The effects of conjugating anti-MUC1 aptamers on gold nanobipyramids and nanostars for photothermal cancer ablation.

Aim: To ascertain the impact of shape and surface modification of anisotropic nanoparticles on the toxicity and photothermal efficiency toward cancerous cell lines.Methods: Gold nanobipyramids and nanostars surface modified with MUC1 aptamer were used in the current study to explore the toxicity and photothermal efficiency on MCF7 breast cancer cell lines via MTT assay.Results: Surface functionalization with MUC1 aptamer showed significant reduction in % cytotoxicity and increase in % specific internalization of nanostructures into MCF7 cell lines. Further, the photothermal studies accomplished at IC50 concentration for 6 h of treatment and laser exposure for 15 min reported that aptamer-conjugated nanobipyramids were more effective and specific toward MCF7 cell lines than aptamer-conjugated nanostars.Conclusion: This work establishes a platform for the development of tailored photoablation based gold nanostructures for in vivo studies.

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Supramolecular dye nanoassemblies for advanced diagnostics and therapies.

Dyes have conventionally been used in medicine for staining cells, tissues, and organelles. Since these compounds are also known as photosensitizers (PSs) which exhibit photoresponsivity upon photon illumination, there is a high desire towards formulating these molecules into nanoparticles (NPs) to achieve improved delivery efficiency and enhanced stability for novel imaging and therapeutic applications. Furthermore, it has been shown that some of the photophysical properties of these molecules can be altered upon NP formation thereby playing a major role in the outcome of their application. In this review, we primarily focus on introducing dye categories, their formulation strategies and how these strategies affect their photophysical properties in the context of photothermal and non-photothermal applications. More specifically, the most recent progress showing the potential of dye supramolecular assemblies in modalities such as photoacoustic and fluorescence imaging, photothermal and photodynamic therapies as well as their employment in photoablation as a novel modality will be outlined. Aside from their photophysical activity, we delve shortly into the emerging application of dyes as drug stabilizing agents where these molecules are used together with aggregator molecules to form stable nanoparticles.

Brain-Mimicking Phantom for Photoablation and Visualization.

While the use of tissue-mimicking (TM) phantoms has been ubiquitous in surgical robotics, the translation of technology from laboratory experiments to equivalent intraoperative tissue conditions has been a challenge. The increasing use of lasers for surgical tumor resection has introduced the need to develop a modular, low-cost, functionally relevant TM phantom to model the complex laser-tissue interaction. In this paper, a TM phantom with mechanically and thermally similar properties as human brain tissue suited for photoablation studies and subsequent visualization is developed. The proposed study demonstrates the tuned phantom response to laser ablation for fixed laser power, time, and angle. Additionally, the ablated crater profile is visualized using optical coherence tomography (OCT), enabling high-resolution surface profile generation.

Salmonella typhimurium detection and ablation using OmpD specific aptamer with non-magnetic and magnetic graphene oxide.

Foodborne diseases have increased in the last few years due to the increased consumption of packaged and contaminated food. Major foodborne bacteria cause diseases such as diarrhea, vomiting, and sometimes death. So, there is a need for early detection of foodborne bacteria as pre-existing detection techniques are time-taking and tedious. Aptamer has gained interest due to its high stability, specificity, and sensitivity. Here, aptamer has been developed against Salmonella Typhimurium through the Cell-Selex method, and to further find the reason for specificity and sensitivity, OmpD protein was isolated, and binding studies were done. Single molecular FRET experiment using aptamer and graphene oxide studies has also been done to understand the mechanism of FRET and subsequently used for target bacterial detection. Using this assay, Salmonella Typhimurium can be detected up to 10 CFU/mL. Further, Magnetic Graphene oxide was used to develop an assay to separate and ablate bacteria using 808 nm NIR where temperature increase was more than 60 °C within 30 s and has been shown by plating as well as a confocal live dead assay. Thus, using various techniques, bacteria can be detected and ablated using specific aptamer and Graphene oxide.

Ultrafast and Resist-Free Nanopatterning of 2D Materials by Femtosecond Laser Irradiation.

The performance of two-dimensional (2D) materials is promising for electronic, photonic, and sensing devices since they possess large surface-to-volume ratios, high mechanical strength, and broadband light sensitivity. While significant advances have been made in synthesizing and transferring 2D materials onto different substrates, there is still the need for scalable patterning of 2D materials with nanoscale precision. Conventional lithography methods require protective layers such as resist or metals that can contaminate or degrade the 2D materials and deteriorate the final device performance. Current resist-free patterning methods are limited in throughput and typically require custom-made equipment. To address these limitations, we demonstrate the noncontact and resist-free patterning of platinum diselenide (PtSe2), molybdenum disulfide (MoS2), and graphene layers with nanoscale precision at high processing speed while preserving the integrity of the surrounding material. We use a commercial, off-the-shelf two-photon 3D printer to directly write patterns in the 2D materials with features down to 100 nm at a maximum writing speed of 50 mm/s. We successfully remove a continuous film of 2D material from a 200 µm × 200 µm substrate area in less than 3 s. Since two-photon 3D printers are becoming increasingly available in research laboratories and industrial facilities, we expect this method to enable fast prototyping of devices based on 2D materials across various research areas.

Esterase-Activated, pH-Responsive, and Genetically Targetable Nano-Prodrug for Cancer Cell Photo-Ablation.

Activatable prodrugs have drawn considerable attention for cancer cell ablation owing to their high specificity in drug delivery systems. However, phototheranostic prodrugs with dual organelle-targeting and synergistic effects are still rare due to low intelligence of their structures. Besides, the cell membrane, exocytosis, and diffusional hindrance by the extracellular matrix reduce drug uptake. Moreover, the up-regulation of heat shock protein and short singlet-oxygen lifetime in cancer cells hamper photo-ablation efficacy, especially in the mono-therapeutic model. To overcome those obstacles, we prepare an esterase-activated DM nano-prodrug, which is conjugated by diiodine-substituted fluorogenic malachite green derivative (MG-2I) and phototherapeutic agent DPP-OH via hydrolyzable ester linkage, having pH-responsiveness and genetically targetable activity for dual organelles-targeting to optimize photo-ablation efficacy. The DM nanoparticles (NPs) present improved pH-responsive photothermal/photodynamic property by the protonation of diethylaminophenyl units in acidic environment. More importantly, the MG-2I and DPP-OH moieties can be released from DM nano-prodrug through overexpressed esterase; then specifically target lysosomes and mitochondria in CT-26 Mito-FAP cells. Hence, near-infrared DM NPs can trigger parallel damage in dual-organelles with strong fluorescence and effective phototoxicity, thus inducing serious mitochondrial dysfunction and apoptotic death, showing excellent photo-ablation effect based on esterase-activated, pH-responsive, and genetically targetable activities.

Quantifying Strain-Sensing Protein Recruitment During Stress Fiber Repair.

A family of proteins have been identified that recognize damaged, strained actin filaments in stress fibers. These proteins are often referred to as strain- or force-sensing and trigger downstream signaling mechanisms that can facilitate repair at these strain sites. Here we describe a method using high-resolution microscopy to screen and quantify the mechanosensitive recruitment of proteins to these stress fiber strain sites. Strain sites are induced using spatially controlled illumination of UV light to locally damage actin stress fibers. Recruitment of potential strain-sensing proteins can then either be compared to (Blanchoin, Physiol Rev 94, 235-263, 2014) a known control (e.g., zyxin-GFP) or (Hoffman, Mol Biol Cell 23, 1846-1859, 2012) the pre-damaged stress fiber protein distribution. With this method, strain-sensing proteins and their dynamic association with stress fiber strain sites can be reproducibly measured and compared.

Sequential intracorneal ring segment implantation followed by transepithelial phototherapeutic keratectomy and corneal cross-linking.

PURPOSE: To evaluate the safety and visual outcomes of intrastromal corneal ring segment (ICRS) implantation followed by transepithelial phototherapeutic keratectomy (te-PTK) and corneal cross-linking (CXL) in patients with mild keratoconus. METHODS: Patients with mild keratoconus and contact lens intolerance who underwent sequential ICRS implantation followed by phototherapeutic keratectomy and corneal CXL between April 2015 and July 2018 were retrospectively included in the study. Refractive and visual outcomes, satisfaction questionnaire and complications were recorded at the last follow-up (mean 9.5 months postoperatively). RESULTS: Twenty eyes of 17 patients were enrolled, including 5 women and 15 men. The mean time between the two procedures was 16 months. Based on values before the first procedure and 9.5 months after the second procedure, significant improvements were noted in uncorrected distance visual acuity (UDVA) (0.80±0.35 logMAR vs. 0.46±0.38 logMAR), corrected distance visual acuity (CDVA) (0.38±0.23 logMAR vs. 0.13±0.16 logMAR), maximal K (56.11±4 diopters [D] vs. 50.6±3.56 D), mean K (51.87±3.43 D vs. 48.45±2.91 D), cylinder (7.99±3.94 D vs. 4.23±3.49 D), and spherical equivalent (-3.84±3.36 D vs. -0.99±2.15 D) (P<0.01). Among the outcomes, we noted 5 (25%) superficial corneal scarring (haze); 75% of eyes gained>=1 logMAR line of CDVA. In all, 94.5% of patients reported that they were satisfied with their outcomes. CONCLUSION: Combining ICRS implantation followed by te-PTK and corneal CXL appears to be a safe and effective approach for improving visual outcomes and quality of life in keratoconus patients.

MiniSOG2-mediated Specific Photoablation of Motor Neurons in Ascidian Embryos.

When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light-oxygen-voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A ( Ciona robusta) , a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera. Graphical abstract.

Light-Stimulated Carbon Dot Hydrogel: Targeting and Clearing Infectious Bacteria In Vivo.

Infectious bacteria evolve fast into resistance to conventional antimicrobial agents, whereas treatments for drug resistance bacteria progress more slowly. Here, we report a universally applicable photoactivated antimicrobial modality through light-responsive carbon dot-embedding soft hyaluronic acid hydrogel (CDgel). Because of the innate nature of the infectious bacteria that produce hyaluronidase, applied hyaluronic acid-based CDgel breaks down via bacteria and releases carbon dots (CDs) into the infectious sites. The released CDs possess photodynamic capabilities under light irradiation, inducing 1O2 generation and growth inhibition of the infectious bacteria, S. aureus and E. coli (∼99% and ∼97%, respectively), in vitro. In particular, these photodynamic effects of CDs from CDgel have been shown to accelerate the healing of infected wounds in vivo, showing a higher wound regeneration rate as compared to that of untreated wounds. Our work demonstrates that the biocompatible and shape-controllable CDgel possesses therapeutic potential as a treatment modality for the light-driven control of drug-resistant bacterial infections.

Photoablation at single cell resolution and its application in the Drosophila epidermis and peripheral nervous system.

Tissues contain diverse cell populations that, together, make up physiologically functional units. A remarkable example is the animal epidermis, where neuronal and non-neuronal cells intermingle to allow somatosensory perception. In the peripheral nervous system (PNS), the tight association between heterogenous cell types poses challenges when the structural and physiological contributions of neuronal and surrounding cells need to be dissected with suitable precision. When genetic tools for cell-specific, spatiotemporally controlled gene expression are not available, targeted cell ablation represents a considerable obstacle. Here, we describe an efficient method to overcome this limitation and demonstrate its application to the study of the differentiating Drosophila epidermis and PNS. This methodology relies on the use of near infrared (NIR) femtosecond (fs) laser pulses for ablation of the desired cells at the desired time. We show how to confine the photodamage to the targeted cell to induce its death, without harming neighbouring tissues or structures. We validated our approach in the Drosophila PNS by studying the responses of photo-ablated neurons, non-neuronal cells, and the surrounding epidermis. Diverse cellular behaviours including cell extrusion, cell rearrangements and cell shape changes can be monitored in vivo immediately after damage, as well as for several hours post-ablation with high optical resolution using confocal microscopy. This methodology provides a flexible tool to ablate individual cells with high precision and study morphological responses to cell loss in targeted areas or neighbouring structures. We anticipate that this protocol can be easily adapted to other model systems and tissues.

Outpatient 180 W XPS GreenLight Laser Photoselective Vaporization of the Prostate: 7-Year Experience.

Purpose: GreenLight XPS Laser System (GL-XPS) photoselective vaporization of the prostate (PVP) is not only noninferior to transurethral resection of the prostate, but also with shorter rates of hospital stay and length of catheterization. Scarce literature has been published about the feasibility and safety of performing GL-XPS PVP in an outpatient setting. Our aim is to report our 7-year experience with outpatient GL-XPS PVP. Methods: Medical charts of all patients who underwent GL-XPS PVP between 2013 and 2020 were reviewed. Patients were discharged after careful monitoring in the recovery room and the catheter was removed either at home or at a scheduled hospital visit. We used the Shapiro-Wilk test to assess for normal distribution, and the evaluation of homoscedasticity was performed with the SD test. For qualitative variables, the comparison between groups was carried using the chi-square test and for the quantitative variables we used the nonparametric Mann-Whitney U test. Results: A total of 537 patients were treated, 517 in an outpatient basis. Median age was 68 years (interquartile range [IQR] 62-76), median prostate volume 50 cc (IQR 40-70). 22.8% were on anticoagulants or antiplatelet therapy. Median operative time was 100 minutes (IQR 75-125), length of hospital stay 5 hours (IQR 4-6.45), mean length of catheterization 48 hours (SD 14.92). Readmission rate within 90 days of surgery was 11.7%, 58 (10.8%) for surgical-related complications. Median time for readmission was 4 days (IQR 2-24). Immediate readmission (within 10 days) was recorded in 7.6% of patients, urinary retention was the main cause. No significant differences were found trying to find perioperative predictors for readmission. Conclusions: Our experience suggests that outpatient GL-XPS PVP could be performed safely with a predefined outpatient pathway, with a low readmission and complication rate. (IRB number CEI-521).

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes.

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.

Multiphoton Laser Fabrication of Hybrid Photo-Activable Biomaterials.

The possibility to shape stimulus-responsive optical polymers, especially hydrogels, by means of laser 3D printing and ablation is fostering a new concept of "smart" micro-devices that can be used for imaging, thermal stimulation, energy transducing and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. In light of the increasing demand for micro-devices for nanomedicine and personalized medicine, interest is growing in the combination of composite and hybrid photo-responsive materials and digital micro-/nano-manufacturing. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. Less often, the two approaches have been combined and active nanomaterials have been embedded in the microstructures. The aim of this review is to give a short overview of the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photothermal activity, chemical swelling and chemical sensing.

Bubble Forming Films for Spatial Selective Cell Killing.

Photodynamic and photothermal cell killing at the surface of tissues finds applications in medicine. However, a lack of control over heat dissipation following a treatment with light might damage surrounding tissues. A new strategy to kill cells at the surface of tissues is reported. Polymeric films are designed in which iron oxide nanoparticles are embedded as photosensitizers. Irradiation of the films with pulsed laser light generates water vapor bubbles at the surface of the films. It is found that "bubble-films" can kill cells in close proximity to the films due to mechanical forces which arise when the bubbles collapse. Local irradiation of bubble-films allows for spatial selective single cell killing. As nanosurgery becomes attractive in ophthalmology to remove superficial tumors, bubble-films are applied on the cornea and it is found that irradiation of the bubble-films allows spatial and selective killing of corneal cells. As i) the photosensitizer is embedded in the films, which reduces its uptake by cells and spreading into tissues and ii) the bubble-films can be removed from the tissue after laser treatment, while iii) a low laser fluence is sufficient to generate vapor bubbles, it is foreseen that bubble-films might become promising for safe resection of superficial tumors.

Physiologically triggered injectable red blood cell-based gel for tumor photoablation and enhanced cancer immunotherapy.

Hydrogels are widely used for drug delivery and tissue engineering. Here we developed a simple injectable red blood cells (RBCs)-based gel for cancer photo-immunotherapy. We find that subcutaneous injected homologous RBCs could form hydrogel-like composition in mice, due to the infiltrated platelets and thrombin under physiological environment. In addition, the formed RBC-gel has photothermal effect under NIR laser exposure on account of deep reddish color. In mice bearing CT26 tumors, we demonstrate photo-immunotherapy of cancer by local injection of imiquimod (R837) adjuvant engineered RBCs. The photothermal effect of the in situ formed RBC-gel effectively burns tumor to release tumor-associated antigens (TAAs), promotes the release of R837 from RBCs to the tumor draining lymph node, thereby activating the lymph node-resident antigen-presenting cells (APCs) remarkably. A durable systemic immune response is induced following the combination treatment of the primary tumor. 100% mice rejected tumor rechallenge and are survived at least 250 days without any detectable tumors. Our strategy highlights the RBCs, the most common type of cell in our blood, as the hydrogel for drug delivery and cancer photo-immunotherapy.

Treatment outside the Recommended Guidelines for Retinopathy of Prematurity (ROP): Prevalence, Characteristics, and Issues.

This study aims to assess the prevalence and characteristics of preterm infants with retinopathy of prematurity (ROP) treated outside the recommended guidelines. In this retrospective monocentric cohort, we included all premature children treated in our department for ROP by laser photoablation or anti-VEGF intravitreal injection. The main outcome was treatment of both eyes for ROP less severe than pre-threshold type 1, treated outside ETROP guidelines. A total of 114 children received treatment for ROP in our department, among whom 32 (28.1%) children received treatment for indications outside the ETROP guidelines for both eyes. The indications outside the guidelines were persistent stage 2 or 3 ROP that showed no evidence of regression after 41 weeks of corrected gestational age (11 children; 34.4%), pre-plus stage (11; 34.4%), difficulties in disease staging (7; 21.9%), type 2 ROP with plus disease (2; 6.2%), and treatment due to logistical difficulties (1; 3.1%; hospitalized in neonatal units hundreds of miles away from our department, with no fundus examination possible in the neonatal unit). To resume, in our cohort, 28.1% of children received treatment for ROP less severe than pre-threshold type 1 both eyes. The main indications for off-label treatment were the persistence of active ROP during follow-up and the presence of pre-plus-stage disease. Our data suggest the need to update ROP treatment criteria to reflect real-life practices. Additional studies are required in order to evaluate the long-term benefits and side effects of treatments outside the recommended indications, and to establish revised treatment guidelines.

Laser-assisted nucleic acid delivery: A systematic review.

Gene therapy has become an effective treatment modality for some conditions. Laser light may augment or enhance gene therapy through photomechanical, photothermal, and photochemical. This review examined the evidence base for laser therapy to enhance nucleic acid transfection in mammalian cells. An electronic search of MEDLINE, Scopus, EMBASE, Web of Science, and Google Scholar was performed, covering all available years. The preferred reporting items for systematic reviews and meta-analyses guideline for systematic reviews was used for designing the study and analyzing the results. In total, 49 studies of laser irradiation for nucleic acid delivery were included. Key approaches were optoporation, photomechanical gene transfection, and photochemical internalization. Optoporation is better suited to cells in culture, photomechanical and photochemical approaches appear well suited to in vivo use. Additional studies explored the impact of photothermal for enhancing gene transfection. Each approach has merits and limitations. Augmenting nucleic acid delivery using laser irradiation is a promising method for improving gene therapy. Laser protocols can be non-invasive because of the penetration of desirable wavelengths of light, but it depends on various parameters such as power density, treatment duration, irradiation mode, etc. The current protocols show low efficiency, and there is a need for further work to optimize irradiation parameters.

Subcellular Singlet Oxygen and Cell Death: Location Matters.

We developed a tool for targeted generation of singlet oxygen using light activation of a genetically encoded fluorogen-activating protein complexed with a unique dye molecule that becomes a potent photosensitizer upon interaction with the protein. By targeting the protein receptor to activate this dye in distinct subcellular locations at consistent per-cell concentrations, we investigated the impact of localized production of singlet oxygen on induction of cell death. We analyzed light dose-dependent cytotoxic response and characterized the apoptotic vs. necrotic cell death as a function of subcellular location, including the nucleus, the cytosol, the endoplasmic reticulum, the mitochondria, and the membrane. We find that different subcellular origins of singlet oxygen have different potencies in cytotoxic response and the pathways of cell death, and we observed that CT26 and HEK293 cell lines are differentially sensitive to mitochondrially localized singlet oxygen stresses. This work provides new insight into the function of type II reactive oxygen generating photosensitizing processes in inducing targeted cell death and raises interesting mechanistic questions about tolerance and survival mechanisms in studies of oxidative stress in clonal cell populations.