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The design of nanomedicines targeting specific tumor microenvironment (TME) characteristics, such as low pH, hypoxia, and elevated glutathione (GSH) concentration, holds significant potential for selectively killing cancer cells. In this study, we have developed a TME-responsive nanoparticle, i.e., MIL-53(Fe)@MnO2 stabilized with polyethylene glycol (FMP), which is capable of depleting GSH and sustainably generating hydroxyl radical (â¢OH) for enhanced chemodynamic therapy of cancer. After endocytosis, the FMP nanoparticles undergo gradual decomposition, resulting in GSH depletion while releasing Fenton-active Fe2+ and Mn2+ ions. These ions then subsequently catalyze a Fenton-like reaction to generate highly toxic â¢OH, ultimately inducing oxidative cell death. As a synergistic effect, the depletion of GSH further enhances therapeutic efficacy by inhibiting intracellular â¢OH scavenging. In vivo experiments demonstrated that this synergistically chemotherapeutic approach efficiently suppressed tumor growth without inducing significant systemic toxicity. This work presents a promising nanotherapeutic strategy that simultaneously depletes intracellular GSH and sustains cytotoxic â¢OH generation for enhanced tumor treatment.
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Antineoplásicos , Glutationa , Radical Hidroxila , Compostos de Manganês , Estruturas Metalorgânicas , Óxidos , Microambiente Tumoral , Glutationa/metabolismo , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Radical Hidroxila/metabolismo , Radical Hidroxila/química , Microambiente Tumoral/efeitos dos fármacos , Animais , Óxidos/química , Óxidos/farmacologia , Humanos , Camundongos , Antineoplásicos/química , Antineoplásicos/farmacologia , Camundongos Endogâmicos BALB C , Nanopartículas/química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sobrevivência Celular/efeitos dos fármacos , Tamanho da Partícula , Linhagem Celular Tumoral , Propriedades de Superfície , Estruturas Metalorgânicas/químicaRESUMO
Reactive oxygen species (ROS)-driven chemodynamic therapy has emerged as a promising anti-tumor strategy. However, the insufficient hydrogen peroxide (H2O2) supply in tumor microenvironment results in a low Fenton reaction rate and subsequently poor ROS production and therapeutic efficacy. Herein, we report on a new nanocomposite MIL-53@ZIF-67/S loaded with doxorubicin and glucose oxidase, which is decomposed under the acidic tumor microenvironment to release Fe3+, Co3+, glucose oxidase, and doxorubicin. The released content leads to synergistic anti-tumor effect through the following manners: 1) doxorubicin is directly used for chemotherapy; 2) Fe3+and Co3+ result in glutathione depletion and Fenton reaction activation through Fe2+ and Co2+ generation to achieve chemodynamic therapy; 3) glucose oxidase continuously catalyzes glucose consumption to induce starvation of the cancer cells, and 4) at the same time the produced gluconic acid and H2O2 significantly promote Fenton reaction and further boost chemodynamic therapy. This work not only demonstrates the high anti-tumor effect of the new nanocomposite, but also provides an innovative strategy for the development of a multi-in-one nanoplatform for cancer therapy.
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Cobalto , Doxorrubicina , Ferro , Estruturas Metalorgânicas , Nanocompostos , Nanocompostos/química , Cobalto/química , Cobalto/farmacologia , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Estruturas Metalorgânicas/síntese química , Ferro/química , Humanos , Doxorrubicina/farmacologia , Doxorrubicina/química , Catálise , Animais , Camundongos , Peróxido de Hidrogênio/química , Glucose Oxidase/metabolismo , Glucose Oxidase/química , Microambiente Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Propriedades de Superfície , Tamanho da Partícula , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
OBJECTIVE: This study aims to identify clinical factors that may predict failed endoscopic lumbar spine surgery to guide surgeons with patient selection during the initial learning curve. METHODS: This is an Australasian prospective analysis of the first 105 patients to undergo lumbar endoscopic spine decompression by 3 surgeons. Modified MacNab outcomes, visual analog scale (VAS) and Oswestry Disability Index (ODI) scores were utilized to evaluate clinical outcomes at 6 months postoperatively. Descriptive statistics and ANOVA t tests were performed to measure statistically significant (P < 0.05) associations between variables using GraphPad Prism v10. RESULTS: Patients undergoing endoscopic lumbar surgery via an interlaminar or transforaminal approach have overall good/excellent modified MacNab outcomes and a significant reduction in postoperative VAS and ODI scores. Regardless of the anatomic location of disc herniations, good/excellent modified MacNab outcomes and significant reductions in VAS and ODI were reported post-operatively, however, not in patients with calcified disc herniations. Patients with central and foraminal stenosis overall reported poor/fair modified MacNab outcomes, however, there were significant reductions in VAS and ODI scores postoperatively. Patients with subarticular stenosis or an associated spondylolisthesis reported good/excellent modified MacNab outcomes and significant reductions in VAS and ODI scores postoperatively. Patients with disc herniation and concurrent degenerative stenosis had generally poor/fair modified MacNab outcomes. CONCLUSIONS: The outcomes of endoscopic spine surgery are encouraging with low complication and reoperation rates. However, patients with calcified disc herniations, central canal stenosis, or disc herniation with concurrent degenerative stenosis present challenges during the initial learning curve and may benefit from traditional open or other minimally invasive techniques.
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Deslocamento do Disco Intervertebral , Humanos , Deslocamento do Disco Intervertebral/cirurgia , Constrição Patológica , Curva de Aprendizado , Estudos Retrospectivos , Endoscopia/métodos , Vértebras Lombares/cirurgia , Resultado do TratamentoRESUMO
The introduction of new therapeutics requires validation of Good Manufacturing Practice (GMP)-grade manufacturing including suitable quality controls. This is challenging for Advanced Therapy Medicinal Products (ATMP) with personalized batches. We have developed a person-alized, cell-based gene therapy to treat age-related macular degeneration and established a vali-dation strategy of the GMP-grade manufacture for the ATMP; manufacturing and quality control were challenging due to a low cell number, batch-to-batch variability and short production duration. Instead of patient iris pigment epithelial cells, human donor tissue was used to produce the transfected cell product ("tIPE"). We implemented an extended validation of 104 tIPE productions. Procedure, operators and devices have been validated and qualified by determining cell number, viability, extracellular DNA, sterility, duration, temperature and volume. Transfected autologous cells were transplanted to rabbits verifying feasibility of the treatment. A container has been engineered to ensure a safe transport from the production to the surgery site. Criteria for successful validation and qualification were based on tIPE's Critical Quality Attributes and Process Parameters, its manufacture and release criteria. The validated process and qualified operators are essential to bring the ATMP into clinic and offer a general strategy for the transfer to other manufacture centers and personalized ATMPs.
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Aim: Gene-based immunotherapy against cancer is limited by low gene transfer efficiency. In the literature, interleukin-12 (IL-12) encoding plasmid associated with sonoporation has been shown to enhance antitumoral activity. Moreover, non-viral carriers and high-frequency ultrasound have both been shown to promote immune response activation. Here, IL-12 encoding plasmid, non-viral carrier stimulating the immune response and focused ultrasound were combined in order to improve the antitumoral efficiency. Methods: In order to enhance a gene-based antitumoral immune response, home-made lipids Toll-like receptor 2 (TLR2) agonists and plasmid free of antibiotic resistance version 4 (pFAR4), a mini-plasmid, encoding the IL-12 cytokine were combined with high-intensity focused ultrasound (HIFU). The lipid composition and the combination conditions were selected following in vitro and in vivo preliminary studies. The expression of IL-12 from our plasmid construct was measured in vitro and in vivo. The combination strategy was evaluated in mice bearing colon carcinoma cells (CT26) tumors following their weight, tumor volume, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) levels in the serum and produced by splenocytes exposed to CT26 tumor cells. Results: Lipid-mediated cell transfection and intratumoral injection into CT26 tumor mice using pFAR4-IL-12 led to the secretion of the IL-12 cytokine into cell supernatant and mice sera, respectively. Conditions of thermal deposition using HIFU were optimized. The plasmid encoding pFAR4-IL-12 or TLR2 agonist alone had no impact on tumor growth compared with control mice, whereas the complete treatment consisting of pFAR4-IL-12, TLR2 lipid agonist, and HIFU limited tumor growth. Moreover, only the complete treatment increased significantly mice survival and provided an abscopal effect on a metastatic CT26 model. Conclusions: The HIFU condition was highly efficient to stop tumor growth. The combined therapy was the most efficient in terms of IL-12 and IFN-γ production and mice survival. The study showed the feasibility and the limits of this combined therapy which has the potential to be improved.
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Chemodynamic therapy has become an emerging cancer treatment strategy, in which tumor cells are killed through toxic reactive oxygen species (ROS), especially hydroxyl radicals (ËOH) produced by the Fenton reaction. Nevertheless, low ROS generation efficiency and ROS depletion by cellular antioxidant systems are still the main obstacles in chemodynamic therapy. In the present work, we propose a dually enhanced chemodynamic therapy obtained by inhibiting ËOH consumption and promoting ËOH production based on the administration of bimetallic sulfide Co3-xCuxS4 nanoparticles functionalized by polyethylene glycol. These bimetallic nanoparticles display glutathione depleting and photothermal properties. The nanoparticles are gradually degraded in a tumor microenvironment, resulting in Co2+ and Cu2+ release. The released Co2+ triggers a Fenton-like reaction that turns endogenous hydrogen peroxide into highly toxic ËOH. In the cellular environment, Cu2+ ions are reduced to Cu+ by endogenous GSH, which decreases the intracellular antioxidant capacity and additionally up-regulates ËOH production via the Cu+-induced Fenton-like reaction. Moreover, under near-infrared light irradiation, the bimetallic nanoparticles display a photothermal conversion efficacy of 46.7%, which not only improves chemodynamic therapy via boosting a Fenton-like reaction but results in photothermal therapy through hyperthermia. Both in vitro cancer cell killing and in vivo tumor ablation experiments show that the bimetallic nanoparticles display outstanding therapeutic efficacy and negligible systemic toxicity, indicating their anticancer potential.
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Hipertermia Induzida , Neoplasias , Antioxidantes , Cobre/farmacologia , Cobre/uso terapêutico , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Neoplasias/tratamento farmacológico , Polietilenoglicóis , Espécies Reativas de Oxigênio , SulfetosRESUMO
Robotic assistance technologies are being incorporated into minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) to minimize radiation exposure to the patient and operating staff. However, they introduce new issues including increased operating time and difficult incorporation into surgical workflow. This study, conducted with 42 patients under the care of one neurosurgeon in Sydney, Australia, investigates the operating time increase with three different robotic modalities, and the learning curves they pose to the surgeon. Between the comparable modalities of freehand MIS-TLIF and Mazor Renaissance® CT to Fluoro, there was a significant increase in time from patient draping to insertion of the final K-wire (p = 0.0019), and a non-significant increase in time per K-wire (p = 0.55) using Mazor Renaissance®. Comparing the ROSA® and Mazor Renaissance® Scan and Plan, there were significant increases in drape to final K-wire time and time per K-wire using ROSA® assistance (p = 0.000068 and p = 0.011). ROSA® also had a steeper learning curve compared to both Mazor Renaissance® modalities, which were similar. Our study shows that Mazor Renaissance® modalities are superior to ROSA® in minimizing extra operating time, and also have easier learning curves; however, both modalities increase operating time compared with freehand MIS-TLIF. This study, to our knowledge, is the first to compare multiple robotic techniques in MIS-TLIF. Though these results highlight important differences between robotic modalities that are crucial for spinal surgeons to understand, the low sample size and variability in data reveal the need for larger, multi-centre studies in this field.
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Parafusos Pediculares , Robótica , Fusão Vertebral , Humanos , Curva de Aprendizado , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Estudos Retrospectivos , Fusão Vertebral/métodos , Resultado do TratamentoRESUMO
Microfluidization has been investigated as a new, scalable, and basic component saving method to produce cationic lipid nanoparticles, in particular for the delivery of short interfering RNAs (siRNAs). The design of experiment (DoE) allowed to reach optimized characteristics in terms of nanocarrier size reduction and low polydispersity. The structure of cationic liposomes and siRNA-lipoplexes was characterized. The optimized preparation parameters were identified as three microfluidization passages at a pressure of 10,000 psi, with a thin film hydration volume of 4 ml. Microfluidized liposomes mean size was 160 nm, with a polydispersity index of 0.2-0.3 and a zeta potential of +40 mV to +60 mV. Positive versus negative charge ratio between the charges of the cationic lipid and the phosphate charges of the siRNAs is a key factor determining the structure and silencing efficacy of siRNA lipoplexes. At a (+/-) charge ratio of 8, a proportion of 88% of the siRNA was associated to microfluidized lipoplexes, which remained stable for one month. These lipoplexes exhibited moderate cytotoxicity and gene silencing efficacy, which should be further optimized.
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Lipídeos , Nanopartículas , Cátions , Lipossomos , RNA Interferente Pequeno , TransfecçãoRESUMO
Macrophages possess an innate ability to scavenge heterogenous objects from the systemic circulation and to regulate inflammatory diseases in various organs via cytokine production. That makes them attractive targets for nanomedicine-based therapeutic approaches to inflammatory diseases. In the present study, we have prepared several different poly(lactic-co-glycolic acid) (PLGA) polymer nanospheres for macrophage-targeted drug delivery using both nanoprecipitation and emulsification solvent evaporation methods. Two experimental linear PLGA polymers with relatively low molar weight, one experimental branched PLGA with unique star-like molecular architecture, and a commercially available PLGA, were used for nanosphere formulation and compared to their macrophage uptake capacity. The nanosphere formulations labelled with loaded fluorescent dye Rhodamine B were further tested in mouse bone marrow-derived macrophages and in hepatocyte cell lines AML-12, HepG2. We found that nanospheres larger than 100 nm prepared using nanoprecipitation significantly enhanced distribution of fluorescent dye selectively into macrophages. No effects of nanospheres on cellular viability were observed. Additionally, no significant proinflammatory effect after macrophage exposure to nanospheres was detected as assessed by a determination of proinflammatory cytokines Il-1ß and Tnfα mRNA. All experimental PLGA nanoformulations surpassed the nanospheres obtained with the commercially available polymer taken as a control in their capacity as macrophage-specific carriers.
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BACKGROUND: A bacterial cause of disc degeneration has evoked several controversies and, if true, would lead to a major shift in treatment paradigm. Earlier studies analyzing the relationship of bacterial disc infection within a degenerative cohort featured prolonged cultures susceptible to contamination. The degenerate-disc infection study with contaminant control (DISC) trial aims to investigate this theory further by examining infection rates using a non-degenerative control cohort in comparison to a degenerative internal control cohort and a sham cohort (sampling only sterile paraspinal tissue). To our knowledge, the current study is the largest evaluating the growth of organisms (or possible contamination rate) in paraspinal tissue if prolonged cultures are performed. Protocols on methodology have been previously published. PURPOSE: (1) To investigate the infection rates across cohorts (degenerative vs. nondegenerative control; paraspinal and/or disc controls vs. combined sampling cohorts) using stringent standardized aseptic surgical technique and laboratory processing. (2) To compare our findings to that of the literature and make a statement in support and/or against a possible contamination theory to positive cultures. STUDY DESIGN: Multicenter, multisurgeon case-control trial. PATIENT SAMPLE: In all, 812 surgical samples were retrieved across a 3.5-year period (2013-2016) including 25 trauma controls (nondegenerative), 550 "disc and paraspinal" samples (degenerative cases with internal control), 190 disc-only samples (degenerative cases without internal control), and 46 paraspinal only controls (sham group). OUTCOME MEASURES: Growth and/or Contamination rate (%) per cohort. Chi-square of growth in disc versus paraspinal samples as a means of examining the distribution of false positive and contaminant growth. The impact of previous injections and/or surgery on positive disc or paraspinal growth. Correlation of Modic changes with positive growth rates analyzed with the Kruskal-Wallis Test. The distribution of species in positive samples were also analyzed. METHODS: The DISC trial is registered under Australian and New Zealand clinical trials registry-ACTRN12616000541404. Institutional ethics review was obtained (HREC northern sector 13/218) at the primary center and further centers (n=6) were recruited. Patients undergoing spinal surgery with discectomy were eligible for trial entry with tissue specimens obtained using strict aseptic technique for microbiological examination. All specimens were handled with sterile instruments only and by a fresh instrument to a sterile pot that was closed immediately. Separate pots were used for the disc and paraspinal tissue respectively with similar stringent processing during microbiological assessment. A cohort of the degenerative cases at one single institution also underwent an additional histopathological examination. RESULTS: There was an expected significant difference in gender and age associated with the non-degenerative control group (due to trauma patients) compared with other cohorts. There was a higher percentage of positive-growth in the control group in comparison to the disc and paraspinal and disc only groups across positive disc growth (48% vs. 27% vs. 17%, p<.001). A similar infection rate was observed in the paraspinal samples across the equivalent controls (44% vs. 36% vs. 37%, p=.739). There was a significant difference in the proportions of positive growth with a large proportion of false positives (growth in both disc and paraspinal samples; p<.001). There was no difference in true positive growth between the case and control groups (16.0 vs. 7.7%, p=.112). These trends were preserved across all cohorts and when stratifying by spinal segment (cervical or lumbar). There was no correlation between Modic changes and positive disc culture growth (p=.398, n=144 samples). Cutibacterium (formerly Propionibacterium) acnes was the most dominant pathogen isolated, representing between 50% and 70% of positive disc and paraspinal specimens, followed by staphylococcal species. CONCLUSIONS: Our study failed to find a difference in true infection rates between the nondegenerative and degenerative disc populations. These findings are suggestive of a contamination theory and against a common infective etiology in the setting of discogenic back and neck pain. We believe the rationale for antibiotic therapy in the management of discogenic back pain warrants further evidence to establish efficacy.
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Degeneração do Disco Intervertebral , Deslocamento do Disco Intervertebral , Disco Intervertebral , Austrália , Ensaios Clínicos como Assunto , Humanos , Degeneração do Disco Intervertebral/cirurgia , Vértebras Lombares , Nova Zelândia , Propionibacterium acnes , Estudos ProspectivosRESUMO
Liver-directed gene therapy, using mainly viral vectors for the genetic cell modification, is a promising therapeutic approach for many genetic and metabolic liver diseases. The recent successful preclinical trials with AAV vectors expose the benefits as well as the limitations of the system. We focused on the development of an alternative non-viral episomal gene transfer system, by inserting the DNA element Scaffold/Matrix Attachment Region (S/MAR) into the free of antibiotic resistance gene miniplasmid vector (pFAR4). We produced pFAR4 derivative experimental vectors, carrying the eGFP gene driven by the composite HCRHPi liver-specific promoter and either lacking (pFAR4-noS/MAR) or containing the S/MAR element in an upstream (pFAR-S/MAR-IN) or downstream (pFAR4-S/MAR-OUT) configuration in relation to the poly-A signal of the eGFP expression cassette. Upon transfer into Huh7 cells by lipofection, vector pFAR4-S/MAR IN showed significantly higher transfection efficiency and eGFP expression than the control vector or the pFAR4-S/MAR-OUT (p < 0.005), estimated by fluorescent microscopy and flow cytometry. Stable transfections were produced only with cultures containing vector pFAR4-S/MAR IN, through the expansion of single colonies, which displayed sustained GFP expression and plasmid copy number per cell of 2.3 ± 0.4, at 3 months of culture. No vector integration events were detected in these cultures by FISH analysis, while the presence of free, circular plasmids was documented by plasmid rescue assay. The presence of S/MAR renders pFAR4 miniplasmid substantially more efficient regarding episomal gene transfer and is suitable for liver-directed studies towards gene therapy applications.
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Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hepatócitos/metabolismo , Plasmídeos , Linhagem Celular Tumoral , Células Cultivadas , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Fígado/metabolismo , TransfecçãoRESUMO
BACKGROUND: Direct lateral interbody fusion (DLIF) mitigates many of the vascular complications and bony resections associated with other interbody fusion techniques. However, there are concerns regarding postoperative neural complications and that indirect decompression of the foramen has not been consistently demonstrated. This study prospectively assessed the clinical and radiological outcomes and the complication rates of the DLIF approach. METHODS: A prospective review was conducted of the first 50 consecutive DLIF cases of a single neurosurgeon between 2010 and 2014. Clinical outcomes were assessed using Visual Analogue Scale (VAS), Oswestry Disability Index (ODI) and Roland Morris Disability Questionnaire (RMDQ) surveys. Radiological outcomes, including spondylolisthesis, disc height, local disc angle, lumbar lordosis and foraminal height and width, were measured using Surgimap Spine software at the preoperative, 6 weeks, 6 months, and 12 months postoperative follow-up. Complication rates were also reported. RESULTS: A total of 50 patients (84 levels) were treated with DLIF. The mean patient age was 68.2±9.8 years and 62.0% were female. At latest follow-up, mean VAS pain score improved from 7.7±1.5 to 1.9±0.9 (P<0.0001), mean ODI improved from 42.1±14.5 to 16.9±6.7 (P<0.0001) and mean RMDQ score improved from 12.1±5.2 to 6.2±4.7 (P<0.0001). Mean spondylolisthesis reduced from 7.5%±6.5% to 1.3%±1.1% at 6 weeks (P<0.0001), 0.95%±0.74% at 6 months (P<0.0001) and recurred to 1.9%±1.7% at 12 months postoperatively (P=0.0006). Mean anterior disc height improved from 7.3±3.2 to 11.6±2.5 mm at 6 weeks (P<0.0001), 12.2±3.3 mm at 6 months (P<0.0001) and 9.8±2.1 mm at 12 months (P=0.0032) postoperatively. Mean posterior disc height improved from 4.4±2.0 to 6.8±2.1 mm at 6 weeks (P<0.0001), 6.6±2.5 mm at 6 months (P=0.0003), and 5.9±1.4 mm at 12 months (P=0.0039) postoperatively. Mean local disc angle improved from 7.0°±3.7° to 9.2°±3.3° at 6 weeks (P=0.0072), 10.4°±3.9° at 6 months (P=0.0013) and 8.2°±2.9° at 12 months (P=0.2487) postoperatively. No significant postoperative changes in lumbar lordosis were observed. Mean foraminal height improved from 18.3±3.5 to 21.5±3.9 mm at 6 weeks (P=0.0004), 20.6±3.4 mm at 6 months (P=0.0266), and 18.7±1.9 mm at 12 months (P=0.8021) postoperatively. Mean foraminal width improved from 7.9±2.0 to 10.2±2.8 mm at 6 weeks (P=0.0001), 9.4±2.6 mm at 6 months (P=0.0219) and 8.3±1.6 mm at 12 months (P=0.5734) postoperatively. Fusion rate at 6 and 12 months was 62.2% and 89.2%, respectively. A total of 6 patients (12%) had postoperative complications. Three patients (6%) had pain-related psoas muscle weakness and 3 patients (6%) had sensory neural complications that had resolved entirely by 8 and 16 weeks postoperatively, respectively. CONCLUSIONS: The study provides encouraging short and medium-term clinical and radiological results for DLIF. In this patient series, there was a low complication rate with no permanent neural injury reported.
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Ultrasound-mediated gene delivery is an interesting approach, which could help in increasing gene transfer in deep tissues. Moreover, it allows for performing experiments guided by the image to determine which elements are required. Microbubbles complexed with a eukaryotic expression cassette are excellent agents as they are responsive to ultrasounds and, upon oscillation, can destabilize membranes to enhance gene transfer. Here, we describe the preparation of positively charged microbubbles, plasmid free of antibiotic resistance marker, their combination and the conditions of ultrasound-mediated liver transfection post-systemic administration in mice. This association allowed us to obtain a superior liver gene expression at least over 8 months after a single injection.
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Microbolhas , Transfecção/métodos , Ondas Ultrassônicas , Animais , Permeabilidade da Membrana Celular/efeitos da radiação , Terapia Genética/métodos , Células HeLa , Humanos , Fígado/citologia , Fígado/metabolismo , Fígado/efeitos da radiação , Camundongos , Camundongos Endogâmicos BALB C , Ácidos Nucleicos/genéticaRESUMO
Lactosylated albumin is currently used as a radiopharmaceutical agent to image the liver asialoglycoprotein receptors and quantify hepatic liver function in various diseases. A lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to non-lactosylated protein and a high signal to noise ratio, based on the biodistribution in mice using 99mTc-scintigraphy. However, in the laboratory, it is useful to have a method that can be used in daily practice to quantify cellular targeting or biodistribution. We propose a methodology from synthesis validation to pre-clinical demonstration and introduce a new practical detector (LACTAL.Eu) of the LACTAL molecule in biological media. We confirmed the purity and colloidal stability of the sample through physical analytical techniques, then showed the absence of in vitro toxicity of the agent and demonstrated in vitro targeting. Taking advantage of the fluorescence decay of the lanthanide, we performed measurements directly on the cell media without any further treatment. Finally, biodistribution in mice was confirmed by ex vivo measurements.
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Európio/química , Lactose/química , Albumina Sérica Humana/química , Coloração e Rotulagem , Aglutininas/metabolismo , Animais , Feminino , Glicosilação , Células Hep G2 , Humanos , Camundongos Endogâmicos BALB C , Ricina/metabolismo , Distribuição TecidualRESUMO
Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50µg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.
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DNA/administração & dosagem , Fígado/metabolismo , Microbolhas , Ondas Ultrassônicas , Animais , Técnicas de Transferência de Genes , Células HeLa , Humanos , Lipídeos/química , Fígado/diagnóstico por imagem , Luciferases/genética , Luciferases/metabolismo , Camundongos Endogâmicos BALB C , Plasmídeos , Transgenes , UltrassonografiaRESUMO
Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers.
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Inflamação/induzido quimicamente , Lipídeos/imunologia , Poliaminas/imunologia , Receptor 2 Toll-Like/imunologia , Linhagem Celular , Células HEK293 , Humanos , Inflamação/imunologia , Lipídeos/efeitos adversos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Simulação de Acoplamento Molecular , Ácidos Nucleicos/administração & dosagem , Ácidos Nucleicos/genética , Poliaminas/efeitos adversos , Transfecção , Fator de Necrose Tumoral alfa/imunologiaRESUMO
PURPOSE: The traditional surgical approach to treat multi-level cervical disc disease (mCDD) has been anterior cervical discectomy and fusion (ACDF). There has been recent development of other surgical approaches to further improve clinical outcomes. Collectively, when elements of these different approaches are combined in surgery, it is known as hybrid surgery (HS) which remains a novel treatment option. A systematic review and meta-analysis was conducted to compare the outcomes of HS versus ACDF for the treatment of mCDD. METHODS: Relevant articles were identified from six electronic databases from their inception to January 2016. RESULTS: From 8 relevant studies identified, 169 patients undergoing HS were compared with 193 ACDF procedures. Operative time was greater after HS by 42 min (p < 0.00001), with less intraoperative blood loss by 26 mL (p < 0.00001) and shorter return to work by 32 days (p < 0.00001). In terms of clinical outcomes, HS was associated with greater C2-C7 range of motion (ROM) preservation (p < 0.00001) and less functional impairment (p = 0.008) after surgery compared to ACDF. There was no significant difference between HS and ACDF with respect to postoperative pain (p = 0.12). The postoperative course following HS was not significantly different to ACDF in terms of length of stay (p = 0.24) and postoperative complication rates (p = 0.18). CONCLUSIONS: HS is a novel surgical approach to treat mCDD, associated with a greater operative time, less intraoperative blood loss and comparable if not superior clinical outcomes compared to ACDF. While it remains a viable consideration, there is a lack of robust clinical evidence in the literature. Future large prospective registries and randomised trials are warranted to validate the findings of this study.
Assuntos
Vértebras Cervicais/cirurgia , Discotomia , Degeneração do Disco Intervertebral/cirurgia , Deslocamento do Disco Intervertebral/cirurgia , Fusão Vertebral , Perda Sanguínea Cirúrgica , Humanos , Duração da Cirurgia , Dor Pós-Operatória , Complicações Pós-OperatóriasRESUMO
BACKGROUND: Surgical approaches for multi-level cervical spondylotic myelopathy (CSM) include posterior cervical surgery via laminectomy and fusion (LF) or expansive laminoplasty (EL). The relative benefits and risks of either approach in terms of clinical outcomes and complications are not well established. A systematic review and meta-analysis was conducted to address this topic. METHODS: Electronic searches were performed using six databases from their inception to January 2016, identifying all relevant randomized controlled trials (RCTs) and non-RCTs comparing LF vs EL for multi-level cervical myelopathy. Data was extracted and analyzed according to predefined endpoints. RESULTS: From 10 included studies, there were 335 patients who underwent LF compared to 320 patients who underwent EL. There was no significant difference found postoperatively between LF and EL groups in terms of postoperative JOA (P = 0.39), VAS neck pain (P = 0.93), postoperative CCI (P = 0.32) and Nurich grade (P = 0.42). The total complication rate was higher for LF compared to EL (26.4 vs 15.4 %, RR 1.77, 95 % CI 1.10, 2.85, I 2 = 34 %, P = 0.02). Reoperation rate was found to be similar between LF and EL groups (P = 0.52). A significantly higher pooled rate of nerve palsies was found in the LF group compared to EL (9.9 vs 3.7 %, RR 2.76, P = 0.03). No significant difference was found in terms of operative time and intraoperative blood loss. CONCLUSIONS: From the available low-quality evidence, LF and EL approaches for CSM demonstrates similar clinical improvement and loss of lordosis. However, a higher complication rate was found in LF group, including significantly higher nerve palsy complications. This requires further validation and investigation in larger sample-size prospective and randomized studies.
Assuntos
Vértebras Cervicais/cirurgia , Laminectomia , Laminoplastia , Doenças da Medula Espinal/cirurgia , Fusão Vertebral , Humanos , Complicações Pós-OperatóriasRESUMO
To provide long circulating nanoparticles able to carry a gene to tumors, we have designed anionic pegylated lipoplexes which are pH sensitive. Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. The neutralization of the particle surface charge as a function of the pH was monitored by light scattering in order to determine the ratio between anionic and cationic lipids that would give pH sensitive complexes. This ratio has been optimized to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes is checked by DNA accessibility to picogreen. The transfection efficiency and pH sensitive property of these formulations has been shown in vitro using bafilomycin, a vacuolar H+-ATPase inhibitor.