sistema de Sinalização das MAP Quinases , Magnésio/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
Leaching of toxic or reactive chemicals from polymeric materials can adversely affect the quality and safety of biopharmaceuticals. It was therefore the aim of the present study to analyze leachables from a disposable clinical administration syringe using a polysorbate-containing surrogate solution and to assess their chemical reactivity. Analytical methods did include (headspace) GC-MS, Fourier-transform-infrared spectroscopy, a ferrous oxidation-xylenol orange assay, and nuclear magnetic resonance analysis. In the syringe leachables solution, the carcinogenic 1,1,2,2-tetrachloroethane (TCE) was detected in concentrations above the ICH M7-derived analytical evaluation threshold. TCE was shown to be an oxidation product of dichloromethane used during sample preparation. Since TCE was only isolated from incubations with the contained rubber stopper, we hypothesized that a stopper-derived leachable acted as a reactive oxidant promoting this chemical reaction. Subsequently, the leachable was identified to be the polymerization initiator Luperox® 101. Combining different analytical approaches led to the structural elucidation of a chemical reactive oxidant, which has the potential to interact and alter drug products. We conclude that chemically reactive compounds, such as the newly identified rubber stopper leachable Luperox® 101, may be of concern and therefore should be routinely considered if a prolonged exposure of polymers with drug products can be anticipated.
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Borracha , Seringas , Contaminação de Medicamentos , Embalagem de Medicamentos , OxirreduçãoRESUMO
There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6-9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.
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Quitosana/química , Técnicas de Transferência de Genes , Polietilenoimina/química , Transfecção , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Fenômenos Químicos , Técnicas de Química Sintética , Coloides/química , DNA/química , Expressão Gênica , Genes Reporter , Vetores Genéticos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Transfecção/métodosRESUMO
Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/terapia , Terapia Genética , Neoplasias Hepáticas/terapia , Camundongos , Plasmídeos , ProteínasRESUMO
Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.
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Portadores de Fármacos/farmacologia , Terapia Enzimática/métodos , Enzimas/administração & dosagem , Lipopeptídeos/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Portadores de Fármacos/química , Embrião não Mamífero , Enzimas/farmacocinética , Células HEK293 , Hepatócitos/metabolismo , Humanos , Lipopeptídeos/química , Fígado/citologia , Fígado/metabolismo , Camundongos , Modelos Animais , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Simportadores/metabolismo , Distribuição Tecidual , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Buprenorphine is a frequently used analgetic agent in veterinary medicine. A major drawback, however, is the short duration of action requiring several daily administrations. We therefore designed a poly-lactic-co-glycolic acid (PLGA) based microparticulate drug formulation for sustained parenteral drug release. Particles were designed to allow for a fast onset of action and a duration of the analgesic effect of at least two days in laboratory mice. Microparticles were produced using a solvent evaporation technique. Release rate was dependent on polymer type and particle size. Spherical particles used for subsequent animal studies had a mean size of 50 µm and contained 4.5% of buprenorphine. Drug release was characterized by an initial burst release of 30% followed by complete release over seven days. In vivo pharmacokinetic experiments in female C57BL/6 J mice confirmed prolonged exposure in plasma and brain tissue and correlated with the pharmacological effect in the hot plate assay or after minor abdominal surgery. No adverse side effects with respect to food and water intake, body weight, local tolerability, or nesting behavior were observed. Our formulation is an attractive alternative to established immediate release formulations. A use for prolonged pain management in laboratory animals is proposed.
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Analgésicos , Buprenorfina , Composição de Medicamentos/métodos , Composição de Medicamentos/veterinária , Desenho de Fármacos , Manejo da Dor/veterinária , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Animais , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Fatores de TempoRESUMO
Cellular delivery of DNA vectors for the expression of therapeutic proteins is a promising approach to treat monogenic disorders or cancer. Significant efforts in a preclinical and clinical setting have been made to develop potent nonviral gene delivery systems based on lipoplexes composed of permanently cationic lipids. However, transfection efficiency and tolerability of such systems are in most cases not satisfactory. Here, we present a one-pot combinatorial method based on double-reductive amination for the synthesis of short-chain aminolipids. These lipids can be used to maximize the DNA vector delivery when combined with the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). We incorporated various aminolipids into such lipoplexes to complex minicircle DNA and screened these systems in a human liver-derived cell line (HuH7) for gene expression and cytotoxicity. The lead aminolipid AL-A12 showed twofold enhanced gene delivery and reduced toxicity compared to the native DOTAP:cholesterol lipoplexes. Moreover, AL-A12-containing lipoplexes enabled enhanced transgene expression in vivo in the zebrafish embryo model.
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DNA has been widely used as a key tether to promote self-organization of super-assemblies with emergent properties. However, control of this process is still challenging for compartment assemblies and to date the resulting assemblies have unstable membranes precluding in vitro and in vivo testing. Here we present our approach to overcome these limitations, by manipulating molecular factors such as compartment membrane composition and DNA surface density, thereby controlling the size and stability of the resulting DNA-linked compartment clusters. The soft, flexible character of the polymer membrane and low number of ssDNA remaining exposed after cluster formation determine the interaction of these clusters with the cell surface. These clusters exhibit in vivo stability and lack of toxicity in a zebrafish model. To display the breadth of therapeutic applications attainable with our system, we encapsulated the medically established enzyme laccase within the inner compartment and demonstrated its activity within the clustered compartments. Most importantly, these clusters can interact selectively with different cell lines, opening a new strategy to modify and expand cellular functions by attaching such pre-organized soft DNA-mediated compartment clusters on cell surfaces for cell engineering or therapeutic applications.
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DNA/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Animais , Catálise , Linhagem Celular Tumoral , Membrana Celular/metabolismo , DNA/metabolismo , Células HEK293 , Humanos , Lacase/química , Lacase/metabolismo , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Polímeros/química , Polímeros/metabolismo , Polímeros/farmacocinética , Polímeros/toxicidade , Receptores Depuradores/antagonistas & inibidores , Receptores Depuradores/metabolismo , Distribuição Tecidual , Peixe-ZebraRESUMO
The concept of triggered drug release offers a possibility to overcome the toxic side effects of chemotherapeutics in cancer treatment by reducing systemic exposure to the active drug. In the present work, the concept foresees the use of the extracellular enzyme MMP9 as an enzymatic trigger for drug release in the proximity of tumor cells. METHODS: A paclitaxel-hemisuccinate-peptide conjugate as a building block for self-assembling nanoparticles was synthesized using standard conjugation approaches. The building block was purified via preparative HPLC and analyzed by LC-MS. Nanoparticles were formed using the nanoprecipitation method and characterized. For selection of a suitable in vitro model system, common bioanalytical methods were used to determine mRNA expression, enzyme amount, and activity of MMP9. RESULTS: The MMP9-labile prodrug was synthesized and characterized. Nanoparticles were formed out of MMP9-labile conjugate-building blocks. The nanoparticle's diameter averaged at around 120 nm and presented a spherical shape. LN-18 cells, a glioblastoma multiforme derived cell line, were chosen as an in vitro model based on findings in cancer tissue and cell line characterization. The prodrug showed cytotoxicity in LN-18 cells, which was reduced by addition of an MMP9 inhibitor. CONCLUSION: taken together, we confirmed increased MMP9 in several cancer tissues (cervical, esophageal, lung, and brain) compared to healthy tissue and showed the effectiveness of MMP9-labile prodrug in in vitro tests.
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Desenho de Fármacos , Espaço Extracelular/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Paclitaxel/química , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Química Sintética , Humanos , Metaloproteinase 9 da Matriz/genética , Paclitaxel/metabolismo , Tamanho da Partícula , Peptídeos/química , Polietilenoglicóis/química , RNA Mensageiro/genéticaRESUMO
Hepatocellular carcinoma is the most common type of primary malignancy in the liver and one of the most common types of cancer worldwide. Its readily increasing mortality rate highlights the urgent need for the development of efficient therapeutic strategies. Tyrosine kinase inhibitors (TKIs) such as sorafenib and sunitinib are used as efficient angiogenesis inhibitors for this purpose. However, despite their pharmacological effects, their transfer into clinical practice is characterized by their poor aqueous solubility and accumulation in off-target tissues, resulting in unfavorable side effects. Here, we report a nanocomposite made of amine-functionalized mesoporous silica nanocomposites (MSNs) that are surface-coated with cerium oxide nanoparticles (CNPs) for the controlled delivery and release of TKIs. Amine-functionalized MSNs were prepared using a sol-gel method and loaded with TKIs. To trap drug molecules into the mesoporous structure, CNPs were covalently conjugated to the surface of MSNs. The synthesis and functionalization steps were controlled using different characterization methods, confirming the desired morphology and structure, the identity of functional groups on the surface, successful coating, and appropriate loading efficiency. Under physiological conditions, CNP-capped MSNs demonstrated a sustained drug release over time as a result of CNPs' gatekeeping effect on the payloads. Strong cellular interactions with different liver cancer cells and enhanced cellular uptake were also observed in vitro for the gate-capped MSNs. Internalization of nanocomposites induced cell death via the production of reactive oxygen species, and subsequent activation of apoptosis pathways. This study demonstrates that gate-capped MSNs are promising chemotherapeutic vehicles characterized by a sustained drug release profile and high cellular internalization.