Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Graphene And Carbon Nanotube Nanocomposite For Gene Transfection.

Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Identification Of Corynebacterium Spp. Isolated From Bovine Intramammary Infections By Matrix-assisted Laser Desorption Ionization-time Of Flight Mass Spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Morphological Aspects And Cox-2 Expression After Exposure To 780-nm Laser Therapy In Injured Skeletal Muscle: An In Vivo Study.

The effectiveness of low-level laser therapy in muscle regeneration is still not well known. To investigate the effects of laser irradiation during muscle healing. For this purpose, 63 rats were distributed to 3 groups: non-irradiated control group (CG); group irradiated at 10 J/cm(2) (G10); and group irradiated at 50 J/cm(2) (G50). Each group was divided into 3 different subgroups (n=7), and on days 7, 14 and 21 post-injury the rats were sacrificed. Seven days post-surgery, the CG showed destroyed zones and extensive myofibrillar degeneration. For both treated groups, the necrosis area was smaller compared to the CG. On day 14 post-injury, treated groups demonstrated better tissue organization, with newly formed muscle fibers compared to the CG. On the 21(st) day, the irradiated groups showed similar patterns of tissue repair, with improved muscle structure at the site of the injury, resembling uninjured muscle tissue organization. Regarding collagen deposition, the G10 showed an increase in collagen synthesis. In the last period evaluated, both treated groups showed statistically higher values in comparison with the CG. Furthermore, laser irradiation at 10 J/cm(2) produced a down-regulation of cyclooxygenase 2 (Cox-2) immunoexpression on day 7 post-injury. Moreover, Cox-2 immunoexpression was decreased in both treated groups on day 14. Laser therapy at both fluencies stimulated muscle repair through the formation of new muscle fiber, increase in collagen synthesis, and down-regulation of Cox-2 expression.

Evaluation Of Photodynamic Therapy Using A Diode Laser And Different Photosensitizers Against Enterococcus Faecalis.

Photodynamic therapy (PDT) has been proven to be effective in disinfecting root canals. The aim of this present study was to evaluate the effects of PDT on the viability of Enterococcus faecalis using methylene blue (MB) and malachite green (MG) as photosensitizers. Solutions containing E. faecalis (ATCC 29212) were prepared and harvested by centrifugation to obtain cell suspensions, which were mixed with MB and MG. Samples were individually irradiated by the diode laser at a distance of 1mm for 30, 60, or 120 seconds. Colonyforming units (CFU) were determined for each treatment. PDT for 60 and 120 seconds with MG reduced E. faecalis viability significantly. Similar results were obtained when MB was used as photosensitizer. PDT using MB and MG have antibacterial effect against E. faecalis, showing potential to be used as an adjunctive antimicrobial procedure in endodontic therapy.

Laser Printer Attribution: Exploring New Features And Beyond.

With a huge amount of printed documents nowadays, identifying their source is useful for criminal investigations and also to authenticate digital copies of a document. In this paper, we propose novel techniques for laser printer attribution. Our solutions do not need very high resolution scanning of the investigated document and explore the multidirectional, multiscale and low-level gradient texture patterns yielded by printing devices. The main contributions of this work are: (1) the description of printed areas using multidirectional and multiscale co-occurring texture patterns; (2) description of texture on low-level gradient areas by a convolution texture gradient filter that emphasizes textures in specific transition areas and (3) the analysis of printer patterns in segments of interest, which we call frames, instead of whole documents or only printed letters. We show by experiments in a well documented dataset that the proposed methods outperform techniques described in the literature and present near-perfect classification accuracy being very promising for deployment in real-world forensic investigations.

A Metabolomic Protocol For Plant Systematics By Matrix-assisted Laser-desorption/ionization Time-of Flight Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) has been widely used for the identification and classification of microorganisms based on their proteomic fingerprints. However, the use of MALDI-TOF MS in plant research has been very limited. In the present study, a first protocol is proposed for metabolic fingerprinting by MALDI-TOF MS using three different MALDI matrices with subsequent multivariate data analysis by in-house algorithms implemented in the R environment for the taxonomic classification of plants from different genera, families and orders. By merging the data acquired with different matrices, different ionization modes and using careful algorithms and parameter selection, we demonstrate that a close taxonomic classification can be achieved based on plant metabolic fingerprints, with 92% similarity to the taxonomic classifications found in literature. The present work therefore highlights the great potential of applying MALDI-TOF MS for the taxonomic classification of plants and, furthermore, provides a preliminary foundation for future research.

Effect Of Low-level Laser Therapy (lllt) On Peripheral Nerve Regeneration Using Fibrin Glue Derived From Snake Venom.

The purpose of this study was to assess whether the adhesive permits the collateral repair of axons originating from a vagus nerve to the interior of a sural nerve graft, and whether low-level laser therapy (LLLT) assists in the regeneration process. Study sample consisted of 32 rats randomly separated into three groups: Control Group (CG; n=8), from which the intact sural nerve was collected; Experimental Group (EG; n=12), in which one of the ends of the sural nerve graft was coapted to the vagus nerve using the fibrin glue; and Experimental Group Laser (EGL; n=12), in which the animals underwent the same procedures as those in EG with the addition of LLLT. Ten weeks after surgery, the animals were euthanized. Morphological analysis by means of optical and electron microscopy, and morphometry of the regenerated fibers were employed to evaluate the results. Collateral regeneration of axons was observed from the vagus nerve to the interior of the autologous graft in EG and EGL, and in CG all dimensions measured were greater and presented a significant difference in relation to EG and EGL, except for the area and thickness of the myelin sheath, that showed significant difference only in relation to the EG. The present study demonstrated that the fibrin glue makes axonal regeneration feasible and is an efficient method to recover injured peripheral nerves, and the use of low-level laser therapy enhances nerve regeneration.

Low-level Laser Therapy To The Mouse Femur Enhances The Fungicidal Response Of Neutrophils Against Paracoccidioides Brasiliensis.

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.

Application Of Laser Microdissection Icp-ms For High Resolution Elemental Mapping In Mouse Brain Tissue: A Comparative Study With Laser Ablation Icp-ms.

Mapping of elements in biological tissue by laser induced mass spectrometry is a fast growing analytical methodology in life sciences. This method provides a multitude of useful information of metal, nonmetal, metalloid and isotopic distribution at major, minor and trace concentration ranges, usually with a lateral resolution of 12-160 µm. Selected applications in medical research require an improved lateral resolution of laser induced mass spectrometric technique at the low micrometre scale and below. The present work demonstrates the applicability of a recently developed analytical methodology - laser microdissection associated to inductively coupled plasma mass spectrometry (LMD ICP-MS) - to obtain elemental images of different solid biological samples at high lateral resolution. LMD ICP-MS images of mouse brain tissue samples stained with uranium and native are shown, and a direct comparison of LMD and laser ablation (LA) ICP-MS imaging methodologies, in terms of elemental quantification, is performed.

Influência da redução medicamentosa da pressão intra-ocular na medida da espessura da camada de fibras nervosas da retina de olhos hipertensos e glaucomatosos pela polarimetria de varredura a laser

PURPOSE: To evaluate changes in retinal nerve fiber layer thickness as measured by scanning laser polarimetry (SLP) after the use of medication to reduce intraocular pressure (IOP) in glaucomatous or ocular hypertensive patients. METHODS: The authors prospectively enrolled 37 eyes of 37 patients in whom IOP was reduced by more than 25% after the use of medication. The images were obtained before and 15 to 30 days after the introduction of medication. The SLP parameters measured before and after the use of medication were compared using paired Student's t Test. RESULTS: The mean IOP was significantly reduced from 26.57±4.23 mmHg to 16.54 ±2.92 mmHg after the use of medication (p<0.05). None of the 10 SLP analyzed parameters was significantly affected by the reduction of IOP with medication (p>0.05). CONCLUSION: The retinal nerve fiber layer thickness, as measured by SLP, is not affected by the reduction of IOP with medication in patients with glaucoma or ocular hypertension.

Assessment in vitro of brushing on dental surface roughness alteration by laser interferometry

Noncarious cervical lesions (NCCLs) are considered to be of multifactorial origin, normally associated with inadequate brushing. This study assessed the influence in vitro of simulated brushing on NCCL formation. Fifteen human premolars were submitted to brushing in the cementoenamel junction region, using hard-, medium- and soft-bristled brushes associated with a toothpaste of medium abrasiveness under a 200 g load, at a speed of 356 rpm for 100 minutes. The surface topography of the region was analyzed before and after brushing, by means of a laser interferometer, using "cut-off" values of 0.25 and considering roughness values in mm. The initial roughness (mm) results for dentin (D / bristle consistency: 1 - soft, 2 - medium and 3 - hard) were as follows: (D1) 1.25 ± 0.45; (D2) 1.12 ± 0.44; (D3) 1.05 ± 0.41. For enamel (E / bristle consistency: 1 - soft, 2 - medium and 3 - hard), the initial results were: (E1) 1.18 ± 0.35; (E2) 1.32 ± 0.25; (E3) 1.50 ± 0.38. After brushing, the following were the values for dentin: (D1) 2.32 ± 1.99; (D2) 3.30 ± 0.96; (D3) Over 500. For enamel, the values after brushing were: (E1) 1.37 ± 0.31; (E2) 2.15 ± 0.90; (E3) 1.22 ± 0.47. Based on the results of the ANOVA and Tukey statistical analyses (a = .05) it was concluded that soft, medium and hard brushes are not capable of abrading enamel, whereas dentin showed changes in surface roughness by the action of medium- and hard-bristled brushes.

Effect of laser welding on the titanium composite tensile bond strength

The aim of this study was to analyze the shear bond strength between commercially pure titanium, with and without laser welding, after airbone-particle abrasion (Al2O3) and 2 indirect composites. Sixty-four specimens were cast and divided into 2 groups with and without laser welding. Each group was divided in 4 subgroups, related to Al2O3 grain size: A - 250 µm; B - 180 µm; C- 110 µm; and D - 50 µm. Composite rings were formed around the rods and light polymerized using UniXS unit. Specimens were invested and their shear bond strength at failure was measured with a universal testing machine at a crosshead speed of 2.0 mm/min. Statistical analysis was carried out with ANOVA and Tukey's test (α=0.05). The highest bond strength means were recorded in 250 µm group without laser welding. The lowest shear bond strength means were recorded in 50 µm group with laser welding. Statistically significant differences (p<0.05) were found between all groups. In conclusion, airborne particle abrasion yielded significantly lower bond strength as the Al2O3 particle size decreased. Shear bond strength decreased in the laser welded specimens.

Measurements of jaw movements and TMJ pain intensity in patients treated with GaAlAs laser

The aim of this study was to evaluate the effectiveness of low-level laser therapy (LLLT) on the improvement of the mandibular movements and painful symptoms in individuals with temporomandibular disorders (TMD). Forty patients were randomly divided into two groups (n=20): Group 1 received the effective dose (GaAlAs laser ? 830 nm, 40 mW, 5J/cm2) and Group 2 received the placebo application (0 J/cm2), in continuous mode on the affected condyle lateral pole: superior, anterior, posterior, and posterior-inferior, twice a week during 4 weeks. Four evaluations were performed: E1 (before laser application), E2 (right after the last application), E3 (one week after the last application) and E4 (30 days after the last application). The Kruskal-Wallis test showed significant more improvements (p<0.01) in painful symptoms in the treated group than in the placebo group. A significant improvement in the range of mandibular movements was observed when the results were compared between the groups at E4. Laser application can be a supportive therapy in the treatment of TMD, since it resulted in the immediate decrease of painful symptoms and increased range of mandibular movements in the treated group. The same results were not observed in the placebo group.